Standard Operation Procedures for evaluating ...

Standard Operation Procedures for evaluating COMPRO Products The procedures are based on the microorganisms in the most common in commercial products. Included are products containing symbiotic nitrogen fixation, free living nitrogen fixation (Azospirillum and Azotobacter), Phosphorus Solubilizing Rhizobacteria (Bacillus and Pseudomonas), Arbuscular mycorrhiza fungi (nutrient uptake) and Trichorderma (biocontrol).

A. SOP for Testing the Quality of Commercial Symbiotic Nitrogen fixation Products 1.0.

Definitions

For the purpose of this standard the following definitions shall apply: The following are definition of terms used in synonym and those in the SOP. The terminologies Biofertilizer, Bioenhancer, Biostimulant and Bioinoculant are sometimes used synonymously. Biofertilizers: is a substance which contains living microorganisms which are applied to seed, plant surface or product colonizes the rhizosphere or the interior of the plant or soil and promotes growth by increasing supply or availability of nutrients to the host plant. It is a term widely used term meaning ―bacterial inoculant‖ usually refer to preparations of microorganisms that may be a partial or complete substitute for chemical fertilization (like rhizobial inoculants). The use of word fertilizer allows easier registration for commercial. It is a formulation containing one or more beneficial strains (or species) in an easy to use and economical carrier either organic, inorganic or synthesized from defined molecules. It contains living microorganisms embedded in a carrier material which are applied to seed, plant surface or product colonizers the rhizosphere or the interior of the plant or soil and promotes growth by increasing supply or availability of nutrients to the host plant, or preparations containing living cells or organisms that enrich the nutrient quality of product. Bioenhancers: Substances that increase the bioavailability of active ingredients, vitamins and nutrients Biostimulants: Dormant strains of positive product microorganisms that come alive when introduced into the product system. They contain strains of specific bacteria, fungi or algae which take up nutrients and make it available to plants, collect and store available nutrients, enhance uptake of nutrients, provide physical barriers against pathogens, stimulate growth, decompose organic residues Bacteria: A Kingdom of single celled microorganisms whose cells lack membrane-bound nucleus (prokaryotic). Microorganisms that inhabit soil, water, plants and animals.

Symbiotic bacteria: Species of bacteria living in a mutual relationship with plants where they convert atmospheric nitrogen to nitrates which are absorbed by the plants. They in turn obtain sugars from the plant. Rhizobia: Soil bacteria that fix nitrogen (diazotrophs) after becoming established inside root nodules of legumes (Fabaceae). Rhizobia require a plant host: They cannot independently fix nitrogen. In general they are gram-negative, motile non-sporulating rods. They are unique in that they are the only living nitrogen-fixing bacteria living in a symbotic relationship with legumes. Common crop and forage legumes are peas, beans, clover and soy. Contamination: In other microorganism other than bacteria not declared on the label. Aseptic Conditions/Aseptically: Environment where no microorganisms is present. It can be obtained by using a Bunsen burner to create an aseptic zone which is the aspheric area around the flame with a diameter of approximately 15 cm or a Laminar flow hood with sterile air continuously produced and present in the hood. The high pressure of air flow from the hood prevents the air from outside to come inside and contaminate the environment in the hood; aseptic conditions are also created by cleaning surface with 70% ethanol. Contaminated microorganisms: Every plate, tube, pipette, or other instruments (glassware, pestles, eppendorff tube…) which has been in contact with microorganisms and cannot be sterilized by the flame of a Bunsen burner is considered as contaminated. Contaminated by toxic chemicals: Every tube, flasks, pipette or other instruments which has been in contact with toxic chemicals is considered contaminated Good Laboratory Practices (GLP): The principles of GLP have been developed to promote the quality and validity of results and of the analysis conducted in a laboratory. It is a management concept covering the organization and the conditions under which laboratory studies are planned, performed, monitored, recorded and reported. Its principle also includes the protection of man and the environment. Mother tube/plate/products: Tube/plate/product which the bacteria are picked from. The result of the growth of this inoculation is considered as the daughter, which can become the mother for the next inoculation. Substrate: Any material, that serves as source of energy for an organism. Carrier: Delivery vehicle of live microorganisms from the factory to the field. The carrier is the major portion of inoculant. Materials and types of formulations of carriers vary: Slurry, powder, peat, liquid.

2.0.

Introduction

Farmers have knowledge on benefits on improved yield of non-legumes derived from mixing crops with legume crops. Inoculation of plants with beneficial bacteria can be traced back for centuries. For over 100 years rhizobial inoculants have been produced around the world primarily by small companies. Rhizobial inoculants have been produced small companies for over 100 years, but is only recently that the first commercial preparations have appeared in

the market. The rhizobial products still have less penetration compared to the chemical and pesticide market. Major impacts of inoculaton are seen in soybean in Brazil, USA, Argentina, Australia, South Africa. Rhizobia have legally established standards although the inoculant quality in Africa is not properly regulated. Inoculants may vary in formulations with carriers of different characteristics, physical and chemical composition and quality. The carrier may be sterile or the most commonly used non sterile carriers. In most countries there are no regulations on the population levels in the most commonly used non sterile peat preparations. There are therefore no common standards in rhizobial inoculants. The Rhizobial inocula level varies worldwide between 107 and 4 x 99 cfu/g inoculant with inoculants in Canada ranging from 103 – 105 nodulating Rhizobia per seed (size dependent) or 1011 rhizobia per ha. A survey in Canada showed level sometimes exceeding by several orders of magnitude. The contaminant species could have detrimental effects on Rhizobia. It is not clear or known whether contaminants present health hazards to humans and animals and plants. France does not tolerate contamination, Australia allows 0.1% of total bacterial population and requires very high rhizobial populations. In some countries inoculum quality is left to the market forces and manufacturers discretion. To guarantee farmers quality, it is paramount that standards are established to regulate quality of products. There is presently no universal carrier or formulation available for release of microorganism into soil. A good carrier should have the capacity to deliver the right number of viable cells in good physiological condition at the right time. A carrier may be sterile or non-sterile 1. Purpose The Standard Operation Procedure (SOP) is intended to guide laboratories appointed by regulatory agencies in screening of Nitrogen fixing (Rhizobial products) commercial products for quality. Based on the information on the product label, the screening is to confirm the strains, populations and ability to produces effective nodules. This is to guarantee customers quality product devoid of contaminants. This exercise is also to verify if there are any contaminants that can reduce the efficacy of the product or are harmful to plants, human, animals and the environment. Proper sampling and handling of the product at port of entry and careful dispatch to the testing laboratory is to be guaranteed to avoid any claims of contamination by the product proponent. The final outcome is to guide regulatory agencies on the quality of the product and its suitability for use by farmers. The SOP outlines the procedures provided by the regulatory agency to generate quality data as stipulated on the label and of acceptable standards set by regulatory agencies. 2. Requirements for Quality Control Good laboratory practices and precautionary statements (i.e. safety) Precaution Autoclave: The autoclave can pose a great danger if not used correctly because of high pressure and temperature. Before using, visually check the general aspect (no corrosion, no leak), the quality of water and settings. Strictly follow the instructions to start it. During heating phase, the pressure increases. Be sure that the door is correctly closed so that there is no leakage. Don‘t try to force the autoclave to open. The autoclave has a safety devise and will refuse to open if the temperature is still high or if the pressure is not bak to atmospheric pressure. The maintenance has to be done regularly and the results recorded in a specific file (maintenance file, available in the office).

Bunsen Burner: The risk of fire can be minimised by following a few simple rules. If the hood is being used, turn on the fire only when it is needed, don‘t cross your arms, don‘t pass your arm on the burner. Laminar flow hood: If he hood is not working properly, it can lead to a fire risk. The maintenance has to be done regularly and results recorded in a specific file (maintenance file). Biological Hazards: Manipulating microorganisms poses a risk not only to the one who is working, but also to other people in the lab and potentially to the environment in case of dissemination. The rulers of safety have to be well understood and respected in order to avoid any contamination of staff and/or the environment. In case of accidental contamination, clean and disinfect properly before the activities can be restarted. Dispose contaminated waste. Chemicals: Before using a new chemical, the information about toxicity, conditions of use, risks and safety phrases… have to be understood and observed. Use the equipment of protection if needed (gloves, masks, hood…). The Material Safety Data Sheet (MSDS) of different chemicals are also available to get more details about the products.

3. Quality control management (General procedures) 1. A specific protocol is established before screening is set up. It includes the number of samples, the details about the inoculants (origin, composition, rate of application...), and any other relevant information. 2. Calculations, weights, records (of temperature, humidity...), observations, measurements, preparation of stock solutions (nutritive solutions for example) and any other relevant information are recorded in the lab book on a daily basis. 3. The stock solutions are labelled with the date of reception, name or initials of the person who received it, the number of the container (x of n), date of opening, name or initial of the person who opened it. 4. The reagents are labelled with the name of the contents, date of preparation, name or initials of the person who prepared them and any other relevant information. 5. All samples are clearly identified and records are taken about the different steps of analysis (when they have been put in the oven, grinded, sent for analysis). 6. After harvesting, samples might need to be decontaminated before elimination. Solarization may be considered. 7. In case of contamination of the bench, floor, user, ..., it has to be cleaned and disinfected before the work can be continued (cf. ―Hygiene and Safety rules in a laboratory‖ document). 8. Non contaminated or decontaminated items are cleaned with soap, and rinsed with water and eventually rinsed with distilled water. 9. Waste management: a. Non contaminated waste is eliminated in the normal bin. b. Non contaminated glass waste are put in a separate container labelled with the mention ―Broken glass‖ c. Anything contaminated by microorganisms should be decontaminated before appropriate elimination/cleaning. d. Waste may be put into a special autoclave bags for 20 mins at 121 ºC. e. Solid and liquid waste contaminated by toxic chemicals are placed in a separate container to be collected by special disposable service providers.

f. Disposable glass instruments are ut into a container containing Sodium Hypochlorite solution (commercial Jik) for decontamination before being eliminated. Solid (including broken glass) and liquid waste contaminated by toxic chemicals are placed in separate containers for elimination by waste disposal company. g. Contaminated glassware is properly rinsed with tap water and water collected in specific items (consult MSDS) on product disposal. 10. Equipment‘s are regularly cleaned to remove dust, samples or other waste. 11. Room is cleaned daily. Disinfect every surface with disinfectant and or with 70% ethanol before starting work. The bench and the floor is to be cleaned and disinfected before the work begins and in case of any contamination during the working session it should be disinfected before work continues.

4. General laboratory practices 1. Always wear a lab coat when working in the laboratory. 2. Never put on gloves when working close to flames. In case of burn, the glove would stick on your skin and make the wound worse. 3. To maintain good hygiene, clean your hands with soap and work under aseptic conditions. Clean bench of hood and any surface of operation with a disinfectant; pour some disinfectant on the bench, leave for I min and wipe with gauze/cotton wool. It is also possible to clean the bench with 70% ethanol. 4. Always have a seat when working to avoid accidents. Avoid useless movement in the lab during work. Organize your work space to ensure a smooth run to the analysis. Some equipment need to be switched on some time before use (incubator, laminar flow hood). 5. Plan your work to give them enough time. Organize everything you need in the hood before starting to work so that you don‘t have to cross hands or have to have put your hand out of the hood. Never put items close to the back of the hood because it can affect the flow hence affect the sterility of air. Use preferably the middle and sides of the bench. 6. Every aseptic manipulation has to be done in the middle of the hood, never outside of it and not at the side or the back of the hood. 7. Air contaminations are common. To avoid them, don‘t speak, run or move it it is not necessary and keep the door and windows closed. 8. A burner is used to sterilize items such as loops, tubes, or forceps. The hottest part of the flame is the top of the blue part, so this is the part which has to be used. If you have to work without a hood but just with a burner, leave it on throughout the manipulation and consider that the aseptic zone is spherical area about 15 cm around the flame. 9. Remember to label every tube, bottle, flask, plate… before you start. Always note the date on everything that you will incubate, keep or autoclave (tubes, plates..) label the plate so as to be able to read what is written during incubation (upside down).

5. Method of Analysis for Rhizobium 1.    

Instruments/Apparatus/Equipment Balance Bunsen burner Incubator and rotative incubator Hot air Oven

3.

         

Laminar hood Microscope (Dissecting and compound) pH meter Spectrophotometer Vortex Washer Colony counter Autoclave Magnetic stirrer Hand tally or mechanical counting device

2.                      

Materials Coton wool Cryoboxes Dispenser Dropper and Pipette Ependorff tubes and racks Foil Forceps Gauze Glassware: Flaks. Bottles, volumetric flask, measuring cylinder, beakers Loops Microtubes Parafilm Petri plates/dishes Pestles Pipetteboy Pipettes (Pasteur pipettes and Graduated and volumetric pipettes) Plastic box for incubation Slides and cover glasses Test tube and racks Paper towels Graduated pipettes – 1 and 10 ml Dilution bottles or flasks

Reagents Required 1. Crystal Violet: (commercial product diluted by ½) made up t 100 ml of crystal violet from 50ml commercial Violet mixed with 50 ml of distilled water. It is flammable; depending on the concentration and the frequency of use, limited evidence of a carcinogenic effect and harmful to aquatic organisms (may cause long-term effects in the aquatic environment) 2. Distilled water 3. Ethanol: (Absolute and General purpose) 4. Fuschine: Commercial product to be diluted by 1/10 to prepare 100ml. Measure 10ml of commercial Fucshine and mix with 90ml of distilled water. Fuschine is harmful by inhalation, contact with skin or if swallowed. It causes burns. 5. Glycerol: 40% made from 100 % Glycerol and distilled water 6. Lugol (Iodine)

7. Physiological water: 9g of Sodium chloride (NaCl) mixed with 800 ml distilled water and mixed with magnetic stirrer and then topped up to 1000ml 8. Savlon antiseptic 9. Congo red – 1 percent aqueous solution

4.

Media Preparation

If commercial bases for media are used, refer to the instruction of the supplier (indicated on the bottle). A plating medium with the following composition must be used; Agar

20 g

Yeast Extract

1g

Mannitol

10 g

Potassium hydrogen phosphate (K2HPO4)

0.5g

Magnesium sulphate (MgSO4 7H2O)

0.2g

Sodium Chloride (NaCl)

0.1g

Congo red

2.5 ml

Distilled water

1000 ml

pH

7.0

5.

Sterilizing and Preparation Procedure for Plates

If plates are glass, sterilize the sampling and plating equipment with dry heat in a hot air oven at not less than 160 oC for not less than 2 hours Sterilize the media by autoclaving at 120 oC for 20 min. To permit passage of steam into and from closed container when autoclaved, keep stoppers slightly loosened or plugged with cotton. Air from within the chamber of the sterilizer should be ejected allowing steam pressure to rise. 6.

Preparation of Plating Medium and Pouring 1. Add approximately 2/3 of distilled water into beaker add all chemicals except the agar and vitamins and other nutrients which are sensitive to heat. Place on magnetic stirrer and allow to mix well while heating. Filter (0.2 µm) under aseptic conditions. The pH of the mixture adjusted with either HCL or NaoH if necessary. 2. Store plates in closed plastic box at room temperature label he box with name of the media, the date of preparation and name of persons who prepared. 3. Agar is weighed and placed in a Schott bottle and the contents from the beaker are poured into the bottle. Close the bottle loosely with cotton wool, cover with an

4. 5.

6.

7.

Aluminium foil and label: nature of the content, date of preparation, name or initials of who prepared it. Add a piece of autoclave tape so as to hold both the cap and the bottle. Autoclave at 121 ºC for 20mins. Remove the media from autoclave. Melt the required amount of medium in boiling water or by exposure to flowing steam in partially closed container but avoid prolonged exposure to unnecessarily high temperature during and after melting. Melt enough medium which will be used within 3 hours. Re-sterlization of the medium may cause partial precipitation of ingredients. When holding time is less than 30 min promptly cool the molten medium to about 45 o C, and store until used, in a water bath or incubator at 43 oC to 45 oC. Introduce 12 to15 ml of liquefied medium or appropriate quantity depending on size of the Petri dish at 42 to 44 oC into each plate. Gently lift the cover of the dish just enough to pour in the medium. a. Sterilize the lips of the medium container by exposure to flame; b. Immediately before pouring. periodically during pouring, and when pouring is completed for each batch of plates, if portion of molten medium remain in containers and are to be used without subsequent sterilization for pouring additional plates. c. As each plate is poured thoroughly mix the medium with test portions in the Petri dish. By rotating and tilting the dish and without splashing the medium over edge, spread the medium evenly over the bottom of the plate. Provide conditions so that the medium solidifies with reasonable promptness (5-10 min) before removing the plates from surface.

6. Preparation of Serial Dilution for Plate Count A laminar air-flow chamber is used in order to achieve serial dilution of the broth culture of the strain or sample suspension. For plate counts, the countable range is generally 30–300 cells/ml. The procedure is as follows: 1. Set out 8 tubes, each containing 9 ml of sterile water. 2. Dilute 1 ml of broth culture or sample suspension (1 g of sample in 9 ml of water) in steps (10-1 to 10-8) with a sterilized 1 ml serological pipette equipped with a rubber bulb of 1 ml capacity. 3. Suck up broth culture or sample suspension from tube 1 to the 1 ml mark. 4. Immediately expel the broth culture or sample suspension back into the tube with sufficient vigour to effect a thorough mixing. 5. Repeat sucking up and expelling 5 times, and then transfer 1 ml to tube 2. 6. Take a new sterile pipette, attach the rubber bulb, and remove 1 ml to tube 3. 7. Repeat this procedure using a fresh sterile pipette each time until the dilution series is completed. 6.1 Incubation of plates 1. Put 1.0 g of the sample in a test-tube. Add 9 ml of water and make a suspension. Serially dilute this suspension (section 1.5). Use the diluted suspension to grow the bacteria by plating techniques (section 1.7 below). 2. Label the plates and incubate at 28 ± 2 °C for 3–5 days for fast-growing rhizobia and 5–10 days for slow-growing ones.

3. Count the colonies with the aid of a magnifying lens under uniform and properly controlled artificial illumination. Use a colony counter, equipped with a guide plate and rules in square centimeters. Record the total number of colonies with the hand tally. 4. Count all plates, but for the purpose of calculation consider plates showing more than 30 and fewer than 300 colonies per plate. Disregard colonies that absorb Congo red and stand out as reddish colonies. Rhizobium stands out as white, translucent, glistening and elevated colonies. Count such colony numbers, and calculate the figure in terms of per milliliter of suspension used for plating. Relate it to the original sample (1 g), taking into account the dilution factor. Also check for being free from contamination at 10-6 dilution. 5. Take uncontaminated Rhizobium cells and multiply them further for use in nodulation tests in pot culture trials. 6.2 Viable cell count There are many techniques for counting viable cells that are able to divide and form offspring. The usual method is to determine the number of cells in a given sample of forming colonies on a suitable agar media. The method involves serial dilution and spreading of diluted suspensions on plates, and counting of colony-forming units on plates. Sterilized molten medium is kept ready in conical flasks placed in a water-bath at a constant temperature of 48 °C by pour, spread or drop plate methods. The procedure for the pour plate method is as follows: 1. Remove dry sterilized Petri dishes from paper packs and stack them in a laminar airflow chamber. 2. For each dilution, three Petri dishes will be required. Stack the plates in sets of three each and label them with the help of a glass marker pen. 3. Using a fresh sterile pipette, pour 1 ml of aliquot from the last dilution (eg., 10-8) into each of the three Petri dishes. 4. Using the same pipette, pour similar aliquots from the next two dilutions (say, from 10-7 and 10-6) into three Petri dishes for each dilution. Pour about same volume of molten medium into each of the plates. 5. Immediately after pouring, move the plates gently in a whirling motion to mix the contents. Allow the medium to solidify, and incubate at 28 °C for 3–5 days. 6. Count the colonies after 3–5 days. Multiply the average number of colonies by the dilution factor. If the average number of colonies at 10-8 dilution is 60, then the sample had a concentration of 60 × 108 = 6 × 109 cells/ml. 6.3 Pot culture test for nodulation and N fixation  Composition of plant nutrient solution for pot culture tests S. No a b c d e

Composition Potassium chloride Di Potassium hydrogen phosphate(K2HPO4) Calcium sulphate (CaSO4 2H2O) Magnesium sulphate (MgSO4 7H2O) Trace elements solution

Conc. 0.001M 0.001M 0.002M 0.001M -

g/l 0.0745 0.175 0.344 0.246 0.5 ml

1. Copper sulphate (CuSO4 5H2O) 0.01 mg/kg 0.78 2. Zinc sulphate (ZnSO4 7H2O) 0.25 mg/kg 2.22 3. Ammonium molybdate ((NH4)6Mo7O24 0.0025 mg/kg 0.01 4H2O) 2.03 4. Magnesium sulphate (MgSO4 7H2O) 0.25 mg/kg 5. Boric acid (H3BO4) 0.125 mg/kg 1.43 6. Water 1 litre Prepare the solution no (e) consisting of trace elements in one liter of stock solution and add to final nutrient solution at the rate of 0.5 ml per liter. f

Iron solution: 0.5 ml Ferrous sulphate 5 Citric acid 5 Water 100 ml Prepare the solution no. (f) As 100 ml of stock solution and add final nutrient solution at the rate of 0.5 ml per liter.

Prepare the nutrient solution by dissolving potassium chloride (0.0745 g), potassium hydrogen phosphate (0.175 g) and magnesium sulphate (0.246 g) in 1 litre of water. To this solution, add 0.5 ml of trace elements solution (respective amounts of micronutrient salts as given in table above dissolved in 1 litre of water) and 0.5 ml of iron solution (respective amounts are dissolved in 1 litre). In a mortar, grind 0.344 g of calcium sulphate to a fine consistency and add it to the final nutrient solution (keep the pH of the solution at 6.0) and autoclave the nutrient solution at 120 °C for 20 minutes. 6.3.1 Procedure for pot culture Immerse legume seeds in 95 percent alcohol, and wash with chlorine water and then with 0.1 percent mercuric chloride solution for 2–3 minutes. Wash the seeds with sterile water in order to remove the sterilant. 1. Fill glazed pots of 2 kg capacity with soil (2 parts soil and 1 part sand), and autoclave for 2 hours at 120 °C. 2. Inoculate the surface of the sterilized seeds with water slurry of inoculant taken from a culture packet (biofertilizer sample). Depending on the size of the seeds, 1 ml of inoculant inoculates 15–100 g of seeds. 3. Keep a set of pots sown with non-inoculated seeds as control, another set with inoculated seeds and a third set with ammonium nitrate at 100 kg N/ha. Take four replications of each treatment. Keep the pots in the growth room. 4. Add nutrient solutions to the pots at the start to attain WHC and subsequently with sterile water to keep the soil moist. After 2–3 weeks of growth, thin down the number of plants in each pot to four uniform plants. 5. After 6–8 weeks, harvest the plants separately from each set, and separate the plants carefully from the soil under slow running water. Observe the number, colour and mass of nodules for each treatment. 6. If good effective pink nodulation is obtained in inoculated plants together with absence (or sometimes presence) of stray nodules in controls, and if there is at least a 50‑ percent increase in the dry matter

yield of plants compared with non-inoculated controls, it may be concluded that the culture is of the required quality. The growth and dry matter yield with ammonium nitrate treatment enables a comparison between the N used through fertilizers and the N fixed through inoculation, i.e. the extent of contribution in terms of N fixation by Rhizobium. 6.4 Maintenance and preparation of pure culture and quality control at Broth stage Maintain pure culture of rhizobia on yeast extract mannitol agar (YEMA) slants to the following composition; Mannitol

10.0g

Potassium hydrogen phosphate (K2HPO4)

0.5 g

Magnesium sulphate (MgSO4 7 H2O)

0.2g

Sodium chloride (NaCl)

0.1 g

Calcium carbonate (CaCO3)

1.0 g

Yeast extracts

1.0g

Agar

18g

Distilled water

1 liter

pH

6.8- 7.0

6.4.1 Procedure  Transfer a loopful of the pure culture to each of the agar slant aseptically in an inoculation room and incubate at 28+/- 2 oC for 3 to 10 days depending upon the species of Rhizobium. Always keep culture at 4 ºC.  Preparation of inoculums cultures  Prepare yeast extract mannitol broth minus agar.  Transfer a loop full of the culture. 6.4.2 Quality Control Test Recommended at Broth Stage Qualitative Tests  Check for freedom from the visible contaminants  The pH of the bacterial broth shall normally be between 6.5 and 7.5.  Smear and Gram stain Reagents  Ammonium oxalate crystal violet stain- weigh 0.2 g of crystal violet and dissolve in 20 ml of 95 percent ethyl alcohol. Dissolve separately 0.8 g of ammonium oxalate in 30 ml of distilled water. Mix the two solutions and filter through a filter paper. Iodine solution

Iodine

1.00 g

Potassium Iodide

2.00 g

Distilled water

300 ml

Weigh the ingredient and dissolve in water. Filter through a filter paper. Erythrosine solution Erythrosine

1.00 g

Phenol

5.00 g

Distilled water

100 ml

Weigh the ingredient, dissolve in distilled water and filter through a filter paper. 6.4.3 Procedure  Prepare a smear on a clean microscope slide, fix over a flame by gently and intermittent heating, air cool and flood with ammonium, oxalate crystals violet stain for 1 min. After removing the excess of ammonium oxalate crystals violet, wash the slide under a gentle stream of running tap water.  Flood the slide with iodine solution for half a minute; remove excess stain wash 95 percent ethyl alcohol and finally wash under a gentle stream of running tap water. Flood the slide with erythrosine stain for about 3min, wash under a gentle stream of running tap water and dry between the folds of a filter paper.  Examine the slide under a compound microscope using an oil immersion objective. Note: A smear prepared from undiluted broths should be free from Gram positive cells. The presence of a few gram positive cells in occasional fields which may be due to dead cells in the medium may be disregarded. 6.4.4 Quality Assessment Absence of Growth on Glucose - Peptone Agar The composition of the glucose –peptone agar is as follows: Glucose

10.0 g

Peptone

20.0 g

Sodium chloride (NaCl)

5.0 g

Agar (IS6850)

15.0 g

Distilled water

100 ml

Bromocresol purple

10 ml

Alcoholic solution

(1.6%)

pH

7.2

Note:- when a loopful of the broth is streaked in to this Medium and incubated at 28+/- 2 ºC for 24 h, the purple-violet colour of the medium( due to the indicator bromo-cresol purple) shall Not change . If the colour changes to yellow (acidic reaction) or blue (alkaline reaction) the broth is grossly contaminated. Hence the broth should be rejected. Streak on yeast extract mannitol agar with congo-red When a loopful of broth culture is streaked to a plate of this medium and incubated at 28± 2 ºC for 3 - 10 days, it shows colonies of bacteria with growth characteristics same as that of the pure culture used in the preparation of the broth, otherwise the broth should be rejected. Quantitative Test Viable or plate counts Serially dilute one milliliter of the broth to obtain dilutions of the order of 106 to 109. Plate 0.2 ml aliquots of the dilution on YEMA plates and incubate at 28±2 ºC for 2 to 6 days, depending on the species of Rhizobium. The counts of viable Rhizobium in the final broth from shake culture or fermenters shall be not less than 108 to 109 cells/ ml. Otherwise, the broth should be rejected. Bradyrhizobium strains are slow growing, gram negative sol bacteria. The genus Bradyrhizouim represents a heterogenous group of nodulating bacteria. The Bradyhizobium currently consists six species. Isolation of Rhizobium strains Root nodules collected are presented in a vial with desiccated silica gel. Nodules from same plant represent 1 unit. Dried root nodules are kept in a refrigerator of 4ºC until isolation of Bradyrhizobia. 7.0. Conclusion The carrier is the major portion of inoculant. There is presently no universal carrier or formulation available for release of microorganism into soil. Materials and types of formulations of carriers vary: Slurry, powder, peat, liquid. A good carrier should have the capacity to deliver the right number of viable cells in good physiological condition at the right time. 7.1.

Type of carriers 1. Soil: peat, coal, clay and inorganic soil 2. Plant waste material: Compost, farmyard manure, soybean meal, soybean and peanut oil, wheat bran, press mud, agricultural waste material, spent mushroom compost and plant debris 3. Inert Materials: Vermiculite, perlite, ground rock phosphate, Calcium sulphate, polyacrylamide gel and alginate beads 4. Plain lyophilized microbial cultose. 5. Sterile carrier is the best but commercially expensive to produce. Nono-sterile carrier is the most widely used in larger markets. 6. Formulation is the crucial issue for inoculants containing effective bacterial strain and can determine the success or failure of the product. 7. Formulation is the industrial ―Art‖ of converting a promising laboratory proven bacterium into a commercial product

8. a. Long shelf life and stability: over the range of -50C to 30C within the marketing distribution systems. Products lacking this range of temperature tolerance will be unacceptable in the agricultural market b. Ease of use c. Resistance to abuse by farmers d. Overcome: loss of the viability during shor storage in the growers warehouse (In developing counties usually lack refrigeration). Increased standards ensure that the farmer is provided with effective inoculants but are also in the best interest of the inoculation industry. 7.2. Additional desirable characteristics of a good inoculant: 1. Chemical and physical characteristics. Inoculants should be nearly sterile, easily sterilized and chemically and physically uniform as possible. They should be of consistent quality high water holding capacity (for wet carrier) and suitable for as many bacterial species as possible. 2. Manufacturing qualities: The inoculant should be easily manufactured and mixed with existing industry, it should allow for the addition of nutrients, have an easily adjustable pH, and can be made of a reasonable priced raw material in adequate supply. 3. Farm handling qualities: A good inoculant allows for ease of handling (a major concern for the farmer), provide rapid and controlled release of bacteria into the soil and can be applied with standard agrotechnical machinery. 4. Environmental characteristics: The inoculant should be nontoxic, biodegradable, non polluting and should minimize environmental risks such as the dispersal of cells to the atmosphere or ground water. 5. Storage Qualities: The inoclant should have sufficient shelf life (one or two years at room temperature is often necessary for successful integration into the agricultural distribution system in some countries). 6. A good carrier should have as many of these qualities as possible. 7.3.

Forms of Inoculants  Powder: used as seed coating. The standard size of particles: 0.75 to 0.25mm Slurries : powder type of inoculants suspended in liquid (Usually water), in oil or organic oils  Granulars: Size range 0.35 to 1.18 mm. Bead like forms – macros (13mm diam) or micro size used as a powder for seed coating.  Peat formulations: An inoculum should contain a ;level of bacteria sufficient to inoculate plants and produce an economic gain. The level of bacteria cannot be established as a general standard because it varies from one bacterial species to another.

B. SOP for Testing the Quality of Commercial Non-Symbiotic Nitrogen fixation Products - Azospirillum 1.0.

Definitions

For the purpose of this standard the following definitions shall apply: The following are definition of terms used in synonym and those in the SOP. The terminologies Biofertilizer, Bioenhancer, Biostimulant and Bioinoculant are sometimes used synonymously. . Biofertilizers: is a substance which contains living microorganisms which are applied to seed, plant surface or product colonizes the rhizosphere or the interior of the plant or soil and promotes growth by increasing supply or availability of nutrients to the host plant. It is a term widely used term meaning ―bacterial inoculant‖ usually refer to preparations of microorganisms that may be a partial or complete substitute for chemical fertilization (like rhizobial inoculants). The use of word fertilizer allows easier registration for commercial. It is a formulation containing one or more beneficial strains (or species) in an easy to use and economical carrier either organic, inorganic or synthesized from defined molecules. It contains living microorganisms embedded in a carrier material which are applied to seed, plant surface or product colonizers the rhizosphere or the interior of the plant or soil and promotes growth by increasing supply or availability of nutrients to the host plant, or preparations containing living cells or organisms that enrich the nutrient quality of product.

Bioinoculants: Living organisms containing specific strains of specific bacteria, fungi and algae which fix atmospheric nitrogen, make nutrients soluble and available, collect and store nutrients, provide physical barriers to pests and pathogens, stimulate plant growth, and decompose organic residue. Bioenhancers: Substances that increase the bioavailability of active ingredients, vitamins and nutrients Biostimulants: Dormant strains of positive product microorganisms that come alive when introduced into the product system. They contain strains of specific bacteria, fungi or algae which take up nutrients and make it available to plants, collect and store available nutrients, enhance uptake of nutrients, provide physical barriers against pathogens, stimulate growth, decompose organic residues Bacteria: A Kingdom of single celled microorganisms whose cells lack membrane-bound nucleus (prokaryotic) microorganisms that inhabit soil, water, plants and animals. Non Symbiotic nitrogen fixing bacteria: Represents a range of bacteria including saprophytic living on plant residues, bacteria living in close association with rhizosphere of plant roots and bacteria which live entirely within plants (endophytes). They fix nitrogen by reducing gaseous nitrogen in the air to ammonia by a process catalysed by enzyme complex nitrogenase. Plant Growth Promoting Rhizobacteria (PGPR): The Indispensable part of rhizosphere biota that when grown in association with the host plants can stimulate the growth of the host. Azospirillum: Gram negative free living (Non Symbiotic) nitrogen fixing diazotroph bacteria in family Rhodospirilliaceae. It is associated with rhizosphere of Monocots particularly in the

Gramineaeae (grass family) amongst which are the common cereals maize (corn), wheat and rice. Contamination: In other microorganism other than AMF not declared on the label. Aseptic Conditions/Aseptically: Environment where no microorganisms is present. It can be obtained by using a Bunsen burner to create an aseptic zone which is the aspheric area around the flame with a diameter of approximately 15 cm or a Laminar flow hood with sterile air continuously produced and present in the hood. The high pressure of air flow from the hood prevents the air from outside to come inside and contaminate the environment in the hood; aseptic conditions are also created by cleaning surface with 70% ethanol. Contaminated microorganisms: Every plate, tube, pipette, or other instruments (glassware, pestles, eppendorff tube…) which has been in contact with microorganisms and cannot be sterilized by the flame of a Bunsen burner is considered as contaminated. Contaminated by toxic chemicals: Every tube, flasks, pipette or other instruments which has been in contact with toxic chemicals is considered contaminated Good Laboratory Practices (GLP): The principle of GLP have been developed to promote the quality and validity of results and of the analysis conducted in a laboratory. It is a management concept covering the organization and the conditions under which laboratory studies are planned, performed, monitored,, recorded and reported. Its principle also include the protection of man and the environment. Mother tube/plate/products: Tube/plate/product which the bacteria are picked from. The result of the growth of this inoculation is considered as the daughter which can become the mother for the next inoculation. Substrate: Any material, that serves as source of energy for an organism. Carrier: Delivery media of live microorganisms from the production to the field. The carrier is the major portion of inoculant. There is presently no universal carrier or formulation available for release of microorganism into soil. Materials and types of formulations of carriers vary: Slurry, powder, peat, liquid. A good carrier should have the capacity to deliver the right number of viable cells in good physiological condition at the right time. 2.0. Introduction Azospirillum are associated with non symbiotic nitrogen fixation and phytostimulation by producing plant growth promoting hormones. It is commonly associated with cereals, grasses and tuber crops. Azospirillum excrete phytohomones such as gibberellins, cytokinins and auxin (IAA). The Azospirillum- plant interaction produces no visible phenotypes in the root system as is the case with Rhizobia-plant interactions. Azospirillum was tested as biofertilizer in the 1970‘s with the inception of commercialization in 1980s and 1990s. It has been used for over 20 years. Latin America had more interest in Azospirillum inoculants with over 1.5 million hactares application of Azospirillum. It was was considered to be an equivalent to Rhizobia, but to be more economically important to cereal plants. Azospirillum inoculants are available for maize, rice and wheat in America, Asia, Europe and Africa. Just as in mineral fertilizers, variability exists resulting to inconsistency and unpredictable and variability in yield response. Commercialization is as limited by the inconsistency and variability in yield response caused by environmental conditions, type of crop, soil type and physiological state

of bacterial cells. Differences exist between different strains of Azospirillum in their ability to promote plant growth. The effectiveness is therefore affected by the bacterial strain of a particular species as well as the number and physiological state of the cell. A single strain cannot therefore be universally successful under all soil conditions and with all hosts. 2.1.

Quality control management (General procedures) 1. A specific protocol is established before screening is set up. It includes the number of samples, the details about the inoculants (origin, composition, rate of application...), and any other relevant information. 2. Calculations, weights, records (of temperature, humidity...), observations, measurements, preparation of stock solutions (nutritive solutions for example) and any other relevant information are recorded in the lab book on a daily basis. 3. The stock solutions are labelled with the date of reception, name or initials of the person who received it, the number of the container (x of n), date of opening, name or initial of the person who opened it. 4. The reagents are labelled with the name of the contents, date of preparation, name or initials of the person who prepared them and any other relevant information. 5. All samples are clearly identified and records are taken about the different steps of analysis (when they have been put in the oven, grinded, sent for analysis). 6. After harvesting, samples might need to be decontaminated before elimination. Solarization may be considered. 7. In case of contamination of the bench, floor, user, ..., it has to be cleaned and disinfected before the work can be continued (cf. ―Hygiene and Safety rules in a laboratory‖ document). 8. Non contaminated or decontaminated items are cleaned with soap, and rinsed with water and eventually rinsed with distilled water. 9. Waste management: a. Non contaminated waste is eliminated in the normal bin. b. Non contaminated glass waste are put in a separate container labelled with the mention ―Broken glass‖ c. Anything contaminated by microorganisms should be decontaminated before appropriate elimination/cleaning. d. Waste may be put into a special autoclave bags for 20 mins at 121 ºC. e. Solid and liquid waste contaminated by toxic chemicals are placed in a separate container to be collected by special disposable service providers. f. Disposable glass instruments are ut into a container containing Sodium Hypochlorite solution (commercial Jik) for decontamination before being eliminated. Solid (including broken glass) and liquid waste contaminated by toxic chemicals are placed in separate containers for elimination by waste disposal company. g. Contaminated glassware is properly rinsed with tap water and water collected in specific items (consult MSDS) on product disposal. 10. Equipment‘s are regularly cleaned to remove dust, samples or other waste. 11. Room is cleaned daily. Disinfect every surface with disinfectant and or with 70% ethanol before starting work. The bench and the floor is to be cleaned and disinfected before the work begins and in case of any contamination during the working session it should be disinfected before work continues.

2.2.

General laboratory practices

1. Always wear a lab coat when working in the laboratory. 2. Never put on gloves when working close to flames. In case of burn, the glove would stick on your skin and make the wound worse. 3. To maintain good hygiene, clean your hands with soap and work under aseptic conditions. Clean bench of hood and any surface of operation with a disinfectant; pour some disinfectant on the bench, leave for I min and wipe with gauze/cotton wool. It is also possible to clean the bench with 70% ethanol. 4. Always have a seat when working to avoid accidents. Avoid useless movement in the lab during work. Organize your work space to ensure a smooth run to the analysis. Some equipment need to be switched on some time before use (incubator, laminar flow hood). 5. Plan your work to give them enough time. Organize everything you need in the hood before starting to work so that you don‘t have to cross hands or have to have put your hand out of the hood. Never put items close to the back of the hood because it can affect the flow hence affect the sterility of air. Use preferably the middle and sides of the bench. 6. Every aseptic manipulation has to be done in the middle of the hood, never outside of it and not at the side or the back of the hood. 7. Air contaminations are common. To avoid them, don‘t speak, run or move it it is not necessary and keep the door and windows closed. 8. A burner is used to sterilize items such as loops, tubes, or forceps. The hottest part of the flame is the top of the blue part, so this is the part which has to be used. If you have to work without a hood but just with a burner, leave it on throughout the manipulation and consider that the aseptic zone is spherical area about 15 cm around the flame. 9. Remember to label every tube, bottle, flask, plate… before you start. Always note the date on everything that you will incubate, keep or autoclave (tubes, plates..) label the plate so as to be able to read what is written during incubation (upside down). 3.0 Method of Analysis for Azospirillum 3.1 The apparatus required:  Graduated pipettes – 1 and 10 ml  Dilution bottles or flasks  Petri dishes – uniform, flat-bottomed  Hot-air oven  Autoclave  Incubator  Hand tally or mechanical counting device  pH meter. 3.2 Reagents 3.2.1 Medium Use N-free semisolid medium (Nfb) of the following composition for preparation of MPN tubes. DL-Malic acid

5.0g

K2HPO4

0.5g

MgSO4 7H2O

0.2g

NaCl

0.1g

CaCl2

0.02g

Trace element Soln.

2.0 ml

Fe EDTA (1.64% Soln.)

4.0 ml

Vitamin soln.

1.0 ml

KOH

4.0 ml

Bromothymol blue (0.5% aq.)

2.0 ml

Adjust pH

6.8-7.0 with KOH

For semi solid add agar

1.75 g

For solid medium add agar

15.0 g

3.2.1.1 Trace element solution (g/litre)

g/litre Na2MoO4 2H2O

0.2

MnSO4 H2O

0.235

H3BO3

0.28

CuSO4 5H2O

0.008

ZnSO4 7H2O

0.024

Distilled water

1000 ml

Use 2 ml of this solution in one litre of Nfb media.

3.3. Sterilization and preparation of MPN tubes Prepare Nitrogen free Bromothymol Blue malate medium. Boil to dissolve agar. Quickly dispense 10 ml molten media in 15 x 150 ml test tubes or screw capped culture tubes and close either with cotton plugs or screw caps. Minimum of 25 such tubes shall be needed for each sample. Sterilize the tubes by autoclaving at 121 oC for 20 minutes, as in Rhizobium. 3.4. Preparation of serial dilution for MPN count Dispense 30 g of Azospirillum biofertilizers in 270 ml of sterile water and shake for 10 minutes on a reciprocal shaker. Make serial dilutions up to 10-8 dilution. Pipette out 1 ml aliquots of 10-4 to 10-8 dilution and deliver it to screw cap tubes or test tubes containing Nfree semi solid Nfb media. 3.5. Incubation of tubes Label the tubes and incubate at 36 + 10C for 3-4 days in vertical position in a test tubes stand. Do not disturb the medium during the entire period of incubation. 3.6. Counting

Count the tubes which have turned blue and have developed typical white subsurface pellicle. Count the tubes as +ve or –ve for the presence of sub-surface pellicle and consider for the purpose of calculation. 3.7 Method for Estimating MPN Count  To calculate the most probable number of organisms in the original sample, select as P1 the number of positive tubes in the least concentrated dilution in which all tubes are positive or in which the greatest number of tubes is +ve, and let P2 and P3 represent the numbers of positive tubes in the next two higher dilutions (refer to Appendix 5.1).  Then find the row of numbers in Table 1 in which P1 and P2 correspond to the values observed experimentally. Follow that row of numbers across the table to the column headed by the observed value of P.  The figure at the point of intersection is the most probable number of organisms in the quantity of original sample represented in the inoculum added in the second dilution. Multiply this figure by the appropriate dilution factor to obtain the MPN value.

Appendix 1: Most Probable Number Table P1

P2

Most probable number for indicated values of P 3 0 1 2 3

4

5

0 0 0 0 0 0 1 1 1 1 1 1

0 1 2 3 4 5 0 1 2 3 4 5

0.018 0.037 0.056 0.075 0.094 0.020 0.040 0.061 0.089 0.11 0.13

0.018 0.036 0.055 0.074 0.094 0.11 0.040 0.061 0.082 0.10 0.13 0.15

0.036 0.055 0.074 0.093 0.11 0.13 0.060 0.081 0.10 0.13 0.15 0.17

0.054 0.073 0.092 0.11 0.13 0.15 0.080 0.10 0.12 0.16 0.17 0.19

0.072 0.091 0.11 0.13 0.15 0.17 0.10 0.12 0.16 0.17 0.19 0.22

0.090 0.11 0.13 0.15 0.17 0.19 0.12 0.14 0.17 0.19 0.22 0.24

2 2 2 2 2 2 3 3 3 3 3 3

0 1 2 3 4 5 0 1 2 3 4 5

0.046 0.068 0.093 0.12 0.15 0.17 0.078 0.11 0.14 0.17 0.21 0.25

0.068 0.092 0.12 0.14 0.17 0.20 0.11 0.14 0.17 0.21 0.24 0.29

0.091 0.12 0.14 0.17 0.20 0.23 0.13 0.17 0.20 0.24 0.28 0.32

0.12 0.14 0.17 0.20 0.23 0.26 0.16 0.20 0.24 0.28 0.32 0.37

0.14 0.17 0.19 0.22 0.25 0.29 0.20 0.23 0.27 0.31 0.36 0.41

0.16 0.19 0.22 0.25 0.28 0.32 0.23 0.27 0.31 0.35 0.40 0.45

4 4 4 4 4 4 5 5 5 5 5 5

0 1 2 3 4 5 0 1 2 3 4 5

0.13 0.17 0.22 0.27 0.34 0.41 0.23 0.33 0.49 0.79 1.3 2.4

0.17 0.21 0.26 0.33 0.40 0.48 0.31 0.46 0.70 1.1 1.7 3.5

0.21 0.26 0.32 0.39 0.47 0.56 0.43 0.64 0.95 1.4 2.2 5.4

0.25 0.31 0.38 0.45 0.54 0.64 0.58 0.84 1.2 1.8 2.8 9.2

0.30 0.36 0.44 0.52 0.62 0.72 0.76 1.1 1.5 2.1 3.5 16.0

0.36 0.42 0.50 0.59 0.69 0.81 0.95 1.3 1.8 2.5 4.3 --

3.8 Maintenance and preparation of pure culture and quality control at Broth stage 1. Maintain pure culture of Azospirillum on nitrogen free bromothymol blue medium and maintain as solid medium. 2. Transfer a loopful of pure culture to each of the agar culture tube aseptically in an inoculation room and incubate 37+ 2 oC for three days and keep in undisturbed. Always keep pure culture below 5 oC. Preparation of Inoculums culture and Mass culture: Inoculums culture and mass culture of this standard shall be prepared as described for Rhizobium of this standard. 3.9 Quality Control Test Recommended at Broth Stage 3.9.1 Quality Test  Check for free from contaminants by preparing slide and observing under microscope.  The pH of bacterial broth shall normally be between 7.0 to 8.0.  Gram staining test shall be carried out as descried for Rhizobium of this standard.  See the colour change in the media after 24 hours from inoculation. The colour will change from green to blue.  Watch the pellicle just below the surface of the media. It is checked on the third day after keeping inoculated broth undisturbed. 3.9.2 Quantitative Test Most probable Number (MPN). The counts of Azospirillum in the final broth from shake culture or fermenter shall be not less than 108 to 109 cells/ ml. Otherwise the broth should be rejected. Conclusion The type of carrier determines the shelf life, contamination level, quality and the efficacy under field conditions. The formulations are either solid or liquid based. The most commonly used solid based carriers are peat, vermiculite, lignite, cured compost coal or charcoal. The quality of sold formulations is known to vary in shelf life, higher probability of contaminants and are more unpredictable and inconsistent. Azospirillum functions best when nitrogen

fertilizer is not applied and also alleviates saline stress. The optimal concentration that produces effect is 1 x 107 cfu per plant or 1 x 109 cfu mL-1 The common Azospirillum species for biofertilizer are A. lipoferum and A. brasilense. Other species isolated in association with graminaceous plants are A. oryzae, A. amazonense, A. Irakense, A. largimobile, A. deobereinerae. Due to variability and inconsistency in effectiveness in different environmental conditions and host plants, it is recommended to be applied in mult-strain formulations. It is a common ‗helper bacteria‘ used in Co-Inoculation improve beneficial association between Rhizobium and plants as different plants and mycorrhizae and plants.

C. SOP for Testing the Quality of Commercial Non-Symbiotic Nitrogen fixation Products - Azotobacter 1.0.

Definitions

For the purpose of this standard the following definitions shall apply: The following are definition of terms used in synonym and those in the SOP. The terminologies Biofertilizer, Bioenhancer, Biostimulant and Bioinoculant are sometimes used synonymously. . Bofertilizer Biofertilizers: is a substance which contains living microorganisms which are applied to seed, plant surface or product colonizes the rhizosphere or the interior of the plant or soil and promotes growth by increasing supply or availability of nutrients to the host plant. It is a term widely used term meaning ―bacterial inoculant‖ usually refer to preparations of microorganisms that may be a partial or complete substitute for chemical fertilization (like rhizobial inoculants). The use of word fertilizer allows easier registration for commercial. It is a formulation containing one or more beneficial strains (or species) in an easy to use and economical carrier either organic, inorganic or synthesized from defined molecules. It contains living microorganisms embedded in a carrier material which are applied to seed, plant surface or product colonizers the rhizosphere or the interior of the plant or soil and promotes growth by increasing supply or availability of nutrients to the host plant, or preparations containing living cells or organisms that enrich the nutrient quality of product.

Bioinoculants: Living organisms containing specific strains of specific bacteria, fungi and algae which fix atmospheric nitrogen, make nutrients soluble and available, collect and store nutrients, provide physical barriers to pests and pathogens, stimulate plant growth, and decompose organic residue. Bioenhancers: Substances that increase the bioavailability of active ingredients, vitamins and nutrients Biostimulants: Dormant strains of positive product microorganisms that come alive when introduced into the product system. They contain strains of specific bacteria, fungi or algae which take up nutrients and make it available to plants, collect and store available nutrients,

enhance uptake of nutrients, provide physical barriers against pathogens, stimulate growth, decompose organic residues Bacteria: A Kingdom of single celled microorganisms whose cells lack membrane-bound nucleus (prokaryotic) microorganisms that inhabit soil, water, plants and animals. Non Symbiotic nitrogen fixing bacteria: Represents a range of bacteria including saprophytic living on plant residues, bacteria living in close association with rhizosphere of plant roots and bacteria which live entirely within plants (endophytes). They fix nitrogen by reducing gaseous nitrogen in the air to ammonia by a process catalysed by enzyme complex nitrogenase. Azotobacter: belongs to the family Azotobacteraceae, It is a gram negative areobic heterotrophic, catalase positive, free living diazotrophic bacteria. Contamination: In other microorganism other than AMF not declared on the label. Aseptic Conditions/Aseptically: Environment where no microorganisms is present. It can be obtained by using a Bunsen burner to create an aseptic zone which is the aspheric area around the flame with a diameter of approximately 15 cm or a Laminar flow hood with sterile air continuously produced and present in the hood. The high pressure of air flow from the hood prevents the air from outside to come inside and contaminate the environment in the hood; aseptic conditions are also created by cleaning surface with 70% ethanol. Contaminated microorganisms: Every plate, tube, pipette, or other instruments (glassware, pestles, eppendorff tube…) which has been in contact with microorganisms and cannot be sterilized by the flame of a Bunsen burner is considered as contaminated. Contaminated by toxic chemicals: Every tube, flasks, pipette or other instruments which has been in contact with toxic chemicals is considered contaminated Good Laboratory Practices (GLP): The principle of GLP have been developed to promote the quality and validity of results and of the analysis conducted in a laboratory. It is a management concept covering the organization and the conditions under which laboratory studies are planned, performed, monitored,, recorded and reported. Its principle also include the protection of man and the environment. Mother tube/plate/products: Tube/plate/product which the bacteria are picked from. The result of the growth of this inoculation is considered as the daughter which can become the mother for the next inoculation. Substrate: Any material, that serves as source of energy for an organism. Carrier: Delivery media of live microorganisms from the production to the field. The carrier is the major portion of inoculant. There is presently no universal carrier or formulation available for release of microorganism into soil. Materials and types of formulations of carriers vary: Slurry, powder, peat, liquid. A good carrier should have the capacity to deliver the right number of viable cells in good physiological condition at the right time.

2.0.

Introduction

Azotobacter is an aerobic soil microbe which facilitates nitrogen fixation and also increases germination of seed. It is more abundant in the rhizosphere of plants than the surrounding soil with the abundance depending on the plant species. The strain Azotobacter choococcum is the most common species in the soil. The mode of action of Azotobacter is direct mechanism of plant growth improvement by biological nitrogen fixation under free-living conditions and production of phytohormones and indirect mechanism of plant growth improvement by biocontrol. Since 1926, Azotobacter was recommended for use to improve agricultural plants and soil properties. The species A. chroococum and A. vinelandii have long been used as soil and seed inoculants. Inoculation with nonsymbiotic associative rhizosphere bacteria like Azotobacter was used in large scale in Russia in the 1930s and 1940s. It was abandoned and the interest as inoculant for agriculture has only recently been revived. The predominant species used for biofertilizer are Azotobacter choococcum and Azotobacter vinelandii. Azotobacter is a broad spectrum biofertilizer and can be used as inoculants for most agricultural crops and soils. Azotobacter requires large amounts of organic carbon for growth and is a high respiring organisms hence works well in the soils having sufficient organic matter. It is an important inoculant especially in organic farming and is less effective in soils with poor organic matter. It thrives even in alkaline soil and is tolerant to high salts. The products are in either liquid form or lignite carrier based. Suitable carriers for Azotobacter are lignite cool, compost and peat soil with Lignite recommended as the most suitable, cheaper, keeps organisms living longer period and does not lower quality of biofertilizer. The fitness of lignite has to be 250 – 300 microns and the pH of the carrier adjusted by adding CaCO3 to neutral. Naturally lignite has a variety of microorganisms and hence it is sterilized in autoclave at 30Ibs pressure for 30 minutes. It is applied as seed treatment, seedling root/tuber dip, soil application and drip irrigation. Azotobacter is sensitive to temperature and storage conditions of the products affecting the viability. The quality control of Azotobacter products requires adequate microbiological laboratory and qualified microbiologist, all contents and cell counts specified and the quality of carrier also specified.

2.1. Purpose The Standard Operation Procedure (SOP) is intended to guide laboratories appointed by regulatory agencies in screening of Free living nitrogen fixing (Azotobacter products) commercial products for quality. Based on the information on the product label, the screening is to confirm the strains, populations and ability to fix nitrogen. This is to guarantee customers quality product devoid of contaminants. This exercise is also to verify if there are any contaminants that can reduce the efficacy of the product or are harmful to plants, human, animals and the environment. Proper sampling and handling of the product at port of entry and careful dispatch to the testing laboratory is to be guaranteed to avoid any claims of contamination by the product proponent. The final outcome is to guide regulatory agencies on the quality of the product and its suitability for use by farmers. The SOP outlines the procedures provided by the regulatory agency to generate quality data as stipulated on the label and of acceptable standards set by regulatory agencies.

2.2.

Requirements for Quality Control

Good laboratory practices and precautionary statements (i.e. safety) Precaution

Autoclave: The autoclave can pose a great danger if not used correctly because of high pressure and temperature. Before using, visually check the general aspect (no corrosion, no leak), the quality of water and settings. Strictly follow the instructions to start it. During heating phase, the pressure increases. Be sure that the door is correctly closed so that there is no leakage. Don‘t try to force the autoclave to open. The autoclave has a safety devise and will refuse to open if the temperature is still high or if the pressure is not back to atmospheric pressure. The maintenance has to be done regularly and the results recorded in a specific file (maintenance file, available in the office). Bunsen Burner: The risk of fire can be minimised by following a few simple rules. If the hood is being used, turn on the fire only when it is needed, don‘t cross your arms, don‘t pass your arm on the burner. Laminar flow hood: If he hood is not working properly, it can lead to a fire risk. The maintenance has to be done regularly and results recorded in a specific file (maintenance file). Biological Hazards: Manipulating microorganisms poses a risk not only to the one who is working, but also to other people in the lab and potentially to the environment in case of dissemination. The rulers of safety have to be well understood and respected in order to avoid any contamination of staff and/or the environment. In case of accidental contamination, clean and disinfect properly before the activities can be restarted. Dispose contaminated waste. Chemicals: Before using a new chemical, the information about toxicity, conditions of use, risks and safety phrases… have to be understood and observed. Use the equipment of protection if needed (gloves, masks, hood…). The Material Safety Data Sheet (MSDS) of different chemicals are also available to get more details about the products.

2.3.

Quality control management (General procedures) 1. A specific protocol is established before screening is set up. It includes the number of samples, the details about the inoculants (origin, composition, rate of application...), and any other relevant information. 2. Calculations, weights, records (of temperature, humidity...), observations, measurements, preparation of stock solutions (nutritive solutions for example) and any other relevant information are recorded in the lab book on a daily basis. 3. The stock solutions are labelled with the date of reception, name or initials of the person who received it, the number of the container (x of n), date of opening, name or initial of the person who opened it. 4. The reagents are labelled with the name of the contents, date of preparation, name or initials of the person who prepared them and any other relevant information. 5. All samples are clearly identified and records are taken about the different steps of analysis (when they have been put in the oven, grinded, sent for analysis). 6. After harvesting, samples might need to be decontaminated before elimination. Solarization may be considered. 7. In case of contamination of the bench, floor, user, ..., it has to be cleaned and disinfected before the work can be continued (cf. ―Hygiene and Safety rules in a laboratory‖ document). 8. Non contaminated or decontaminated items are cleaned with soap, and rinsed with water and eventually rinsed with distilled water. 9. Waste management:

 

Non contaminated waste is eliminated in the normal bin. Non contaminated glass waste are put in a separate container labelled with the mention ―Broken glass‖  Anything contaminated by microorganisms should be decontaminated before appropriate elimination/cleaning.  Waste may be put into a special autoclave bags for 20 mins at 121 ºC.  Solid and liquid waste contaminated by toxic chemicals are placed in a separate container to be collected by special disposable service providers.  Disposable glass instruments are ut into a container containing Sodium Hypochlorite solution (commercial Jik) for decontamination before being eliminated. Solid (including broken glass) and liquid waste contaminated by toxic chemicals are placed in separate containers for elimination by waste disposal company.  Contaminated glassware is properly rinsed with tap water and water collected in specific items (consult MSDS) on product disposal. 10. Equipment‘s are regularly cleaned to remove dust, samples or other waste. 11. Room is cleaned daily. Disinfect every surface with disinfectant and or with 70% ethanol before starting work. The bench and the floor is to be cleaned and disinfected before the work begins and in case of any contamination during the working session it should be disinfected before work continues.

2.4.

General laboratory practices 1. 2. 3.

4.

5.

6. 7. 8.

Always wear a lab coat when working in the laboratory. Never put on gloves when working close to flames. In case of burn, the glove would stick on your skin and make the wound worse. To maintain good hygiene, clean your hands with soap and work under aseptic conditions. Clean bench of hood and any surface of operation with a disinfectant; pour some disinfectant on the bench, leave for I min and wipe with gauze/cotton wool. It is also possible to clean the bench with 70% ethanol. Always have a seat when working to avoid accidents. Avoid useless movement in the lab during work. Organize your work space to ensure a smooth run to the analysis. Some equipment need to be switched on some time before use (incubator, laminar flow hood). Plan your work to give them enough time. Organize everything you need in the hood before starting to work so that you don‘t have to cross hands or have to have put your hand out of the hood. Never put items close to the back of the hood because it can affect the flow hence affect the sterility of air. Use preferably the middle and sides of the bench. Every aseptic manipulation has to be done in the middle of the hood, never outside of it and not at the side or the back of the hood. Air contaminations are common. To avoid them, don‘t speak, run or move it it is not necessary and keep the door and windows closed. A burner is used to sterilize items such as loops, tubes, or forceps. The hottest part of the flame is the top of the blue part, so this is the part which has to be used. If you have to work without a hood but just with a burner, leave it on throughout the manipulation and consider that the aseptic zone is spherical area about 15 cm around the flame.

9.

3.0.

Remember to label every tube, bottle, flask, plate… before you start. Always note the date on everything that you will incubate, keep or autoclave (tubes, plates..) label the plate so as to be able to read what is written during incubation (upside down).

Method of Analysis for Azotobacter

Azotobacter is grown on a solid medium free of nitrogen. After some time (6 months) old growth of Azotobacter is transferred to fresh solid medium to renew growth (vigor). 3.1 Instruments/Apparatus/Equipment Required:  Balance  Bunsen burner  Incubator and rotative incubator  Hot air Oven  Laminar hood  Microscope (Dissecting and compound)  pH meter  Vortex  Washer  Colony counter  Autoclave  Magnetic stirrer  Hand tally or mechanical counting device

3.2. Materials  Cotton wool  Cryoboxes  Dispenser  Dropper and Pipette  Ependorff tubes and racks  Foil  Forceps  Gauze  Glassware: Flaks. Bottles, volumetric flask, measuring cylinder, beakers  Loops  Microtubes  Parafilm  Petri plates/dishes  Pestles  Pipetteboy  Pipettes (Pasteur pipettes and Graduated and volumetric pipettes)  Plastic box for incubation  Slides and cover glasses  Test tube and racks  Paper towels

 Graduated pipettes – 1 and 10 ml  Dilution bottles or flasks 3.3. The reagents required: Use a plating medium of the following composition: Agar

20g

Sucrose (C12 H 22O11)

20g

Ferric sulphate Fe2 (SO4) 3

0.1g

Dibasic potassium phosphate (K 2HPO4)

1.0g

Magnesium sulphate (MgSO4, 7H 2O)

0.5g

Sodium chloride (NaCl)

0.5g

Calcium carbonate (CaCO3)

2.0g

Sodium molybdate (Na 2MoO4)

0.005g

Distilled water

1000ml

pH

3.4.

6.8 to 7.2

Sterilizing and Preparation Procedure for Plates

If plates are glass, sterilize the sampling and plating equipment with dry heat in a hot air oven at not less than 160 oC for not less than 2 hours Sterilize the media by autoclaving at 120 oC for 20 min. To permit passage of steam into and from closed container when autoclaved, keep stoppers slightly loosened or plugged with cotton. Air from within the chamber of the sterilizer should be ejected allowing steam pressure to rise.

3.5.

Preparation of plating medium and pouring

Follow serial dilution procedure (Section 1.5) and prepare diluents for plating prior to preparation of plating medium for incubation of microorganism. 3.6.

Preparation of Serial Dilution for Plate Count

A laminar air-flow chamber is used in order to achieve serial dilution of the broth culture of the strain or sample suspension. For plate counts, the countable range is generally 30–300 cells/ml. The procedure is as follows: 1. Set out 8 tubes, each containing 9 ml of sterile water. 2. Dilute 1 ml of broth culture or sample suspension (1 g of sample in 9 ml of water) in steps (10-1 to 10-8) with a sterilized 1 ml serological pipette equipped with a rubber bulb of 1 ml capacity. 3. Suck up broth culture or sample suspension from tube 1 to the 1 ml mark. 4. Immediately expel the broth culture or sample suspension back into the tube with sufficient vigour to effect a thorough mixing. 5. Repeat sucking up and expelling 5 times, and then transfer 1 ml to tube 2. 6. Take a new sterile pipette, attach the rubber bulb, and remove 1 ml to tube 3.

7.

Repeat this procedure using a fresh sterile pipette each time until the dilution series is completed.

3.7.

Incubation of plates 1. Put 1.0 g of the sample in a test-tube. Add 9 ml of water and make a suspension. Serially dilute this suspension (section 3.6). Use the diluted suspension to grow the bacteria by plating techniques (section 3.9 below). 2. Label the plates and incubate at 28 ± 2 °C for 3–5 days for fast-growing rhizobia and 5–10 days for slow-growing ones. 3. Count the colonies with the aid of a magnifying lens under uniform and properly controlled artificial illumination. Use a colony counter, equipped with a guide plate and rules in square centimeters. Record the total number of colonies with the hand tally. 4. Count all plates, but for the purpose of calculation consider plates showing more than 30 and fewer than 300 colonies per plate. Disregard colonies that absorb Congo red and stand out as reddish colonies. Rhizobium stands out as white, translucent, glistening and elevated colonies. Count such colony numbers, and calculate the figure in terms of per milliliter of suspension used for plating. Relate it to the original sample (1 g), taking into account the dilution factor. Also check for being free from contamination at 10-6 dilution. 5. Take uncontaminated Rhizobium cells and multiply them further for use in nodulation tests in pot culture trials.

3.8. Viable cell count There are many techniques for counting viable cells that are able to divide and form offspring. The usual method is to determine the number of cells in a given sample of forming colonies on a suitable agar media. The method involves serial dilution and spreading of diluted suspensions on plates, and counting of colony-forming units on plates. Sterilized molten medium is kept ready in conical flasks placed in a water-bath at a constant temperature of 48 °C by pour, spread or drop plate methods. The procedure for the pour plate method is as follows: 1. Remove dry sterilized Petri dishes from paper packs and stack them in a laminar airflow chamber. 2. For each dilution, three Petri dishes will be required. Stack the plates in sets of three each and label them with the help of a glass marker pen. 3. Using a fresh sterile pipette, pour 1 ml of aliquot from the last dilution (eg., 10-8) into each of the three Petri dishes. 4. Using the same pipette, pour similar aliquots from the next two dilutions (say, from 10-7 and 10-6) into three Petri dishes for each dilution. Pour about same volume of molten medium into each of the plates. 5. Immediately after pouring, move the plates gently in a whirling motion to mix the contents. Allow the medium to solidify, and incubate at 28 °C for 3–5 days. 6. Count the colonies after 3–5 days. Multiply the average number of colonies by the dilution factor. If the average number of colonies at 10-8 dilution is 60, then the sample had a concentration of 60 × 108 = 6 × 109 cells/ml. 3.9.

Preparation of Plating Medium and Pouring

1. Add approximately 2/3 of distilled water into beaker add all chemicals except the agar and vitamins and other nutrients which are sensitive to heat. Place on magnetic stirrer and allow to mix well while heating. Filter (0.2 µm) under aseptic conditions. The pH of the mixture adjusted with either HCL or NaoH if necessary. 2. Store plates in closed plastic box at room temperature label he box with name of the media, the date of preparation and name of persons who prepared. 3. Agar is weighed and placed in a Schott bottle and the contents from the beaker are poured into the bottle. Close the bottle loosely with cotton wool, cover with an Aluminium foil and label: nature of the content, date of preparation, name or initials of who prepared it. Add a piece of autoclave tape so as to hold both the cap and the bottle. Autoclave at 121 ºC for 20mins. 4. Remove the media from autoclave. 5. Melt the required amount of medium in boiling water or by exposure to flowing steam in partially closed container but avoid prolonged exposure to unnecessarily high temperature during and after melting. Melt enough medium which will be used within 3 hours. Re-sterlization of the medium may cause partial precipitation of ingredients. 6. When holding time is less than 30 min promptly cool the molten medium to about 45 o C, and store until used, in a water bath or incubator at 43 oC to 45 oC. Introduce 12 to15 ml of liquefied medium or appropriate quantity depending on size of the Petri dish at 42 to 44 oC into each plate. Gently lift the cover of the dish just enough to pour in the medium. 7. Sterilize the lips of the medium container by exposure to flame; 8. Immediately before pouring. periodically during pouring, and when pouring is completed for each batch of plates, if portion of molten medium remain in containers and are to be used without subsequent sterilization for pouring additional plates. 9. As each plate is poured thoroughly mix the medium with test portions in the Petri dish. 10. By rotating and tilting the dish and without splashing the medium over edge, spread the medium evenly over the bottom of the plate. Provide conditions so that the medium solidifies with reasonable promptness (5-10 min) before removing the plates from surface. 11. Dispense 30 g of inoculants to 270 ml of sterile distilled/ demineralized water and shake for 10 min on a reciprocal shaker or homogenizer. Make serial dilutions up to 10-9 by suspending 10 ml aliquot of previous dilution to 90 ml of water. Take 0.1 ml or suitable aliquots of 10-5 to 10-9 dilutions using sterile pipettes and deliver to Petri dishes containing set medium (Section 2.1) and spread it uniformly with a spreader. Label the plates, invert the plates and promptly place them in the incubator at 28+/- 3 ºC for 4 to 6 days. Azotobacter chroococcum colonies are gummy, raised with or without striations, viscous and often sticky. The pigmentation varies from very light brown to black. Count the colony number and observe the cyst formation as given below and calculate number per gram of the carrier material. Grow the vegetative cells at 30 ºC on Burks agar medium which comprises Sucrose, 20 g; Dipotassium hydrogen phosphate, 0.64 g; Dihydrogen potassium phosphate, 0.20 g; Sodium chloride 0.20 g; Calcium sulphate, 0.05 g; Sodium molybdate, 0.001 g; Ferric sulphate, 0.003 g; Agar, 20 g; and distilled wate,r 1000 ml. Look for vegetative cells after 18 to 24 h either by simple staining method or through phase contrast microscopy. Grow the cyst cells on Burks agar medium as given above with 0.3 percent n- butanol in place of the carbon source. Look for cyst formation after 4 to 5 days incubation. 3.9. Test for Nitrogen fixation in pure cultures

3.9.1. Pure culture medium Prepare medium as given for Azotobacter excluding agar. 3.9.2. Procedure 1. Select from each Azotobacter colony, of the type that has been counted as Azotobacter chroococcum. Pick up one colony and plate on the medium given. 2. Use this pure culture for inoculating the broth for nitrogen fixation. For this purpose, take 50 ml aliquots of broth in 250 ml conical flasks for inoculation. 3. After 12 days growth at 28 ºC, test the contents of the flasks for purity by streaking on fresh medium and concentrating over water bath (50 to 60 ºC) to dryness. Wash the dried culture and take it as a sample. The contents of the flasks in inoculated control series should be similar manner. 3.10. Determination by Kjeldahl Method 3.10.1. Reagents (i) Sulphuric acid-93-98 percent, N-free (ii) Digestion mixture- Mix copper sulphate and potassium sulphate in the ratio 1: 10 and grind them to a fine powder. (iii) Sodium hydroxide pellets or solution, N-free- For solution, dissolve about 450 g of sodium hydroxide in water, cool, and dilute 1 liter (sp gr of the solution should be at least 1.36) (iv) Zinc granules-reagent grade. (v) Indicators: - (a) Methyl red indicator - Dissolve 1g of methyl red in 200 ml of Ethanol and (b) Mixed indicator - Prepare mixed indicator by Dissolving 0.8 of methyl red and 0.2 g of methyl blue in 500 ml of ethanol. (vi) Hydrochloric or sulphuric acid – Standard solution 0.5 or 0.1 N when amount of nitrogen is small. (vii) Sodium hydroxide standard solution – 0.1 N (or other specified concentration) Note - Ratio of salt to acid (m/v) should be about 1:1 at the end of the digestion for proper temperature control. Digestion may be incomplete at a lower ratio, and nitrogen may be lost at higher ratio. Each gram of fat consumes 10 ml of sulphuric acid and each gram of carbohydrate 4.0 ml of sulphuric acid during digestion. 3.10.2 Apparatus (i) For digestion - Use Kjeldahl‘s flasks of hard, moderately thick, well annealed glass with total capacity approximately 500 to 800 ml. Conduct digestion over heating device adjust to bring 250 ml of water at 25 ºC to rolling boil in about 5 minutes. To test the heaters, preheat for 10 minutes in the case of gas burners and for 30 minutes in the case of electric heaters. Add 3 to 4 boiling chips to prevent superheating. (ii) For distillation - Use 500 to 800 ml Kjeldahl‘s flask fitted with rubber stopper through which passes the lower end of an efficient scrubber bulb or trap to prevent mechanical carryover of sodium hydroxide during distillation. Connect the upper end of the bulb tube to a condenser by rubber tubing. Trap the outlet of the condenser in such a way as to ensure absorption of ammonia distilled over with the receiver. 3.10.3 Procedure (a) Place 0.25 g of the sample in the digestion flask. Add 0.7 g mercuric oxide, 15 g potassium sulphate followed by 25 ml of sulphuric acid. Shake, let stand for about 30 minutes

and heat carefully until frothing ceases. Boil briskly until the solution clears and continue boiling further for 90 minutes. Cool, add about 200 ml of water cool to room temperature and add a few zinc granules. (b) Tilt the flask and carefully add 50 ml of sodium hydroxide solution without agitation. Immediately connect the flask to the distillation bulb on the condenser whose tip is immersed in 50 ml of standard 0.1 N acid in the receiving flasks. Rotate the digestion flask carefully to mix the content. Heat until 150 ml of the distillate collects and titrate excess acid with 0.1 N base using methyl-red or mixed indicator. Carry out blank determination on reagents. Note: Check the ammonia recording periodically, using inorganic nitrogen control, for example, ammonium sulphate. Calculation: (i) Nitrogen content, percent by mass =

(ii) Total nitrogen in culture = Total dry mass of sample x percent nitrogen. 3.11. Determination of Glucose From the supernatant, draw suitable aliquots and estimate reducing sugars (glucose) as follows; Reagents (i) Soxhelt modification of Fehling solution: - Prepare by mixing equal volumes of solution A and solution B immediately before using. (ii) Copper sulphate solution (Solution A)- Dissolve 34.639 g of copper sulphate crystals (CuSO4 5H2O) in water, dilute to 500 ml and filter through glass wool or filter paper. Standardization of copper sulphate solution: - Using separate pipettes, pipette accurately 5 ml of solution A and 5 ml of solution B into a conical flask of 250 ml capacity. Heat this mixture to boiling on asbestos gauze and add standard invert sugar solution from a burette, about 1 ml less than the expected volume, which will reduce the Fehling solution completely (about 48 ml). Add 1 ml of methylene blue indicator while keeping the solution boiling. Complete the titration within 3 min, the end point being indicated by change of colour from blue to red. From the volume of invert sugar solution used, calculate the strength(s) of the copper sulphate solution by multiplying the titre value by 0.001 (mg/ml of the standard invert sugar solution). This would give the quantity of invert sugar required to reduce the copper in 5 ml of copper sulphate solution. (iii) Potassium sodium tartrate (Rochelle salt) solution (solution B): - Dissolve 173 g of potassium sodium tartrate and 50 g of sodium hydroxide in water, and dilute to 500 ml. Let the solution stand for a day, and filter. (iv) Hydrochloric acid – sp gr 1.18 at 20 oC (approximately 12 N). (v) Standard invert sugar solution –Weigh accurately 0.95 g of sucrose and dissolve it in 500 ml of water. Add 32 ml of concentrated hydrochloric acid, boil gently for 30 min and keep aside for 24 hours. Neutralize with sodium carbonate and make the final volume to 1000ml; 50 ml of this solution contains 0.05 g of invert sugar. (vi) Methylene blue indicator - 0.2 percent in water.

3.11.1. Procedure: Place about 1 g(M), accurately weighed, of the prepared sample of AI into a 250ml volumetric flask and dilute with about 150 ml of water. Mix thoroughly the contents of the flask and make the volume of 250 ml with water. Using separate pipettes, take accurately 5 ml each of solution A and solution B in a porcelain dish. Add about 12 ml of AI solution from a burette and heat to boiling over an asbestos gauze. Add 1 ml of methylene blue indicator and while keeping the solution boiling complete the titration within 3 minutes, the end point being indicated by change of colour from blue to red. Note the volume (H) in ml of AI solution required for the titration. Calculation

Where S = strength of copper sulphate solution, H = volume in ml of AI solution required for titration, and M = mass in g of AI taken for the test. 3.11.2. Determination of sucrose (i) Procedure: - To 100 ml of the stock AI solution, add 1 ml of concentrated hydrochloric acid and heat the solution to near boiling. Keep aside overnight. Neutralize this solution with sodium carbonate and determine the total reducing sugars as described in calculation; (a) Sucrose, % by mass = (reducing sugars after inversion,% by mass)–(reducing sugars before inversion, % by mass) x 0.95 (b) Nitrogen (mg per gram of sucrose consumed) = 2(a-b)-C Where a= initial quantity of sucrose taken for the test b=mass of sucrose as calculated in (a),and c= amount of nitrogen fixed per gram of glucose.

3.11.3. Test for nitrogen fixation by pure culture With pure culture medium, the procedure is: 1. Prepare Beijerinckia medium as per the composition given above. 2. Take Azotobacter from a suitable colony. Multiply and use this culture for inoculating the broth for N fixation. For this purpose, put 50‑ml aliquots of broth in a 250‑ml conical flask, and inoculate with Azotobacter. Keep a non-inoculated flask as a control. After12 days‘ growth at 28 °C, test the contents of the flask for purity by streakingon fresh medium and concentrating over a water-bath (50–60 °C) to dryness.Wash the dried culture, and take it as a sample. Process the contents of the flask in the non-inoculated controlled series in the same manner. 4. Determine the N in the sample using the Kjeldahl method. The difference in the N content between the control and the inoculated flasks will indicate the N-fixing capacity of Azotobacter. 3.11.4 Maintenance and preparation of pure culture and quality control at Broth stage

Maintain pure culture of Azotobacter on slants of the following composition; Agar

20 g

Sucrose

20 g

Ferrous Sulphate

0.1 g

Dibasic Potassium Phosphate

1.0 g

Magnesium Sulphate

0.5 g

Calcium carbonates

2.0g

Sodium Molybdate

0.005g

Transfer a loopful pure culture to each of agar slants aseptically in an inoculation room and incubate at 28±2 oC for 3 to 10 days depending up on the species of Azotobactor. Always keep pure culture at 5 o C. 3.11.5. Preparation of inoculums culture 1. Prepare Jensen's media broth minus the agar. 2. Transfer a loop full of the culture into a 100 ml/250 ml conical flask containing the broth. Incubate the flasks at 28±2o C on a rotary shaker for 2 to 6 days. 3.11.6. Quality control Tests recommended at Broth stage 1. Check for free from contaminants by preparing slide and observing under microscope. 2. The pH by bacterial broth shall normally be between 6.5 to 7.0 3. Gram staining test shall be carried out as described for Rhizobium of this standard. 3.11.7. Quantitative Test Viable or plate counts Serially dilute one milliliter of the broth to obtain dilutions of the order of 106 to 109. Plate 0.2 ml aliquots of the dilution on YEMA plates and incubate at 28±2 ºC for 2 to 6 days, depending on the species of Rhizobium. The counts of viable Rhizobium in the final broth from shake culture or fermenters shall be not less than 108 to 109 cells/ ml. Otherwise, the broth should be rejected.

4. Conclusions / Recommendations Fertilizer stored for 15 – 20ºC then it will remain effective for six months, however at 0 to 4ºC (cold storage) the bacteria will remain active for 2 years. Critical factors responsible for effectiveness of Azotobacter: 1. Suitability of the strains to target crops. Some strains are specific but generally are broad spectrum 2. Identification of strains as suited to agro-ecosytem, particularly soil pH and moisture conditions 3. The aseptic conditions of manufacturing, the cell count of living organisms present in the carrier material, purity and level of contamination 4. The condition of carrier material in which the culture is packed and the quality of the packing material which determine the shelf life.

5. The conditions in which the packed materials are stored, distributed and kept with farmers before it is applied. 6. Soil conditions particularly pH, organic matter content, moisture level and agronomic practices 4.4.

Indian Standard Specifications for Azotobacter Parameter

Azotobater Inoculants

1

Base

Carrier based

2

Viable cells

107 cells/g of carrier within 15 days of manufucture

3

Cell number at time of 106 cells/g of carrier within 15 days before expiry date expiry

4

Shelf life or expiry period 6 months from the date of manufucture

5

Permissible

No contamination at 105 dilution

contamination 6

pH

6.0 – 7.5

7

Moisture %

35 – 40%

8

Strain

9

Carrier

Should pass through 100 micron IS sieve

10

Efficiency test

Minimum amount of N-fixation not less than 10 mg/g

A. chroococum

of sucrose utilized

D. SOP for Testing the Quality of Commercial Phosphorus Solubilizing Bacteria - Bacillus 1.0.

Definitions

For the purpose of this standard the following definitions shall apply: The following are definition of terms used in synonym and those in the SOP. The terminologies Biofertilizer, Bioenhancer, Biostimulant and Bioinoculant are sometimes used synonymously. . Bofertilizer is a substance which contains living microorganisms which are applied to seed, plant surface or product colonizes the rhizosphere or the interior of the plant or soil and promotes growth by increasing supply or availability of nutrients to the host plant. It is a term widely used term meaning ―bacterial inoculant‖ usually refer to preparations of microorganisms that may be a partial or complete substitute for chemical fertilization (like

rhizobial inoculants). The use of word fertilizer allows easier registration for commercial. It is a formulation containing one or more beneficial strains (or species) in an easy to use and economical carrier either organic, inorganic or synthesized from defined molecules. It contains living microorganisms embedded in a carrier material which are applied to seed, plant surface or product colonizers the rhizosphere or the interior of the plant or soil and promotes growth by increasing supply or availability of nutrients to the host plant, or preparations containing living cells or organisms that enrich the nutrient quality of product. Biopesticides: Living organisms or natural products derived from these organisms which suppress pathogen populations microbial pesticides, other organisms (nematodes, insects) used to control pests; natural products derived from living organisms (biochemical pesticides) and plant incorporated protectants (genetically modified plants) – Bacillus thuringiensis in Bt maize and Bt cotton. Biopesticides decompose quickly in the environments; and are less toxic towards non-target species.

Bioinoculants: Living organisms containing specific strains of specific bacteria, fungi and algae which fix atmospheric nitrogen, make nutrients soluble and available, collect and store nutrients, provide physical barriers to pests and pathogens, stimulate plant growth, and decompose organic residue. Bioenhancers: Substances that increase the bioavailability of active ingredients, vitamins and nutrients Biostimulants: Dormant strains of positive product microorganisms that come alive when introduced into the product system. They contain strains of specific bacteria, fungi or algae which take up nutrients and make it available to plants, collect and store available nutrients, enhance uptake of nutrients, provide physical barriers against pathogens, stimulate growth, decompose organic residues Bacteria: A Kingdom of single celled microorganisms whose cells lack membrane-bound nucleus (prokaryotic) microorganisms that inhabit soil, water, plants and animals. Non Symbiotic nitrogen fixing bacteria: Represents a range of bacteria including saprophytic living on plant residues, bacteria living in close association with rhizosphere of plant roots and bacteria which live entirely within plants (endophytes). They fix nitrogen by reducing gaseous nitrogen in the air to ammonia by a process catalysed by enzyme complex nitrogenase. Bacillus: Very common Gram positive mesophillic, aerobic heterotrophs that produce heatresistant endospore. Contamination: In other microorganism other than Bacillus not declared on the label. Aseptic Conditions/Aseptically: Environment where no microorganisms is present. It can be obtained by using a Bunsen burner to create an aseptic zone which is the aspheric area around the flame with a diameter of approximately 15 cm or a Laminar flow hood with sterile air continuously produced and present in the hood. The high pressure of air flow from the hood

prevents the air from outside to come inside and contaminate the environment in the hood; aseptic conditions are also created by cleaning surface with 70% ethanol. Contaminated microorganisms: Every plate, tube, pipette, or other instruments (glassware, pestles, eppendorff tube…) which has been in contact with microorganisms and cannot be sterilized by the flame of a Bunsen burner is considered as contaminated. Contaminated by toxic chemicals: Every tube, flasks, pipette or other instruments which has been in contact with toxic chemicals is considered contaminated Good Laboratory Practices (GLP): The principle of GLP have been developed to promote the quality and validity of results and of the analysis conducted in a laboratory. It is a management concept covering the organization and the conditions under which laboratory studies are planned, performed, monitored,, recorded and reported. Its principle also include the protection of man and the environment. Mother tube/plate/products: Tube/plate/product which the bacteria are picked from. The result of the growth of this inoculation is considered as the daughter which can become the mother for the next inoculation. Substrate: Any material, that serves as source of energy for an organism. Carrier: Delivery media of live microorganisms from the production to the field. The carrier is the major portion of inoculant. There is presently no universal carrier or formulation available for release of microorganism into soil. Materials and types of formulations of carriers vary: Slurry, powder, peat, liquid. A good carrier should have the capacity to deliver the right number of viable cells in good physiological condition at the right time.

2.0 Introduction Genus Bacillus encompasses a large genetic diversity present in wide range of environmental conditions. The genus Bacillus is in the family Bacillaceae with distinguishing characteristic in the production of endospores, which is not a reproductive structure but rather a resistant, dormant survival form of the organism. Members of the genus Bacillus are characterized as rod-shaped, gram-positive, aerobic and facultative anaerobic, endospore-forming bacteria. Many Bacillus produce extracellular hydrolytic enzymes that break down complex polymers such as polysaccharides, nucleic acids, and lipids, permitting the organisms use these products as carbon sources and electron donors. Numerous species of Bacillus are common in soil. Species of Bacillus usually grow well on defined media containing any of a number of sources. The Genus Bacillus comprises a diverse and commercially useful variety of species. Bacillus are amongst the most powerful phosphate solubilizing bacteria. The principal mechanism for mineral phosphate solubilization is the production of organic acids, and acid phosphatases which play a major role in the mineralization of organic phosphorus in soil. Strains of Bacillus were found to produce mixtures of lactic, isovaleric,isobutyric and acetic acids. Other organic acids, such as glycolic, oxalic, malonic, and succinic acid, have also been identified among phosphate solubilizers. Bacillus subtillis, Bacillus pumilus, B. licheniformis

produce phytohormones. Bacillus megaterium and Bacillus subtillis is a phosphate solubilizer used as a soil inoculant in agriculture and horticulture. Many Bacillus spp. produce antibiotics, of which bacitracin, polymyxin, tyrocidin, gramicidin, and circulin are examples. Half of the commercially available biocontrol agents are Bacillus based. Bacillus subtilis commercialized as biocontrol – biopesticides and the products represent the majority of the microorganisms based biopesticides and has been granted safe by USDA and non-pathogenic. Spore preparations derived from B.thuringiensis and B. popilliae are commercially available as biological pesticides. Among the bacterial products. , biocontrol agent Bacillus thuriengiensis accounts for more than 70% of total sales. molecules. The Baclillus spp – B. amyloliquefaciens, B. subtilis, B. cereus, B. licheniformis, B. megaterium, B. mycoides and B. pumilus are efficient producers of antibiotic. The first product named bacteriological fertilizer for inoculation of cereals was marketed as early as 1897 by a company in Germany today the Bayer AG based on Bacillus species known as Bacillus subtillis. In the 1990‘s several Plant growth based products became commercially available in the USA: Bacillus puilus, B. amyloquefaciens, B. subtilis, B. megaterium, B. mucilaginosus and B. cereus. The future of biofertilizers based on hormoneproducing bacteria seems very promising.

2.1 Quality control management (General procedures) 1. A specific protocol is established before screening is set up. It includes the number of samples, the details about the inoculants (origin, composition, rate of application...), and any other relevant information. 2. Calculations, weights, records (of temperature, humidity...), observations, measurements, preparation of stock solutions (nutritive solutions for example) and any other relevant information are recorded in the lab book on a daily basis. 3. The stock solutions are labelled with the date of reception, name or initials of the person who received it, the number of the container (x of n), date of opening, name or initial of the person who opened it. 4. The reagents are labelled with the name of the contents, date of preparation, name or initials of the person who prepared them and any other relevant information. 5. All samples are clearly identified and records are taken about the different steps of analysis (when they have been put in the oven, grinded, sent for analysis). 6. After harvesting, samples might need to be decontaminated before elimination. Solarization may be considered. 7. In case of contamination of the bench, floor, user, ..., it has to be cleaned and disinfected before the work can be continued (cf. ―Hygiene and Safety rules in a laboratory‖ document). 8. Non contaminated or decontaminated items are cleaned with soap, and rinsed with water and eventually rinsed with distilled water. 9. Waste management: a. Non contaminated waste is eliminated in the normal bin. b. Non contaminated glass waste are put in a separate container labelled with the mention ―Broken glass‖ c. Anything contaminated by microorganisms should be decontaminated before appropriate elimination/cleaning. d. Waste may be put into a special autoclave bags for 20 mins at 121 ºC.

e. Solid and liquid waste contaminated by toxic chemicals are placed in a separate container to be collected by special disposable service providers. f. Disposable glass instruments are ut into a container containing Sodium Hypochlorite solution (commercial Jik) for decontamination before being eliminated. Solid (including broken glass) and liquid waste contaminated by toxic chemicals are placed in separate containers for elimination by waste disposal company. g. Contaminated glassware is properly rinsed with tap water and water collected in specific items (consult MSDS) on product disposal. 10. Equipment‘s are regularly cleaned to remove dust, samples or other waste. 11. Room is cleaned daily. Disinfect every surface with disinfectant and or with 70% ethanol before starting work. The bench and the floor is to be cleaned and disinfected before the work begins and in case of any contamination during the working session it should be disinfected before work continues. General laboratory practices 1. Always wear a lab coat when working in the laboratory. 2. Never put on gloves when working close to flames. In case of burn, the glove would stick on your skin and make the wound worse. 3. To maintain good hygiene, clean your hands with soap and work under aseptic conditions. Clean bench of hood and any surface of operation with a disinfectant; pour some disinfectant on the bench, leave for I min and wipe with gauze/cotton wool. It is also possible to clean the bench with 70% ethanol. 4. Always have a seat when working to avoid accidents. Avoid useless movement in the lab during work. Organize your work space to ensure a smooth run to the analysis. Some equipment need to be switched on some time before use (incubator, laminar flow hood). 5. Plan your work to give them enough time. Organize everything you need in the hood before starting to work so that you don‘t have to cross hands or have to have put your hand out of the hood. Never put items close to the back of the hood because it can affect the flow hence affect the sterility of air. Use preferably the middle and sides of the bench. 6. Every aseptic manipulation has to be done in the middle of the hood, never outside of it and not at the side or the back of the hood. 7. Air contaminations are common. To avoid them, don‘t speak, run or move it it is not necessary and keep the door and windows closed. 8. A burner is used to sterilize items such as loops, tubes, or forceps. The hottest part of the flame is the top of the blue part, so this is the part which has to be used. If you have to work without a hood but just with a burner, leave it on throughout the manipulation and consider that the aseptic zone is spherical area about 15 cm around the flame. 9. Remember to label every tube, bottle, flask, plate… before you start. Always note the date on everything that you will incubate, keep or autoclave (tubes, plates..) label the plate so as to be able to read what is written during incubation (upside down).

2.0 Materials and Methods Most Bacillus species can be grown in defined or relatively-simple complex media. For a few bacilli (e.g. B. subtilis, B. megaterium), minimal media have been established. Primary

isolations can be performed on either nutrient agar (peptone 5g/l, beef extract 3g/l, agar15g/l, pH6.8) or plates of J-agar (tryptone 5g/l, yeast extract 15g/l, K2HPO4 3g/l, glucose 2g/l, agar20g/l, pH7.4). Stock cultures can be maintained in the laboratory on soil extract agar or on special sporulation media.

2.1. 0. Isolation of various species of Bacillus The procedure involves extreme heat which is designed to eliminate vegetative cells of Bacillus which have not already formed endospores and reproductive spores. This will leave only colony-forming units, the endospores when a heated product sample is plated. The variety of different types of colonies resulting after aerobic incubation will represent many different species of Bacillus which have survived by having already formed endospores in the product habitat prior to the heat treatment. The endospores seen microscopically when these colonies are stained will show how the endospore cycle continues on in the artificial habitat of the petri dishes. Most aerobic spore-forming species are easily isolated and readily grown in the bacteriology laboratory. The simplest technique that enriches for aerobic spore formers is to pasteurize a diluted product sample at 80 ᵒC for 15 minutes, then plate onto nutrient agar and incubate at 37 degrees for 24 hours up to several days. The plates are examined after 24 hours for typical colonies identified as catalase-positive, Gram-positive, endospore-forming rods. Direct isolation of particular species requires selective medium or selective conditions that are available for few species. The methods in the SOP are therefore representative of species commonly found in biofertilizer products.

2.1.1. STEP I: Plating and Isolation Materials 1. Product samples 2. One screw-capped tube containing about 12-15 ml of saline (1% NaCl) 3. Water bath set at 80°C 4. Eight 9 ml saline dilution blanks 5. Pipettors and sterile tips 6. Eight plates of Nutrient Agar Composition of Nutrient Agar: Peptone Yeast Dextrose (PYD) plates  Add to 1 liter of distilled water:  2g Peptone  2g Yeast extract  5g Dextrose  15g Agar Procedure 1. Place about half a teaspoonful of product in the screw-capped tube of saline and mix well. Record the details about the sample you are using. 2. Prepare four, serial dilutions of the product suspension 3. Pipette 0.5 mL of the suspension into test tube 1. This bacterial suspension should be mixed thoroughly (using the vortex on each bench) before proceeding to the next step.

4. Obtain a clean pipette and withdraw 0.5 mL of the diluted bacterial suspension from the first test tube and pipette that into the second test tube. Continue in this fashion until you have serially diluted the original bacterial suspension into test tube 7. 5. In test tube 1 you have diluted the bacteria 10 fold, a 1:10 or 1 x 10-1 dilution, in test tube 5 you have diluted the bacteria from the original tube to obtain a 1 x 10-5 dilution, in test tube 10 you have diluted the bacteria from the original tube to obtain a 1 x 10 -10 dilution. 6. The following dilutions are obtained: 1 x 10-1, 1 x 10-2, 1 x 10-3, 1 x 10-4, 1 x 10-5, 1 x 10-6, 1 x 10-7, 1 x 10-8, 1 x 10-9 and 1 x 10-10. 7. Inoculate 0.1 ml from each of the four dilutions onto a separate spread plate of Nutrient Agar. Label the plates NOT HEAT-SHOCKED 8. Screw the cap of the tube on tightly and label the cap top with an identifying mark. Completely immerse the tube in the 80°C water bath for 10 minutes. 9. Remove the tube (with forceps) and cool it in a glass of cold tap water for a few minutes. This heating and cooling constitute the heat-shocking procedure. This is suppose to eliminate the vegetative cells and reproductive spores. 10. After mixing the suspension, prepare plates from dilutions as you did in step 2. Label the plates HEAT-SHOCKED. 11. Incubate the plates at 30°C for 2 or more days Note the use of the term heat-shocking for the heating/cooling process applied to the product suspensions. Consider the fact that two oxygen relationships are represented within the genus Bacillus: Some species are strictly aerobic while others are facultatively anaerobic.

2.1.2. STEP II: Diagnosis of cultures for Bacillus spp. Materials 1. Three Durham tubes of Glucose Fermentation Broth 2. One plate of Starch Agar 3. Dropper bottle of malachite green (5% aqueous solution, filtered) Composition of Glucose Fermentation Broth  1.0% Glucose for source of carbon  0.2% Peptone for supply of amino acids as source of nitrogen, carbon, sulfur and energy  pH indicator 0.003% Bromo-thymol blue which turns yellow with net acidic pH and blue with net alkaline pH  0.5% NaCL2  0.03% KH2HPO4  0.3% Agar adjusted to pH 7.1 Composition of Starch Agar  Beef extract  Soluble Starch  Agar  Distilled water  Adjusted pH

3g 10g 12g 1L 7.5±0.2 at 25ºC

Composition of endospore-staining reagents Malachite green stain (0.5% (wt/vol) aqueous solution)    

0.5 g of malachite green 100 ml of distilled water Decolorizing agent Tap water

Procedure 1. Compare two sets of plates (labeled heat-shocked and not heat-shocked) 2. Check if the heat-shocking causes any noticeable effect in the variety of different types of colonies Note: As Bacillus colonies tend to be light or tan-colored, any brightly-pigmented colonies would be of other genera, and fuzzy, filamentous colonies would probably be molds. (Any such colonies on the heat-shocked plates may indicate faulty aseptic technique or contaminants!). 3. Determine the number of CFUs per ml of the suspension for both sets of plates and note any difference. When counting colonies, remember to choose one plate (having between 30 and 300 colonies) to determine the CFU/ml count. (Don't count all the plates) Any large, spreading colony - such as the spiral-colony-forming Bacillus mycoides which can cover an entire plate in time - should be counted as one colony. 4. On the plates marked heat-shocked proceed as follows, recording your observations in table form. (Save your plates until you are satisfied with your microscopic observations in Step III. You may wish to continue incubating them.) 5. Pick out at least three different, well-isolated colonies. Label them by number or letter and record their colonial appearances. 6. Then, prepare heat-fixed smears from each numbered colony for subsequent staining (Step III, below). For one of the larger colonies, prepare two smears - one from the center of the colony and one from the edge. 7. From each numbered colony, inoculate a tube of Glucose Fermentation Broth, and also spot-inoculate a sector of the plate of Starch Agar. (A separate plate should be obtained for any wide-spreading colony.) Incubate at 30°C. 8. When time permits, stain the smears by the endospore-staining procedure 9. Look for the presence of rod-shaped vegetative cells (red) and circular or oval endospores (green). The endospores may be found within the vegetative cells and/or free. (If no endospores are seen, consider time of incubation so far, and re-incubate your plates if necessary.) 10. For the smears you made from the edge and center of the same colony, explain any noticeable difference in the apparent ratio of endospores to vegetative cells.

2.1.3. STEP III : Characterization of Bacillus spp. Materials 1. Dropper bottle of 3% hydrogen peroxide 2. General purpose media 3. Empty plastic petri dishes for the slide catalase test Procedure 1. From each of the isolates growing on the Starch Agar plates, perform the slide catalase test (3% Hydrogen peroxide) and record the results. Discard the slide into

the disinfectant and the plastic petri dish in the usual place. (Refer to Appendix below for catalase test) 2. For each of the isolates, note the reaction(s) of the Glucose Fermentation Broth and the amylase reaction (Refer to Appendix below for amylase reaction?).

2.1.4. STEP IV Gram Staining Gram staining is a simple, differential staining method which helps to group bacteria according to their Gram character (Gram positive or Gram negative). The Gram stain is a very important preliminary step in the initial characterization and classification of bacteria. It is also a key procedure in the identification of bacteria based on staining characteristics, enabling the bacteria to be examined using a light microscope. The bacteria present in an unstained smear are invisible when viewed using a light microscope. Once stained, the morphology and arrangement of the bacteria may be observed as well. It is also an important step in the screening of infectious agents in clinical specimens such as direct smears from a patient.The Gram stain procedure enables bacteria to retain color of the stains, based on the differences in the chemical and physical properties of the cell wall. Apparatus and Equipment required  Inoculating loop  Bunsen Burner  Glass microscope slide  Pipette (disposable)  Clasp (Close pin or tongs)  Microscope (preferably with oil immersion lens) Reagents      

Medium     

L-Glutamic acid Citric acid Dipotassium hydrogen phosphate Ferric ammonium citrtate Magnesium sulphate Gram stains reagent set  Crystal Violet  Grams Iodine  Decolorize reagent (95% alchohol)  Safranin

Peptone Yeast Destrose (PYD) plates: Peptone Yeast extract Dextrose Aagar

Procedure 1. Draw circle in the center of a glass slide . The circle indicates where the sample will be placed. Turn slide over so that the markings so the markings and the sample will be on the opposite sides. Label slide.

2. Place 1 ml of diluent (water) into test tube using a dosposable pipette Thoroughly mix the sample. 3. Sterilize the inoculation loop in the flame of the Bunsen Burner. With a cooled inoculation loop obtain a small sample of product from the container. 4. Transfer the sample into the previously measured diluent (water). Cover test tube with the screw cap and gently shake for 5 to 10 sec. To thoroughly mix the sample throughout the diluent. 5. Sterilize the inoculation loop in the flame. Transfer a small amount (two loops) of sample from the last tube to previously prepared slide. Tilt the slide to spread the drop out slightly about the size of a penny 6. ―Heat-fix‖ the slide with the specimen by holding the slide with a clasp and pass it over a heat source, such as a flame, several times using a forceps. The slide should be passed very quickly through the flame and not be heated excessively. Dont place the slide directly in the flame so that the bacteria wont be burned. Place slide on the staining tray. 7. Flood the fixed smear with crystal violet solution and allow to remain for 1 minute. Rinse off the crystal violet with distilled or tap water. 8. Flood the slide with iodine solution. Allow to remain for one minute. Rinse off the iodine solution with distilled or tap water. 9. Flood the slide with decolorizer for one to five seconds. Rinse off the decolorizer with distilled or tap water. 10. Flood the slide with safranin. Allow to remain for 30 seconds. Rinse off the safranin with distilled or tap water. 11. Dry the slide on absorbent paper and gently pass the paper down for few times. Place in an upright position. 12. Microscopically examine the slide for bacterial organisms under a 100X objective. Observe several fields on the slide for bacterial organisms. Describe the gram reaction of any organisms seen. Gram-positive bacteria stain deep violet to blue and gramnegative bacteria stain pink to red.

2.20. Isolation of specific Bacillus spp (i) Isolation of three Bacillus strains (Bacillus subtillis, Bacillus licheniformis, and Bacillus amyloliquefaciens) Luria Bertani (LB) Medium Preparing LB Medium (Makes 1000 mL) Equipment:  1000 ml graduated cylinder  stir bar  pH meter, with 7.0 and 10.0 pH solutions  1000 mL autoclavable bottle  autoclave machine LB Agar (Luria Bertani Agar) Composition of Ingredients Grams/Litre  deionized water  Tryptone  Yeast extract

1000ml 10 5g

      

NaCl 5g 5 M NaOH several drops 1 M HCl several drops Agar 15g 1000x ampicillin 1mg Final pH 7.2 at 37°C Store prepared media below 8°C, protected from direct light. Store dehydrated powder, in a dry place, intightly-sealed containers at 2-25°C.

Procedure 1. Measure about 700 ml deionized water into a graduated cylinder. 2. Mix following into water: tryptone, yeast, and NaCl. 3. Use stir bar to mix well. While stirring, add 300 ml deionized water, making sure to wash sides of cylinder. 4. Use stir bar to mix well. 5. Calibrate pH meter with 7.0 and 10.0 pH solutions. Never leave meter out of solution too long. Rinse meter with deionized water after dipping into and gently. 6. While broth mixes, adjust its pH to 7.5 with dropwise additions of NaOH and HCl. 7. Pour broth into a 1000 mL autoclavable bottle, 8. Add agar to large autoclavable bottle. Over agar pour pH-adjusted plating solution. Mix by swirling. 9. Autoclave agar solution on liquid cycle at 121°C for 15 minutes. 10. Cool in water bath for up to an hour. 11. Once it reaches room termperature, store in cold room. Plating and Identification 1. Isolation of bacteria from sample 90ml 2. Sterilized saline solution (NaCl 0.85%, w/v) is added to 10g sample and homogenized for 2 minutes with a blender. 3. Sample solution is diluted serially ten-fold with 0.85% NaCl (101 - 108) 4. Pipette 0.5 ml of the suspension into test tube 1. This bacterial suspension should be mixed thoroughly (using the vortex on each bench) before proceeding to the next step. 5. Obtain a clean pipette and withdraw 0.5 ml of the diluted bacterial suspension from the first test tube and pipette that into the second test tube. Continue in this fashion until you have serially diluted the original bacterial suspension into test tube 7. 6. In test tube 1 you have diluted the bacteria 10 fold, a 1:10 or 1 x 10-1 dilution, in test tube 5 you have diluted the bacteria from the original tube to obtain a 1 x 10-5 dilution, in test tube 10 you have diluted the bacteria from the original tube to obtain a 1 x 10-10 dilution. 7. Spread on LB agar plates and incubate for 16h at 37C. 8. Bacterial colonies are isolated according to morphological characteristics of individual colonies. 9. Individual colonies are selected randomly and purified by single colony isolation after triple re-streaking on LB agar medium. Morphological characteristics observation is done with a phase contrast microscope.

Principle and Interpretation:Tryptone and Yeast extract serve as a source of nitrogen, sulfur and carbon while Yeast extract also contains Vitamin B complex. Sodium chloride provides sodium ions for the membrane transport and maintains osmotic equilibrium of the medium.

3.0.

Qualitative and Quanititative analysis of Bacillus spp.

1. Colony Morphology: a. Baciillus subtillis: Colonies are larger, but not as large as B. cereus. The margin is undulate, with circular form and flat elevation b. Bacilus cereus: Colonies are large, irregular and flat with an undulate margin c. Bacillus polymyxa: Colonies are large, irregular and flat with an undulate margin 2. Gram staining (Refer to procedure above): a. Bacillus subtillis and Bacillus cereus: This microbe forms gram-positive rods. Endospores may be visible in older cultures as clear areas inside the cells. Older cultures may stain pink, due to the deterioration of cell walls. b. Bacillus polymyxa: This microbe may appears as gram-negative short rods. However, the large amount of capsule surrounding these cells often interferes with the de-staining process, causing them to stain Gram positive. Older cultures (more than 24 hours) may appear as cocci. 3. Catalase Test: This test is used to identify organisms that produce the enzyme, catalase. This enzyme detoxifies hydrogen peroxide by breaking it down into water and oxygen gas. The bubbles resulting from production of oxygen gas clearly indicate a catalase positive result (See method on Appendix 1D below). a. Bacillus subtillis, B. cereus and B. polymyxa: Note the bubble formation. Catalase positive 4. Starch hydrolysis test: This test is used to identify bacteria that can hydrolyze starch (amylose and amylopectin) using the enzymes a-amylase and oligo-1,6-glucosidase. Often used to differentiate species from the genera Bacillus. In order to use these starches as a carbon source, bacteria must secrete a-amylase and oligo-1,6-glucosidase into the extracellular space. In order to interpret the results of the starch hydrolysis test, iodine must be added to the agar. The iodine reacts with the starch to form a dark brown color. Thus, hydrolysis of the starch will create a clear zone around the bacterial growth. Bacillus subtilis is positive for starch hydrolysis (Refer to procedure below Appendix 1E) a. Bacillus subtillis, B. cereus and B. polymyxa: Note the zone of clearing. All the starch in the medium near the microbe has been hydrolyzed by extracellular amylases. 5. Nitrate test (Nitrate Borth) Recommended for detecting nitrate-reducing and indole-producing microorganisms. Ingredients Grams/Litre  Peptone 5.0  Meat extract 3.0  Potassium nitrate 1.0

 Final pH 7.0 ± 0.2 at 25°C  Store prepared media below 8°C, protected from direct light. Store dehydrated powder, in a dry place, in tightly-sealed containers at 5°C. 

Preparation of Reagents:  Sulfanilic acid solution (Reagent A): Dissolve 8 g of sulfanilic acid (Fluka 86090) in 1 litre 5N acetic acid. Store reagent A at room temperature for up to 3 months, in dark. Reagents may be stored in dark brown glass containers; bottles may be wrapped in aluminum foil to ensure darkness.  _-Naphthylamine solution (Reagent B): Dissolve 6 g of N,N-Dimethyl-1naphthylamine (Fluka 40860) in 1 litre 5N acetic acid. Store Reagent B at 2 to 8°C for up to 3 months, in dark. Reagents may be stored in dark brown glass containers; bottles may be wrapped in aluminum foil to ensure darkness. Procedure:  Dissolve 9 g in 1 litre distilled water. Dispense in tubes and sterilize by autoclaving at 121°C for 15 minutes.  Inoculate the tubes heavily with a fresh culture of the suspect organism. Inoculate at least 1 ml sample in a tube or take a big part of a colony with an inoculating loop. Do not forget a negative control without any bacteria. For special organisms the media has to be modified. Nitrate reaction occurs only under anaerobic conditions. The medium is dispensed in tubes to give a low surface area to depth ratio which limits the diffusion of oxygen into the medium. Most bacteria use the oxygen in the medium and rapidly produce anaerobic conditions. To reach faster an anaerobic condition it is recommendable to give about one centimetre of paraffin oil on the surface of the media or over-gassing with e.g. carbon dioxide and seal the tube with parafilm.  Incubate the tubes at 35 to 37°C (bacilli at 30°C) for 24 to 48 h in an incubator with or without supplemental carbon dioxide.  Put a 5 drops of reagent A and 5 drops of reagent B into the tube containing culture to be tested. Shake the tube well to mix reagents with medium. A distinct red or pink colour, which should develop within a few minutes, indicates nitrate reduction.  If the suspension turns pink-red before the addition of Zn powder, the reaction is positive and the test is completed.  If the suspension is colorless after the addition of reagents A and B, add a small amount (―sharp knife point") of zinc powder to the medium. Shake the tube vigorously and allow it to stand at room temperature for 10-15 min.  If the medium remains colorless after the addition of Zn powder, the test result is positive.  If the medium turns pink after the addition of Zn powder, the result is negative.  The negative control should also be tested. There should be no pink colour formation after adding reagent A and B and if zinc powder is added the colour should change to pink. Addition of too much zinc powder can results in falsenegative reaction. a. Bacillus subtillis, B. cereus and B. polymyxa: This microbe can reduce nitrate to nitrite

6. Motility Test Motility test medium  Beef extract  Pancreatic digest of casein  Sodium chloride  Agar

3.0 g 10.0 g 5.0 g 4.0 g

Preparation of Media 1. Bring to 1 liter with distilled water and heat to boiling to melt agar. 2. Add 5 ml of 1% triphenyltetrazolium chloride (TTC) solution. 3. Dispense in 5-ml aliquots into tubes and autoclave at 121°C under 15 psi pressure for 15 minutes. Cool upright in racks. 4. After inoculation, incubate at 35°C for 18 hours or until growth is evident. Motility test medium is commercially available in premixed forms and prepoured tubes from biological supply companies. Motility test Protocol 1. To test for motility, use a sterile needle to pick a well-isolated colony and stab the medium to within 1 cm of the bottom of the tube. 2. Be sure to keep the needle in the same line it entered as it is removed from the medium. Incubate at 35°C for 18 hours or until growth is evident (Fig. 1). 3. A positive motility test is indicated by a red turbid area extending away from the line of inoculation. A negative test is indicated by red growth along the inoculation line but no further . a. Bacillus subtillis, B. cereus and B. polymyxa: This microbe is motile. Note how the microbe swims to the top of the tube where there is more oxygen. 7. Fermentation of carbohydrate (Both Glucose Fermentation Broth) Testing whether an organism can ferment glucose is one of the basic, primary tests in the identification of chemoheterotrophic bacteria. For this test we routinely use a "Glucose Fermentation Broth." Fermentation of glucose results in the abundant production of acidic end products, the presence of which can be detected by the pH indicator in the medium. Many organisms produce gas – either CO2 alone or a mixture of H2 and CO2. H2 is insoluble and is detected by bubble formation in a Durham tube placed in the medium.  Glucose – a sugar from which most common chemo heterotrophic bacteria can obtain energy by fermentation and/or respiration. Glucose can also be utilized as a source of carbon, but these media include a large number of potential carbon sources (amino acids as well as glucose).  Peptone – a commonly-used medium ingredient which mainly supplies amino acids (sources of nitrogen, carbon, sulfur and energy for many bacteria). It is a crude preparation of a partially-digested protein, and a peptone solution can serve as a complete medium for a number of organisms. If too much peptone (relative to glucose) is incorporated in the medium, detection of acidic products of fermentation or respiration may not be possible, as overabundance of ammonium (which is alkaline) released from the breakdown of amino acids can neutralize the acids.

 pH indicator – the pH indicators employed in these media turn yellow under acidic conditions. Brom-cresol purple is in Glucose Fermentation Broth (which also contains the Durham tube). Protocol for testing fermentation A. Inoculation of media 1. Aseptically inoculate each test tube with the test microorganism using an inoculating needle or loop. 2. Make sure to avoid the Durham tube, if present. Swirl the tube gently to mix contents. 3. Avoid contact of liquid with tube cap. 4. Alternatively, inoculate each test tube with 1 to 2 drops of an 18- to 24-hour broth culture of the desired organism . 5. Examples of broth media that could be used include brain heart infusion, nutrient agar, and tryptic soy agar. B. Incubation 6. Incubate tubes at 35 to 37°C for 18 to 24 hours . Longer incubation periods may be required to confirm a negative result . 7. C. Interpretation of results 1. When using phenol red as the pH indicator, a yellow color indicates that enough acid products have been produced by fermentation of the sugar to lower the pH to 6.8 or less. A delayed fermentation reaction may produce an orange color. In such cases, it is best to re-incubate the tube . Refer to Table for other commonly used pH indicators and their corresponding colors. 2. Gas production results: Bubbles trapped within the Durham tube indicate the production of gas. Even a single bubble is significant and denotes evidence of gas production. No bubbles within the Durham tube indicate a non-gas-producing or anaerogenic organism. 3. Negative results: A reddish or pink color indicates a negative reaction. In negative tubes, the presence of turbidity serves as control for growth. A reddish or pink color in a clear tube could indicate a false negative. a. Bacillus subtillis, B. cereus and B. polymyxa: This microbe cannot ferment glucose. 8. Indole from tryptophan Tryptophan is an amino acid that can undergo deamination and hydrolysis by bacteria that express tryptophanase enzyme. The chief requirement for culturing an organism prior to performing the indole test is that the medium contains a sufficient quantity of tryptophan. The presence of indole when a microbe is grown in a medium rich in tryptophan demonstrates that an organism has the capacity to degrade tryptophan. Detection of indole, a by-product of tryptophan metabolism, relies upon the chemical reaction between indole and p-dimethylaminobenzaldehyde (DMAB) under acidic conditions to produce the red dye rosindole. tryptophan + water = indole + pyruvic acid + ammonia The main requirement for a suitable indole test medium is that it contain a sufficient amount of tryptophan. Although many media meet this criterion, tryptone broth is commonly used. Tryptone broth Ingredient

Amount

Tryptone Sodium Chloride

10.0g 5.0g

Procedure for Media preparation 1. Dissolve the ingredients in 1 liter of sterile water. 2. Dispense 4 ml per tube. 3. Cap tube and autoclave at 121oC under 15 psi pressure for 15 minutes. 4. Store the tubes in the refrigerator at 4 to 10°C. Kovács reagent Ingredients Amyl or isoamyl alcohol, reagent grade (butyl alcohol may be substituted) p-dimethylaminobenzaldehyde (DMAB) HCL (Concentrated)

Amount 150.0ml 10g 50.0ml

Procedure (Media preparation) 1. Dissolve DMAB in the alcohol. Gentle heating might be required to get the aldehyde into solution. 2. Slowly add the acid to the aldehyde-alcohol mixture. The solution should be a pale yellow color and is only stable for a short time. 3. Store the mixture in a brown glass bottle in the refrigerator. ( Kovács reagent also is commercially available). Protocol for Testing 1. Inoculate the tube of tryptone broth with a small amount of a pure culture. Incubate at 35°C (+/- 2°C) for 24 to 48 hours. 2. To test for indole production, add 5 drops of Kovács reagent directly to the tube. 3. A positive indole test is indicated by the formation of a pink to red color ("cherry-red ring") in the reagent layer on top of the medium within seconds of adding the reagent . 4. If a culture is indole negative, the reagent layer will remain yellow or be slightly cloudy.

a. Bacillus subtillis, B. cereus and B. polymyxa: This microbe is negative for indole production

3.7 Quantitative Analysis Method for Estimating MPN Count 1. To calculate the most probable number of organisms in the original sample, select as P1 the number of positive tubes in the least concentrated dilution in which all tubes are positive or in which the greatest number of tubes is +ve, and let P2 and P3 represent the numbers of positive tubes in the next two higher dilutions (refer to Appendix 5.1). 2. Then find the row of numbers in Table 1 in which P1 and P2 correspond to the values observed experimentally. Follow that row of numbers across the table to the column headed by the observed value of P. 3. The figure at the point of intersection is the most probable number of organisms in the quantity of original sample represented in the inoculum added in the second dilution. Multiply this figure by the appropriate dilution factor to obtain the MPN value. 4. Most Probable Number (MPN) Serial dilution Counts 1. Crush product in sterile motor and pestle and shake with 100ml of sterile distilled water for 10 – 20 minutes to obtain a standard suspension.

2. Pipette 0.5 mL of the suspension into test tube 1. This bacterial suspension should be mixed thoroughly (using the vortex on each bench) before proceeding to the next step. 3. Obtain a clean pipette and withdraw 0.5 mL of the diluted bacterial suspension from the first test tube and pipette that into the second test tube. Continue in this fashion until you have serially diluted the original bacterial suspension into test tube 7. 4. In test tube 1 you have diluted the bacteria 10 fold, a 1:10 or 1 x 10-1 dilution, in test tube 5 you have diluted the bacteria from the original tube to obtain a 1 x 10-5 dilution, in test tube 10 you have diluted the bacteria from the original tube to obtain a 1 x 10-10 dilution. Plating the serially diluted cells: 1. Obtain 10 King Agar B plates and label with initials and the dilution factor. 2. The following dilutions will be plated: 1 x 10-1, 1 x 10-2, 1 x 10-3, 1 x 10-4, 1 x 10-5, 1 x 10-6, 1 x 10-7, 1 x 10-8, 1 x 10-9 and 1 x 10-10. 3. Obtain a beaker containing a loop and pour a small volume of alcohol into the beaker. The loop is used to spread the diluted bacterial suspension evenly over the surface of the plate. 4. For the cell suspension that will be plated onto the King Agar B plate label 1 x 10-1, pipette 0.5 mL of the diluted suspension from the appropriately diluted test tube onto the surface of the plate. 5. Dip the loop into the alcohol solution and flame until the alcohol has burned off. Do not heat the loop to long; you only need to flame the loop to burn off the alcohol—that will be sufficient for sterilizing. 6. After sterilizing the loop, use it to spread the bacterial suspension evenly over the entire surface of the plate. Allow the plate to dry. Continue this process with the remainder of the bacterial dilutions. 7. Tape all of plates together and incubate the plates, upside down, at 37oC for 24 hours. During the next period count the number of colony forming units for each dilution and calculate the number of bacteria in the original suspension.

Bacillus count per gram of carrier = Viable MPN table x Dilution level Dry mass of product Most Probable Number Table P1

0 0 0 0 0 0

P2

Most probable number for indicated values of P 3 0 1 2 3 4 0 1 2 3 4 5

0.018 0.037 0.056 0.075 0.094

0.018 0.036 0.055 0.074 0.094 0.11

0.036 0.055 0.074 0.093 0.11 0.13

0.054 0.073 0.092 0.11 0.13 0.15

5 0.072 0.091 0.11 0.13 0.15 0.17

0.090 0.11 0.13 0.15 0.17 0.19

1 1 1 1 1 1

0 1 2 3 4 5

0.020 0.040 0.061 0.089 0.11 0.13

0.040 0.061 0.082 0.10 0.13 0.15

0.060 0.081 0.10 0.13 0.15 0.17

0.080 0.10 0.12 0.16 0.17 0.19

0.10 0.12 0.16 0.17 0.19 0.22

0.12 0.14 0.17 0.19 0.22 0.24

2 2 2 2 2 2 3 3 3 3 3 3 4 4 4 4 4 4 5 5 5 5 5 5

0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5

0.046 0.068 0.093 0.12 0.15 0.17 0.078 0.11 0.14 0.17 0.21 0.25 0.13 0.17 0.22 0.27 0.34 0.41 0.23 0.33 0.49 0.79 1.3 2.4

0.068 0.092 0.12 0.14 0.17 0.20 0.11 0.14 0.17 0.21 0.24 0.29 0.17 0.21 0.26 0.33 0.40 0.48 0.31 0.46 0.70 1.1 1.7 3.5

0.091 0.12 0.14 0.17 0.20 0.23 0.13 0.17 0.20 0.24 0.28 0.32 0.21 0.26 0.32 0.39 0.47 0.56 0.43 0.64 0.95 1.4 2.2 5.4

0.12 0.14 0.17 0.20 0.23 0.26 0.16 0.20 0.24 0.28 0.32 0.37 0.25 0.31 0.38 0.45 0.54 0.64 0.58 0.84 1.2 1.8 2.8 9.2

0.14 0.17 0.19 0.22 0.25 0.29 0.20 0.23 0.27 0.31 0.36 0.41 0.30 0.36 0.44 0.52 0.62 0.72 0.76 1.1 1.5 2.1 3.5 16.0

0.16 0.19 0.22 0.25 0.28 0.32 0.23 0.27 0.31 0.35 0.40 0.45 0.36 0.42 0.50 0.59 0.69 0.81 0.95 1.3 1.8 2.5 4.3 --

OR Counting colony forming units (CFU) and calculating the amount of bacteria in the original solution. 1. Using a counter for each dilution, count the number of colony forming units on your plates. Typically numbers between 30 and 800 are considered to be in the range where one‘s data is statistically accurate. If the number of CFUs on your plate are greater than 1000, you may record in your table TNTC (too numerous to count). 2. Alternatively, if your numbers are greater than 1000 AND you have evenly distributed the diluted bacterial suspension on the surface of the plate AND you can discern individual colonies; divide the plate into 4 sectors, count the number of bacteria in one sector and multiply by four. 3. If the number of CFUs on your plate is below 10, record the number of CFUs, but do not use this in your calculations. T=Trial

Dilution Number of bacterial colonies (CFUs) factor T1 1:10

-1

T2

T3

T4

T5

T6

Avg Avg # # bacteria/ml CFU T7

T8

T9

1:10-2 1:10-3 1:10-4 1:10-5 1:10-6 1:10-7 1:10-8 1:10-9 1:10-10

Calculating the number of bacteria per mL of serially diluted bacteria: To calculate the number of bacteria per mL of diluted sample one should use the following equation: Number of CFU

Number of CFU

Volume plated (mL) x total dilution used

mL

For example, if for the 1x 10-8 dilution plate you plated 0.1 mL of the diluted cell suspension and counted 200 bacteria, then the calculation would be: 200/0.1 mL x 10-8 or 200/10-9 or 2.0 x 1011 bacteria per mL. OR?

Conclusion Bacillus are divided into 11 groups and due to physiological diversity, the direct isolation of a particular (Bacillus) species requires selective medium or other selective conditions that are available for only a few species. Inoculants are applied to seed and seedling treatment, soil application and drip irrigation. Carriers are solid and liquid form, with the solid in organic or clay mineral form. The most common formulation most products is wettable powder. The products are produced in bulk commercially by fermentation. The CFU counts for example for product with Bacillus amyloliquefaciens for different formulations in China are: liquid (0.5 – 1.5 x 109), powder (0.1 -0.3 x x 109) and granular (0.1 -0.3 x x 109). The Bacillus genus is diverse in functions with species with antibiotics activity (Bacillus amyloliquefaciens and B. pumilus), Biopesticides (Bacillus thuringiensis and Bacillus subtillis), Biofertilizer (B. megatarium, B. polymyxa and B. licheniformis). The pathogenic Bacillus anthracis, a causal agent of Anthrax, is of clinical importance. Species important in food spoilage are Bacillus coagulans, B. cereus, B. subtillis, B. licheniformis and B. pumilus.

Appendix 1 A. Starch Agar (g/L) Reagents Beef extract Soluble Starch

3g 10g

Agar Distilled water Adjusted pH

12g 1L 7.5±0.2 at 25ºC

Procedure 1. Suspend the first 3 ingredients in 1 litre distilled water 2. Mix thoroughly 3. Heat with frequent agitation and carefully bring to adjust boiling 4. Do not allow to boil as excessive boiling may hydrolyze the starch 5. Autoclave at 121ºC for 15 minutes psi 6. Final pH of the medium should be 7.5±2 at 25C 7. After sterilization pour the melted medium into sterilized petri plates (approximately 20 to 30ml per plate) and let it solidify before use./ Medium color is light amber to slightly opalscent 8. The prepared starch agar plates become opaque if refrigerated 9. The prepared medium can be dispensed into screw-cap agar and stored for up to 2 weeks 10. After 2 weeks the starch changes and reddish purple spots may develop upon addition of iodine 11. The stored medium in the tubes should be melted into boiling water bath, poured into individual plates and brough to room temperature before use.

B. Endospore-staining procedure Schaeffer-Fulton method for staining endospores Malachite green stain (0.5% (wt/vol) aqueous solution) 0.5 g of malachite green 100 ml of distilled water Decolorizing agent Tap water Safranin counterstain Stock solution (2.5% (wt/vol) alcoholic solution) 2.5 g of safranin O 100 ml of 95% ethanol Working solution 10 ml of stock solution 90 ml of distilled water Protocol Note: the protocols that follow are those reported by Gerhardt et al. in the 1981 and 1994 editions of the textbooks noted here. Many common modifications of these protocols are used by professionals around the world. Some of the modifications are addressed in the Tips and Comments section as a result of valuable input from the reviewers. Schaeffer-Fulton method for staining endospores

1. Air dry and heat fix the organism on a glass slide and cover with a square of blotting paper or toweling cut to fit the slide. 2. Saturate the blotting paper with malachite green stain solution and steam for 5 minutes, keeping the paper moist and adding more dye as required. Alternatively, the slide may be steamed over a container of boiling water. 3. Wash the slide in tap water. 4. Counterstain with safranin for 30 seconds. Wash with tap water; blot dry. 5. Examine the slide under the oil immersion lens (1,000X) for the presence of endospores. Endospores are bright green and vegetative cells are brownish red to pink.

C. Catalase Test The purpose is to see if the microbe has catalase , a protective enzyme capable of destroying the dangerous chemical hydrogen peroxide Reagent 1. Hydrogen peroxide 2. General purpose medium Composition of General purpose Medium Trypticase soy agar is a bacterial growth medium. TSA is a general purpose medium, providing enough nutrients to allow for a wide variety of microorganisms to grow. It is used for a wide range of applications including; culture storage, enumeration (counting), isolation of pure cultures or simply general culture. e.g. Tryptocase Soy Agar (TSA) Tryptocase Soy Broth (TSB) Nutrient Agar Trypticase soy agar (g/L)  Casein peptone (pancreatic)  Soya peptone (papainic)  Sodium chloride  Agar  Final pH

15.0 5.0 5.0 15.0 7.3 +/- 0.2 at 25°C

Store prepared media below 8°C, protected from direct light. Store dehydrated powder, in a dry place, in tightly-sealed containers at 2-25°C. .  The medium contains enzymatic digests of casein and  Soybean meal which provides amino acids and other nitrogenous substances making it a nutritious medium for a variety of organisms.  Glucose is the energy source.  Sodium chloride maintains the osmotic equilibrium,  dipotassium phosphate acts as buffer to maintain pH.  Agar extracted from any number of organisms is used as a gelling agent.  Care should be taken to avoid using blood agar, as blood cells contain catalase and trace contamination of medium can lead to false positive results. Procedure

1. Remove cells from an isolated colony on a streak plate. 2. Streak an inoculum from a pure culture on a sterile plate of general purpose growth medium 3. Incubate inoculated plate at 35-37 C for 24 hours. 4. Smear the growth from the plate on a microscope slide and place a drop of 3% hydrogen peroxide on the smear. 5. Copious bubbles liberated in the hydrogen peroxide by the growth indicates presence of catalase

D.

Amylase Reaction List of Reagents and Instruments A. Equipment •Erlenmeyer flasks •Beakers •Graduated cylinder •Pipets, 1ml, 10ml •Test tubes •Temperature bath •Thermometer •Balance •Syringe Filter holder and filter paper Spectrophotometer •Brookfield viscometer B. Reagents •Enzymes ◦Bacterial amylase solution, 3000 SKB units/ml •Corn starch •HCl Stopping Solution, 0.1N HCl •Iodine Reagent Stock Solution (in aqueous solution) See Note 1. ◦Iodine: 5 g/l ◦KI: 50 g/l •Potassium Phosphate Buffers ◦KH2PO4 (monobasic phosphate) (FW=136.1) ◦K2HPO4•3H2O (dibasic phosphate) (FW=228.23) •CaCl2•2H2O, 0.1M solution •Reagents for the analysis of reducing sugars Procedures 1. Prepare a 20 g/l starch solution. 1.Mix 20 g of soluble potato starch in approx. 50 ml of cold water. 2. While stirring, add the slurry to approx. 900 ml of gently boiling water in a large beaker. 3. Mix well and cool the gelatinized starch solution to room temperature. 4. Add more water to bring the total volume to 1 liter. 5. Put a few drops of the starch solution on a glass plate. Add 1 drop of the iodine reagent and see that a deep blue color is developed. The blue color indicates the presence of starch in the solution. 6. Effect of the pH 1.Prepare 0.1M pH buffer solutions ranging from pH=4.5 to pH=9 in increments of one pH unit. (Note that phosphate buffer is only good for ph=4.5--9 due to the dissociation constant.)

Before coming to the lab, review how to make a pH buffer solution in a freshman chemistry textbook and calculate the relative amounts of KH2PO4 (monobasic phosphate) and K2HPO4•3H2O (dibasic phosphate) needed to make these phosphate buffer solutions. 7. Add an equal volume of one of the above buffer solutions to 5.0ml of the 20g/l starch solution prepared in Step 1. The resulting solution should contain 10g/l of starch in a buffered environment. 8. Start the enzymatic digestion process by adding 0.5 ml of the bacterial amylase solution; shake and mix. 9. Let the hydrolysis reaction proceed for exactly 10 minutes at 25ºC. 10. Add 0.5 ml of the reacted starch solution to 5ml of the HCl stopping solution (0.1N) 11. Add 0.5 ml of the above mixture to 5ml iodine solution to develop color. Shake and mix. The solution should turn deep blue if there is any residual, unconverted starch present in the solution. 12. The solution is brown-red colored for partially degraded starch, while it is clear for totally degraded starch. 13. Measure the absorbance with a spectrophotometer at 620nm. See Note 14. Carry out the same procedure for the other starch solutions buffered at different pH's.

E. SOP for Testing the Quality of Commercial Phosphorus Soulubilizing Bacteria – Pseudomonas (Gram negative) 1.0.

Definitions

For the purpose of this standard the following definitions shall apply: The following are definition of terms used in synonym and those in the SOP. The terminologies Biofertilizer, Bioenhancer, Biostimulant and Bioinoculant are sometimes used synonymously. Bofertilizer is a term widely used term meaning ―bacterial inoculant‖ usually refer to preparations of microorganisms that may be a partial or complete substitute for chemical fertilization (like rhizobial inoculants). The use of word fertilizer allows easier registration for commercial. It is a formulation containing one or more beneficial strains (or species) in an easy to use and economical carrier either organic, inorganic or synthesized from defined molecules. It contains living microorganisms embedded in a carrier material which are applied to seed, plant surface or product colonizers the rhizosphere or the interior of the plant or soil and promotes growth by increasing supply or availability of nutrients to the host plant, or preparations containing living cells or organisms that enrich the nutrient quality of product. Bioinoculants: Living organisms containing specific strains of specific bacteria, fungi and algae which fix atmospheric nitrogen, make nutrients soluble and available, collect and store nutrients, provide physical barriers to pests and pathogens, stimulate plant growth, and decompose organic residue.

Bioenhancers: Substances that increase the bioavailability of active ingredients, vitamins and nutrients Biostimulants: Dormant strains of positive product microorganisms that come alive when introduced into the product system. They contain strains of specific bacteria, fungi or algae which take up nutrients and make it available to plants, collect and store available nutrients, enhance uptake of nutrients, provide physical barriers against pathogens, stimulate growth, decompose organic residues Bacteria: A Kingdom of single celled microorganisms whose cells lack membrane-bound nucleus (prokaryotic) microorganisms that inhabit soil, water, plants and animals. Non Symbiotic nitrogen fixing bacteria: Represents a range of bacteria including saprophytic living on plant residues, bacteria living in close association with rhizosphere of plant roots and bacteria which live entirely within plants (endophytes). They fix nitrogen by reducing gaseous nitrogen in the air to ammonia by a process catalysed by enzyme complex nitrogenase. Rhizosphere: is defined as the area influenced by the root system. In comparison with root-free soil, the rhizosphere forms a nutrient-rich niche for bacteria as a result of exudation of compounds such as organic acids, sugars and amino acids Plant Growth Promoting Rhizobacteria (PGPR): are free-living diazotrophs associated with plant roots. Rhizosphere bacteria termed as PGPR. Pseudomonas: . Bacteria called pseudomonads are gram-negative, aerobic, flagellated, and straight to slightly curved rods that catabolize carbohydrates by the Entner-Doudoroff and pentose phosphate pathways. These organisms are noted for their ability to break 5 down numerous organic compounds. Pseudomonas, Burkholderia, Zymomonas, and Xanthomonas are some examples of this group Contamination: In other microorganism other than AMF not declared on the label. Aseptic Conditions/Aseptically: Environment where no microorganisms is present. It can be obtained by using a Bunsen burner to create an aseptic zone which is the aspheric area around the flame with a diameter of approximately 15 cm or a Laminar flow hood with sterile air continuously produced and present in the hood. The high pressure of air flow from the hood prevents the air from outside to come inside and contaminate the environment in the hood; aseptic conditions are also created by cleaning surface with 70% ethanol. Contaminated microorganisms: Every plate, tube, pipette, or other instruments (glassware, pestles, eppendorff tube…) which has been in contact with microorganisms and cannot be sterilized by the flame of a Bunsen burner is considered as contaminated. Contaminated by toxic chemicals: Every tube, flasks, pipette or other instruments which has been in contact with toxic chemicals is considered contaminated Good Laboratory Practices (GLP): The principle of GLP have been developed to promote the quality and validity of results and of the analysis conducted in a laboratory. It is a management concept covering the organization and the conditions under which laboratory

studies are planned, performed, monitored,, recorded and reported. Its principle also include the protection of man and the environment. Mother tube/plate/products: Tube/plate/product which the bacteria are picked from. The result of the growth of this inoculation is considered as the daughter which can become the mother for the next inoculation. Substrate: Any material, that serves as source of energy for an organism. Carrier: Delivery media of live microorganisms from the production to the field. The carrier is the major portion of inoculant. There is presently no universal carrier or formulation available for release of microorganism into soil. Materials and types of formulations of carriers vary: Slurry, powder, peat, liquid. A good carrier should have the capacity to deliver the right number of viable cells in good physiological condition at the right time.

2.0. Introduction Pseudomonas are rod-shaped, Gram negative obligate aerobe bacteria in the family Pseudomonadaceae. It is a large group with most of them saprophytic. Pseudomonas species occur in and on plants, in water and soil and have important functions as chemical transformations, mineralization of organic compounds, and interactions with plants that affect their productivity. Pseudomonas which are aerobes could utilize a large number of organic compounds. Some of the Pseudomonas spp. are phosphate solubilizing rhizocacteria and produce phytohormones such as phytase that can release P from organic acids and in soil and make P more available to plants. It is referred to as Plant Growth Promoting Rhizobacteria (PGPR). With other rhizosphere organisms such as Rhizobia and mycorrhizal fungi, Pseudomonas is used as a ‗helper‘bacteria which improves interaction of these organisms and plants. It is non-specific and affects a large variety of plants. The ssuppression of soilborne plant pathogen though competition for niche and nutrients has been demonstrated for Pseudomonas spp. There are over 200 species of Pseudomonas spp, most commonly saprophytic. Since the 1980‘s, certain member of Pseudomonas genus have been applied directly to soil as a way of biocontrol. The aerobic pseudomonads are bacteria of considerable scientific and practical importance. They are among the most active participants in the process of mineralization of organic matter in nature, a role that can be easily inferred from their widespread occurrence in soil and water. Many members (Pseudomonas fluorescence, Pseudomonas putida) of the group are endowed with the capacity of attacking a large variety of organic compounds. Pseudomonas fluorescens, P. protegens strain and P. chlororaphis also has antibiotic and antagonistic effects against pathogen while P. putida also has Bioremediation activity, whereby they degrade pollutants (P. alcaligenes, P. mendocina, P. pseudoalcaligenes, P. resinovorans, P. veronii and P. putida). . Pseudomonas is clinically relevant with majority resistant to antibiotics amongst which are Plant pathogens (Pseudomonas syringae and P. tolasii and P. agarici which cause problems in mushrooms), oppotunistic human pathogenic species (Pseudomonas aeruginosa, P. oryzihabitans and P. plecoglossicida). Pseudomonas aeruginosa is one of the most commonly isolated pathogens. This organism is a significant cause of burn and nosocomial infections. The divserse physiological functions of the Pseudomonas genus is a concern to commercial production and consumption by end-user. It is therefore vital to authenticate species to guarantee release of products that are not harmful.

2.1.

Quality control management (General procedures) 1. A specific protocol is established before screening is set up. It includes the number of samples, the details about the inoculants (origin, composition, rate of application...), and any other relevant information. 2. Calculations, weights, records (of temperature, humidity...), observations, measurements, preparation of stock solutions (nutritive solutions for example) and any other relevant information are recorded in the lab book on a daily basis. 3. The stock solutions are labelled with the date of reception, name or initials of the person who received it, the number of the container (x of n), date of opening, name or initial of the person who opened it. 4. The reagents are labelled with the name of the contents, date of preparation, name or initials of the person who prepared them and any other relevant information. 5. All samples are clearly identified and records are taken about the different steps of analysis (when they have been put in the oven, grinded, sent for analysis). 6. After harvesting, samples might need to be decontaminated before elimination. Solarization may be considered. 7. In case of contamination of the bench, floor, user, ..., it has to be cleaned and disinfected before the work can be continued (cf. ―Hygiene and Safety rules in a laboratory‖ document). 8. Non contaminated or decontaminated items are cleaned with soap, and rinsed with water and eventually rinsed with distilled water. 9. Waste management: i. Non contaminated waste is eliminated in the normal bin. ii. Non contaminated glass waste are put in a separate container labelled with the mention ―Broken glass‖ iii. Anything contaminated by microorganisms should be decontaminated before appropriate elimination/cleaning. iv. Waste may be put into a special autoclave bags for 20 mins at 121 ºC. v. Solid and liquid waste contaminated by toxic chemicals are placed in a separate container to be collected by special disposable service providers. vi. Disposable glass instruments are ut into a container containing Sodium Hypochlorite solution (commercial Jik) for decontamination before being eliminated. Solid (including broken glass) and liquid waste contaminated by toxic chemicals are placed in separate containers for elimination by waste disposal company. vii. Contaminated glassware is properly rinsed with tap water and water collected in specific items (consult MSDS) on product disposal 10. Equipment‘s are regularly cleaned to remove dust, samples or other waste. 11. Room is cleaned daily. Disinfect every surface with disinfectant and or with 70% ethanol before starting work. The bench and the floor is to be cleaned and disinfected before the work begins and in case of any contamination during the working session it should be disinfected before work continues.

2.2.

General laboratory practices 1. Always wear a lab coat when working in the laboratory. 2. Never put on gloves when working close to flames. In case of burn, the glove would stick on your skin and make the wound worse.

3. To maintain good hygiene, clean your hands with soap and work under aseptic conditions. Clean bench of hood and any surface of operation with a disinfectant; pour some disinfectant on the bench, leave for I min and wipe with gauze/cotton wool. It is also possible to clean the bench with 70% ethanol. 4. Always have a seat when working to avoid accidents. Avoid useless movement in the lab during work. Organize your work space to ensure a smooth run to the analysis. Some equipment need to be switched on some time before use (incubator, laminar flow hood). 5. Plan your work to give them enough time. Organize everything you need in the hood before starting to work so that you don‘t have to cross hands or have to have put your hand out of the hood. Never put items close to the back of the hood because it can affect the flow hence affect the sterility of air. Use preferably the middle and sides of the bench. 6. Every aseptic manipulation has to be done in the middle of the hood, never outside of it and not at the side or the back of the hood. 7. Air contaminations are common. To avoid them, don‘t speak, run or move it it is not necessary and keep the door and windows closed. 8. A burner is used to sterilize items such as loops, tubes, or forceps. The hottest part of the flame is the top of the blue part, so this is the part which has to be used. If you have to work without a hood but just with a burner, leave it on throughout the manipulation and consider that the aseptic zone is spherical area about 15 cm around the flame. 9. Remember to label every tube, bottle, flask, plate… before you start. Always note the date on everything that you will incubate, keep or autoclave (tubes, plates..) label the plate so as to be able to read what is written during incubation (upside down).

3.0.

Materials and Methods

Pseudomonas has very simple nutritional requirements. It is often observed "growing in distilled water", which is evidence of its minimal nutritional needs. In the laboratory, the simplest medium for growth of Pseudomonas consists of acetate as a source of carbon and ammonium sulfate as a source of nitrogen. Pseudomonas grows even on simple agar. A petri incubated at 37°C and left for a while at 25°C and the colonies will get a green color if it is Pseudomonas. Pseudomonas is distinguished into opportunistic human pathogens (P. aeruginosa), plant pathogens (P. syringae), soil bacterium (P. putida) and plant growth promoting (P. fluorescens). Characteristics of Pseodomonas spp. is the secretion of pyoverdine, a fluorescent yellow green siderophore, pyocyanin by P. aeruginosa and thioquinolobactin by P. fluorescences.

3.1.

Isolation of Broad spectrum Pseudomonads

Peptone mixture in Pseudomonas selective agar base allows growth of broad spectrum Pseudomonas. The amount of potassium sulfate and magnesium chloride supports forming of pigments.

Materials

Reagents Pseudomonas Agar Base – Typical Formula (g/l)  Pancreatic Digest of Gelatin  Acid Digest of Casein  Magnesium Chloride  Potassium Sulphate  Agar

16.0 10.0 1.4 10.0 11.5

CN Pseudomonas Supplement (vial contents for 500 ml of medium)  Cetrimide 100 mg  Nalidixic Acid 7.5 mg CFC Pseudomonas Supplement (vial contents for 500 ml of medium)  Cetrimide 5 mg  Acido fusidico 5 mg  Cefalosporina 25 mg Pseudomonas CN Selective Agar (ready to use plates 55 and 90 mm)  Typical Formula (g/l)  Pancreatic Digest of Gelatin 16.0  Acid Digest of Casein 10.0  Magnesium Chloride 1.4  Potassium Sulphate 10.0  Agar 14.0  Cetrimide 200 mg  Nalidixic Acid 15 mg  Glycerol 10 ml Principles Peptone provides carbon and nitrogen for bacterial growth. It enhances the production of the water soluble blue pigment pyocyanin by the addition of magnesium chloride and potassium sulpahte and inhibit the formation of fluorescin. Both pigments diffuse from Pseudomonas colonies into the medium on which they grow. Some Pseudomonas strains show both pigments, while others show only one of the two. Pyocyanin on Pseudomonas Agar is blue in colour. Glycerol serves as an energy source and also helps to promote pyocyanin production. Procedure Media preparation 1. Suspend 24.2g in 500ml of sterile water 2. Add 5ml glycerol and heat to boil until dissolved completely 3. Autoclave (15 min at 121C) to sterilse 4. Cool the medium to 45-50C and aseptically add the contents to one vial of Pseudomonas (CFC) selective supplements or Pseudomonas CN selective supplements. Mix thoroughly and pour plates. 5. pH 7.1 ±0.2 6. The prepared plates are clear and colorless and can be stored for up to 4 weeks at 2 – 8C in the refrigerator 7. Protect from light and drying.

8. Do not keep the liquid medium (40-50C) longer than 4 hours and do not re-melt the medium several times. 9. From suspension serially diluted product take aliquot of 0.5ml for inoculation 10. Inoculate the CFC selective medium using surface spread method 11. Incubate 44 ± 4 hours at 25±1C 12. All grown colonies are suspect Pseudomonas spp. and are counted as such 13. The suspect colonies must be confirmed. 14. Colonies which show a positive oxidase reaction but not glucose fermentation are confirmed Pseudomonas spp. Colonies.

Most Probable Number (MPN) Serial dilution 1. Crush product in sterile motor and pestle and shake with 100ml of sterile distilled water for 10 – 20 minutes to obtain a standard suspension. 2. Pipette 0.5 mL of the suspension into test tube 1. This bacterial suspension should be mixed thoroughly (using the vortex on each bench) before proceeding to the next step. 3. Obtain a clean pipette and withdraw 0.5 mL of the diluted bacterial suspension from the first test tube and pipette that into the second test tube. Continue in this fashion until you have serially diluted the original bacterial suspension into test tube 7. 4. In test tube 1 you have diluted the bacteria 10 fold, a 1:10 or 1 x 10-1 dilution, in test tube 5 you have diluted the bacteria from the original tube to obtain a 1 x 10-5 dilution, in test tube 10 you have diluted the bacteria from the original tube to obtain a 1 x 10 -10 dilution. 5. The following dilutions are obtained: 1 x 10-1, 1 x 10-2, 1 x 10-3, 1 x 10-4, 1 x 10-5, 1 x 10-6, 1 x 10-7, 1 x 10-8, 1 x 10-9 and 1 x 10-10.

3.2.

Procedure for Isolation of Pseudomonas strains (Pseudomonas flourescens and Pseudomonas putida)

Reagents P1 Composition of Media for Pseudomonas producing Fluorescent pigment KH2PO4 1g MgSO4.7H2O 0.5g KCL 0.2g NaNO3 5g Desoxycholate 1g Bataine 5g Agar 15g Distilled water 1000ml pH 7.2 -7.4

P2 Composition of selective media (P2) for Pseudomonas putida strains KH2PO4 1g MgSO4.7H2O 0.5g KCL 0.2g NaNO3 5g Desoxycholate 1g Hippurate 1g

Agar Distilled water pH

15g 1000ml 7.2 -7.

P3 Composition of selective media for Pseudomonas fluorescens KH2PO4 1g MgSO4.7H2O 0.5g KCL 0.2g NaNO3 1g Desoxycholate 2g Trehalose 5g NACL 5g Agar 15g Distilled water 1000ml pH 7.2 -7.

P4 Composition of selective media for Pseudomonas fluorescens KH2PO4 1g MgSO4.7H2O 0.5g KCL 0.2g NaNO3 1g Desoxycholate 2g Inositol 5g NACL 5g Agar 15g Distilled water 1000ml

Procedures Most Probable Number (MPN) Serial dilution 5. Crush product in sterile motor and pestle and shake with 100ml of sterile distilled water for 10 – 20 minutes to obtain a standard suspension. 6. Pipette 0.5 mL of the suspension into test tube 1. This bacterial suspension should be mixed thoroughly (using the vortex on each bench) before proceeding to the next step. 7. Obtain a clean pipette and withdraw 0.5 mL of the diluted bacterial suspension from the first test tube and pipette that into the second test tube. Continue in this fashion until you have serially diluted the original bacterial suspension into test tube 7. 8. In test tube 1 you have diluted the bacteria 10 fold, a 1:10 or 1 x 10-1 dilution, in test tube 5 you have diluted the bacteria from the original tube to obtain a 1 x 10-5 dilution, in test tube 10 you have diluted the bacteria from the original tube to obtain a 1 x 10-10 dilution. 9. The following dilutions are obtained: 1 x 10-1, 1 x 10-2, 1 x 10-3, 1 x 10-4, 1 x 10-5, 1 x 10-6, 1 x 10-7, 1 x 10-8, 1 x 10-9 and 1 x 10-10.

Inoculations 1. Aliqots from each product suspension are spread on the plates of each of the selective medium (P1, P2, P3 and P4) with a glass rod or sterile loop. 2. After inoculation, the plates are incubated at 28C to 30C for 2 to 4 days 3. Flourescent pigment is dtected by ultra violet light. Yellow-green or blue fluorescent in and around the colonies indicates the presence of Pseudomonas strains producing fluorescent pigment. Maximum intensity of fluorescence is usually apparent after 3 days.

Qualitative Identification 1. Bacterial strains are cultivated at 30 ᵒC without shaking. Beef extract-peptone agar (BP: beef extract 0.5%; Peptone, 0.5%; NaCl, 0.25%; agar , 1.5%; pH 6.8 -7.2) is used for preculture. Observe cell forms and Gram reaction on BP agar at 30 ᵒC. 2. Production of fluorecent pigment is tested on P1, P2, P3, P4 and King B medium (see on King B medium procedure below). 3. Conduct tests (See tests below on Section 4).

Quantitatve analysis Method for Colony Counts (See Section 4: Counting colony forming units (CFU) and calculating the amount of bacteria in the original solution).

3.3.

Isolation of Pseudomonas fluorescens

The product thus obtained is crushed in a sterile mortar and pestle and shaken with 100 ml of sterile distilled water for 10-20 min. to obtain standard product suspension. Isolation of product is made by following the serial dilutions and pour plate method using the specific King‘s B medium. Reagents King’s B Medium King Agar B (Pseudomonas Agar (for Fluorescein) Composition: Ingredients Grams/Litre 1. Mixed peptone 20.0 2. Dipotassium hydrogen phosphate 1.5 3. Magnesium sulfate 1.5 4. Agar 10.0 5. Final pH 7.2 +/- 0.2 (at 25°C) 6. Store prepared media below 8°C, protected from direct light. Store dehydrated powder, in a dry place, in tightly-sealed containers at 2-25°C. Procedure 1. Dissolve 33 g in 990 ml distilled water and add 10 ml glycerol (Fluka 49769). Sterilize by autoclaving at 121°C for 15 minutes. Optional CFC Selective Supplement (Fluka 53477) can be added to make the media selective.

CFU Selective media: An antibiotic supplement for the selective isolation of Pseudomonas species a. Composition: (per vial, sufficient for 500 ml medium): Cetrimide 5mg Fucidin 5mg; Cephaloridine 25mg b. Procedure: Rehydrate the contents of 1 vial aseptically with 2 ml of sterile distilled water. Mix well and aseptically add it to 500 ml of sterile, molten King Agar B (Fluka 60786). Mix well and pour into sterile petri plates. 2. King‘s B medium, a selective one is used for the isolation of Pseudomonas fluorescens

Most Probable Number (MPN) Serial dilution 1. Pipette 0.5 mL of the suspension into test tube 1. This bacterial suspension should be mixed thoroughly (using the vortex on each bench) before proceeding to the next step. 2. Obtain a clean pipette and withdraw 0.5 mL of the diluted bacterial suspension from the first test tube and pipette that into the second test tube. Continue in this fashion until you have serially diluted the original bacterial suspension into test tube. 3. In test tube 1 you have diluted the bacteria 10 fold, a 1:10 or 1 x 10-1 dilution, in test tube 5 you have diluted the bacteria from the original tube to obtain a 1 x 10-5 dilution, in test tube 10 you have diluted the bacteria from the original tube to obtain a 1 x 10-10 dilution. 4. The following dilutions are obtained: 1 x 10-1, 1 x 10-2, 1 x 10-3, 1 x 10-4, 1 x 10-5, 1 x 10-6, 1 x 10-7, 1 x 10-8, 1 x 10-9 and 1 x 10-10.

Plating and counts 3. One ml of product suspension from aliquot dilutions (105 to 108) is aseptically added to sterile Petri plates containing 20 ml of sterile Kings B medium and incubated at 28±20C for 48h 4. After incubation, well separated individual colonies with yellow green and blue white pigments are marked and detected by viewing under UV light. 5. Determine the count of the fluorescing bacteria under the UV lamp and the total microbial count. 6. Simultaneously individual colonies are picked up with sterile loop and transferred to fresh King‘s B slants and the pure cultures so obtained are stored in refrigerator at 4ºC for further use. Principle and Interpretation: 7. King Agar B enhances the elaboration of fluorescein and inhibits the pyocyanin formation. 8. Mixed peptone provides the essential nitrogenous nutrients, carbon, sulphur and trace elements. 9. Glycerol serves as a Carbon-source 10. Dipotassium hydrogen phosphate buffers the medium. 11. Magnesium sulfate is necessary for the activation of fluorescein production. Distinguishing P. fluorescens from the pathogenic P. aeruginosa

Pseudomonas aeruginosa build colonies surrounded by a yellow to greenish-yellow zone due to fluorescein production which fluoresces under UV light. If pyocyanin is also synthesized, a bright green colour is produced. For the confirmation of pyocyanin the coloured pigments can be extracted with chloroform. Most pyocyanin-producing Pseudomonas strain synthesize also fluorescein and others produce just one pigments. The temperature can be a determining factor as most fluorescent strains will not grow at 35°C. Rather, they grow 25-35°C. A typical pyocyanin-negative, fluorescein positive P. aeruginosa strains can also be differentiated from Pseudomonas fluorescens and Pseudomonas putida. Pseudomonas aeruginosa can be differentiated by following cultivation on Mac Conkey Agar (Fluka 70143, because P. putida and P. fluorescence do not fluorescen under UV light and grow poorly. CFC Selective Supplement is an antibiotic supplement for the selective isolation of Pseudomonas species. Cultural characteristics after up to 72 hours at 20-25°C: Organisms Growth Yellow-green pigment in daylight Fluorescense at 366 nm. Mac Conkey Agar (Fluka 70143) Ingredients Peptone Lactose Bile salts Sodium chloride Neutral red Agar Final pH

Grams/Litre 20.0 10.0 5.0 5.0 0.075 12.0 7.4 +/- 0.2 at 25°C

Store prepared media below 8°C, protected from direct light. Store dehydrated powder, in a dry place, in tightly-sealed containers at 2-25°C. Procedure: 1. Suspend 52 g in 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilize by autoclaving at 121°C for 15 minutes. 2. Pour into sterile petri plates. Dry the surface of the gel before inoculation. Principle and Interpretation:  Peptone provides carbon, nitrogen, vitamines and other essential growth nutrients.  Lactose is the fermentable sugar which causes acid production and a color change of the indicator, neutral red, to red. Lactose-positive bacteria like E. coli build pink to red colonies and are often surrounded by a turbid zone due to the precipitation of bile acids.  Most of the gram positive organisms are inhibited by bile salts.  Sodium chloride maintains the osmotic equilibrium. Cultural characteristics are observed after 18-24 hours at 35-37°C . 4.0. Identification and Biochemical tests of Pseudomonas spp.

A. Qualitative characterization I. Morphological characterization 1. Pure cultures of the selected isolates are streaked on King‘s B agar Petri plates separately for colony development. 2. The individual colonies are examined for shape, size, structure of colonies and pigmentation.

a. Pseudomonas fluorescens: Colonies are circular, convex with an entire margin II. Gram staining 1. Draw circle in the center of a glass slide . The circle indicates where the sample will be placed. Turn slide over so that the markings so the markings and the sample will be on the opposite sides. Label slide. 2. Place 1 ml of diluent (water) into test tube using a dosposable pipette Thoroughly mix the sample. 3. Sterilize the inoculation loop in the flame of the Bunsen Burner. With a cooled inoculation loop obtain a small sample of product from the container. 4. Transfer the sample into the previously measured diluent (water). Cover test tube with the screw cap and gently shake for 5 to 10 sec. To thoroughly mix the sample throughout the diluent. 5. Sterilize the inoculation loop in the flame. Transfer a small amount (two loops) of sample from the last tube to previously prepared slide. Tilt the slide to spread the drop out slightly about the size of a penny 6. ―Heat-fix‖ the slide with the specimen by holding the slide with a clasp and pass it over a heat source, such as a flame, several times using a forceps. The slide should be passed very quickly through the flame and not be heated excessively. Dont place the slide directly in the flame so that the bacteria wont be burned. Place slide on the staining tray. 7. Flood the fixed smear with crystal violet solution and allow to remain for 1 minute. Rinse off the crystal violet with distilled or tap water. 8. Flood the slide with iodine solution. Allow to remain for one minute. Rinse off the iodine solution with distilled or tap water. 9. Flood the slide with decolorizer for one to five seconds. Rinse off the decolorizer with distilled or tap water. 10. Flood the slide with safranin. Allow to remain for 30 seconds. Rinse off the safranin with distilled or tap water. 11. Dry the slide on absorbent paper and gently pass the paper down for few times. Place in an upright position. 12. Microscopically examine the slide for bacterial organisms under a 100X objective. Observe several fields on the slide for bacterial organisms. Describe the gram reaction of any organisms seen. Gram-positive bacteria stain deep violet to blue and gramnegative bacteria stain pink to red. a. Pseudomonas fluorescens: This microbe forms gram-negative rods

B. Biochemical charaterization III. Starch hydrolysis Starch hydrolysis test: This test is used to identify bacteria that can hydrolyze starch (amylose and amylopectin) using the enzymes a-amylase and oligo-1,6-glucosidase. Often used to differentiate species from the genera Bacillus. In order to use these starches as a carbon source, bacteria must secrete a-amylase and oligo-1,6-glucosidase into the extracellular space. In order to interpret the results of the starch hydrolysis test, iodine must be added to the agar. The iodine reacts with the starch to form a dark brown color. Thus, hydrolysis of the starch will create a clear zone around the bacterial growth. Bacillus subtilis is positive for starch hydrolysis (Refer to procedure below)

Procedure 1. Filter paper is dipped in a dry old culture suspension and is placed on Petri dishes containing starch agar medium and incubated for two days. 2. The plates are than flooded with one per cent iodine solution. 3. A colorless halo around the growth and blue color in the rest of the plates shows utilization of starch by the microorganism. a. Pseudomonas fluorescens: Note the lack of a zone of clearing. This microbe is unable to hydrolyze starch and does not produce amylase.

IV. Gelatin liquefaction Gelatin Nutrient Agar Medium Nutrient Gelatin is used for the differentiation of microorganisms on the basis of gelatinase production. Gelatin was the first gelling agent used to solidify culture media. The incubation requirement for gelatin is at 20°C, a temperature that is lower than optimum for growing many microorganisms, and many organisms metabolize (liquefy) gelatin. Identifying fermentative and non-fermentative Gram-negative bacteria include testing for gelatin liquefaction. If the proteolytic enzyme gelatinase is present, gelatin is hydrolyzed and loses its gelling characteristic. The nitrogen, carbon, vitamins, and amino acids are provided by Enzymatic Digest of Gelatin and Beef Extract for general growth requirements in Nutrient Gelatin. Gelatin is the substrate for determining if microorganisms elaborate the proteolytic enzyme to hydrolyze (liquefy) gelatin. Formula / Liter Enzymatic Digest of Gelatin 5g Beef Extract 3g Gelatin 120 g Final pH: 6.8 ± 0.2 at 25ºC Formula may be adjusted and/or supplemented as required to meet performance specifications or Agar 15.0g/L Gelatin 10.0g/L Meat extract 10.0g/L Meat peptone 10.0g/L Sodium chloride 5.0g/L

Procedure for testing 1. Filter paper discs is dipped in a day old culture suspension and is placed on Petri dishes containing gelatin nutrient agar medium. 2. The Petri dishes is incubated at 300C for two days and then flooded with 12.5 per cent HgCl2 solution. 3. The development of yellow halo around the growth indicates utilization of gelatin. V. Fluorescent pigment

1. The test tubes containing sterilized Kings B medium are inoculated with the isolate of Pseudomonas sp. incubated for five days and observed. 2. Yellowish green fluorescent pigment observed under UV light (365 nm) indicated positive results. OR Pseudomonas Agar (For Fluorescein) 1. Pseudomonas Agar (For Fluorescein) is recommended for the detection of fluorescein production by Pseudomonas species. Composition Ingredients Gms / Litre Casein enzymic hydrolysate 10.0 Proteose peptone 10.0 Dipotassium phosphate 1.50 Magnesium sulphate 1.50 Agar 15.0 Final pH ( at 25°C) 7.0±0.2 **Formula adjusted, standardized to suit performance parameters Procedures 1. Suspend 38 grams in 1000 ml distilled water containing 10 ml glycerol. Heat to boiling to dissolve the medium completely. 2. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.  Pseudomonas Agar (For Fluorescein) is based on the formula described by and as modified in the U.S. The medium enhances the elaboration of fluorescein by Pseudomonas and inhibits the pyocyanin (a blue-green) pigment secreted by Pseudomonas aeruginosa) formation. The fluorescein pigment diffuses from the colonies of Pseudomonas into the agar and shows yellow fluorescent colouration. Some Pseudomonas strains produce small amounts of pyocyanin resulting in a yellowgreen colouration.  The Casein enzymic hydrolysate and proteose peptone provide the essential nitrogenous nutrients, carbon, sulphur and trace elements for the growth of Pseudomonas.  Dipotassium phosphate buffers the medium while magnesium sulphate provides necessary cations for the activation of fluorescein production. Salt concentration exceeding 2% affects pigment production.  UV illumination may be bactericidal, so make sure that there is good growth before placing culture under UV light.  A pyocyanin-producing Pseudomonas strain will usually also produce fluorescein. It must, therefore, be differentiated from other simple fluorescent pseudomonads by other means. Temperature can be a determining factor as most other fluorescent strains will not grow at 35°C. Rather, they grow at 25-30°C.

VI. Estimation of IAA 1. Indole acetic acid (IAA) in the methanol fraction is determined by employing Salper reagent.

2. To 1.5 ml of distilled water in a test tube, 0.5 ml of methanol residue was mixed, four ml fresh Salper‘s reagent (1 ml of 0.5 M FeCl3 in 50 ml of 35 % (v/v) per-choleric acid) was rapidly added, kept in complete darkness for one hour and read in spectrophotometer at 535 nm. 3. From a standard curve prepared with known concentration of IAA, the quantity of IAA in the filtrate is calculated (1 division =0.307 μg of IAA) VII. Extraction of siderophore from the medium 1. The spent culture fluid is separated from the cells by centrifugation at 7000 rpm for 15 min. 2. The supernatant was concentrated to one fifth of the original volume by the flash evaporation at 450C. 3. Catechol type phenolates were extracted with ethyl acetate from the culture supernatant twice with an equal volume of solvent at pH 2.0. 4. The ethyl acetate layer was removed and evaporated to dryness and the residues were dissolved in a minimum quantity of distilled water, while hydroxamate types were measured from the untreated culture supernatant. OR 1. Pseudomonas assayed for siderophore production on chrome azurole agar (CAS). 2. Chrome azurole S agar plates are prepared and spot inoculated with Pseodomonas and incubated for 5 days. 3. The development of yellow-orange halo around the colony was considered as positive for siderophore production.

VI. HCN production 1. Production of HCN is determined as per Wei et al. (1996). Bacteria are grown on Trypticase (Tryptic) soy agar (TSA – 15g Tryptone; 5g soytone –enzyme digest of soybean meal; 5g sodium chloride; 15g agar) supplemented with 4.4g/l of glycine, white filter paper strips soaked in picric acid solution (2.5 g of Na2CO3 and 1 lit. of water) are placed in the lid of each Petri dishes, sealed with parafilm and incubated for two to three days at 28±20C. 2. After incubation HCN production is indicated by the presence of a coloured zone around the bacteria. VIII.

Catalase Test: This test is used to identify organisms that produce the enzyme, catalase. This enzyme detoxifies hydrogen peroxide by breaking it down into water and oxygen gas. The bubbles resulting from production of oxygen gas clearly indicate a catalase positive result (See methodon Appendix below). a. Pseudomonas flourescens: Note the bubble formation. Catalase positive

IX. Nitrate test (Nitrate Borth) Recommended for detecting nitrate-reducing and indole-producing microorganisms. Ingredients Peptone Meat extract Potassium nitrate

Grams/Litre 5.0 3.0 1.0

Final pH

7.0 ± 0.2 at 25°C

Store prepared media below 8°C, protected from direct light. Store dehydrated powder, in a dry place, in tightly-sealed containers at 5°C. Preparation of Reagents:  Sulfanilic acid solution (Reagent A): Dissolve 8 g of sulfanilic acid (Fluka 86090) in 1 litre 5N acetic acid. Store. Reagent A at room temperature for up to 3 months, in dark. Reagents may be stored in dark brown glass containers; bottles may be wrapped in aluminum foil to ensure darkness.  _-Naphthylamine solution (Reagent B): Dissolve 6 g of N,N-Dimethyl-1naphthylamine (Fluka 40860) in 1 litre 5N acetic acid. Store Reagent B at 2 to 8°C for up to 3 months, in dark. Reagents may be stored in dark brown glass containers; bottles may be wrapped in aluminum foil to ensure darkness.   

      

Procedure: Dissolve 9 g in 1 litre distilled water. Dispense in tubes and sterilize by autoclaving at 121°C for 15 minutes. Inoculate the tubes heavily with a fresh culture of the suspect organism. Inoculate at least 1 ml sample in a tube or take a big part of a colony with an inoculating loop. Do not forget a negative control without any bacteria. For special organisms the media has to be modified. Nitrate reaction occurs only under anaerobic conditions. The medium is dispensed in tubes to give a low surface area to depth ratio which limits the diffusion of oxygen into the medium. Most bacteria use the oxygen in the medium and rapidly produce anaerobic conditions. To reach faster an anaerobic condition it is recommendable to give about one centimetre of paraffin oil on the surface of the media or over-gassing with e.g. carbon dioxide and seal the tube with parafilm. Incubate the tubes at 35 to 37°C (bacilli at 30°C) for 24 to 48 h in an incubator with or without supplemental carbon dioxide. Put a 5 drops of reagent A and 5 drops of reagent B into the tube containing culture to be tested. Shake the tube well to mix reagents with medium. A distinct red or pink colour, which should develop within a few minutes, indicates nitrate reduction. If the suspension turns pink-red before the addition of Zn powder, the reaction is positive and the test is completed. If the suspension is colorless after the addition of reagents A and B, add a small amount (―sharp knife point") of zinc powder to the medium. Shake the tube vigorously and allow it to stand at room temperature for 10-15 min. If the medium remains colorless after the addition of Zn powder, the test result is positive. If the medium turns pink after the addition of Zn powder, the result is negative. The negative control should also be tested. There should be no pink colour formation after adding reagent A and B and if zinc powder is added the colour should change to pink. Addition of too much zinc powder can results in false-negative reaction. b. Pseudomonas fluorescens: Pseudomonas fluorescens reduces nitrate all the way to nitrogen gas N2. This will cause the nitrate broth to not turn red when reagents are added. Subsequent addition of zinc, which will chemically reduce nitrate to nitrite, will not occur, since there is no nitrate to act upon. Thus the tube will not turn red

X. Motility Test Motility test medium Beef extract Pancreatic digest of casein Sodium chloride Agar

3.0 g 10.0 g 5.0 g 4.0 g

Preparation of Media 5. Bring to 1 liter with distilled water and heat to boiling to melt agar. 6. Add 5 ml of 1% triphenyltetrazolium chloride (TTC) solution. 7. Dispense in 5-ml aliquots into tubes and autoclave at 121°C under 15 psi pressure for 15 minutes. Cool upright in racks. 8. After inoculation, incubate at 35°C for 18 hours or until growth is evident. Motility test medium is commercially available in premixed forms and prepoured tubes from biological supply companies. Motility test Protocol 4. To test for motility, use a sterile needle to pick a well-isolated colony and stab the medium to within 1 cm of the bottom of the tube. 5. Be sure to keep the needle in the same line it entered as it is removed from the medium. Incubate at 35°C for 18 hours or until growth is evident (Fig. 1). 6. A positive motility test is indicated by a red turbid area extending away from the line of inoculation. A negative test is indicated by red growth along the inoculation line but no further . b. Pseudomonas fluorescens: This microbe is motile. Note how the microbe swims to the top of the tube where there is more oxygen. XI. Fermentation ofcarbohydrate (Both Glucose Fermentation Broth) Testing whether an organism can ferment glucose is one of the basic, primary tests in the identification of chemoheterotrophic bacteria. For this test we routinely use a "Glucose Fermentation Broth." Fermentation of glucose results in the abundant production of acidic end products, the presence of which can be detected by the pH indicator in the medium. Many organisms produce gas – either CO2 alone or a mixture of H2 and CO2. H2 is insoluble and is detected by bubble formation in a Durham tube placed in the medium.  Glucose – a sugar from which most common chemo heterotrophic bacteria can obtain energy by fermentation and/or respiration. Glucose can also be utilized as a source of carbon, but these media include a large number of potential carbon sources (amino acids as well as glucose).  Peptone – a commonly-used medium ingredient which mainly supplies amino acids (sources of nitrogen, carbon, sulfur and energy for many bacteria). It is a crude preparation of a partially-digested protein, and a peptone solution can serve as a complete medium for a number of organisms. If too much peptone (relative to glucose) is incorporated in the medium, detection of acidic products of fermentation or

respiration may not be possible, as overabundance of ammonium (which is alkaline) released from the breakdown of amino acids can neutralize the acids.  pH indicator – the pH indicators employed in these media turn yellow under acidic conditions. Brom-cresol purple is in Glucose Fermentation Broth (which also contains the Durham tube). Protocol for testing fermentation A. Inoculation of media 1. Aseptically inoculate each test tube with the test microorganism using an inoculating needle or loop. 2. Make sure to avoid the Durham tube, if present. Swirl the tube gently to mix contents. 3. Avoid contact of liquid with tube cap. 4. Alternatively, inoculate each test tube with 1 to 2 drops of an 18- to 24-hour broth culture of the desired organism . 5. Examples of broth media that could be used include brain heart infusion, nutrient agar, and tryptic soy agar. B. Incubation Incubate tubes at 35 to 37°C for 18 to 24 hours . Longer incubation periods may be required to confirm a negative result . C. Interpretation of results 1. When using phenol red as the pH indicator, a yellow color indicates that enough acid products have been produced by fermentation of the sugar to lower the pH to 6.8 or less. A delayed fermentation reaction may produce an orange color. In such cases, it is best to re-incubate the tube . Refer to Table for other commonly used pH indicators and their corresponding colors. 2. Gas production results: Bubbles trapped within the Durham tube indicate the production of gas. Even a single bubble is significant and denotes evidence of gas production. No bubbles within the Durham tube indicate a non-gas-producing or anaerogenic organism. 3. Negative results: A reddish or pink color indicates a negative reaction. In negative tubes, the presence of turbidity serves as control for growth. A reddish or pink color in a clear tube could indicate a false negative. b. Pseudomonas fluorescens: Pseudomonas is an obligate respirer and cannot ferment glucose XII. Indole from tryptophan Tryptophan is an amino acid that can undergo deamination and hydrolysis by bacteria that express tryptophanase enzyme. The chief requirement for culturing an organism prior to performing the indole test is that the medium contains a sufficient quantity of tryptophan (5). The presence of indole when a microbe is grown in a medium rich in tryptophan demonstrates that an organism has the capacity to degrade tryptophan. Detection of indole, a by-product of tryptophan metabolism, relies upon the chemical reaction between indole and p-dimethylaminobenzaldehyde (DMAB) under acidic conditions to produce the red dye rosindole (5, 8). tryptophan + water = indole + pyruvic acid + ammonia

The main requirement for a suitable indole test medium is that it contain a sufficient amount of tryptophan. Although many media meet this criterion, tryptone broth is commonly used. Tryptone broth (2) Ingredient Tryptone Sodium Chloride

Amount 10.0 5.0

Dissolve the ingredients in 1 liter of sterile water. Dispense 4 ml per tube. Cap tube and autoclave at 121oC under 15 psi pressure for 15 minutes. Store the tubes in the refrigerator at 4 to 10°C. Kovács reagent Ingredients Amyl or isoamyl alcohol, reagent grade (butyl alcohol may be substituted) p-dimethylaminobenzaldehyde (DMAB) HCL (Concentrated)

Amount 150.0ml 10g 50.0ml

Procedure (Media preparation) 1. Dissolve DMAB in the alcohol. Gentle heating might be required to get the aldehyde into solution. 2. Slowly add the acid to the aldehyde-alcohol mixture. The solution should be a pale yellow color and is only stable for a short time. 3. Store the mixture in a brown glass bottle in the refrigerator. ( Kovács reagent also is commercially available). Protocol for Testing 1. Inoculate the tube of tryptone broth with a small amount of a pure culture. Incubate at 35°C (+/- 2°C) for 24 to 48 hours. 2. To test for indole production, add 5 drops of Kovács reagent directly to the tube. 3. A positive indole test is indicated by the formation of a pink to red color ("cherry-red ring") in the reagent layer on top of the medium within seconds of adding the reagent 4. If a culture is indole negative, the reagent layer will remain yellow or be slightly cloudy. b. Pseudomonas fluorescens: This microbe is negative for indole production. C. Quantitative analysis XIII. Method for Estimating MPN Count 1. To calculate the most probable number of organisms in the original sample, select as P1 the number of positive tubes in the least concentrated dilution in which all tubes are positive or in which the greatest number of tubes is +ve, and let P2 and P3 represent the numbers of positive tubes in the next two higher dilutions (refer to Appendix 5.1). 2. Then find the row of numbers in Table 1 in which P1 and P2 correspond to the values observed experimentally. Follow that row of numbers across the table to the column headed by the observed value of P.

3. The figure at the point of intersection is the most probable number of organisms in the quantity of original sample represented in the inoculum added in the second dilution. Multiply this figure by the appropriate dilution factor to obtain the MPN value. Most Probable Number (MPN) Serial dilution Counts 1. Crush product in sterile motor and pestle and shake with 100ml of sterile distilled water for 10 – 20 minutes to obtain a standard suspension. 2. Pipette 0.5 mL of the suspension into test tube 1. This bacterial suspension should be mixed thoroughly (using the vortex on each bench) before proceeding to the next step. 3. Obtain a clean pipette and withdraw 0.5 mL of the diluted bacterial suspension from the first test tube and pipette that into the second test tube. Continue in this fashion until you have serially diluted the original bacterial suspension into test tube 7. 4. In test tube 1 you have diluted the bacteria 10 fold, a 1:10 or 1 x 10-1 dilution, in test tube 5 you have diluted the bacteria from the original tube to obtain a 1 x 10-5 dilution, in test tube 10 you have diluted the bacteria from the original tube to obtain a 1 x 10-10 dilution. Plating the serially diluted cells: 1. Obtain 10 King Agar B plates and label with initials and the dilution factor. 2. The following dilutions will be plated: 1 x 10-1, 1 x 10-2, 1 x 10-3, 1 x 10-4, 1 x 10-5, 1 x 10-6, 1 x 10-7, 1 x 10-8, 1 x 10-9 and 1 x 10-10. 3. Obtain a beaker containing a loop and pour a small volume of alcohol into the beaker. The loop is used to spread the diluted bacterial suspension evenly over the surface of the plate. 4. For the cell suspension that will be plated onto the King Agar B plate label 1 x 10-1, pipette 0.5 mL of the diluted suspension from the appropriately diluted test tube onto the surface of the plate. 5. Dip the loop into the alcohol solution and flame until the alcohol has burned off. Do not heat the loop to long; you only need to flame the loop to burn off the alcohol—that will be sufficient for sterilizing. 6. After sterilizing the loop, use it to spread the bacterial suspension evenly over the entire surface of the plate. Allow the plate to dry. Continue this process with the remainder of the bacterial dilutions. 7. Tape all of plates together and incubate the plates, upside down, at 37oC for 24 hours. During the next period count the number of colony forming units for each dilution and calculate the number of bacteria in the original suspension.

Pseudomonas count per gram of carrier = Viable MPN table x Dilution level Dry mass of product Most Probable Number Table P1

0 0 0 0 0 0

P2

Most probable number for indicated values of P 3 0 1 2 3 4 0 1 2 3 4 5

0.018 0.037 0.056 0.075 0.094

0.018 0.036 0.055 0.074 0.094 0.11

0.036 0.055 0.074 0.093 0.11 0.13

0.054 0.073 0.092 0.11 0.13 0.15

5 0.072 0.091 0.11 0.13 0.15 0.17

0.090 0.11 0.13 0.15 0.17 0.19

1 1 1 1 1 1

0 1 2 3 4 5

0.020 0.040 0.061 0.089 0.11 0.13

0.040 0.061 0.082 0.10 0.13 0.15

0.060 0.081 0.10 0.13 0.15 0.17

0.080 0.10 0.12 0.16 0.17 0.19

0.10 0.12 0.16 0.17 0.19 0.22

0.12 0.14 0.17 0.19 0.22 0.24

2 2 2 2 2 2 3 3 3 3 3 3 4 4 4 4 4 4 5 5 5 5 5 5

0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5

0.046 0.068 0.093 0.12 0.15 0.17 0.078 0.11 0.14 0.17 0.21 0.25 0.13 0.17 0.22 0.27 0.34 0.41 0.23 0.33 0.49 0.79 1.3 2.4

0.068 0.092 0.12 0.14 0.17 0.20 0.11 0.14 0.17 0.21 0.24 0.29 0.17 0.21 0.26 0.33 0.40 0.48 0.31 0.46 0.70 1.1 1.7 3.5

0.091 0.12 0.14 0.17 0.20 0.23 0.13 0.17 0.20 0.24 0.28 0.32 0.21 0.26 0.32 0.39 0.47 0.56 0.43 0.64 0.95 1.4 2.2 5.4

0.12 0.14 0.17 0.20 0.23 0.26 0.16 0.20 0.24 0.28 0.32 0.37 0.25 0.31 0.38 0.45 0.54 0.64 0.58 0.84 1.2 1.8 2.8 9.2

0.14 0.17 0.19 0.22 0.25 0.29 0.20 0.23 0.27 0.31 0.36 0.41 0.30 0.36 0.44 0.52 0.62 0.72 0.76 1.1 1.5 2.1 3.5 16.0

0.16 0.19 0.22 0.25 0.28 0.32 0.23 0.27 0.31 0.35 0.40 0.45 0.36 0.42 0.50 0.59 0.69 0.81 0.95 1.3 1.8 2.5 4.3 --

OR Counting colony forming units (CFU) and calculating the amount of bacteria in the original solution. 4. Using a counter for each dilution, count the number of colony forming units on your plates. Typically numbers between 30 and 800 are considered to be in the range where one‘s data is statistically accurate. If the number of CFUs on your plate are greater than 1000, you may record in your table TNTC (too numerous to count). 5. Alternatively, if your numbers are greater than 1000 AND you have evenly distributed the diluted bacterial suspension on the surface of the plate AND you can discern individual colonies; divide the plate into 4 sectors, count the number of bacteria in one sector and multiply by four. 6. If the number of CFUs on your plate is below 10, record the number of CFUs, but do not use this in your calculations. T=Trial

Dilution Number of bacterial colonies (CFUs) factor T1 1:10

-1

T2

T3

T4

T5

T6

Avg Avg # # bacteria/ml CFU T7

T8

T9

1:10-2 1:10-3 1:10-4 1:10-5 1:10-6 1:10-7 1:10-8 1:10-9 1:10-10

Calculating the number of bacteria per mL of serially diluted bacteria: To calculate the number of bacteria per mL of diluted sample one should use the following equation: Number of CFU Volume plated (mL) x total dilution used

Number of CFU mL

For example, if for the 1x 10-8 dilution plate you plated 0.1 mL of the diluted cell suspension and counted 200 bacteria, then the calculation would be: 200/0.1 mL x 10-8 or 200/10-9 or 2.0 x 1011 bacteria per mL.

5.0. Conclusion Pseudomonas spp. could be conserved efficiently until the end of 2 months at 20 °C. Initial cell number of 1.6x1010 cells/ml increased abruptly in 1 month and then began to decrease and dropped to 2.2x107 cells/ml at the end of 3 months. Storage at 20 ºC is not appropriate for long terms but it can be used for short term storages such as 2 months. Pseudomonas products are applied to seed treatment, seedling dipping and soil application. The stability of a product will depend on the carriers and type of formulations. The most common carriers include Rock phospate, vermicompsot, perlite, peat, bentonite. Commercial formulations include liquid inoculant formulations, solid formulations of wettable powder containing dried bacteria, seed coating prepared through peletization, granulation and film coating and Ca-alginate encapsulation. One key aspect in viability is viable culturability of the formulation which is a better indicator of both quality and quantity. Measuring quantity of cells through counts may not necessarily distinguish living from dead individuals. However, CFU counts of 107 to 108 g-1 is regarded as more appropriate to improve yield.

F. SOP for Testing the Quality of Commercial Arbuscular mycorrizal fungal (AMF) Products

1.0.

Definitions

The following are definition of terms used in synonym and those in the SOP

Biofertilizers: is a substance which contains living microorganisms which are applied to seed, plant surface or product colonizes the rhizosphere or the interior of the plant or soil and promotes growth by increasing supply or availability of nutrients to the host plant. It is a term widely used term meaning ―bacterial inoculant‖ usually refer to preparations of microorganisms that may be a partial or complete substitute for chemical fertilization (like rhizobial inoculants). The use of word fertilizer allows easier registration for commercial. It is a formulation containing one or more beneficial strains (or species) in an easy to use and economical carrier either organic, inorganic or synthesized from defined molecules. It contains living microorganisms embedded in a carrier material which are applied to seed, plant surface or product colonizers the rhizosphere or the interior of the plant or soil and promotes growth by increasing supply or availability of nutrients to the host plant, or preparations containing living cells or organisms that enrich the nutrient quality of product. Biopesticides: Living organisms or natural products derived from these organisms which suppress pathogen populations microbial pesticides, other organisms (nematodes, insects) used to control pests; natural products derived from living organisms (biochemical pesticides) and plant incorporated protectants (genetically modified plants) – Bacillus thuringiensis in Bt maize and Bt cotton. Biopesticides decompose quickly in the environments; and are less toxic towards non-target species. Bioinoculants: Living organisms containing specific strains of specific bacteria, fungi and algae which fix atmospheric nitrogen, make nutrients soluble and available, collect and store nutrients, provide physical barriers to pests and pathogens, stimulate plant growth, and decompose organic residue. Bioenhancers: Substances that increase the bioavailability of active ingredients, vitamins and nutrients Biostimulants: Dormant strains of positive product microorganisms that come alive when introduced into the product system. They contain strains of specific bacteria, fungi or algae which take up nutrients and make it available to plants, collect and store available nutrients, enhance uptake of nutrients, provide physical barriers against pathogens, stimulate growth, decompose organic residues Fungi: Multicellular organism, separate Kingdom from plants, animals, protists and bacteria. They have cells that contain chitin and derive nutrition through breakdown of dead organic materials (saprophytic), obtain carbohydrates from living plants through mutual exchange (symbiosis) or killing the plant or animal (parasitism). The organisms is hidden and the fruit may be visible e.g mushrooms, toadstools and bread mold or invisible Symbiosis: A mutually beneficial partnership formed between two living organisms Mycorrhiza: A beneficial symbiosis between fungus and a living plant root. A group of fungi that include a number of types based on the different structures formed inside or outside the root. There are two types of mycorrhiza Ectomycorrhiza and Endomycorrhiza (Arbuscular mycorrhiza (AM), Ericaceous mycorrhiza, Orchidaceous mycorrhiza, Ectoendomycorrhiza).

Arbuscular mycorrhiza fungi (AMF): A group of fungi that inhabit roots of plants and forms mutually beneficial obligate relationship with the plants and exchange nutrients and water for carbohydrates. Arbuscules: A branched treelike organ, one of the treelike huastorial organs in certain mycorrhizal fungi. Arbuscules are the sites of nutrient and sugar exchange between plant and fungus, and arbuscular development is often most intense in the innermost cortical cells, nearest the endodermis. Vesicles: sacs that are globose, ovoid thin-walled storage organs filled with lipids and glycolipids and may have a function in storage, survival and reinfection of new roots. Vesicle development often occurs later in the development of AM infection. Vesicles are not formed by Gigaspora spp. or Scutellospora spp. Those formed by Acaulospora spp. are typically thin-walled and irregularly shaped. Root infection by other non-mycorrhizal endophytes can be mistaken for AM infection. Auxilliry cells: Swollen structure produced terminally by extraradical hyphae of only Gigaspora, Pacispora and Scutellospora spp. Auxilliary cells formed by Gigaspora spp. (spiny) or Scutellospora spp. (nodulose) often remain attached to the root by hyphae after staining. Spores: Multinucleate single reproductive cells mainly produced blastically at the tip of a sporogenous hyphae. Mycelia: It is the vegetative part of the fungus. Mycelia is either hidden in the product, wood or any other food source (living or dead organic material). It is the bridge that connects the root with the surrounding product microenvironment. Hyphae: The web of tiny filaments/threads that make up the mycelia. Coiled hyphal structures develop throughout the cortical cells, and can also occur in the epidermal cells. Coiled hyphae often develop smaller side branches and may have a similar function to the arbuscules. Inoculum potential: The ability of total infective propagules (spores, mycelia, infected root fragments) to initiate root colonization. Infectivity: The mycorrhizal potential within a product. Contamination: Microorganism other than AMF not declared on the label.

2.0. Introduction Arbuscular mycorrhizae (AM) is the most common mycorrhizal symbiosis type associated with agricultural systems. These common, soil-borne fungi belong to phylum Glomeromycota and produce fungal structures (arbuscules, vesicles, coils and hyphae) in the cortex of the roots and spores, mycelia and auxiliary cells in the product. The AMF benefits mineral nutrition particularly phosphate nutrition, plant water potential under drought stress, bioprotection against various pests and pathogens and improvement of product structure. Inoculation with AMF commercial products in agricultural, forest and degraded landscapes is

a common practice. The AMF inoculum typically produced in the presence of host and soil, soil less cultures and root organ cultures, encompasses the entrapment and immobilization of mycorrhizal root pieces, vesicles and spores. The inoculant contains highly selected strains of low host specificity. The quality of the inoculant is determined by the stains, abundance of infective propagules and the formulation. Amongst the product inoculant carriers are solid (granular and powder) and liquid based. The desired characteristics of inoculants is defined by high propagule densityand long shelf life (months to years). The mycorrhizal inoculant technology appears to be a promising avenue for sustainable agriculture and forestry. Commercial inoculum is produced in pots, nursery plots, containers with different substrates and plants, aeroponic systems, and by nutrient film techniques or in vitro. Different formulated products are marketed and hence the need to establish standards for widely accepted quality. Product quality assess whether product is fit for the purpose. The product must meet or exceed customer and regulatory requirements. It is the customer and the applicable regulatory bodies who sets quality standards in terms of their requirements or expectations. Customers‘ definition of quality characteristics for mycorrhizal inoculum includes: formulation, handling, weight, safety, and functionality. In general, the expected amount spores > 8 x 109.

Purpose The Standard Operation Procedure (SOP) is intended to guide laboratories appointed by regulatory agencies in screening of Arbuscular mycorrhiza commercial products for quality. Based on the information on the product label, the screening is to confirm the strains, propagule density and ability to colonize. This is to guarantee customers viable quality product devoid of contaminants. This exercise is also to verify if there are any contaminates that are harmful to plants, human, animals and the environment. Proper sampling and handling of the product at port of entry and careful dispatch to the testing laboratory is to be guaranteed to avoid any claims of contamination by the product proponent. The final outcome is to guide regulatory agencies on the quality of the product and its suitability for use by farmers. The SOP outlines the procedures provided by the regulatory agency to generate quality data as stipulated on the label and of acceptable standards set by regulatory agencies. Requirements for Quality Control Good laboratory practices and precautionary statements (i.e. safety) Caution (Safety) Heavy items to carry: Care is taken to transport heavy items (such as tubes, pots, products...) using the necessary tools (trolley.) to ensure that one doesn‘t get hurt. Sharps items: Blades, scalpels or any other sharp tool used in the greenhouse is handled properly, stored in a particular place and always returned in its protective packaging. Centrifuge: Before starting the centrifuge, make sure that the tubes are well equilibrated in the rotor. A non equilibrated centrifuge can cause great damages to the centrifuge and can also lead to a ―flying‖ centrifuge. Chemicals: Before using a new chemical, the information about the toxicity, conditions of use, risks and safety phrases... have to be understood and observed. Use the equipments for

protection if needed (gloves, masks, hood...). The MSDS (Material Safety Data Sheet) of the different chemicals are also available to get more details about the products. MSDS files are available in the Preparation Room and in the office. Chloral hydrate: is toxic if swallowed and irritating to eyes and skin. Users should realize that chloral hydrate is a legally controlled substance (U.S. Deprtment of Justice, Drug Enforcement Administration) and can be obtained only with a proper permit or a prescription. Iodine: Harmful by inhalation and in contact with skin. Lactic acid: Causes eye and skin burns. Causes digestive and respiratory tract burns. Sodium azide: is a respiratory inhibitor and therefore should be handled with care (wearing gloves) Avoid Ringer's and other solutions that provide an isotonic environment; buffering appears to serve no useful purpose and in some cases, causes plasmolysis. Quality control management 1. A specific protocol is established before screening is set up. It includes the number of samples, the details about the inoculants (origin, composition, rate of application...), and any other relevant information. 2. Calculations, weights, records (of temperature, humidity...), observations, measurements, preparation of stock solutions (nutritive solutions for example) and any other relevant information are recorded in the lab book on a daily basis. 3. The stock solutions are labelled with the date of reception, name or initials of the person who received it, the number of the container (x of n), date of opening, name or initial of the person who opened it. 4. The reagents are labelled with the name of the contents, date of preparation, name or initials of the person who prepared them and any other relevant information. 5. All samples are clearly identified and records are taken about the different steps of analysis (when they have been put in the oven, grinded, sent for analysis). 6. After harvesting, samples might need to be decontaminated before elimination. Solarization may be considered. 7. In case of contamination of the bench, floor, user, ..., it has to be cleaned and disinfected before the work can be continued (cf. ―Hygiene and Safety rules in a laboratory‖ document). 8. Non contaminated or decontaminated items are cleaned with soap, and rinsed with water and eventually rinsed with distilled water. 9. Equipments are regularly cleaned to remove dust, samples or other waste. 3.0. Arbuscular Mycorrhizal Fungal:Spore Characterization 3.1. Minimum materials and equipment for characterization of spores 1. 2. 3. 4. 5.

Bench CentrifugesCentrifuge test tubes: 50 ml, round bottom tubes clinical centrifuge tubes: 15 ml Water bottles, Sieves: 2 mm Nalgene

6. 710 um mesh sieves 7. 250 um mesh sieves (Optional) 8. 38 or 45 um mesh sieves 9. Beakers: 1L, 250 ml and 100 ml 10. Petri dishes, 90 and 50 mm or watchglass 11. Glass Pasteur pipette (1 ml) 12. Compound microscopes 13. Dissecting microscopes 3.2. Preparation of reagents for Isolation and diagnosis of spores Spores are the most important diagnostic parts of the arbuscular mycorrhizal fungi. Most common reagents required are for spore extraction (sucrose), mounting of specimens (PolyVinyl Lactophenol Glycerol (PVLG) and Melzers reagent for spore staining. The stock solutions are labelled with the date of reception, name or initials of the person who received it, the number of the container (x of n), date of opening, name or initial of the person who opened it. The reagents are labelled with the name of the contents, date of preparation, name or initials of the person who prepared them and any other relevant information.

3.2.1 Spore Extraction: 227 g sucrose (Normal Sugar) dissolved in 500 ml water 3.2.2 Mounting Reagents 3.2.2.1 Polyvinyl-Lacto-Glycerol (PVLG) PVLG is used to permanently mount whole or broken spores on glass slides. For best results, mounted specimens should be studied 2-3 days after they are mounted to give time for spore contents to clear. Whole spores will change color, generally darkening to varying degrees, and shrink or collapse with plasmolysis of spore contents. Discrete layers of the spore wall or flexible inner walls of broken spores will swell to varying degrees and appear fused after long storage in some instances. PVLG stores well in dark bottles for approximately one year. Ingredients and Quantity 1. 2. 3. 4.

Distilled water 100 ml Lactic acid 100ml Glycerol 10 ml Polyvinyl alcohol 16.6g

3.2.2.2 Melzer's Reagent Melzer's reagent is important in diagnosis of AMF spore morphology showing dextrinoid reactions of spore wall and inner germination walls. In freshly extracted spores, intensity of the staining reaction mainly depends on the length of carbohydrate chains present in the components. In stored spores, the reactivity of their subcellular structures usually decreases or completely disappears. Iodine staining reactions vary from pale pink (weak reaction) to dark

red-brown (moderate reaction) to dark reddish-purple (intense dextrinoid reaction. When Melzer's reagent is used according to the recipe, staining reactions will be most intense. Mounts are temporary even when a coverslip is sealed, and often dries out within 1-2 years of storage and, thereby, usually are unsuitable for further studies. More permanent mounts are made by mixing Melzer's reagent with PVLG in a volume ratio of 1:1 (and storing the mixture in a dark bottle). The staining reaction is diminished slightly, but not enough to cause any confusion as to the intensity of the reaction. In structures staining weakly, the color reaction fades within a year or two of storage. The solution should be stored in a tight‐capped bottle and is most easily dispensed from some form of a dropping bottle and the mixture should be stored in a dark bottle.

Melzer's reagent is a stain used to study the cellular structure of fungi. 1. 2. 3. 4.

5.0 g Potassium Iodide ( KI) 1.5 g Iodine 100 g Chloral hydrate 100 ml Distilled water

3.2.3 Methods of preserving spores 3.2.3.1 Sodium azide for voucher specimens If spores are not examined immediately spores are stored in Sodium azide. Spores die in the sodium azide and degrade naturally over time. They float, contents are often darken or lose their integrity (appearing either cloudy or vacant). However, subcellular structures largely retain their integrity. Other preservative solutions such as FAA (Formalin + Acetic Acid + Alcohol) and lactophenol (lactic acid + phenol) have been used extensively in the past, but evidence from type specimens indicates they can cause major changes or degradation of subcellular structure of spores. Spores stored in sodium azide or any other preservative turn red-brown to brown within 10 days. Solutions and vials are stored at 4oC as an added precaution to optimize safety of the workplace.

3.2.3.2 Distilled water and sand for voucher specimens Fungal species with small (< 150 µm) light-colored spores tolerate these conditions best (large darkly pigmented spores tend to degrade no matter what), and they have been successfully stored this way for over a year. Store washed spores in distilled water at 4oC. The drawback with storing in water is that parasitized spores (often not detectable at the time of extraction) are sources of spread of colonizing fungal saprophytes or actinomycetes to adjacent spores. Store in distilled water or water agar, or on 0.1% MgSO4.7H2O solidified with gellan gum in Petri dishes and glass vials and stored in the fridge at 4˚C. 3.3 Diagnosis of specimen and identification of species 3.3.1

Example: Isolation of spores from a clay carrier based commercial product

Extraction 1. The product unlike soil does not have plant debris as is the case with field soils and pot cultures. Measure the volume or dry weight of the sample. 50 g product samples are recommended for extraction. 2. If possible pre-soak the product in water before processing. This is particularly important if products are dry or high in clay content. For products with carriers of significant clay content, the clay is likely to cause blockage. To prevent blockage of the finer sieve, sieve quickly and tap the base of that sieve to encourage excess water to drain through or soak product in sodium hexametaphosphate to disperse the clay fraction. 3. Mix the product in water in a 1 L plastic beaker, stir thoroughly and decant through 710µm and 45µm micron sieves (other sieves may be included in this series e.g. 500, 250, 100µm sieves, but each collection will need to be examined separately). 4. Use a jet of tap water to wash the spores and finer product particles through the 710µm sieve. This water will quickly pass through the 710µm sieve, but it is difficult to get the water to drain through the 45 um sieve because of the build up of fine particles. The two sieves are separated and a jet of water on the surface of the 45 um sieve permits the water to drain at that spot. Then the two sieves are stacked together again, and the process is repeated until the water washing through the two connecting sieves leaves the bottom sieve colourless. Generally this means the particles in the top sieve is washed 3 times. 3 In case some of the strains declared are sporocarpic or in Gigasporaceae or with other infective propagules (infected root fragments and mycelia), before discarding the content, examine the collection on the 710µm sieve (and If more sieves are used as well) for sporocarps, auxiliary cells and spores before discarding. Root fragments and mycelia may also be collected from this sieve for staining. Note: The water drains slowly through the lower sieve and may overflow from the 45um sieve, therefore keep checking visually the height of the water by carefully separating the two sieves; If the water does overflow the lower sieve, spores are lost. 4 A concentration of AMF spores remains in the pellet on the 45um sieve. Collect the particles from the 45um sieve by back washing them into a beaker using a small stream from a wash bottle. The contents in a beaker are swirled and pour into two or four or more 50 ml centrifuge tubes (1/3 soil and 2/3 tap water up to within 2.5 cm of the top of the tube). 5 Then balance the tubes by weight and thoroughly stir the mixture, balance the tubes and then centrifuge for 5 minutes at 1750 rpm. Carefully decant the supernatant containing without disturbing the pellet at the bottom of the centrifuge tube. 6 Suspend the pellet containing the spores in a 48% sucrose (227 g dissolved in 500 ml water) solution and is mixed well. Balance the tubes by weight and mix thoroughly immediately before centrifuging for 15 seconds at 1750 rpm. 7 Immediately after centrifugation, carefully decant the supernatant of sucrose solution that contains the spores through a small 45µm sieve. Immediately Rinse the spores retained on the sieve thoroughly with tap water to wash out the sucrose. 8 Wash the AMF spores into a beaker of water using a wash water bottle.

9 Transfer the spores from the 45 µm sieve into a small petri dish (5cm diameter) with grid lines for examination under a dissecting microscope at x40. Note: The dissecting microscope is a Stereoscopic zoom microscope with bifurcated illuminator of fiber arm and transmitted illumination system. 10 Using a dissecting microscope, pick morphologically similar spores, aggregates, and sporocarps by means of fine glass Pasteur pipette or needle and enumerate and mount on slides for further diagnosis. Note: Fine glass pipettes: soften tip of disposable glass Pasteur pipette (1 ml) with fame of Bunsen burner and sharpen by pulling the malleable (soft) tip with forcep and breaking the tip to make various sizes of tips to fit different sizes of spores. A Light fine tweezers (forceps) is preferable the most convenient with good handling. 11 Spores may immediately be mounted and characterized for identification or stored to consult expert diagnosis. 12 Characterization and identification is by a Compound microscope: biological compound microscope preferably with a Nomarsky‘s DIC illuminator system. The morphology of spores is in the basis of identification. They have characteristics shapes and colour. It is however recommended that those who have not yet observed spores should learn from experts how the spores look like. Use pictures in text books and websites to recognize AMF fungal spores. 13 Spores are put in watch glass or small petri dish and their shape, colour and the attachment to spore observed. 14 Spores are classified into spore types based on morphology and detailed observation is conducted. 15 For colour description a standard soil colour chart or colour chart of Glomelean fungi (INVAM) or colour chart of fungi should be used. The colour chart should be under the same illumination as used for spore observation because colour itself is greatly affected by the characteristics of illumination. 3.3.2

Procedures for preparation of diagnostic slides, mounting of slides and observations of spores

Voucher specimens of permanent slides (slides sealed with colourless nail varnish at 5-10 days after mounting) are prepared as reference material. For description of spores refer to INVAM WEBSITE and Schenck and Perez, 1990. Mount spores on slides with PVLG on glass slide. Several slides are made: intact spores mounted on water are to be observed immediately for colour, intact spores mounted on PVLG, crushed spores mounted on PVLG, crushed spores mounted on PVLG containing Melzers reagent. For spores 40 – 50 spores should be examined in PVLG. Mounting slides and observations of spores: a. Voucher specimens of permanent slides (slides sealed with colourless nail varnish at 5-10 days after mounting) are prepared as reference material. b. Prepare three slides: On the first one, place two drops of water at opposite ends of a microscope slide. On the second, place one drop of PVLG on each end and on the third one, put PVLG + Melzers (ratio 1:1) on both ends. (Slides frosted

at one end are preferred for labelling purposes but in case they are not available, a sticker may be used). c. Pick carefully spores with a fine forcep or micro-pipette and place in each drop. d. Lower a cover-slip, preferably circular with diameter of 13 mm onto each drop of mounting reagent + spore. e. For each slide, observe the spore as whole for diagnosis of appearance on one end and on the other end, crush the spore gently and severely. A needle or pin may be used to crush the spores. Caution must be taken not to break the coverslip. f. In case of bubbles, gently place ethanol though the space between slide and cover-slip to clear bubble, absorb ethanol with tissue and then gently place the mounting reagent again. g. Slides are properly labelled to indicate the sample identity, mounting reagent and date and link slide specimen with information in database on site of collection, soil type, collector name, institution and spore morphological characteristics. h. For description of spores refer to INVAM WEBSITE and Schenck and Perez, 1990: Manual for Identification. G. Morphological characteristics are recorded to identify genus of the target fungus. The genus Archeospora needs both morphology characteristics and sequence data. H. Species identification is undertaken with comparison with species description in original references. Species description and pictures are available in INVAM website. To completely verify a species consultation with experts in AM fungal taxonomy is necessary. 3.3.3 Procedures for spore storage for reference 1. Sterilization and storage of spores: Concentrate the spores on the 45µm sieve and then with a squeeze bottle, wash the spores into the sterilization solution. Soak the spores in this solution for 20 minutes. Pour the spores onto the filter which is sticking on the funnel, and rinse with distilled water 5 times. Make a hole in the filter paper and wash the spores into a 100 ml graduated cylinder. Add more water until the volume is 100 ml. Samples can be removed to determine the spore number. 2. Short term storage: Store the isolated, non-sterilized spores for 2 days in the refrigerator in a beaker of water sealed with parafilm. 3 Long term storage: Fill a clean petri dish 3/4 full of clean, dry sand and pour the sterilized and rinsed spore suspension over the sand. Allow the water to evaporate from the sand by leaving the cover off. This may take overnight to several days. When the sand is dry, cover the petri dish and seal it with parafilm. Store the sealed and labelled dish at 4º c until the spores are used. 4 Retrieval of long term stored spores: Pour the dry sand/spore mixture onto the 250 µm sieve (top), which has been stacked on the 45 µm sieve (bottom). Using distilled water, rinse the spores through the top sieve and collect them from the bottom one (45 µm) with a squeeze bottle containing distilled water. The spores can be washed into a petri dish. 5 Isolated spores may be identified and/or used to inoculate other plants. 3.4. Estimation and quantification of total spore abundance and infective propagules

1. Total viable spore counts under a dissecting microscope from 50 g sample of product. Depending on the carrier material, spore isolation methods may vary. 2. There is more than spores in the products that serve as infective propagules. Other propagules are infected root fragments and mycelia. Enumerating spores abundance may therefore underestimate or overestimate the ability of AMF to colonize. Other methods such as the Most probable Number (MPN) method and Mycorrhizal Product Infectivity (MSI) test have been used to detect viable infective propagules. 3. The method is used for enumeration of AMF viable infective propagules in the product. The infective propagules include spores, hyphae and infected root fragments. The procedure described is for four fold dilution. Other dilutions such as two fold and tenfold may be used depending on the inoculums potential of products, the former for low inoculums potential and the latter for high inoculums potential 3.4.1 1. 2. 3. 4. 5.

Materials Plant tubes (150cm x 2.5 cm) Stands for keeping plant tubes/Polythene bags (30cm x 20cm) Sterilized sand: product (1:1) mix Physical balance Plant material: Plant is chosen depending on its mycorrhizal potential and on the aim of the study. Onion seed. 6. Products: Product is serially diluted in sand previously sterilized.

For 10-fold dilutions,  Five replication or 4 possible with a 35 g (or 50 g) per chosen small pot/ bag.  Product diluted in sand: 40, 4-1, 4-2……4-8, where 100 is undiluted product.  Test product 65 g is mixed with 195g of sterile sand (autoclaved) = 260 g mix thoroughly to get a ten fold dilution ( 10-1).  Place 35g in each replicate (175 g). Use 65g for the next dilution (100 fold) and throw away 10g. Make futher dilutions until 10-8 .  Calculate dry weight of the test product per fresh weight used to give the final propagules number in gram in dry product (72 hrs at 70oC).  Sow 5 pre-germinated seeds, preferably leek seeds (surface sterilized) per pot and after emergence, trim to only one plant per tube. Grow plants in green house or growth chamber for six weeks.  At harvest, wash soil from the roots and stain root system with trypan blue and assess presence or absence of colonization under a stereoscope and check if necessary colonization under the compound microscope (mount roots on a glass slide).  For each of the 5 replicates in each of the four dilutions (4-1, 4-2, 4-3, and 10-4), one might obtain a combination of numbers such as 5, 5, 3, 2.  This means that all 5 replicate tubes are positive for AM colonization in dilutions 4-1 and 4-2, three are positive tubes in dilution 4-3 and 2 positive tubes in dilution 4-4. For the calculation of MPN of propagules only three numbers of the given combination are required.  The first number (N1) is that corresponding to the least dilution which all the (or the greatest number of) tubes are positive for AMF colonization.  The two other numbers (N3 and N4) are those corresponding to the next two higher dilutions. In our example it would be the combination N1 N3 N4

5 3 2  The most probable number of AM propagules can then be calculated using MPN table (Appendix 1). Example of MPN Calculation If using 50 g per pot set out the table of results like this: Dilution

g of product per dilution from Infected reps per dilution test product

40

50

5

4-1

12.5

5

4-2

3.13

4

4-3

0.78

3

4-4

0.20

2

4-5

0.05

1

4-6

0.01

0

4-7

0.00

0

4-8

0.00

0

Formula used: Log λ = ( x Log a) – K λ = number of infective propagules X = mean number of plants infected NO. OF TOTAL INFECTED REPS NO. OF REPS PER DILUTION 20 = 4 5 y = No. of dilutions (s) = s – x (s is dilutions from 40 to 4-8) 9–4=5 a = dilution factor = 4 (four fold dilution) K=

K = Constant found from tables of Fishcer & Yates (1970) for the 4-fold dilution series using (x) or (y) for the number of dilutions (s).

N.B. Where the neither (x) or (y) has a value in the table use the (*) value indicated. In this example it is the * value = 0.552 Log a (dilution factor = 4) = 0.602 Formula Calculation: Log λ = 4 x 0.602 – 0.552 = 1.86 λ = 6.4 in 50g fresh weight (F.W) soil.

Confidence Limits (C.L.) Log C.L. = log λ ± S λ

x Z

√n λ = no. of infective propagules s λ = √0.201 for 4-fold dilutions n = no. of reps per dilution z = tabulated values of probability

90% = z=1.282 95% = z= 1.645 – this is the level that is normally used 99% =z= 2.326 Therefore in this example, Log C.L. = 1.86 + √0.201 x 1.645 √5 = 1.86 ± 0.329 log λ upper limit = 2.19 Log λ lower limit = 1.53 λ upper limit = 8.9 λ lower limit = 4.6

Summary

So in this example the number of infective propagules (λ = 6.4) in 50 g F.W. Products falls between 8.9 and 4.6 in 95% of cases (quote as dry weight (D.W) of product). Appendix 1 Table (Fisher and Yates) for 4-fold dilutions x or y values 4 x 0.4 0.704 0.6 0.615 0.8 0.573 1.0 0.555 1.5 0.545 2.0 0.537 2.5

Number of dilutions 5

6 or more

0.706 0.617 0.576 0.558 0.551 0.548 0.545

0.707 0.618 0.517 0.559 0.553 0.551 0.552 0.552*

y 3.5 3.0 2.5 2.0 1.5 1.0 0.8 0.6 0.4

0.537 0.522 0.488 0.464 0.431 0.375

0.545 0.537 0.522 0.488 0.464 0.431 0.375

0.550 0.548 0.545 0.537 0.522 0.488 0.464 0.431 0.373

When x and y fall outside the range of the table use the value marked with the asterik (*)

Points to note: The last dilution must contain no infection and the dilutions must be well mixed.

4.0 Methods in Root Analysis for AMF The preparation of staining reagents is done under a fume cupboard. Strict measures are observed in the preparation process and caution taken in the handling and disposal of reagents. The process involves sampling, clearing, Staining and analysis of roots to see AM Structures Materials and Equipment:  Water bath heat to 90º c/ Oven/Autoclave  Test tubes and holders  Tweezers  Slides

 Coverslips  Dissecting or low magnification microscope  Compound microscopes Preparation of Solutions:  2.5% KOH (25g KOH in 1000 mls water)  10% HCL  Lactic Acid Solution  875 ml lactic acid  63 ml glycerin  63 ml distilled water The reagents used in the preparation of the staining solution are KOH (caustic), alkaline hydrogen peroxide (irritant), hydrochloric acid (toxic), Glycerol and trypan blue (toxic and carcinogenic). This comprises of preparation of staining reagents, root staining, slide preparation and assessment of AMF colonization. In the process of staining many samples, it is recommended that perforated plastic tubes be used. The tubes are placed on racks and the rack immersed into the solutions. Staining Reagents 1. Trypan Blue: 0.05% trypan blue (500 ml glycerol, 450 ml water, 50 ml of 1% HCL and 0.5g trypan blue) staining reagent was added and placed in the oven for 1 hour at 70 °C. 2. Add 0.1 g. acid fuchsin to 1 liter lactic acid solution (0.01% acid fuchsin-lactic acid solution); Heat at 90º C. for 10 to 60 minutes. 3. Chlorazol Black E (0.03 % w/v) stain in lactoglycerol solution (1:1:1 lactic acid:glycerol:water) (Brundrett et al. 1983). 4. Ink (black = Shaeffer, blue = Pelikan) One could use either of them. Not all them are required at the same time

4.1 Root Clearing and Staining Before AM colonization can be observed, root samples need to be taken through a series of stages during which roots are cleared in potassium hydroxide, bleached in alkaline hydrogen peroxide, acidified in hydrochloric acid and stained. Phenol: Do not add to lactic acid solution. It is not necessary and is potentially carcinogenic.

4.1.1 Alternative staining Procedure Root Staining with Ink and vinegar, a simple staining technique for AMF (Vierheilig et al., 1998) Materials 1. 10% KOH (w/v) (10 mg KOH in 100 ml H2O) 2. Ink (black = Shaeffer, blue = Pelikan) 3. Household vinegar

4. 5% ink-vinegar solution (50 ml ink +950 ml vinegar) 4.2. Methods for assessment of Arbuscular Mycorrhizal Fungal (AMF) Infection 4.2.1. Assessment of arbuscular mycorrhizal infection using gridline intersect method After staining, AM structures can be observed in the cortical cells of the roots using a low power microscope (Dissecting (Stereo-) microscope).

Total infected X 100

= % infection

Total uninfected

4.2.2 Assessment of Arbuascular Mycorrhiza under a compound microscope Analysis of mycorrhizal colonization using slide method Under the dissecting microscope, intracellular structures such as arbuscules and coiled hyphae and hyphae are difficult to differentiate; only intraradical spores and vesicles are easily identified. AM structures (appressoria, intercellular hyphae, intracellular coiled hyphae, arbuscules and vesicles) can be observed in the root cells using a compound microscope. This method enables different AM structures within the root to be quantified and related to function. A minimum sample size of 100 cm for the gridline intersect method is recommended).

5.0 Conclusion Basic information on chemical and physical characteristics such as nutrient content, pH of substrate and different additives like fertilizer or gels apart from AMF is necessary. This should be done by an independent conventional soil analytic official laboratory to provide valid data. To separate the effects of AMF from the nutrients included in the product, the additives should aim to support mycorrhiza development and therefore be specifically compatible with AMF. Inoculation under differing pH regimes from that specified by product proponents may often require a longer term to show desired mycorrhizal effects and the carrier materials depend on the target use of the product. Spore dormancy, spore maturity, activity of extraradical mycelium activity and colonized root fragments affect root colonization. In MPN, the number of infective propagules depend on the number of spores, colonized root fragments and mycelial fragments which will lead to root colonization under favorable conditions. The MPN method is not a constant and stable measure compared to spores. Products dominated by extraradical mycelia and infected root fragments have therefore shorter shelf life compared to products dominated by spores.

Compared to spores, it is difficult to compare MPN from different laboratories unless identical biotic and abiotic conditions are considered. However, the methods for spore extraction from type of carrier will influence the number of spores counted. Carriers that retain spores and do not allow extraction will under estimate the spores extracted. It is more difficult to extract spores from clay based formulations than standard soil and quartz based formulations. Variations in procedures for spore extraction like preparation of product samples, centrifugation times, sieves mesh width etc influence extraction results. The adaptability of AMF strains to exotic conditions may result to slow or no spore germination. It is therefore important to identify the end user of the product as some products will function in the field situation (landscape use) and not in greenhouse assay or horticultural production. The outcome of products depends on environmental factors hence it is not possible for producers to predict effectiveness. Mixed consortia are therefore usually recommended since they increase potential benefits. Examine product out for possible contaminants such pathogenic and saprophytic Fusarium, Pythium, Rhizoctonia and Thielaviopsis. The personnel should have ability to recognize and identify pathogens as well as to judge their relative significance. Trap plants that are highly susceptible or plants that the buyer will use may be used to detect the pathogen. The ultimate goal is to provide the best service to the buyer. Facts that the product proponent must provide to consumers of products: 1. What they proved, which method was used and what result was found 2. Gives recommendations of maximum and/or minimum dilution factors for inoculation process but the use of the optimum amount can be specified to ensure mycorrhization 3. Any relevant adaptations of the strains in the product to guide end-user‘s choice Note: If marketing strategy points out against phytopathogens, the mycorrhizal product would have to be treated as a biological plant protection product.

G. SOP for Testing the Quality of Commercial Trichorderma Products 1.0. Definitions For the purpose of this standard the following definitions shall apply: The following are definition of terms used in synonym and those in the SOP. The terminologies Biofertilizer, Bioenhancer, Biostimulant and Bioinoculant are sometimes used synonymously. . Bofertilizer is a term widely used term meaning ―bacterial inoculant‖ usually refer to preparations of microorganisms that may be a partial or complete substitute for chemical fertilization (like rhizobial inoculants). The use of word fertilizer allows easier registration for

commercial. It is a formulation containing one or more beneficial strains (or species) in an easy to use and economical carrier either organic, inorganic or synthesized from defined molecules. It contains living microorganisms embedded in a carrier material which are applied to seed, plant surface or product colonizers the rhizosphere or the interior of the plant or soil and promotes growth by increasing supply or availability of nutrients to the host plant, or preparations containing living cells or organisms that enrich the nutrient quality of product. Biopesticides: Living organisms or natural products derived from these organisms which suppress pathogen populations microbial pesticides, other organisms (nematodes, insects) used to control pests; natural products derived from living organisms (biochemical pesticides) and plant incorporated protectants (genetically modified plants) – Bacillus thuringiensis in Bt maize and Bt cotton. Biopesticides decompose quickly in the environments; and are less toxic towards non-target species. Bioinoculants: Living organisms containing specific strains of specific bacteria, fungi and algae which fix atmospheric nitrogen, make nutrients soluble and available, collect and store nutrients, provide physical barriers to pests and pathogens, stimulate plant growth, and decompose organic residue. Bioenhancers: Substances that increase the bioavailability of active ingredients, vitamins and nutrients Biostimulants: Dormant strains of positive product microorganisms that come alive when introduced into the product system. They contain strains of specific bacteria, fungi or algae which take up nutrients and make it available to plants, collect and store available nutrients, enhance uptake of nutrients, provide physical barriers against pathogens, stimulate growth, decompose organic residues Fungi: Multicellular organism, separate Kingdom from plants, animals, protists and bacteria. They have cells that contain chitin and derive nutrition through breakdown of dead organic materials (saprophytic), obtain carbohydrates from living plants through mutual exchange (symbiosis) or killing the plant or animal (parasitism). The organisms is hidden and the fruit may be visible e.g mushrooms, toadstools and bread mold or invisible Trichorderma:. A saprophyte fungus, which belongs to the family Hypocreaceae, the division Ascomycota. Contamination: In other microorganism other than AMF not declared on the label. Aseptic Conditions/Aseptically: Environment where no microorganisms is present. It can be obtained by using a Bunsen burner to create an aseptic zone which is the aspheric area around the flame with a diameter of approximately 15 cm or a Laminar flow hood with sterile air continuously produced and present in the hood. The high pressure of air flow from the hood prevents the air from outside to come inside and contaminate the environment in the hood; aseptic conditions are also created by cleaning surface with 70% ethanol. Contaminated microorganisms: Every plate, tube, pipette, or other instruments (glassware, pestles, eppendorff tube…) which has been in contact with microorganisms and cannot be sterilized by the flame of a Bunsen burner is considered as contaminated.

Contaminated by toxic chemicals: Every tube, flasks, pipette or other instruments which has been in contact with toxic chemicals is considered contaminated Good Laboratory Practices (GLP): The principle of GLP have been developed to promote the quality and validity of results and of the analysis conducted in a laboratory. It is a management concept covering the organization and the conditions under which laboratory studies are planned, performed, monitored,, recorded and reported. Its principle also include the protection of man and the environment. Mother tube/plate/products: Tube/plate/product which the bacteria are picked from. The result of the growth of this inoculation is considered as the daughter which can become the mother for the next inoculation. Substrate: Any material, that serves as source of energy for an organism. Carrier: Delivery media of live microorganisms from the production to the field. The carrier is the major portion of inoculant. There is presently no universal carrier or formulation available for release of microorganism into soil. Materials and types of formulations of carriers vary: Slurry, powder, peat, liquid. A good carrier should have the capacity to deliver the right number of viable cells in good physiological condition at the right time. Growth Medium: growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms Agar: Agar consists of a mixture of agarose and agaropectin. Agar is used to provide a solid surface containing medium for the growth of bacteria and fungi. Aseptic : an environment or procedure that is free of contamination by pathogens Abbreviations °C: degrees Celsius mg : milligram g: gram

2.0.

Introduction

Trichoderma spp. are filamentous fungi widely distributed in the soil, plant material, decaying vegetation, and wood. They are facultative anaerobes growing saprophytically or as a parasite on other fungi. As parasites, they grow toward hyphae of other fungi, coils around them, and attach to host mycelium and gain nutrition from other fungi. Trichoderma colonize roots enhancing their growth and development which resultus in plants tolerance to stress and induced resistance to disease. Trichoderma grows and proliferate fast making them rhizosphere competent thus compete well for space and nutrients solubilizing and sequestrating inorganic nutrients. Trichoderma synthesizes a lot of antibiotics (gliotoxin, viridine, trichodermin and others). They destroy the cell walls of phytopathogene fungi and produce biologically active substances, which stimulate plant growth and development. Trichoderma is able to suppress more than 60 species of pathogens (Fusarium, Rhizoctonia, Pythium, Botrytis, Sclerotinia and Armillaria, Phoma, , Ascochyta, Alternaria and others) on

different plants (cucumbers, tomatoes, cabbages, peppers, various ornamentals, cereals and grain legume crops). Other two known mechanisms of genus Trichoderma biological control are rhzosphere competence and an induced system resistance which provide longtime plant defense. The use of Trichorderma has been common since 1930. It is one of the oldest and most widely used fungi-based pesticide in the world. Different varieties of this fungus are used in agriculture against various phytopathogenes of crops in outdoor planting and green house. Such species as T. harzianum, T. hamatum, T. lignorum, T. viride, T. koningii and their biotypes have the most biological and commercial importance. The active components of biopesticides made on the base of this fungus-antagonist are their spores, mycelium and products of metabolism. The procedure below describes how you can recover Trichoderma from substrates. Different media are used to isolate and grow Trichoderma. No medium exists for the selective growth of particular Trichoderma species. You can however, use a Trichoderma Selective Medium (TSM) to suppress growth of other fungi/bacteria and obtain Trichoderma colonies that can then be differentiated by morphology. It is crucial to prepare monospore (single spore) cultures for identification purposes since several Trichoderma spp. may occupy one ecological niche. Quality control management (General procedures) 1. A specific protocol is established before screening is set up. It includes the number of samples, the details about the inoculants (origin, composition, rate of application...), and any other relevant information. 2. Calculations, weights, records (of temperature, humidity...), observations, measurements, preparation of stock solutions (nutritive solutions for example) and any other relevant information are recorded in the lab book on a daily basis. 3. The stock solutions are labelled with the date of reception, name or initials of the person who received it, the number of the container (x of n), date of opening, name or initial of the person who opened it. 4. The reagents are labelled with the name of the contents, date of preparation, name or initials of the person who prepared them and any other relevant information. 5. All samples are clearly identified and records are taken about the different steps of analysis (when they have been put in the oven, grinded, sent for analysis). 6. After harvesting, samples might need to be decontaminated before elimination. Solarization may be considered. 7. In case of contamination of the bench, floor, user, ..., it has to be cleaned and disinfected before the work can be continued (cf. ―Hygiene and Safety rules in a laboratory‖ document). 8. Non contaminated or decontaminated items are cleaned with soap, and rinsed with water and eventually rinsed with distilled water. 9. Waste management: i. Non contaminated waste is eliminated in the normal bin. ii. Non contaminated glass waste are put in a separate container labelled with the mention ―Broken glass‖ iii. Anything contaminated by microorganisms should be decontaminated before appropriate elimination/cleaning. iv. Waste may be put into a special autoclave bags for 20 mins at 121 ºC. v. Solid and liquid waste contaminated by toxic chemicals are placed in a separate container to be collected by special disposable service providers.

vi. Disposable glass instruments are ut into a container containing Sodium Hypochlorite solution (commercial Jik) for decontamination before being eliminated. Solid (including broken glass) and liquid waste contaminated by toxic chemicals are placed in separate containers for elimination by waste disposal company. vii. Contaminated glassware is properly rinsed with tap water and water collected in specific items (consult MSDS) on product disposal. 10. Equipment‘s are regularly cleaned to remove dust, samples or other waste. 11. Room is cleaned daily. Disinfect every surface with disinfectant and or with 70% ethanol before starting work. The bench and the floor is to be cleaned and disinfected before the work begins and in case of any contamination during the working session it should be disinfected before work continues. General laboratory practices 1. Always wear a lab coat when working in the laboratory. 2. Never put on gloves when working close to flames. In case of burn, the glove would stick on your skin and make the wound worse. 3. To maintain good hygiene, clean your hands with soap and work under aseptic conditions. Clean bench of hood and any surface of operation with a disinfectant; pour some disinfectant on the bench, leave for I min and wipe with gauze/cotton wool. It is also possible to clean the bench with 70% ethanol. 4. Always have a seat when working to avoid accidents. Avoid useless movement in the lab during work. Organize your work space to ensure a smooth run to the analysis. Some equipment need to be switched on some time before use (incubator, laminar flow hood). 5. Plan your work to give them enough time. Organize everything you need in the hood before starting to work so that you don‘t have to cross hands or have to have put your hand out of the hood. Never put items close to the back of the hood because it can affect the flow hence affect the sterility of air. Use preferably the middle and sides of the bench. 6. Every aseptic manipulation has to be done in the middle of the hood, never outside of it and not at the side or the back of the hood. 7. Air contaminations are common. To avoid them, don‘t speak, run or move it it is not necessary and keep the door and windows closed. 8. A burner is used to sterilize items such as loops, tubes, or forceps. The hottest part of the flame is the top of the blue part, so this is the part which has to be used. If you have to work without a hood but just with a burner, leave it on throughout the manipulation and consider that the aseptic zone is spherical area about 15 cm around the flame. 9. Remember to label every tube, bottle, flask, plate… before you start. Always note the date on everything that you will incubate, keep or autoclave (tubes, plates..) label the plate so as to be able to read what is written during incubation (upside down). 3.0.

Procedures for isolation of Trichorderma spp.

Media Trichoderma Selective Media (TSM) Oat Flour Agar (OFA) Czapek Dox Agar (CDA) Rose Bengal Agar (RBA)

Potato Dextrose Agar (PDA) Materials Petri dishes (you can use disposable type) Media preparation bottles Weighing boats Instruments /Equipments Safety cabinet Autoclave Incubator Bunsen burner Weighing balance Calibrated Compound microscope Dissecting microscope Pestle and mortar Isolating needle Spatula

3.1.

Isolation of Trichoderma from commercial products and other

substrates Isolation Techniques

1.

2. 3. 4.

5. 6.

7.

(i) Warcup’s Soil Plate Technique Take 5mg of product and distribute evenly over the surface of the solidified media (Wracup, 1950). You can grind the product (if in granular formulation) sample in a mortar with a pestle and pass the product through a 1.7mm sieve if the product is not fine. This helps in separation and recovery of colonies. The isolation media can be any of the following: Potato Dextrose Agar (PDA), Malt Extract Agar (MEA), Oat Flour Agar (OA), Rose Bengal Agar (RBA), Czapex Dox Agar (CDA), or Trichoderma Selective Media (TSM) (Appendix 1). The last is selective for against other fungi and bacteria. Incubate the plates for 5 days at 28oC. Transfer all colonies determined to be Trichoderma according to Watts et al., (1988) and Rifai (1969), onto fresh media for purification. Incubate the plates for 5 days at 28oC. From the bacteria free cultures you now have, prepare single spore cultures for use in identification (Appendix 2). Colonies are usually first white then develop yellowish tints until they become various deep shades of green. Conidiophores will arise as branches of aerial mycelia, septate, and grow up to 70 cm in height. If you used TSM, transfer Trichoderma colonies to PDA. Incubate the plates for 5 days at 28oC. Observe characteristics of the colony such as colour of colony, pigmentation diffusing into growth medium, liquid droplets. The identification keys listed below will guide you on the characters to look for. Prepare slides for examination further examination and measurements of features.

Use recommended identification keys by Watts et al., (1988) and Rifai (1969). The website below also provides an interactive key by Samuels GJ: http://nt.ars-grin.gov/taxadescriptions/keys/TrichodermaIndex.cfm This link will lead you to a morphological key with descriptions and over 500 images for the 32 species of Trichoderma compiled by Dr. Gary J. Samuels from USDA, Beltsville, USA. The key also includes species of Hypocrea that have named Trichoderma stages. 8. In cases where the key requires growth rates for identification, measure radial growth daily for 14 days. Trichoderma grows fast and sporulates heavily. This requires very careful preparation of slides for meaningful examination. Pick very thin growth for your slide preparation (Appendix 3). Conidiophore branching is an important identification characteristics. In case you are unable to prepare good slides from your Petri dishes, prepare slide cultures for this (Appendix 4) 9. It is well known that due to homoplasy of morphological characters it is often impossible to discriminate species. For this reason it is advisable to do molecular identification using the following phylogenetic markers (a) ITS 1 and ITS 2 (internal transcribed spacer 1 and 2 of rRNA gene cluster (b) Tef 1 (translation elongation factor 1-alpha encoding gene) (ii) Product Dilution Plate Technique The dilution media is 0.05% WA (Appendix 1). 1. Grind the product sample in a mortar with a pestle. 2. Dissolve 1g by agitation in 9 ml of sterile 0.05 % WA while liquid is viscous enough to be evenly distributed. 3. Subject the suspension to a ten-fold dilution series in sterile 0.05 % WA up to 10-4 dilution, Figure 1.

Figure 1: Serial dilution 4. Transfer 1ml of the product suspension from the 10-2, 10-3 and 10-4 dilutions to a 100-mm diameter Petri dish using a pipette and spread evenly across the agar surface. Use a glass ‗hockey stick‘ applicator to spread the suspension. Use the various isolation media described above. Petri dishes containing agar to be used with product dilutions should be allowed to dry for 3-5 days before use. The drier media more quickly soaks up excess water in the suspension, which helps minimize bacterial contamination. 5. Follow procedure outlined in section (i) no. 2-8 above.

4.0. Conclusion The fungus Trichoderma is harmless and pesticides don't cause any harm to plants and the environment. Trichorderma is applied to (i) seed to increase germination, seedlings to accelerate root growth, increase root mass (iii) at transplant to reduce transplant stress (iv) as an additive to greenhouse and potting mixes and (v) mature trees to reduce production decline, increase water and nutrient absorption and reduce root oxidation. It is commonly applied in granular and powder formulations. Some of the limitations for the application of Trichoderma biopesticides (i) At first they are preventive only because biopesticides are

usually not able to control the diseases, which have already developed (2) Scientific researches have shown that the development of Trichoderma isolates is suppressed by the high density of phytopathogen population (ii) Trichoderma biopesticides are recommended to apply as a component of the integrated system of plant defense (iv) The pesticides containing Trichoderma are effective at temperatures more than 14°C (the optimal threshold of development is observed at 24–28°C). (v) The using of the conidium forms of the pesticides makes their application independent on the conditions of relative air humidity.

APPENDIX 1 MEDIA FOR ISOLATION AND GROWTH Potato Dextrose Agar (PDA) The medium contains: 20 g dextrose 20 g PDA 1 L distilled water Add all these to a media preparation bottle and autoclave at Autoclave at 15 lbs pressure (121°C) for 15 minutes. Let to cool until you can hold by hand. Dispense 10-20ml of the sterile molten agar into Petri dishes aseptically. Rose Bengal Medium (RBM) Mycological Peptone Glucose di-Potassium hydrogen phosphate Magnesium sulfate Rose Bengal Chloramphenicol Bacteriological agar

5.0g 10.0g 1.0g 0.5g 0.05g 0.1g 15.5g

Dissolve ingredients in water and autoclave for 15 min at 121°C. Czapek Dox Agar (CDA) Sucrose Sodium nitrate Dipotassium phosphate Magnesium sulphate Potassium chloride Ferrous sulphate Agar Distilled water

30g 2g 1g 0.5g 0.5g 0.01g 15g 1L

Final pH ( at 25°C) should be 7.3±0.2

If using commercial product, suspend 49.01 g in 1000 ml distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Mix well and pour into sterile Petri plates Oatmeal Agar (OA) Oatmeal or oat flour 60 g Agar 12.5 g Distilled water 1L Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Final pH is 6.0 and requires no adjustment Malt Extract Agar (MEA) Malt extract 30g Mycological peptone 5g Agar 15g Distilled water 1L Suspend 50g in 1 litre of distilled water and boil to dissolve. Sterilise by autoclaving at 15 lbs pressure (121°C) for 15 minutes Trichoderma Selective Medium (TSM) (Elad et al., 1981) Basal medium MgSO4 (7H:O) K:HPO KCl N H4NO3 D glucose anhydrous Agar Distilled water

0. 2 g 0 .9 g 0.15 g 10 g 3g 20 g 950ml

Autoclave at 121 C for 15 min. The biocidal ingredients Chloramphenicol (crystallized) p-dimethylaminobenzenediazo sodium sulfonate Pentachloronitrobenzene Rose bengal

0.25 g 0.3 g 0.2g 0. 15 g

Mix in 50 ml of sterilized (autoclaved at 121 C for 15 min) distilled water and add to the autoclaved basal medium.

APPENDIX 2 SINGLE-SPORE TECHNIQUE EQUIPMENT REQUIRED Dissecting microscope (30 – 40X) total magnification; with light source below the observation stage) or a micro-manipulator. Sharp needle; or flattened, arrow-shaped needle

2-3 test tubes (per culture) filled with 10-15 ml sterile, distilled water 2-3 Difco Bact0 agar plates (per culture), thin-medium thickness agar, 90 mm diam. 4 Difco plates (per culture), thinly poured agar, 90 mm diam, no additives or antibiotics. 5 ml syringe, sterile, disposable. GENERAL METHOD NB!! Single-sporing should only be done from Trichoderma cultures which are free of bacterial and/or mite contamination. Contaminated cultures should first be cleaned up, e.g. plating onto PDA ring plates o remove bacteria. Single-sporing is useful to separate ‗mixed‘ cultures and is absolutely essential for Trichoderma strains which are to be deposited in any Culture Collection or used for identification, mycotoxin, genetic or molecular studies. Under aseptic conditions, remove a needle-point of spore mass and place in the first of three tubes of water. Shake for approx. 2 – 4 sec. From this first tube, transfer 1 ml suspension to the second tube with a sterile syringe and shake. Make a similar dilution from the second tube into the third tube. Pour most of the content of each tube onto the surface of a thin Bacto agar plate. Swirl plate gently and pour off the excess suspension. Keeping these Bacto agar plates inverted, incubate them at a slight angle, at 20 – 25oC for 16 – 24 hours. The angle allows water to drain away from the spores attached to the agar surface. Examine the plates under the dissecting microscope and select the dilution with the best spread of germinated spores. The best magnification to work is at 30-35 X. Adjust the light source so that the germinated spores are clearly visible. The exact plane of view of the spores can be established by touching the agar surface with a needle, then focus on this hole and find the spores lying nearby. Germinated spores should lie well clear of each other, with no overlapping of germ tubes or developing hyphae! Select each ‗germling‘ and while viewing under the microscope, make four incisions to obtain a± 1 mm2 block. Lift out the agar block, making sure that the conidium is not dislodged from the agar in the process. Place each block in the centre of a PDA plate. Make two plates per culture/strain. Incubate the plates, preferably inverted, at 25oC.

APPENDIX 3 MAKING A BASIC MICROSCOPE PREPARATION Observing Trichoderma Place a small drop of stain or mountant in the middle of the slide. If the material is dark, use colorless lactophenol; if it is colourless use a stain e.g lactophenol cotton blue.

Using a fine needle pick off a small portion of the mycelium of the fungus from the substrate or Petri plate provided and place on the mountant. Tease it out with as little disturbance as possible. Take a clean cover slip and hold it on one side, then let it slip onto the liquid from one side without trapping air bubbles underneath, Fig 2. Press the cover slip down gently. There must be just enough mountant or stain to suspend the cover slip. Observe using a compound microscope.

Fig 2 for microscopic examination

Preparation of a slide

APPENDIX 3 SLIDE CULTURE TECHNIQUE FOR FUNGI Materials Required Culture: 7-10 day old fungal culture Media: PDA Equipments: Sterile Petri dish Filter paper (9cm diameter) U-shaped glass rod Microscope slides and coverslips (Sterile) PDA plate with mixed culture of fungi Sterile PDA plate Lactophenol cotton blue stain Glass capillary tube Scalpel Inoculating needle Sterile distilled water 95% ethanol Forceps Procedure: A) Slide Culture Preparation 1. Aseptically, with a pair of forceps, place a sheet of sterile filter paper in a Petri dish.

2. Place a sterile U-shaped glass rod on the filter paper. (Rod can be sterilized by flaming, if held by forceps.) 3. Pour enough sterile water (about 4 ml) on filter paper to completely moisten it. 4. With forceps, place a sterile slide on the U-shaped rod 5. Gently flame a scalpel to sterilize, and cut a 5 mm square block of the medium from the plate of PDA (Fig 3). 6. Pick up the block of agar by inserting the scalpel and carefully transfer this block aseptically to the centre of the slide. 7. Inoculate four sides of the agar square with spores or mycelial fragments of the fungus to be examined. Be sure to flame and cool the loop prior to picking up spores. 8. Aseptically, place a sterile cover glass on the upper surface of the agar cube. 9. Place the cover on the Petri dish and incubate at room temperature for 48 hours. 10. After 48 hours, examine the slide under low power. If growth has occurred there will be growth of hyphae and production of spores. If growth is inadequate and spores are not evident, allow the mold to grow for another 24–48 hours before making the stained slides.

Fig 3: Preparation of slide culture B) Application of Stain

1. 2. 3.

Place a drop of lactophenol cotton blue stain on a clean microscope slide. Remove the cover glass from the slide culture and discard the block of agar. Add a drop of 95% ethanol to the hyphae on the cover glass. As soon as most of the alcohol has evaporated place the cover glass, mold side down, on the drop of lactophenol cotton blue stain on the slide. Examine the slide under microscope Advantages of slide culture:

1. 2. 3.

It is a rapid method of preparing fungal colonies for examination and identification. Permits fungi to be studied virtually in situ with as little disturbance as possible Fungi are identified mostly by close examination of its morphology and the characteristics it possess. In slide cultures, we are growing the fungi directly on the slide on a thin film of agar. By doing this, there is no need to remove a portion of the fungus from a culture plate and transfer it to the slide. So there is less chance for the features that are key to identification, notably the spore-bearing structures, to be damaged.