Laboratory Experiments in General Microbiology

Laboratory Experiments in General Microbiology Second Edition

Yahya Rashid Faidy Assistant Professor of Microbiology, Department of Medical Laboratory Sciences, An-Najah University

Mohammed Saleem Ali-Shtayeh Professor of Biological Sciences, Department of Biological Sciences, An-Najah University

Department of Medical Laboratory Sciences An-Najah National University, Nablus

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Copyright  2000, by Yahya Rashid Faidy & Mohammed Saleem AliShtayeh All rights are reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopy, recording or otherwise without the prior permission of the copyright owner.

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TABLE OF CONTENTS Preface laboratory Rules of Safety UNIT 1. Equipment Used in Microbiology 1. Media Preparation UNIT 2. Preparation and Staining of Smears 2. Preparation and Fixation of a Bacterial Smear and Simple Staining 3. Negative Staining Technique 4. Gram Staining 5. Acid-Fast Staining 6. Structural Stains (Endospores, Capsule, and Flagella) 7. Morphology Unknown UNIT 3. Cultivation of Bacteria 8. Microbes in the Environment 9.Transfer of Bacteria: Aseptic Technique 10. Isolation of Bacteria by Dilution Technique 11. Special Media for isolating Bacteria 12. Cultivation of Anaerobic Bacteria UNIT 4. Microbial Metabolism 13. Action of Microorganisms on Carbohydrates 14. Action of Microorganisms on Proteins, Amino Acids and Urea 15. Respiratory Enzymes 16. Rapid Identification Methods UNIT 5. Quantitative Measurements of Bacterial Growth 17. Quantitative Measurements of Bacterial Growth UNIT 6. Control of Microbial Growth 18. Sterilization and Disinfection 19. Antimicrobial Agents Susceptibility UNIT 7. The Microbial World 20. Unknown Identification and Bacterial Taxonomy References 21. Fungi: Yeast 22. Fungi: Molds 23. Bacteriophage UNIT 8:Microbiology and the Environment 24. Microbes in Water: Multiple - Tube Technique 25. Microbes in Water: Membrane Filter Technique 26. Microbes in Food: Contamination REFERENCES APPENDICES Appendix A. Media and Stains Appendix B. Keys to Bacteria

V VII 1 3 7 9 13 17 21 25 31 35 37 41 47 53 57 61 63 71 77 81 87 87 91 93 97 101 103 107 113 119 123 125 129 133 139 141 143 147

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PREFACE The second edition of Laboratory Experiments in General Microbiology includes updated exercises that cover basic microbiological techniques with application for undergraduate students in areas, including, but not limited to, biological and allied health sciences; pharmacy, and environmental sciences. The manual contains 26 exercises covering different areas of microbiology. To meet the needs of a particular course, the instructor may choose an appropriate combination of exercises that provide the required learning experience. The manual is divided into 8 units. Unit 1, Equipment Used in Microbiology, familiarizes student with basic equipment and their uses including media preparation. Unit 2, Preparation and Staining of Smears, explains the handling of bacterial cultures, preparation of smears, use of most common stains including preparation of stained samples and their examination. Unite 3, Cultivation of Bacteria, emphasizes aseptic technique, the isolation of bacterial strains and maintenance of bacterial cultures. Unit 4, Microbial Metabolism, explains main aspects of biochemical processes in bacteria. Unit 5, Quantitative Measurements of Bacterial Growth, deals with bacterial growth and its quantitative determination. Unit 6, Control of Microbial Growth, explains some methods of controlling unwanted microbes in food or in a clinical environment. Unit 7, The Microbial World, demonstrates the diversity of microorganisms. Unit 8, Microbiology and the Environment, includes standard methods for the examining the microbiological quality of water and food. Each exercise is followed by a Laboratory Report that provides a space to record results. Questions in the report are usually aimed at the interpretation of results. Finally we would like to thank Miss Rana M. Jamous for her editorial skill and patience.

The authors Nablus August, 2000

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LABORATORY RULES OF SAFETY When working with pathogenic microorganisms, there is always the possibility of acquiring an infection, or disseminating it to community. For this reason, precautions should be taken in handling laboratory materials by all students in laboratory. The following rules for safety must be complied with. Any infraction of these rules may make the students liable to infection. 1. Do not put cloth, bags, or books on the bench. Leave these things outside the laboratory. 2. Wear clean coats during the laboratory period. Autoclave the coats after handling an infectious material. 3. Smoking, eating, drinking and chewing gums are completely forbidden in the laboratory. 4. Keep pens, pencils, hands and other objects away from the face. 5. If a living culture is broken or spilled, cover immediately with a disinfectant, and notify the instructor. 6. If your fingers come in contact with living culture material, rinse with disinfectant (70% alcohol or diluted dettol) and scrub with soap and water. 7. Avoid cutting or picking the fingers or spreading infective material or culture especially in the eye. 8. Put Petri dishes, culture tubes or any infected material in autoclavable sacs, so that they can be autoclaved after the class session. 9. Place contaminated cotton applicators, pipettes, slides and covers-slides in jar containing diluted dettol. 10. The practice of mouth pipetting of infectious material is completely forbidden. 11. Before leaving the laboratory, clean the hands by thorough scrubbing with soap and water followed by washing with a disinfectant. Make sure that you clean the disk, and shut off the gas, water, lights and instruments. 12. All cultures should be incubated at 37 oC, unless otherwise specified. Examine cultures 18 - 24 hours after inoculation, or earlier if necessary. 13. Make sure that all windows and doors are closed during media preparation and culture inoculation, as currents of air may contaminate your culture.

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Laboratory Experiments in Microbiology 1 1

UNIT 1 1. Media Preparation

Equipment Used in Microbiology 1. Platinum Loops and Needles

6. Autoclave

A needle has a straight wire attached to a holder. It is used to pick up colonies from solid media and make stab cultures. A loop has a small ring at the apex of the wire, which is attached to a holder. It is used for culturing and transferring of bacteria sent for bacteriological examination.

Steam under pressure is the sterilizing agent in this apparatus. It is used for sterilizing media, contaminated material, glassware, cloth and other materials, which are not decomposed by heat. Exposure to temperature of 121 oC under 15 pounds pressure (above atmospheric pressure) for 15-30 minutes is sufficient to kill all organisms or spores. Other temperature pressure relations in the autoclave are given below.

2. Swabs These are composed of wooden sticks which have small cotton plugs at one of their ends. Swabs are used for obtaining specimens from the throat, nose, vagina, wounds, rectum and elsewhere. The same swabs are used for inoculating suitable media to detect the presence of microorganisms which may be present in these areas.

3. Glassware

Temperature (oC) 108 116 121 127

Pressure in pounds 5 10 15 20

131 134

25 30

Glassware should be thick, free from organic matter, acids and alkalines, and resist sterilization under pressure. Pipettes are plugged with cotton at the mouth end and placed top downward in a metal can.

7. Filters

4. Petri Dishes

8. Hot-Air Oven

Microbiological filters are used for sterilizing fluids which are decomposed by heat. Porcelain, diatomaceous and Seitz filters are used.

These are circular flat plastic or glass dishes of different sizes, each has an overlapping cover. Glass Petri dishes are sterilized in a hot air oven or in an autoclave.

A temperature of 160 oC for 2 hours is necessary for complete destruction of bacterial spores. This method is used for sterilizing glassware.

5. Incubators

9. Centrifuge

These are doubly insulated boxes, having the inside temperature maintained constant by a thermo-regulator. Some incubators are provided with CO2 to grow anaerobic bacteria. Glass-door incubators are used for culturing photosynthetic bacteria. Usually 37 oC and 20 oC incubators are used in microbiology. Incubators with magnetic stirring or shakers are specific types of incubators for providing air to aerobic bacteria during incubation.

Ordinary centrifuge and high speed centrifuges incorporating a refrigerating unit are used in microbiology. Speeds up to 400 rpm are usually used to sediment cells and microorganisms.

10. Lyophilizer Lyophilization is the most effective procedure for the preservation of cultures. Many species of bacteria preserved by this technique have remained viable and unchanged for more than 20 years.

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Lyophilized bacteria can be kept for more than 20 years. In the lyophilizer, cells are dried rapidly, while they are frozen. Vials of lyophilized bacteria can be kept for years at room temperature.

11. Microscope The microscope is an important tool in microbiology and it must be used carefully and correctly. The following are general guidelines that could be helpful while using this tool. The microscope should be carried with both hands. To avoid eye strain, slides are observed with both eyes open. Focusing is better carried out by moving the lens slowly and carefully away from the slide. Good contrast can be achieved when using the low-power lens, by barely opening the iris diaphragm. More light is needed with higher magnification. Before using the oil immersion lens, slide is always focused with low power first, then under high power. The stage and the lenses, except the oil immersion lens, should be kept free of oil. Lenses are kept clean. Lens paper only is used to clean lenses. Oil should be wiped

off the oil immersion lens before putting the microscope away. Lenses are not touched with hands. Ocular lens is cleaned with lens paper. After use the slide is removed, oil is wiped off, dust cover is put on, and the microscope is returned to the designated area. In microbiology, usually the light microscope is used, which use yellow light of a wavelength of 0.4 nm. Thus with oil immersion lens, particles of 0.2 m in diameter are magnified to about 0.2 mm and become clearly visible. Dark field microscope is used for observing microorganisms such as spirochetes which is < 0.2 in diameter and therefore cannot be observed with direct light. A confocal microscope uses intense laser light beams and computer-assisted image enhancement to image from thick fluorescent specimens. Confocal microscopy has made significant contributions to the field of cell biology.

Laboratory Experiments in Microbiology 3

EXERCISE 1 Media Preparation Objectives 1. Prepare different media for the cultivation of bacteria using aseptic techniques as demonstrated by the instructor. 2. Prepare agar plates and slants.

Introduction

require the addition to these media of substances such as serum, blood, or extracts of plant or animal tissues, minerals and growth promoting factors. It is called enriched media. The addition of certain chemical substances (crystal violet) to nutrient agar will prevent the growth of one group of microorganisms without inhibiting others, such media are called selective media. Commercially prepared types of media are available for the different types of microorganisms. In addition to nutrients, several important factors should be considered for producing maximum multiplication of microorganisms on culture media: oxygen, pH, temperature, moisture, sterility and sterile techniques.

A prerequisite to a satisfactory study of microorganisms is their cultivation under laboratory conditions. To do this one must know what food nutrients and physical conditions they require. Bacteria exhibit wide differences with respect to its nutritional and physical conditional requirements, which favor its growth. Extensive research resulted in the development of numerous media (singular, Materials medium) for their cultivation. Petri plates; culture tubes; media Bacteria can be divided into two major ingredients (nutrient agar; nutrient groups on the basis of their nutritional broth; Avery's broth; blood agar; requirements: autotrophs and heterotrophs. chocolate agar). See appendix A for Autotrophs require only carbon dioxide for recipes. their carbon source, and thus exhibit the simplest requirement. A medium composed of known chemical compounds is used for Techniques / Equipment Required the cultivation of these microorganisms. Media preparation; aseptic techniques; Such a medium is known as chemically autoclave; water bath; incubator; defined or synthetic medium. Heterotrophes refrigerator. vary considerably in the specific nutrients required for their growth. A satisfactory Procedure culture medium must contain available 1. Preparation of Nutrient Broth sources of carbon, nitrogen, inorganic salts, (NB): and in some cases, vitamins or other growth To make 100 ml of nutrient broth: Mix, promoting substances. These substances are the required ingredients (Appendix A), heat supplied usually in the form of meat infusion gently to boiling. Sterilize in the autoclave at (ground lean extracted with water) or beef 121 oC for 15 minutes. extract and peptones (partially hydrolyzed proteins). Preparation of Avery's Broth: A solution of peptones and meat extracts 2. To sterile cold 95-ml nutrient broth, 5is the basic component of many culture ml sterile animal or human blood are added, media and is commonly called nutrient broth. using aseptic techniques demonstrated by the Usually sodium chloride is added to nutrient instructor. broth to make it isotonic. Agar may be added to nutrient broth to make it solid or semisolid Preparation of Nutrient Agar and the medium is known as nutrient broth. 3. (NA): Nutrient broth and nutrient agar can Make 100 ml of nutrient agar in a flask support the growth of many bacteria. as follows: Mix, the required ingredients However certain fastidious microorganisms

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1.

Preparation

of

Blood

Agar

(Appendix A), and leave for a short period, Plates: then heat gently to boiling with continuous Prepare nutrient agar as previously shaking. Make agar slants by pouring 5ml mentioned. Cool in a water bath at 50 oC. media in test tubes. Plug the flask and the Add 5 ml of human or animal blood, mix and tubes and sterilize in the autoclave. After pour in Petri dishes. Keep one plate autoclaving, keep the tubes in an incline overnight in the incubator, to check for position to have slant and butt. Cool the sterility and the remaining plates in the nutrient agar flask to 50 oC in a water bath. refrigerator for future use. Then pour the media in disposable Petri dishes to have 15 ml in each Petri dish. Put Preparation of Chocolate Agar: one plate in the incubator to check for 2. Heat blood agar to 70 oC, then pour in sterility. Keep the remaining plates and tubes Petri dishes as previously mentioned. in the refrigerator for future use.


EXERCISE 1 Media Preparation

Laboratory Report Name:_____________________ Date:______________________ Section:____________________

Questions 1.

Define autotrophs; heterotrophes; chemically defined medium, enriched medium.

2.

What is agar? What is its composition? Does it have any nutritive value?

3. What is the purpose of cooling nutrient agar to 50 oC before addition of blood?

4. Why is gelatin not used as a solidifying agent in nutrient agar?


5. What is the purpose of shaking bacterial culture during incubation?

6. How can you improve the resolution power of the microscope?

Laboratory Experiments in Microbiology

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UNIT 2 Preparation and Staining of Smears Objectives 1. Prepare and stain a smear. 2. Know the advantages of staining microorganisms. 3. Understand the basic mechanisms of staining; and the rational and procedures for different stains.

Introduction

when basic stain permeates the cell wall and adheres by weak ionic bonds to the negative charges of the bacterial cell. Staining procedures that use only one stain are called simple stains. A simple stain that stains the bacteria is a direct stain, and that stains the background, but leaves the bacteria unstained, is a negative stain. Simple stains can be used to determine cell morphology, size, and arrangement. A number of stains are now available to the microbiologist:

The morphology of bacteria can be tested by examining living, unstained microorganisms, or by examining dead cells that are stained with dyes. Living bacteria are almost colorless and lack sufficient contrast 1. with water to be clearly seen. Staining these organisms increases their contrast with their surroundings so that they become more visible. Stains unite chemically with the 2. bacterial protoplasm. The staining process will kill bacteria, and this may produce artifacts. Most stains used in microbiology are synthetic aniline (coal tar derivative) dyes 3. derived from benzene. The dyes are usually salts, although a few are bases or acids, composed of charged colored ions (chromatophores). If the chromatophore is a positive ion (e.g., methylene blue), the stain is considered a basic stain, if a negative ion, g. it is an acidic stain. Most bacteria are stained

Simple stains: a. Basic dyes. b. Acidic dyes.

Differential stains a. Gram stain. b. Zeihl Neelson stain (acid fast stain).

Structural stains a. b. c. d. e. f.

Flagella stain. Capsule stain. Endospore stain. Cell wall stain. Cell membrane stain. Granules stain. Nucleic acid stain.

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Faidy & Ali-Shtayeh


EXERCISE 2 Preparation and Fixation of a Bacterial Smear and Simple Staining Before bacteria can be stained, a smear must be made and fixed. A smear may be made by spreading a bacterial suspension on a clean slide and allowing it to air dry. The dry smear is passed several times through a flame of a Bunsen burner to heat fix the bacteria. This process may not kill all the bacteria. Alternatively, the dry smear can be placed on a hot plate at 60 oC for 10 minutes or chemically fixed. To chemically fix the bacteria, the smear is covered with 95 % methyl alcohol for 1 minute. Fixing denatures bacterial enzymes, preventing them from digesting cell parts, which causes the cell break (autolysis). Fixing also enhances the adherence of bacterial cells to the microscope slide.

Materials

3. 4. 5.

6. 7.

Methylene blue; wash bottles of distilled water; slide; inoculating loop.

Cultures (as assigned) Agar and broth cultures of: Escherichia coli Bacillus subtilis Staphylococcus epidermidis

Techniques / Equipment Required

8.

Compound light microscope; simple staining.

Procedure 1.

Clean your slide well with abrasive soap or cleanser; rinse and dry. Handle clean slides by the end edge. 2. With an inoculating loop, place a small drop of a broth culture on a clean slide. For culture on solid media, place 1

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or 2 loopfuls of distilled water in the center of clean slide, and thoroughly mix with it a small bit of a bacterial colony taken by the loop. Spread the drop on the slide with the ring to prepare a thin smear (Figure 2.1). Allow the smear to dry at room temperature. Fix the smear by passing the slide over the flame of a burner two or three times. Too much heating distorts the shape and structure of microorganisms. The purpose of heating is to kill the microorganisms and to cause them to adhere to the slide. Cover the smear with 95 % methyle alcohol for one minute, then allow the smear to air dry. Staining: Hold the slide with a clothespin, or place it on a staining rack. Cover the smear with methylene blue and leave for 30-60 seconds. Carefully wash the excess stain off with distilled water from a wash bottle. Allow water to run down tilted slide. Gently blot the smear with a paper towel or absorbent paper and allow to dry. Examine your stained smear microscopically using the low, high, and oil immersion objectives. Place oil directly on the smear; cover slips are not needed. Record your observations with labeled drawings. Blot the oil from the objective lens with lens paper, and return your microscope to its designated location. Remove the oil from the slide by blotting with a paper towel. Stained slides can be stored in a slide box.

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Figure 2.1. Preparing a bacterial smear.


EXERCISE 2

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Preparation and Fixation of a Bacterial Smear and Simple Staining

Purpose:_______________________________________________________________ ______________________________________________________________________ Data: Draw a few bacteria under 1000 X magnification Cultures Appearance Draw a bacteria (1000X)

few

Morphology, arrangement, relative size

Color

Questions: 1.

What is the purpose of fixing the smear?______________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 2. In heat fixing, what would happen if too much heat were applied? __________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________


3. Methylene blue can be prepared as a basic or an acidic stain. How would the pH affect the

staining bacteria?___________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 4. Bacteria can be seen without staining. Why then was recommending fixation and staining important for microbiology?_________________________________________________

______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________


EXERCISE 3 Negative Staining Technique The technique does not stain the bacteria nigrosin. For cultures on solid media add but stains the background. The bacteria will a loopful of distilled water and mix a appear clear against stained background. The small amount of the culture in the acidic stain and the bacteria both have nigrosin-water drop. Do not spread the negative charges. Hence, the stain does not drop or let dry. stain the bacteria because of ionic repulsion. 2. Using the end edge of another slide, No heat fixing or strong chemicals are used, spread the drop out to produce a smear the bacterial cells are less distorted than in (opaque black to gray in color). other staining procedures. This technique is 3. Let the smear air dry. Do not heat fix. very useful in situations where other staining 4. Examine the stained slides under the low, techniques do not clearly indicate bacterial high, and oil immersion objectives of a cell morphology or size. compound light microscope. Generally if a few short rods are seen with small cocci, the morphology is rod-shaped. The Materials apparent cocci are short rods viewed Nigrosin; clean slides; distilled water; from the end or products of the cell sterile toothpicks. division of small rods. 5. Place a small drop of nigrosin and a Cultures (as assigned) loopful of water at the end of a slide. Agar and broth cultures of: Scrape the base of your teeth and gums Escherichia coli with a sterile toothpick. Mix the Bacillus subtilis nigrosin-water with toothpick to obtain Staphylococcus epidermidis an emulsion of bacteria from your mouth. Follow steps 2 and 3 above to Techniques / Equipment Required complete the negative strain. Compound light microscope 6. Observe the stain and describe your results. Discard the toothpick in Procedure disinfectant. Wash your slides. Wipe oil 1. Place a small drop of nigrosin at the off your microscope and return it to its end of a clean slide. For cultures, mix a designated location. loopful of the culture into the drop of

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EXERCISE 3 Negative Staining Technique

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Purpose________________________________________________________________ ______________________________________________________________________ Data Draw a few bacteria under 1000 X magnification Cultures Appearance Draw a bacteria (1000X)

few


Color

Questions: 1.

Why is the size more accurate in a negative stain than in a simple stain?________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

2.

Could any dye used in place of nigrosin for negative staining?________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

3.

What type of dyes used for negative staining?_____________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________



EXERCISE 4

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Gram Staining The Gram stain is a very useful stain for Gram's iodine; ethyl alcohol; safranin; identifying and classifying bacteria. It is a diluted fuchsin; wash bottle of distilled differential stain that allows the classification water; slide. of bacteria as either gram- positive or gramnegative. This technique was discovered by Cultures (young cultures 18-24 hours): Hans Christian Gram in 1884 when he Escherichia coli attempted to stain cells and found that some Bacillus subtilis lost their color when excess stain was Staphylococcus epidermidis washed off. The staining technique consists of the following steps: Techniques / Equipment Required 1. Application of a primary stain (crystal Compound light microscope; simple violet). All bacteria are stained purple staining; smear preparation. by this stain. 2. Application of a mordant (Gram's Procedure iodine). The iodine combines with the 1. Prepare and fix smears. Clean the crystals violet in the cell to form a slide well and make 3 circles with a crystal violet-iodine complex (CV-I). marker on it. Label each circle for one of 3. Application of decolorizing agent (ethyl the cultures. alcohol or ethyl alcohol-acetone). The 2. Prepare a Gram stain (Figure 4.1). primary stain is washed out Use a clothespin or slide rack to hold the (decolorized) of some bacteria, while slide: others are unaffected. a. Cover the smear with crystal violet 4. Application of a secondary stain or and leave for 30 seconds. counter stain (safranin). This basic dye b. Wash the slide carefully with stains the decolorized bacteria red. distilled water from a wash bottle. Many variations of the stain have been c. Cover the smear with Gram's developed on the basis of the rate of iodine for 10 seconds. decolorization in different bacteria. Bacteria d. Wash off the iodine by tilting the that decolorize easily are referred to as gramslide and squirting water above the negative, whereas those that retain the smear so that the water runs over primary stain are called gram-positive. the smear. Bacteria stain differently because of e. Decolorize with acetone-alcohol (or chemical and physical differences in their 95 % ethyl alcohol). Allow the cell walls. The primary stain (crystal violet) decolorizing agent to run through the is picked up by the cell. Iodine reacts with smear until no large amounts of the dye to form CV-I, which combines with purple wash out (10-20 seconds). The peptidoglycan. In gram-negative cells, the degree of decolorizing depends on the decolorizing agent dissolves the outer thickness of the smear. Do not over lipopolysaccharide layer, and the CV-I decolorize. washes out through the thin layer of f. Immediately wash gently with peptidoglycan. distilled water. The gram stain is most consistant g. Add or dilute fuchsin safranin when done on young cultures of bacteria (< (counter stain) for 30 seconds. 24 hours old). Because Gram stain is usually h. Wash with distilled water and blot the the first step in identifying bacteria, the slide dry with a paper towel absorbent procedure should be memorized. paper. 3. Examine the stained slide using the low, Materials high- dry, and oil immersion objectives of a Gram staining reagents: crystal violet; microscope. Put the oil directly on the smear. Record your results.


Figure 4.1. The Gram stain.


EXERCISE 4 Gram Staining

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Purpose_______________________________________________________________

________________________________________________________________ Data Report, with drawings, Gram reaction, and morphology of the used bacterial cultures. Cultures Appearance Draw a bacteria (1000X)

few


Color Gram reaction

Questions 1.

Why did Gram positive bacteria retain the crystal violet iodine complex, while Gram negative bacteria are easily decolorized with acetone alcohol?

2.

What is the purpose of heating the prepared bacterial smear?


3. 3. Mention five bacterial genera, which can not be stained by Gram's stain.

4. Why it is advisable to do Gram's stain on fresh cultures?

5.

What is the function of iodine in Gram's stain? Can iodine be added before the primary stain in a Gram stain?___________________________________________________________

6.

If you Gram stain human cells, what would happen?

7.

If the cell wall of gram-positive bacteria is removed. What would happen after staining these bacteria with gram stain?______________________________________


EXERCISE 5

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Acid-Fast Staining The acid-fast stain is a differential stain. Mycobacterium tuberculosis, the causative agent of tuberculosis, was found by Etirlich (1882) to retain the primary stain even after washing with an acid-alcohol mixture. Most bacteria are decolorized by acid-alcohol, with only the families of Mycobacteriaceae and Nocardiaceae (Bergey's Manual, 1994) being acid-fast. This technique has a very important value as a diagnostic procedure because both Mycobacterium and Nocardia contain pathogenic species. Acid-fast organisms cell walls contain a wax-like lipid called mycolic acid, which renders the cell wall impermeable to most stain. The most widely used acid-fast stains today, are those that were developed by Franz Ziehl and Friedrich Neelsen and by J. J. Kinyoun. In the Ziehl-Neelsen procrdure, the smear is flooded with carbol fuchsin (a dark red dye containing 5 % phenol), which has a high affinity for a chemical component of the bacterial cell. The smear is heated to facilitate penetrating of the stain into the bacteria, washed with an acid-alcohol mixture that decolorizes most bacteria except acid-fast microbes, and methylene blue is then used as a counter stain to enable the observation of non- acid-fast organisms. In the Kinyoun modification (cold stain), the concentration of phenol and carbolfuchsin are increased so heating is not necessary. The mechanism of the acid-fast stain is probably the result of the relative solubility of carbol uchsin and the impermeability of the cell wall. Fuchsin is more soluble in carbolic acid (phenol) than in water, and phenol solubilizes more easily in lipids than

in acid-alcohol and will remain with the cell wall when washed with acid-alcohol.

Materials Acid-fast staining reagents: Kinyoun carbol fuchsin, acid-alcohol, methylene blue; wash bottle of distilled water; slide; prepared slide of acid fast sputum

Cultures (as assigned): Mycobacterium tuberculosis Escherichia coli Staphylococcus epidermides

Demonstration Slides (as assigned) Techniques /Equipment Required Compound light microscope; simple staining; smear preparation.

Procedure 1. Prepare heat-fixed smears of each culture. 2. Cover the smear with carbol fuchsin and leave for 5 minutes. 3. Gently wash the slide with distilled water from a wash bottle. Do not squirt directly onto the smear. 4. Wash the smear with decolorizer for 1 minute or until no more red color runs off when the slide is tipped. 5. Wash carefully with distilled water. 6. Counter stain for about 1 minute with methylene blue (Loefler's). 7. Wash with distilled water and blot dry with tissue paper. 8. Examine the acid-fast-stained slide microscopically (oil immersion lens) and record your observations. 9. Observe the demonstration slides.



EXERCISE 5

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Acid-Fast staining

Purpose___________________________________________________________

_________________________________________________________________ Data Draw a few bacteria under 1000 X magnification Cultures Appearance Draw a bacteria (1000X)

few

Morphology, arrangement, relative size Acid reaction

fast

Questions 1.

What is the decolorizing agent in the Gram stain? In the acid - fast stain? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 2. What diseases are diagnosed using the acid-fast procedure? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________


3. What is phenol (carbolic acid), and what is its usual application?

_____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 4.

What is the color of M. tuberculosis and S. aureus stained by acid fast stain? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 5. What is the purpose of heating in acid fast stain? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 6. What is the Gram reaction of M. tuberculosis?____________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 7. Is S. aureus decolorized by acid alcohol?_________________________________________ _____________________________________________________________________________ 8. What is the effect of overheating and dryness on the smear?__________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________


EXERCISE 6

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Structural Stains (Endospores, Capsule, and Flagella) The main objectives of this exercise are to learn preparation and interpretation of endospore, capsule, and flagella stains; recognition of the different types of flagellar arrangements; and identification of functions of endospore, capsules, and flagella. Structural stains are used to identify and study the structure of bacteria, especially endospores, capsule, and flagella.

background, leaving the capsules unstained (a 'negative' capsule stain). Capsules have an important role in the disease causing ability (virulence) of some bacteria. Because capsules prevent body's white blood cells to phagocytize efficiently, and disease occurs. When bacteria lack a capsule they are easily engulfed and are not virulent.

Endospores

Flagella

Endospores are formed by members of ten genera of bacteria (endospore-forming gram-positive rods and cocci), the most familiar of which are Bacillus and Clostridium. Endospores (resting bodies) do not metabolize and are resistant to heating, various chemicals, and many harsh

environmental conditions of (e. g., lack essential nutrients or water). Once an endospore forms in a cell, the cell will disintegrate. Endospores can remain dormant for long periods of time. However, they may return to their vegetative or growing states. Endospore formation and position is taxonomically helpful. They are impermeable to most stains, so heat is usually applied to drive the stain into the endospore. Once stained, endospores do not readily decolorize.

Capsule Many bacteria secrete chemicals that adhere to their surfaces, forming a visous coat. This coat is called capsule when it is round or oval in shape, and is called a slime layer when it is irregularly shaped and loosely bound to the bacterium. The ability to form capsules is determined genetically, but the size is influenced by the medium on which the bacterium is grown. Most capsules are composed of polysaccharides, which are water-soluble and uncharged (non-ionic). Hence, simple stains do not adhere to the capsule. Most capsule staining techniques stain the bacteria and the

Many bacteria are motile. Most motile bacteria possess flagella, but others forms of motility occur (gliding motion in Myxobacteria, and undulation using axial filaments in spirochetes). Flagella are thin proteinaceous structures that originate in the cytoplasm and project out from the cell wall. They are very fragile and are not visible with a light microscope. They can be stained after coating them with a mordant, which increases their diameter. The presence and location of flagella are helpful in the identification and classification of bacteria. Flagella are of two main types: peritichous (all around the bacterium) and polar (at one or both ends of the cell). Motility can be determined by observing hanging-drop or wet drop preparations of unstained bacteria, flagella stains, or inoculation of semisolid (soft) agar.

Materials Slides; coverslip; paper towels; wash bottle of distilled water; forceps; scalpel; endospore stain reagents: malachite green and safranin; capsule stain reagents: congo red, acid alcohol, and acid fuchsin; flagella stain reagents: flagella mordant and Ziehl's carbol fuchsin.

Cultures (as assigned): Endospore stain: Bacillus subtilis (24hour), B. subtilis (72-hour)


Capsule stain: Klebsiella pneumoniae, Streptococcus salivarius, Enterobacter aerogenes Flagella stain: Proteus vulgaris (18-h)

Capsule stain

1. Make a thick smear of bacteria in a loopful of Congo red. Air dry and then fix with acid-alcohol for 15 Demonstration Slides seconds. Endospore stain, capsule stain, flagella 2. Wash with distilled water and cover stain. the smear with acid fuchsin for 1 min. 3. Wash with distilled water, blot dry, Techniques / Equipment Required and examine microscopically. The Compound light microscope; smear bacteria will stain red, and the preparation; simple staining; negative capsule will be colorless against a staining dark blue background. Record your observations. Procedure 4. Observe the demonstration slides of Endospore stain capsule stains. 1. Prepare smears of cultures presented to you each on 1 or 2 slides, air dry, and heat fix. Flagella stain 2. Place small pieces of paper towel on 1. Using a scalpel and forceps, cut out a each smear to reduce evaporation of piece of agar on which Proteus is the stain. growing. Place the agar, culture-side 3. Flood the smears and paper with down, on a clean glass slide. malachite green; steam for 5 Carefully remove the agar with minutes. Add more stain as needed. forceps. Place the piece of agar in a Keep the smear wet. petri dish. 4. Remove the paper towel pieces and 2. Allow the slide to air dry. Do not discard carefully. Wash the stained heat fix. smears well with distilled water. 3. Cover the slide with flagella mordant 5. Counter stain with safranin for 30 and allow it to stand for 10 minutes. seconds. 4. Gently rinse off the stain with 6. Wash with distilled water and blot distilled water. dry. 5. Cover the slide with Ziehl's carbol 7. Examine under the microscope and fuchsin for 5 minutes. Rinse gently with record your observations. distilled water. 8. Observe the demonstration slides of 6. Allow the smear to air dry and bacterial endospores. examine microscopically for flagella. 7. Observe the demonstration slides for flagella.


EXERCISE 6 Structural Stains (Endospores, Capsule, and Flagella Stain)

16


Purpose:_______________________________________________________________ ______________________________________________________________________ Data Endospores Sketch results and label each diagram as to color. Label the vegetative cells and endospores. Demonstration slide

Bacteria Total magnification Endospore position

Capsules Sketch and label the capsules and bacterial cells. Demonstration slide

Bacteria Total magnification

Cell morphology

17 Faidy & Ali-Shtayeh Flagella

Sketch and label the flagella and bacteria. Demonstration slide

Bacteria Total magnification Flagella position

Questions 1-

How might a capsule contribute to pathogenicity?________________________________ ___________________________________________________________________________

2-

Of what advantage to Clostridium is an endospore?_______________________________ ___________________________________________________________________________

3-

Sketch each of the following flagellar arrangements: a. Monotrichous

b. Lophotrichous

c. Amophitrichous

d. Petritrichous

4-

How would appearance of 24- hour and 72- hour Bacillus cultures differ? How do you account for this difference?____________________________________________________


__________________________________________________________________________

18

_____________________________________________________________________________ _____________________________________________________________________________ 5-

Of what morphology are most bacteria possessing flagella?__________________________ _____________________________________________________________________________ Which morphology usually does not have flagella?__________________________________ _____________________________________________________________________________ _____________________________________________________________________________

6-

What prevents the cell from appearing green in the finished endospore stain? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

7-

What is the function of the mordent?____________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

8-

What is the difference between prokaryotes and eukaryotes flagella?___________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

9-

What is the purpose of slide cleaning for flagella staining? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________


10- Why it is difficult to see flagella in Gram's stain?

_____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 11- Why are spores resistant to dryness? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 12- What is the difference between endospores and spores of fungi? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ ____________________________________________________________________________ 13- What is the difference between E. coli and K. pneumoniae? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________


EXERCISE 7

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Morphology Unknown

Objectives Identification of the morphology and staining characteristics of unknown organism.

Introduction

24-h unknown slant culture of bacteria.

Techniques / Equipment Required Compound light microscope; hanging drop and wet mount; smear preparation; simple staining; negative staining; gram staining; acid-fast staining; and endospore, capsule, and flagella staining.

The first step in the identification of bacteria is differential staining. Morphology and structural characteristics obtained from microscopic examination are also useful for identification. Additional biochemical testing Procedure is needed to identify bacterial species. 1. Record the number of your You will be given an unknown culture unknown. of bacteria. Determine its morphology and 2. Determine the morphology, Gram structural characteristics. The culture reaction, and cellular arrangement of contains one species (rod or coccus) and is your unknown. Perform a Gram stain, less than 24 hours old. and if needed an endospore stain, acid fast stain, flagella stain, wet mount techniques, or capsule stain. Materials 3. Record your results in the laboratory Staining reagents report.

Cultures (as assigned):

Faidy & Ali-Shtayeh


EXERCISE 7 Morphology Unknown

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Purpose:_______________________________________________________________ ______________________________________________________________________ Data: Sketch of the Gram stain Unknown #:________________

Gram reaction:__________________ Morphology:______________________ Predominant arrangement:_______________ Endospore present?_____________________ Motile?_____________

Sketch if present

Capsule present?_______________________

Sketch if present

Motile?_____________ How did you determine motility?_______________________________ Acid fast?________________________________

Faidy & Ali-Shtayeh

Questions 1. Using Bergey's Manual and your textbook, place the following genera Bacillus, Corynebacterium, Escherechia, Mycobacterium, Neisseria, Sporosarcina, and Staphylococcus in this flow chart Gram Reaction -

+

Morphology

Morphology

Rod

Coccus

Rod

Coccus

Acid-fast -

Endospore +

-

+

Endospore -

+

2. Using your textbook and lab manual, fill in the morphology column in the table shown below. Then use the information to construct a flow chart of these bacteria Gram Morphology Motile Capsule Arrangement Endospore reaction Clostridium + + Pairs, chains + Enterobacter + Singly, pairs Klebsiella + Singly, pairs Lactobacillus + Rarely Chains Staphylococcus + Pairs, clusters Some Streptococcus + Pairs, chains species


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UNIT 3 Cultivation of Bacteria

Introduction In order to characterize and identify bacteria, they must be grown on culture media. Before attempting to culture bacteria, their nutritional requirements must be considered. Bacteria require sources of energy, carbon, nitrogen, minerals, and growth factors. A culture medium whose exact chemical composition is known is called chemically defined medium. Most bacteria are routinely grown on complex media (i. e., their composition varies slightly from batch to batch). Organic carbon, energy, and nitrogen sources are usually supplied by protein (e. g., meat

extracts, and partially digested proteins called peptones). Nutrient broth is a commonly used liquid complex medium. When agar is added, it becomes a solid medium called nutrient agar (Appendix A). Agar, an extract from marine red algae, has some unique properties that make it useful in culture media. Few microbes can degrade agar, so it remains solid during microbial growth. It liquefies at 100 oC and remains in a liquid state until cooled to 40 o C. The use of aseptic techniques is essential for the prevention of unwanted microorganisms in laboratory and medical procedures.



EXERCISE 8 Microbes in the Environment Objectives To describe colony morphology using accepted terms; and compare bacterial growth on solid and liquid culture media.

Introduction Microbes occupy ecological niches on all forms of life and in environment. Ubiquitous microorganisms are harmless, however, in microbiology, work must be done carefully to avoid contaminating sterile media and materials with these microbes. In this exercise, you will attempt to culture some microbes. Culture media can be prepared in various forms, depending on the desired use. Petri dishes containing solid media provide a large surface area for examination of colonies. The microbes may be inoculated, or intentionally introduced, onto nutrient agar or into nutrient broth. The bacteria that are inoculated into culture media increase in number during an incubation period. After suitable incubation, liquid media become turbid, or cloudy, due to bacterial growth. On solid media visible colonies are formed. A colony is a population of cells that arises from a single bacterial cell.

Materials Nutrient agar (NA) in Petri plates (4); Nutrient broth in culture tubes; sterile cotton swabs; sterile water.

Procedure 1.

Inoculate 2 NA plates from the environment by wetting a cotton swab in sterile water, swabbing the floor or workbench, and then swabbing the surface of the agar. Discard the swab in

2.

3. 4. 5.

the container of disinfectant. Inoculate one NA tube using a swab. After swabbing the agar surface, place the swab in the nutrient broth and leave it there during incubation. Incubate the plates and tube at the approximate temperature of the environment sampled. Inoculate 2 plates from your body. You could touch the plate with your fingers, place a hair on the agar, or obtain an inoculum by swabbing part of your body with a wet swab (step 1). Incubate the plates at 37 oC. Incubate all plates inverted, so water will condense in the lid instead of on the surface of the agar. Incubate all inoculated media until the next laboratory period. Observe and describe the resulting growth on the plates. Note colony morphology and describe it using the characteristics given in Figure 8.1. Determine the approximate number of each type of colony. When many colonies are present, record TNTC (too numerous to count) to designate number of colonies. 6. Describe the appearance of the nutrient broth (i. e., uniformly cloudy or turbid). Compare your nutrient broth with an uninoculated broth tube. Look for clumps of microbial calls, called flocculant. Is there a membrane (pellicle) across the surface of the broth? See whether microbial cells have settled on the bottom of the tube forming sediment. 7. Discard the plates properly by placing them in the biohazard bag for autoclaving.

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Figure 8.1Colony descriptions.


EXERCISE 8

39

Laboratory Report

Microbes in the Environment

Name:_____________________ Date:______________________ Section:____________________

Purpose________________________________________________________________ ______________________________________________________________________ Data Describe bacterial colonies using a separate line for each different appearing colony in the following table Colony Description Diameter Appearance Margin Elevation Color Number Area sampled Incubation _______oC for _______ days

Diameter

Appearance

Colony Description Margin Elevation

Color

Number

Diameter

Appearance


Color

Number

Diameter

Appearance


Color

Number

Area sampled Incubation _______oC for _______ days

Area sampled Incubation _______oC for _______ days

Area sampled Incubation _______oC for _______ days Description of the nutrient broth: Area sampled:______________________Incubated at_________ oC for _______________days Is it turbid?_________________


Is a flocculant present?_________________ Is a sediment present?_______________________ Is a pellicle present?_________________________ Has the color changed?________________ Conclusions:__________________________________________________________________ ____________________________________________________________________________

Questions 1.

What observations can you make about the variety and number of colonies observed on the different NA plates?______________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

2.

How can one tell whether or not there is bacterial growth in NB?__________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

3.

Of what advantage is a solid medium over a liquid medium?______________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

4.

What is the value of Petri plates in microbiology?______________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________


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EXERCISE 9 Transfer of Bacteria: Aseptic Technique Objectives 1. To provide rational for aseptic techniques; 2. To differentiate among broth culture, agar slant, and agar deep; 3. To learn how aseptically transfer bacteria from one form of culture medium to another.

Introduction In order to facilitate their identification and examine their growth and metabolism, bacteria must be cultured in the laboratory. Bacteria are inoculated into various forms of culture media in order to keep them alive and to study their growth. Inoculation must be done without introducing unwanted microbes, or contaminants, into the media. Aseptic technique is used in microbiology to exclude contaminants. All culture media are sterilized (i. e., rendered free of all life) before use. Sterilization is accomplished using an autoclave. Broth cultures provide large numbers of bacteria in a small space and are easily transported. Agar slants are test tubes containing solid culture media that were left at an angle while the agar solidified. Agar slants, like Petri plates, provide a solid growth surface, but slants are easier to store and transport than Petri plates. Agar is allowed to solidify in the bottom of a test tube to make an agar deep, which can be used to grow bacteria that prefer less oxygen than is present on the surface of the medium. semisolid agar deeps containing 0.5-0.7 % agar instead of the usual 1.5 % agar can be used to determine whether a bacterium is motile. Motile bacteria will move away from the point of inoculation, giving an inverted "Christmas tree" appearance. Transfer and inoculation are usually performed with a sterile, heat resistant, noncorroding nichrome wire attached to an insulated handle. When the end of the wire is bent into a loop, it is called inoculating loop;

when straight, it is an inoculating needle. For special purposes, cultures may be transferred with sterile cotton swabs, pipettes, glass rods, and syringes.

Materials NB in tubes (3); NA slants in tubes (3); Nutrient semisolid deeps in tubes (3); inoculating loop; iInoculating needle; Gram staining reagents.

Cultures (as assigned) Lactococcus lactis broth Pseudomonas aeruginosa broth Proteus vulgaris slant Staphylococcus aerues

Techniques / Equipment Required Compound light microscope; preparation; Gram staining.

smear

Procedure a. Nutrient broth (NB) 1. Sterilize the loop by holding the wire in a Bensen Burner flame. Heat to redness. 2. While holding the loop like a pencil, curl the little finger of the same hand around the cap of the broth culture. Gently pull the cap off the tube while turning the culture tube. If cotton stopper is used grasp the stopper with your finger. 3. Holding the tube at an angle (to minimize the amount of dust that could fall into it), pass the mouth of the tube through the flame. 4. Immerse the sterilized, cooled loop into the broth culture to obtain a loopful of culture. Remove the loop, and while holding the loop, flame the mouth of the tube and recap by turning the tube rack. 5. Remove the cap from a tube of sterile NB as previously described, and flame the mouth of the tube. Immerse the inoculating loop into the sterile broth and then withdraw it from the tube.


Flame the mouth and replace the cap. Return the tube in the test tube rack. 6. Reflame the loop until it is red and let it cool. 7. Label the inoculated loop with your name, the name of the bacteria, the date, and lab section.

b. NA slant Repeat steps 1-4, and inoculate the slant by moving the loop gently across the agar surface from the bottom of the slant to the top, being careful not to gouge the agar. Flame the mouth of the tube and replace the cap. Flame your loop and let it cool. Label the tube.

c. Nutrient semisolid agar deep Repeat steps 1-4 and using an inoculating needle inoculate the semisolid agar deep by plunging the needle straight down the middle of the deep, then pull out through the same stab. Flame the mouth of

Figure 9.1 Patterns of Growth on agar slants.

the tube and replace the cap. Flame your needle and let it cool. Label the tube. Using the other broth culture, inoculate a broth tube, agar slant, and semisolid agar deep, as described in steps a, b, & c, using your inoculating needle. To transfer Proteus vulgaris, flame your loop and allow it to cool. Flame the mouth of the tube and use your inoculating loop to carefully scrape a small amount of the culture from the agar. Flame tube's mouth and replace cap. Inoculate a broth and slant as described previously. Inoculate a semisolid agar deep with an inoculating needle as previously described. Label the inoculated tubes. Incubate all tubes at 35 oC until the next lab period. Record the appearance of each cultures referring to Figure 9.1. Make a smear of Lactococcus broth and slant cultures. Gram stain both smears and compare them.


EXERCISE 9 Transfer of Bacteria: Aseptic Techniques

43


Purpose________________________________________________________________ ______________________________________________________________________ Data Nutrient Broth Describe the nutrient broth cultures. Bacteria Is it turbid?

Is flocculant, pellicle, or sediment present?

Pigment?

Nutrient Agar Slant Sketch the appearance of each culture. Bacterium: _____________ Type of growth:___________

Bacterium:______________ Bacterium:______________ Bacterium:______________ Type of growth:__________ Type of growth:__________ Type of growth:__________


Nutrient Semisolid Agar Deep Show the location of bacterial growth and note any pigment formation.

Bacterium:________________ Bacteria:__________________ Bacteria:__________________ Comparison of Broth and Slant Cultures Broth Culture

Lactococcuc lactis Slant Culture

Gram stain Morphology Arrangement

Questions 1. Did growth occur at different levels in the agar deep? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 2. Were any of the bacteria growing in the semisolid agar deeps motile? Explain ____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 3. What other methods can be used to determine motility?______________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________


45

4. What is the primary use of slants? of deeps? of broth?________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 5. Can you determine whether a broth culture is pure by visually inspecting it without a microscope?_____________________ An agar deep culture?____________________________ An agar slant culture?___________________________________________________________ 6. When is a loop preferable for transferring the loop before use? After use?________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 7. What is the purpose of flaming the loop before use? After use? ____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 8. Why must the loop be cool before touching it to a culture? Should you set down to let it cool? How do you determine when it is cool? ____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 9. Why is aseptic technique important?______________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________



47

EXERCICE 10 Isolation of Bacteria by Dilution Technique Objectives 1.

Isolate bacteria by the streak plate and pour plate techniques. 2. Prepare and maintain a pure culture.

Introduction

than 25 colonies in inaccurate because a single contaminant causes at least a 4 % error. A plate with > 250 colonies is difficult to count. The number of bacteria in the original sample is calculated using the following equation:

Microbes in nature grow in Bacteria per ml = Number of colonies / environments that contain many different Dilution organisms. Mixed cultures are nevertheless of little use in studying microorganisms Dilution refers to the dilution of sample. because of the difficulty they present in For example if 50 colonies were present on determining which organism is responsible the 1: 1000 plate, the calculation would be: for any observed activity. A pure culture (one Bacteria per ml = 50 / 103 = 50 x 103 = containing a single kind of microbes, is 50,000 = 5 x 10 4 required in order to study concepts such as growth characteristics, pathogenicity, Materials metabolism, and antibiotic susceptibility. NA plates (2); melted NA in tubes (3); Since bacteria are too small to separate sterile Petri dishes (3); 250-ml beaker; directly, without sophisticated micromanisterile 1-ml pipettes (3); propipette or pulation equipment, indirect methods of pipette bulbe; NA slant (second period) separation must be used. There are three dilution methods commonly used for the isolation of bacteria: Cultures the streak plate, the spread plate, and the Mixed broth of bacteria, pour plate. In the streak technique the most Turbid nutrient broth (from part A of common isolation in use today, a loop is used this exercise). to streak the mixed sample several times over the surface of a solid culture medium in a Techniques / Equipment Required Petri plate. It is assumed that by streaking the Inoculating loop; compound light loop repeatedly over the agar surface, the microscope; aseptic technique; bacteria fall off the loop one by one and are pipetting; serial dilution techniques. ultimately distributed over the agar surface, where each cell develops into a colony. The spread plate and pour plate are Procedure quantitative methods that allow determinStreak Plate ation of the number of bacteria in a sample. 1. Label the bottoms of 2 NA plates to In the spread plate technique, a small amount correspond to the two broth cultures: of a previously diluted specimen is spread mixed culture and turbid broth, your over the surface of a solid medium using a name, lab section, and date. spreading rod. In the pour plate technique, a 2. Flame the inoculating loop to small amount of diluted sample is mixed redness, allow to cool, and aseptically with melted agar and poured into empty obtain a loopful of one broth culture. sterile Petri dishes. After incubation, bacteria 3. Streak the two plates: one of the growth is visible as colonies in and on the mixed culture provided and of the turbid agar of a pour plate. To determine the broth from environmental sample. Streak number of bacteria in the original sample, a may be done as follows (Figure 10.1 ): plate with 25-250 colonies is selected. Fewer


One. Lift one edge of the Petri plate cover and streak the first sector by making as many streaks as possible with out overlapping previous streaks. Hold the loops as you would hold a pencil and gently touch the surface of the agar. Two. Flame your loop and let it cool. Turn the plate so the next sector is on top. Streak through one area of the first sector, then streak a few times away from the first sector. Three. Flame your loop, turn the plate again, and streak through one area of the second sector, and streak the third sector. Four. Flame your loop, streak through one area of the third sector, being careful not to make additional contact with any streaks in the previous sectors. Flame your loop before setting it down. 4. Incubate the plates in an inverted position at a 35 oC until discrete, isolated colonies developed (usually 24 - 48 hours). 5. After incubation, record your results using proper terms to describe the colonies.

6.

Prepare a subculture of one colony. Flame your needle. Let it cool. Touch the center of a small isolated colony located on a streak line, and then aseptically streak a sterile NA slant. Incubate the slant at 35 oC until good growth is observed. Describe the growth pattern. Pour Plate (Figure 10.2) 1. Label the bottoms of 3 empty sterile Petri dishes with your name, lab section and date. Label one plate "undiluted", another "1:20", and third one, "1: 400". Place the labeled plates on your workbench right-side up. 2. Place three tubes of melted nutrient agar (19 ml/tube) in a water bath at 50 oC. 3. Select a mixed broth culture. Remove a pipette, attach a bulb, and aseptically transfer 1 ml to the third tube. Pour the contents of the second tube into dish 1:20. Mix the third tube and pour into the remaining dish 1:400. 4. Discard the tubes properly. Let the agar solidify in the plates, then incubate at 35 oC in an inverted position until growth is seen. 5. After incubation, count the number of colonies on the plates.


49

Figure 10.1. Streak plate technique for pure culture isolation of bacteria. The direction of streaking is indicated by the arrows.

Figure 10.2. Pour-plate technique. Bacteria are diluted through a series of tubes containing a 90ml of melted nutrient agar.



EXERCISE 10 Isolation of Techniques

Bacteria

51

Laboratory Report by

Dilution


Purpose________________________________________________________________ ______________________________________________________________________ Data Streak Plate Sketch the appearance of the streak plates

Mixed Culture

Turbid broth

Fill on the following table using colonies from the most isolated streak areas Colony Description (Each different appearing colony should be described) Culture Diameter Appearance Margin Elevation Color Mixed culture

Turbid broth

Number of different colonies in Mixed culture________________ Turbid broth ____________


Pour plate Dilution Number of colonies Undiluted 1:20 1:400 _______________ Bacteria / ml Calculate the number of bacteria / ml in the mixed culture. Show your caculations.

Subculture Describe the growth on your slant._________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

Questions 1. What is a contaminant? _______________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 2. How would you determine whether a colony was a contaminant on a streak plate? _________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 3. On a pour plate?______________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 4. What is mixed culture?________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________


53

EXERCISE 11 Special Media for Isolating Bacteria Objectives Differentiate between selective and differential media. Provide an application for enrichment and selective media.

Introduction

Materials Phenylethyl alcohol agar Petri plates; tryptose agar in Petri plates; Gram reagents.

Cultures (as assigned)

To help isolate organisms present in Staphylococcus epidermidis; limited, various enrichment and selective Escherichia coli; mixed culture of culturing methods may be used to either Staphylococcus & Escherechia. enhance the growth of some organisms or inhibit the growth of other organisms. Techniques / Equipment Required Selective media contain chemicals that Compound light microscope; smear prevent the growth of unwanted bacteria preparation, Gram staining; aseptic without inhibiting the growth of the desired technique; inoculating loop. organisms. Enrichment media contain chemicals that enhance the growth of the Procedure desired bacteria. Other bacteria will grow, 1. Using a marker divide each plate into but the growth of the desired bacteria will be 3 sections by labeling the bottoms. Label increased. one section on each plate for each Another category of media useful in culture. identifying bacteria are differential media, 2. Streak each culture on the agar. which contain various nutrients that allow us 3. Incubate the plates in inverted to destinguish one bacterium from another by position at 35 oC. Record the results after how they mtabolize or change the media. 24 hours, and after 48 hours, of Two culture media will be compared in incubation. Gram stain differentthis exercise: tryptose agar, and phenylethyl appearing colonies. alcohol agar (Appendix A).



EXERCISE 11

55

Laboratory Report

Special Media for Isolating Bacteria


Purpose________________________________________________________________ ______________________________________________________________________ Data

Organisms

Tryptose Agar 24 hr 48-72 hr

Phenylethyl alcohol Agar 24 hr 48-72 hr

Gram stain results

Questions 1. What do your results indicate? What difference did you see between 24 and 48 to 72 h? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 2. Which medium is selective? ____________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 3. How did the media composition affect bacterial growth?______________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 4.

How did the results observed on the phenylethyl alcohol agar plate correlate to the

Gram stain?____________________________________________________________ __________________________________________________________________________ _______________________________________________________________________


5. From your study in microbiology, mention the agents which are used for preparing media. _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 6.

Give examples for selective, enriched and differential media.

_____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________


57

EXERCISE 12 Cultivation of Anaerobic Bacteria Anaerobic agar plates should be prepared fresh and free of moisture. 2Fluid media are put in deep tubes and containing either fresh animal tissue (e.g., chopped cooked meat) or 0.1 % agar and a reducing agent such as sodium Introduction thioglycolate (e.g., fluid thioglycolate). Bacteria vary considerably in their Such tubes can be handled like aerobic requirement for gaseous oxygen. Many media, and within 15 mm of the surface bacteria are obligate aerobes, specifically exposed to air. Tubes should be prepared requiring oxygen as hydrogen acceptor; some fresh. Old tubes can be heated in a are facultative, able to live aerobically or boiling water bath followed by anaerobically; still others are obligate immediate cooling. anaerobes, requiring a substance other than oxygen as hydrogen and being sensitive to Materials oxygen inhibition. Obligate anaerobes will Petri plates with blood agar; culture not grow in the presence of oxygen and are tubes with fluid thioglycolate. killed by oxygen or toxic oxygen radicals, because usually they lack superoxide dismutase and catalase enzymes to get rid of Cultures oxygen radicals and hydrogen peroxide. Bacteroides fragilis Microaerophelic bacteria require the Pseudomonas aeroginosa presence of little amount of oxygen (5 %). Anaerobic conditions can be established Techniques / Equipment Required by one of the following means:Aseptic techniques; Gaspak jar. 1Agar plates or culture tubes are placed in an airtight jar from which air is Procedure removed and replaced by nitrogen and 10 1. The instructor will explain to the % CO2. This can be achieved by using a students the components and theory of pressure vaccum pump. Oxygen also can the Gaspak jar (Figure 12.1). Students be removed by other means, for example; will be provided with a culture of each of Gaspak jar. This jar has hydrogen Bacteroides fragilis, Pseudomonas envelop which generatess hydrogen upon aeruginosa and blood agar plates. Streak addition of water. It reacts with oxygen two blood agar plates with the previously of the atmosphere of the jar in the mentioned microorganisms. One plate presence of a pallidum catalyst. will be incubated in the Gaspak jar and Anaerobic conditions can be detected the other under aerobic conditions at 37 o by a colorless strip of methylene blue C for 48 hours. Read and record your placed in the jar. Anaerobes grow most result. readily on comples media such as 2. Students will be provided with fluid trypticase soy agar base, Schaedler thioglycolate tubes. Each of B. fragilis blood agar, Brucella agar, Brain Heart and P. aeruginosa will be inoculated into Infusion agar, and other's each highly a separate tube. Incubate under aerobic supplemented with hemin, vitamin K conditions at 37 oC for 48 hours. Read and blood. A selective complex medium and record your results. can be prepared from the above mentioned media with the addition of antibiotics such as Kanamycin.

Objectives

1. 2.

Cultivate anaerobic bacteria. Compare between aerobic anaerobic bacteria.


Figure 12.1 Gaspak jar (brewer jar).

60

Faidy & Ali-Shtayeh


EXERCISE 12 Cultivation of Anaerobic Bacteria

59


Purpose________________________________________________________________ ______________________________________________________________________ Data

Questions 1.

Explain why anaerobic bacteria can't tolerate the presence of oxygen? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

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Faidy & Ali-Shtayeh

2. What is the effect of boiling and immediate cooling of fluid thioglycolate medium? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 3. Explain the theory of Gaspak jar? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 4. From the literature, write a list containing the names, morphology and Gram's reaction of anaerobic bacteria. _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 5.

At least 2/3 of the tube is filled with anaerobic culture media. Why? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

Laboratory Experiments in Microbiology16 13. Action of Microorganisms on Carbohydrates

UNIT 4 Microbial Metabolism Introduction The different biochemical changes which result from the activities of bacteria are the most clear evidence of their presence. All cellular activities are mediated by enzymes. Micro-organisms produce many types of enzymes whose activities have been extensively used as a method for the biological differentiation of bacteria. The method of differentiation may consist of a test of presence of a reaction product which is characteristic of the type of hydrolysis affected by the organism. Exoenzymes are mainly hydrolytic enzymes (e. g., amylase which hydrolyzes starch into smaller carbohydrates) that leave

14. Action of Microorganisms on Protein, Amino Acids and Urea 15. Respiratory Enzymes 16. Rapid Identification Methods

the cell and break down by addition of water, large substrates into smaller components that can then be transported into the cell. Glucose, a monosaccharide, can be released by hydrolysis of starch. In the laboratory the presence of an exoenzyme is indicated by a change in the substrate outside of bacterial colony. Glucose can enter a bacterial cell and be catabolized (in the presence of molecular oxygen, O2) oxidatively, producing carbon dioxide and water. However, most bacteria ferment glucose without using oxygen. Fermentative catabolism does not require oxygen but may occur in the presence of oxygen.

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Laboratory Experiments in Microbiology16

EXERCISE 13 Action of Microorganisms on Carbohydrates Objectives 1.

Define the terms: carbohydrates; catabolism; hydrolytic enzymes; fermentation. 2. Differentiate between oxidative and fermentative catabolism. 3. Perform and interpret microbial starch hydrolysis and OF tests and fermentation of sugars. 4. Perform and interpret MR and V-P tests. Most microorganisms use various carbohydrates as their main source of energy. These include polysaccharides, disaccharides and monosacchorides.

1- Fermentation of sugars: Microorganisms usually attack carbohydrates present in the medium to obtain energy for their life processes. This results in what is called fermentation. Fermentation is entirely anaerobic; facultative and aerobic dissimulation is called respiration. Facultative and aerobic microorganisms usually are capable of anaerobic dissimulation of glucose but continue the degradation aerobically if oxygen is present. Among the common products of carbohydrates breakdown by microorganisms are organic acids (e.g. acetic acid and lactic acid) and gases (carbon dioxide and hydrogen). The types and proportion of products formed depend on the species of microorganisms and the carbohydrates being dissimilated. Some microorganisms are homofermentative and produce one product from the dissimilation of a carbohydrate. Others are heterofermentative and produce a variety of products during fermentation. Thus it is possible to use degradation of carbohydrates for the identification of microorganisms. Whether an organism is oxidative or fermentative can be determined by using OFglucose deeps (Rudolph Hughan Einar Leifson's OF basal media) with the desired carbohydrate added. OF medium is a nutrient

semisolidagar deep containing a high concentration of carbohydrate and a low concentration of peptone. The peptone will support the growth on nonoxidative nonfermentive bacteria. Two tubes are used: one open to the air and one sealed to keep air out. OF medium contains the indicator bromothymol blue, which turns yellow in the presence of acids, indicating catabolism of carbohydrates. Alkaline conditions, due to the use of peptone and not the carbohydrate, are indicated by the dark blue color. If the carbohydrate is metabolized in both tubes, fermentation has occurred. Some bacteria produce gases from the fermentation of carbohydrates. An organism that can only use the carbohydrate oxidatively will produce acid in the open tube only. OF medium is used to determine the carbohydrate catabolism of gram-negative bacteria.

Materials OF- glucose deeps; fermentation tubes with phenol red glucose or lactose or sucrose broth containing Drahm tube.

Cultures Escherichia coli (broth) Proteus mirabilis (broth) Alcaligenes odorans (broth)

Techniques / Equipment Required Aseptic technique; inoculating loop and needle.

Procedure 1. Students will be provided with the following tubes: a. Phenol red glucose broth containing Drahm tube. b. Phenol red lactose broth containing Drahm tube. c. Phenol red sucrose broth containing Drahm tube. d. Glucose oxidative fermentative agar (OF).

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gently. Add 4 drops of 0.3% creatine dissolved in 40% KOH solution. Shake the tube vigorously several times. Allow the tube to stand at room temperature for 10-30 minutes. If the reaction is positive a pink colour will develop.

2. Inoculate single tubes of phenol red broth with each of the following microorganisms: a. Escherichia coli. b. Proteus mirabilis. c. Alcaligenes odorans. 3. Inoculate two pairs of OF-Glucose tubes; one pair with E. coli and the other pair with Pseudomonas aeruginoosa. Cover one tube of each pair with sterile liquid parafin. 4. Incubate all tubes overnight at 37 oC. Read, record and interpret your results. Motility can also be ascertained from the OF tubes.

3- Hydrolysis of Starch: Many microorganisms are capable of elaborating alpha and beta amylase enzymes, which are capable of hydrolyzing starch to dextrin and maltose. The amylase are extracellular enzymes secreted by organisms to convert the indiffusible compound into diffusible substances. These substances are then capable of entering the cell where they are utilized. They are further hydrolyzed to E-glucose by the enzyme maltase. A qualitative test for the presence of starch is the appearance of a blue color upon addition of a solution of iodine. After starch is hydrolyzed, the product dextrins, maltose and glucose do not give this color reaction. This principle is used in testing for starch hydrolysis in this exercise.

2. Methyl - Red / Voges -Proskauer Test (MR - VP): These tests are used for the identification of gram-negative nonspore forming rods and some species of Bacillus. Some bacteria convert the metabolic intermediate, pyruvic acid, to neutral products and CO2. Acetylmethylcarbinol (CH3COCHOHCH3) is one such neutral product that is easily detected by the VP test. The MR test detects microorganisms that do not convert acidic products to neutral products and therefore remain acidic. The methyl red indicator changes to a red colour, which is a positive test.

Materials Petri plate containing nutrient starch agar; Grams iodine.

Cultures Bacillus subtilis Escherichia coli Pseudomonas aerugenosa

Materials Tubes with MR-VP medium; methyl red indicator; 5 % alphanaphthol in absolute ethanol; 0.3 % creatine in 40 % KOH solution.

Techniques / Equipment Required Inoculating loop and needle; aseptic technique.

Culture Escherichia coli Enterobacter aerogenes

Procedure 1.

Using a marker, divide the starch agar into three sectors by labeling the bottom of Procedure the plate. Inoculate two tubes of MR-VP medium 2. Streak a short single line of Bacillus, with E. coli and two other tubes with Escherichia and Peudomonas. o Enterobacter aerogenes. Incubate at 37 C 3. Incubate, inverted at 35 oC, for 24 hours. for 4 days. After growth occurs, the plate may be refrigerated at 5 oC until the next lab period. A. MR reaction 4. Record any bacterial growth then flood Add 5 drops of methyl red indicator to the plate with Gram's iodine. Areas of starch each tube and observe color change. hydrolysis will appear clear, while unchanged starch will stain dare blue. B. VP - reaction Record your results. To 1 ml of culture add 0.6 ml of 5% alpha-naphthol in absolute ethanol. Shake


4- Citrate Utilization: This test is used to identify microorganisms that utilize sodium citrate as their sole carbon source and inorganic ammonium salts as their sole nitrogen source. Growth on citrate agar medium usually turns thymol blue indicator from green to blue. However, some microorganisms may show growth and colony formation while the indicator remains unchanged.

Materials Tubes of Simmons citrate agar

Cultures Escherichia coli Enterobater aerogenes

Procedure Inoculate a tube of Simmons citrate agar with a culture of E. coli. Inoculate another tube with a culture of Enterobacter aerogenes. Incubate at 37 oC for 24 hr. Examine the tubes for the presence of growth and color changes of the indicator.

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EXERCISE 13

Laboratory Report

Action of Microorganisms Carbohydrates

on

Name:____________________ Date:_____________________ Section:___________________

Purpose_______________________________________________________________ ______________________________________________________________________ Data 1. Fermentation Tubes Color of uninoculated medium:________________________________

Organism Escherichia coli

Glucose Growth Color Acid

Gas

Carbohydrate Lactose Growth Color Acid

Gas

Sucrose Growth Color Acid

Gas

24 hr 48 hr

Alcaligenes faecalis 24 hr 48 hr Proteus vulgaris

24 hr 48 hr

Results of OF-Glucose tubes: __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ ______________________________________________________________________________

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2. MRVP Tests MR Organism

Growth

Color

V-P + or -

Color

+ or -

Escherichia coli Enterobacter aeroginenes Controls

-

-

-

Questions 1.

Why are fermentation tubes evaluated at 24 and 48 hours?_____________________________ __________________________________________________________________________________ What would happen if an organism used up all the carbohydrates in a fermentation tube?___________ __________________________________________________________________________________ What would it use for energy___________________________________ What color would the indicator be then____________________________________________________________________

2.

If an organism oxidatively metabolizes glucose, what result will occur in the fermentation tubes? __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________

3.

Were these media differential or selective?_________________________________________

4.

Could an organism be MR and V-P positive? Explain.________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________

5.

Could an organism be a fermenter and be MR and V-P negative? Explain.________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________

6.

How could you determine whether a bacterium fermented the following carbohydrates: mannitol, sorbitol, adonitol, or arabinose?____________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________


7. If a bacterium cannot ferment glucose, why not test its ability to ferment other carbohydrates? __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ 8. What is the purpose of Drahm tubes?__________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ 9. How can you interpret the results of (OF) medium?______________________________________ __________________________________________________________________________________ __________________________________________________________________________________ 10. Some microorganisms use carbohydrates without forming detectable levels of acids. How can you determine that carbohydrates were used?______________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ 11. Are amylases exo- or endoensymes? __________________________________________________________________________________ __________________________________________________________________________________ 12. What is the advantage of these enzymes to microorganisms? __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ 13. What is the advantage of using semisolid agar in OF medium?_____________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________

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EXERCISE 14

17

Action of Microorganisms on Proteins, Amino Acids and Urea 1.

Proteus vulgaris

Indole Production

Some microorganisms are able to hydrolyze the amino acid tryptophane to Procedure indole and serine. The indole test is 1. Stab the butt and streak the slant of important because some microorganisms lysine agar with a culture of E. form indole, and it can be detected aerogenes. Stab and streak a second tube chemically. Tryptophane is found in a wide of the same medium with a culture of P. variety of proteins, however the easier digest vulgaris. broth tryptone, is usually used as an abundant 2. Incubate at 37 oC for 48 hours. source of tryptophane. 3. Read and record your results after incubation. If there is decarboxylation no change in the color of the medium will Materials occur. The presence of a red ring Tubes of 1% tryptone broth; Kovac's between the slant and butt indicates reagent. positive deamination.

Cultures

3. Hydrogen Sulfide Production:

Escherichia coli Enterobater aerogenes

Certain microorganisms act on protein with the production of H2S gas. This action is helpful in the characterization of these microorganisms. The sulfur-containing amino acids methionine, cystine and cysteine are dissimilated to yield H2S.

Procedure 1.

Inoculate two tubes of 1% tryptone broth, one with E. Coli and the other with E. aerogenes. 2. Incubate at 37 oC overnight. 3. Test each tube for the presence of indole using the Kovac's reagent Add 0.3 ml of this reagent to the tryptone broth, shake well. The alcohol layer will separate from the aqueous layer upon standing. The appearance of a red color in the alcohol layer indicates the presence of indole.

Materials Triple sugar iron (TSI) agar slants (2).

Cultures Escherichia coli Proteus vulgaris

Procedure

Inoculate one slanted tube of triple sugar iron agar (TSI) with Proteus vulgaris and a second tube with E. coli. The decarboxylation of an amino acid is Stab the butt and streak the slant. the splitting of its carboxyl group to yield an 2. Incubate at 37 oC for 48 hours. amine and CO2 (Figure 14.1 a). 3. After incubation examine the tubes The deamination of an amino acid is the for any blackening of the medium. TSI is enzymatic splitting of the amino group to also used for characterization of bacteria yield NH3 and usually the corresponding by looking for acid and gas production in the slant and butt. This is due to the keto acid (Figure 14.1 b). fermentation of sugars (lactose, sucrose, and glucose) present in the medium. Materials Acid production is detected from the Lysine agar slant (2) change in the color of medium from red to yellow. Gas production is detected Cultures from the presence of cracking in the Enterobacter aerogenes medium. Read and report your results.

2. Decarboxylation and Deamination of Amino Acids:

1.

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17

4. Gelatin Hydrolysis:

Gelatin is obtained from hydrolyzing collagen, a component of connective tissue and tendons of animals. Some microorganisms produce proteolytic enzymes (gelatinases) which can hydrolyze gelatin. Hydrolysis of gelatin results in the formation of smaller molecules so that liquification occurs.

5. Hydrolysis of Urea: Some microorganisms possess the enzyme urease which breakdown urea into carbon dioxide and ammonia. The alkaline reaction of the medium will cause change of color in the indicator (phenol red) from yellow to red.

Materials Urea agar slant (2)

Materials Nutrient gelatine slants (2)

Cultures Proteus vulgaris Escherichia coli

Cultures Bacillus subtilis Escherichia coli

Procedure 1.

Procedure 1.

Stab one tube of nutrient gelatin with E. coli. Stab a second tube of the same medium with. B. subtilis. 2. Incubate at 37 oC overnight. 3. After incubation keep the tubes in an ice bath. Water solutions of gelatin at the concentrations used in this exercise are liquid at room temperature, but solidify at lower temperatures.

Streak a slanted tube of urea agar medium with a loopful of culture of culture of P. vulgaris. Streak a second tube of the same medium with a culture of E. coli. 2. Incubate at 37 oC for 24 hours. 3. Start to observe the change in the color of the medium after 2-4 hours. Record your results.

(a) R - CH - COOH 

NH2 an amino acid.

Decarboxylase ---------------------------------

R - C - CH2 + CO2 an amine

(b) R - CH - COOH Deaminase 

NH2 an amino acid.

----------------------------

+ H2O

O  R - C - COOH + NH3 a keto acid

ammonia

Figure 14.1 Decarboxylation and deamination of an amino acid. a. Decarboxylation, b. Deamination of an amino acid.


EXERCISE 14

37

Laboratory Report Name:_____________________ Date:______________________ Section:___________________

Action of Microorganisms on Protein, Amino Acids and Urea

Purpose____________________________________________________________________ ___________________________________________________________________________ Data 1. Indole Production Results ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ 2. 3.

Dexarboxylation and Deamination of Amino Acids Hydrogen Salfide Production Results __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ Fill in the following table. Test Phenylalanine deaminase

Growth Organism

Hydrogen sulfied

Original color:

Original color:

____________

____________

Color

Reaction

Growth

Color

(+ or -)

(+ or -)

Escherichia coli Pseudomonas aeruginosa

Not tested

Proteus vulgaris Enterobacter aerogenes

Reaction

Not tested

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4-5 Gelatine and Urea Hydrolysis Fill in this table and the one on the next page. Controls Appearance of uninoculated tube Gelatin Urea agar

Diagram the appearance of the gelatin

Bacillus subtilis

Escherichia coli Results

Test Gelatine hydrolysis at ________ days growth Hydrolysis? at _________ days growth Urea agar growth hydrolysis?

Bacillus subtilis

Escherichia coli


37

Questions 1.

Nutrient gelatine can be incubated at 35 oC. What would have to be done to determine

hydrolysis after incubation at 35 oC?________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ 2.

What happens to milk when the suspended (colloidal) proteins are hydrolyzed?____________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________

3.

What is the source of urea in the body?____________________________________________ __________________________________________________________________________________ __________________________________________________________________________________

76 Faidy & Ali-Shtayeh 6. Urea hydrolysis is recommended as a test for the diagnosis of Proteus species. Why? __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________

7. What does blackening of lysine iron agar indicate? __________________________________________________________________________________ __________________________________________________________________________________

8. What pH would take place with deamination? Would it be as much of a pH change as occurs with decarboxylation? __________________________________________________________________________________ __________________________________________________________________________________ 9. What is the chemical nature of gelatin? Why is gelatin can't be used as a solidifying agent in microorganisms media? __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________


EXERCISE 15 Respiratory Enzymes Objectives 1.

Compare and contrast the following terms: aerobic respiration, anaerobic respiration, and fermentation. 2. Perform catalase and oxidase tests.

Introduction Catalase Activity: Many microorganisms are obligate aerobes, requiring oxygen as hydrogen receptor, some are facultative, able to live aerobically or inaerobically and others are obligate anearobes, requiring a substance other than oxygen as hydrogen acceptor and being sensitive to oxygen inhibition. The toxicity of O2 results from its reduction by enzymes in the cell (such as flavoprotiens) to hydrogen peroxide and by ferrous ion to the even more free radical, superoxide (O2). Aerobes and aerotolerant anaerobes are protected from these products by the presence of supereoxide dismutase, an enzyme that catalyzes the reaction. 2O2 +2H ------- O2 + H2O2 And by the presence of catalase, an enzyme that catalyzes the reaction. catalase 2H2O2 + 2H --------- 2H2O + O2 Lactic acid bacteria that do not contain catalase, relies instead on peroxidases, which reduce H2O2 to H2O. All strict anaerobes lack both superoxide disutase and catalase.

Materials H2O2 (3%)

Cultures Streptococcus fecalis Staphylococcus aureus

Techniques / Equipment Required Aseptic technique; inoculating loop.

Procedure Catalase Test: 1. Students will be provided with cultures of Streptococcus fecalis and Staphylococcus aureus. 2. Two drops of 3% H2O2 are placed each at the periphery of a slide. 3. Take part of a colony with the loop from each microorganism and emulusify it with H2O2 on the slide. 4. The immediate formation of bubbles (The release of O2) indicates positive test. Record your results. 5. The test can also be performed by pouring H2O2 solution over a heavy growth of the bacteria on an agar slant or broth.

Oxidase Test: This test is used to differentiate microorganisms which oxidize some aromatic amines, for example, tetramethyl p-phenyline diamine dihydrochloride. As a result of oxidation, coloured end products are produced. This oxidation correlates with the cytochrome oxidase activity of some bacteria, including the genera Pseudomonas and Neisseria. The test is also important for the identification of the Enterobacteriaceae which are oxidase negative.

Materials NA plates; 1% tetranethyl P-phenyline diamine dihydrochloride; slide.

Cultures Pseudomonas aeuriginosa Eschirechia coli

Techniques / Equipment Required Aseptic technique; inoculating loop.

Procedure Students will be provided with nutrient agar plates of P. aeruginosa and E. coli.

33

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1. Flood each plate with freshly prepared 1% tetranethyl P-phenyline diamine dihydrochloride (oxidase reagent). 2. Note the formation of a violet color immediately upon addition of the reagent to the P. aeruginosa plate, while no change in

the color of E. coli colonies. The violet colors will turn into black later on due to the death of bacteria. 3. The test could be repeated using ready made oxidase reagent discs.


EXERCISE 15

37

Laboratory Report Name:_______________________ Date:________________________ Section:______________________

Respiratory Enzymes

Purpose____________________________________________________________________ __________________________________________________________________ Data Oxidase test Organism Escherichia coli

Color after reagent

Oxidase reaction

Color after reagent

Oxidase reaction

Pseudomonas aeruginosa

Catalase test Organism Staphylococcus aureus Streptococcus fecalis

Questions 1.

Why it is not advisable to do the catalase test on blood agar culture?___________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ ______________________________________________________________

2.

What is the function of superoxide dismutase?____________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ ______________________________________________________________ 3. The catalase test is valuable for the differentiation of

the Gram positive cocci

_________________________ And the gram positive cocci,______________________ 4. Why must the oxidase reaction be read immediately?________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ _________________________________________________________

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5. Mention some oxidase positive bacteria. __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ _________________________________________________________ 6. What other enzymes can give a positive oxidase test? __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ _________________________________________________________ 6.

Differentiate between aerobic and anaerobic respiration._____________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ _________________________________________________________

7.

Differentiate between fermentation and anaerobic respiration.________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ _________________________________________________________

8.

Why does hydrogen peroxide bubbles when it is poured on a skin cut? __________________________________________________________________________________ __________________________________________________________________________________ __________________________________________________________________________________ ___________________________________________________________________________________________________________________________________________

18


EXERCISE 16 Rapid Identification Methods Objectives 1. Evaluate three methods of identifying enterics. 2. Know the advantages of the "systems approach" over conventional tube methods.

Introduction Clinical microbiology laboratories must identify bacteria quickly and accurately. Accuracy is improved by using a series of standardized tests. The IMViC tests were developed as a means of separating enterics, particularly the coliforms, using a standard combination of four tests. Each capital letter in IMViC represents a test; the i is added for easier pronunciation. The tests are: I, for indole production from tryptophan; M, for methyl red rest for acid production from glucose; V, for the Vogas-Proskauer test for production of acetoin from glucose; C, for the utilization of citrate as the sole carbon source. The Simmons citrate agar used in this exercise contains the indicator bromthymol blue. Citric acid will be the only source of carbon; therefore, only organisms capable of utilizing citric acid as a source of carbon will grow. When the citric acid is metabolized, an excess of sodium and ammonium ions results, and the indicator turns from green to blue, indicating alkaline conditions. Although variation among strains does exist. IMViC reactions for selected species of entirecs are given in Table 16.1. Rapid identification systems provide a large number of results from one inoculation. Examples are Enterotube II and API 20E for identifying oxidasenegative, gram-positive bacteria belonging to the family Enterobacteriacea. Enterotube II is divided into twelve compartments, each containing a different substrate in agar. No culturing beyond the initial isolation is necessary with these systems. Comparisons between these rapid identification methods show that they are as accurate as conventional test tube method.

Table 16.1 IMViC reactions for selected species enterics Species

Indole Methyl red Voges-Pros- Citrate kauer + (v) + -

Escherichia coli Cirobacter + freundii Enterobacter aerogenes Enterobacter aerogenes Serratia + or marcescens -* Proteus + + vulgaris Proteus + mirabilis v= variable * Majority of strains give + results ** Majority of strains give - results

-

+

+

+

+

+

+

+

-

- (v)

- or + **

+ (v)

Computerized analysis of test results increases accuracy because each test is given a point value. Tests that are more important than others get more points. The IMViC tests are four tests with equal value. As commercial identification systems are developed they can provide greater standardization in identification because they overcome the limitations of hunting through a key, differences in media preparation, and evaluation of tests within a laboratory or between different laboratories. They are also time-, cost-, and labo-saving.

Materials Petri plates containing nutrient agar; IMViC tests and reagents; enterotube II and reagents; tube containing 5.0 ml sterile saline; 5-ml pipette; oxidase reagent; mineral oil; sterile Pasteur pipette; tryptone broth tubes; Simmons citrate agar plates; MRVP broth in tubes; Kovacs reagent.

Cultures Unknown enteric #_________

Techniques / Equipment Required Inoculating loop and needle; Aseptic technique; MRVP tests; protein catabolism; respiration.

82 Faidy & Ali-Shtayeh Procedure

4.

Isolation 1.

Streak the nutrient agar plate with your unknown for isolation and to determine purity of the culture. Incubate inverted at 35 o C for 24 to 48 hours. Record the appearance of the colonies. 2. Determine the oxidase reaction of one of the colonies remaining on the plate. Record your results.

5. 6.

IMViC Tests 1.

Inoculate tubes of tryptone broth (indole test), MRVP broths, and Simmons citrate agar with your unknown. 2. Incubate the tubes at 35 oC for 48 hours or longer; perform the appropriate tests, and record your results.

Enterotube II

Remove both caps from the Enterotube II. One end of the wire is straight and is used to pick up the inoculum; the end of the wire is the handle. 2. Inoculate the Enterotube  II by holding the bent end of the wire and twisting; then withdraw the needle through all twelve compartments using a turning motion. 3. Reinsert the needle into the Enterotube II, using a turning motion, through all twelve compartments until the notch on the wire is aligned with the opening of the tube. The tip of the wire should be visible in the citrate compartment. Break the needle at the notch by bending. The portion of the needle remaining in the tube maintains anaerobic conditions necessary for fermentation, production of gas, and decarboxylation. 1.

7.

8.

Punch holes with the broken-off wire through the foil covering the air inlets of the last eight compartments (adonitol through citrate) to provide aerobic conditions. Replace the caps on both ends of the tube. Incubate the tube lying on its flat surface at 35 oC for 24 hours. Interpret and record all reactions (see Table X) in the laboratory report. Read all other tests before the indole and V-P tests, which follow: Indole test. Place the EnterotubeII horizentally and melt a small hole in the plastic film covring the H2S/ indole compartment using a warm inoculating loop. Add 1 to 2 drops of Kovacs reagent, and allow the reagent to contact the agar surface. A positive test is indicated by a red color within 10 seconds. V-P test. Add 2 drops of 20% KOH containing 5% -nephthol to the V-P compartment. A positive test is indicated by the development of a red color within 20 minutes. Indicate each positive reaction by circuling the number appearing below the appropriate compartment of the Enterotube II outlined in the Laboratory Report. Add the circled numbers only within each bracketed section and enter this sum in the space provided below the arrow. Note that the V-P test is used as a confirmatory test only. If available, read the five numbers in these spaces across as a five-digit number in the Computer Coding and Identification System. Dispose of the EnterotubeII by placing in the autoclave basket.


Table 16.2 . Enterotube II Biochemical Reactions Test GLU GAS LYS ORN H2S IND ADON LAC ARAB SORB V-P DUL PA UREA CIT

Indicator changed To

Comments

From

Acid from glucose Gas produced from fermentation of glucose trapped in this compartment, causing separation of the wax Lysine decarboxylase Ornithine decarboxylase Ferrous ion reacts with sulfide ions forming a black precipitate Kovacs reagent is added to the H2s/IND compartment to detect indole Adonitol fermentation Lactose fermentation Arabinose fermentation Sorbitol fermentation Voges - Proskauer reagents detect acetoin Dulcitol fermentation Phenylpyruvic acid released from phenylalanine after its deamination combines with iron salts to form a black precipitate Ammonia changes the pH of the medium Citric acid used as a carbon source

Red

Yellow

Yellow Yellow

Purple Purple

Beige

Red

Red Red Red Red Beige Green

Yellow Yellow Yellow Yellow Red Yellow

Yellow Green

Pink Blue

Source: Becton Dickinson Microbiology Systems, Cockeysville, Md 21030.

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Laboratory Report

EXERCISE 16 Rapid Identification Methods

Name:____________________ Date:_____________________ Section:___________________

Purpose________________________________________________________________ ______________________________________________________________________ Data Unknown #______________ Appearance on nutrient agar:____________________________ Oxidase reaction:_____________________________________

IMViC Indicate positive (+) and negative (-) results for each test. Indole:_________________________ Methyl red:___________________ V-P:________________ Citrate:__________________

Enterotube II Circle the number corresponding to each positive reaction below the appropriate compartment.

Questions 1.

What species was identified in unknown # _______________________? By the Enterotube II__________________________________________________________

Faidy & Ali-Shtayeh


UNIT 5 Quantitative Measurements of Bacterial Growth reported as cfu (colony forming unit) per milliliter rather than number of bacteria per Growth in unicellular organisms leads milliliter. to an increase in the number of individuals making up a population or culture. There is an increase in all components of an organism. Cell manipulation is a Exercise 17: Quantitative consequence of growth. The increase in size Measurements of Bacterial which results when a cell takes up water or Growth deposits lipid or polysaccharides is not a true growth. Objectives Growth can be determined by numerous 1. Measure bacterial growth techniques based on one of the following types turbidometrically. of measurments: 2. Identify the four phases of typical 1. Cell count: directly by microscopy growth curve. or an electronic particle counter or 3. Determine the effect of temperature indirectly by a colony count. on bacterial growth. 2. Cell mass: directly by weighing or measuring of cell nitrogen or Materials indirectly by turbidity. Petri plates with NA; spectrophotometer 3. Cell activity: indirectly by relating tubes (2); flask containing nutrient broth the degree of biochemical activity to + 1.5 % NaCl; sterile 1 ml, 5 ml, and 10 the size of the population. ml pippetes; tubes with NA; Petri plates. In studies of microbial genetics or the inactivation of cells, cell count is the significant quantity. In studies on microbial Cultures biochemistry or nutrition, cell mass is the Escherichia coli significant quantity.

Introduction

The viable cell count is usually considered the measure of cell concentration, however, for many purposes the turbidity of a culture, measured by photoelectric means, may be related to the viable count in the form of a standard curve. Students will do the plate count technique using either the streak plate or pour plate. The technique is based on the principal that each viable organism will grow into one colony. The bacterial suspension used should be homogeneous and no aggregates of cells are present. If the cells have a tendency to aggregate, e.g., cocci in clusters (Staphyloccoi) or cocci in chains (Streptococci), the resulting counts will be lower than the number of individual cells, since each aggregate will produce only one colony. For this reason the counts are

Techniques / Equipment Required Aseptic techniques; spectrophotometer; inocilating loop; pipetting.

Procedure A.

Streak Plate: 1. Students will be provided with nutrient agar plates, sterile saline tubes each contains 9-ml saline, sterile1 ml pipettes, bent glass rods in beakers of absolute ethanol, and a broth culture of E. coli. 2. Prepare ten fold dilutions of E. coli saline as follows: under aseptic conditions add 1 ml from E. coli broth culture to 9 ml sterile saline tube to make 10 -1 dilution; mix. Transfer 1 ml from the mixture to another 9-ml sterile saline tube to make a 10 -2 dilution. Repeat this

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step to make 10 -3, 10 -4 and 10 dilutions. 3. Transfer under aseptic conditions 0.1-ml broth culture suspension from each dilution to a nutrient agar plate. Use three plates for each dilution. Spread the suspension with the alcohol flamed glass rod. 4. Incubate plates aerobically overnight at 37 oC. 5. Choose plates containing 25-250 colonies. Count the number of colonies of three plates of the dilution. Find the cfu/ml according to the following formula:

-6

2. Pour the suspension in Petri plates, leave to solidify. Incubate overnight at 37 oC. 3. Find the cfu/ml as mentioned above.

C. Turbidimetric Estimation Bacterial Growth:

of

1. Each group of students will be provided with a flask of a 12-14 hour culture of E. coli growing in nutrient broth. The flask will be in a shaker water bath at 37 oC. 2. Read and record the absorbance of the culture at 650 nm using the spectrophotometer provided, against nutrient broth blank. Find the viable CFU/ml= Average number of colonies X count using the streak plate mentioned dilution X10. above. Record your results for discussion. 3. Continue taking absorbance reading and viable counts at 15 minutes intervals until the absorbance no longer increases. B. Pour Plate: After each reading reincubate the culture. 1. Prepare 10 fold dilutions of E. coli 4. Plot the absorbance readings versus culture in 9-ml sterile liquid nutrient viable count on semilog paper. agar tubes (kept at 45 oC water bath) as mentioned above.


EXERCISE 17 Quantitative Measurements of Bacterial Growth

89


Purpose________________________________________________________________ ______________________________________________________________________ Data

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Questions 1.

How can you use the curve of absorbance versus viable count for the estimation of E. coli viable count in future exercises? Can you estimate the viable count of other bacteria using the same curve?________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

2.

Can you measure all kinds of microbes using the colony count technique? Why not? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

3.

What are the differences between the turbidimetric method and the colony plate method? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 4. Why do we prefer to use cfu rather than bacterial cell in reporting number of bacteria?______________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________


UNIT 6

18. Sterilization and Disinfection

Control of Microbial Growth

19. Antimicrobial Agents Susceptibility

19

This unit includes exercises on the effectiveness of different physical and chemical methods for killing microbes.

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EXERCISE 18 Sterilization and Disinfection Objectives Examine the effect of dry heat, moist heat, ultraviolet radiation, antiseptics, and disinfectants on bacteria.

Introduction Sterilization is the process of killing all living microorganisms in or on a material. Disinfection is the process of killing pathogenic microorganisms in or on a material. Usually disinfection is applied to nonliving objects. Bacteriostatic agent is that which inhibits the multiplication of microorganisms. Growth is resumed when this agent is removed or diluted. Bactericidal agent is that which kills microorganisms. Growth is not resumed when this agent is removed or diluted. Antiseptics are inhibitory chemicals which are used to cleanse the skin. These agents are usually bacteriostatic Disinfectants are bactericidal. Sterile - free of life of every kind. Septic - characterized by the presence of pathogenic microbes in living tissue. Aseptic - characterized by the absence of pathogenic microorganisms.

Agents of Sterilization and Disinfection: 1.Physical Agents: A. Heat: Heat is the simplest means of sterilizing materials, provided that the material is itself resistant to heat damage.

a. Dry heat: Flamming of loops and needles. Hot air oven - 160 oC for 1-2 hours. It is used for sterilization of glassware and surgical equipments.

b.Moist Heat: Boiling - surgical equipment. Autoclaving - 121 oC for 15 minutes under 15/un2 pressure.

Sterilization of surgical equipment, culture media, glassware and cloth.

B. Radiation: Ultraviolet light and ionizing radiation have various applications as sterilizing agents. Ultraviolet light is not a satisfactory means of sterilization because of the wavelength of the ultraviolet portion of spectrum. Gamma rays, are ionizing radiation from a cobalt - 60 or cesium - 139 source and cathode rays from electron generators and accelerators, Radiation is used for sterilization of certain materials such as pharmaceuticals and disposable Petri dishes. Radiation causes damage of DNA.

C. Filtration: The principle method of sterilizing liquids that contain heat-sensitive components such as vitamins, proteins, and antibiotics is by filtration. Old diatomaceous earth, asbestos filters of sintered glass membrane filters held in stainless steel, glass, plastic, and disposable plastic funnels. Suction can be used to draw the liquid through the filter. Another filtering device is the Swinney filter, which is used to sterilefilter small amounts of heat-labile liquids such as antibiotics and serum components, as they are added to cultures or media. A pore size of 0.2 mm to 0.45 mm is conventionally used for sterilization.

II. Chemical Agents: Because antibacterial agents must be safe for the host organism under the conditions employed, the number of commonly used antibacterial agents is much less than the number of cell poisons and inhibitors available. Thus cyanide and arsenicals are not included.

A. Alcohols: Ethyl alcohol CH3CH2OH and isopropyl alcohol (CH3)2CHOH at 70% concentrations,

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are generally used. They act as protein denaturants.

B. Phenols: Phenol and many phenolic compounds are strong antibacterial agents. These are generally employed as 1-2% aqueous solutions. They denature protein.

Cultures Pseudomonas aeruginosa Staphylococcus epidermidis Escherichia coli

Techniques / Equipment Required Autoclave; UV lamp (265 nm); aseptic technique.

C. Heavy Metal Ions: Mercury, copper and silver salts are commonly used at very low concentrations. All are protein denaturants. Mercury can be made safer by combining it with organic compounds such as mercurochrome and merthiolate.

D. Oxidizing Agents: Inactivate cells by oxidizing free sulfahydryl groups. These include hydrogen peroxidae, iodine, hypochlorite, and chlorine.

E. Alkylating Agents: Two agents are commonly used: formaldehyde (sold as the 37% aqueous solution formalin) and ethylene oxide. Ethylene oxide gas, rendered inexplosive by mixture with 90% CO2 or a flourocarbon, is the most reliable disinfectant available for dry surfaces. Ethylene oxide is the gas most commonly used in gaseous autoclave. This autoclave is used for sterilizing heat-labile materials such as disposable syringes, petri dishes, culture tubes, plastic bags, plastic filtration devices and adhesive bandages.

F. Detergents: Surface active agents are of two types: anionic and cationic agents such as soaps.

Materials Petri plates containing NA; antiseptics disinfectants.

Procedure 1. The instructor will demonstrate to the students how to use the autoclave. 2. The instructor will demonstrate sterilization of glassware in the hot air oven. 3. Streak four nutrient agar plates each with a loopful of E. coli, replace the cover by an aluminium foil which has a hole in the middle. Expose these plates to ultraviolet light for 5 sec, 10 sec, 15 sec, and 20 sec. Incubate these plates overnight at 37 oC. Examine the plates after incubation for the presence or absence of growth in the areas exposed to ultraviolet light. Record your results. 4. Obtain two nutrient agar plates. Streak one with a loopful of Pseudomonas aeruginosa and the other with a loopful of Staphylococcus epidermidis. With alcohol-flamed forceps distribute six filter-paper discs around each seeded plate. Using the pipettes provided in the antiseptics and disinfectants solutions, add 0.1 ml of the various agents to the discs on each plate and label the bottom of the plate with the identity of each agent. Incubate overnight at 37 oC. Examine the plates and record your results.


EXERCISE 18 Sterilization and Disinfection

19


Purpose________________________________________________________________ ______________________________________________________________________ Data

Questions 1-

What is phenol coefficient?________________________________________________ _____________________________________________________________________________ ____________________________________________________________________________

2-

What is the difference between moist and dry heat in sterilization?________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

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What is the method of choice for sterilizing each of the following: forceps, disposable syringes, cloth, culture media, body fluids and loops? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

4-

Design an experiment to distinguish between inhibition and killing? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

5-

What

are

the

possible

modes

of

action

of

disinfectants?_____________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________


19

EXERCISE 19 Antimicrobial Agents Susceptibility Colistin, Imidazoles, Polyenes and Polymuxins. Define the following terms: antibiotic, Antimicrobial agents that inhibit chemotherapy, and MIC; perform antibiotic 3protein synthesis: Chloramphenicol, sensitivity test; provide the rational for the Erythromycins, Lincomycins, disc diffusion technique. Tetracyclins and Aminoglycosides. 4Antimicrobial agents that inhibit Introduction nucleic acids synthesis: Quinolones, Microbial species in a common Pyrimethamine, Rifampin, Sulfonamides environment often inhibit each other's and Trimethoprim. growth. Besides excreting end products of You will do the in vitro susceptibility carbohydrates metabolism (such as alcohol testing of microorganisms to antimicrobial and acids) that can limit growth, some agents (Kirby-Bauer method). This is a disc microorganisms (bacteria and fungi) produce diffusion antimicrobial susceptibility test more complex and quite different chemicals which measure the inhibition of growth of a antibiotics such as penicillin. Some microorganism on the surface of an antimicrobial agents can be prepared in the inoculated agar plate by an antimicrobial laboratory such as sulfonamides. agent diffusing into the surrounding medium Although various chemicals have been from an impregnated disc. The size of the used for the treatment of infectious diseases inhibition zone of a given bacterial strain is since the 17th century, chemotherapy as a inversely proportional to the minimum science began with Paul Ehrlich in 1900. He inhibitory concentration (MIC) of a given was the first to put the rules of planned antibiotic as determined by the tube dilution chemotherapy. The rapid development in test. Susceptibility testing should always be antimicrobial chemotherapy began in 1935, done on pure cultures and not on mixed with the discovery of sulfonamides by cultures. Domagk. In 1940, Chain and Florey demonstrated that penicillin, which had been Materials observed in 1929 by Fleming, could be made Petri plate containing Mueller Hinton into an effective chemotherapeutic substance. agar; culture tubes containing trypticase This is followed by the development of soy broth; barium chloride; sulfuric acid streptomycin, tetracyclines, chloramphenicol (conc.); sterile cotton swab; forceps; and many other agents. In recent years, antimicrobiala discs; alcohol chemical modification of molecules by biosynthesis has been a prominent method of Cultures new drug development. Staphylococcus epidermidis An ideal antimicrobial agent exhibits Eschirechia coli selective toxicity, that is to say the drug is harmful to the parasite without being harmful Techniques / Equipment Required to the host. Aseptic techniques; autoclave; According to their mechanism of action, incubator; inoculating loop. antimicrobial agents are classified into: 1Antimicrobial agents that inhibit cell wall synthesis: Penicillins, CephalProcedure osporines, Vancomycin, Bacitracin and 1Transfer with a wire loop five Cycloserine. colonies of E. coli and S. epidermidis 2Antimicrobial agents that inhibit each into a test tube containing 4-ml memebrane function: Amphotericin B, tryticase soy broth. Incubate broth 2-5 hr

Objectives

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to produce bacterial suspension similar in 3The plates are left to dry for about 3turbidity 5 minutes. Then the antimicrobial disks to a barium sulfate standard, prepared by are placed on the agar with alcohol adding 0.5ml of 1.017% barium chloride flamed forceps or a disk applicator and (BaCl2.2H2O) to 99.5 ml of 1% sulfuric gently press down to ensure contact. acid (0.36N). This standard should be 4Plates are aerobically incubated prepared monthly and be kept in the dark. within 15 minutes. Incubate overnight at Dilute the suspension if necessary with 37 oC. sterile water or saline. After incubation, measure zone 2Using a cotton applicator, swab 15diameters (including the 6-mm disc) with a cm Mueller Hinton Petri dishes, with ruler or a caliper from the back of the plate. each of the bacterial broth suspension. A reading of 6 mm indicates no zone. The swabbing should be done evenly in Measurements of the zone of inhibition three planes and circularly around the diameters are then interpreted according to periphery of the medium. Use blood agar the provided table as sensitive or resistant. for fastidious organisms.


EXERCISE 19 Antimicrobial Agents Susceptibility

11


Purpose__________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ Data

Conclusion_____________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________ ______________________________________________________________________________

_________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________

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Questions 1.

Mention the antimicrobial agents which are used for Gram positive and Gram negative bacteria?__________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________

2.

What are the factors which affect the results of this test?___________________________ _______________________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________

3.

What are the factors which affect the diameter of the zone of inhibition around the disc? _______________________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________ 4. Define MIC and MBC._________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________ 5. The increase in the diameter inhibition zone around the antibiotic does not mean that the antimicrobial agent is more effective than others. Why?__________________________________ _______________________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________ _______________________________________________________________________________


UNIT 7 The Microbial World

20. Unknown Identification and Bacterial Taxonomy References 21. Fungi: Yeast 22. Fungi: Molds 23. Bacteriophage

Eucaryotic microorganisms include fungi, algae, and cyanobacteria, and protozoa. This unit includes exercises on yeasts, and mycelial fungi (molds). Bacteriophage (virus) is also dealt with in exercise 23.

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EXERCISE 20 Unknown Identification and Bacterial Taxonomy References Objectives

Techniques / Equipment required

1.

Explain how bacteria are characterized and classified. 2. Use Bergey's Manual. 3. Identify an unknown bacterium.

Compound light microscopes; hanging drop; wet mount; negative staining; Gram staining; endospores; capsule; and flagella staining; inoculation loop and needle; aseptic technique, plate streaking; OF test; starch hydrolysis; MVR tests; fermentation tests; protein catabolism; catalase test; nitrate reduction test; oxidation test.

Introduction Microbiologists use a system of classification to categorize and classify organisms. Such a universal system is also necessary for scientist to communicate among each other. The taxonomy of bacteria is difficult because few definite anatomical or visual differences exist. Therefore, most bacteria are characterized by evaluation of primary characteristics, such as metabolism and serology. Bergey's Manual is considered the most important reference for bacterial taxonomy. In this manual bacteria are grouped into numbered sections on the basis of Gram stain reaction, cell nutritional and metabolic properties. You will be provided with a culture of an unknown heterotrophic bacterium to characterize and identify with aid of a taxonomic key (Appendix B). The provided key is a dichotomous classification system; that is, a population is repeatedly divided into two parts until a description identifies a single member. You may want to check your conclusion with the species description given in Bergey's Manual. To obtain your identification, ascertain the purity of the culture you have been given and prepare stock and working cultures. Note growth characteristics and Gram stain reaction for clues about how to proceed.

Materials Trypticase soy agar in Petri plates (2), and agar slants (2); stains, reagents, and media previously used.

Culture Unknown bacterium # ___________

Procedure 1.

2.

3.

4.

5.

Streak your unknown onto the agar plates for isolation. Incubate one plate at 35 oC and the other at room temperature, from 24 to 48 hr. Note growth characteristics and the temperature at which each one grew best. Aseptically inoculate 2 trypticase soy agar slants. Incubate for 24 hr. describe the resulting growth. Keep one slant culture in the refrigerator (stock culture); the other is your working culture. Subculture onto another slant for your stock culture when your working culture becomes contaminated or not viable. Working culture is kept in the refrigerator while not in use. Use your working culture for all identification procedures. When a new pure slant is made, discard the old working culture. Follow the keys in Appendix B and develop ideas on how to proceed. Determining staining characteristics might be a good place to start. After determining its staining and morphology characteristics, determine which biochemical tests you will need.

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EXERCISE 20


Unknown Identification and Taxonomy References

Purpose________________________________________________________________ ______________________________________________________________________ Data Write NT (not tested) next to tests that were not performed. Unknown #_________________ Morphological, staining, and cultural characteristics Sketches: Label and give magnification The cell Staining characteristics Gram_______Age__ Other_______Age__ Shape____________ Size_____________ Arrangement______ Endospore position_ _________________ Motility__________ Essential biochemical characteristics results Determined by_____ Colonies on Petri dishes Diameter_________ Appearance_______ Color____________ Elevation_________ Margin___________ Consistency_______ Agar slant Age

Glucose Lactose Mannitol Catalase Oxidase H2 S Nitrate reduction Amount of growth__ Indole Pellicle___________ Methyl red Flocculant________ V-P Sediment_________ Citrate

24

Time (hr) 48

..

Temp.____ oC

Abbreviations: A = Acid G = Gas a = slight acid alk = alkaline + = positive - = negative ng = no grpwth

Other characteristics, special media, etc. __________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________

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Conclusion Organism unknown #__________ is _____________________________________________ Write your rational for arriving at your conclusion. _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________


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EXERCISE 21 Fungi: Yeast Sabouraud agar (SDA) is commonly used in the laboratory to isolate yeast (Appendix A). This medium is low in nutrients and pH and hence does not allow the growth of most other organisms. Many of the techniques useful in working with bacteria can be applied to yeasts.

Objectives 1. 2.

Culture and identify yeasts. Differentiate between yeast bacteria

and

Introduction Fungi are "eucaryotic, spore-producing, achlorophyllous organisms with absorptive nutrition that generally reproduce both sexually and asexually and whose usually filamentous, branched somatic structures, known as hyphae, typically are surrounded by cell walls." (Allexopoulos et al., 1996). Unicellular yeasts, multicellular molds and macroscopic species, as mushrooms are included in the Kingdom Fungi. Fungi generally prefer more acidic conditions and tolerate higher osmotic pressure and lower moisture than bacteria. The are larger than bacteria, with more cellular and morphologic detail. In contrast to bacterial characterization, primary characteristics, such as morphology and cellar detail, are used to classify fungi, with little attention given to secondary characteristics, such as metabolism and antigenic composition. Fungi are structurally more complex than bacteria but are less diverse metabolically. Yeasts are nonfilamentous, unicellular fungi that are typically spherical or oval in shape. They are widely distributed in nature, frequently found on fruits and leaves as a white powdery coating. Yeasts reproduce asexually by budding (a process in which a new cell forms as a protuberance or bud, from the parent cell). When buds fail to detach themselves, a short chain of cells called pseudohypha forms. When yeasts produce sexually, they may produce one of several types of sexual spores. Metabolic activities may also be used for the identification of yeasts genera. Yeasts are facultative anaerobes. Their metabolic activities are used in many industrial processes. Yeasts are therefore used to prepare many foods, including bread, and beverages such as wine and beer.

Materials Glucose fermentation tubes (2); sucrose fermentation tubes (2); SDA plates (2); bottle containing glucose-yeast extract broth (GYEB); sterile cotton swab; cover slip; test tube; methylene blue; balloon; fruit or leaves.

Cultures (as assigned) Beaker's yeast Rhodotorula rubra Candida albicans Saccharomyces cerevisiae

Techniques / Equipment Required Wet mount; plate streaking; fermentation tests; compound light microscope.

Procedure Yeast Students will work in pairs Prepare beaker's yeast suspension in a small amount of warm water in a test tube. 2. Each pair of students will use one of the yeasts cultures and the beaker's yeast suspension. a. Divide one SDA plate in half. Streak one half with a known yeast culture and the other half with the beaker's yeast suspension. b. Inoculate each organism into glucose fermentation tube and a sucrose fermentation tube. 3. Incubate all media at 35 o C until growth is seen. 4. Prepare a wet mount in a drop of methylene blue of each culture. Record your observations. 1.

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After the yeasts have grown, record your results. Examine cultures of the yeasts you did not culture, and record pertinent results.

Yeast Isolation 1.

Cut the fruit or leaves into small pieces. Place them in the GYEB bottle. Cover the bottle's mouth with a balloon. Incubate at room temperature until growth has occurred. Record the appearance of the broth after incubation. 2. Divide a SDA plate in half. Each partner inoculates half of the medium, following step a or b.

a. Swab the surface of your tongue with a sterile swab. Inoculate one half of the agar surface with the swab. b. Using a sterile inoculating loop, streak one half of the agar surface with a loopful of broth from the bottle prepared in step 1. 3. Incubate the plate inverted at room temperature until growth has occurred. Prepare wet mounts with methylene blue from different appearing colonies. Record your results.


EXERCISE 21


Fungi: Yeasts

Purpose________________________________________________________________ ______________________________________________________________________ Data Use the tables to record your results.

Yeasts Fermentation tubes: Organism Acid

Glucose Gas Fermentation?

Acid

Sucrose Gas Fermentation?

Rhodotorula rubra Candida albicans Saccharomyces cerrerisiae Baker's yeast Sabouraud agar plate: Organism Rhodotorula rubra Color_______________

Candida albicans Color_______________

Draw a Typical Colony

Wet Mounts

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Organism Saccaromyces cerevisiae Color_______________

Draw a Typical Colony

Wet Mounts

Baker's yeast Color_______________

Yeast Isolation Plants used:___________________________________________________________ Describe the appearance of glucose broth after ___________ days' incubation_______ _____________________________________________________________________ Was gas produced? _____________________________________________________ Sabouraud agar: Colony appearance Mouth:

Plants:

Color

Size

Wet mounts


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Any bacteria seen?______________________________________________________ If so, which colonies?___________________________________________________

Questions Compare and contrast yeast and bacteria regarding their appearance both on solid media and under the microscope?_______________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ ____________________________________________________________________________

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EXERCISE 22 Fungi: Molds Objectives 1. Characterize and classify fungi. 2. Compare and contrast fungi and bacteria. 3. Identify common saprophytic molds. 4. Explain dimorphism.

Introduction Multicellular filamentous fungi are usually called molds. Molds are morphologically very diverse, and hence morphology is very useful in classifying these fungi. A mold colony is called thallus and is composed of a mass of strands or filaments called mycelia. Each filament is a hypha, with the vegetative hyphae growing in or on the surface of the growth medium. Aerial hyphae originate from the vegetative hyphae and produce a variety of asexual reproductive spores. The hyphal filaments of most molds (mycelial fungi) are composed of individual cells seperated by crosswalls or septa (sing. Septum) (septate hyphae). A few fungi, have hyphae that lack septa and are continuous mass of cytoplasm with multiple nuclei (coencytic hyphae). Fungi are characterized and classified by their colony appearance (color, size, etc.), hyphal organization (septate or nonseptate), and the structure and organization of reproductive spores. Members of the division (phylum) Zygomycota are saprophytic molds that have nonseptate hyphae. Saprophytes (e. g., Rhizopus, bread mold) obtain their nutrients from dead organic matter. Asexual spores are formed inside a sporangium (spore sac), and are called sporangiospores. Sexual spores called zygospores are formed by the fusion of two cells. The Oomycota are generally found in aquatic habitats and form sexual spores called oospores (result of gametangial contact), and motile asexual spores called zoospores. The Ascomycota include molds with septate hyphae and some yeasts. They are

called sac fungi because their sexual spores (ascospores) are produced in a sac (ascus). Mycelial ascomycetous fungi usually produce conidiospores asexually. Some of these fungi also reproduce asexually by budding. The arrangement and other characteristics of the conidiospores are used to identify these fungi (e. g., Penicillium and Aspergillus). The Basidiomycota include the fleshy fungi, or mushrooms, and have sexual spores called basidiospores. Asexual reproduction may be achieved by fragmentation of hyphae. The Deuteromycota include mycelial, septate fungi in which sexual spores have not been demonstrated as yet. The majority of fungi are saprophytic, but each fungal division contains few genera that are pathogens, causing disease in plants, man, and animals. Some fungi are important clinically, industrially, and as biocontrol agents of other pathogenic fungi and pests. Spores in the air are also the most common source of contamination in the laboratory. Some pathogenic fungi exhibit dimorphism, that is, they have two growth forms, depending on growing condition temperature.

Materials Cultivation of mycelial fungi Petri plates containing SDA (2); melted SDA; sterile petri dish; cover slips; bent glass rod; Pasteur pipette.

Dimorphic Gradient SDA, 5 ml, melted at 48 oC; 5-ml small paper cups (2); razor blade; tape.

Cultures (as assigned) Rhizopus stolonifer Aspergillus niger Penicillium notatum Mucor rouxii

Demonstrations Prepared slides of the following: Zygospores, Oospores, Ascospores, Basidiospores.

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Techniques / Equipment Required Compound light microscope, dissecting microscope; aseptic techniques.

Procedure First period 1.

Expose one SDA plate to the air for 15-30 minutes. Incubate the plate inverted at room temperature for 5-7 days. 2. Inoculate a second SDA plate with the mold culture assigned to you. Using a sterile inoculating needle or a scalpel, transfer spores or fragments of the mycelium from the mold culture, to the center of the SDA plate. 3. Prepare a slide culture of your assigned mold (Ali-Shtayeh et al., 1998): a. Prepare a suitable agar medium (e.g. MEA, SDA, CMA, and PDA), and pour into a plate to a depth of about 2-mm. b. When it has set completely, cut out a small block, about 1-cm square using a sterile scalpel and transfer to the center of a sterile slide. c. Inoculate the block with the fungus on all four edges. d. Place a sterile coverslip on top of the block of agar. e. Incubate the slide in a moist chamber. This may be made from a sterile Petri dish with filter paper in the bottom. Two sterile glass rods (or a bent glass rod) are placed on the filter paper to function as supports for the slide. About 10 ml of a sterile 20 % solution of glycerol is poured into the dish. This will keep the agar moist. The dish with the inoculated slide is incubated until the growth reaches a desired stage (until the mycelium formed from each edge of the block has reached the edge of the coverglass). The fungus usually spreads out from the agar block and tends to attach itself to the two glass surfaces. f. When the desired stage of fungal growth has been reached, lift off the coverslip and carefully lower

fungus side down onto a drop of mounting fluid such as lactophenol (with or without stain) on a clean slide. Carefully remove the agar block, add a drop of mountant (e.g., lactophenol) to the growth adhering to the slide, and apply a clean coverslip. Then seal the slides using nail varnish. 4. Observe the prepared slides showing sexual spores. Diagram each of the spores formations in the space provided in the laboratory report.

Dimorphic Gradient 1.

Flick the fungal culture to resuspend and culture. Inoculate the melted agar with 2 or 3 loopful of the Mucor (or as assigned) culture. Mix the tube by rolling it between your hands, and quickly pour the contents into an empty beaker before it hardens. 2. Place a piece of wet paper towel in the remaining empty beaker, and invert over the beaker containing the agar. Tape the edges. 3. Incubate at room temperature until growth occurs (5-7 days).

Second Lab Period 1.

2. 3. 4. 5.

6. 7.

Usually examine plate cultures of each mold, and describe their color, and appearance. Then examine with a dissecting microscope. Look at the top and the underside. Examine your contaminated (airexposed) plate and describe the results. Examine slide cultures of each mold using a dissecting microscope. Record your observations. To examine your dimorphic gradient, remove the tape and cut the beaker in half vertically with a razor blade. Cut a thin (1 mm thick) vertical slice of the inoculated agar with a razor blade. Carefully place the agar slice on a slide and cover with a cover slip. Observe under low and high power. Scan the agar from the bottom of the slice to the top. Discard your beaker and disinfect your slide as instructed.


EXERCISE 22


Fungi: Molds

Purpose________________________________________________________________ ______________________________________________________________________ Observations Plant Cultures Organism Rhisopus stolonifer Colony appearance

Macroscopic Hyphae color Spore color Underside color Microscopic Diagram

Aspergillus niger Penicillium notatum

Unknown from contaminated plant

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Slide Culture Characteristics Rhisopus stolonifer

Organism Aspergillus niger

Penicillium notatum

Coenocytic? Septate? Type of spores Color of spores Diagram hyphal and spore arrangement

Prepared slides Sexual spores Zygospores

Ascospores

Genus:_______________________________ Genus:_______________________________ ____________X ____________X Oospores

Basidiospores

Genus:_______________________________ Genus:_______________________________ ____________X ____________X


Dimorphic Gradient Fungus:_____________________________________________________________ Top of agar: (Draw the structures seen)

Magnification _________ X

___________ X

___________ X

___________ X

___________ X

___________ X

___________ X

Middle of agar

Magnification _________ X Bottom of agar

Magnification _________ X

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Questions 1.

fill in the following table Organism

Asexual Spores

Sexual spores

Division (Phylum)

Rhizopus Aspergillus Penicillium 2.

How do mold spores differ from bacterial endospores? __________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

3.

What determines the form of mucor seen in the dimorphic gradient?________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ ____________________________________________________________________________

4.

Why do media used to culture fungi contain sugars?____________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

5.

Why are antibiotics frequently added to Sabouraud agar for isolation of fungi from clinical sample?____________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________


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EXERCISE 23 Bacteriophage reproduction and lysis of the population of infected bacterial cells in the area.

Objectives 1. Isolate a bacteriophage from a natural environment. 2. Describe the cultivation of bacteriophages.

Materials Raw sewage (45 ml); NA plates; gloves; 10X nutrient broth (5 ml); 50 ml graduated cylinder; funnel

Introduction The bacteriophages or phages are viruses that live on or parasitize bacteria. They are host specific. These viruses are reproduced readily by young actively multiplying susceptible bacteria. Phages are obligate intracellular parasites that live only on their specific host. The invasion and destruction of susceptible cells in broth (bacteriolysis) or by plaque formation in semisolid agar. Lysogeny or inapparent infection occasionally occurs; such an infection sometimes confers new characteristic to the host. For the isolation of phages of enteric microorganisms, raw sewage would be a source. Soil would be a source of phages of spore formers and other soil-dwelling bacteria. For the propagation of phages, they are cultured with their specific bacterial cells on the appropriate media. As the bacterial cells increase, viral particles are formed; the cells eventually disrupt and release the virions into the medium. The sample containing the phage is centrifuged to remove coarse material, then the sample is filtered and treated with chloroform to remove the remaining bacterial cells. The clear fluid now contains phages. Phages now can be detected by mixing some of the liquid with a young culture of uninfected specific bacterial cells. This mixture is layered on the surface of an agar plate. After incubation at 37 oC for 24 hours, the film of bacterial growth will be mottled by clear circular areas called plaques. These plaques represent areas of phage

Cultures Escherichia coli broth

Techniques /Equipment Required Pour plate; pipetting; serial dilution technique; sterile membrane filter apparatus.

Procedure 12-

34-

5-

678-

Students will try to isolate phages of Escherichia coli from raw sewage. To 5 ml concentrated broth add 45 ml of raw sewage. To the mixture add 5 ml of 24 hours culture of E. coli and incubate at 37 oC for 24 hours. This step is for increasing the number of phages. Centrifuge 10 ml of the mixture at 2500 rpm for 10 minutes. Remove the remaining microorganisms by filtration through a bacteriological filter or treat the mixture with chloroform. To 3 ml 0.7 ml soft agar tube add 0.1 ml of a 24 E. coli culture and one drop sewage culture filtrate, mix and pour on to the surface of one of a nutrient agar plate. Repeat step 5 using 5 drops sewage culture filtrate in addition to the other constituents. Allow the soft agar to solidify and incubate the plates at 37 oC overnight. Examine the plates for the presence of plagues. These are the areas in which phage particles have lysed young growing of E. coli.

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EXERCISE 23 Bacteriophages

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Purpose________________________________________________________________ ______________________________________________________________________ Data

Questions 1.

What is a plaque? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

2.

Design an experiment to count the number of phages in a suspension._______________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

3.

Is it possible for E. coli phage to infect Pseudomonas aeruginosa?_________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

122 4.

Faidy & Ali-Shtayeh

Design an experiment to isolate E. coli phage.________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ ____________________________________________________________________________ _____________________________________________________________________________

5.

How can you detect the presence of a bacteriophage?____________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

6.

What are filamentous phages? Give examples.________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

7.

Can phage infect mammalian cells?______________________________________________ If not, why?____________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________


UNIT 8 Microbiology and the Environment

321

24. Microbes in Water: Multiple - Tube Technique 25. Microbes in Water: Membrane Filter Technique 26. Microbes in Food: Contamination

Microorganisms are considered important components of different ecosystems where they play an essential role in the maintenance of life. However, the presence of some microorganisms in certain places, such as water or food, may be undesirable. Harmful microorganisms are only a very small fraction of the total microbial population present in the environment. The activities of most microbes are in fact beneficial. This unit includes exercises on microbial contamination of water and food.



321

EXERCISE 24 Microbes in Water: Multiple - Tube Technique The fecal coliform to fecal streptococci ratio in a sample, is an indicator for animal source of fecal pollution. The standard water analysis for the presence of coliform is divided into three parts: 1. The presumptive test: look for microorganisms that ferment lactose with Introduction the production of gas (colifoorms). Due to increasing population and developing technology, the resources of 2. The confirmed test: transfer water become more liable to microbial cultures that show growth and gas contamination as well as chemical production the presumptive test on to contamination. media that are selective and differential Instances of the spread of diseases for the coli-aerogenes group. such as typhoid fever, dysentery and hepatitis 3. The completed test: isolate and by fecal pollution of water supplies are grow pure cultures of the organisms that familiar. Diseases transmitted by water gave reactions typical of coliforms on the supply create other problems. confirmatory media. The quality of water is determined In this method (mulyiple-tube largely by bacteriological analyses. The technique), coliforms can be detected and important objective of these analyses is to numerated in three stages. In the presumptive determine whether a water supply contains test, dilutions from a water sample are added fecal organisms (not necessarily pathogens) to lactose fermentation tubes. Lactose broth whose presence is indicative of pollution by can be made selective for gram-negative human or animal wastes. The organisms bacteria by the addition of lauryl sulfate or sought are usually coliform bacteria, or the brilliant green and bile. Fermentation of coli - aerogenes group, which includes all lactose to gas Is a positive reaction. aerobic and facultative Gram-negative, nonSamples from the positive tube at the spore forming rods that ferment lactose with highest dilutions are examined for coliforms gas formation. These are indicative by inoculating differential medium in the microorganisms of water quality. confirmed test. The presence of such microorganisms is The number of coliforms is determined an indication of water contamination by fecal by a statistical estimation called the most matter, which is also an indication for the probable number (MPN) method. In the presence of pathogens such as the typhoid presumptive test, tubes of lactose broth are and dysentery bacillus. It is not advisable to inoculated with samples of the water being culture water for the isolation of such tested. A count of the number of tubes pathogens, because it is not easy to isolate showing acid and gas is then taken, and the them, as they are present in low number. figure compared to statistical tables, shown Some coliforms are found in nonfecal in Table X. the number is the most probable sources. Fecal coliforms are typically number of coliform per 100 ml of water. Escherichia coli of the IMVC (indole, methy red, Voges-Proskauer, citrate) types (++--) as Materials well as Enterobacter aerogenes (--+ +). The Double-strength lactose fermentation fecal Streptococci are more numerous tubes (3); single-strength lactose inhabitants of the animal intestine than fermentation tube (6); Endo or EMB human intestine.

Objectives 1.

Define coliform. 2. Provide the rational for determining the presence of coliform. 3. List and explain each step in the multiple-tube technique.

321 Faidy & Ali-Shtayeh agar plates; sterile 1-ml and 10-ml pipettes; Gram staining reagents; water sample; 50 ml(pond or stream).

Note: see table I for the most probable numbers of bacteria per 100 ml of sample, using three tubes of each dilution.

Techniques / Equipment Required

Confirmed Test

Gram staining; aseptic technique; spore staining.

This test is applied to samples which give positive or doubtful results. 1Streak EMB agar or endo agar from positive lactose broth. Procedure Incubate plates at 37 oC for 48 hours. Students will collect tap water using 2If typical colonies of coliforms appeard, then 500-ml capacity sterile flasks. the confirmed test is positive.

Presumptive test 1.

Inoculate three 10 ml portions of water sample each into a double strength 10 ml of lactose broth to allow for the dilution (one set of three). 2. Inoculate 1.0 ml and 0.1 ml of water into small tubes (two sets of three each) of single strength lactose broth. 3. Incubate at 37 oC for 48 hours. 4. Observe after 24 and 48 hours. The presence of gas in any tube after 24 hours is a positive presumptive test. The formation of gas during a second 24-hour period is a doubtful test. The absence of gas formation after 4 hours incubation constitutes, a negative test, indicating that the water supply does not contain coliforms.

Completed Test 1-

Pick two colonies from the EMB or Endo plates and transfer to an agar slope and to a lactose broth tube. Coliform organisms on EMB or Endo agar form darkish colonies that usually have a greenish metallic sheen. 2Incubate at 37 oC for 48 hours. 3From the agar slope, make a Gram stain and a spore stain. 4The formation of gas in the lactose broth and the demonstration of Gram negative, non-spore-forming rods in the agar culture constitute a positive test. This reveals that the water sample contains coliform bacteria, which indicates that water is polluted.

Table 1. Most probable numbers (MPN)* of bacteria per 100 ml of sample, using three tubes of each dilution Number of tubes giving positive Number of tubes giving positive reaction out of reaction out of 3 of 10 ml 3 of 1 ml 3 of 0.1 ml MPN Index 3 of 10 ml 3 of 1 ml 3 of 0.1 ml MPN Index Each Each Each per 100 ml Each Each Each per 100 ml 0 0 0 2,400 2 2 1 28 * Standard methods for the examination of water and wastewater, (1971).


EXERCISE 24

321


Microbes in Water: Multiple-Tube Technique

Purpose________________________________________________________________ _________________________________________________________________ Data Water sample source: ________________________________________________________ Number of tubes with: Growth

Acid

Gas

Possible coliforms present?

Inoculum 0.1 ml 1.0

ml 10 ml

Confirmed Test Tube:__________ Growth_____________________________________________________ Appearance of colonies:_______________________________________________________ _____________________________________________________________________________ _______________________________________________________________________ Are coliforms present?________________________________________________________ Data from water samples tested by other students: Sample

MPN

Coliforms present?

Conclusions 1.

What coliforms present in your water sample?_________________________________ __________________________________________________________________________

321 Faidy & Ali-Shtayeh 2. What is the MPN of your water sample?_________________ per_____________ml

Questions 1.

Could the water have a high concentration of the pathogenic bacterium Vibrio cholerae and give negative results in the multiple-tube technique? Briefly explain-____________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ ________________________________________________________

2.

Why are coliforms used as indicator organisms if they are not usually pathogens? _____________________________________________________________________________ _____________________________________________________________________________ ____________________________________________________________________

3.

Why is not a pH indicator needed in the lactose broth fermentation tubes? _____________________________________________________________________________ _____________________________________________________________________________ ____________________________________________________________________

4.

Mention other methods for bacteriological examination of water.__________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _________________________________________________________________

5.

Why is it advisable to isolate pathogens in water examination?____________________ __________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ ____________________________________________________________________

6.

How can you differentiate between fecal and non-fecal coliforms?_________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________


321

EXERCISE 25 Microbes in Water: Membrane Filter Technique Objectives 1.

Explain the principle of the membrane filter technique. 2. Perform a coliform count using the membrane filter technique.

Procedure 1.

Introduction Fecal contamination of water can be determined by the number of coliforms present in a water sample, by the multipletube technique. Coliforms can be detected by the membrane filter technique, in which the water is drawn through a thin (millipore) filter. Filters with a variety of pore sizes are available. Pores of 0.45 m are used for filtering out most bacteria. Bacteria are retained on the filter, which is then placed on a pad of suitable nutrient medium. Nutrients that diffuse through the filter can be metabollized by bacteria trapped on the filter. Each bacterium that is trapped on the filter will develop into a colony. Bacterial colonies growing on the medium can then be counted. When a selective or differential medium is used, desired colonies will have a distinctive appearance. EMB (eosin methylene blue) agar is frequently used as a selective and differential medium with the membrane filter technique. The composition of EMB agar is shown in Appendix A. EMB is selective because the EMB dyes inhibit the growth of gram-positive organisms, allowing the growth of gramnegative bacteria. EMB is differential in that colonies of lactose-fermenting bacteria have a pink to blue color, black center, or metallic sheen from the eosin and methylene blue.

Materials 47-mm Petri plate containing EMB agar; sterile membrane filter apparatus; sterile 0.45 m filter; forceps; alcohol; sterile pipette or graduated cylinder, as needed; water sample (from a pond or stream).

Techniques Required Pipetting; membrane filtration.

2.

3.

4. 5.

Set up filtration equipment. a. Attach the filter trap to the vacuum source. b. Place the filter holder base (with stopper) on the filtering flask. Attach the flask to the filter trap. c. Using the sterile forceps, place a filter on the filter holder. The filter must be centered exactly on the filter holder. d. Set the funnel on the filter holder and fasten in place Filtering a. Shake the water sample and pour pr pipette a measured volume into the funnel. Your instructor will help you determine the volume. (For samples of 10 ml or less, pour 20 ml sterile water unto the funnel first). b. Turn on the vacuum and allow the sample to pass into the filtering flask. Leave the vacuum on. c. Pour sterile rinse water into the funnel. (Use the same volume as the sample). Allow the rinse water to go through the filter. Turn the vacuum off. Inoculation (Figure 25.1): a. Carefully remove the filter from the filter holder using sterile forceps. b. Carefully place the filter on the culture medium. Do not bend the filter; place one edge down first, then carefully set the remainder down. Place the filter on the plate as it was in the filter holder. Invert the plate and incubate for 24 hours at 35 oC. Examine the plates for the presence of coliforms. On EMB, coliforms will form colored colonies, and some may have a green mattalic sheen. Count the number of coliform colonies: Number of coliform per 100 ml water= 100 X Number of coliform colonies Volume of sample filtered


Figure 25.1 Membrane-filter technique: Inoculation.


EXERCISE 25 Membrane Filter Technique

313


Purpose________________________________________________________________ _________________________________________________________________ Data Sample tested: ______________________________________________________________ Describe the general appearance of the colonies on EMB medium:_____________________ _____________________________________________________________________________ _____________________________________________________________________________ ____________________________________________________________________ Number of coliform colonies:________________________________ Number of coliforms / 100 ml water:____________________________ Use the following space for your calculations:

Data from samples tested by other students: Sample Coliforms / 100 ml

Conclusions 1.

Which water sample(s) is (are) potable?___________________________________ __________________________________________________________________________Wh ich water sample (s) is (are) contaminated with fecal material?__________________ _____________________________________________________________________________ _____________________________________________________________________________

312 Faidy & Ali-Shtayeh Questions 1.

What basic assumption is made in this technique, if the number of bacteria is determined from the number of colonies?_______________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

2.

Why can filtration be used to sterilize culture media?____________________________ ____________________________________________________________________________ Sewage?______________________________________________________________________ _____________________________________________________________________________ Air?_________________________________________________________________________ ____________________________________________________________________________

3.

If you did the multiple-tube technique, list one advantage and one disadvantage of each method of detecting coliforms.________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

4.

Why is the membrane filter technique useful for a sanitarian working in the field? _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ 5. Mention other media used in membrane filter technique._____________________________

6.

Why it is not advisable to culture water for pathogens as an indicator of water contamination?____________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________

7.

If water culture proved negative to indicator organism, does this mean that water is absolutely not contaminated?__________________________________________________

8.

What are the differences between total coliforms and fecal coliforms?______________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________


311

EXERCISE 26 Microbes in Food: Contamination explanation). The microbial population in the original food sample can then be calculated using the following equation: Colony-forming units/gram or ml of sample= Number of colonies . Amount plated X dilution* A limitation of the standard plate count is that only bacteria capable of growing in the culture medium and environmental conditions provided will be counted. A medium that supports the growth of most heterotrophic bacteria is used.

Objectives 1.

Determine the number of bacteria in a food sample using a standard plate count. 2. Provide reasons for monitoring the bacteriologic quality of foods. 3. Explain why the standard plate count is used in food quality control.

Introduction Illness and food spoilage can result from microbial growth in foods. The sanitary control of food quality is concerned with testing foods for the presence of pathogens. During processing (grinding, washing, and packaging), food may be contaminated with soil microbes and microbiota from animals, food handlers, and machinery. Foods are the primary vehicle responsible for the transmission of diseases of the digestive system. For this reason, they are examined for the presence of coli-forms because the presence of coliforms indicates fecal contamination. Standard plate counts are routinely performed on food and milk by food processing companies and public health agencies. The standard plate count is used to determine the total number of viable bacteria in a food sample. The presence of large numbers of bacteria is undesirable in most foods because it increases the likelihood that pathogens will be present, and it increases the potential for food spoilage. In a standard plate count, the number of colony forming units (cfu) is determined. Each colony may arise from a group of cells rather than from one individual cell. The initial sample is diluted through serial dilutions in order to obtain a small number of colonies on each plate. A known volume of the diluted sample is placed in a sterile Petri plate, and melted cooled nutrient agar is poured over the inoculum. After incubation, the number of colonies is counted. Plates with between 25 to 250 colonies are suitable for counting (see exercise 3C for

Materials Melted standard plate count or nutrient agar, cooled to 45 oC; sterile 1ml pipettes; sterile Petri plates; sterile 99ml dilution blanks; food samples, diluted 1:10.

Techniques Required Aseptic technique; pour plates; serial dilution techniques.

Procedure First Period. I: Bacteriologic Examination of Milk 1.

Obtain a sample of either raw or pasteurized milk that has been diluted 1:10. 2. Using a sterile 1-ml pipette, aseptically transfer 1 ml of the 1:10 milk sample into a 99-ml dilution blank; label the bottle "1:103" and discard the pipette (Figure 26.1). Shake the bottle twenty times, with your elbow resting on the table. 3. Label the bottoms of four sterile Petri plates with the dilutions: "1:10," "1:102," "1:103," and "1:104". 4. Using a 1-ml pipette, aseptically transfer 0.1 ml of the 1:103 dilution into the bottom of the 1:104 dilution of the original sample. Using the same pipette, transfer 1.0 ml of the 1:103 into the plate labeled 1:103. Pipette 0.1 ml and 1.0 ml

311 Faidy & Ali-Shtayeh from the 1:10 dilution into the 1:10 2 and 1:10 plates. 5. Check the temperature of the water bath containing the nutrient agar. Test the temperature of the outside of the agar container with your hand. It should be "baby bottle" warm. 6. Pour the melted nutrient agar into one of the plates (to about on-third full). Cover the plate and swirl it gently to distribute the milk sample evenly through the agar. Continue until the plates are poured. 7. When each plate has solidified, invert it, and incubate all plates at 35 oC for 24 to 48 hours.

1. 2.

3. 4.

5.

container with your hand. It should be "baby bottle" warm. 6. Pour the melted nutrient agar into one of the plates (to about on-third full). Cover the plate and swirl it gently to distribute the sample through the agar evenly. Continue until the plates are poured. 7. When each plate has solidified, invert it, and incubate all plates at 35 oC for 24 to 48 hours

Second Period 1.

Arrange each plate in order from lowest to highest dilution. 2. Select the plate with 25 to 250 colonies. Record data for plates with First Period. II: Bacteriologic fewer than 25 colonies as (too few to count TFTC) and those with more than Examination of Hamburger and Frozen 250 colonies (too numerous to count Vegetables TNTC). Obtain a sample of raw hamburger or 3. Count the number of colonies on the frozen vegetables diluted 1:10. plate selected. Using a sterile 1-ml pipette, 4. Multiply the number of colonies by aseptically transfer 1ml of the 1:10 the dilution of the plate to determine the sample into 99-ml dilution blank; label 3 number of bacteria in the original food. the bottle "1:10 " and discard the pipette For example, if 129 colonies were (Figure 26.2). Shake the bottle twenty 3 counted on a 1:10 dilution: times, with your elbow resting on the 129 colonies= 129,000 table. Make a 1:105 dilution using 1 ml X 103 another 99-ml dilution blank. Shake as before. = 1.29 X 105 colony-forming units/ Label the bottom of four sterile Petri ml or gram of food plates with the dilutions: "1:103," 4 5 6 "1:10 ," "1:10 ," and "1:10 ". III. The Detection in Milk of the Using a 1-ml pipette, aseptically transfer 0.1 ml of the 1:105 dilution into Presence of Pathogens the 1:106 plate. Note: 0.1 ml of a 1:10 5 1. Students will be provided with blood 6 dilution results in a 1:10 dilution of the agar and MacConkey plates. original sample. Using the same pipette, 2. Using a calibrated loop, take a repeat this procedure with the 1:103 loopful of milk and streak it on blood dilution until all the plates have been agar and MacConkey plates. inoculated. 3. Incubate at 37 oC for 48 hours. Check the temperature of the water Read and record your results. bath containing the nutrient agar. Test Note:- Specific media and procedures are the temperature of the outside of the agar used for other pathogens such as Mycobacterium tuberculosis.


Figure 26.1. A: Make serial dilutions of a milk sample. B Label for Petri plates for the dilution.

Figure 26.2. Standard plate count of food. A. Prepare serial dilutions of a food sample. B. Label for Petri plates for the dilutions.



EXERCISE 26

Laboratory Report

Microbes in Food: Contamination

Name:_______________________ Date:________________________ Section:______________________

Purpose_____________________________________________________________ ____________________________________________________________________ Data Sample_____________________________________ Dilution

Milk

Colonies per plate Hamburger

Frozen Vegetables

1: 1: 1: 1: Number of colony-forming units per ml (or gram) of original food): Milk:____________________________________________________________ Frozen Vegetables:_________________________________________________ Do your calculations in the space below:

Record data for other food tested by other students. Food

cfu/ml (or gram)


Questions 1.

What could you do to ensure that the bacteria present in foods do not pose a health hazard?____________________________________________________________________ __________________________________________________________________________ _________________________________________________________________

2.

Why are plates with 25 to 250 colonies used for calculations?_____________________ __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________

3.

In a quality-control laboratory, each dilution is plated in duplicate or triplicate. Why would this increase the accuracy of a standard plate count?________________________ __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________

4.

There are other techniques for counting bacteria, such as a direct microscopic count and turbidity. Why is the standard plate count preferred for food?______________________ __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ _________________________________________________________________________

5.

Why is ground beef a better bacterial growth medium than a steak or roast? __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________

6.

Why does repeated freezing and thawing increase bacterial growth meat? __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________

7.

What are the microorganisms which are transmitted through milk from animals to man? __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________


REFERENCES Alexopoulos, C. J., Mims, C. W., & Blackwell,

M.

(1996).

Introductory

th

mycology, 4 ed. New Yourk: John Wiley & Sons, Inc.

Cruickshank, Microbiology, 11

R. th

(1970).

Medical

ed. London: E. and S.

Iiviingstone. Holt, J. G., (ed.). (1994). Bergey's

Ali-Shatyeh, M. S., Jamous, Rana M., & Yaghmour, Reem M-R. (1998). Mycology manual. Nablus: Authors.

manual of determinative bacteriology, (9 th ed.). Baltimore: Williams & Wilkins. Jawetz, E., Melnick, J. L., & Adelberg,

Ali-Shtayeh, M. S., & Abu-Ghsaid, S. I. (1999). Antifungal activity of twenty –two

E. A (1995). Medical microbiology, 20th ed. USA: Appleton and Lange, Los Altos.

medicinal plants used in folkloric medicine

Johnson, T. R., & Case, C. L. (1998).

in the Palestinian area. Mycoses, 42, 665-

Laboratory experiments in microbiology, 5 th

672.

ed. Reading: The Benjamin / Cummings

Ali-Shtayeh, M. S., Al-Nuri, M. A., Yaghmour, R. M. R., & Faidi, Y. R. (1997). Antimicrobial

activity

of

Micromeria

Publishing Company, Inc. Mims, C. A, Playfair, J. HL., Roitt, I. M. Wakelin, D., & Williams, R. (1993).

nervosa from Palestinian area. Journal of

Medical

Microbiology.

Ethnopharmacology, 58, 143-147.

Limited, St. Louis.

Mosby

Europe

Ali-Shtayeh, M. S., Yaghmour, R. M. R.,

Pelczar, M. J., Reid, R. D. and Chan, E.

Faidi, Y. R., Salem, K., & Al-Nuri, M. A., &

C. S. Microbiology, 4th ed. Tata McGraw-

(1998). Antimicrobial activity of 20 plants

Hill, New York, 1972.

used in folkloric medicine in the Palestinian

Seeley, H. W, Vandemark, P. J., & Lee,

area. Journal of Ethnopharmacology, 60,

J. J. (1991). Microbes in Action: A

265-271.

laboratory Manual of Microbiology, 4th ed.

American Public Health Association. (1971),

Standered

methods

for

the

examination of water and waste water, 13 ed. New York: Author. Balows, A., Hausler, W. J., Herrmann, K. L., Isenberg, H. D., & Shadomy J. H. (1991). Manual of Clinical Microbiology, 5th ed. ASM, Washington D. C: ASM. Bauer, J. D. (1982). Clinical Laboratory Methods, 9th ed. Missouri: Mosby, St. Louis.

New York: Freeman and Company.



APPENDICES

Appendix A: Media & Stains Appendix B: Keys to Bacteria

141



141

APPENDIX A Media and Stains Acid Alcohol (decolorizing agent) Concentrated HCL 95 % ethyl alcohol Add the HCL to the alcohol.

3 ml 97 ml

Basic fuchsin Basic fuchsin (certified flagellar stain Ethyl alcohol, 95%

for

0.6 g 50 ml

Distilled water

1000 ml

Eosin - Methylene Blue Agar (EMB) Peptone Lactose Sucrose K2HPO4 Eosin Y Methylene blue Agar Distilled water

10 g 5g 5g 2g 0.4 g 0.06 g 15 g 1000 ml

Burke Iodine Iodine crystals Potassium iodide Distilled water

1g 2g 100 ml

Carbol Fuchsin (Concentrated Fuchsin) Basic fuchsin 95% ethanol Phenol Distilled water

0.3 g. 10 ml. 5 ml. 95 ml.

Chrlich Reagent Paradimethylaminobenaldehyde Absolute ethanol Concentrated HCl

4g 380 ml 80 ml

Gelatin medium Beef extract Peptone Bacto dextrose Lead acetate Sodium thiosulfate Sodium chloride Bacto agar Distilled water

3g 5g 1g 0.2 g 0.08 g 5g 15 g 1000 ml

Hucker Crystal Violet Crystal violet Ethyl alcohol 95% Ammonium oxalate Distilled water

2g 20 ml 0.8 g 80 ml

Corn Meal Agar (CMA) Cornmeal (Dehydrated 17.0 g infusion from corn) Agar 15.0 g Distilled water

1000 ml

Czapek Dox Agar (Modified) (Oxoid) (CzA) Sucrose Agar Potassium sulphate Ferrous sulphate Sodium nitrate Potassium chloride Magnessium glycerophosphate Distilled water

30 g 12.0 g 0.35 g 0.01 g 2g 0.5 g 0.5 g 1000 ml

Endo Agar Peptone Lactose K2HPO4 Sodium sulfite Basic fuchsin Agar

10 g 10 g 3.5 g 2.5 g 0.5 g 15 g

Indole Test Broth or Tryptophan Broth peptone Sodium chloride Distilled water

20 g 5g 1000 ml

Kovacs Reagent Paradimethylaminobenzaldehyde Amyl alcohol Concentrated HCl

5g 75 g 25 ml

Lactose Broth Beef extract Peptone Lactose Distilled water

3g 5g 5g 1000 ml

Loefler's methylene blue stain (Counter Stain) Methylene blue Ethyl alcohol KOH Distilled water

8.9 g 600 ml 2g 2000 ml


Lysine Iron Agar Bacto peptone Yeast extract Dextrose L-lysine Ferric ammonium citrate Sodium thiosulfate Bromocresol purple Agar Distilled water

5g 3g 1g 10 g 0.5 g 0.04 g 0.02 g 15 g 1000 ml

MacConky Agar Bacto peptone or gelysate pepton Lactose Bile salts Sodium chloride Agar Neutral red Crystal violet Distilled water

17 g 10 g 1.5 g 5g 13 g 0.03 g 0.001 g 1000 ml

Malt Extract Agar (MEA) Malt extract Agar Peptone Dextrose Distilled water

20 g 20 g 10 g 20 g 1000 ml

Mordent Potassium alum, saturated aqueous solution Tannic acid, 20% aqueous solution Mercuric chloride, saturated aqueous solution

5 ml 2 ml 2 ml

peptone Sodium chloride Dipotassium phosphte Bromothymol blue Agar Distilled water

2g 5g 0.3 g 0.03 g 3g 1000 ml

Phenol Red Broth Base Beef extract Proteose peptone No. 3 Sodium chloride Phenol red Distilled water

1g 10 g 5g 0.018 g 1000 ml

Phenylethyl alcohol Agar Tryptose Beef extract NaCl Phenylethyl alcohol Agar Distilled water

10 g 3g 5g 2.5 g 15 g 1000 ml

Potato Dextrose Agar (PDA) Potato infusion Dextrose Agar Distilled water

200 g 20 g 15 g 1000 ml

Sabouraud Dextrose Agar (SDA) Dextrose Peptone Agar Distilled water

20 g 10 g 17 g 1000 ml

Simmons Citrate Agar

MR - VP Medium Buffered peptone Bacto dextrose Dipottasium phosphate Distilled water

Oxidation - Fermentation Medium (OF) (Basal Medium)

7g 5g 5g 1000 ml

Magnesium sulfate Monoammonium phosphate Dipottasium phosphate Sodium citrate Bacto agar Bacto bromthymol blue Distilled water

0.2 g 1g 1g 2g 15 g 0.08 g 1000 ml

Nutrient Broth (NB) Beef extract Peptone Sodium chloride Distilled or deionised water

3g 5g 5g 1000 ml

Nutrient Agar (NA) Beef extract Peptone Sodium chloride Agar Distilled or deionised water

3g 5g 5g 15 g 1000 ml

Starch Agar Beef extract Peptone or tryptone Starch (soluble) agar Distilled water

3g 5g 2g 15 g 1000 ml


Triple Sugar Iron Agar (TSI)

Urea Agar

Beef extract Yeast extract Peptone Components Dextrose Lactose Ferrous sulphate or ferrous Ammonium sulphate Sodium chloride Sodium thiosulfate Agar Phenol red Distilled water

Bacto yeast extract Mono potassium phosphate Urea, difco Bacto-phenol red Agar Distilled water

3g 3g 1g 10 g 10 g 0.2 g G 5g 0.3 g 12 g 0.024 g 1000 ml

141

1g 9.5 g 20 g 0.01 g 15 g 1000 ml

Prepare all the previously mentioned media according to the manufacturer instructions.



APPENDIX B Keys to Bacteria

141

Gram-positive Cocci

Rods Catalase

Catalase +

-

Acid from glucose

Growth in 6.5% NaCl; at pH 9.6

+ Acid from manitol; grow in 0.5% NaCl

+

-

-

+

Pigment

Acid from arabinose

Red

Growth at 10 oC

+ Acid from lactose

Yellow +

Staphylococcus aureus

-

Micrococcus Microcuus roseus luteus Enterococcus Acid from fructose faecium

Gas from glucose

+

-

Acid from mannitol

Acid from glucose

-

V-P test

Bacillus cereus

+ Lactococcus Lactococcus plantarum lactis

+

+ Streptococcus pyogenes

Staphylococcus saprophyticus

-

Bacillus megaterium

Corynebacterium xerosis

Inhibited by optochin + Streptococcus pneumoniae

Acid from inulin

+ Streptococcus salivarius

+ Lactobacillus fermentum -

Bacillus subtilis

Figure B.1 Key to selected Gram-positive heterotrophic bacteria.

Endospore

+

Enterococcus feacelis

+ Staphylococcus epidermidis

-

+

- hemolysis

-

+

Streptococcus mitis

Corynebacterium psedodiphtheriticum

Acid from mannitol

+ Lactobacillus Lactobacillus casei acidophilus

Gram-negative Cocci

Rods Oxidase

Glucose fermented + Nitrate reduced + Neisseria mucosa

+

-

Glucose oxidized

Acid and gas from lactose

Neisseria flavescens +

-

Litmus milk

Gelatin hydrolyzed

Neisseria sicca Peptonized reduced

Alkaline

+

-

Citrate utilized

Red pigment at 25 oC

+

Pseudomonas aeruginosa

Pseudomonas fluorescens

+ Deleya aquamarina

-

+ Serratia marcescens

White colonies

Yellow colonies

Indole produced

+

Alcaligenes feacalis

-

Alcaligenes paradoxis Escherechia coli

H2S produced

Ptoteus mirabilis

MRVP test + MR negative V-P positive

Proteus vulgaris Enterobacter aerogenes

Figure B.2 keys to selected Gram-negative heterotrophic bacteria.

-

MR positive V-P negative Citrobacter freundii

Morganella morganii