Objective: To familiarize with biosafety issues in Microbiology laboratory and .....
Atlas, Ronald M. 1989. Microbiology Fundamentals and Applications.
LABORATORY-1 Biosafety Issues and Basic Techniques in Microbiology
Manvika Sahgal Objective: To familiarize with biosafety issues in Microbiology laboratory and instruments A. Biosafety Issues Despite a greater awareness of biosafety and biocontainment practices, handling infectious microorganisms remains a source of infection, and even mortality, among laboratory workers. Incidents of secondary transmission of disease to the public at large, which may be due to possible contamination of the environment or personnel, are also prevalent. Laboratory workers can minimize the risks associated with work involving these infectious agents through the application of appropriate biosafety and containment principles and practices. Regulatory authorities can minimize this through strict implementation of biosecurity plan. While biosafety deals with all aspects of containment to prevent any exposure to and accidental release of pathogens, biosecurity is implemented to prevent the theft, misuse or intentional release of pathogens. Risk Assessment, deciding containment levels, hazard analysis and decontamination constitute a complete biosafety plan. Biological safety issues to be managed may include the following: •
Development and delivery of biosafety training program, performing risk assessments and developing recommendations for procedural or physical laboratory modifications.
•
Auditing the effectiveness of the biosafety program and its associated management system on a regular basis.
•
Participating in accident investigations and promoting the reporting of incidents within laboratory.
•
Distributing new and relevant biosafety information to laboratory staff.
L1
•
Coordinating and monitoring the decontamination, disinfection and disposal procedures for infectious materials.
•
Coordinating the receipt, shipment and transport within the facility of infectious material.
•
Maintaining records and secure storage system for all infectious material entering the facility.
•
Co-ordinating emergency response activities.
•
Maintaining liaison with support and housekeeping staff on matters related to facility biosafety.
1.
Risk Assessment A risk group to which an organism falls is based upon the characteristics of the organism, such as pathogenicity, infectious dose, and mode of transmission, host range, availability of effective preventive measures and effective treatment. Four levels of risk have been defined as follows. •
Risk Group 1 (low individual and community risk) Any biological agent that is unlikely to cause disease in healthy workers or animals.
•
Risk Group 2 (moderate individual risk, low community risk) These pathogens cause human disease but are unlikely to be a serious hazard to laboratory workers, the community, livestock or the environment. Laboratory exposures rarely cause infection leading to serious disease; effective treatment and preventive measures are available, and the risk of spread is limited.
•
Risk Group 3 (high individual risk, low community risk) Any pathogen that usually causes serious human disease, results in serious economic consequences but does not spread by casual contact from one individual to another. Disease is treatable by antimicrobial agents.
•
Risk Group 4 (high individual risk, high community risk) Any pathogen that usually causes very serious human disease, often untreatable, and may be readily transmitted from one individual to another, or from animal to human or vice-versa, directly or indirectly, or by casual contact. In addition to the Risk Group classifications, the L2
following factors associated with the laboratory operation should also be examined: potential for aerosol generation quantity concentration agent stability in the environment type of work (e.g., in vitro , in vivo , aerosol challenge studies) use of recombinant organisms 2.
Containment Levels Risk assessment is a critical step in the selection of an appropriate containment level for the microbiological work to be carried out. The containment level is based on the manipulations generally associated with laboratory scale research and clinical procedures. If a particular procedure, such as preliminary identification, poses a lower hazard than manipulation of a live culture, then a lower containment level may be appropriate. An increase in containment may be required once a facility begins large scale production (manipulations in volume excess of 10L).The specific safety procedures are available for large scale production and handling facility. A hazard analysis may indicate that, because of high pathogenicity, the route of transmission and the low infectious dose, a particular study involving volumes of < 10 L may pose a greater hazard than research scale quantities and therefore may require increased levels
of
physical
and
operational containment. Four containment levels are described as follows:
Containment Level 1 (CL1) Biological safety cabinets (BSCs) are not required. Work may be done on an open bench top, and containment is achieved through the use of practices normally employed in a basic microbiology laboratory.
Containment Level 2 (CL2) Biological safety cabinets (BSCs) and centrifuges with sealed rotors or safety cups, appropriate personal protective equipment (i.e., gloves, laboratory coat and protective eyewear) must be used. An environmental contamination must be minimized by the use of hand washing sinks and autoclaves. L3
Containment Level 3 (CL3) Infectious agents transmitted by the airborne route have a low infectious dose and can cause serious or lifethreatening disease. An appropriate respiratory protection, HEPA filtration of exhausted laboratory air and strictly controlled laboratory access are required.
Containment Level 4 (CL4) Infectious agents have the potential for aerosol transmission, have a low infectious dose and produce very serious and often fatal disease; there is generally no treatment or vaccine available. CL4 emphasizes maximum containment of the infectious agent by complete sealing of the facility perimeter with confirmation by pressure decay testing; isolation of the researcher from the pathogen by his or her containment in a positive pressure suit or containment of the pathogen in a Class III BSC line; and decontamination of air and other effluents produced in the facility.
3.
Decontamination It is a basic biosafety principle that all contaminated materials be decontaminated
prior
to
disposal.
Decontamination
includes
both
sterilization (the complete destruction of all microorganisms, including bacterial spores) and disinfection (the destruction and removal of specific types of micro-organisms). It is the responsibility of all laboratory workers to ensure the effective use of products for decontamination of materials, equipment, and samples from containment zones; of surfaces and rooms; and of spills of infectious materials. These procedures represent a critical containment barrier which in case of failure results in occupational exposure to infectious agents and/or the unintentional release of agents from a containment facility. Employee infection with M. tuberculosis as a result of exposure to contaminated waste has been documented. Employees should leave their laboratory clothing for laundering in a designated area. Employees must be trained in all decontamination procedures and factors influencing the effectiveness of the treatment procedure, as discussed briefly below. (i)
Autoclaves Infectious laboratory wastes (petri dishes, pipettes, culture tubes, glassware, etc.) can be effectively decontaminated in L4
autoclave.
The
effectiveness
of
decontamination
by
steam
autoclaving depends upon various loading factors that influence the temperature to which the material is subjected and the contact time. Tight-fitting containers do not permit steam penetration. Piling containers above one another and overloading can result in decontamination failure. Monitoring : Ampoules of Bacillus sterothermophilus are sent to the designated person in each laboratory biannually for validation of the equipment. Instructions are provided for placement of ampoule in the autoclave during the run. This ampoule is returned to the Biological safety agency for incubation to determine if autoclave has provided a proper microbiological kill. Results are sent back for maintaining as long as machine is functional (ii)
Chemical Disinfection. Chemical disinfectants are used for the decontamination of surfaces and equipment that cannot be autoclaved, such as specimen containers and clean up of spills of infectious materials, rooms. The initial choice of a chemical disinfectant depends upon the resistance of the microorganisms of concern. The most susceptible are vegetative bacteria, fungi. Mycobacteria and non-enveloped viruses are less susceptible; bacterial spores and protozoan cysts are generally the most resistant. Consideration should also be given to practicability, stability, compatibility with materials and health hazards. Class of chemicals (e.g., hypochlorites, quaternary ammonium compounds, phenolics, iodines, alcohols) could be used as disinfectants.
(iii)
Gaseous Decontamination of Rooms Vaporized hydrogen peroxide has
been
proposed
as
a
safer
alternative
to
gaseous
decontamination with formaldehyde. 30% liquid hydrogen peroxide is vaporized to yield approximately 1200 ppm. The vapour breaks down into nontoxic oxygen and water. Successful gaseous decontamination requires an ambient temperature of at least 21o C and a relative humidity of 70%. Biological indicators should be used to monitor the effectiveness procedure. L5
(iv)
Liquid Effluent Treatment Systems are used in containment level 4 laboratories for decontaminating liquid waste streams from sinks, showers, autoclave chambers and other drains. This represents a secondary treatment system, as no infectious micro-organisms are disposed off directly into the drain without prior treatment. Decontaminated liquids released from the treatment system must meet
all
applicable
regulations
(e.g.
municipal
bylaws
for
temperature, chemical/metal content, suspended solids, oil/grease and biochemical oxygen demand). (v)
Irradiation Gamma irradiation (e.g., 60 Co) can be used for the decontamination of heat-sensitive chemicals and solvents removed from a containment facility.
(vi)
Incineration has traditionally been the chosen method for processing anatomical biomedical waste and animal carcasses. In most cases, wastes to be incinerated have to be packaged and transported off-site in accordance with territorial legislation. Materials removed from containment laboratories for off-site incineration should initially be treated at the containment barrier, preferably by autoclaving.
4.
Biosecurity Plan A primary component to a biosecurity plan must be a detailed risk assessment. The biosecurity risk assessment should review and list the relevant assets, define the threats,
outline
the
vulnerabilities,
and
determine the counter measures or mitigation strategies specific for each facility. The biosecurity plan should then address the following
factors:
physical protection; personnel suitability/reliability; pathogen accountability; and related incident and emergency response.
L6
GENERAL LABORATORY QUALITY- CONTROL PRACTICES General sterility and cleanliness Sterility and cleanliness of the laboratory is necessary to ensure the integrity of samples and analytical procedures •
Traffic through the laboratory is restricted to those doing work in the laboratory, especially when analytical work is being done.
•
The tabletops are wiped down with 70 percent ethanol, before and after use.
•
Antimicrobial soap is available at various laboratory sinks to facilitate hand washing before and after laboratory work.
•
Clean and sterile glassware that is free of detergent residue is crucial to ensure valid results in microbiology.
•
Dirty dishes are placed separately after use and are not to be stored on tabletops. Dishes are washed with hot water and laboratory-grade phosphate-free detergent.
Dishes are rinsed with tap water and then
deionized water. Autoclaves Sterilization is the process that eliminates living organisms from substances or objects. Glasswares should be are wrapped in aluminum foil or paper and placed in the autoclave for moist heat sterilization. Clean and sterile glassware are stored in closed cupboards until use. The autoclaves are operated at 15 lb/in2 steam pressure, producing an inside temperature of 121 to 124oC. Do not overload the autoclave. Autoclave time depends on the type and amount of equipment as follows: Glassware and up to 250 ml of liquid—15 min •
500 to 2,000 ml liquid - 30 min
•
Greater than 2,000 ml to 6,000 ml liquid - 15 min per 1,000 ml
•
Greater than 6,000 ml liquid - 90 min
•
Carbohydrate-containing media -15 min (250 ml)
•
Contaminated materials and discarded cultures - 45 to 90 min
Operating temperature and pressure are checked once a week. Heatsterilizing tape is used with each run to identify supplies that have been properly sterilized and checks the performance of the autoclave. If the autoclave does not reach the specified temperature, service the autoclave and re-sterilize all L7
glassware and reagents that were insufficiently sterilized. The autoclaves are operated using deionized water. At the end of the day, autoclaves are drained. Twice a month, autoclaves are cleaned with mild soap, rinsed with water, and drained. The condensate holding tank is drained daily or as needed. Laboratory water Aspirator bottles used to store single and triple distilled water are drained completely and washed with soap solution every week.
Analytical balances Analytical balances are used for accurate weighing of reagents and media. They are checked and calibrated annually. Balances must rest on a firm, level surface. Balance trays are wiped off daily with water or a surface disinfectant such as 70 % ethanol.
A laminar-flow bench •
Laminar flow hoods have magnehelic pressure gauges (MAG) that are used to monitor operation of the hoods. When using, check that the pressure gauge is
reading at a level approximately equal to the annually
recorded MAG level on the calibration sticker. A significant increase in pressure indicates that the filters are dirty whereas decrease in pressure indicates an electrical problem. •
A laminar-flow bench must be free from contamination by live organisms.
•
The working surfaces of the laminar-flow bench are wiped with 70 % ethanol before and after use.
•
The ultraviolet lights in the laminar flow bench are cleaned quarterly by wiping with a soft cloth.
•
Biannually, nonselective agar plates are exposed to airflow in the laminarflow bench. The plates are incubated at 35oC for 24 h and examined for contamination.
L8
pH meters With each use of the specific conductance, pH, or turbidity meter, calibrate the instrument according to the manufacturer’s instructions. Use a calibrated solution that is within the range of the water sample to be measured. Label specific conductance and pH buffer solutions with the date opened and discard working solution weekly. Each piece of equipment has daily logbook; record all calibrations in the appropriate logbook. Micropipettors Micropipettors are used for the accurate delivery of small volumes. Pipettors are cleaned, calibrated and adjusted annually, if necessary. Vacuum pump The vacuum pump is mainly used for membrane filtration. The oil is changed in the pump every 2 years. Incubators, water baths, refrigerators, freezers, and thermometers The temperatures of the laboratory incubators, water baths, refrigerators, and freezers are checked quarterly with laboratory. During period of heavy use, the temperatures are checked and recorded weekly. Deep freezers (-700 C) are used to store samples and microbiological cultures. Its filter is cleaned and fans behind the filter are checked. Water baths are filled with 50% deionized water and 50% tap water and are cleaned with mild soap quarterly, or more often as needed. Microscope The microscope is used for general laboratory work. The microscope is cleaned and the ocular micrometer is calibrated yearly. Centrifuges They
are
used
for
processing
bacterial
extractions,
purifications,
concentrations, and phase separations. •
Each run of the centrifuge is recorded in the centrifuge log book.
•
The temperature is monitored quarterly with the digital thermometer (acceptance criteria is 4+ 3ºC).
•
The buckets are cleaned with soap and water quarterly.
•
Rotors and adapters are checked for deterioration, as needed.
•
Lubrication is done annually, or as needed.
L9
Master Cycler The Master Cycler is used to amplify bacterial DNA through a series of temperatures changes. It detects enteric viruses in water by reverse-transcriptase polymerase chain reaction (RT-PCR). It is also used in bacterial source tracking methods. •
The temperature is monitored twice a year.
L 10
LABORATORY-2 Media Preparation & PA Coliform Test
Mahejibin Khan Part-I Preparation & Sterilization of Media Objective: To prepare & sterilization the nutrient media for use in water analysis Principle: A medium serves as a source of nutrient for in-vitro growth of various types of microbes in the laboratory. Each medium has four basic constituents-C, N, H, & O. Besides this, some other macro and microelements are also required by fastidious organisms. a)
Carbon source: Based on carbon requirement, microorganisms can be divided into two categories
i)
Autotrophs-require CO2 as sole carbon source.
ii)
Heterotrophs-require organic form of carbon.
b)
Nitrogen: Required for synthesis of amino acids and proteins.
c)
Hydrogen & Oxygen: Forms part of many organic compound
d)
Sulphur: Biosynthesis of amino acids like cysteine, cystine, methionine.
e)
Phosphorous: Nucleic acids and ATP
f)
Mineral salts: Macroelements like Sodium & Ferrous help the enzyme while microelements like Zinc, Manganese, Cobalt, Molybdenum, Copper are required in trace quantity for activation of enzymes.
g)
Energy source: Light or chemical compounds.
Types of media Media can be of two types: a). Complex nutritional media: not defined quantitatively or qualitatively e.g. nutrient agar b). Chemically defined media: Media defined qualitatively as well as quantitatively e.g. defined media. On the basis of physical condition, media can be categorized into three categories as: a). Solid media: Media having 2% agar-agar (solidifying agent) L 11
b). Semi-solid media: Contains 0.75-1% agar-agar c). Liquid media: Media not having solidifying agent i.e. agar-agar Requirements: Autoclave 250 ml. conical flasks Weighing balance Butter paper Spatula Laminar air flow bench Petriplates Measuring cylinder Test tubes Test tube stand Cotton bundle EMB Agar Pepton Lactose Sucrose K2HPO4 Eosin Y Methylene blue Agar Distill water
g/l 10 g 5g 5g 2g 0.4 0.06 20.00 1000 ml
MacConkey Agar Peptone Lactose Bill Salts Sodium chloride Neutral Red pH
g/l 20.0 10.0 5.0 5.0 0.075 7.4
Alternatively, Hi-media supplies readymade EMB & MacConkey agar and depending upon the grade, an amount of powder (as mentioned on the container) is dissolved in 1 l of distilled water and autoclaved to get the specific agar medium ready. Methodology Preparation of broth medium (test tubes) 1) Prepare the broth media as given above. 2) Transfer nutrient broth (liquid) to the test tubes before sterilization to minimize chances of contamination while transferring after sterilization. 3) Test tubes are ready for sterilization after placing cotton plugs. L 12
Preparation of agar slants (test tubes) 1) Add 2% agar-agar to the respective broth. 2) Homogenise the medium by heating it on hot plate or microwave oven. 3) Transfer 5-10 ml of Nutrient agar into test tubes for sterilization & put cotton plugs. 4) Sterilize these by autoclaving. After autoclaving the tubes are placed at an angle of 45ºC and allowed to solidity so that the slants are prepared. Preparation of EMB or MacConkey agar for petriplates 1) Prepare respective media as given above. 2) Cover the conical flask with cotton plug and the flask is ready for sterilization after covering the cotton plug with paper. Note: 1) Sterilization in all three cases above is carried out at 15 lb pressure for 20-30 min. 2) Check the dye strip after sterilization to ensure proper sterilization of media. Observations and result (to be filled in by trainees) QUESTIONS: 1. What is a complex medium? 2. Why are buffers added to the growth media for microbes? 3. How can the temperature in the autoclave go above boiling temperature of 212 F? 4. Why do you have boil the agar solution BEFORE dispensing it into tubes? 5. At what temperature does agar solidify?
L 13
Part-II PA Coliform Kit (HiMedia Laboratories) A qualitative test to detect the presence or absence of coliform bacteria in water for treatment plants or distribution systems. Material Provided 1 sterile disposable bottle
= 100 ml capacity
Dehydrated Medium (PA broth)
= 3x Concentration
Ziplock bag
= 1 No.
Procedure: Collect 100 ml water to be tested in ziplock bag and transfer to sterile disposable bottle. Add entire quantity of dehydrated medium (PA broth) slowly to water by swirling to dissolve the powder completely. After dissolution, incubate the bottle for 24-48 hr. at 30-350C. Observe the colour change indicating the presence of coliform bacteria.
L 14
LABORATORY-3 Microbiological Analysis of Water and Wastewater
Lakshmi Tewari Objective: To test bacteriological quality of drinking water and microbial analysis of wastewater Principle:
With
increasing
industrialization,
water
sources
available
for
consumption and recreation have been adulterated with industrial as well as animal and human wastes. As a result, water has become a formidable factor in disease transmission. It may act as a potential common source of pathogenic and non-pathogenic microorganisms. Water purification is, therefore, the most important measure available for ensuring public health. In India more than 70% of the epidemic emergencies are either water borne or are water related. Over 80 genera of bacteria that are non-pathogenic for humans have their natural habitat in water. Several enteric bacteria, coliforms and several other pathogenic bacteria, (Pseudomonas, Streptomyces,
Serratia, Vibrio
Aeromonas,
cholerae,
Enterobacter,
Cryptosporidium,
Escherichia
Yersinia
coli,
enterocolitica,
Campylobacter sp., streptococci etc.) Water in relation to human consumption is classified as (i) the potable water (drinking water) and (ii) the unpotable water or waste water (sewage). Potability refers to the pollution level or drinking quality of water. It is therefore, highly desirable that the water, before its wide circulation for consumption purposes, should be undergone for proper bacteriological testing. Potable water can be defined as any water that is clear, free from undesirable flavours, odors, of reasonable temperature, neither corrosive nor scale forming, free from minerals that could produce undesirable physiological effect and does not contain pathogenic microorganisms capable of causing human diseases. World Health Organization (WHO) has recommended following bacteriological standards for treated water: (i)
Coliform count in any sample of 100 ml should be zero or in 90% samples examined throughout any year. L 15
(ii)
Coliform organisms not more than 10/100 ml water samples shall be present in any water and
(iii)
Coliform organisms should not be detectable in 100 ml of any two consecutive samples or more than 50% of the samples collected for the year.
Microbiological analysis of water: The bacteriological analysis of water is done primarily to determine its potability i.e. fitness for drinking and for waste-water to determine the most probable number of bacteria in the sample. The coliforms (E. coli & related organisms), Streptococcus faecalis and Clostridium perfringens which are normal inhabitants of the large intestine of animals and humans enter water supplies through faecal contamination. Presence of any of these bacterial species in water is evidence of sewage or faecal pollution. Techniques are available by which presence of these specific groups can be easily identified. However, detection of specific pathogenic organisms in water supply is much more difficult because of their small number. Therefore, E. coli, which outnumbers the pathogens in faecal discharges of animals & human beings, can serve as good indicator bacteria. Thus E. coli is used as an indicator to assess contamination from sewage in case of drinking water; while Enterobacter aerogenes, which originated from soil or plant materials, and contaminate water can be easily distinguished from E. coli. Higher number of E. coli may indicate high probability of pathogens presence. The routine bacteriological examination of water involves three types of tests: 1) The standard plate count (total bacterial count) test. 2) Test for coliforms (the standard bacteriological test) 3) Membrane filter techniques Requirements: Lactose broth Test tubes Inoculating loop Durham tubes EMB agar (readymade, Hi-media) L 16
IMViC Hi-media test kit Water sample Bacterial filters Filtration assembly Methodology: I. The standard plate count method for total bacterial counts (ACC): This test provides an estimate of the total number of bacteria in a water sample which will grow and develop in a particular culture medium at 370C under laboratory conditions. I) Water sample is first diluted with sterile water and then plated on agar medium (Cappuccino and Sherman, 1996). II) The bacterial colonies developed on plates are counted to give the total bacterial counts in the sample under test. III) Total cfu counts/ml are calculated using the following formula: cfu counts/ml = Dilution factor of original sample × No. of colonies Water of good quality has low bacterial counts of less than 100/ml II. Tests for Coliforms for assessing water safety (Drinking and Swimming pool water): Two procedures are commonly used to test for coliforms in water sample I). The Most probable number (MPN) technique and II). The Membrane filter (MF) technique. The conventional test (used as standard analysis test) for the detection of faecal contamination in water was given by Eijackman and is based on determination of most probable number of coliforms. Assessment of the microbiological load in different water samples viz. drinking water (municipal water supply), surface water, waste water, ground water, swimming pool water, mineral water etc. include a). Total Aerobic Colony Counts (Heterotrophic Plate Count) (ACC/HPC), b). Total Coliform Count (TCC), c). Faecal Coliform Count (FCC) and d). Faecal Streptococcal Counts (FSC).
L 17
These indicator parameters should be studied by standard methodologies recommended by the American Public Health Association (APHA) or WHO. In case of swimming pools, water is to be examined monthly for presence of indicator microorganisms. Samples should be tested for aerobic colony counts (ACC) or plate count, coliform (both FC & NFC), E. coli, Pseudomonas aeruginosa. The aerobic colony count should normally be 10 or less cfu/ml of pool water. Coliforms are sensitive to disinfectant and should be absent in 100 ml of pool water. Pseudomonas aeruginosa can grow within untreated water and forms biofilms. It can cause skin; ear and eye infections when present in large number and outbreaks of skin infections have been linked to swimming pool and spa pools. Well operated pools should usually not contain P. aeruginosa, if the count is 10 cfu/100ml, repeat testing and filtration and disinfection processes should be done and if counts exceed 50/100 ml (high contamination) the pool should be closed to prevent illness in pool areas. Following should be considered as gross contamination of swimming pool water: a.
b.
> 10 E.coli cfu/100 ml in combination with one or both of the following: 1.
An unsatisfactory aerobic colony count (> 10 cfu/ml)
2.
An unsatisfactory P. aeruginosa count (> 10 cfu/100ml)
> 50 P. aeruginosa / 100 ml in combination with high aerobic colony count (> 100/ml) Additional microbiological testing in the event of an outbreak of illness
associated with a swimming pool may be considered necessary and tests should be carried out quarterly for Legionella, Staphylococcus aureus, Cryptosporidium, Giardia (Fig.3.2 a-b) and viruses. I. Most Probable Number (MPN) Technique for Detection of Coliforms: This test includes (a) presumptive test (b) confirmatory test and (c) completed test. Schematic presentation of these tests is given in Fig-1. A. Presumptive Test: (i) Inoculate each of 3 test tubes containing 10.0 ml of double strength lactose broth of a set aseptically with 10.0 ml of water sample. (ii) Similarly inoculate 1.0 ml and 0.1 ml of water samples into each of three small tubes of 2nd and 3rd sets respectively containing single-strength lactose broth using aseptic conditions. L 18
(iii) Incubate all tubes at 370C for 2 days. (iv) Observe for gas production after 24 and 48 hours. The presence of gas in any tube after 24 hr is a positive presumptive test. Find out number of tubes in each set showing gas production and determine MPN counts /100 ml of the water sample by comparing with Mac Greedy Chart (Annexure-IV) following the standard methods for examination of water and waste water given by APHA. The formation of gas during a second 24-hr period is a doubtful test. The absence of gas formation after 48 hr incubation shows negative presumptive test indicating that the water supply does not contain coliforms. B. Confirmed Test: This test should be applied to all samples that give a positive or doubtful presumptive test. (i) From the lactose-broth tubes showing positive presumptive test with smallest water sample, streak a plate of Eosine-methylene blue (EMB) agar (ii) Incubates at 370C for two days. (iii) If typical dark coloured colonies with metallic sheen develop on the plate within this period, the confirmed test can be considered positive. The colonies of E.coli are small and flat, and show a definite metallic green sheen. While Enterobacter aerogenes also grows on EMB agar but it does not produce as much acid as E. coli. Thus, E. aerogenes colonies are mucoid and slightly pink. Endo-agar is another selective medium that can be used in the confirmatory test. C. Completed Test: i).
From the EMB-agar plates pick up one colony, which is most likely to be organism of coliform group (E. coli), transfer half of the colony on agar-slant and the other half to lactose-broth tube.
ii).
Incubate the tubes at 370C for two days.
iii).
From agar-slant, make a slide and perform gram staining and spore staining.
iv).
Observe lactose broth tubes for gas production. Gram (-), non spore forming, short rods in the agar culture constitute a positive test, showing presence of coliform group of bacteria, and indicating that the water sample was polluted.
L 19
II. Biochemical tests for Differentiation of Faecal (Escherichia coli) and NonFaecal (Enterobacter sp.) coliform present in water samples: IMViC Test: Since, E. coli and E aerogenes bear a close resemblance to each other in their morphological and cultural characteristics, four biochemical tests are performed to differentiate them. These tests are collectively known as the IMViC tests. Each letter of IMViC stands for a reaction/property or a product, which can be used for both to characterize E. coli and to differentiate it from E. aerogenes (I - indole production, M - methyl red test, Vi - Voges-Proskauer reaction, and C - citrate utilization). Colonies from the nutrient agar slant of the completed test, described above, are used to inoculate Hi Media IMViC test kit with 50 microlitre/loopful of culture and the kit is incubated at 370C for 24-48 hours. 1. Indole test (I): E. coli produce/synthesize an enzyme, tryptophanase, which forms indole, pyruvic acid and ammonia from tryptophan, whereas E. aerogenes cannot catabolize tryptophan and do not produce indole. 2. Methyl red test (M): Methyl red is an acid-base indicator that turns red in a slightly acid medium. Both the organisms produce acid from glucose, E. coli produce large amount of acids thus a low pH, which turn the indicator (methyl red) to red colour
whereas E. aerogenes cultures produce only small amounts of
organic acids and consequently do not produce the colour change. 3. Voges-Proskauer (Vi) test: It detects the presence of acetyl methyl carbinol. E. coli do not produce acetyl-methyl-carbinol in glucose peptone medium but E. aerogenes do. 4. Citrate utilization test (C): E. aerogenes is capable of utilizing sodium citrate as its sole source of carbon, i.e. it will grow in a chemically defined medium in which sodium citrate is the only source of carbon. E. coli does not grow under these circumstances. (ii) Membrane Filter Technique: This technique has become common and is preferred. It involves the use of a sterile filter disk having pores fine enough to retain bacteria (
