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Experiment 14V – Introduction to Microbial Water Analysis

Water is essential to life and our way of life. We drink it, cook with it, bathe in it, and use it to process sewage and other wastes. Pathogenic organisms such as those causing typhoid, cholera, hepatitis, polio and protozoal infections are shed in feces and can be discharged into the environment with improperly-treated sewage. The stream receiving this discharge can be used by downstream residents for drinking or recreational purposes, and the pathogen enters a new infectious cycle if ingested. Strict standards have been imposed by the Environmental Protection Agency (EPA) to regulate the bacteriological quality of industrial and municipal waste discharges and to set safe microbial limits for drinking water, swimming pools and beaches.

I. THE CONCEPT OF INDICATOR ORGANISMS

Due to the wide variety of intestinal pathogens (bacteria, viruses, protozoa) that could be found in feces, sewage and (ultimately) water, it would not be cost-effective to test specifically for the presence of each different pathogen. This is where the concept of the indicator organism applies. Generally, an “innocent” organism which often finds itself associated with (i.e., in the same natural habitat as) problem-causing organisms can be, when isolated from a site, an indication that the problem organisms may also be present at that site. Such an associated organism is therefore an indicator of the possible problem.

Specifically, if pollution of a water resource by sewage or other fecal pollution (and the associated intestinal pathogens) is suspected, the general attributes of an indicator organism will be adapted to follow these criteria:

1. It is always present in the feces from both normal and infected individuals.

2. It is not found anywhere in the environment except where there is contamination by fecal pollution.

3. It survives longer in the environment than pathogens, but not so long as to indicate “historical” contamination.

4. It is easy to detect in the laboratory in that its characteristics can be exploited to make it easily enriched for and isolated (according to principles we learned in Exp. 11).

5. It should not cause a “problem” itself and pose a health risk for laboratory workers.

II. COLIFORMS: THEIR DEFINITION, selective ENRICHMENT

and INITIAL detection

Coliforms are considered indicator organisms in food and water analysis – not because they are “bad” in themselves, but because they are often associated with potential health problems. Two examples:

1. Identification of an isolate from any source as Escherichia coli would then implicate the source

as being contaminated by intestinal waste (fecal material), which is the habitat of E. coli. It is much easier to find whether or not E. coli is present through the enrichment and isolation procedure than it is to test for each specific intestinal pathogen (bacterial, protozoan and viral).

2. Coliforms are abundant in soil, and if they are found in well water, that would be an indication

of probable surface soil contamination of the water. Usually such coliforms are identified as Klebsiella and Enterobacter.

The basic definition one encounters about coliforms is that they are gram-negative rods that ferment lactose rapidly with the production of gas – i.e., insoluble gas that is detectable in a Durham tube. A more precise definition would specify that they can grow and show gas production from fermentation in Lactose Lauryl Tryptose Broth and Brilliant Green Bile Broth at 35°C. Those which additionally do so in EC Broth at 44.5°C belong to the subset of coliforms called “fecal coliforms.” Many “old school” references include “aerobic or facultatively anaerobic” in the definition, but – as we found out in Virtual Experiment 5A – no one organism can be both. All coliforms are facultatively anaerobic, being able to aerobically respire and also ferment at least glucose.

With these characteristics in mind, coliforms happen to be easy to detect with the appropriate selective-differential media in the enrichment process. These media tend to inhibit gram-positive bacteria, and the presence of coliforms (among the other organisms which would also be present) is suggested by gas from lactose fermentation in the Durham tube.

1. As the bacterial cells in the sample to be tested may have been impaired from osmotic incom-

patability, starvation, chlorination or other harmful conditions, it would be additionally harmful to subject them to an enrichment medium with harsh selective agents. Thus we use a two-stage enrichment where the initial medium (Lactose Lauryl Tryptose Broth) aims primarily to “revive” the damaged coliform cells such that they can increase their numbers and be detected by gas resulting from lactose fermentation.

2. The subsequent enrichment media (Brilliant Green Lactose Bile Broth and EC Broth) can then

select more effectively against gram-positive organisms without putting undue stress on the coliform population.

The enrichment media in the following table increase in selectivity for the desired organisms from left to right. We never expect a pure culture in any enrichment medium (selective or otherwise), so the subsequent isolation (on EMB Agar, below) is important as in any enrichment-isolation procedure, as noted in Experiment 11. For a tube that shows growth and gas, suggesting the presence of coliforms, all it might have taken for a population of coliforms to develop in the tube could have been just one coliform cell in the inoculum. Cells of non-coliforms may or may not be able to grow in any of these media. However, one way or another in the enrichment and subsequent isolation process, they will be eliminated from consideration.

Organisms in the water sample that may grow in LLTB.	Note that growth with gas indicates the probable presence of coliforms in LLTB and is confirmatory in the other two media that are inoculated from LLTB
LLTB	BGLB	EC Broth at 44.5°C
The true coliforms: “fecal” and others.	growth & gas	growth & gas	growth & gas for fecal coliforms (no growth for other coliforms)
Occasional strains of Bacillus and Clostridium that ferment lactose to acid and gas (“false coliforms”).	growth & gas	no growth	no growth
Some Gram-negative bacteria other than coliforms. (Pseudomonas may persist in any of these media.)	growth with no gas	growth of fewer kinds (no gas)	growth of still fewer kinds (no gas)
Some rare Gram-positive bacteria other than those in the 2nd row.	growth with no gas	no growth	no growth

III. COLIFORMS: THEIR ISOLATION AND IDENTIFICATION

With the use of EMB Agar (which contains lactose and gram-positive inhibitors) in the isolation process, coliforms are detected by their acidic colonies from which isolates are chosen for identification by the use of the appropriate differential media.

One must realize that coliforms do not constitute a discrete taxonomic group. Usually they are ultimately identified as various species of enteric bacteria of the genera Escherichia, Enterobacter, Klebsiella and Citrobacter, and a coliform isolate from an EC Broth enrichment is usually identified as E. coli. That does not mean that these genera are to be therefore categorized as coliforms! Out in the real world, one may encounter many “exceptional” strains of these genera that do not conform to the definition of coliform. Many pathogenic E. coli strains do not ferment lactose, and it is sometimes very difficult to differentiate them from Shigella. Also, many strains of Citrobacter slightly ferment lactose (or do not do so at all) and may be initially confused with Salmonella when isolations are made from clinical or natural samples. So, considering the absolute definition of this non-taxonomic term, there can be no such thing as a non-lactose-fermenting coliform.

IV. BACTERIOLOGICAL STANDARDS FOR WATER

The bacteriological standards set by the EPA for water depend on the highest use of that water, with drinking water requirements receiving the strictest controls. Drinking water is monitored for the presence of total rather than just fecal coliforms. The reasoning behind this is that ground water, from which most drinking water is obtained, has been filtered as it percolated through the soil to enter the water table. This movement should effectively filter out organisms that are routinely encountered in surface runoff water. The presence of any coliform would then tend to indicate that water is entering the ground water from such things as abandoned wells or poorly-functioning septic tanks. A drinking water sample must contain fewer than 2.2 coliforms per 100 ml to be considered bacteriologically “safe.” (How one goes about quantifying coliforms is addressed in the next section.)

Swimming pools are monitored for coliforms to assess the relative efficacy of filtering and disinfecting procedures. Pools and hot tubs also must contain fewer than 2.2 coliforms per 100 ml to be considered “safe.” In these circumstances, total plate counts and counts of Staphylococcus aureus and Pseudomonas aeruginosa are also made to assess the potential for skin, eye, ear, nose and throat infections by these organisms.

Natural bathing beaches are monitored throughout the swimming season for fecal rather than total coliforms. Total coliform counts in surface waters are subject to a wide range of fluctuations having little sanitary significance. Beaches are closed when fecal coliforms in a single sample rise to greater than 400 per 100 ml or when the monthly geometric mean is more than 200 per 100 ml.

Adequacy of municipal and industrial waste treatment techniques can be evaluated through total plate counts of samples taken from various points in the treatment design. When organic materials and aquatic nutrients (nitrate and phosphate) are removed from wastewater, the bacterial load is also greatly decreased. The EPA and state regulatory agencies have set a discharge limit of 200 fecal coliforms per 100 ml of effluent sample for municipal sewage treatment plants and certain industrial dischargers.

EXP 14V. Multiple Tube Fermentation Test and MPN Analysis of a Water Sample

SUGGESTED DILUTIONS FOR VARIOUS EXPECTED DEGREES OF CONTAMINATION

sample type	COLIFORM MPN
Tap water Drinking water	10 ml, 1 ml, 0.1 ml
Clean surface water	100, 10–1, 10–2, 10–3, 10–4
Moderately-polluted surface water Treated sewage	10–2, 10–3, 10–4, 10–5, 10–6
Heavily-polluted surface water Raw sewage	10–3, 10–4, 10–5, 10–6, 10–7

Period 1

Materials

1 water sample labeled as one of the several types in the table at the bottom of this page

2 or 3 saline dilution blanks (99 ml)

15 tubes of Lactose Lauryl Tryptose Broth (LLTB; with Durham tube)

Pipettors and sterile tips

Procedure

1. We will now set up the presumptive test. Consulting the table above, label 3 tubes of LLTB for each of the five dilutions.

2. Inoculate 3 tubes for each plated dilution by inoculating either 1 ml or 0.1 ml from the appropriate dilution bottle into each tube.

3. Incubate all tubes at 37°C for 1-2 days. (The standard temperature is 35°C, but 37°C is OK for our purposes.)

Period 2

Materials (per pair)

Up to 15 Tubes of Brilliant Green Lactose Bile (BGLB) Broth and EC Broth (each with Durham tube)

44.5°C water bath

Procedure

For the MPN method we will be reading the presumptive test and starting the confirmed test.

· For each set of 3 tubes, determine the number of positive tubes. Growth and gas must both be present for a positive tube! Note the results on the data sheet.

· Calculate the presumptive, most probable number of coliforms/ml of the sample using the MPN table in virtual experiment

· For each positive tube, procure an equal number of tubes of BGLB and EC Broths. From each positive tube, inoculate (by loop) a tube of each medium. Incubate the BGLB Broth at 37°C and the EC Broth in the 45°C water bath, each for 2 days.

· If all of the tubes were negative, would you then conclude that there were zero coliforms in the sample? Using the MPN table, which 3 sets of tubes would you use to find the actual solution? Similarly, how would you interpret a case in which all tubes are positive?

Period 3

Materials

2 plates of Eosin-Methylene Blue (EMB) Agar (per pair)

Procedure (reading Confirmatory Tests and beginning Completed Test for coliforms)

1. For each of your broth media, record the number of positive (growth and gas) tubes as in the previous period. In each case, you are still dealing with 15 tubes; any missing tube is scored as negative, as any negative Presumptive Test tube would automatically yield a negative result in either Confirmatory Test. Indicate the results on the data sheet.

2. For the BGLB Broth tubes, calculate the confirmed, most probable number of coliforms/ml of the sample. For the EC Broth tubes, calculate the confirmed, most probable number of fecal coliforms/ml of sample. Record these values on the data sheet.

3. From a BGLB Broth tube of the highest dilution showing growth and gas, streak a plate of EMB Agar for isolated colonies. Do the same for the EC Broth. Incubate the plates at 37°C for 1-2 days.

Period 4

Materials

Tubes of Lactose Fermentation Broth

Tryptone Broth,

MR-VP Broth

Simmons Citrate Agar

1. Observe your plates of EMB Agar and note the demonstration plates. Colonies of gram-negative, lactose-fermenting bacteria will show a relatively dark color. Of these colonies, one usually notes either or both of the following classical types of coliform colonies:

· Coli-type

· colonies are very dark, almost black, when observed directly against the light. Usually a green sheen is seen by reflected light. This sheen is due to the precipitation of methylene blue in the medium, a result of the very high amount of acid produced from fermentation. Those that form this type of colony are methyl red-positive organisms including E. coli and those strains of Citrobacter which ferment lactose rapidly.

· Aerogenes-type

· colonies are less dark. Usually a dark center is seen surrounded by a wide, light-colored, mucoid rim. Those that form this type of colony are methyl red-negative organisms including Klebsiella and Enterobacter.

2. Choose one or more different colonies and inoculate each into Lactose Fermentation and Tryptone Broths (as for Experiment 7) and also MR-VP Broth and Simmons Citrate Agar (as for Experiment 11). The latter 3 media are used for the IMViC tests. We will only be doing the methyl red test on the MR-VP Broth culture.

3. Incubate at 37°C for 1-2 days (30°C if longer). Recall that the MR-VP Broth must be incubated for at least 2 days before performing any test on it.

Period 5

Materials

Dropper bottles of Kovacs reagent and methyl red

Procedure

1. For each coliform isolate, perform the necessary tests or observations on each medium as in

previous experiments. Each isolate must have fermented lactose to acid and visible gas in order to be considered a coliform.

2. Record your results on page 85 and compare them with those seen in the table below.

KEY FOR TENTATIVE IDENTIFICATION OF THE COLIFORM ISOLATES

Note: All coliforms will produce acid and gas in Lactose Fermentation Broth.

1 Voges-Proskauer Test was not run. Usually it gives the opposite reaction of the Methyl Red Test.

2 The symbol ± indicates “equivocal” (orange) reaction in the Methyl Red Test. See Exp.14.6.

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