Microbial Water Analysis

Introduction to microbial water analysis

Water, the universal solvent, is essential to life. In drier climes, people will even fight over it (look in a newspaper and read about water rights in Colorado and California). Critical to our modern civilization is the availability of a clean water supply for bathing, drinking and cooking. Unfortunately, many pathogens are transmitted through the water supply. Some of these disease-causing pests enter water from the feces of ill individuals and are then ingested and thereby transmitted to others. Diseases such as polio, typhoid, cholera, hepatitis, shigellosis, salmonellosis and others can spread in this manner. To assure a safe water supply, it is critical to monitor for the presence of these pathogens. However, it would be expensive and time consuming to check the water supply for all of them; instead, an indicator organism is used to assay for fecal contamination. Indicator organisms must have four properties to be useful for water analysis.

1. The only natural environment of the microbe should be in association with feces and it should always be present.
2. It should not grow outside of its natural environment.
3. The bacterium should survive longer than the most viable pathogen, but not so long so that historical events are detected.
4. It should be easy to detect.

Coliforms come closest to fulfilling all these criteria and are the standard indicator organisms used to test for the biological pollution of water. Enterobacter and Klebsiella are able to survive and multiply in the environment and are therefore not the best indicators of fecal pollution. The sole habitat of E. coli and K. pneumoniae, termed fecal coliforms, is the intestines of warm blooded animals. Thus, fecal coliforms are good indicators of fecal pollution and can be differentiated from other coliforms by incubating on selective media at 44.5°C.

Using coliforms, the EPA has developed standards for clean, safe water. These standards vary, depending upon the waters intended use. Drinking water and the water in swimming pools must be of the highest purity. There can be no more than one positive sample (>1 coliform/100 ml) in 40 samples tested in a month and the concentration of fecal coliforms must be zero. But wait, we just said that some coliforms are present in the environment. How can these standards be met? In good quality well water most microorganisms are filtered out as the water percolates from the surface to the well. Unusually high numbers of coliforms in well water may indicate run-off from a polluted area. In the case of surface waters (rivers and lakes), filtration through a sand bed and chlorination remove most microbes. There may also be further steps that need to be taken to insure water safety depending on the treatment plant. Swimming pools, being open surface water, are often contaminated by organisms in the air or by swimming, bacteria-infested humans. Chlorine is added to keep numbers low. Note that the number of permissible coliforms is not zero. This would be difficult to achieve and would provide no additional gain in safety.

Natural bathing beaches and treated sewage are assayed for numbers of fecal coliforms. Total coliform counts are not used as a measure due to the near ubiquitous presence of Enterobacter and Klebsiella in the environment. If the count reaches > 400 fecal coliforms per 100 ml or a monthly geometric average of > 200 per 100 ml, it may indicate a problem in the sewage treatment process or that the beach should be closed. The latter often happens in heavily utilized beaches during the summer.

Testing for coliforms

Presently, several tests are in use to assay for coliforms in water, The oldest of these is the multiple tube fermentation test. In this test three steps are performed; the presumptive, confirmed, and completed tests. A moderately selective lactose broth medium (Lactose Lauryl Tryptose Broth), containing a Durham tube, is first used in the presumptive test to encourage the recovery and growth of potentially stressed coliforms in the sample. If harsher selective conditions are used, a deceptively low count may result. A tube containing both growth and gas is recorded as a positive result. It is possible for non-coliforms (Clostridium or Bacillus) to cause false positives in this medium and therefore all positive tubes are then inoculated into a more selective medium (Brilliant Green Lactose Broth or EC Broth) to begin the confirmed test.

The confirmed test medium effectively eliminates all organisms except true coliforms or fecal coliforms, depending upon the medium and incubation conditions. If a positive result is recorded in these tubes the completed test is begun by first streaking a loopful of the highest dilution tube which gave a positive result onto highly selective Eosin Methylene Blue (EMB) agar. After incubation, subsequent colonies are evaluated for typical coliform reactions.

The multiple tube fermentation test has the great disadvantage of taking 3-5 days to complete. If a municipality has a drinking water crisis, this is too long to wait. This has lead to development of faster, less complex tests. In the membrane filter technique 100 ml or greater of a test sample is passed through a filter with pores small enough to retain all bacteria in the sample. The filter is then placed on a selective medium that allows for the detection of coliforms. The advantages of this technique are the shorter time needed to complete the test (1 day vs. 3 to 4 days), its low cost, the higher accuracy in counting, since the colonies can be enumerated directly from the plate, and its simplicity. Disadvantages are that particulate samples (containing silt or other organic matter) quickly clog the filter, metals and phenols can stick to the filter inhibiting growth, and non-coliforms in the test sample may interfere with the formation of coliform colonies on the plate.

Recently, less complex tests for the detection of coliforms have become available. In the presence-absence test (P-A test), a large water sample (100 ml) is mixed with triple strength LLTB in a single culture bottle. Brom cresol purple is added as a pH indicator. If present, coliforms will ferment the lactose to acid and gas, turning the medium from purple to yellow. To detect coliforms and E. coli, the Colilert defined substrate test can be used. A 100 ml sample of water is mixed with a medium containing ortho-nitrophenyl-β,D-galactoside (ONPG) and 4-methyl umbelliferyl-β,D-glucoronide (MUG) as the only nutrients. If coliforms are present ONPG is metabolized, resulting in a yellow color. If E. coli is present, it will degrade MUG to a fluorescent product that can be detected by observation under long wave length UV-light. Both the P-A test and the Colilert defined substrate test are preliminary and any positive results will warrant further analysis of the offending sample.

The most probable number method of enumeration (MPN)

In this experiment we will detect coliforms in a water sample using the multiple tube fermentation method. Enumeration of coliforms using this method involves inoculating multiple tubes with a 10-fold dilution series of the water sample and uses the most probable number (MPN) technique to estimate the population. To understand this technique, let us imagine preparing a 10^-1 to 10^-6 dilution series of a culture and inoculating 1 ml portions into tubes containing nutrient broth. After incubation, growth is observed in the tubes inoculated with the 10^-1 to 10^-5 dilutions, but not in the 10^-6 tube. The number of organisms in the original sample is estimated to be less than 10⁶, but greater than 10⁵ bacteria per ml. By inoculating 3 or 5 tubes per dilution and using statistical analysis, a more accurate estimate of the bacterial concentration can be made. This is the basis of the MPN method. Realize that if a 10 ml solution contains 20 organisms, each 1 ml sample will probably not contain exactly 2 organisms. There will be some variation (0, 1, 2, 3 or 4 organisms/ml), but the total of ten 1 ml portions will add up to 20. This is why at lower dilutions, one tube inoculated with a certain dilution blank may show growth (receiving 1 or 2 organisms when inoculated) while other tubes inoculated with the same dilution do not (no organisms in the 1 ml). The number of organisms is assessed by counting the number of positives in the last three dilutions showing growth and then determining the MPN by following the directions on an appropriate table. An excellent explanation of the MPN method has been created by John Lindquist from UW-Madison.

Water analysis procedure

We will use two techniques to determine microbial density in our water samples. The so-called total aerobic plate count will be used to get a general estimate of colony-forming units per ml of the sample. Remember from Experiment 4 that this technique is neither total nor aerobic when strictly applying these terms to the microorganisms, but we will be able to enumerate most common chemoheterotrophic bacteria able to grow in the all-purpose medium (Plate Count Agar) in the presence of air at 30°C. We will also perform the most probable number method of enumeration as discussed in the introduction for thie experiment for a specific morphological/physiological group, the coliforms (and specifically the fecal coliform subgroup).

Observation of proper aseptic technique, efficient mixing and accurate pipetting are the keys to this experiment!

Period 1

Materials

Work in Pairs

1 water sample

2 or 3 saline dilution blanks (99 ml)

8 sterile petri dishes

2 bottles of melted Plate Count Agar (PCA; 100 ml/bottle) - in 50°C water bath

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

Pipettors and sterile tips

Table 1: Plated Dilutions (or Amounts of Undiluted SAMPLE) Suggested For Various Expected Degrees of Contamination

Sample Type	Total Plate Count	Coliform MPN
Tap water or Drinking water	100, 10-1, 10-2, 10-3	10 ml, 1 ml, 0.1 ml
Clean surface water	10-1, 10-2, 10-3, 10-4	100, 10-1, 10-2, 10-3, 10-4
Moderately-polluted surface water or Treated sewage	10-2, 10-3, 10-4, 10-5	10-2, 10-3, 10-4, 10-5, 10-6
Heavily-polluted surface water or Raw sewage	10-4, 10-5, 10-6, 10-7	10-3, 10-4, 10-5, 10-6, 10-7

Use this table to figure out how far you need to dilute your sample.

1. Record the source and type of water sample you have received (e.g., moderately-polluted surface water, treated sewage).
2. Label the 99 ml water blanks with even-numbered dilutions.
3. Mix the water sample vigorously and prepare centimal (1/100) dilutions in the 99 ml dilution blanks. Be sure each dilution is mixed thoroughly before making inoculations from it. Looking forward to steps 2 and 3, it is possible to inoculate the plates and tubes while making the dilutions. Be sure to obtain a new pipette tip when you are about to work with a new dilution.
4. Total aerobic plate count. Label 8 sterile petri dishes with the plated dilutions* and your identifying marks. You will be plating 4 dilutions in duplicate. Regarding plated dilutions, remember that this term ultimately refers to the equivalent amount of undiluted sample which is being plated.
5. Inoculate 2 plates for each plated dilution by inoculating either 1 ml or 0.1 ml from the appropriate dilution bottle into each plate. (Having gone through dilution theory and plating exercises before, this should be old stuff. But, review Appendix C if necessary.) To save some time and pipette tips, you can do your tube inoculations (step 3) at the same time.
6. Obtain 2 bottles of melted PCA from the water bath (1 bottle per 4 plates) and wipe them dry with a paper towel. Following the instructor's demonstration, pour enough medium into each plate such that they are about a third to half-filled. Immediately after the plates are poured, gently rotate each plate (reversing the direction a few times) to mix the inoculum thoroughly with the medium.
7. When the medium has hardened, invert the plates and incubate them at 30°C.
8. We will now set up the presumptive test. Consulting the table above, label 3 tubes of LLTB for each of the five plated dilutions.
9. Inoculate 3 tubes for each plated dilution by inoculating either 1 ml or 0.1 ml from the appropriate dilution bottle into each tube. Note comments regarding dilution theory and pipetting in step 2b, above.
10. Incubate all tubes at 37°C for 1-2 days. (The standard temperature is 35°C, but 37°C is OK for our purposes.) Note: If the next period is more than 2 days away, place the tubes into the baskets on the stage and be sure the tubes are labeled such that you will be able to retrieve them easily next period. The tubes will be refrigerated until 2 days before the next period, at which time they will be incubated as required.

Period 2

Materials

(per pair)

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

44.5°C water bath

Figure 1: Total aerobic count

Results of the total aerobic count of the water. Be sure to count the plate with between 30 and 300 colonies. To get a better look at a plate, click on the image.

Figure 2: LLTB

Typical growth results in lactose lauryl tryptose broth. Use these tubes to calculate an MPN.

1. For the total aerobic plate count, find the pair of plates where between 30 and 300 colonies can be counted on each plate.
2. Note the variety of different sizes of colonies on and in the medium. All colonies must be counted. As previously, the colonies can be counted quickly by first dividing the bottom of the plate into sectors, then scanning the sectors. If none of your dilutions have the proper number of colonies, use the set that most nearly approaches the 30-300 range.
3. Record your results in your notebook.
4. Calculate the total aerobic plate count as no. of CFUs/ml of the (undiluted) sample.
5. For the MPN method we will be reading the presumptive test and starting the confirmed test.
6. 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.
7. Calculate the presumptive, most probable number of coliforms/ml of the sample using the MPN table in Figure 15-12
8. 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 4
9. 5°C water bath, each for 1-2 days. If the next period is more than 2 days away, place the tubes in the baskets on the stage.
10. 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?

Table 2: The MPN table

No of Tubes Positive In				No of Tubes Positive In
first	middle	last	MPN	first	middle	last	MPN
0	0	0		2	0	0	0.091
0	0	1	0.03	2	0	1	0.14
0	0	2	0.06	2	0	2	0.20
0	0	3	0.09	2	0	3	0.26
0	1	0	0.03	2	1	0	0.15
0	1	1	0.061	2	1	1	0.20
0	1	2	0.092	2	1	2	0.27
0	1	3	0.12	2	1	3	0.34
0	2	0	0.062	2	2	0	0.21
0	2	1	0.093	2	2	1	0.28
0	2	2	0.12	2	2	2	0.35
0	2	3	0.16	2	2	3	0.42
0	3	0	0.094	2	3	0	0.29
0	3	1	0.13	2	3	1	0.36
0	3	2	0.16	2	3	2	0.44
0	3	3	0.19	2	3	3	0.53
1	0	0	0.036	3	0	0	0.23
1	0	1	0.072	3	0	1	0.39
1	0	2	0.11	3	0	2	0.64
1	0	3	0.15	3	0	3	0.95
1	1	0	0.073	3	1	0	0.43
1	1	1	0.11	3	1	1	0.75
1	1	2	0.15	3	1	2	1.2
1	1	3	0.19	3	1	3	1.6
1	2	0	0.11	3	2	0	0.93
1	2	1	0.15	3	2	1	1.5
1	2	2	0.5	3	2	2	2.1
1	2	3	0.24	3	2	3	2.9

This MPN table is for calculating MPN using 3 growth medium tubes per dilution.

Period 3

Materials

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

Demonstration of membrane filter method of coliform enumeration

Figure 3: Results in BGLB

Typical growth results observed for brilliant green lactose bile broth. Use these tubes to determine an MPN

Figure 4: EC broth

Typical growth results observed for EC broth. Use these tubes to determine an MPN

1. Note the demonstration of the membrane filter method of enumerating coliforms.
2. 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.
3. 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.
4. 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 (or place on stage for special incubation).

Period 4

Materials

Tubes (as needed) of Lactose Fermentation Broth, Tryptone Broth, MR-VP Broth and Simmons Citrate Agar

Demonstration plates of various colony types on EMB Agar

Figure 5: EMB agar

Colony types observed on EMB agar. Both fish-eye-type and coli-type colonies are shown. Photos courtesy of John Lindquist.

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 which 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 which 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

Figure 6: Lactose fermentation broth

Typical reactions in LFB. For coliforms, all tubes should be positive. Why?

Figure 7: Indole reaction

The classic indole reaction. Both a positive and negative are shown.

Figure 8: Methyl red reactions

Reaction of the methyl red test

Figure 9: Simmon citrate medium

Reactions in Simmons citrate medium

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. Compare your results for the indole, methyl red and citrate tests to the table below which shows the IMViC reactions (including the Voges-Proskauer test expected for typical coliforms.
3. Time permitting, more complete characterization may be accomplished with the use of MIO Medium and other media used in Experiment 14
4. To practice determining the genus of water analysis isolates, use the following web page. Write down the number of your unknown then compare it to tht table. See if you can work out what the identity of the microbe

Table 3: Correlation of IMViC Results with Probable Identification

indole	methyl red	Voges-Proskauer	citrate	probable identification
+ (-)	+	-	-	Escherichia coli
- (+)	- (+)	+	+	Enterobacter or Klebsiella
- (+)	+	-	+	Citrobacter

Reactions for the tests performed and their likely species identification. (Occasional reactions are shown in parentheses.)

References:

1. http://inst.bact.wisc.edu/inst/index.php?module=Book&func=toc&book_id=3

Note: For the media composition, Please read the next post -The Media Formulation