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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

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

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?

Period 3

Materials

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

Demonstration of membrane filter method of coliform enumeration

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

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

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

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