Chapter 8   Fecal Coliform return to Table of Contents

 

                 An improperly treated or unprotected water supply may contain microorganisms that are pathogenic, that is, capable of producing disease.  Testing for specific pathogenic microorganisms (pathogens which cause diseases such as typhoid or dysentery) is very time-consuming and requires special techniques and equipment.  So instead of testing for pathogens, water is generally analyzed for the presence of an “indicator organism”, the coliform group of bacteria.

                 Coliform bacteria, or rather their absence, is a good index of the degree of bacteriologic safety of a water supply.  In general, coliform bacteria can be divided into a fecal and a non-fecal group.  Fecal coliform bacteria occur normally in the intestines of humans and other warm-blooded animals.  They are discharged in great numbers in human and animal wastes.  Coliforms are generally more hardy than true pathogenic bacteria and their absence from water is thus a good indication that the water is bacteriologically safe for human consumption.  The presence of coliforms, however, indicates the potential presence of pathogenic organisms that may have entered the water with them, and suggests that the water is not safe to drink.  No organism or group of organisms satisfies all of the criteria for an indicator; however, this group, the coliforms, satisfies most of the requirements.

                 The coliform group of bacteria comprise all the aerobic and faculative anaerobic gram negative, nonspore forming rod shaped bacteria that ferment lactose (a sugar) within 48 hours at 35^º C. In general, coliform bacteria can be divided into a fecal and a non-fecal group.  The fecal coliform can grow at a higher temperature (45^º C) than the non-fecal coliform.

               Total coliform tests are used for potable water supplies.  Fecal coliform tests usually are performed on untreated (nonpotable) water, wastewater, bathing water and swimming water.   Heterotrophic Plate Count measures a broad group of bacteria including non pathogens, pathogens, and opportunistic pathogens.  The significance of HPC the lies in its indication of poor general biological quality of the drinking water.  Five hundred (500) colonies per milliliter has been suggested as an upper level above which corrective action should be taken.

                 EPA is evaluating four analytical methods for testing water in distribution systems to determine whether total coliforms are present.  These are the Membrane Filter Technique, Multiple-Tube Fermentation Technique, Presence-Absence Coliform Test, and the Mixed Media ONPG-MUG test.

                 In the Membrane Filter (MF) Technique, a vacuum pulls 100 mL of water sample through a membrane filter held in place by a filter-holding device.  Total coliform and other bacteria are retained on the filter.  The filter is then placed on a special medium which allows the growth of total coliform and incubated at 35 ± 0.5^º C for 22 to 24 hours.  If a total coliform-like colony is observed on the membrane, the lab should make sure that it is a total coliform by using another EPA approved test.

                 The Multiple Tube Fermentation Technique (MPN) involves adding the water sample to a set of tubes, each of which contains either lactose broth or lauryl tryptose broth and an inverted tube.  The tubes are then incubated at 35 ± 0.5^º C for 24 to 48 hours (fecal coliforms are incubated in a water bath at 44.5 ± 0.2^º C).  If gas production is observed in an inverted tube after incubation, the sample contains total coliforms. 

                 The Presence/Absence (P/A) method is a qualitative test that indicates only the presence or absence or organisms, not the number of organisms.  In the Presence/Absence Coliform Test, 100 mL of the water sample is added to a bottle containing P/A Broth, and incubated at 35 ± 0.5^º C for 24 to 48 hours.  If the broth becomes yellow-colored, total coliforms are present.

                 The Mixed Media ONPG-MUG (MMO-MUG) test is the simplest of the EPA approved tests for total coliforms.  A 100 mL water sample is added to a flask containing the MMO-MUG powder, mixed, and incubated at 35 ± 0.5^º C for 24 hours.  The formation of a yellow color denotes the presence of total coliforms.

                     Under the total coliform rule, the laboratory must test all total coliform-positive samples for the presence of either fecal coliform or E. coli.

                 Fecal coliforms are the indicator organisms for possible pathogenic bacteria contamination.  The most common methods employed for the detection of fecal coliforms are the Most Probable Number (MPN) fermentation tubes, the Membrane Filtration (MF) procedure and the Colilert^tm version of the MPN.  The MPN test is described in SM₁₈ Method 9221, and the MF technique is SM₁₈ Method 9222.  The Colilert^tm procedure is not listed in the approved list of EPA methods for NPDES monitoring (40 CFR 136, Table 1A, as amended 31Jan94 in Federal Register  Vol 59 #20, pp. 4504-4515).  The MPN procedure remains the reference method when conflicting results are obtained by the two methods.

                 The multiple tube coliform test has been a standard method for determining coliform group bacteria since 1936.  In this procedure tubes of lactose broth or lauryl tryptose broth are inoculated with dilutions of a wastewater or water sample.  The coliform density is then calculated from statistical probability formulas that predict the most probable number, MPN, of coliforms necessary to produce certain combinations of gas-positive and gas-negative tube in the series of inoculated tubes.

                 The incubator time and temperature are critical when using the Membrane Filtration (MF) procedure in addition to the sterility of the dilution water and tools.  Liberal use of the autoclave is indicated, but DO NOT sterilize the culture media, membrane filters or disposable plastic petri plates in the autoclave.  The sample collection containers must be sterilized and contain the dechlorinating preservative, sodium thiosulfate.  The correct amount of sodium thiosulfate will give a 0.008% solution in the final sample.  This is 8 mg in 100 mL, or as commonly performed, 0.1 mL of a 10% sodium thiosulfate hydrate solution to a 120 mL sample for residual chlorine levels up to 15 mg/L.

                 The M-FC media at 44.5 ± 0.2^º C is highly selective for fecal coliforms.  The give varying shades of blue colonies.  Counts should be made with a 10X to 15X wide view binocular dissecting microscope.  Appropriate colony densities are between 0 and 60 colonies.  Normal ranges of filtered sizes are:

                 Source                                     Volume filtered

Lakes, reservoirs                              50 to 100 mL

Wells, springs                                    50 to 100 mL

Water intake supplies                         1 to   50 mL

Natural bathing waters                       1 to   50 mL

Secondary effluent                            0.1 to  10 mL

Rivers, farm ponds                           0.01 to     1 mL

Stormwater                                        0.01 to     1 mL

Raw sewage                                    0.001 to   0.1 mL

Feedlot runoff                                    0.001 to   0.1 mL

EPD requires filtering samples at three different dilutions for NPDES reporting.

                 Blanks should be run with each batch of samples to insure freedom from laboratory contamination.  Blanks are most easily prepared by filtering at least 100 mL of the sterile dilution water through a membrane and the treating it as a regular sample.  Positive controls must be run with each new batch of media and at least quarterly.  Positive E. coli control samples are available from the American Type Culture Collection, 1-800-638-6597.

                 Good laboratory technique is always important, but microbiological procedures are particularly fussy.  A clean lab, a clean work area, tight temperature control, and quality equipment and consumables can help assure reliable results.

                 The total coliform bacteria test includes both Escherichia and Aerobacter coliform bacteria groups.  Aerobacter and some Escherichia can grow in soil.  Therefore, not all coliforms found in the total coliform test come from human wastes.  Escherichia coli (E. coli) apparently are all of fecal origin.  However, it is difficult to determine E. coli without measuring soil coliforms too.  The fecal coliform and Escherichia coli tests are used in an attempt to more specifically determine the extent of human wastes in water.

 

                                           Materials

 

MF Fecal Coliform Checklist (9222, D Standard Methods 18th Edition)

          ___  1.  M-FC broth medium.

          ___  2.  45 mm disposable pre-sterilized culture dishes (Petri dishes).

          ___  3.  Water bath incubator set at 44.5 ± 0.2^º C.

          ___  4.  Vacuum filtration apparatus.

___  5.  Gridded 45 mm 0.45 lm pore size membrane filters, pre-sterilized.

___  6.  Stock phosphate buffer:  3.40 g KH₂PO₄ (potassium phosphate monobasic) is dissolved in 50 mL of reagent water.  The pH is adjusted to 7.2 ± 0.5 SU with 1 M sodium hydroxide, then diluted to 100 mL with reagent water.

___  7.  Magnesium chloride stock solution:  Dissolve 8.11 g MgCl₂ • 6H₂O) (magnesium chloride hexahydrate) in 100 mL of reagent water.

___  8.  Sterile buffered dilution water:  Dilute 1.25 mL stock phosphate buffer and 5.0 mL stock magnesium chloride to 1 L with reagent water.  Sterilize in the autoclave prior to use.

 

 

                   Procedure

 

          ___  1.  Sterilize all glassware and dilution water in autoclave, 121^º C and 14 psi for at least 15 minutes (not more than 45).  Sterilize filtration apparatus in autoclave.

          ___  2.  Assemble filtration apparatus with gridded side of the membrane uppermost.

          ___  3.  Seat membrane by filtering about 5 mL of sterile dilution water and filtering with vacuum.

          ___  4.  Add sample to filter funnel.  If less than 20 mL sample is being filtered, add 10 mL sterile dilution water to filter funnel prior to the sample to aid in dispersion of the organisms over the surface of the filter.

          ___  5.  Rinse filter and funnel with three 20 to 30 mL portions of sterile dilution water.

        ___  6.  Add 2 mL M-FC broth to absorbent pad in culture dish.

          ___  7.  Using sterile forceps, place membrane on top of the pad, gridded side uppermost, so that no air is trapped under the membrane.  Place lid tightly on the culture dish, label dish with wax pencil and place in incubator bag upside down.

          ___  8.  Prior to the next sample, submerge the filtration apparatus in boiling water for 5 minutes or place unit in UV sterilization cabinet for 2 minutes.

          ___  9.  After all samples are prepared, seal the incubator bag and submerge in the incubator.  All the samples must be in the incubator within 30 minutes of being filtered.

          ___ 10. Record the time, date and temperature of the incubator on the lab work sheet.

          ___ 11. After 24 ± 2 hours incubation, remove the plates from the incubator and record the time, date and temperature of the incubator on the benchsheet.

          ___ 12. Count the colonies on each membrane and record the number on the benchsheet.

          ___ 13. Calculate results for the samples and update precision control chart with RPD from duplicate. 

        ___ 14. Sterilize used plates in autoclave prior to disposal.

 

Questions for Chapter 8

1.  What is the difference between the presumptive and confirmed tests?

2.  Why do we check for coliform bacteria?

3.  How much gas needs to be present in the MPN tube in order to consider it positive?

4.  What do you do if the test is positive?

5.  If the MFT sample has a count of 5 colonies/100 mL, it means that the water is ...

6.  If you use 4 mL of sample and get a count of 24 colonies, how would you record it as ... per 100 mL of sample?

7.  At what temperature must the incubator be kept for coliform testing?

8.  How long and at what temperature can M-Endo agar plates be stored?

9.  At what temperature and pressure do you operate the autoclave?

10.  At what temperature and pressure do you operate the hot air sterilizer?

11.  What is the difference between the MFT and MPN?

12.  What is the HPC and what is it’s purpose?

13.  What is the minimum amount of time the MF funnels must remain under the UV sterilizer lights in between samples?

14.  What is the difference between the MFT and Colilert (MMO-MUG)?

15.  Media should be ordered to last no more than ...

16.  What are the approved methods for detecting coliform bacteria?

17.  You must use ... mL of sample regardless of the method used.

18.  The minimum number of samples that a water system must collect is based on ...

19.  If a routine distribution sample is total coliform positive, how long do you have to collect your repeat samples upon being notified of the positive result?

20.  Where must the repeat samples be collected?

21.  The MCL for total coliforms is based on the ... of total coliforms.

22.  For a system that collects at least 40 samples/month, the system is in compliance if no more than ... % of the samples are coliform positive.

23.  A system that collects fewer than 40 samples/month can have ... samples total coliform positive in order to remain in compliance.

24.  How many repeat samples must be collected?

25.  What must the chlorine residual be at the furthermost point in the distribution system?

26.  The turbidity of the finished water should not exceed ... NTU.  Why?

       Three types of algae are:  Cymbella, Spirogyra, and Asterionella.  How will they affect the water treatment process?

       Why do we use sodium thiosulfate in the sample bottles?

       What are the sterilization times for:  a) glassware, MF assemblies, sample bottles; b) dilution blanks and rinse water; c) filters and pads; d) kill run, using the autoclave?

On to Chapter 9	My question sets for self exam	errors?