Lesson 17:

Fecal Coliforms

 

 

Objective

In this lesson we will learn the following:

 

 

 

Reading Assignment

Read the online lecture, also read in your textbook Chapter 16, lesson 7 : Operation of Wastewater Treatment Plants Vol. I1

 

 

 

Lecture

Fecal Coliforms: General

As in any type of industry, some form of process monitoring is required to ensure that the quality of the final product is maintained at the highest possible level.  In the wastewater treatment industry, laboratory testing is one of the methods used to ensure that a high quality effluent is maintained.  Accurate and reliable laboratory analyses are absolutely necessary to monitor effluent characteristics and to provide a basis for making operational changes in the treatment process itself.

 

 

Introduction to Fecal Coliform Testing

Fecal coliform bacteria are non‑disease causing organisms which are found in the intestinal tract of all warm‑blooded animals.  Each discharge of body wastes contains large amounts of these organisms.  The presence of fecal coliform bacteria in a stream or lake indicates the presence of human or animal wastes.  The number of fecal coliform bacteria present is a good indicator of the amount of pollution present in the water.

Most waterborne disease-causing organisms originate in human or animal bodies and are discharged as part of body wastes.  Due to the relatively small numbers of disease‑causing organisms, it is very difficult to isolate and identify specific disease‑causing bacteria.  Since fecal coliform bacteria originate in the same location, they are used as an indicator of possible disease hazards in a body of water.  The presence of very few fecal coliform bacteria would indicate that a water source probably contains no disease‑producing organisms, while the presence of large numbers of fecal coliform bacteria would indicate a very high probability that the water source could contain disease‑producing organisms.  For this reason, regulatory agencies with responsibility for protection of public health have established water quality standards which include maximum levels of fecal coliform bacteria.

 

 

 

Glossary

Aerobic:  A condition in which “free” or dissolved oxygen is present in the aquatic environment.

Aerobic Bacteria:  Bacteria which can live and reproduce only in the presence of “free” or dissolved oxygen.

Aseptic Conditions:  Free of contamination by living microorganisms, i.e. bacteria.

Bacteria:  Single cell organisms which can only be seen by means of a microscope.

Bacterial Culture:  A group of bacteria.

Broth:  A mixture of chemicals which will encourage the growth of a specific organism or group of organisms.

Buffer:  A chemical which has the ability or capacity to neutralize acids or bases.

Coliform:  A group of bacteria which can be used as an indicator of pollution.  A major portion of this group live in the intestinal tract of warm blooded animals, including human beings.  They are easy to identify and count in the laboratory because of their ability to ferment lactose.

Colony:  A group of bacteria growing on a supporting surface.  The colony is considered to be the result of the growth and reproduction of a single cell.

Disinfection:  To destroy most (but not necessarily all) of the harmful or objectionable microorganisms by means of chemicals, heat, ultraviolet light, etc.

Fermentation:  The process by which bacteria convert organic matter into carbon dioxide and water.

Fermentation Tube:  A container designed to allow easy identification of gas production.

Fecal Coliform:  A subclass of the coliform bacteria which originate almost exclusively in the intestinal tract of warm blooded animals.

Inoculation:  The process by which a sample or seed culture is introduced into a system.

MPN:  The most probable number (MPN) of coliform or fecal coliform bacteria per unit volume of a sample.  It is expressed as the number of organisms which are most likely to have produced the laboratory results noted in a particular test.

Medium (Media):  A substance (or substances) used to provide nutrients for the growth of bacteria.

Microorganisms:  Very small organisms which can only be seen through a microscope.

Pathogenic Organisms:  Bacteria, viruses, protozoa, etc. which can cause disease in animals or human beings.  There are many types of organisms which do not cause disease and are essential to plant operation.  Typical pathogenic organisms in wastewater include hepatitis and polio viruses, the bacteria which can cause cholera and typhoid, and various parasites (i.e., worms, amoebae, Giardia, etc.).

Sterilization:  Destruction or removal of all viable or living organisms.

 

 

 

Approved Methods

The 18th Edition of “Standard Methods for the Examination of Water and Wastewater” includes two methods for the determination of fecal coliform in wastewater:  the multiple tube fermentation (MPN) procedure and the membrane filter (MF) procedure.  These methods are also described in the US EPA publication “Microbiological Methods for Monitoring the Environment, Water and Waste.”  Because the MF procedure can yield low or highly variable results for chlorinated wastewater, the US EPA requires verification of results using the MPN procedure to resolve any controversies.  Each of these procedures is discussed in this lesson.

 

 

 

Safety and Hygiene

Whenever samples of wastewater are handled, it is very important that operators wash their hands before eating or smoking.  While some laboratory chemicals are not dangerous, many of them are poisonous or harmful to skin and clothing.  Rubber gloves and safety glasses should be used.  It is important to wash thoroughly with soap and water after handling laboratory chemicals, especially if chemicals come into contact with the skin.  Keep bench areas free of clutter and clean bench surfaces with disinfectant before and after bacteriological testing.

Read the labels carefully and know what to do in case of an accidental spill.  Always clean up spills quickly and in the safest possible manner using disposable rags or towels.  Sterilize all inoculated tubes, filters, and culture dishes prior to disposal of bacteriological test waste materials.  Always put stoppers or tops back on containers when not in use.

 

 

Equipment and Reagents

Whenever microbiological testing of water samples is performed, certain general considerations and techniques will be required. Since these are basically the same for every microbiological test procedure covered by this text, they will be discussed prior to the specific instructions for each test method.

 

 

Reagents

All reagents and media utilized in performing microbiological tests on water examples must meet the standards specified in Section 9000 of “Standard Methods for the Examination of Water and Wastewater”, 18th Edition.

 

 

Reagent Grade Water

Preferably deionized water that has been tested annually and found free of dissolved metals and bactericidal or inhibitory compounds should be used for the preparation of culture media and test reagents, although distilled water may be used.  The reagent grade water should also be free of contaminating nutrients.  The deionizer cartridges should be replaced in accordance with the manufacturer’s instructions.  An annual analysis of the reagent grade water must be performed to determine the absence of toxic materials.  A test to determine the suitability of the water for bacteriological testing should be performed as well.  The chemical analysis and suitability test of reagent grade water may exceed the capabilities of most wastewater laboratories.  To ensure accurate assessment of the reagent grade water, the plant operator or laboratory analyst may wish to consult a reputable contract laboratory service.

 

 

Chemicals

All chemicals used in fecal coliform monitoring must be ACS reagent grade or equivalent.

 

 

Media

To ensure uniformity in the test procedures, the use of dehydrated media is recommended.  It is not advisable to prepare media from the basic ingredients when suitable dehydrated media are available.  Sterilized prepared media in sealed test tubes, ampules, or in dehydrated media pads are also acceptable for use in this test.  The dates of receipt and opening of dehydrated media bottles should be recorded in the laboratory’s Quality Assurance log.  Unopened dehydrated media should not be kept for longer than two years from date of receipt.  Bottles of dehydrated media must be used or discarded within six months of being opened.  Storage practices for prepared media in sealed test tubes, ampules, or dehydrated media pads must conform to the manufacturer’s instructions.

 

 

Glassware

All glassware used in microbiological testing should be made of borosilicate glass and should conform with Section 9000 of “Standard Methods for the Examination of Water and Wastewater”, 18th Edition.

 

 

Pipettes

Pipettes may be of any convenient size, provided that they deliver quickly and accurately.  The error of calibration for any given manufacturing lot should not exceed 2.5%.  It is recommended that the mouth end of all bacteriological pipettes be protected with a cotton plug.  This should serve to protect the analyst from possible health hazards and the sample from possible contamination.  Pre-sterilized, disposable plastic or glass pipettes are acceptable.

 

 

Dilution Bottles

Dilution bottles must be made of resistant glass, preferably borosilicate glass, closed with glass stoppers or plastic screw caps which are equipped with liners that do not produce toxic or inhibitory compounds when sterilized.  Do not use cotton plugs to close dilution bottles.  Graduation levels should be indelibly marked on the side of the bottles.  Autoclavable, plastic bottles which are non‑toxic may be substituted.

 

 

Fermentation Tubes

Test tubes of any type may be used as fermentation tubes if their design permits conformance to the requirements for concentrations of nutritive ingredients.  Where tubes are to be used for a test of gas production, a smaller test tube should be inverted in the fermentation tube.  The sizes of the two tubes should be such that the inverted smaller tube is completely filled with medium and partially submerged in the larger tube.

 

 

Sampling Bottles

Bottles made of glass resistant to the solvent action of water and capable of being sterilized may be used for sampling water for bacteriological examination.  These bottles may be of any suitable size and shape.  Wide mouthed, ground‑glass stoppered or screw capped bottles are recommended, although autoclavable plastic containers may be used if preferred.  Plastic liners on screw capped bottles must not produce toxicity on sterilization. Commercially available, sterilized plastic bags with a suitable watertight closure are also acceptable.

 

 

 

Equipment

All equipment utilized in performing microbiological tests on water samples must meet the standards specified in Section 9000 of “Standard Methods for the Examination of Water and Wastewater”, 18th Edition.  The temperatures of all thermally regulated equipment (refrigerators, incubators, autoclaves, etc.) must be recorded at least daily in the laboratory’s Quality Assurance log.  To ensure comparability of instrument temperatures, data readings should be taken at the same time each day.  Thermometers must be graduated in increments of 0.2°C or less, and should be checked against a thermometer traceable to the National Bureau of Standards semiannually.

 

 

Refrigerators

Refrigerators used for storing bacteriological media or samples must be able to maintain the temperature in the refrigeration chamber in the range of 2‑10°C.

 

 

Incubators

Incubators used in the fecal coliform tests must maintain a uniform and constant temperature at all times.  They must not vary by more than +/‑0.5°C in the areas used for the tests.  Adequate temperature control of an air incubator operated at 44°C is not ordinarily possible.  To maintain this temperature within +/‑0.2°C, a water bath or heat sink incubator must be used.

 

 

Hot Air Sterilizing Oven

Hot air sterilizing ovens must be of sufficient size to prevent internal crowding.  They should be constructed to give uniform and adequate sterilizing temperatures, and equipped with suitable thermometers capable of registering accurately in the range of 160 to 170°C.

 

 

Autoclaves

Autoclaves must be of sufficient size to prevent internal crowding.  They should be constructed to provide uniform temperatures within the chamber (up to and including the sterilizing temperature of 121°C) and equipped with an accurate thermometer.  The thermometer bulb must be located on the exhaust line to record the minimum temperature within the sterilizing chamber.  Autoclaves should be equipped with a pressure gauge and properly adjusted safety valves connected directly to the saturated‑steam power lines or steam generator.  Autoclaves should be capable of reaching the desired temperature within 30 minutes.  Pressure cookers may be substituted for autoclaves, provided they are equipped with efficient pressure gauges and thermometers, the bulbs of which are 2.5 cm (1 in.) above the water level.

 

 

Microscope and Light Source

Membrane filter colonies can best be counted using a binocular wide field dissecting microscope with a magnification of 10X to 15X with a diffused light developed by cool white fluorescent lamps.

 

 

Pipette Containers

Pipette cans should be constructed of aluminum or stainless steel.  They should be 2‑3 inches in diameter and approximately 16 inches long.  If pipette cans are not available, pipettes should be wrapped in aluminum foil.

 

 

Inoculation Equipment

Wire loops made of 22 or 24 gauge chromel, nichrome, or platinum‑iridium should be used for inoculation needles where flame sterilization is used.  The diameter of all inoculation loops must be 3 mm.  Dry heat or steam may also be used for sterilization.  Single‑use aluminum, stainless steel or plastic loops, or hard wood applicators may be substituted if preferred and if properly sterilized.

 

 

 

 

Preparation of Glassware

All glassware used for bacteriological testing must be thoroughly cleaned using a suitable detergent and hot water. The glassware should be rinsed with hot water to remove all traces of residual from the detergent and finally rinsed with distilled water.

Some detergents may leave a residue which could inhibit the bacteria growth.  The procedure for determination of the possible inhibiting effect of detergent residues is outlined in “Standard Methods for the Examination of Water and Wastewater”, 18th Edition.  Since many laboratories performing this test may not be equipped to perform this test, it is recommended that laboratories use a detergent certified to meet bacteriological standards, or, at a minimum, rinse all glassware after washing with 2 tap water rinses followed by 5 distilled water rinses.

 

 

Sterilization

Hot Air Sterilizer

All equipment should be wrapped in high quality (Kraft) paper or placed in containers prior to hot air sterilization.  All glassware, except those in metal containers, should be sterilized for  a minimum of 60 minutes at 170°C.  Sterilization of glassware in metal containers should require a minimum of 2 hours.  Hot air sterilization should not be used for glassware containing media or other liquids.  The caps or stoppers of glassware sterilized in a hot air sterilizer should be partially loosened to prevent pressure buildup during sterilization.

 

 

Autoclave

Sample bottles, dilution water, culture media and glassware may be sterilized by autoclaving at 121°C for 15 minutes.  The autoclaving process uses steam and pressure (15 psig) for sterilization.  Care should be taken that the pressure has returned to zero prior to opening the autoclave to prevent injuries or loss of sterilized liquids.  The caps or stoppers of glassware sterilized in an autoclave or pressure cooker should be partially loosened to prevent pressure buildup inside the containers.

 

 

Preparation of Sterile Dilution Water

The dilution water used for making sample serial dilutions is prepared as follows:

  1. Prepare stock buffer solution:

    Dissolve 34 g of potassium dihydrogen phosphate (KH2PO4) in 500 mL of distilled water.  Adjust the pH of the stock solution to 7.2 with 1 N sodium hydroxide (NaOH).  Dilute to 1 L with distilled water.

  2. Prepare magnesium chloride solution:

    Dissolve 38 g of magnesium chloride (MgCl2) in 1 L of distilled water.

  3. Prepare buffered dilution water by adding 1.25 mL of stock buffer solution and 5.0 mL of magnesium chloride solution to each liter of distilled water.  Dispense buffered dilution water into the dilution bottles in large enough volumes to obtain 9 or 99 mL after the sterilization.

  4. Sterilize as noted above.





 

Serial Dilution Procedure

At times the density of the organisms in a sample makes it difficult to accurately determine the actual number of organisms present.  When this occurs, the sample size may need to be reduced to as little as one millionth of a milliliter.  In order to obtain such small volumes, a technique known as serial dilution has been developed.  In a serial dilution, successive volumes of diluted sample are further diluted until the desired dilution range is obtained.

The following steps describe a serial dilution procedure:

  1. Prepare dilution bottles by placing sufficient volume of dilution water in each bottle to have 99 mL after autoclaving.

  2. Pipette 11 mL of sample into dilution bottle #1 and gently swirl to mix.

  3. Pipette 11 mL from bottle #1 into bottle #2 and gently swirl to mix.

  4. Pipette 11 mL from bottle #2 into bottle #3 and swirl to mix.

  5. Pipette 11 mL from bottle #3 into bottle #4 and swirl to mix.

 

At this point, the sample has been diluted to such a degree that as little as 0.0001 mL of original sample can be measured accurately by pipetting 1 mL of the dilution in bottle #4.  Additional reductions in sample size can be accomplished by further dilutions.

 

 

 

Bacteriological Sampling

Proper technique, equipment and sample preservation are critical to obtaining valid test results which can be used in the evaluation of process efficiency or water quality.  These factors are especially critical in bacteriological sampling. The sample containers, sampling procedure, and handling after sampling can all be sources of errors long before the bacteriological testing actually begins.  To ensure proper samples are collected, the instructions on cleaning and sterilizing sample bottles must be strictly followed.

 

 

Sample Dechlorination

When samples of chlorinated effluents are to be collected and tested, the sample must be dechlorinated.  Chlorine remaining in the sample can further disinfect the sample during any holding time after sample collection.  Since sterile sampling procedures must be followed for a valid bacteriological test, the dechlorination steps cannot be performed after the sample is collected.

The procedure for dechlorination of bacteriological samples is as follows:

  1. Prior to sterilization, place enough sodium thiosulfate solution (10%) in a clean sample container to produce a concentration of 100 mg/L in the sample.  For a 120 mL sample bottle, 0.1 mL is usually sufficient.

  2. Sterilize sample bottles treated as above according to the procedures described above.

 

 

Sampling Procedures

To obtain an aseptic, representative sample for bacteriological examination:

  1. Keep the sample bottle unopened after sterilization until the sample is to be collected.

  2. Remove the bottle stopper and hood or cap as one unit.  Do not touch or contaminate the cap or neck of the bottle.

    NOTE:  If manual sampling is performed, hold the bottles near the base during sampling.

  3. Submerge the sample bottle in the water to be sampled.

    NOTE:  If the bottle is hand‑held, sample against the current and keep the hand downstream from the neck of the bottle.

  4. Fill the sample bottle approximately ¾ full, but not less than 100 mL.  Leave ample air space to allow the sample to be mixed by shaking prior to testing.

  5. Aseptically replace the stopper or cap on the bottle.

  6. On a sample tag or field data sheet, record the date, time, and location of sampling, as well as the sampler’s name and any other descriptive information pertaining to the sample.

 

 

Sample Preservation and Storage

Examination of bacteriological water samples should be performed immediately after collection.  If testing cannot be started within one hour of sampling, the sample should be iced or refrigerated at 4°C or less.  If samples are iced during transport or storage, use only enough ice to maintain the required preservation temperature.  Excess ice can submerge the sample bottles after melting and potentially contaminate the sample.

The maximum recommended holding time for fecal coliform samples from wastewater is 6 hours.  If the shipping time of the samples is consistently greater than the recommended holding time, consider doing on‑site testing for fecal coliforms.  The storage temperature and holding time should be recorded as a part of the test data.

 

 

 

Multiple Tube Fermentation Technique

The Multiple Tube Fermentation (MPN) technique for fecal coliform testing is useful in determining the fecal coliform density in most water, solid or semisolid samples.  It is recognized as the method of choice for any samples which may be controversial (enforcement related).  The technique is based on the most probable number of bacteria present in a sample which produces gas in a series of fermentation tubes with various volumes of diluted sample.  The MPN is obtained from charts based on statistical studies of known concentrations of bacteria.

The technique utilizes a two‑step incubation procedure.  The sample dilutions are first incubated in lauryl (sulfonate) tryptose broth for 24‑48 hours.  Positive samples are then transferred to EC broth and incubated for an additional 24 hours.  Positive samples from this second incubation are used to statistically determine the MPN from the appropriate reference chart.

The sampling procedures for this technique are outlined above.

 

 

Equipment and Reagents

Equipment

The following equipment and glassware will be needed to perform the MPN procedure.  (Descriptions and specifications for those items marked with an asterisk are given in previous sections.)

 

 

Reagents

The following broths and chemicals will be needed to perform the MPN procedure. (Descriptions and specifications for these items are given in previous sections.)

 

Reagent grade water

Dehydrated lauryl sulfonate tryptose (LST) broth

 

Dehydrated EC broth

Potassium dihydrogen phosphate (KH2PO4)

 

 

 

Preparation of Sterile Media Broths

Lauryl Sulfonate Tryptose (LST) Broth

For most wastewater effluent samples, the lauryl sulfonate tryptose broth can be prepared by dissolving 35.6 grams of dehydrated media in 1 liter of distilled water.  If the volume of sample being tested is greater than 1 mL per fermentation tube, the strength of the broth must be increased to maintain the correct proportions.

 

EC Broth

The EC broth can be prepared by dissolving 37.0 g of dehydrated EC media in 1 liter of distilled water.

 

 

 

Preparation of Fermentation Tubes

After the broths are prepared, the fermentation tubes should be prepared by dispensing 10 mL of broth into each fermentation tube.  This volume should be sufficient to partially cover the inverted, inner test tube after sterilization.  Sterilization procedures for culture media are discussed above.  After sterilization, refrigerate the prepared fermentation tubes at 10°C or less until they are needed.  Incubate fermentation tubes prepared and stored in this manner at 35 +/‑0.5°C for 24 hours prior to use.  Discard any tubes in which the inverted, inner test tube is not completely filled.

 

 

Presumptive Test

The first step of the MPN procedure for fecal coliform testing is called the presumptive test.  In this test, samples or serial sample dilutions are inoculated into a series of fermentation tubes.  The fermentation tubes are then incubated at 35 +/‑0.5°C. The tubes are observed at the end of 24 and 48 hours for gas production.  Any tube showing gas production during this test indicates the possible presence of coliform group bacteria and is recorded as a positive presumptive tube.  All positive presumptive tubes are transferred to EC broth fermentation tubes to confirm the presence of fecal coliform bacteria.

 

 

Test Procedure

  1. Prepare a series of decimal dilutions of the sample to be tested for fecal coliform using the procedure outlined in the next section.

  2. For each dilution, inoculate 5 fermentation tubes containing lauryl sulfonate tryptose (LST) broth (10 mL/tube).  Mark each tube for identification using a non‑water soluble marker or grease pencil.

    NOTE 1:  In steps 1 and 2, care should be taken to ensure that the sample or sample dilutions are well mixed before the inoculum is withdrawn from the sample or dilution bottle.

    NOTE 2:  The volume of sample and the number and degree of serial dilution will vary with the nature of the water being tested.  In no case should less than 3 dilutions of 5 tubes each be used.

  3. Incubate the inoculated tubes at 35 +/‑0.5°C for 24 (+/‑2) hours.

  4. Check each tube for the presence of gas in the inner shell vials.  If gas production is not readily apparent, shake the tubes gently and check for rising gas bubbles.

    NOTE:  DO NOT confuse gas production with possible air bubbles. Gas production should be accompanied by a change in the appearance of the broth; it may become cloudy.

  5. Record positive presumptive results (gas produced) on the MPN test data sheet.

  6. Return all of the negative tubes to the incubator at 35°C (+/‑0.5°C).

  7. Repeat steps 4 and 5 at the end of the remaining 24 (+/‑2) hours.

  8. Discard any negative tubes left after step 7, using appropriate safety precautions.

    All positive presumptive tubes should be carried into the fecal coliform confirming test procedure.  Transfers should be made as soon as the gas production is noted in a fermentation tube.

    NOTE:  DO NOT hold 24 hour positive presumptive tubes until the 48 hour total incubation time is completed.


 

 

 

Fecal Coliform Confirming Test

In the confirming test procedure for fecal coliform bacteria, the positive presumptive cultures are transferred to EC broth, which is specific for fecal coliform bacteria.  Any presumptive tube transfer which shows gas production after 24 (+/‑2) hours incubation at 44.5°C (+/‑0.2°C) confirms the presence of fecal coliform bacteria in that tube and is recorded as a positive confirmed tube.

 

 

Test Procedure

  1. Pair each positive presumptive fermentation tube with a fermentation tube containing EC broth.  Mark each EC tube to match its paired presumptive tube.

  2. Using a sterile transfer loop, transfer a portion of the liquid from each presumptive tube to its paired EC broth fermentation tube.

    NOTE:  Flame sterilize metal loops before each transfer or use individual pre‑sterilized loops or wood splints for each transfer.

  3. Discard the positive presumptive tubes after transferring using appropriate safety precautions.

  4. Place all of the inoculated EC broth tubes in a water bath incubator maintained at 44.5° +/‑0.2°C.

    NOTE:  The tubes should be placed in the water bath within 30 minutes of inoculation.

  5. Incubate the EC broth tubes for 24 (+/‑2) hours.

  6. Remove the tubes from the water bath, shake gently and inspect for gas production.

  7. Record all fermentation tubes showing gas production as positive on the test data sheet.

  8. Calculate the test results and record as Most Probable Number (MPN)/100 mL.

  9. Discard the fermentation tube contents using appropriate safety precautions.

 

 

 

Calculation of Most Probable Number (MPN)/100 mL

The calculation of the MPN test results requires the selection of a valid series of 3 consecutive dilutions.  The number of positive tubes in each of the three selected dilution inoculations is used to determine the MPN/100 mL.  In selecting the dilutions to be used in the calculation, each dilution is expressed as a ratio of positive tubes per tubes inoculated in the dilution, i.e. 3 positive/5 inoculated (3/5).  There are several rules to follow in determining the most valid series of dilutions.  In the following examples, four dilutions were used for the test.

 

  1. Select the highest dilution showing all positive results (no lower dilution showing less than all positive) and the next two higher dilutions.

    Example #1

          1 mL                  0.1 mL                         0.01 mL                       0.001 mL                  Selected Series
           5/5                       5/5                                3/5                                1/5                              5‑3‑1



  2. If a series shows all negative values with the exception of one dilution, select the series that places the only positive dilution in the middle of the selected series.

    Example #2

          1 mL                  0.1 mL                         0.01 mL                       0.001 mL                  Selected Series
           0/5                       0/5                                3/5                                0/5                              0‑3‑0


  3. If a series shows a positive result in a dilution higher than the selected series (using rule #1), it should be incorporated into the highest dilution of the selected series.

    Example #3

          1 mL                  0.1 mL                         0.01 mL                       0.001 mL                  Selected Series
           5/5                       3/5                                2/5                                1/5                              5‑3‑3

 

 

After selecting the valid series, the MPN/100 mL is determined by matching the selected series with the same series on the MPN reference chart (See Table below).  If the selected series does not match the sample dilution series at the top of the MPN reference chart, the results must be calculated using the following formula:

MPN/100 mL = MPN from chart x (mL sample for first column of chart/mL sample in first dilution of the selected series)

 

MPN Reference Table (MPN/100mL)

Sample Volume                   Sample Volume

 

10 mL

 

1 mL

 

0.1 mL

 

MPN

 

10 mL

 

1 mL

 

0.1 mL

 

MPN

0

0

0

       0

4

2

0

        22

0

0

1

       2

4

2

1

        26

0

1

0

       2

4

3

0

        27

0

2

0

       4

4

3

1

        33

1

0

0

       2

4

4

0

        34

1

0

1

       4

5

0

0

        23

1

1

0

       4

5

0

1

        31

1

1

1

       6

5

0

2

        43

1

2

0

       6

5

1

0

        33

 

 

 

 

5

1

1

        46

2

0

0

       5

5

1

2

        63

2

0

1

       7

5

2

0

        49

2

1

0

       7

5

2

1

        70

2

1

1

       9

5

2

2

        94

2

2

0

       9

5

3

0

        79

2

3

0

     12

5

3

1

      110

 

 

 

 

5

3

2

      140

3

0

0

       8

5

3

3

      180

3

0

1

     11

5

4

0

      130

3

1

0

     11

5

4

1

      170

3

1

1

     14

5

4

2

      220

3

2

0

     14

5

4

3

      280

3

2

1

     17

5

4

4

      350

 

 

 

 

5

5

0

      240

4

0

0

     13

5

5

1

      350

4

0

1

     17

5

5

2

      540

4

1

0

     17

5

5

3

      920

4

1

1

     21

5

5

4

    1,600

4

1

2

     26

5

5

5

  >2,400

 

 

 

 

Sample Calculation

Using the following example’s results, calculate the MPN/100 mL of the example.

mL of Sample in Each Serial Dilution  Positive Tubes/Tubes Inoculated
10 mL 5/5
1.0 mL 5/5
0.1 mL 3/5
0.01 mL 1/5

 

  1. Select the highest dilution with all positive tubes (1.0 mL dilution) and the next two higher dilutions (0.1 mL and 0.01 mL).  This step will give a selected series of 5‑3‑1.

  2.   Include any positive results in dilutions higher than the selected series (0.001 mL dilution 1/5).  This step changes the selected series to 5‑3‑2.

  3. Locate the 5‑3‑2 horizontal series on the MPN reference chart in the first three columns.

  4.   Read the MPN value from the fourth column (MPN/100 mL = 140).

  5. Since Column #1 is marked 10 mL of sample and the first dilution for the selected series for this sample was 1.0 mL, the final MPN/100 mL for this sample must be calculated using the formula from Section 15(f).

    Final MPN/100 mL = 140 MPN/100 mL x (10 mL/1.0 mL)
    Final MPN/100 mL = 1400 MPN/100 mL.

 

 

 

 

Membrane Filtration Technique

The membrane filtration technique for fecal coliform testing is useful in determining the fecal coliform density in wastewater effluents, with the exception of those which have received only primary treatment prior to chlorination, or wastewaters containing toxic metals or phenols.  Chlorinated secondary or tertiary effluents may be tested using this method.  However, the results are subject to verification by the MPN technique.

The membrane filter technique utilizes a specially designed filter pad with uniformly sized pores (openings).  The pores of the membrane filter are small enough to prevent bacteria from passing through the filter.  In fact, the bacteria cannot travel into the filter at all, but must remain on the filter’s surface. Another unique characteristic of the filter allows liquids placed under the filter to pass upward through the filter.  This characteristic lets media placed under the filter provide nourishment for bacterial growth.

The sampling procedures for this technique are outlined in above.

 

 

 

Interferences

Large amounts of turbidity, algae, or suspended solids may interfere with this technique by blocking the filtration of the sample through the membrane filter.  Dilution of these samples to prevent this problem may make the test inappropriate for samples with low fecal coliform densities since the sample volumes after dilution may be too small to give representative results.  The presence of large amounts of non‑coliform group bacteria in the sample may also prohibit the use of this method.

 

 

Equipment and Reagents

Equipment

The following equipment and glassware will be needed to perform the membrane filter procedure.  (Descriptions and specifications for those items marked with an asterisk are given in previous sections.)

 

 

Reagents

The following broths and chemicals will be needed to perform the membrane filter procedure.  (Descriptions and specifications for those items marked with an asterisk are given in previous sections.)

 

 

 

Preparation of Dilution Water

Broth and Reagents

Dilution Water

A section above describes the procedures for preparing sterile buffered dilution water.  Since the dilution water will be used as rinse water during the filtration procedure, additional portions of sterile dilution water should be prepared with those needed for serial dilutions.  The additional dilution water should provide enough volume for three 20‑30 mL rinses of the filter funnel and membrane filter after filtration of the sample.

 

 

Rosolic Acid

Rosolic acid reagent is required in the MFC broth used for the membrane filter procedure to inhibit the growth of background organisms which might interfere in the test.  The rosolic acid may be omitted if background colony counts are low and comparable results have been obtained.  Rosolic acid will decompose if autoclaved.  Therefore, MFC broth made with rosolic acid in it should not be sterilized by autoclaving.  Stock rosolic acid solution should be refrigerated (2‑10°C) and discarded if the color changes to muddy brown.  The stock rosolic acid solution must be prepared fresh every two weeks.

The procedure for preparing rosolic acid is as follows:

  1. Dissolve 1 gram of rosolic acid powder in 100 mL of 0.2 N Sodium Hydroxide (NaOH).

  2. Mix thoroughly and store at 2‑10°C.

 

 

MFC Broth

The MFC broth used in the membrane filter procedure for fecal coliform can be prepared by dissolving 37.1 g of dehydrated media in distilled water which contains 10 mL of 1% rosolic acid solution.  This mixture is diluted to 1 liter with distilled water.  Heat the broth until it just begins to boil, then immediately cool the broth to 45°C.  DO NOT STERILIZE BY AUTOCLAVING!  Cover the container and store the MFC broth in a refrigerator (2‑10°C) until ready for use.  Discard any unused broth after 96 hours.

 

 

 

Membrane Filter Procedure

Sample Filtration

  1. Using sterile forceps, place a sterile membrane filter on the filter support assembly.

  2. Place the funnel portion of the assembly over the filter, making sure the filter is properly aligned during this step.

  3. Clamp or lock the assemble in place.

  4. Mix the sample (or sample dilution) thoroughly by shaking at least 30 times.

  5. Pour the undiluted sample into the funnel assembly to the 100 mL mark OR pour 100 mL of a serially diluted sample into the funnel assembly OR pour a small amount of sterile dilution water into the funnel assembly and pipette a suitable volume of sample into the funnel assembly.  Sample volumes less than 1.0 mL must be serially diluted as outlined in Section 13. 

    NOTE:  The sample size and/or necessary serial dilution should be selected to grow 20‑60 fecal coliform colonies after incubation.

  6. Apply vacuum and filter the entire volume of sample or dilution through the membrane filter.

  7. Rinse the funnel assembly and membrane filter with three 20‑30 mL portions of sterile buffered dilution water.  (Allow the entire volume of each portion to pass through the filter before adding the next portion.)

  8. Carefully remove the funnel assembly and immediately remove the membrane filter, using sterile forceps.

    NOTE:  Filtration units should be sterile at the start of each filtration series and should be sterilized again if the series is interrupted for 30 minutes or more.  A rapid interim sterilization can be accomplished by 2 minutes exposure to ultraviolet light, flowing steam or boiling water.

 

 

Incubation

  1. Using sterile forceps, carefully place a sterile absorbent pad in the bottom portion of a sterile culture dish.

  2. Transfer 2.0 mL of MFC broth or the contents of a prepared media ampule with a sterile pipette onto the pad.

  3. Drain off any broth not absorbed by the pad.

  4. Using sterile forceps, carefully place the sample filter on the absorbent pad using a rolling motion to avoid catching air bubbles under the filter.

  5. Cover the culture dish and mark the top of the cover to identify the sample.

  6. Seal the culture dish in a plastic bag or by using electrical tape and place in a water bath incubator at 44.5° (+/‑0.2°) C.

    NOTE:  All of the prepared culture dishes should be placed in the water bath within 30 minutes after filtration.  The plastic bags must be anchored or weighted to ensure the culture dishes are kept completely submerged during the entire incubation period.

  7. Incubate the culture dishes for 24 (+/‑2) hours.

  8. At the end of the incubation period, remove the culture dishes from the water bath and count the blue colored colonies on the surface of the filter.

  9. Discard the membrane filters and absorbent pads using the appropriate safety precautions.

 

 

 

Colony Counting

Upon completion of the incubation period, the surface of the filter will have growths of both fecal coliform and non‑fecal coliform bacterial colonies.  The rosolic acid present in the MFC media will normally reduce the number of non‑fecal coliform colonies to a minimum.  The fecal coliform colonies will appear blue in color, while non‑fecal coliform colonies will appear gray or cream colored.

When counting the colonies, the entire surface of the filter should be scanned using a 10X‑15X binocular, wide field dissecting microscope.   Colonies may be counted by scanning across one row and back across the next, etc.  This should ensure that all areas of the filter are observed.

The desired range of colonies for the most valid fecal coliform determination is 20 to 60 colonies per filter.  If multiple sample dilutions are used for the test, counts for each filter should be recorded on the laboratory data sheet.

NOTE:  Filters which show a growth over the entire surface of filter with no individually identifiable colonies should be recorded as TNTC (too numerous to count).

 

 

Calculation of Colonies/100 mL

The fecal coliform density can be calculated using the following guidelines.

  1. For samples with one or more volumes with colony counts in the range of 20 to 60 colonies, the correct daily average calculation is as follows:

    Arithmetically average only the samples with counts in the acceptable (20 to 60) range.

                     Example:                        Volume                           Colony count
                                                            50 mL                                    59
                                                            25 mL                                    30
                                                            20 mL                                    18

     

    Calculate the colony count per 100 mL for each sample in the acceptable range using the following formula:


    Colonies/100 mL = (100 mL x colony count)/volume used

    50 mL = (100 x 59)/50 = 118 colonies/100 mL

    25 mL = (100 x 30)/25 = 120 colonies/100 mL

    (Reject 20 mL sample since count is less than 20.)

    Average the results arithmetically.

    (118 + 120)/2 = 119 colonies/100 mL




  2. For samples with colony counts for all volumes less than 20 and greater than zero, the correct daily average calculation is as follows:

    Select the most acceptable count (usually the largest volume used) to avoid additional variability due to low counts.  Calculate the colony count per 100 mL for that sample.

                     Example:                        Volume                           Colony count
                                                            50 mL                                  19
                                                            25 mL                                  10
                                                            10 mL                                    4



    50 mL = (100 x 19)/50 = 38 colonies/100 mL estimated

    The result is estimated because all counts were less than 20.  The results are to be included in the monthly average without the “less-than” signs.  If the “less-than” value is equal to or greater than the permit limit, the laboratory must flag the associated DMR data and indicate the number of times this occurs.  If this occurs more than 2 times in a month, additional dilutions must be routinely analyzed (using more volume of sample filtered).



  3. For samples with all colony counts less than 20 and one or more counts of zero, the correct daily average calculation is as follows:

    Select the most acceptable count (usually the largest volume used) to avoid additional variability due to low counts.  Calculate the colony count per 100 mL for that sample.

                     Example:                        Volume                           Colony count
                                                            50 mL                                  17
                                                            25 mL                                  10
                                                            10 mL                                    0



    50 mL = (100 x 17)/50 = 34 colonies/100 mL estimated

    The result is estimated because all counts were less than 20.  The results are to be included in the monthly average without the “less-than” signs.  If the “less-than” value is equal to or greater than the permit limit, the laboratory must flag the associated DMR data and indicate the number of times this occurs.  If this occurs more than 2 times in a month, additional dilutions must be routinely analyzed (using more volume of sample filtered).



  4. For samples with all colony counts greater than 60, but still countable (have not grown together into a mass of poorly defined colonies), the correct daily average calculation is as follows:

    Select the count from the smallest volume filtered and calculate the colony count per 100 mL for that sample.  (If the colonies have all grown together, report as TNTC.)

                     Example:                        Volume                           Colony count
                                                            50 mL                                199
                                                            25 mL                                110
                                                            10 mL                                  65



    10 mL = (100 x 65)/10 = greater than 650 colonies/100 mL

    The result is reported as “greater than” because all counts were greater than 60.  Greater than values are to be avoided by analyzing multiple dilutions.  If such results are obtained more than 2 times per month, the number of dilutions routinely analyzed must be increased.  The DMR data associated with these results must be flagged with a statement that includes the number of “greater than” or “TNTC” occurrences and what corrective measures have been performed to avoid such results in the future.  These results must be included in the monthly average (geometric mean) without the “greater than” sign.

 

 

 

 

Comparison of Test Methods

For many years there has been a controversy regarding the use of the membrane filter technique for fecal coliform testing on samples from chlorinated secondary and tertiary effluents.  At one point, the federal regulations on acceptable methods for testing wastewater effluents proposed elimination of the membrane filter technique.  This action was contemplated due to the possible slow recovery of fecal coliform bacteria after exposure to chlorine.  It was felt that the shorter incubation period of the membrane filter technique did not allow sufficient time for the organisms to recuperate and grow on the filter media.

Research at that time had revealed that test results from the membrane filter technique were consistently lower than those from the MPN procedure.  This was a major concern to the regulatory agencies and placed the acceptable use of the procedure in jeopardy.  Further research has indicated that for most applications, the membrane filter procedure will allow the organisms to grow satisfactorily.  From this research the membrane filter technique has been accepted in all but controversial or questionable situations.

 

The table below illustrates the various uses and limitations as well as the main advantages and disadvantages of the MPN procedure for fecal coliform.

 

Multiple Tube Fermentation (MPN) Method

USES

LIMITATIONS

 

 

Potable waters

No major limitations

Surface waters
Primary treated effluents
Secondary treated effluents
Tertiary treated effluents
Chlorinated effluents
Saline or brackish waters
Estuarine or other waters
capable of propagating
shellfish
Mud, sediments, and sludges

 

 

ADVANTAGES

DISADVANTAGES

 

 

Does not require correlation with other methods for use with other methods for use

Higher initial equipment costs

 

Requires longer time for determination of results

Lower operating costs

 

 

More preparation time required

 

 

 

More laboratory technique required

 

 

The table below illustrates the various uses and limitations as well as the main advantages and disadvantages of the membrane filter technique for fecal coliform testing.


Membrane Filter Technique

USES

LIMITATIONS

 

 

Potable waters (after application ability has been established)

Cannot be used for highly turbid samples

Non-potable waters

Cannot be used for samples with large amounts of algae

Secondary treated effluents

Cannot be used for chlorinated primary effluents

Tertiary treated effluents

Cannot be used for samples with toxic wastes

Chlorinated effluents (may not be acceptable in controversial situations)

Cannot be used for samples with phenols

 

Cannot be used for samples from estuarine waters capable of propagating shellfish

 

 

 

ADVANTAGES

DISADVANTAGES

 

 

Lower initial costs

Higher operating costs

Results available faster

Not acceptable for certain sample type

Less training required such as solids or semi-solids

 

 

Results may not be accepted in controversial situations

 

 

 

 

Geometric Mean Calculation

Many NPDES permitted facilities must test for and report fecal coliform bacteria densities.  For most of these facilities, the required data includes a geometric mean (average) of all the test results obtained during a reporting period.  A geometric mean, unlike an arithmetic mean, tends to dampen the effect of very high or low values which might bias the mean if a straight average (arithmetic mean) were calculated.

Calculation of the Geometric Mean can be performed by either of two procedures.  The use of either method requires special functions on your calculator.  The general formula for the Geometric Mean is:

Geometric Mean = n-th root of (X1)(X2)...(Xn)

 

Where:  X is any data point and the subscripts indicate which point n is the number of individual data points used in the calculation.

This equation stated simply means that the Geometric Mean can be found by multiplying all of the data points for the given report period together and taking the n‑th root of this product.

 

 

Example #1:

                                     Fecal Coliform

                  Date                                  (colonies/100 mL)

               02/01/96                                   5 col/100 mL
               02/08/96                                   7 col/100 mL
               02/15/96                                   90 col/100 mL
               02/22/96                                1000 col/ 100 mL

 

Geometric Mean = 4th root of (5)(7)(90)(1000)

      = 4th  root of 3,150,000

 

Geometric Mean = 42.13 colonies/100 mL

The Geometric Mean can also be calculated using the logarithms of each data point.  In order to use this calculation procedure you must have a calculator which will give logarithms and antilogarithms.  The first step in calculating the Geometric Mean using this method is to determine the logarithm of each data point using your calculator.  Next add all of the data point logarithms together and divide this sum by the number of data points (n).  Reverse the procedures used to determine the logarithms to find the antilogarithm of the resulting value.

 

 

Example #2:  (using previous data)

                                       Logarithm from

             Fecal Coliform                                       Calculator or
          (colonies/100 mL)                                  reference tables

                       5                                                     0.69897
                       7                                                     0.84510
                      90                                                     1.95424
                     1000                                                   3.00000

             Total:  6.49831

 

The logarithm of the Geometric Mean = 6.49831/4 = 1.62458

 

From your calculator determine the number whose logarithm is 1.62458 (antilogarithm).  Geometric Mean = 42.13 colonies/100 mL

For each of these examples, the arithmetic mean (average) of the data points is:

Arithmetic Mean = (5 + 7 + 90 + 1000)/4 = 1102/4

Arithmetic Mean = 275.5 colonies/100 mL


The calculation of the Geometric Mean is more complicated if one or more of the data points is zero (0) colonies/100 mL.  In these cases, a value of ‘1’ should be used for each zero data point in the calculation.  This substitution does not affect the result of the calculation, but just ensures that the data is entered into the calculation in a usable form.

 

 

Example #3:  Using the data points:

0 colonies/100 mL
1000 colonies/ 100 mL
20 colonies/ 100 mL
17000 colonies/ 100 mL
0 colonies/100 mL

 

Geometric Mean = 5th  root of 1 X 1000 X 20 X 17000 X 1

= 5th root of 340,000,000

 

Geometric Mean = 50.85 colonies/100 mL

OR

Log      1 = 0.00000
Log      1000 = 3.00000
Log      20 = 1.30103
Log      17000 = 4.23045
Log      1 = 0.00000
Total     8.53148

 

The logarithm of the Geometric Mean = 8.53148/5 = 1.70630

 

From your calculator determine the number whose logarithm is 1.70630 (antilogarithm).

For Example #3 the arithmetic mean is:

Arithmetic Mean = (0 + 1000 + 20 + 17000 + 0)/5

Arithmetic Mean = 3604 colonies/100 mL

 

A step-by-step method that can be used on most scientific calculators for determining geometric mean by the log/antilog method is as follows:

 

 

 

Calculating Geometric Mean

  1. Enter fecal coliform result into calculator.

  2. Press the (Log) function key.  Record the result.

  3. In the same manner as (2) above, calculate the log for each sample result.

  4. Add the log of each sample and divide the result by the number of sample.

  5. Record the result obtained in (4) above.

  6. Clear the calculator.

  7. Enter the number 10.

  8. Press the (In) or (Inx) function key.

  9. Press the (X) key.

  10. Enter the number recorded in (5) above.

  11. Press the (=) key.

  12. Press the (ex) function key.  The number which appears is the geometric mean of the series of
            fecal coliform results.

 

 

 

 

QA/QC

A Quality Assurance/Quality Control program is required by the NPDES permit.  Quality Assurance (QA) is a set of operating principles that must be followed during sample collection and analysis.  Lab bench sheets must be maintained that document when the sample was collected, how it was preserved and what results were obtained.

Quality Control (QC) includes any testing which is done to prove that the results are reliable.  One of every ten samples analyzed should be a QC check.  This may include duplicate samples, spike samples, reagent blank analyses and known QC samples obtained from outside sources.

Duplicate sample analysis involves analyzing the same sample twice and comparing the results.  The closer the results, the more accurate the analysis.  Results should not differ by more than 10%.  Spike sample analysis involves adding known amounts of analyte to a sample and calculating the percent recovery.  These are discussed further in Chapter 10.

In fecal coliform testing, duplicate samples should be run every tenth sample to test for variability.  The MPN method should be used to confirm the membrane filtration method.

A sterility check and growth check should be run on the MFC broth each time a new batch is made.  The sterility check proves that the broth is not contaminated with fecal coliforms and is performed by placing broth on a filter pad in the culture dish without any sample.  There should be no growth after 24 hours of incubation.  The growth check proves the MFC broth is capable of sustaining colonies and is performed by filtering several milliliters of plant influent through a filter.  Colonies should form after incubating for 24 hours.  Sample bench sheets are included in this lesson.

 

 

 

Sources

Standard Methods for the Examination of Water and Wastewater, 18th Edition, AWWA, APHA, WPCF; Water Pollution Control Federation, Washington, DC, 1992.

Microbiological Methods for Monitoring the Environment, Water, and Waste, EPA‑600/8‑78‑017, US Environmental Protection Agency, Pages 124 and 132.

Greeson, P.E., et al.  Methods for Collection and Analysis of Aquatic Biological and Microbiological Samples, US Geological Survey, Techniques of Water Resources Investigations, Book 5, Chapter A4, Laboratory Analysis, 1977, Method #B‑0050‑77.

Letter to Don Caldwell (Q.A. Program Leader) from Joseph Slayton and Patricia Sosinski of U.S.E.P.A., May 25, 1993, regarding the Fecal Coliform Counting Procedure.

 

 

Assignment

Answer the following questions and email your answers to rcooke@mecc.edu.

  1. Why are fecal coliform bacteria referred to as indicator organisms?
  2. How can fecal coliform samples be preserved?
  3. Why has there been some controversy over the use of the membrane filter technique for fecal coliform analysis of wastewater samples?
  4. What is the maximum allowable holding time for wastewater fecal coliform samples?
  5. Why is dehydrated media recommended for the fecal coliform procedures?

 

 

Quiz

Answer the questions in the Lesson 17 quiz. When you have completed the quiz, print it or either email or fax it to the instructor. You may also take the quiz online and directly submit it into the database for a grade.