Bacteriological Sampling and Examination


Photo Credit:  Virginia Department of Health
Sampling followed by testing for bacteria can reveal whether a public drinking water supply is safe. 
Problems which may otherwise go unnoticed are often detected early enough to prevent the development of a major problem.  Sources of contamination can be determined through proper sampling and testing.

Representative samples are collected from designated sampling points and tested.  The results of these tests reveal the quality of the drinking water and should be filed.  This permanent record of bacteriological quality is public information and must be maintained by the water works operator for at least 5 years.  When a sample indicates the water is unsatisfactory, ADEM requests that check samples be collected and submitted.  Many times the follow-up check samples must be submitted because the water works operator contaminated the sample.  This contamination problem is both costly and time consuming and many times could be avoided by closely following instructions. 

The laboratory will not test samples which are submitted in bottles other than those furnished for this purpose by the laboratory, and water samples over 30 hours old will not be acceptable.  Since the sample bottle leaves the laboratory clean and sterile, it must be assumed that either the water was polluted or the sample was improperly collected. 

The laboratory uses a standard bottle.  It is washed, a very small amount of sodium thiosulfate added, capped, and then heat-sterilized in an autoclave (which is similiar to a large pressure cooker).  The sodium thiosulfate is added to react with the chlorine which, in most cases, will be in the public water supply sample.  If the chlorine were not neutralized and was allowed to remain in the sample, it would kill all bacteria which might have been present when collected.  This would cause a false report to be made, and the water works operator would not know the true quality of the water. 

The samples are tested for chlorine in the laboratory.  When chlorine is found in the sample, it usually indicates the bottle was rinsed by the collector, or that chlorine was added to try to assure a good report.  DO NOT RINSE THE SAMPLE BOTTLE WHEN COLLECTING SAMPLES AND DO NOT ADD CHLORINE TO THE SAMPLE.  The purpose of collecting samples is to find "bad" water if it exists in the system.  Due to several reasons there may be sections of the distribution system where the water is becoming contaminated, and these should be found to maintain public health.  Therefore, samples should be collected from various representative spigots over the entire system.  Cross-connections, back siphonage, leaks and other unknown conditions may cause contamination in any section of any system.  Part of the job of the water works operator is to seek, find and correct deficiencies.  Perfect samples time after time for long periods of time should give rise to several questions:  Is this water always perfect?  Is this distribution system perfect?  Is the technique of collecting samples perfect?  Who is being fooled?  What is wrong, all samples are always good? 

  *All the informaton on this page came from the Alabama Water Works Operator's Manual of 1989. 

Their references were the APHA, AWWA, and WPCF, Standard Methods for the Examination of Water and Wastewater, 16th ed. Port City Press, Baltimore, 1985. 


Sampling Procedure

Step by step instructions necessary for the proper collection of water samples are given below: 

Seek a sampling station which could give representative results of water in the area.  If possible, collect all samples from a yard tap or faucet.  Chrome plumbing fixtures, usually found indoors, may be damaged or made unsightly by properly flaming the fixture to sterilize it.  Avoid taking samples from garages, filling stations, kitchens or other places where grease may collect on the tap.  Samples should not be collected from fire plugs.  Fire plugs have weep holes and large barrels which may be easily contaminated and are very difficult to disinfect.  The mouth of the fire plug cannot be disinfected easily; nor can a bottle be properly filled. 

After the spigot is selected the following steps should be followed: 

  1. Let the water run for several minutes to insure fresh water from the system.  Shut the water off. 

  2. Flame the faucet in order to destroy any bacteria which may be clinging to the faucet.  If available, a blowtorch can be used for this purpose.  There are small "easy to light" gas torches available which are excellent for this purpose.  The flame should be directed against the inside edge of the faucet as much as possible.  A good torch can be made by tightly rolling a piece of cotton or a rag, wrapping it with a stiff wire on a handle.  Soak this in alcohol.  This will light easily, makes a good flame, and can be lit and put out several times before more fuel must be added.  A rolled up newspaper can be used.  Do not overheat the fixture. 

  3. Open the faucet and let the water run in a stream about the size of a pencil. 

  4. While the tap is running, remove the bottle stopper by first pulling back foil or paper and lifting the stopper. 

  5. Hold the bottle in one hand and with the other hand remove the top.  Care should be excercised in removing the stopper so that the fingers do not touch the top or the inside of the bottle.  Hold  the top in such a position that the paper covering will shield the top from falling dust particles.  Do not put the top down.  Next, hold the bottle under the faucet, allowing only a small, steady stream to flow into the faucet.  Do not splash water on the lip of the bottle any more than necessary.  Immediately remove the bottle from underneath the faucet and replace the top.  Press the paper down around the neck of the bottle and tie it back into position with the cord.  Remember, when removing the top and replacing it in the bottle, do not touch either the stopper or the lip of the bottle with the cord or paper which  could be knocked or washed into the bottle while collecting the sample.  Also, do not put the top down at any time.  Leave 5 to 10 of the bottle empty to allow air-space; but make sure the bottle contains at least 100 ml. of sample. 

  6. Fill out the identification card completely, tie the stub to the neck of the bottle, and replace the bottle in the shipping case. 

  7. The samples should be collected as near shipping time as possible since the sample must reach the testing laboratory and be examined within 30 hours after collection. Transportation time should not exceed 6 hours.  Ship the case prepaid to the proper laboratory serving the district in which the water supply is located.  If the case and a supply of clean, sterile bottles are to be returned by mail, postage must be included.  The shipping container will be returned at the water work's expense.  Samples do not need to be iced during shipment.  Prompt shipment cannot be overstressed, since the sample must be at the laboratory and examination begun within 30 hours after it is collected. 

The above directions must be followed explicitly in order to get reliable results. 

After the sample arrives at the laboratory, the bacteriological examination is made and the report of examination is sent to the water works. 

*All the informaton on this page came from the Alabama Water Works Operator's Manual of 1989. 

Their references were the APHA, AWWA, and WPCF, Standard Methods for the Examination of Water and Wastewater, 16th ed. Port City Press, Baltimore, 1985. 


Water Sample Submittal Form and Sample Tags

Disinfection of Potable Water System

New or repaired potable water systems shall be purged of deleterious matter and disinfected prior  to use.  The method to be followed shall be that prescribed by the health authority having jurisdiction, or, in the absence of a prescribed method, the procedure described in either AWWA C651 or AWWA C652 listed in Appendix A or as described below.  This requirement shall apply to "onsite" or "in-plant" fabrication of a system or a modular portion of a system. 

  1. The pipe system shall be flushed with clean, potable water until dirty water does not appear at the points of outlet.
  2. The system or part thereof shall be filled with a water/chlorine solution containing at least 50 parts per million (50 mg/l) of chlorine, and the system or part thereof shall be valved off and allowed to stand for 24 hours;  or the system or part thereof shall be filled with a water/chlorine solution containing at least 200 parts per million (200 mg/l) of chlorine and allowed to stand for 3 hours. 
  3. Following the allowed standing time, the system shall be flushed with clean potable water until chlorine does not remain in the water coming from the system. 
  4. The procedure shall be repeated if it is shown by a bacteriological examination by the authority that contamination is still present in the system. 

Bacteriological Analysis Input Form

Bacteriological Examination

When the water sample arrives at the laboratory, the bacteriological examination is accomplished by the membrane filter method. 

The membrane filter procedure has been approved by U.S. EPA Standards and by the American Water Works Association.  It was first described as a full alternate procedure in the Twelfth Edition of Standard Methods for the Examination of Water and Wastewater, (1965).  The membrane filter procedure has been used for all routine bacteriological examinations of drinking water since February, 1966. 

All operators should be familiar with the principles of the bacteriological tests as performed by the laboratory.

Photo Credit:  Virginia Department of Health
This knowledge should help the operator to interpret the results reported to him by the laboratory.  In order that the operator may become acquainted with these principles, a brief discussion is given below on both the membrane filter method and the multiple tube fermentation method.  The multiple tube method is the procedure that was universally used prior to the development of the membrane filter method and is still an approved method for bacteriological examination.  The multiple tube method is still used by ADEM to confirm doubtful results obtained occasionally by the membrane filter method.

Membrane Filters

Membrane or MF Millipore Filters have come into use in many fields of bacteriology and have supplemented or replaced some of the older methods of isolating and identifying bacteria.  These filters are thin membranes made of bonded cellulose ester sheet material approximately 150 microns thick (0.006 inch).  The structure of the filters are such that they have uniform pores which compose 80 to 85 percent of  the membrane surface.  By varying the size of the cellulose ester particles, the size of the pores can be controlled and the membranes can be made to be used for many different purposes.  The filters are used for bacteriological examinations by forcing liquid samples through the membrane with pressure or suction.  The pores of the membrane will permit water to pass through;  however, they are of such size the bacteria cannot pass but are retained on the surface of the filter.  For example, the Type HA filters which are used by certified laboratories have pores which are 0.45 microns (20 millionths of an inch) in size;  and they will retain nearly all known non-viral microorganisms. 

Photo Credit:  Virginia Department of Health
Extensive work with the membrane filter on the examination of water samples for coliform bacteria has been completed;  and it is now possible to filter a sample of water through a membrane filter, then grow coliform bacteria on the surface of the filter and get a coliform count for a definite volume of water.

The membrane filter method offers several advantages over the multiple tube fermentation procedures: 

  1. Time required for the test for coliform bacteria is reduced from 48 or 96 hours to 20 hours.
  2. A larger and more representative sample of water can be subjected to the test, thus increasing accuracy.
  3. The test can be made in the field as well as in the laboratory.
  4. Direct reading of the bacteria count is made rather than arriving statistically at the most probable number.
  5. Because of the enormously large sample size which can be analyzed using the membrane filter  method, specific pathogens can be isolated with a relative ease not possible in the past.
  6. The simplicity inherent in the use of the membrane filter and of the analysis of results has permitted a sharp reduction in the technician hours required to eastablish adequate water controls and has assured greater reliability of results.
Perhaps the outstanding feature of the procedure is the availability of results in a very short period of time so that prompt initiation of corrective measures can be implemented.  Previous methods often served only as a historical record of seasonal and other large variations in water conditions. Using the membrane filter, the laboratory tests of a sample consist of three simple steps: 
  1. Filtration
  2. Incubation
  3. Observation of results
Water samples are collected in a sterile container from the water source.  A portion, usually 100 ml., of each sample is then filtered through the membrane filter held in a standardized filter holder.  In many situations, a one liter sample may be collected.  Then four 250 ml. samples can be run and total coliforms for one liter can be determined.  Then by dividing by 10, the colonies per 100 ml. can be more accurately determined. The filters and the apparatus are easily sterilized by any of several simple procedures;  however, care must be exercised in their use.  While the water passes easily through the filter, bacteria are unable to do so and are deposited on its surface.  A 100 ml. water sample would pass through a filter in about one minute. 

The filter bearing the bacteria on its surface is then placed on a nutrient pad (food for bacteria) in a plastic petri dish.  The nutrient solution must be in accordance with Standard Methods and can be bought already prepared.  For total coliform, the petri dish holding the filter and the nutrient solution is then placed in an incubator at 35°C (95°F) for 20 hours. 

For fecal coliform, a different nutrient solution is used and the petri dish is waterproofed and placed in a water bath for 24 hours at 44.5°C (112°F). 

Fecal streptococcus is grown on a different medium at 35°C for 48 hours. 

During the incubation period, a visible bacterial colony will grow wherever a single organism was caught by the filter.  After incubation, the filter disc is removed from the dish and allowed to dry for one hour on absorbent paper.  The colonies are then counted. 

The colonies are easily distinguished from other bacteria that may be presented by color and/or sheen.  The medium helps to bring out this difference and also suppresses the growth of most other bacteria.  The number of colonies on the filter disc reveals the number of coliform bacteria in the volume of the filter sample.  The laboratory reports the results in terms of the number of coliforms per 100 ml. of water.  If 200 ml. of water are filtered and two "sheen" colonies develop, the report shows one coliform per 100 ml. 

Bacterial examination of water in the field has been made practicable by the development of a field testing kit.  In this kit, the membrane filter disc and absorbent pad are assembled in a two-part sterile plastic device which serves as a filter holder and culturing dish.  This assembly is called a "field monitor".  To use the field monitoring set, a sample of water is forced through a membrane that has been properly prepared and enclosed in a plastic holder to protect it.  The filter is then saturated with medium (or food) for growing bacteria and the two openings in the monitor are plugged.  It is then incubated in an inverted position in a portable incubator that  is available, or the filter can be mailed to the laboratory for incubation and analysis of coliform colonies.  The use of these sets may make present methods of shipping water samples to the laboratory unnecessary. 

*All the informaton on this page came from the Alabama Water Works Operator's Manual of 1989.

Their references were the APHA, AWWA, and WPCF, Standard Methods for the Examination of Water and Wastewater, 16th ed. Port City Press, Baltimore, 1985.



Plate Count and Multiple Tube Method

Prior to the membrane filter method, the multiple tube method was used for bacteriological examination of water.  In conjunction with this test, a total plate count was also conducted.  These tests are discussed here since they are still considered standard tests in the water works field. 

When a water sample arrives at the laboratory, two tests, the plate count and the coliform test by the multiple tube method, are made and reported to the operator.  The coliform test actually consists of two steps known as the presumptive test and the confirmed test.  Under certain conditions, it is necessary to go one step further and make a completed test;  however, this step is not always necessary.  To make the tests, small portions of the water sample are used in accordance with the following procedures. 

The Plate Count

The plate count is a test made by the laboratory to determine the total number of bacteria present in the sample.  This test does not differentiate between the many different types of bacteria and is thought of as giving index to the general "housekeeping" practices.  A "high" count indicates that some type of contamination is present and is undesirable. 

The test is made by placing a portion of agar in a petri dish.

Photo Credit:  Virginia Department of Health

Agar is a type of plant food that favors the growth of most bacteria.  A portion of the water sample is placed in the petri dish along with the agar.  It is then placed in an incubator with the temperature at 37°C or 98.6°F, which is body temperature.  After 24 hours, the plate is removed, examined, and the colonies in and on the agar are counted and recorded on the report form as "Bacteria per ml at 37°C".

It is assumed that each single bacteria in the agar starts to grow and due to the short generation time, it develops so that a spot or colony visible to the eye can be seen on the plate. These are the colonies that are counted.  Each colony is considered to represent an original bacterium. 

 The Multiple Tube Test

The following tests determine if coliform organisms are present and are much more significant than the plate count in determining if fecal contamination has occurred.  The presence of these organisms may be an indication that harmful bacteria are entering the water supply. 

The presumptive test

Coliform bacteria are grown in test tubes containing Lactose Broth in which a water sample is placed.  The Lactose Broth provides the moisture needed for growth, and few other organisms grown in the broth.  The test tubes are placed in an incubator at a temperature of 98.6°F. 

If food, moisture, and proper temperature are provided for the proper time, the organisms, if present, will grow.  If coliform organisms or the few other types which will grow in Lactose are not present in the water, then no growth occurs.  However, if organisms are present, they will grow under these ideal conditions and will ferment the Lactose Broth and produce gas.  The gas indicates the presence of the organisms. 

After 24 hours in the incubator, the tubes are examined for gas.  If no gas has formed, they are given an additional 24 hours time.  If, after 48 hours, gas has not formed,  then no organisms were present and the report reads "Absent" and the water is considered safe for drinking purposes. 

If organisms that reproduce in Lactose Broth are present in the sample, gas will be produced in any or all of the tubes within 18 hours.  Because of the presence of gas, it is known that some type organism is present;  and it is presumed that the organisms are coliform.  That is the reason the test is called the "presumptive test", it is merely presumed that coliform organisms are present if gas is produced in Lactose Broth. 

The presumptive test would have been completed if coliform were the only organism which grew in Lactose Broth.  Unfortunately, other organisms will ferment the broth and produce gas.  Thus, it becomes necessary to transfer a small amount of the contents of the Lactose Broth tubes showing gas into another, more selective food called Brilliant Green Bile.  Since coliform organisms are able to grow in the human intestine, bile is a preferred food and other bacteria which are present are unable to grow in this medium. 

The confirmed test

The tubes of Brilliant Green Bile containing some of the organisms from the Lactose Broth are then placed in an incubator.  At the end of 24 and 48 hours, they are examined for gas.  If gas is present, the confirmed test is positive and the results are reported as "Present".  The water sample contained coliforms, so the water supply becomes questionable as a safe drinking water.  If no gas is present, the confirmed test is "negative" and the results are reported as "Absent," and the water is considered safe. 

The presence of the Coli-aerogenes group of bacteria in the above tests does not definately mean that harmful bacteria are present.  Coliform bacteria are normally present in great numbers in the human intestine and, except in unusual circumstances, are not harmful to humans.  When present in a water sample, they do, however, indicate the presence of fecal contamination and the possibility that harmful (pathogenic) organisms, such as typhoid fever germs, may be present.  Therefore, the tests are not measures of actual disease-producing organisms, but rather are indicators of the possibility that they are present. 

*All the informaton on this page came from the Alabama Water Works Operator's Manual of 1989.

Their references were the APHA, AWWA, and WPCF, Standard Methods for the Examination of Water and Wastewater, 16th ed. Port City Press, Baltimore, 1985.
The most recent edition of "Standard Methods" should be used as a reference in setting up a laboratory and making bacteriological tests. 

*All the informaton on this page came from the Alabama Water Works Operator's Manual of 1989.

Their references were the APHA, AWWA, and WPCF, Standard Methods for the Examination of Water and Wastewater, 16th ed. Port City Press, Baltimore, 1985