Appendix B: Bacteriological Sampling and Analysis

San Diego State University
Environmental Health and Safety Department

Water Main and Building Water Disinfection and Testing

AWWA §C651 provides that two consecutive sets of samples, taken at least 24 hours apart, shall be collected from the main and analyzed for bacteriological contamination. If initial bacteriological samples are unsatisfactory, the new main should be reflushed and additional samples collected and analyzed. According to AWWA §C651, if any of the check samples are also unsatisfactory, the main must be rechlorinated, reflushed, and resampled until satisfactory results are achieved.

AWWA Standard §C651 provides that bacteriological testing of the disinfected main should be conducted in accordance with Standard methods for the Examination of Water and Wastewater and show the absence of coliform organisms. Total coliform is the indicator group of bacteria for the use in monitoring drinking water. The Maximum Contaminant Level for total coliform as determined by the Safe Drinking Water Act is now based on the presence or absence of the indicator bacteria, not on density or direct count. There are four standard laboratory techniques that are approved for coliform analysis:

MMO-MUG
Multiple tube fermentation (MTF)
Presence-absence (PA)
Membrane filtration (MF)

The MMO-MUG method is a new technique that is able to confirm the presence of total coliforms in a shorter time frame than the other approved methods. This technique is based on a color change that occurs as the result of the reaction of an enzyme produced by the coliform group of bacteria with the MMO-MUG media. There are several formats currently available for this method. They all involve inoculation in the laboratory of the drinking water sample with a specific quantity of MMO-MUG media. The inoculated sample is incubated at 35°C for 24 hours and observed for development of yellow coloration. If the yellow color is equal to or darker than the minimal standard supplied with the product, the sample contained confirmed total coliforms. The confirmed presence of total coliforms should be interpreted as unsatisfactory requiring reflushing of the main and additional bacteriological samples collected and analyzed.

The laboratory that is chosen to perform the analysis will typically provide the sampler with appropriate containers for sample collection.

AWWA §C651 provides that at least one set of samples for bacteriological analysis should be collected from every 1,200 ft of the new water main, one set from the end of the line, and at least one set from each branch. If trench water or excessive quantities of dirt has entered the new main during construction, AWWA Standard §C651 specifies that samples should be taken at intervals of approximately 200 ft and shall be identified by location.

Use only sterile bottles furnished by the laboratory. Keep the bottles sealed until used. Each sample bottle should contain a dechlorinating agent (typically, sodium thiosulfate) in sufficient amount to neutralize any residual chlorine in the water sample. Do not rinse the bottle prior to taking the sample as such rinsing will remove the dechlorinating agent and render the subsequent sample invalid.
Try to avoid collecting the sample from a hose or fire hydrant. Such outlets will often contaminate the sample leading to unsatisfactory results. AWWA Standard §C651 recommends the use of a specially installed sampling tap consisting of a smooth, unthreaded, ½ inch hose bib. A corporation stop installed in the main equipped with a copper-tube goose-neck assembly can also function as a sampling tap.
Be sure that the heavily chlorinated water has been thoroughly flushed from the main before sampling. Run water through the sampling tap at a steady rate 3 to 5 minutes before beginning sampling procedures.
Wash hands thoroughly. Remove the bottle lid must before filling, holding the lid in your free hand. Do not contaminate the inner surface of the cap of the bottle with your hands. Fill the bottle to the shoulder or fill line. Do not overflow the bottle or splash water on the outside of the bottle. Replace the lid and tighten securely.
Complete the appropriate sample documentation provided by the laboratory. This will typically include a sample label and chain of custody form.
Deliver the samples to the laboratory promptly after collection. There are strict time limits on the amount of time that may elapse between sample collection and analysis before the sample is considered too old to analyze. Check with the laboratory on sample holding time requirements. Unless special arrangements are made, avoid having the sample arrive at the laboratory on weekends or holidays.
Samples should be held at a temperature of 4°C. If practicable, place samples in an iced cooler for storage during transport if transport time will exceed 1 hour. At no time, however, should the sample container be allowed to become immersed or submerged in the ice or melted ice water. Check with the laboratory for specific packaging and transport recommendations.


	Business and Financial Affairs > Environmental Health & Safety > Sanitation > Potable Water >
	Environmental Health & Safety Potable (Drinking) Water San Diego State University Environmental Health and Safety Department Water Main and Building Water Disinfection and Testing Appendix B: Bacteriological Sampling and Analysis AWWA §C651 provides that two consecutive sets of samples, taken at least 24 hours apart, shall be collected from the main and analyzed for bacteriological contamination. If initial bacteriological samples are unsatisfactory, the new main should be reflushed and additional samples collected and analyzed. According to AWWA §C651, if any of the check samples are also unsatisfactory, the main must be rechlorinated, reflushed, and resampled until satisfactory results are achieved. Analysis Method AWWA Standard §C651 provides that bacteriological testing of the disinfected main should be conducted in accordance with Standard methods for the Examination of Water and Wastewater and show the absence of coliform organisms. Total coliform is the indicator group of bacteria for the use in monitoring drinking water. The Maximum Contaminant Level for total coliform as determined by the Safe Drinking Water Act is now based on the presence or absence of the indicator bacteria, not on density or direct count. There are four standard laboratory techniques that are approved for coliform analysis: MMO-MUG Multiple tube fermentation (MTF) Presence-absence (PA) Membrane filtration (MF) The MMO-MUG method is a new technique that is able to confirm the presence of total coliforms in a shorter time frame than the other approved methods. This technique is based on a color change that occurs as the result of the reaction of an enzyme produced by the coliform group of bacteria with the MMO-MUG media. There are several formats currently available for this method. They all involve inoculation in the laboratory of the drinking water sample with a specific quantity of MMO-MUG media. The inoculated sample is incubated at 35°C for 24 hours and observed for development of yellow coloration. If the yellow color is equal to or darker than the minimal standard supplied with the product, the sample contained confirmed total coliforms. The confirmed presence of total coliforms should be interpreted as unsatisfactory requiring reflushing of the main and additional bacteriological samples collected and analyzed. The laboratory that is chosen to perform the analysis will typically provide the sampler with appropriate containers for sample collection. Number of Samples AWWA §C651 provides that at least one set of samples for bacteriological analysis should be collected from every 1,200 ft of the new water main, one set from the end of the line, and at least one set from each branch. If trench water or excessive quantities of dirt has entered the new main during construction, AWWA Standard §C651 specifies that samples should be taken at intervals of approximately 200 ft and shall be identified by location. Sample Collection Procedures Use only sterile bottles furnished by the laboratory. Keep the bottles sealed until used. Each sample bottle should contain a dechlorinating agent (typically, sodium thiosulfate) in sufficient amount to neutralize any residual chlorine in the water sample. Do not rinse the bottle prior to taking the sample as such rinsing will remove the dechlorinating agent and render the subsequent sample invalid. Try to avoid collecting the sample from a hose or fire hydrant. Such outlets will often contaminate the sample leading to unsatisfactory results. AWWA Standard §C651 recommends the use of a specially installed sampling tap consisting of a smooth, unthreaded, ½ inch hose bib. A corporation stop installed in the main equipped with a copper-tube goose-neck assembly can also function as a sampling tap. Be sure that the heavily chlorinated water has been thoroughly flushed from the main before sampling. Run water through the sampling tap at a steady rate 3 to 5 minutes before beginning sampling procedures. Wash hands thoroughly. Remove the bottle lid must before filling, holding the lid in your free hand. Do not contaminate the inner surface of the cap of the bottle with your hands. Fill the bottle to the shoulder or fill line. Do not overflow the bottle or splash water on the outside of the bottle. Replace the lid and tighten securely. Complete the appropriate sample documentation provided by the laboratory. This will typically include a sample label and chain of custody form. Deliver the samples to the laboratory promptly after collection. There are strict time limits on the amount of time that may elapse between sample collection and analysis before the sample is considered too old to analyze. Check with the laboratory on sample holding time requirements. Unless special arrangements are made, avoid having the sample arrive at the laboratory on weekends or holidays. Samples should be held at a temperature of 4°C. If practicable, place samples in an iced cooler for storage during transport if transport time will exceed 1 hour. At no time, however, should the sample container be allowed to become immersed or submerged in the ice or melted ice water. Check with the laboratory for specific packaging and transport recommendations.
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