Is there additional information on methods for counting indicator and opportunistic organisms?

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Document ID TE3312


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Published Date 03/07/2019
Is there additional information on methods for counting indicator and opportunistic organisms?

Counting Indicator and Opportunistic Organisms

Membrane Filtration
      The Membrane Filtration (MF) method requires filtering a sample of appropriate volume through a membrane filter of sufficiently small pore size to retain the organism(s) sought. Then the filter is placed on an appropriate agar medium, or pad saturated with an appropriate broth medium, and incubated. If the organisms sought are present, colonies will grow on the membrane filter. Colonies are examined at 10–15X magnification with a stereoscopic microscope and then identified by size, color, shape and sheen. Typical colonies are counted and the number is reported as the number of colonies per 100 mL of sample.
Total Coliform Bacteria
      The standard definition of total coliform bacteria must be altered slightly when using the membrane filter method for detection and enumeration, because gas production from lactose cannot be detected on a filter surface. Therefore, when using certain types of media, such as m-Endo broth or m-Endo LES agar, it is assumed all colonies producing acid or aldehyde also produce gas. Another type of media, m-ColiBlue24®*, which was recently developed for the simultaneous detection of total coliforms and E. coli, relies on the presence of inhibitors in the medium which allow only total coliform bacteria to grow. The total coliform colonies are identified by a nonspecific color indicator, and E. coli colonies are identified by a specific enzymatic reaction.

      Until recently, in the United States there were only two media, m-Endo broth or m-Endo LES agar, approved for the presumptive detection of total coliforms by MF. On Dec. 1, 1999 m-ColiBlue24® was promulgated in the Federal Register as an acceptable method for the simultaneous detection of total coliforms and E. coli in drinking water for monitoring under the Total Coliform Rule. Confirmation of total coliforms and E. coli, required with the use of m-Endo media, is unnecessary when using m-ColiBlue24®. Other countries have approved the use of lactose tergitol agar and lactose TTC (2,3,5-triphenyltetrazolium chloride) tergitol agar.5 For both of these media, coliforms ferment lactose in the media and produce an acid-aldehyde complex. As with the m-Endo media, a specified number of suspect total coliform bacteria from wastewater or potable water samples must be transferred to liquid media for confirmation.2 In media like m-Endo, a fuchsin sulfite indicator is used to indicate the presence of aldehyde from the fermentation of lactose. The indicator is red in the presence of the aldehyde. A suitably prepared membrane filter is placed on m-Endo medium and incubated for 24 hours at 35 to 37 °C. (For reporting to USEPA, 35 ± 0.5 °C must be used.) Coliforms (lactose-fermenting colonies) appear red with a golden-green metallic sheen on membranes. The number of representative colonies is counted and reported as presumptive coliform organisms. For drinking water samples, atypical coliform colonies must be verified to make sure they are coliforms. Lauryl tryptose and brilliant green lactose bile broth tubes are inoculated with growth from the colonies. Gas production verifies that the suspected organisms are indeed coliforms. The procedure described in the MPN Methods section for confirmation of total coliforms is used with an additional inoculum in a single-strength lauryl tryptose broth tube. Gas production in 24 to 28 hours constitutes a positive test.

      Organisms often are exposed to adverse environments such as water treatment processes. When these organisms grow slowly or not at all under bacteriological testing conditions, they are called stressed, or injured, organisms. Stressed organisms are found in chlorinated effluents, saline waters, and natural waters polluted with substances such as heavy metal ions or toxic organic wastes. Sodium deoxycholate, used as an inhibitory substance in m-Endo type media, and sodium lauryl sulfate have been shown to inhibit as many as 70% of the injured coliforms.6 Using m-Endo LES agar, Evans, et al., identified 25% of the falsenegative colonies as Citrobacter, Enterobacter, Escherichia, or Klebsiella species.7 An enrichment technique to overcome some of the problems in the detection of injured coliforms has been described in Standard Methods. The membrane filter is incubated on a pad saturated with lauryl tryptose broth for 1.5 hours at 35 ± 0.5 °C in a relative humidity of at least 90%. Then the membrane is transferred to a pad which has been saturated with m-Endo broth and incubated for 20 to 22 hours at 35 ± 0.5 °C. The resultant coliform colonies will have the characteristic greenish-gold sheen under 10–15X magnification. Verification is achieved by following the same procedure as the one for unstressed organisms.

      In spite of improved recoveries with the lauryl tryptose broth enrichment technique, LeChevallier, et al.8 reported an improved formulation of Chapman’s Tergitol 7 agar (m-T7)9 recovered 43% more coliforms than recovered by m-Endo agar and 36% more coliforms than recovered by m-Endo agar with lauryl tryptose broth resuscitation. (Appendix B contains instructions for the formulation and preparation of Chapman’s Tergitol 7 agar.) Bromthymol blue and bromcresol purple are used in m-T7 to provide the characteristic yellow, acid-positive colonies and to inhibit the growth of noncoliform bacteria. Penicillin G also is added to inhibit the growth of Staphylococcus and Micrococcus species. Positive results are obtained when smooth, yellow, convex colonies are formed on the membrane after anaerobic incubation for 24 hours at 35 °C. A level of 0.5% false negatives was reported. Confirmation (gas production from lactose) was 70.6% compared to 69.6% for m-Endo agar.

     The m-ColiBlue24® broth was developed at Hach Company to provide a membrane filtration method that would allow the determination of both total coliforms and E. coli in a single 24 hour incubation step.10 It is a nutritive, lactosebased medium, containing specific inhibitors that selectively eliminate the growth of noncoliform bacteria, allowing only the total coliform bacteria to grow. The total coliforms become visible by reducing a non-selective dye, TTC (2,3,5-triphenyltetrazolium chloride) present in the medium. The reduction of TTC results in the formation of red colonies which can be easily seen on the membrane filter. E. coli are distinguishable from other total coliforms by a visible blue color which forms in these colonies. This color formation is the result of the enzymatic cleavage of a substrate, BCIG (5-bromo-4-chloro-3-indolyl-β-D-glucuronide), by the enzyme, β-glucuronidase, which is predominantly produced by E. coli. The colony color remains closely associated with the colonies and does not diffuse away from target colonies, therefore, blue colonies are readily distinguishable even when background total coliform colonies are too numerous to count. This medium was specifically designed to maximize the growth rate of total coliforms and provide for optimal recovery of stressed organisms. In addition, it does not contain deoxycholate or bile acids, which have been proven to inhibit the growth.

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