WATER QUALITY: Analytical Methods: "Confirmation of fecal streptococcal bacteria" by G. G. Ehrlich February 18, 1976 QUALITY OF WATER BRANCH TECHNICAL MEMORANDUM NO. 76.11 Subject: WATER QUALITY: Analytical Methods: "Confirmation of fecal streptococcal bacteria" by G. G. Ehrlich Attached is a copy of the subject provisional method. It may be used to confirm the actual presence of fecal streptococcal bacteria obtained on a membrane filter during the procedure specified in Methods for the Collection and Analysis of Aquatic Biological and Microbiological Samples (Slack and others, 1973, TWRI, Bk. 5, Chap. A4, p. 50-54). The method is similar to that stated in Standard Methods (American Public Health Association and others, 1971, p. 690-691). Questions or comments pertaining to the provisional method should be directed to the Chief, Quality of Water Branch (Mail Stop 412, Reston, Virginia). R. J. Pickering Chief, Quality of Water Branch Attachment WRD Distribution: A,B,FO-LS,PO PROVISIONAL METHOD CONFIRMATION OF FECAL STREPTOCOCCAL BACTERIA M-Enterococcus and KF agar media are selective for the growth of fecal streptococci. A few other types of bacteria, chiefly non- fecal streptococci, may appear occasionally on these media. Colonies of non-fecal streptococci are typically very small but exhibit the characteristic red or pink coloration and would be counted as fecal streptococci in the membrane filter method. In case of doubt, identity of material from suspected colonies may be confirmed according to this procedure. The fecal streptococcal bacteria are distinguished from other bacteria by having the following three characteristics: (l) they lack the enzyme catalase; (2) they can grow at 45!C; (3) they grow in 40 percent bile. The confirmation procedure uses these three characteristics as criteria for identification. The proccdure is similar to that given by the American Public Health Association and others (1971, p. 690-691). 1. Summary o# method Cells from colonies to be testcd are streaked on brain-heart infusion agar slants. Cells from the slants are tested for the presence of catalase and for the ability to grow at 45!C and in the presence of 40 percent bile. Absence of catalase, growth at 45!C and in 40 percent bile broth constitute a positive test for fecal streptococci. Presence of catalase or failure to grow at 45!C or in bile broth indicate that the original colony was not of the fecal streptococcal group. 2. Application The confirmation test is applicable to fecal streptococcal colonies produced by the membrane filter method. Confirmation must be made as soon as possible after completion of thc membrane filter method, but not later than 24 hours. 3. Interferences As far as is known, only fecal streptococci show the pattern of results described below. 4. Apparatus 4.1 Inoculating loop, platinum-iridium wire, 3 mm, Brown and Sharpe gage 26, A. H. Thomas Co. (7012-E20) or equivalent. 4.2 Bunsen burner, for sterilizing inoculating loops. 4.3 Culture tubes, flint glass, 16 x 150 mm, Kimble (73500) or equivalent and test tube caps, 16 mm, Scientiflc Apparatus (9468) or equivalent. 4.4 Culture tube rack, galvanized, for 16 mm tubes, Thomas - Kolmer or equivalent. 4.5 Incubator, capable of maintaining temperature of 35-45! +/- 0.5!C, National Appliance (320) or equivalent. 4.6 Sterilizer, steam autoclave, Market Forge Sterilmatic or equivalent. 4.7 Microscope slides, glass, 76 x 25 mm. 5. Reagents 5.1 Brain-heart infusion agar: Add 52.0g of brain-heart infusion agar, Difco (0418) or equivalent, to 1000 ml of distilled water. Heat with vigorous stirring until solution becomes clear. Remove from heat immediately upon clearing. Place 5 ml of hot solution in each of about twelve 16 x 150 mm tubes. (Caution: do not allow solution to cool below 45!C or it will solidify.) Cap each tube. Sterilize at 121!C at 15 psi for 15 minutes. Remove from sterilizer and set tubes of molten agar at an angle of about 20 degrees from the horizontal (fig. 1). Allow to cool until solid. 5.2 Brain-heart infusion broth: Add 37g of brain-heart infusion, Difco (0037) or equivalent, to 1000 ml of distilled water. Stir until dissolved. Place 6 ml of broth in each of anothcr twelve 16 x 150 mm tubes. Cap each tube. Sterilize at 121!C at 15 psi for 15 minutes. 5.3 Brain-heart infusion -40 percent bile broth: Add 37g brain- heart infusion to 1000 ml of water. Stir until dissolved. Place 6 ml of brain-heart infusion broth in each of another twelve 16 x 150 mm stainless steel capped culture tubes. Sterilize at 121!C at 15 psi for 15 minutes. Add lOOg of oxgall, Difco (0128) or equivalent, to 1000 ml of water. Stir until dissolved. Place 4 ml of 10 percent oxgall solution in each of another twelve 16 x 150 mm stainless steel capped culture tubes. Sterilize at 121!C at 15 psi for 15 minutes. Remove the caps from a tube of sterile 10 percent oxgall solution and a tube of sterile brain-heart infusion broth. Quickly pour the oxgall solution into the brain-heart infusion broth tube and recap. Hydrogen peroxide solution, 3 percent. 5.5 Potassium iodide, crystals. 6. Collection No sample collections are necessary. 7. Analysis 7.1 The membrane filter method for fecal streptococcal bacteria should be conducted according to procedures described by Slack and others (1973, p. 50-54)- 7.2 From the incubated membrane filter select a colony or colonies to be confirmed for fecal streptococcal bacteria. 7.3 Sterilize the inoculating loop by flaming in the burner. The long axis of the wire should be held parallel to the cone of the flame so that the entire end of thc wire and loop are heated to redness. Remove from flame and allow the wire to cool for about 10 seconds. Do not allow the loop to contact any foreign surface during the cooling period. When cool,touch the loop lightly to a single colony. Part of# the colony material will adhere to the wire. 7.4 Uncap a brain-heart infusion agar slant and hold it at an angle of about 45 degrees with the flat surface of the slant upward (fig. 2). Insert the loop with colony material into the tube. Starting at the base of the slant, lightly rub the loop against the agar working toward the top in a zig-zag pattern (fig. 2). 7.5 Recap the tube. Flame the loop and inoculate additional tubes as above until all colonies to be tested have been placed on agar in separate tubes. Place the inoculated tubes in the test tube rack and incubate at 35! +/- 0.5!C for 24-48 hours. 7.6 Remove the tubes from the incubator and examine. Growth will be evident as a translucent, glistening film on the surface of the slant. 7.7 Test the potency of the hydrogen peroxide solution by placing a few millilitres in a test tube and adding a few potassium iodide crystals. A brown coloration and the appearance of bubbles in the solution indicates that the hydrogen peroxide solution is acccptable for use. If otherwise, discard and obtain a fresh hydrogen peroxide solution. 7.8 Flame the loop and allow to cool. Immediately uncap a tube of brain-heart infusion agar having growth. Remove a loopful of growth from the slant and smear on a clean glass slide. Add a few drops of freshly tested 3 percent hydrogen peroxide solution to the material on the slide. Immediately observe the slide for bubble formation. Observation of bubble formation may be facilitated by use of a low power microscope. The absence of bubbles constitutes a negative catalase test indicating a probable fecal streptococcal culture and confirmation should be continued. The presence of bubbles constitutes a positive catalase test indicating the presence of a non-streptococcal bacteria and the test may be terminated at this point. 7.9 Proceed as follows with all catalase negative cultures. Uncap one tube each of brain-heart infusion broth and brain-heart infusion 40 percent bile broth. Using a flamed loop transfer one loopful of material from the agar slant to one of the tubes. Reflame the loop and transfer a loopful of material from the agar slant to the other tube. Recap the tubes. 7.10 Flame thc loop and inoculate additional tubes as above until all catalase negative cultures have been placed in separate tubes of brain-heart infusion broth and brain-heart infusion - 40 percent bile broth. 7.11 Place the inoculated tubes of brain-heart infusion broth in a test tube rack and incubate at 45! +/- 0.5!C for 48 +/- 3 hours. Include tubes of uninoculated broth to serve as controls. 7.12 Place the inoculated tubes of brain-heart infusion - 40 percent bile broth in a test tube rack and incubate at 35! +/- 0.5!C for 72 +/- 4 hours. Include tubes of uninoculated medium to serve as controls. 7.13 Remove tubes from incubator and examine. Appearances of turbidity in the inoculated tubes as compared to the controls constitutes a positive test for growth. Appearance o# growth in both brain-heart infusion broth and brain- heart infusion - 40 percent bile broth constitutes a positive confirmation for the presence of streptococci in the original colony. Absence of growth in either or both tubes indicates that the original colony was not of the fecal streptococcal group. 7.14 All inoculated tubes and smeared slides should be autoclaved at 121!C at 15 psi for 15 minutes before discarding. 8. Calculations No calculations are necessary. 9. Report Results of the fecal streptococcal confirmation test are included in the colony counts for fecal streptococcal bacteria. 10. Precision Absence of bubbling in the catalase test coupled with growth in brain-heart infusion broth at 45!C within 48 hours and in brain- heart infusion 40 percent bile at 35!C within 72 hours constitutes a positive confirmed test. REFERENCES American Public Health Association and others, 1971, Standard methods for the examination of water and wastewater [13th ed.]: New York, Am. Public Health Assoc., 874 p. Slack, K. V., Averett, R. C., Greeson, P. E., and Lipscomb, R. G., 1973, Methods for collection and analysis of aquatic biological and microbiological samples: U.S. Geol. Survey Techniques Water- Resources Inv., Bk. 5, Chap. A4, 165 p.