WATER QUALITY: Analytical Methods - "Nitrifying bacteria (most probable number, MPN, method)" by Gary G. Ehrlich January 24, 1975 QUALITY OF WATER BRANCH TECHNICAL MEMORANDUM NO. 75.13 Subject: WATER QUALITY: Analytical Methods - "Nitrifying bacteria (most probable number, MPN, method)" by Gary G. Ehrlich The attached provisional method is approved for use in the Water Resources Division. Questions or comments pertaining to the 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 Nitrifying Bacteria (most probable number, MPN, method) by Gary G. Ehrlich Nitrification is the biological oxidation of reduced nitrogen compounds to nitrite and nitrate. Most commonly, the initial substance is ammonium, and the final product is nitrate. The process occurs in two distinct steps, each mediated by a specific group of bacteria. The Nitrosomonas group, which includes several genera of bacteria, is able to oxidize ammonium (NH4) only to nitrite (N02) as shown: NH4 ~ 3/2 02 --> N02 + 2H+ + H20 (see hard copy) The Nitrobacter group of bacteria oxidizes nitrite (N02), but not ammonia (NH4+ or any other reduced nitrogen compound, to nitrate (N03-) as shown: N02 ~ 1/2 02 --> N03 (see hard copy) Hydrogen ions produced in the oxidation of ammonia to nitrite may be of some geochemical significance because the excess acid can dissolve minerals and participate in exchange reactions on clays. Nitrification is important in soils because the process controls the supply of nitrate used by higher plants. In surface waters nitrification contributes to oxygen demand. Thc responsible organisms, Nitrosomonas and~Nitrobacter, are autrotrophic bacteria. They obtain their energy from the inorganic oxidations indicated above and use carbon dioxide as a cellular carbon source. The media for enumerating these bacteria are assumed to be free of organic carbon. This assumption is valid to the extent that initially only nitrifiers will grow on the media. Later,as the autotrophs grow and release cell substances to the media, heterotrophs will develop. The medium for enumerating Nitrosomonas contains ~H4. Appearance of NO~ in the inoculated cultures, but not in control cultures, presumptively indicates the presence of Nitrosomonas in the sample. A negative test is not sufficient evidence to prove that Nitrosomonas is absent, because N02 produced by Nitrosomonas can be oxidized to N03 by Nitrobacter. Therefore, a positive test for either NO~ or NO~ in the inoculated cultures indicates the presence of Nitrosomonas. The medium for enumerating Nitrobacter contains NO~; disappearance of NO~ from the inoculated cultures, but not from control cultures, presumptively indicates the presence of Nitrobacter. The method described is similar to that described by Alexander and Clark (1965). 1. Summary of method Decimal dilutions of multiple sample aliquots are inoculated into organic-free media containing ammonium ions for Nitrosomonas enumeration or nitrite ions for Nitrobacter enumcration. The inoculated cultures are incubated at 28~C for 3 weeks, following which the inoculated culturcs and control cultures are tested for the presence of nitrite. The most-probable-number (MPN) of each group of nitrifying bacteria is determined from the distribution of positive and negative responses among the inoculated tubes. 2. Application This method is applicable to all types of fresh and saline waters and soils. 3. Interferences No interferences are known for the procedure. 4. Apparatus All materials used in bacteriological testing must be free of agents which inhibit bacterial growth. 4.1 Water-sampling bottle, samplers for obtaining water samples under sterile conditions as marketed by General Oceanic., Hydro Products, Kahl Scientific Instrument Corp., and others. A metallic water sampler, lowered at a speed of 1 m/sec, may be effective for sterile collection of water samples (Kriss and others, 1966). Metallic water sampling bottles are available from Wildlife Supply Co. (1050 or 1200); Kahl Scientific Instrument Corp. (130WA100); Inter Ocean Systems, Inc. (206); Foerst Mechanical Specialties Co. (Improved Water Sampler, Kemmerer-type); or equivalent. 4.2 Culture tubes and caps, flint glass tubes, 16 x 125 mm, Kimble (73500), Corning (9805), or equivalent; tube caps, 16 mm, Scientific Products (T1390-16) or equivalent. 4.3 Culture tube rack, galvanized for 16 mm tubes, Thomas - Kolmer or equivalent. 4.4 Incubator with temperature range from 5!C above ambient to 60!C. National Appliance (320) or equivalent, or water bath capable of maintaining a temperature of 28 + 1!C, Matheson (65310-10) or equivalent. 4.5 Sterilizer, steam autoclave, Matheson Scientific (59827- 20), Market Forge Sterilmatic, or equivalent. 4.6 Bottles, milk diultion, APHA, Pyrex or Kimax with screwcaps. 4.7 Glass beads, solid, 3 mm, Fisher Scientific (11-312A) or equivalent. 4.8 Sieve, U.S. standard series, 10 mesh, Fisher (408816) or equivalent. 4.9 Pipets, 1.0 ml capacity, presterilized, disposable, glass or plastic with cotton plugs, Millipore (XX63 001 35) or equivalent. 4.10 Pipets, 10.0 ml capacity, Corning (7057) or equivalent. Wrap the pipets in kraft paper and sterilize in the autoclave, or place in pipet box, Matheson Scientific (55930- 20) or equivalent, and heat in an oven at 170!C for 2 hours. 5. Reagents 5.1 Ammonium-calcium carbonate medium for most-probable- number (~N) of Nitrosomonas. To 1000 ml of distilled water, add 0.5 g of ammonium sulfate [( ~ 14)~S04], 1.0 g of potassium phosphate dibasic (K~HP04), 0.03 g of ferrous sulfate (FeS04-7H~O), 0.3 g of sodium chloride (~aCl), 0.3 g of magnesium sulfate (MgS04~7H20), and 7.5 g calcium carbonate tCaC03). Place 3 ml of medium in each culture tube, cap, and autoclave at 121!C at 15 psi for 15 minutes. 5.2 Nitrite-calcium carbonate medium for most-probable-number (MPN) of Nitrobacter. To l000 ml of distilled water, add 0.006 g of potassium nitrite (KNO2), 1.0 g of potassium phosphate dibasic (K2HPO4), 0.3 g of sodium chloride (NaCl), 0.1 g of magnesium sulfate (Mg~04-7H~O), 1.0 g of calcium carbonate (CaC03), and 0.3 g of calcium chloride (CaC12). Place 3 ml of medium in each culture tube, cap, and autoclave at 121!C at 15 psi for 15 minutes. 5.3 Griess - Ilosvay reagent: (a) dissolve 0.6 g sulfanilic acid in 70 ml hot (90+ C) distilled water; cool the solution, add 20 ml of concentrated hydrochloric acid (HCl), dilute the mixture to 100 ml with distilled water, and mix; (b) dissolve 0.6 g of alpha naphthylamine in 10 to 20 ml of distilled water containing 1 ml of concentrated hydrochloric acid (HCl); dilute to 100 ml with distilled water and mix; and (c) dissolve 16.4 g of solium acetate (CH3COONa ~3H20) in distilled water, dilute to 100 ml with distilled water and mix. Store the solutions separately in dark bottles in a refrigerator. Stability of the solutions is unknown; however, storage should not excced l month. 5.4 Zinc-copper-manganese dioxide mixture: Mix together 1 g of powdered zinc metal (Zn), l g of powdered manganese dioxide (MnO2), and 0.1 g of powdered copper (Cu). 5.5 Buffered dilution water: Dissolve 34.0 g potassium dihydrogen phosphate (K1121'04) in 500 ml distilled water. Adjust to pH 7.2 with 1 N sodium hydroxide (NaOH). Dilute to 1 litre with distilled water. Sterilize in dilution bottles at 121!C .at 15 psi for 20 minutes. After opening a bottle of stock solution, refrigerate the unused part. Discard contaminated solution, indicated by slight turbidity or precipitate accumulation. For water sample dilution blanks, add 1.2 ml of sterile stock phosphate buffer solution to 1 litre of distil led water. Dispense in milk dilution bottles in amounts that will provide 99 ml + 2 after autoclaving at 121!C at 15 psi for 20 minutes. Loosen caps prior to sterilizing and tighten when bottles have cooled. For soil sample dilution blanks, place 95 ml of distilled water and about three dozen, 3 mm diameter, glass beads in a milk dilution bottle. For each 95 ml dilution blanl;, prepare also 5 dilution blanks of 90 ml distilled water in milk dilution bottles. Omit the glass beads from the 90 ml blanks. Autoclave at 121!C at 15 psi for 20 minutes. Loosen caps prior to sterilizing and tighten when bottles have cooled. 6. Collection Water samples for bacteriologic examination must be collected in containers that have been sterilized in an autoclave for 20 minutes at 121!C at 15 psi. Sterilized milk dilution bottles are ideal sample containers. When the sample is collected, ample airspace must be left in the bottle to facilitate mixing of the sample' by shaking. Care must be taken to avoid contamination of the sample and sample bottle at the time of collection and in the period prior to analysis. To insure maximum correlation of results, the sample sites and methods used for nitrifying bacteria should correspond to those selected for chemical and other biological sampling. Sampling for bacteria at depth is complicated by the requirement to avoid contamination of the deeper water layers by bacteria carried from shallower depths on the inner walls of the sampler. The sample collection method will be determined by the study objectives.In lakes, reservoirs, deep rivers, and estuaries, bacterial abundance may vary transversely, with depth, and with time of day . To collect a surface sample from a stream or lake, open a sterile milk dilution bottle, grasp it near its base, and plung it, necl; downward, below the water surface. Allow the bottle to fill by slowly turning the bottle until the neck points slightly upward. The mouth of the bottle must be directed into the current. If there is no current, as in the case of a lake, a current should be artificially created by pushing the bottle horizontally forward in a direction away from the hand (American Public Health Association and others, 1971, p. 658). To collect a sample representative of the bacterial concentration at a particular depth, use one of the water sampling bottles discussed in 4.1 above. For small streams, a point sample at a single transverse position located at the centroid of flow is adequate (Goerlitz and Brown, 1972). As soon as possible after collection, prefcrably within 4 hours and not more than 6 hours, inoculate the decimal dilutions of the sample into tubes of ammonium-calcium carbonate medium and nitrite-calcium carbonate medium. Samples must be kept cool during the time between collection and inoculation. If inoculation is delayed, ice or refrigerate the sample but do not freeze. Collect soil samples in a sterile manner and place in polyethylene bags or waxed cardboard containers. Avoid exposing soil samples to heat or drying. If the sample is not processed on the day of collection, it may be stored at 4!C for 1-2 weeks in the closed container, provided that the container is pin holed for aeration. Just prior to processing, pass the entire sample through a 10 mesh sieve and mix thoroughly before taking an aliquot for analysis. If desired, a separate subsample may be taken for determination of dry weight (Clark, 1965). The sizes of inoculums should be such that, after incubation, both positive and negative results are obtained. The method fails if only positive or only negative results are obtained with all volumes tested. The following sample volumes are suggested: 1. For water samples, use volumes of 1, 0.1, 0.01, O.0Ol and 0.0001 ml. 2. For soil samples, use dilutions to 10 6. 7. Analysis 7.1 Before starting the analysis, clear an area of the laboratory bench and swab it with a bit of cotton moistened with 70% ethyl alcohol or undiluted isopropanol. 7.2 Set out 5 tubes of ammonium-calcium carbonate medium and 4 tubes of nitrite-calcium carbonate medium for each volume to be tested. Three of the tubes will be inoculated with a decimal dilution; the fourth tube will be a control tube. 7.3 If the volume of water sample to be tested is greater than 0.1 ml, transfer the measured samples directly to the culture tubes using sterile pipets. Take care when removing caps from sterile culture tubes so as to avoid contamination. When testing water, if the volume of the desired sample aliquot is less than 0.1 ml, proceed as above after preparing appropriate dilutions by adding the sample to a sterile milk dilution bottle in the following amounts: Volume of sample added to 99 ml Dilution dilution bottle Size of inoculum 1:100 1.0 ml original sample 1.0 ml of 1:100 dilution 1:1000 1.0 ml original sample 0.1 ml of 1:1000 dilution 1:104 1.0 ml 1:100 dilution 1.0 ml of 1:104 dilution 1:105 1.0 ml 1:100 dilution 0.1 ml of 1:105 dilution 1:106 1.0 ml 1:104 dilution 1.0 ml of 1:106 dilution 1:107 1.0 ml 1:104 dilution 0.1 ml of 1:107 dilution . NOTE: Use a sterile pipet for each bottle. After each transfer between bottles, close and shake the bottle vigorously 25 times. Diluted samples should be inoculated within 20 minutes after preparation. To prepare a decimal dilution series of a soil sample, proceed by transferring 10 g of moist soil to a sterile water blank containing 95 ml of water and glass beads. Cap the bottle tightly and shake vigorously 25 times. Immediately after shaking, transfer 10 ml from the center of the suspension to a sterile 90 ml water blank. Shake vigorously 25 times and continue the dilutions until a sufficiently dilute sample is obtained. Using this dilution series proceed until all tubes are inoculated. 7.4 Clearly mark each set of inoculated tubes and uninoculated control tubes indicating location, time of collection, sample number, and sample volume. Code each tube for easy identification when recording results. 7.5 Place the inoculated tubes and control tubes in a test tube rack and incubate at 28!C for 21 days. 7.6 After incubation, test each inoculated tube and control tube for nitrite using the Griess-Ilosvay Reagent. Immediately prior to the test, mix together in equal parts the sulfanilic acid reagent, the alpha naphthyl amine reagent, and the sodium acetate reagent. Add e drops of this mixture to each tube. Observe the contents of each tube for the development of a purplish-red color within 5 minutes. 7.7 Record as positive for Nitrosomonas, all inoculated tubes of ammonia-calcium carbonate medium that develop a purplish-red color within 5 minutes. 7.8 To all tubes of ammonia-calcium carbonate medium that do not develop a purplish-red color within 5 minutes, add a small pinch of the Zn-Cu-MnO2 mixture. If a reddish color develops, record the tube as positive for Nitrosomonas on thc basis that the initially negative reading for nitrite indicated that thc nitrite formed by Nitrosomonas was oxidized to nitrate by Nitrobacter. 7.9 Record as positive for Nitrobacter all tubes of nitrite- calcium carbonate medium that do not develop the characteristic purplish red color formed by the reaction of nitrite with the Griess-Ilosvay reagent. 7.10 A positive result in a control tube indicates a contamination of the medium and results of the test, therefore, are invalid. 8. Calculations Record the number of positive inoculated tubes occurring over all sample volumes tested. When more than three volumes are tested, the results from only three of these are used in computing the MPN. To select the three dilutions for the MPN index, proceed as follows: Take as the first member the smallest sample volume in which all tests are positive (no larger sample volume giving any negative results) and the two next succeeding smaller\sample volumes (American Public Health Association and others, 1971, p. 673-674). In the examples given below, the number in the numerator represents positive tubes; the denominator represents the total number of tubes inoculated. Decimal Dilutions Combination Example 1 ml 0.1 ml 0.01 ml 0.001 ml of positives a 3/3 3/3 2/3 0/3 3-2-0 b 0/3 1/3 0/3 0/3 0-1-0 c 3/3 2/3 1/3 1/3 3-2-2 d 3/3 2/3 2/3 0/3 3-2-2 In b the three dilutions should be chosen to place the positive result in the middle dilution. When a positive result occurs in a dilution higher than the three chosen according to the rule, as in c it should be placed in the result for the highest chosen dilution as in d. A table giving MPN for various combinations of positive and negative results when three 10.0 ml dilutions, three 1.0 ml, and three 0.1 ml dilutions is given in Table 1. If a series of decimal dilutions other than 1.0, 0.1, and 0.01 ml is used, record the MPN as the value from the table multiplied by a factor of 10 divided by the volume in which all tests were positive. MPN tables for other combinations of sample volumes and number of tubes are given by the American Public Health Association and others (1971, p. 674-676). 9. Report The concentration of nitrifying bacteria is reported as MPN Nitrosomonas and MPN Nitrobacter per 100 ml for water samples or as MPN per 100 gm for soil samples. The method of expressing unit weight (wet or dry) of soil samples should be indicated. Values less than 10, report whole numbers; 10 or more, report two significant figures. 10. Precision The MPN inherently has a low order of precision. Precision increases as the number of tubes is increased. It increases rapidly as the number of tubes increase from 1 to 5 but then increases at a slower rate so that the gain in using 10 tubes instead of 5 is much less than is achieved by increasing the number of tubes from 1 to 5. Variance as a function of the number of tubes inoculated from a decimal dilution series is given below. No. of tubes at Variance for lO-fold each dilution dilution series 1 0.580 3 0.335 5 0.259 10 0.183 REFERENCES Alexander, Martin, and Clark, E. E., 1965, Nitrifying bacteria in Blach, C. A., ed., Methods of soil analysis: Madison, Wisc., Am. Soc. of Agronomy, Part 2, p. 1477-1483. American Public Health Association and others, 1971, Standard methods for the examination of water and wastewater (13th Ed): New York, Am. Public l~ealth Assoc., 874 p. Clark, F. E., 1965, Agar-plate method for total microbial count, in Blach, C. A., ed. Methods of soil analysis: Madison, Wisc., Am. Soc. of Agronomy, Part 2, p. 1463. Goerlitz, D. T. and Brown, Eugene, 1972, Methods for the investigation of organic substances in water: U.S. Geol. Survey Techniques Water-Resources Inv., Book 5, Chap. A3, 40 p. Kriss, A. E., Lebedeva, M. N., and Tsiban, A. V., 1966, Comparative estimate of a Nansen and microbiological water bottle for sterile collection of watcr samples from depths of seas and oceans: Deep-Sea Research, v. 13, p. 205-212. Table l.--MPN index and 95 percent confidence limits for various combinations of positive and negative results when three 10 ml dilutions, three 1 ml dilutions, and three 0.1 ml dilutions are used. (American Public Health Association and others, 1971, p. 676). . (not transferable) _______________________________________________________