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)

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