I. APPARATUS.

1. Sample Bottles.—Any size, shape or quality of bottle may be used for a bacterial sample, provided it holds a sufficient amount to carry out all the tests required and is such that it may be properly washed and sterilized and will keep the sample uncontaminated until the analysis is made. Four- or eight-ounce, ground-glass-stoppered bottles are recommended. These should be protected by being wrapped in paper, or their necks covered with tin foil, and should be placed in proper boxes for transportation.

2. Pipettes.—Pipettes may be of any convenient size or shape provided it is found by actual test that they deliver accurately the required amount in the manner in which they are used. The error of calibration shall in no case exceed 2 per cent. Protecting the pipettes with a cotton stopper is recommended.

3. Dilution Bottles.—Bottles for use in making dilutions should preferably be of tall form, of such capacity as to hold at least twice the volume of water actually used. Close-fitting ground-glass stoppers are preferable, but tight fitting cotton stoppers may be used, provided due care is taken to prevent contamination and to avoid loss of volume through wetting of the stopper before mixing has been accomplished.

4. Petri Dishes.—Petri dishes ten centimeters in diameter shall be used with glass or porous tops^[211] as preferred. The bottoms of the dishes shall be as flat as possible so that the medium shall be of uniform thickness throughout the plate.

5. Fermentation Tubes.—Any type of fermentation tube^[203] may be used provided it holds at least three times as much medium as the amount of water to be tested.

1. Water.—Distilled water shall be used in the preparation of all culture media and reagents.

2. Meat Extract.—Liebig’s meat extract shall be used in place of meat infusion. Other brands may be substituted for Liebig’s when comparative tests have shown that they give equivalent results.

3. Peptone.—Armour’s, Digestive Ferments Company’s, Fairchild’s, or any other peptone which gives equivalent results may be used.

4. Sugars.—All sugars used shall be of the highest purity obtainable.

5. Agar.—The agar used shall be of the best quality and shall be dried for one-half hour at 105° C. before weighing. Much of the agar on the market contains considerable amounts of sea salts.^{[221][225][228]} These may be removed by soaking in water and draining before use.

6. Gelatin.—The gelatin used shall be of light color, shall contain not more than a trace of arsenic, copper, sulfides, and shall be free from preservatives, and of such a melting point that a 10 per cent. standard nutrient gelatin shall have a melting point of 25° C. or over. Gelatin shall be dried for one-half hour at 105° C. before weighing.

7. Litmus.—Reagent litmus of highest purity (not litmus cubes) or azolitmin (Kahlbaum) shall be used for all media requiring a litmus indicator.

8. General Chemicals.—Special effort shall be made to have all the other ingredients used for culture media chemically pure.

III. METHODS.

1. PREPARATION OF CULTURE MEDIA.

a. Adjustment of Reaction.

aa. Phenol Red Method for adjustment to a hydrogen-ion concentration of P_H+ = 6.8–8.4. Withdraw 5 cc. of the medium, dilute with 5 cc. of distilled water, and add 5 drops of a solution of phenol red (phenol sulphone phthalein). This solution is made by dissolving 0.04 grams of phenol red in 30 cc. of alcohol and diluting to 100 cc. with distilled water.

Titrate with a 1:10 dilution of a standard solution of NaOH (which need not be of known normality) until the phenol red shows a slight but distinct pink color. Calculate the amount of the standard NaOH solution which must be added to the medium to reach this reaction. After the addition check the reaction by adding 5 drops of phenol red to 5 cc. of the medium and 5 cc. of water.

bb. Titration with phenolphthalein. (For the convenience of those who wish to retain the use of this method for the present it is given here, but it is recommended that as soon as possible the more accurate method of determining the hydrogen-ion concentration be substituted.)

In a white porcelain dish put 5 cc. of the medium to be tested, add 45 cc. of distilled water. Boil briskly for one minute. Add 1 cc. of phenolphthalein solution (5 grams of commercial salt to one liter of 50 per cent. alcohol). Titrate immediately with a n/20 solution of sodium hydrate. A faint but distinct pink color marks the true end-point. This color may be precisely described as a combination of 25 per cent. of red (wave length approximately 658) with 75 per cent. of white as shown by the disks of the standard color top made by the Milton Bradley Educational Co., Springfield, Mass.

All reactions shall be expressed with reference to the phenolphthalein neutral point and shall be stated in percentages of normal acid or alkali solutions required to neutralize them. Alkaline media shall be recorded with a minus (-) sign before the percentage of normal acid needed for their neutralization and acid media with a plus (+) sign before the percentage of normal alkali solution needed for their neutralization.

The standard reaction for culture media for water analysis shall be +1.0 per cent., as determined by tests of the sterilized medium. As ordinarily prepared, broth and agar will be found to have a reaction between +0.5 and +1.0. For such media no adjustment shall be made. The reaction of media containing sugar shall be neutral to phenolphthalein. Whenever reactions other than the standard are used, it shall be so stated.

b. Sterilization.

All media and dilution water shall be sterilized in the autoclav at 15 lbs. (120° C.) for 15 minutes after the pressure reaches 15 lbs. All air must be forced out of the autoclav before the pressure is allowed to rise. As soon as possible after sterilization the media shall be removed from the autoclav and cooled rapidly. Rapid and immediate cooling of gelatin is imperative.

Media shall be sterilized in small containers, and these must not be closely packed together. No part of the medium shall be more than 2.5 cm. from the outside surface of the glass. All glassware shall be sterilized in the dry oven at 170° C. for at least 1½ hours.

c. Nutrient Broth. To Make One Liter.

1. Add 3 grams of beef extract and 5 grams of peptone to 1,000 cc. of distilled water.

2. Heat slowly on a steam bath to at least 65° C.

3. Make up lost weight and adjust the reaction to a faint pink with phenol red, or if the phenolphthalein titration is used, and the reaction is not already between +0.5 and +1, adjust to +1.

4. Cool to 25° C. and filter through filter paper until clear.

5. Distribute in test-tubes, 10 cc. to each tube.

6. Sterilize in the autoclav at 15 lbs. (120° C.) for 15 minutes after the pressure reaches 15 lbs.

d. Sugar Broths.

Sugar broths shall be prepared in the same general manner as nutrient broth with the addition of 0.5 per cent. of the required carbohydrate just before sterilization. The removal of muscle sugar is unnecessary as the beef extract and peptone are free from any fermentable carbohydrates. The reaction of sugar broths shall be a faint pink with phenol red or, if on titration with phenolphthalein the reaction is not already between neutral and +1, adjust to neutral. Sterilization shall be in the autoclav at 15 lbs. (120° C.) for 15 minutes after the pressure reaches 15 lbs., provided the total time of exposure to heat is not more than one-half hour; otherwise a 10 per cent. solution of the required carbohydrate shall be made in distilled water and sterilized at 100° C. for 1½ hours, and this solution shall be added to sterile nutrient broth in amounts sufficient to make a 0.5 per cent. solution of the carbohydrate and the mixture shall then be tubed and sterilized at 100° C. for 30 minutes, or it is permissible to add by means of a sterile pipette directly to a tube of sterile neutral broth enough of the carbohydrate to make the required 0.5 per cent. The tubes so made shall be incubated at 37° C. for 24 hours as a test for sterility.

e. Nutrient Gelatin. To Make One Liter.

1. Add 3 grams of beef extract and 5 grams of peptone to 1,000 cc. of distilled water and add 100 grams of gelatin dried for one-half hour at 105° C. before weighing.

2. Heat slowly on a steam bath to 65° C. until all gelatin is dissolved.^[G]

3. Make up lost weight and adjust the reaction to a faint pink with phenol red, or if the phenolphthalein titration is used, and the reaction is not already between +0.5 and +1, adjust to +1.

4. Filter through cloth and cotton until clear.

5. Distribute in test-tubes, 10 cc. to each tube, or in larger containers as desired.

6. Sterilize in the autoclav at 15 lbs. (120° C.) for 15 minutes after the pressure reaches 15 lbs.

f. Nutrient Agar. To Make One Liter.

1. Add 3 grams of beef extract, 5 grams of peptone and 12 grams of agar, dried for one-half hour at 105° C. before weighing, to 1,000 cc. of distilled water. Boil over a water bath until all agar is dissolved, and then make up the loss by evaporation.

2. Cool to 45° C. in a cold water bath, then warm to 65° C. in the same bath, without stirring.

3. Make up lost weight and adjust the reaction to a faint pink with phenol red, or if the phenolphthalein titration is used, and the reaction is not already between +0.5 and +1, adjust to +1.

4. Filter through cloth and cotton until clear.

5. Distribute in test-tubes, 10 cc. to each tube, or in larger containers, as desired.

6. Sterilize in the autoclav at 15 lbs. (120° C.) for 15 minutes after the pressure reaches 15 lbs.

g. Litmus or Azolitmin Solution.

The standard litmus solution shall be a 2 per cent. aqueous solution of reagent litmus. Powder the litmus, add to the water and boil for five minutes. The solution usually needs no correction in reaction and may be at once distributed in flasks or test-tubes and sterilized as is culture media. It should give a distinctly blue plate when 1 cc. is added to 10 cc. of neutral culture medium in a Petri dish.

The standard azolitmin solution shall be a 1 per cent. solution of Kahlbaum’s azolitmin. Add the azolitmin powder to the water and boil for five minutes. The solution may need to be corrected in reaction by the addition of sodium hydrate solution so that it will be approximately neutral and will give a distinctly blue plate when 1 cc. is added to 10 cc. of neutral culture medium in a Petri dish. It may be distributed in flasks or test-tubes and sterilized as is culture media.

h. Litmus-lactose-agar.

Litmus-lactose-agar shall be prepared in the same manner as nutrient agar with the addition of 1 per cent. of lactose just before sterilization. The reaction shall be a faint pink with phenol red, or, if on titration with phenolphthalein the reaction is not already between neutral and +1, adjust to neutral. One cc. of sterilized litmus or azolitmin solution shall be added to each 10 cc. of the medium just before it is poured into the Petri dish, or the mixture may be made in the dish itself.

i. Endo’s Medium.^{[209][214][215]} To Make One Liter.

1. Add 5 grams of beef extract, 10 grams of peptone and 30 grams of agar dried for one-half hour at 105° C. before weighing, to 1,000 cc. of distilled water. Boil on a water bath until all the agar is dissolved and then make up the loss by evaporation.

2. Cool the mixture to 45° C. in a cold water bath, then warm to 65° C. in the same bath without stirring.

3. Make up lost weight, titrate, and if the reaction is not already between neutral and +1 adjust to neutral.

4. Filter through cloth and cotton until clear.

5. Distribute 100 cc. or larger known quantities in flasks large enough to hold the other ingredients which are to be added later.

6. Sterilize in the autoclav at 15 lbs. (120° C.) for 15 minutes after the pressure reaches 15 lbs.

7. Prepare a 10 per cent. solution of basic fuchsin in 95 per cent. alcohol, allow to stand 20 hours, decant and filter the supernatant fluid. This is a stock solution.

8. When ready to make plates melt 100 cc. of agar in streaming steam or on a water bath. Dissolve 1 gram of lactose in 15 cc. of distilled water, using heat if necessary. Dissolve 0.25 gram anhydrous sodium sulphite in 10 cc. water. To the sulphite solution add 0.5 cc. of the fuchsin stock solution. Add the fuchsin-sulphite solution to the lactose solution and then add the resulting solution to the melted agar. The lactose used must be chemically pure and the sulphite solution must be made up fresh.

9. Pour plates and allow to harden thoroughly in the incubator before use.

Samples for bacterial analysis shall be collected in bottles which have been cleansed with great care, rinsed in clean water, and sterilized with dry heat for at least one hour and a half at 170° C., or in the autoclav at 15 lbs. (120° C.) for 15 minutes or longer after the pressure reaches 15 lbs.

Great care must be exercised to have the samples representative of the water to be tested and to see that no contamination occurs at the time of filling the sample bottles.

3. STORAGE AND TRANSPORTATION OF SAMPLES.

Because of the rapid and often extensive changes which may take place in the bacterial flora of bottled samples when stored even at temperatures as low as 10° C., it is urged, as of importance, that all samples be examined as promptly as possible after collection.

The time allowed for storage or transportation of a bacterial sample between the filling of the sample bottle and the beginning of the analysis should be not more than six hours for impure waters and not more than twelve hours for relatively pure waters. During the period of storage, the temperature shall be kept as near 10° C. as possible. Any deviation from the above limits shall be so stated in making reports.

4. DILUTIONS.

Dilution bottles shall be filled with the proper amount of tap water so that after sterilization they shall contain exactly 9 cc. or 99 cc. as desired. The exact amount of water can only be determined by experiment with the particular autoclav in use. If desired, the 9 cc. dilution may be measured out from a flask of sterile water with a sterile pipette.

Dilution bottles shall be sterilized in the autoclav at 15 lbs. (120° C.) for 15 minutes after the pressure reaches 15 lbs.

The sample bottle shall be shaken vigorously 25 times and 1 cc. withdrawn and added to the proper dilution bottles as required. Each dilution bottle after the addition of the 1 cc. of the sample, shall be shaken vigorously 25 times before a second dilution is made from it or before a sample is removed for plating.

All sample and dilution bottles shall be shaken vigorously 25 times before samples are removed for plating. Plating shall be done immediately after the dilutions are made. One cc. of the sample or dilution shall be used for plating and shall be placed in the Petri dish, first. Ten cc. of liquefied medium at a temperature of 40° C. shall be added to the 1 cc. of water in the Petri dish. The cover of the Petri dish shall be lifted just enough for the introduction of the pipette or culture medium, and the lips of all test-tubes or flasks used for pouring the medium shall be flamed. In making litmus-lactose-agar plates, 1 cc. of sterile litmus or azolitmin solution shall be added to each 10 cc. of culture medium either in the Petri dish or before pouring into the Petri dish. The medium and sample in the Petri dish shall be thoroughly mixed and uniformly spread over the bottom of the Petri dish by tilting or rotating the dish. All plates shall be solidified as rapidly as possible after pouring and gelatin plates shall be placed immediately in the 20° C. incubator and the agar plates in the 37° C. incubator. Endo plates shall be made by placing one loopful of the material to be tested on the surface of the plate and distributing the material with a sterile loop or glass rod.

6. INCUBATION.

All gelatin plates shall be incubated for 48 hours at 20 C. in a dark, well-ventilated incubator in an atmosphere practically saturated with moisture.^[227]

All agar plates shall be incubated for 24 hours at 37° C. in a dark, well-ventilated incubator in an atmosphere practically saturated with moisture. Glass covered plates shall be inverted in the incubator. Any deviation from the above described method shall be stated in making reports.

7. COUNTING.

In preparing plates, such amounts of the water under examination shall be planted as will give from 25 to 250 colonies on a plate;^[202] and the aim should be always to have at least two plates giving colonies between these limits. Where it is possible to obtain plates showing colonies within these limits, only such plates should be considered in recording results, except where the same amount of water has been planted in two or more plates, of which one gives colonies within these limits, while the others give less than 25 or more than 250. In such case, the result recorded should be the average of all the plates planted with this amount of water. Ordinarily it is not desirable to plant more than 1 cc. of water in a plate; therefore, when the total number of colonies developing from 1 cc. is less than 25, it is obviously necessary to record the results as observed, disregarding the general rule given above.

Counting shall in all cases be done with a lens of 2½ diameter’s magnification, 3½X. The Engraver’s Lens No. 146 made by the Bausch & Lomb Optical Company fills the requirements, and is a convenient lens for the purpose.

8. THE TEST FOR THE PRESENCE OF MEMBERS OF THE B. COLI GROUP.

It is recommended that the B. coli group be considered as including all non-spore-forming bacilli which ferment lactose with gas formation and grow aËrobically on standard solid media.

The formation of 10 per cent. or more of gas in a standard lactose broth fermentation tube within 24 hours at 37° C. is presumptive evidence of the presence of members of the B. coli group, since the majority of the bacteria which give such a reaction belong to this group.

The appearance of aËrobic lactose-splitting colonies on lactose-litmus-agar or Endo’s medium plates made from a lactose broth fermentation tube in which gas has formed confirms to a considerable extent the presumption that gas-formation in the fermentation tube was due to the presence of members of the B. coli group.

To complete the demonstration of the presence of B. coli as above defined, it is necessary to show that one or more of these aËrobic plate colonies consists of non-spore-forming bacilli which, when inoculated into a lactose broth fermentation tube, form gas.

It is recommended that the standard tests for the B. coli group be either (A) the Presumptive, (B) the Partially Confirmed, or (C) the Completed test as hereafter defined, each test being applicable under the circumstances specified.

A. PRESUMPTIVE TEST.

1. Inoculate a series of fermentation tubes with appropriate graduated quantities of the water to be tested. In every fermentation tube there must always be at least three times as much medium as the amount of water to be tested. When necessary to examine larger amounts than 10 cc. as many tubes as necessary shall be inoculated with 10 cc. each.

2. Incubate these tubes at 37° C. for 48 hours. Examine each tube at 24 and 48 hours, and record gas-formation. The records should be such as to distinguish between:

(a) Absence of gas-formation.

(b) Formation of gas occupying less than ten per cent. (10%) of the closed arm.

(c) Formation of gas occupying more than ten per cent. (10%) of the closed arm.

More detailed records of the amount of gas formed, though desirable for purposes of study, are not necessary for carrying out the standard tests prescribed.

3. The formation within 24 hours of gas occupying more than ten per cent. (10%) of the closed arm of fermentation tube constitutes a positive presumptive test.

4. If no gas is formed in 24 hours, or if the gas formed is less than ten per cent. (10%), the incubation shall be continued to 48 hours. The presence of gas in any amount in such a tube at 48 hours constitutes a doubtful test, which in all cases requires confirmation.

5. The absence of gas formation after 48 hours’ incubation constitutes a negative test. (An arbitrary limit of 48 hours’ observation doubtless excludes from consideration occasional members of the B. coli group which form gas very slowly, but for the purposes of a standard test the exclusion of these occasional slow gas-forming organisms is considered immaterial.)

B. PARTIALLY CONFIRMED TEST.

1. Make one or more Endo’s medium or lactose-litmus-agar plates from the tube which, after 48 hours’ incubation, shows gas formation from the smallest amount of water tested. (For example, if the water has been tested in amounts of 10 cc., 1 cc., and 0.1 cc., and gas is formed in 10 cc., and 1 cc., not in 0.1 cc., the test need be confirmed only in the 1 cc. amount.)

2. Incubate the plates at 37° C., 18 to 24 hours.

3. If typical colon-like red colonies have developed upon the plate within this period, the confirmed test may be considered positive.

4. If, however, no typical colonies have developed within 24 hours, the test cannot yet be considered definitely negative, since it not infrequently happens that members of the B. coli group fail to form typical colonies on Endo’s medium or lactose-litmus-agar plates, or that the colonies develop slowly. In such case, it is always necessary to complete the test as directed under “C” 2 and 3.

1. From the Endo’s medium or lactose-litmus-agar plate made as prescribed under “B,” fish at least two typical colonies, transferring each to an agar slant and a lactose broth fermentation tube.

2. If no typical colonies appear upon the plate within 24 hours, the plate should be reincubated another 24 hours, after which at least two of the colonies considered to be most likely B. coli, whether typical or not, shall be transferred to agar slants and lactose broth fermentation tubes.

3. The lactose broth fermentation tubes thus inoculated shall be incubated until gas formation is noted; the incubation not to exceed 48 hours. The agar slants shall be incubated at 37° C. for 48 hours, when a microscopic examination shall be made of at least one culture, selecting one which corresponds to one of the lactose broth fermentation tubes which has shown gas-formation.

The formation of gas in lactose broth and the demonstration of non-spore-forming bacilli in the agar culture shall be considered a satisfactory completed test, demonstrating the presence of a member of the B. coli group.

The absence of gas-formation in lactose broth or failure to demonstrate non-spore-forming bacilli in a gas-forming culture constitutes a negative test.

APPLICATION OF PRESUMPTIVE, PARTIALLY CONFIRMED, AND COMPLETED TESTS.

A. The Presumptive Test.

1. When definitely positive, that is showing more than 10 per cent. (10%) of gas in 24 hours, is sufficient:

(a) As applied to all except the smallest gas-forming portion of each sample in all examinations.

(b) As applied to the smallest gas-forming portion in the examination of sewage or of water showing relatively high pollution, such that its fitness for use as drinking water does not come into consideration. This applies to the routine examinations of raw water in connection with control of the operation of purification plants.

2. When definitely negative, that is showing no gas in 48 hours, is final and therefore sufficient in all cases.

3. When doubtful, that is showing gas less than 10 per cent. (10%) (or none) in 24 hours, with gas either more or less than 10 per cent. in 48 hours, must always be confirmed.

B. The Partially Confirmed Test.

1. When definitely positive, that is, showing typical plate colonies within 24 hours, is sufficient:

(a) When applied to confirm a doubtful presumptive test in cases where the latter, if definitely positive, would have been sufficient.

(b) In the routine examination of water supplies where a sufficient number of prior examinations have established a satisfactory index of the accuracy and significance of this test in terms of the completed test.

2. When doubtful, that is, showing colonies of doubtful or negative appearance in 24 hours, must always be completed.

C. The Completed Test.

The completed test is required as applied to the smallest gas-forming portion of each sample in all cases other than those noted as exceptions under the “presumptive” and the “partially confirmed” tests.

The completed test is required in all cases where the result of the confirmed test has been doubtful.

9. EXPRESSION OF RESULTS.

In order to avoid fictitious accuracy and yet to express the numerical results by a method consistent with the precision of the work, the numbers of colonies of bacteria per cubic centimeter shall be recorded as follows:^[212]

This applies to the gelatin count at 20° C. and to the agar count at 37° C.

In order that tests for B. coli may have quantitative significance, the following general principles and rules should be observed:

Ordinarily not less than three portions of each sample should be tested, the portions being even decimal multiples or fractions of a cubic centimeter; for example, 10 cc., 1 cc., 0.1 cc., .01 cc., etc. It is essential that the dilutions should be such that the largest amount gives a positive test (unless the water is such as to give negative tests in 10 cc.), and the smallest dilution, a negative result. To insure this result, it is often necessary to plant four or five dilutions, especially in the examination of a sample of entirely unknown quality. The quantitative value of a series of tests is lost, unless all or at least a large proportion of the smallest dilutions tested have given negative results.

In reporting a single test, it is preferable merely to record results as observed, indicating the amounts tested and the result in each, rather than to attempt expression of the result in numbers of B. coli per cc. In summarizing the results of a series of tests, however, it is desirable, for the sake of simplicity, to express the results in terms of the numbers of B. coli per cc., or per 100 cc. To convert results of fermentation tests to this form, the result of each test is recorded as indicating a number of B. coli per cc. equal to the reciprocal of the smallest decimal or multiple fraction of a cubic centimeter giving a positive result. For example, the result: 10 cc. +; 1 cc. +; 0.1 cc. -; would be recorded as indicating one B. coli per cc. An exception should be made in the case where a negative result is obtained in an amount larger than the smallest portion giving a positive result; for example, in a result such as: 10 cc. +; 1 cc. -; 0.1 cc. +. In such case, the result should be recorded as indicating a number of B. coli per cc. equal to the reciprocal of the dilution next larger than the smallest one giving a positive test, this being a more probable result.

Where tests are made in amounts larger than 1 cc., giving average results less than one B. coli per cc., it is more convenient to express results in terms of the numbers of B. coli per 100 cc.

The following table illustrates the method of recording and averaging results of B. coli tests:

The above method of expressing results is not mathematically altogether correct. The average number of B. coli per cc., as thus estimated, is not precisely the most probable number calculated by application of the theory of probability.^[220] To apply this theory to a correct mathematical solution of any considerable series of results involves, however, mathematical calculations so complex as to be impracticable of application in general practice. The simpler method given is therefore considered preferable, since it is easily applied and the results so expressed are readily comprehensible.

In order that results as reported may be checked and carefully valuated, it is necessary that the report should show not only the average number of B. coli per cc., but also the number of samples examined; and, for each dilution, the total number of tests made, and the number (or per cent.) positive.

10. INTERPRETATION OF RESULTS.

While it is not within the province of this report to suggest the proper interpretation of results obtained by the use of the methods herein specified as standard, the committee feels that a word of caution should be given regarding the significance of the presence in a water of members of the B. coli group as defined in this report. Recent work seems to indicate that the B. coli group as herein defined consists of organisms of both fecal and non-fecal origin. Therefore care must be exercised in judging the sanitary quality of a water solely from the determination of the presence of members of the group.

11. DIFFERENTIATION OF FECAL FROM NON-FECAL MEMBERS OF THE B. COLI GROUP.

(1) At least 10 cultures should be used. If possible these should be subcultured from plates made direct from the water since all of the cultures obtained by plating from fermentation tubes may be descendants of a single cell in the water. If cultures from water plates are not available those obtained from plates made as prescribed under B (p. 101) may be used.

(2) Inoculate each culture into dextrose potassium phosphate broth,^[H] adonite broth, and gelatin. For additional confirmatory evidence inoculation may be made into tryptophane broth,^[I] and saccharose broth. The dextrose broth must be incubated at 30°. Other sugar broths may be incubated at 30° or 37° as convenient. Gelatin should be incubated at 20°.

H. (a) Peptone Medium for the Methyl Red Test. To Make One Liter.

1. To 800 cc. of distilled water add 5 grams of Proteose-Peptone, Difco., or Witte’s Peptone (other peptones should not be substituted), 5 grams c. p. dextrose, and 5 grams dipotassium hydrogen phosphate (K₂HPO₄). A dilute solution of the K₂HPO₄ should give a distinct pink with phenolphthalein.

2. Heat with occasional stirring over steam for twenty minutes.

3. Filter through folded filter paper, cool to 20° C. and dilute to 1,000 cc. with distilled water.

4. Distribute 10 cc. portions in sterilized test-tubes.

5. Sterilize by the intermittent method for 20 minutes on three successive days.