Chapter 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]

Footnote G:

The solution of the gelatin will be facilitated by allowing it to soak
in the cold one-half hour before heating.

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.

2. COLLECTION OF SAMPLE.

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.

5. 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.