← The elements of bacteriological technique

Chapter 53

By Physical Methods

Examine cultivations of the organism with reference to its growth and development under the following headings:

Atmosphere:

(a) In the presence of oxygen.

(b) In the absence of oxygen.

Temperature:

(a) Range.

(b) Optimum.

Moist: Vegetative forms.

Spores.

Dry: Vegetative forms.

Spores.

Reaction of medium.

Resistance to lethal agents:

(a) Desiccation.

(b) Light: Diffuse.

Direct.

Primary colours.

(d) Chemical antiseptics and disinfectants.

Vitality in artificial cultures.

I. Atmosphere.—The question as to whether the organism under observation is (a) an obligate aerobe, (b) a facultative anaerobe, or (c) an obligate anaerobe is roughly decided by the appearance of cultivations in the fermentation tubes. Obvious growth in the closed branch as well as in the bulb or in the inverted gas tube as well as in the bulk of the medium will indicate that it is a facultative anaerobe; whilst growth only occurring in the bulb or in the closed branch shows that it is an obligate aerobe or anaerobe respectively. This method, however, is not sufficiently accurate for the present purpose, and the examination of an organism with respect to its behaviour in the absence of oxygen is carried out as follows:

Apparatus Required:

Buchner's tubes.
Bulloch's apparatus.
Exhaust pump.
Pyrogallic acid.
Dekanormal caustic soda.

Media Required:

Glucose formate agar.
Glucose formate gelatine.
Glucose formate bouillon.

Method.—

1. Prepare four sets of cultivations:

(A) Sloped glucose formate agar, and incubate aerobically at 37° C.

Sloped glucose formate gelatine, and incubate aerobically at 20° C.

(B) Sloped glucose agar to incubate anaerobically at 37° C.

Sloped glucose formate gelatine to incubate anaerobically at 20° C.

Glucose formate bouillon to incubate anaerobically at 37° C.

(D) Sloped glucose formate gelatine to incubate anaerobically at 20° C.

Glucose formate bouillon to incubate anaerobically at 20° C.

2. Seal the cultures forming set B in Buchner's tubes (vide page 239).

3. Seal the cultures forming set C in Bulloch's apparatus; exhaust the air by means of a vacuum pump, and provide for the absorption of any residual oxygen by the introduction of pyrogallic acid and caustic soda in solution (vide page 245). Treat set D in the same way.

4. Observe the cultivations macroscopically and microscopically at intervals of twenty-four hours until the completion, if necessary, of seven days' incubation.

5. Control these results.

Gases Other than Oxygen.—

Apparatus Required:

Bulloch's apparatus.
Sterile gas filter (vide page 40).
Gasometer containing the gas it is desired to test (SO2, N2O, NO,
CO2, etc.) or gas generator for its production.

Method.—

1. Prepare at least seven tube cultivations upon solid media and deposit them in Bulloch's apparatus.

2. Connect up the inlet tube of the Bulloch's jar with the sterile gas filter, and this again with the delivery tube of the gasometer or gas generator.

3. Open both stop-cocks of the Bulloch's apparatus and pass the gas through until it has completely replaced the air in the bell jar as shown by the result of analyses of samples collected from the exit tube.

4. Incubate under optimum conditions as to temperature.

5. Examine the cultivations at intervals of twenty-four hours, until the completion of seven days.

6. Remove one tube from the interior of the apparatus each day. If no growth is visible, incubate the tube under optimum conditions as to temperature and atmosphere, and in this way determine the length of exposure to the action of the gas necessary to kill the organisms under observation.

7. Control these results.

II. Temperature.—

(A) Range.—

1. Prepare a series of ten tube cultivations, in fluid media, of optimum reaction.

2. Arrange a series of incubators at fixed temperatures, varying 5° C. and including temperatures between 5° C. and 50° C.

(In the absence of a sufficient number of incubators utilise the water-bath employed in testing the thermal death-point of vegetative forms.)

3. Incubate one tube cultivation of the organism aerobically or anaerobically, as may be necessary, in each incubator, and examine at half-hour intervals for from five to eighteen hours.

4. Note that temperature at which growth is first observed macroscopically (Optimum temperature).

5. Continue the incubation until the completion of seven days. Note the extremes of temperature at which growth takes place (Range of temperature).

6. Control these results—if considered necessary arranging the series of incubators to include each degree centigrade for five degrees beyond each of the extremes previously noted.

(B) Optimum.—

1. Prepare a second series of ten tube cultivations under similar conditions as to reaction of medium.

2. Incubate in a series of incubators in which the temperature is regulated at intervals of 1° C. for five degrees on either side of optimum temperature observed in the previous experiment (A, step 4).

3. Observe again at half-hour intervals and note that temperature at which growth is first visible to the naked eye = Optimum temperature.

Moist—Vegetative Forms:

The t. d. p. here is that temperature which with certainty kills a watery suspension of the organisms in question after an exposure of 10 minutes.

Apparatus Required:

Water-bath. For the purpose of observing the thermal death-point a special water-bath is necessary. The temperature of this piece of apparatus is controlled by means of a capsule regulator that can be adjusted for intervals of half a degree centigrade through a range of 30°, from 50° C. to 80° C. by means of a spring, actuated by the handle a, which increases the pressure in the interior of the capsule. A hole is provided for the reception of the nozzle of a blast pump, so that a current of air may be blown through the water while the bath is in use, and thus ensure a uniform temperature of its contents. Through a second hole is suspended a certified centigrade thermometer, the bulb of which although completely immersed in the water is raised at least 2 cm. above the floor of the bath.

Sterile glass capsules.

Flask containing 250 c.c. sterile normal saline solution.

Case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre).

Special platinum loop.

Test-tubes, 18 by 1.5 cm., of thin German glass.

Case of sterile petri dishes.

Tubes of agar or gelatine.

Method.—

1. Prepare tube cultivations on solid media of optimum reaction; incubate forty-eight hours under optimum conditions as to temperature and atmosphere.

2. Examine preparations from the cultivation microscopically to determine the absence of spores.

3. Pipette 5 c.c. salt solution into each of twelve capsules.

4. Suspend three loopfuls of the surface growth (using a special platinum loop, vide page 316) in the normal saline solution by emulcifying evenly against the moist walls of each capsule.

5. Transfer emulsion from each capsule to sterile 250 c.c. flask, and mix.

6. Pipette 5 c.c. emulsion into each of twelve sterile test-tubes numbered consecutively.

7. Adjust the first tube in the water-bath, regulated at 40° C, by means of two rubber rings around the tube, one above and the other below the perforated top of the bath, so that the upper level of the fluid in the tube is about 4 cm. below the surface of the water in the bath, and the bottom of the tube is a similar distance above the bottom of the bath.

8. Arrange a control test-tube containing 5 c.c. sterile saline solution under similar conditions. Plug the tube with cotton-wool and pass a thermometer through the plug so that its bulb is immersed in the water.

9. Close the unoccupied perforations in the lid of the water-bath by means of glass balls.

10. Watch the thermometer in the test-tube until it records a temperature of 40° C. Note the time. Ten minutes later remove the tube containing the suspension, and cool rapidly by immersing its lower end in a stream of running water.

11. Pour three gelatine (or agar) plates containing respectively 0.2, 0.3, and 0.5 c.c. of the suspension, and incubate.

12. Pipette the remaining 4 c.c. of the suspension into a culture flask containing 250 c.c. of nutrient bouillon, and incubate.

13. Observe these cultivations from day to day. "No growth" must not be recorded as final until after the completion of seven days' incubation.

14. Extend these observations to the remaining tubes of the series, but varying the conditions so that each tube is exposed to a temperature 2° C. higher than the immediately preceding one—i. e., 42° C., 44° C., 46° C., and so on.

15. Note that temperature, after exposure to which no growth takes place up to the end of seven days' incubation, = the thermal death-point.

16. If greater accuracy is desired, a second series of tubes may be prepared and exposed for ten minutes to fixed temperatures varying only 0.5° C., through a range of 5° C. on either side of the previously observed death-point.

Moist—Spores: The thermal death-point in the case of spores is that time exposure to a fixed temperature of 100° C. necessary to effect the death of all the spores present in a suspension.

Note.—If it is desired to retain the time constant 10 minutes and investigate the temperature necessary to destroy the spores, varying amounts of calcium chloride must be added to the water in the bath, when the boiling-point will be raised above 100° C. according to the percentage of calcium in solution. In such case use the bath figured on page 227; the bath figured on page 299 can only be used if the capsule is first removed.

It is determined in the following manner

Apparatus Required:

Steam-can fitted with a delivery tube and a large bore safety-valve tube.

Water-bath at 100° C.

Erlenmeyer flask, 500 c.c. capacity, containing 140 c.c. sterile normal saline solution and fitted with rubber stopper perforated with four holes.

The rubber stopper is fitted as follows:

(a) Thermometer to 120° C., its bulb immersed in the normal saline.

(b) Straight entry tube, reaching to the bottom of the flask, the upper end plugged with cotton-wool.

The nozzle is protected from accidental contamination by passing it through the cotton-wool plug of a small test-tube.

(d) A sickle-shaped piece of glass tubing passing just through the stopper, plugged with cotton-wool, to act as a vent for the steam.

Sterile plates.

Sterile pipettes.

Sterile test-tubes graduated to contain 5 c.c.

Media Required:

Gelatine or agar.

Culture flasks containing 200 c.c. nutrient bouillon.

Method.—

1. Prepare twelve tube cultivations upon the surface (or two cultures in large flat culture bottles—vide page 5) of nutrient agar and incubate under the optimum conditions (previously determined), for the formation of spores.

Examine preparations from the cultures microscopically to determine the presence of spores.

2. Pipette 5 c.c. sterile normal saline into each culture tube or 30 c.c. into each bottle and by means of a sterile platinum spatula emulsify the entire surface growth with the solution.

3. Add the 60 c.c. emulsion to 140 c.c. normal saline contained in the fitted Erlenmeyer flask.

4. Place the flask in the water-bath of boiling water.

5. Connect up the straight tube, after removing the cotton-wool plug, with the delivery tube of the steam can; remove the plug from the vent tube.

6. When the thermometer reaches 100° C., open the spring clip on the syphon, discard the first cubic centimeter of suspension that syphons over (i. e., the contents of the syphon tube); collect the next 5 c.c. of the suspension in the sterile graduated test-tube and pour plates and prepare flask cultures therefrom as in the previous experiments.

7. Repeat this process at intervals of twenty-five minutes' steaming.

8. Observe the inoculated plates and flasks up to the completion, if necessary, of seven days' incubation.

9. Control these experiments, but in this instance syphon off portions of the suspension at intervals of one-half to one minute during the five or ten minutes preceding the previously determined death-point.

Thermal Death-point.—

Dry—Vegetative Forms: The thermal death-point in this case is that temperature which with certainty kills a thin film of the organism in question after a time exposure of ten minutes.

Apparatus Required:

Hot-air oven, provided with thermo-regulator.

Sterile cover-slips.

Flask containing 250 c.c. sterile normal saline solution.

Case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre).

Case of sterile capsules.

Crucible tongs.

Method.—

1. Prepare an emulsion with three loopfuls from an optimum cultivation in 5 c.c. normal saline in a sterile capsule and examine microscopically to determine the absence of spore forms.

2. Make twelve cover-slip films on sterile cover-slips; place each in a sterile capsule to dry.

3. Expose each capsule in turn in the hot-air oven for ten minutes to a different fixed temperature, varying 5° C. between 60° C. and 120° C.

4. Remove each capsule from the oven with crucible tongs immediately after the ten minutes are completed; remove the cover-glass from its interior with a sterile pair of forceps.

5. Deposit the film in a flask containing 200 c.c. nutrient bouillon.

6. Prepare subcultivations from such flasks as show evidence of growth, to determine that no accidental contamination has taken place but that the organism originally spread on the film is responsible for the growth.

7. Control the result of these experiments.

Dry—Spores: The thermal death-point in this case is that temperature which with certainty kills the spores of the organism in question when present in a thin film after a time exposure of 10 minutes.

Apparatus Required:

As for vegetative forms.

Method.—

1. Prepare a sloped agar tube cultivation and incubate under optimum conditions as to spore formations.

2. Pipette 5 c.c. sterile normal saline into the culture tube and emulsify the entire surface growth in it. Examine microscopically to determine the presence of spores in large numbers.

3. Spread thin even films on twelve sterile cover-slips and place each cover-slip in a separate sterile capsule.

4. Expose each capsule in turn for ten minutes to a different fixed temperature, varying 5°C, between 100° C. and 160°C.

5. Complete the examination as for vegetative forms.

III. Reaction of Medium.

(A) Range.—

1. Prepare a bouillon culture of the organism and incubate, under optimum conditions as to temperature and atmosphere, for twenty-four hours.

2. Pipette 0.1 c.c. of the cultivation into a sterile capsule; add 9.9 c.c. sterile bouillon and mix thoroughly.

3. Prepare a series of tubes of nutrient bouillon of varying reactions, from +25 to -30 (vide page 155), viz.: +25, +20, +15, +10, +5, neutral, -5, -10, -15, -20, -25, -30.

4. Inoculate each of the bouillon tubes with 0.1 c.c. of the diluted cultivation by means of a sterile graduated pipette and incubate under optimum conditions.

5. Observe the cultures at half-hourly intervals from the third to the twelfth hours. Note the reaction of the tube or tubes in which growth is first visible macroscopically (probably optimum reaction).

6. Continue the incubation until the completion, if necessary, of seven days. Note the extremes of acidity and alkalinity in which macroscopical growth has developed (Range of reaction).

7. Control the result of these observations.

(B) Optimum Reaction.—The optimum reaction has already been roughly determined whilst observing the range. It can be fixed within narrower limits by inoculating in a similar manner a series of tubes of bouillon which represent smaller variations in reaction than those previously employed (say, 1 instead of 5) for five points on either side of the previously observed optimum. For example, the optimum reaction observed in the set of experiments to determine the range was +10. Now plant tubes having reactions of +15, +14, +13, +12, +11, +10, +9, +8, +7, + 6, +5, and observe as before.

IV. Resistance to Lethal Agents.—

(A) Desiccation.—

Apparatus Required:

Mueller's desiccator. This consists of a bell glass fitted with an exhaust tube and stop-cock (d), which can be secured to a plate-glass base (c) by means of wax or grease. It contains a cylindrical vessel of porous clay (a) into the top of which pure sulphuric acid is poured whilst the material to be dried is placed within its walls on a glass shelf (b). The air is exhausted from the interior and the acid rapidly converts the clay vessel into a large absorbing surface (Fig. 157).

Exhaust pump.

Pure concentrated sulphuric acid.

Sterile cover-slips.

Sterile forceps.

Culture flask containing 200 c.c. nutrient bouillon.

Sterile ventilated Petri dish. This is prepared by bending three short pieces of aluminium wire into V shape and hanging these on the edge of the lower dish and resting the lid upon them (Fig. 158).

Method.—

1. Prepare a surface cultivation on nutrient agar in a culture bottle and incubate under optimum conditions for forty-eight hours.

2. Examine preparations from the cultivation, microscopically, to determine the absence of spores.

3. Pipette 5 c.c. sterile normal saline solution into the flask and suspend the entire growth in it.

4. Spread the suspension in thin, even films on sterile cover-slips and deposit inside sterile "plates" to dry.

5. As soon as dry, transfer the cover-slip films to the ventilated Petri dish by means of sterile forceps.

6. Place the Petri dish inside the Mueller's desiccator; fill the upper chamber with pure sulphuric acid, cover with the bell jar, and exhaust the air from its interior. Ten minutes later connect up the desiccator to a sulphuric acid wash-bottle interposing an air filter so that only dry sterile air enters.

7. At intervals of five hours open the apparatus, remove one of the cover-slip films from the Petri dish, and transfer it to the interior of a culture flask, with every precaution against contamination. Reseal the desiccator and again exhaust, and subsequently admit dry sterile air as before.

8. Incubate the culture flask under optimum conditions until the completion of seven days, if necessary; and determine the time exposure at which death occurs.

9. Pour plates from those culture flasks which grow, to determine the absence of contamination.

10. Repeat these observations at hourly intervals for the five hours preceding and succeeding the death time, as determined in the first set of experiments.

(B) Light.—

(a) Diffuse Daylight:

1. Prepare a tube cultivation in nutrient bouillon, and incubate under optimum conditions, for forty-eight hours.

2. Pour twenty plate cultivations, ten of nutrient gelatine and ten of nutrient agar, each containing 0.1 c.c. of the bouillon culture.

3. Place one agar plate and one gelatine plate into the hot and cold incubators, respectively, as controls.

4. Fasten a piece of black paper, cut the shape of a cross or star, on the centre of the cover of each of the remaining plates (Fig. 159).

5. Expose these plates to the action of diffuse daylight (not direct sunlight) in the laboratory for one, two, three, four, five, six, eight, ten, twelve hours.

6. After exposure to light, incubate under optimum conditions.

7. Examine the plate cultivations after twenty-four and forty-eight hours' incubation, and compare with the two controls. Record results. If growth is absent from that portion of the plate unprotected by the black paper, continue the incubation and daily observation until the end of seven days.

8. Control the results.

(b) Direct Sunlight:

1. Prepare plate cultivations precisely as in the former experiments and place the two controls in the incubators.

2. Arrange the remaining plates upon a platform in the direct rays of the sun.

3. On the top of each plate stand a small glass dish 14 cm. in diameter and 5 cm. deep.

4. Fill a solution of potash alum (2 per cent. in distilled water) into each dish to the depth of 2 cm. to absorb the heat of the sun's rays and so eliminate possible effects of temperature on the cultivations.

5. After exposures for periods similar to those employed in the preceding experiment, incubate and complete the observation as above.

1. Prepare plate cultivations, as in the previous "light" experiments, and incubate controls.

2. Fasten a strip of black paper, 3 cm. wide, across one diameter of the cover of each plate.

3. Coat the remainder of the surface of the cover with a film of pure photographic collodion which contains 2 per cent. of either of the following aniline dyes, as may be necessary:

Chrysoidin (for red).
Malachite green (for green).
Eosin, bluish (for blue).
Methyl violet (for violet).

4. Expose the plates, thus prepared, to bright daylight (but not direct sunlight) for varying periods, and complete the observations as in the preceding experiments. The bactericidal action of light appears to depend upon the more refrangible rays of the violet end of the spectrum and is noted whether the red yellow rays are transmitted or not.

5. Control the results.

Note.—The ultra-violet rays obtained from a quartz mercury vapour lamp destroy bacterial life with great rapidity under laboratory conditions.

(D) Antiseptics and Disinfectants.—The resistance exhibited by any given bacterium toward any specified disinfectant or germicide should be investigated with reference to the following points:

(A) Inhibition coefficient—i. e., that percentage of the disinfectant present in the nutrient medium which is sufficient to prevent the growth and multiplication of the bacterium.

(B) Inferior lethal coefficient—i. e., the time exposure necessary to kill vegetative forms of the bacterium suspended in water at 20° to 25° C, in which the disinfectant is present in medium concentration (concentration insufficient to cause plasmolysis). And if the bacterium is one which forms spores,

(C) Superior lethal coefficient—i. e., the time exposure necessary to kill the spores of the bacterium under conditions similar to those obtaining in B.

The example here detailed only specifically refers to certain of the disinfectants:

viz:—Bichloride of mercury;
Formaldehyde;
Carbolic acid;

investigated with regard to B. anthracis, but the technique is practically similar for all other chemical disinfectants.

Inhibition Coefficient.—

Apparatus Required:

Case of sterile pipettes, 10 c.c. (in tenths).

Case of sterile pipettes, 1 c.c. (in tenths).

Sterile tubes or capsules for dilutions.

Tubes of nutrient bouillon each containing a measured 10 c.c. of medium.

Twenty-four-hour-old agar culture of a recently isolated B. Anthracis.

Germicides:

1. Five per cent. aqueous solution of carbolic acid.

2. One per cent. aqueous solution of perchloride of mercury.

3. One-tenth per cent. aqueous solution of formaldehyde.

Method.—

1. Number six bouillon tubes consecutively 1 to 6. Inoculate each from the stock cultivation of B. anthracis and at once add varying quantities of the carbolic acid solution, viz.:

To tube 1 add 2.0 c.c. (= 1:100)
To tube 2 add 1.0 c.c. (= 1:200)
To tube 3 add 0.6 c.c. (= 1:300)
To tube 4 add 0.5 c.c. (= 1:400)
To tube 5 add 0.4 c.c. (= 1:500)
To tube 6 add 0.2 c.c. (= 1:1,000)

2. Prepare a similar series of tube cultivations numbered consecutively 7 to 12 and add varying quantities of the mercuric perchloride solution, viz.:

To tube 7 add 0.1 (= 1:1,000)
To tube 8 add 0.05 (= 1:2,000)
To tube 9 add 0.03 (= 1:3,000)
To tube 10 add 0.025 (= 1:4,000)
To tube 11 add 0.02 (= 1:5,000)
To tube 12 add 0.01 (= 1:10,000)

3. Prepare a similar series of tube cultivations numbered consecutively 13 to 18 and add varying quantities of the formaldehyde solution, viz.:

To tube No. 13 add 1.0 c.c. (= 1:1,000)
To tube No. 14 add 0.4 c.c. (= 1:2,500)
To tube No. 15 add 0.2 c.c. (= 1:5,000)
To tube No. 16 add 0.1 c.c. (= 1:10,000)
To tube No. 17 add 0.075 c.c. (= 1:15,000)
To tube No. 18 add 0.05 c.c. (= 1:20,000)

4. Incubate all three sets of cultivations under optimum conditions as to temperature and atmosphere.

5. Examine each of the culture tubes from day to day, until the completion of seven days, and note those tubes, if any, in which growth takes place.

6. From such tubes as show growth prepare subcultivations upon suitable media, and ascertain that the organism causing the growth is the one originally employed in the test and not an accidental contamination.

Inferior Lethal Coefficient.—

Apparatus Required:

Highly concentrated solutions of the disinfectants.

Sterile test-tubes in which to make dilutions from the concentrated solutions of the disinfectants.

Hanging-drop slides.

Cover-slips.

Erlenmeyer flask containing 100 c.c. sterile distilled water.

Case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre).

Case of sterile pipettes, 1 c.c. (in tenths of a cubic centimetre).

Method.—

1. Prepare a surface cultivation of the "test" organism B. anthracis upon nutrient agar in a culture bottle and incubate under optimum conditions for twenty-four hours; then examine the cultivation microscopically to determine the absence of spores.

2. Prepare solutions of different percentages of each disinfectant.

3. Make a series of hanging-drop preparations from the agar culture, using a loopful of disinfectant solution of the different percentages to prepare the emulsion on each cover-slip.

4. Examine microscopically and note the strongest solution which does not cause plasmolysis and the weakest solution which does plasmolyse the organism.

5. Make control preparations of these two solutions and determine the percentage to be tested.

6. Pipette 10 c.c. sterile water into the culture bottle and suspend the entire surface growth in it.

7. Transfer the suspension to the Erlenmeyer flask and mix it with the 90 c.c. of sterile water remaining in the flask.

8. Pipette 10 c.c. of the diluted suspension into each of ten sterile test-tubes.

9. Label one of the tubes "Control" and place it in the incubator at 18° C.

10. Add to each of the remaining tubes a sufficient quantity of a concentrated solution of the disinfectant to produce the percentage previously determined upon (vide step 5).

11. Incubate the tubes at 18° C. to 20° C.

12. At hourly intervals remove the control tube and one of the tubes with added disinfectant from the incubator.

13. Make a subcultivation from both the control and the test suspension, upon the surface of nutrient agar; incubate under optimum conditions.

14. Observe these culture tubes from day to day until the completion of seven days, and determine the shortest exposure necessary to cause the death of vegetative forms.

Superior Lethal Coefficient.—

1. Prepare surface cultivations of the "test" organisms upon nutrient agar in a culture bottle, and incubate under optimum conditions, for three days, for the formation of their spores.

2. Transfer the emulsion to a sterile test-tube and heat in the differential steriliser for ten minutes at 80° C. to destroy all vegetative forms.

3. Employing that percentage solution of the disinfectant determined in the previous experiment, and complete the investigations as detailed therein, steps 7 to 14, increasing the interval between planting the subcultivations to two, three, or five hours if considered advisable.

Note.—Where it is necessary to leave the organisms in contact with a strong solution of the disinfectant for lengthy periods, some means must be adopted to remove every trace of the disinfectant from the bacteria before transferring them to fresh culture media; otherwise, although not actually killed, the presence of the disinfectant may prevent their development, and so give rise to an erroneous conclusion. Consequently it is essential in all germicidal experiments to determine first of all the inhibition coefficient of the germicide employed. Under the circumstances referred to above it is usually sufficient to prepare the subcultures in such a volume of fluid nutrient medium as would suffice to reduce the concentration of the germicide to about one hundredth of the inhibition percentage, assuming that the entire bulk of inoculum was made up of that strength of germicide employed in the test. In some cases it is a simple matter to neutralise the germicide and render it inert by washing the organisms in some non-germicidal solution (such for example as ammonium sulphide when using mercurial salts as the germicide). When, however, it is desired to remove the last traces of germicide proceed as follows:

1. Transfer the suspension of bacteria to sterile centrifugal tubes; add the required amount of disinfectant, and allow it to remain in contact with the bacteria for the necessary period.

2. Centrifugalise thoroughly, pipette off the supernatant fluid; fill the tube with sterile water and distribute the deposit evenly throughout the fluid.

3. Centrifugalise again, pipette off the supernatant fluid; fill the tube with sterile water; distribute the deposit evenly throughout the fluid, and transfer the suspension to a litre flask.

4. Make up to a litre by the addition of sterile water; filter the suspension through a sterile porcelain candle.

5. Emulsify the bacterial residue with 5 c.c. sterile bouillon.

6. Prepare the necessary subcultivations from this emulsion.