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.
(c) Bent syphon tube, with pipette nozzle attached by means of rubber tubing and fitted with pinch-cock.
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.
(c) Primary Colours: Each colour—violet, blue, green and red—must be tested separately.
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:
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.
(C) Heat.—(Vide Thermal Death-point, page 298.)
(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:
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[10] of the carbolic acid solution, viz.:
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 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. 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[11] 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.
PATHOGENESIS.
Living Bacteria.—
(a) Psychrophilic Bacteria: When the organism will only grow at or below 18° to 20° C.,
1. Prepare cultivations in nutrient broth and incubate under optimum conditions.
2. After seven days' incubation inject that amount of the culture corresponding to 1 per cent. of the body-weight of a healthy frog, into the reptile's dorsal lymph sac.
3. Observe until death takes place, or, in the event of a negative result, until the completion of twenty-eight days (vide Chapter XVIII).
4. If, and when, death occurs, make a careful post-mortem examination (vide Chapter XIX).
(b) Mesophilic Bacteria: When the organism grows at 35° to 37° C.,
1. Prepare cultivations in nutrient broth and incubate under optimum conditions for forty-eight hours.
2. Select two white mice, as nearly as possible of the same age, size, and weight.
3. Inoculate the first mouse, subcutaneously at the root of the tail, with an amount of cultivation equivalent to 1 per cent. of its body-weight.
4. Inoculate the second mouse intraperitoneally with a similar dose.
5. Observe carefully until death occurs, or until the lapse of twenty-eight days.
6. If the inoculated animals succumb, make complete post-mortem examination.
If death follows shortly after the injection of cultivations of bacteria, the inoculation experiments should be repeated two or three times. Then, if the organism under observation invariably exhibits pathogenic effects, steps should be taken to ascertain, if possible, the minimal lethal dose (vide infra) of the growth upon solid media for the frog or white mouse respectively. Other experimental animals—e. g., the white rat, guinea-pig, and rabbit—should next be tested in a similar manner.
7. If the inoculated mice are unaffected, test the action of the organism in question upon white rats, guinea-pigs, rabbits, etc.
Minimal Lethal Dose (m. l. d.); If the purpose of the inoculation is to determine the minimal lethal dose, a slightly different procedure must be followed. For this and other exact experiments a special platinum loop is manufactured, some 2.5 mm. by 0.75 mm., with parallel sides, and calibrated by careful weighing, to determine approximately the amount of moist bacterial growth, the loop will hold when filled.
1. The cultivation must be prepared on a solid medium of the optimum reaction, incubated at the optimum temperature, and injected at the period of greatest activity and vigour, of the particular organism it is desired to test.
2. Arrange four sterile capsules in a row and label them I, II, III, and IV. Into the first deliver 10 c.c. sterile bouillon by means of a sterile graduated pipette; and into each of the remaining three, 9.9 c.c.
3. Remove one loopful of the bacterial growth from the surface of the medium in the culture tube, observing the usual precautions against contamination, and emulsify it evenly with the bouillon in the first capsule. Each cubic centimetre of the emulsion will now contain one-tenth of the organisms contained in the original loopful (written shortly 0.1 loop).
4. Remove 0.1 c.c. of the emulsion in the first capsule by means of a sterile graduated pipette and transfer it to the second capsule and mix thoroughly. Drop the infected pipette into a jar of lysol solution. This makes up the bulk of the fluid in the second capsule to 10 c.c., and therefore every cubic centimetre of bouillon in capsule II contains 0.001 loop.
5. Similarly, 0.1 c.c. of the mixture is transferred from capsule II to capsule III (1 c.c. of bouillon in capsule III contains 0.00001 loop), and then from capsule III to capsule IV (1 c.c. of bouillon in capsule IV contains 0.0000001 loop).
The dilutions thus prepared may be summarised in a table;
Capsule II = 0.1 c.c. capsule I + 9.9 c.c. water ∴ 1 c.c.=0.001 loop.
Capsule III = 0.1 c.c. capsule II + 9.9 c.c. water ∴ 1 c.c.=0.00001 loop.
Capsule IV = 0.1 c.c. capsule III + 9.9 c.c. water ∴ 1 c.c. = 0.0000001 loop.
6. With sterile graduated pipettes remove the necessary quantity of bouillon corresponding to the various divisors of ten of the loop from the respective capsules, and transfer each "dose" to a separate sterile capsule and label; and to such doses as are small in bulk, add the necessary quantity of sterile bouillon to make up to 1 c.c.
7. Multiples of the loop are prepared by emulsifying 1, 2, 5, or 10 loops each with 1 c.c. sterile bouillon in separate sterile capsules.
8. Inoculate a series of animals with these measured doses, filling the syringe first from that capsule containing the smallest dose, then from the capsule containing the next smallest, and so on. If care is taken, it will not be found necessary to sterilise the syringe during the series of inoculations.
9. Plant tubes of gelatine or agar, liquefied by heat, from each of the higher dilutions, say from 0.0000001 loop to 0.01 loop; pour plates and incubate. When growth is visible enumerate the number of organisms present in each, average up and calculate the number of bacteria present in one loopful of the inoculum.
10. The smallest dose which causes the infection and death of the inoculated animal is noted as the minimal lethal dose.
Toxins.—
Prepare flask cultivations of the organism under observation in glucose formate broth, and incubate for fourteen days under optimum conditions.
(a) Intracellular or Insoluble Toxins:
1. Heat the fluid culture in a water-bath at 60° C. for thirty minutes. (The resulting sterile, turbid fluid is often spoken of as "killed" culture,)
2. Inoculate a tube of sterile bouillon with a similar quantity, and incubate under optimum conditions. This "control" then serves to demonstrate the freedom of the toxin from living bacteria.
3. Inject intraveneously that amount of the cultivation corresponding to 1 per cent. of the body-weight of the selected animal, usually one of the small rodents.
4. Observe during life or until the completion of twenty-eight days, and in the event of death occurring during that period, make a complete post-mortem examination.
5. Repeat the experiment at least once. In the event of a positive result estimate the minimal lethal dose of "killed" culture for each of the species of animals experimented upon.
(b) Extracellular or Soluble Toxins:
1. Filter the cultivation through a porcelain filter candle (Berkefeld) into a sterile filter flask, arranging the apparatus as in the accompanying figure (Fig. 160).
2. Inoculate mice, rats, guinea-pigs, and rabbits subcutaneously with that quantity of toxin corresponding to 1 per cent. of the body-weight of each respectively, and observe, if necessary, until the completion of one month.
3. Inoculate a "control" tube of bouillon with a similar quantity and incubate, to determine the freedom of the filtered toxin from living bacteria.
4. In the event of a fatal termination make complete and careful post-mortem examinations.
5. Repeat the experiments and, if the results are positive, ascertain the minimal lethal dose of toxin for each of the susceptible animals.
The estimation of the m. l. d. of a toxin is carried out on lines similar to those laid down for living bacteria (vide page 316) merely substituting 1 c.c. of toxin as the unit in place of the unit "loopful" of living culture.
It frequently happens, during the course of casual investigations that a bouillon-tube culture is available for a toxin test whilst a flask cultivation is not. In such cases, Martin's small filter candle and tube (Fig. 161) specially designed for the filtration of small quantities of fluid, is invaluable. This consists of a narrow filter flask just large enough to accommodate an ordinary 18 × 2 cm. test-tube. The mouth of the tubular Chamberland candle 15 × 1.5 cm. is closed by a perforated rubber cork into which fits the end of the stem of a thistle headed funnel, whilst immediately below the butt of the funnel is situated a rubber cork to close the mouth of the filter flask. When the apparatus is fixed in position and connected to an exhaust pump, the cultivation is poured into the head of the funnel and owing to the relatively large filtering surface the germ free filtrate is rapidly drawn through into the test-tube receiver.
Raising the Virulence of an Organism.—If it is desired to raise or "exalt" the virulence of a feebly pathogenic organism, special methods of inoculation are necessary, carefully adjusted to the exigencies of each individual case. Among the most important are the following:
1. Passage of Virus.—The inoculation of pure cultivations of the organism into highly susceptible animals, and passing it as rapidly as possible from animal to animal, always selecting that method of inoculation-e. g., intraperitoneal—which places the organism under the most favorable conditions for its growth and multiplication.
2. Virus Plus Virulent Organisms.—The inoculation of pure cultivations of the organism together with pure cultivations of some other microbe which in itself is sufficiently virulent to ensure the death of the experimental animal, either into the same situation or into some other part of the body. By this association the organism of low virulence will frequently acquire a higher degree of virulence, which may be still further raised by means of "passages" (vide supra).
3. Virus Plus Toxins.—The inoculation of pure cultivations of the organism into some selected situation, together with the subcutaneous, intraperitoneal, or intravenous injection of a toxin—e. g., one of those elaborated by the proteus group—either simultaneously with, before, or immediately after, the injection of the feeble virus. By this means the natural resistance of the animal is lowered, and the organism inoculated is enabled to multiply and produce its pathogenic effect, its virulence being subsequently exalted by means of "passages."
Attenuating the Virulence of an Organism.—Attenuating or lowering the virulence of a pathogenic microbe is usually attained with much less difficulty than the exaltation of its virulence, and is generally effected by varying the environment of the cultivations, as for example:
1. Cultivating in such media as are unsuitable by reason of their (a) composition or (b) reaction.
2. Cultivating in suitable media, but at an unsuitable temperature.
3. Cultivating in suitable media, but in an unsuitable atmosphere.
4. Cultivation in suitable media, but under unfavorable conditions as to light, motion, etc.
Attenuation of the virus can also be secured by
5. Passage through naturally resistant animals.
6. Exposure to desiccation.
7. Exposure to gaseous disinfectants.
8. By a combination of two or more of the above methods.
IMMUNISATION.
The further study of the pathogenetic powers of any particular bacterium involves the active immunisation of one or more previously normal animals. This end may be attained by various means; but it must be remembered that immunisation is not carried out by any hard and fast rule or by one method alone, but usually by a combination of methods adapted to the exigencies of each particular case. The ordinary methods include:
A. Active Immunisation.
I. By inoculation with dead bacteria (i. e., bacteria killed by heat; the action of ultra-violet rays, of chemical germicides, or by autolysis).
II. By the inoculation of attenuated strains of bacteria.
III. By the inoculation of living virulent bacteria (exalted in virulence if necessary).
B. Combined Active and Passive Immunisation:
IV. By the inoculation of toxin-antitoxin mixtures.
ACTIVE IMMUNISATION.
The immunisation of the rabbit against the Diplococcus pneumoniæ may be instanced as an example of the general methods of immunisation of laboratory animals.
1. Take a full grown rabbit weighing not less than 1200 to 1500 grammes (large rabbits of 2000 grammes and over are the most suitable for immunising experiments). Observe weight and temperature carefully during the few days occupied in the following steps.
2. Inoculate a small rabbit intraperitoneally with one or two loopfuls of a twenty-four-hour-old blood agar cultivation of a virulent strain of Diplococcus pneumoniæ.
Death should follow within twenty-four hours, and in any case will not be delayed beyond forty-eight hours.
3. Under aseptic precautions, at the post-mortem, transfer a loopful of heart blood to an Erlenmeyer flask containing 50 c.c. sterile nutrient broth. Incubate at 37° C. for twenty-four hours.
4. Prepare also several blood agar cultures from the heart blood of the rabbit, label them all O.C. (original culture). After twenty-four hours incubation at 37° C. place an india-rubber cap over the plugged mouth of the tube of all but one of these cultures and paint the cap with Canada balsam or shellac varnish, dry, and replace in the hot incubator.
This will prevent evaporation, and cultures thus sealed will remain unaltered in virulence for a considerable time.
5. Make a fresh subcultivation on blood agar from the uncapped O.C. cultivation and after twenty-four hours incubation at 37° C. determine the minimal lethal dose of this strain upon a series of mice (see page 316).
6. Suspend the flask containing the twenty-four-hour-old broth culture (step 3) in the water-bath at 60° C. for one hour. Cool the flask rapidly under a stream of cold water.
7. Determine the sterility of this (?) killed cultivation by transferring one cubic centimetre to each of several tubes of nutrient broth, and incubate at 37° C. for twenty-four hours. If growth of Diplococcus pneumoniæ occurs, again heat culture in water-bath at 60° C. for one hour and again test for sterility.
8. Inject the selected rabbit intravenously (see page 363) with 2 c.c. of the killed cultivation, and inject a further 10 c.c. into the peritoneal cavity.
During the next few days the animal will lose some weight and perhaps show a certain amount of pyrexia.
9. When the temperature and weight have again returned to normal—generally about seven days after the inoculation—again inject killed cultivation, this time giving a dose of 5 c.c. intravenously and 20 c.c. intraperitoneally. A temperature and weight reaction similar to, but less marked than that following the first injection will probably be observed, but after about a week's interval the animal will be ready for the next injection.
10. When ready to give the third injection prepare a fresh blood agar subculture from another O.C. tube and after twenty-four hours incubation prepare a minimal lethal dose (as determined in 5) and inject it subcutaneously into the rabbit's abdominal wall.
A slight local reaction will probably be observed as well as the weight and temperature reactions.
11. A week to ten days later inject a similar minimal lethal dose into the peritoneal cavity.
12. Observe the weight and temperature of the rabbit very carefully, and regulating the dates of inoculation by the animal's general condition, continue to inject living cultivations of the pneumococcus into the peritoneal cavity, gradually increasing the dose by multiples of ten.
13. At intervals of two months samples of blood may be collected from the posterior auricular vein and the serum tested for specific antibodies.
14. Under favourable conditions it will be found after some six months steady work that the rabbit may be injected intraperitoneally with an entire blood agar cultivation without any ill effects being apparent; and this characteristic—resistance to the lethal effects of large doses of the virus—is the sole criterion of immunity. Further, the serum separated from blood withdrawn from the animal about a week after an injection, if used in doses of .01 c.c., will protect a mouse against the lethal effects of at least ten minimal lethal doses of living pneumococci.
In the foregoing illustration it has been assumed that complete acquired active immunity has been conferred upon the experimental rabbit in consequence of the formation of antibody, specific to the diplococcus pneumoniac, sufficient in amount to ensure the destruction of enormous doses of the living cocci—the antigen (that is the substance injected in response to which antibody has been elaborated) in this particular case being the bacterial protoplasm of the pneumococcus with its endo-toxins.
But provided death does not immediately follow the injection of the antigen, specific antibody is always formed in greater or lesser amount; and in experimental work a sufficient amount of any required antibody can often be obtained without carrying the process of immunisation to its logical termination.
For instance, if the immunisation of a rabbit toward Bacillus typhosus is commenced on the lines already set out it will often be found, after a few injections of "killed" cultivation that the blood serum of the animal (even when diluted with several hundred times its volume of normal saline) contains specific agglutinin for B. typhosus—and if the sole object of the experiment has been the preparation of agglutinin the inoculations may well be stopped at this point, although the animal is not yet immune in the strict meaning of the word.
Again, antibodies may be formed in response to antigens other than infective particles—thus the injection into suitable animals of foreign proteins such as egg albumin, heterologous blood sera or red blood discs from a different species of animal, will result in the formation of specific antibodies possessing definite affinities for their respective antigens.
The most important antibody of this latter type is Hæmolysin, a substance that makes its appearance in the blood serum of an animal previously injected with washed blood cells from an animal of a different species. The serum from such an animal possesses the power of disintegrating red blood discs of the variety employed as antigen and causing the discharge of their contained hæmoglobin, and is specific in its action to the extent of failing to exert any injurious effect upon the red blood cells of any other species of animal.
The action of this serum is due to the presence of two distinct bodies, complement and hæmolysin.
Complement (or alexine) is a thermo-labile readily oxidised body present in variable but unalterable amount in the normal serum of every animal. It is a substance which exerts a lytic effect upon all foreign matter introduced into the blood or tissues; but by itself is a comparatively inert body, and is only capable of exerting its maximum lytic effect in the presence of and in combination with a specific antibody, or immune body.
Complement is obtained (unmixed with antibody) by collecting fresh blood serum from any healthy normal (that is uninoculated) animal. Guinea-pigs' serum is that most frequently employed for experimental work.
Hæmolysin (immune body, copula, sensitising body, amboceptor) is a thermostable antibody formed in response to the injection of red cells which although in itself inert is capable of linking up complement present in the normal serum to the red cells of the variety used as antigen—a combination resulting in hæmolysis.
Hæmolysin is obtained by collecting fresh blood serum from a suitably inoculated animal and exposing it to a temperature of 56° C. (to destroy the thermo-labile complement) for 15 to 30 minutes before use. It is then referred to as inactivated, and is reactivated by the addition of fresh normal serum—that is serum containing complement.
Hæmolysin is of importance academically owing to the fact that many of the problems of immunity have been elucidated by its aid; but its present practical importance lies in the application of the hæmolytic system (that is hæmolysin, corresponding erythrocyte solution and complement) to certain laboratory methods having for their object either the identification of the infective entity or the diagnosis of the existence of infection.
For use in these laboratory methods of diagnosis it is most convenient to prepare hæmolytic serum specific for human blood—whether the laboratory is isolated or attached to a large hospital. Ox blood, sheep blood or goat blood if readily obtainable, may however be used instead, and although the following method is directed to the preparation of human hæmolysin the same procedure serves in all cases.
THE PREPARATION OF HÆMOLYTIC SERUM.
Apparatus Required: