_Apparatus Required:_
Steam-can fitted with a delivery tube and a large bore
safety-valve tube.
Water-bath at 100 deg. 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 deg. 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.
[Illustration: FIG. 156.--Apparatus arranged for the determination of
the death-point of spores.]
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 deg. 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 deg. C. between 60 deg. C. and
120 deg. 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 deg. C, between 100 deg. C. and 160 deg. 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.
[Illustration: FIG. 157.--Mueller's desiccator.]
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.
[Illustration: FIG. 158.--Petri dish for drying cultivations.]
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.
[Illustration: FIG. 159.--Plate with star for testing effect of light.]
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:
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.
(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 deg. to 25 deg. 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[10] 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 deg. 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 deg. C. to 20 deg. 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 deg. 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 deg. to 20 deg. 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 deg. to 37 deg. 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.