course of a previous experiment (_vide_ page 282, step 3) into four
portions and test for the production of alcohol, acetaldehyde, acetone.
1. Add Lugol's iodine, then a little NaOH solution, and stir with a
glass rod till the colour of the iodine disappears.
Pale-yellow crystalline precipitate of iodoform, with its characteristic
smell, appearing in the cold, indicates acetaldehyde, or acetone;
appearing only on warming indicates alcohol.
The precipitate may be absent even when the odour is pronounced.
2. Add Schiff's reagent.
Violet or red colour = aldehyde.
3. To 10 c.c. of solution add 2.5 c.c., 25 per cent. sulphuric acid, and
a crystal or two of potassium bichromate and distil. Reduction of the
bichromate to a green colour and a distillate, which smells of
acetaldehyde and reacts with Schiff's reagent, shows the presence of
alcohol in the original liquid.
4. Add a few drops of sodium nitroprusside solution, make alkaline with
ammonia, then saturate with ammonium sulphate crystals. Acetone gives
little colour on the addition of ammonia, but after the addition of
ammonium sulphate a deep permanganate colour, which takes ten minutes to
reach its full intensity. Aldehyde gives a carmine red unaltered by
ammonium sulphate.
~5. Indol Production.~--
_Media Required_:
Inosite-free bouillon (_vide_ page 183).
Or peptone water (_vide_ page 177).
_Reagents Required_:
Potassium persulphate, saturated aqueous solution.
Paradimethylamino-benzaldehyde solution. This is prepared by mixing:
Paradimethylamino-benzaldehyde 4 grammes
Absolute alcohol 380 c.c.
Hydrochloric acid, concentrated 80 c.c.
METHOD.--
Prepare several test-tube cultivations of the organism to be tested, and
incubate.
Test for indol by means of the Rosindol reaction in the following
manner. (If the culture has been incubated at 37 deg. C., it must be
allowed to cool to the room temperature before applying the test.)
1. Remove 2 c.c. of the cultivation by means of a sterile pipette and
transfer to a clean tube, then,
2. Add 2 c.c. paradimethylamino-benzaldehyde solution.
3. Add 2 c.c. potassium persulphate solution.
The presence of indol is indicated by the appearance of a delicate
rose-pink colour throughout the mixture which deepens slightly on
standing.
Indol is tested for in many laboratories by the ordinary
nitrosoindol reaction which, however, is not so delicate a
method as that above described. The test is carried out as
follows:
1. Remove the cotton-wool plug from the tube, and run in 1
c.c. pure concentrated sulphuric acid down the side of the
tube by means of a sterile pipette. Place the tube upright
in a rack, and allow it to stand, if necessary, for ten
minutes.
A rose-pink or red colour at the junction of the two liquids
= indol (_plus a nitrite_).
2. If the colour of the medium remains unaltered, add 2 c.c.
of a 0.01 per cent. aqueous solution sodium nitrite, and
again allow the culture to stand for ten minutes.
Red colouration = indol.
NOTE.--In place of performing the test in two stages as
given above, 2 c.c. concentrated _commercial_ sulphuric,
hydrochloric, or nitric acid (all of which hold a trace of
nitrite in solution), may be run into the cultivation. The
development of a red colour within twenty minutes will
indicate the presence of indol.
~5a. Phenol Production.~--
_Medium Required_:
Nutrient bouillon.
_Reagents Required_:
Hydrochloric acid, concentrated.
Millon's reagent.
Ferric chloride, 1 per cent. aqueous solution.
METHOD.--
1. Prepare cultivation in a Bohemian flask containing at least 50 c.c.
of medium, and incubate.
Test for phenol in the following manner:
2. Add 5 c.c., 25 per cent. sulphuric acid to the cultivation and
connect up the flask with a condenser.
3. Distil over 15 to 20 c.c. Divide the distillate into three portions
a, b and c.
4. Add to (a) 0.5 c.c. Millon's reagent and boil.
Red colour = phenol.
5. Add to (b) about 0.5 c.c. ferric chloride solution. Violet colour =
phenol.
(If the distillate be acid the reaction will be negative.)
6. Add to (c) bromine water. Crystalline white ppt. of tribromo-phenol
= phenol.
NOTE.--If both indol and phenol appear to be present in
cultivations of the same organism, it is well to separate
them before testing. This may be done in the following
manner:
1. Prepare inosite-free bouillon cultivation, say 200 or 300 c.c., in a
flask as before.
2. Render definitely acid by the addition of acetic acid and connect up
the flask with a condenser.
3. Distil over 50 to 70 c.c.
Distillate will contain both indol and phenol.
4. Render the distillate strongly alkaline with caustic potash and
redistil.
Distillate will contain indol; residue will contain phenol.
5. Test the distillate for indol (_vide ante_).
6. Saturate the residue, when cold, with carbon dioxide and redistil.
7. Test this distillate for phenol (_vide ante_).
~6. Pigment Production.~--
1. Prepare tube cultivations upon the various media and incubate under
varying conditions as to temperature (at 37 deg. C. and at 20 deg. C.),
atmosphere (aerobic and anaerobic), and light (exposure to and
protection from).
Note the conditions most favorable to pigment formation.
2. Note the solubility of the pigment in various solvents, such as water
(hot and cold), alcohol, ether, chloroform, benzol, carbon bisulphide.
3. Note the effect of acids and alkalies respectively upon the pigmented
cultivation, or upon solutions of the pigment.
4. Note spectroscopic reactions.
~7. Reducing Agent Formation.~--
(a) _Colour Destruction._--
1. Prepare tube cultivations in nutrient bouillon tinted with litmus,
rosolic acid, neutral red, and incubate.
2. Examine the cultures each day and note whether any colour change
occurs.
(b) _Nitrates to Nitrites._--
_Medium Required_:
Nitrate bouillon (_vide_ page 185).
Or nitrate peptone solution (_vide_ page 186).
_Reagents Required_:
Sulphuric acid (25 per cent.).
Metaphenylene diamine, 5 per cent. aqueous solution.
METHOD.--
1. Prepare tube cultivations and incubate together with control tubes
(i. e., uninoculated tubes of the same medium, placed under identical
conditions as to environment).
This precaution is necessary as the medium is liable to take up nitrites
from the atmosphere, and an opinion as to the absence of nitrites in the
cultivation is often based upon an equal colouration of the medium in
the control tube.
Test both the culture tube and the control tube for the presence of
nitrites.
2. Add a few drops of sulphuric acid to the medium in each of the tubes.
3. Then run in 2 or 3 c.c. metaphenylene diamine into each tube.
Brownish-red colour = nitrites.
The depth of colour is proportionate to the amount present.
~8. Gas Production.~--
(A) _Carbon Dioxide and Hydrogen._--
_Apparatus Required_:
Fermentation tubes (_vide_ page 161) containing sugar
bouillon (glucose, lactose, etc.). The medium should be
prepared from inosite-free bouillon (_vide_ page 183).
_Reagent Required_:
n/2 caustic soda.
METHOD.--
1. Inoculate the surface of the medium in the bulb of a fermentation
tube and incubate.
2. Mark the level of the fluid in the closed branch of the fermentation
tube, at intervals of twenty-four hours, and when the evolution of gas
has ceased, measure the length of the column of gas with the millimetre
scale.
Express this column of gas as a percentage of the entire length of the
closed branch.
3. To analyse the gas and to determine roughly the relative proportions
of CO_{2} and H_{2}, proceed as follows:
Fill the bulb of the fermentation tube with caustic soda solution.
Close the mouth of the bulb with a rubber stopper.
Alternately invert and revert the tube six or eight times, to bring the
soda solution into intimate contact with the gas.
Return the residual gas to the end of the closed branch, and measure.
The loss in volume of gas = carbon dioxide.
The residual gas = hydrogen.
Transfer gas to the bulb of the tube, and explode it by applying a
lighted taper.
(B) _Sulphuretted Hydrogen._--
_Media Required_:
Iron peptone solution (_vide_ page 185).
Lead peptone solution.
1. Inoculate tubes of media, and incubate together with control tubes.
2. Examine from day to day, at intervals of twenty-four hours.
The liberation of the H_{2}S will cause the yellowish-white precipitate
to darken to a brownish-black, or jet black, the depth of the colour
being proportionate to the amount of sulphuretted hydrogen present.
Quantitative: For exact quantitative analyses of the gases produced by
bacteria from certain media of definite composition, the methods devised
by Pakes must be employed, as follows:
[Illustration: FIG. 153.--Gas-collecting apparatus.]
_Apparatus Required_:
Bohemian flask (300 to 1500 c.c. capacity) containing from
100 to 400 c.c. of the medium. The mouth of the flask is
fitted with a perforated rubber stopper, carrying an
L-shaped piece of glass tubing (the short arm passing just
through the stopper). To the long arm of the tube is
attached a piece of pressure tubing some 8 cm. in length,
plugged at its free end with a piece of cotton-wool. Measure
accurately the total capacity of the flask and exit tube,
also the amount of medium contained. Note the difference.
Gas receiver. This is a bell jar of stout glass, 14 cm. high
and 9 cm. in diameter. At its apex a glass tube is fused in.
This rises vertically 5 cm., and is then bent at right
angles, the horizontal arm being 10 cm. in length. A
three-way tap is let horizontally into the vertical tube
just above its junction with the bell jar.
An iron cylinder just large enough to contain the bell jar.
About 15 kilos of metallic mercury.
Melted paraffin.
An Orsat-Lunge working with mercury instead of water, provided with two
gas tubes of extra length (capacity 120 and 60 c.c. respectively and
graduated throughout, both being water-jacketed) or other gas analysis
apparatus, capable of dealing with CO_{2}, O_{2}, H_{2}, and N_{2}.
METHOD.--
1. Inoculate the medium in the flask in the usual manner, by means of a
platinum needle, taking care that the neck of the flask and the rubber
stopper are thoroughly flamed before and after the operation.
[Illustration: FIG. 154.--Orsat-Lunge gas analysis apparatus.]
2. Fill the iron cylinder with mercury.
3. Place the bell jar mouth downward in the mercury--first seeing that
there is free communication between the interior of the jar and the
external air--and suck up the mercury into the tap; then shut off the
tap.
4. Plug the open end of the three-way tap with melted wax.
5. Connect up the horizontal arm of the culture flask with that of the
gas receiver by means of the pressure tubing (after removing the
cotton-wool plug from the rubber tube), as shown in Fig. 153.
6. Give the three-way tap half turn to open communication between flask
and receiver, and seal _all_ joints by coating with a film of melted
wax. When the tap is turned, the mercury in the receiver will naturally
fall.
7. Place the entire apparatus in the incubator. (Two hours later, by
which time the temperature of the apparatus is that of the incubator,
mark the height of the mercury on the receiver.)
8. Examine the apparatus from day to day and mark the level of the
mercury in the receiver at intervals of twenty-four hours.
9. When the evolution of gas has ceased, remove the apparatus from the
incubator; clear out the wax from the nozzle of the three-way tap (first
adjusting the tap so that no escape of gas shall take place) and connect
it with the Orsat.
10. Remove, say, 100 c.c. of gas from the receiver, reverse the tap and
force it into the culture flask. Remove 100 c.c. of mixed gases from the
culture flask and replace in the receiver.
Repeat these processes three or four times to ensure thorough admixture
of the contents of flask and receiver.
11. Now withdraw a sample of the mixed gases into the Orsat and analyse.
In calculating the results be careful to allow for the volume of air
contained in the flask at the commencement of the experiment.
For the collection of gases formed under anaerobic conditions a slightly
different procedure is adopted:
1. Fix a culture flask (500 c.c. capacity) with a perforated rubber
stopper carrying an ~L~-shaped piece of manometer tubing, each arm 5 cm.
in length.
2. Prepare a second ~L~-shaped piece of tubing, the short arm 5 cm. and
the long arm 20 cm., and connect its short arm to the horizontal arm of
the tube in the culture flask by means of a length of pressure tubing,
provided with a screw clamp.
3. Fill the culture flask completely with boiling medium and pass the
long piece of tubing through the plug of an Erlenmeyer flask (150 c.c.
capacity) which contains 100 c.c. of the same medium.
4. Sterilise these coupled flasks by the discontinuous method, in the
usual manner.
Immediately the last sterilisation is completed, screw up the clamp on
the pressure tubing which connects them, and allow them to cool.
As the fluid cools and contracts it leaves a vacuum in the neck of the
flask below the rubber stopper.
5. To inoculate the culture flask, withdraw the long arm of the bent
tube from the Erlenmeyer flask and pass it to the bottom of a test-tube
containing a young cultivation (in a fluid medium similar to that
contained in the culture flask) of the organism it is desired to
investigate.
6. Slightly release the clamp on the pressure tubing to allow 4 or 5
c.c. of the culture to enter the flask.
7. Clamp the rubber tube tightly; remove the bent glass tube from the
culture tube and plunge it into a flask containing recently boiled and
quickly cooled distilled water.
8. Release the clamp again and wash in the remains of the cultivation
until the culture flask and tubing are completely filled with water.
9. Clamp the rubber tubing tightly and take away the long-armed glass
tubing.
10. Prepare the gas receiver as in the previous method (in this case,
however, the mercury should be warmed slightly) and fill the horizontal
arm of the receiver with hot water.
11. Connect up the culture flask with the horizontal arm of the gas
receiver.
12. Remove the screw clamp from the rubber tubing, adjust the three-way
tap, seal all joints with melted wax, and incubate.
13. Complete the investigation as described for the previous method.
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.
(c) In the presence of gases other than oxygen.
Temperature:
(a) Range.
(b) Optimum.
(c) Thermal death-point:
Moist: Vegetative forms.
Spores.
Dry: Vegetative forms.
Spores.
Reaction of medium.
Resistance to lethal agents:
(a) Desiccation.
(b) Light: Diffuse.
Direct.
Primary colours.
(c) Heat.
(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 deg. C.
Sloped glucose formate gelatine, and incubate aerobically at 20 deg. C.
(B) Sloped glucose agar to incubate anaerobically at 37 deg. C.
Sloped glucose formate gelatine to incubate anaerobically at 20 deg. C.
(C) Sloped glucose formate agar to incubate anaerobically at 37 deg. C.
Glucose formate bouillon to incubate anaerobically at 37 deg. C.
(D) Sloped glucose formate gelatine to incubate anaerobically at
20 deg. C.
Glucose formate bouillon to incubate anaerobically at 20 deg. 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 (SO_{2}, N_{2}O, NO,
CO_{2}, 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 deg. C.
and including temperatures between 5 deg. C. and 50 deg. 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 deg. 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.
(C) _Thermal Death-point (t. d. p.)_--
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~.
[Illustration: FIG. 155.--Hearson's water-bath.]
_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 deg., from 50 deg. C. to 80 deg. 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 deg. 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 deg. 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
deg. C. higher than the immediately preceding one--i. e., 42 deg. C.,
44 deg. C., 46 deg. 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 deg. C., through a range of 5 deg. 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 deg. 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 deg. 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.