Chapter X: Appendix: 492 (8)

(b) _Spore Germination._--Expose a thick emulsion of the spores to a temperature of 80 deg. C. for ten minutes in the differential steriliser (_vide_ page 257).

Transfer the emulsion to a tube of sterile nutrient broth and incubate.

Remove specimens from the tube culture at intervals of, say, five minutes.

Fix, stain, etc., wet, as under (a), and examine.

(B) ~Fixed.--2.~ In ~stained preparations~.

(a) To determine points in _morphology_:

_Shape_ (_vide_ classification, page 131).

_Size_:

(a) Prepare cover-slip film preparations at the various ages, and fix by exposure to a temperature of 115 deg. C. for twenty minutes in hot-air oven.

(b) Stain the preparations by Gram's method (if applicable) or with dilute carbol-fuchsin, and mount in the usual way.

(c) Measure (_vide_ page 66) some twenty-five individuals in each film by means of the Ramsden's or the stage micrometer and average the result.

_Pleomorphism_; If noted, record--

The predominant character of the variant forms.
On what medium or media they are observed.
At what period of development.

(b) To demonstrate details of _structure_:

_Flagella_: If noted, record--

Method of staining (_vide_ page 101).
Position and arrangement (_vide_ page 136).
Number.

_Spores_: If noted, record--

Method of staining.
Shape.
Size.
Position within the parent cell.
Condition, as to shape, of the parent cell (_vide_
page 139).
Optimum medium and temperature.
Age of cultivation.
Conditions of environment as to temperature,
atmosphere.
Method of germination (_vide_ page 140).

_Involution Forms_: If noted, record--

Method of staining.
Character (e. g., if living or dead).
Shape.
On what medium they are observed.
Age of medium.
Environment.

_Metachromatic Granules_: If noted, record--

Method of staining.
Character of granules.
Number of granules.
Colour of granules.

~3. Staining Reactions.~--

1. _Gram's Method._--Positive or negative.

2. _Neisser's Method._--If granules are noted, record--

1. Position.
2. Number.

3. _Ziehl-Neelsen's Method._--Acid-fast or decolourised.

4. _Simple Aniline Dyes._--(Noting those giving the best results, with details of staining processes.)

Methylene-blue }
Fuchsin } and their modifications.
Gentian violet }
Thionine blue }

BY BIOCHEMICAL METHODS.

Test cultivations of the organism for the presence of--

Soluble enzymes--proteolytic, diastatic, invertase.

Organic acids--(a) quantitatively--i. e., estimate the total acid production; (b) qualitatively for formic, acetic, propionic, butyric, lactic.

Ammonia.

Neutral volatile substances--ethyl alcohol, aldehyde, acetone.

Aromatic products--indol, phenol.

Soluble pigments.

Test the power of reducing (a) colouring matters, (b) nitrates to nitrites.

Investigate the gas production--H_{2}S, CO_{2}, H_{2}. Estimate the ratio between the last two gases.

Prepare all cultivations for these methods of examination under _optimum_ conditions, previously determined for each of the organisms it is intended to investigate, as to

(a) Reaction of medium;
(b) Incubation temperature;
(c) Atmospheric environment;

and keep careful records of these points, and also of the age of the cultivation used in the final examination.

Examine the cultivations for the various products of bacterial metabolism after forty-eight hours' growth, and ~never omit to examine "control" (uninoculated) tube or flask of medium from the same batch, kept for a similar period under identical conditions~.

If the results are negative, test further cultivations at three days, five days, and ten days.

~1. Enzyme Production.~--

(A) _Proteolytic Enzymes._--(Convert proteins into proteose, peptone and further products of hydrolysis; e. g., B. pyocyaneus.)

_Media Required_:

Blood-serum and milk-serum which have been carefully
filtered through a porcelain candle.

_Reagents Required_:

Ammonium sulphate.
Thirty per cent. caustic soda solution.
Copper sulphate, 0.5 per cent. aqueous solution.
One per cent. acetic acid solution.
Millon's reagent.
Glyoxylic acid solution.
Concentrated sulphuric acid.

METHOD.--

1. Prepare cultivations in bulk (50 c.c.) in a flask and incubate.

2. Make the liquid faintly acid with acetic acid, then boil. (This precipitates the unaltered proteins.)

3. Filter.

4. Take 10 c.c. of the filtrate in a test-tube and add 1 c.c. of the caustic soda, then add the copper sulphate drop by drop.

Pink colour which becomes violet with more copper sulphate =
proteose and peptone.

5. Saturate the rest of the filtrate with ammonium sulphate.

Precipitate = proteose.

6. Filter and divide the filtrate into three parts a, b and c.

a. Repeat the copper sulphate test, using excess of caustic soda to displace the ammonia from the ammonium sulphate.

Pink colour = peptone.

b. Boil with Millon's reagent.

Red colour = tyrosine.

c. Add glyoxylic acid solution and run in concentrated sulphuric acid.

Violet ring at upper level of acid = tryptophane.

Both the tyrosine and tryptophane may be either in the free state or in combination as polypeptid or peptone.

(B) _Diastase._--(Converts starch into sugar; e. g., B. subtilis.)

_Medium Required_:

Inosite-free bouillon.

_Reagents Required_:

Starch.
Thymol.
Fehling's solution.

METHOD.--

1. Prepare tube cultivation and incubate.

2. Prepare a thin starch paste and add 2 per cent. thymol to it.

3. Mix equal parts of the cultivation to be tested and the starch paste, and place in the incubator at 37 deg. C. for six to eight hours.

4. Filter.

Test the filtrate for sugar.

Boil some of the Fehling's solution in a test-tube.

Add the filtrate drop by drop until, if necessary, a quantity has been added equal in amount to the Fehling's solution employed, keeping the mixture at the boiling-point during the process.

Yellow or orange precipitate = sugar.

(C) _Invertase._--(Convert saccharose into a mixture of dextrose and laevulose e. g., B. fluorescens liquefaciens.)

_Medium Required_:
Inosite-free bouillon.

_Reagents Required_:
Cane sugar, 2 per cent. aqueous solution.
Carbolic acid.

METHOD.--

1. Prepare tube cultivations and incubate.

2. Add 2 per cent. of carbolic acid to the sugar solution.

3. Mix equal quantities of the carbolised sugar solution and the cultivation in a test-tube; allow the mixture to stand for several hours.

4. Filter.

Test the filtrate for reducing sugar as in the preceding section.

(D) _Rennin and "Lab" Enzymes._--(Coagulate milk independently of the action of acids; e. g., B. prodigiosus.)

_Media Required_:
Inosite-free bouillon.
Litmus milk.

METHOD.--

1. Prepare tube cultivations and incubate.

2. After incubation heat the cultivation to 55 deg. C. for half an hour, to sterilise.

3. By means of a sterile pipette run 5 c.c. of the cultivation into each of three tubes of litmus milk.

4. Place in the cold incubator at 22 deg. C. and examine each day for ten days.

Absence of coagulation at the end of that period will indicate absence of rennin ferment formation.

Fermentation Reactions.

As tested upon carbohydrate substances and organic salts.

_Media Required_:

Peptone water containing various percentages (generally 2 per cent.) of each of the substances referred to under "sugar" media (page 177), also tubes of peptone water containing 1 per cent. respectively of each of the following:

Organic salts: Sodium citrate, formate, lactate, malate,
tartrate.

METHOD.--

1. Prepare tube cultivations in each of the above media.

2. Observe from day to day up to the expiration of ten days if necessary.

3. Note growth, reaction, gas production.

2. Acid Production.

(a) _Quantitative._--

_Medium Required_:
Sugar (glucose) bouillon of known "optimum" reaction.

_Apparatus and Reagents Required_:
As for estimating reaction of media (_vide_ page 150).

METHOD.--

1. Prepare cultivation in bulk (100 c.c.) in a flask; also "control" flask of medium from same batch.

2. After suitable incubation, heat both flasks in the steamer at 100 deg. C. for thirty minutes to sterilise.

3. Determine the _titre_ of the medium in "inoculated" and "control" flasks as described in the preparation of nutrient media (_vide_ page 151).

4. The difference between the titre of the medium in the two flasks gives the total acid production of the bacterium under observation in terms of normal NaOH.

NOTE.--If the growth is very heavy it may be a difficult
matter to determine the end-point. The cultivation should
then be filtered through a Berkefeld filter candle previous
to step 2, and the filtrate employed in the titration.

(b) _Qualitative_ (of all the organic acids present).--

_Medium Required_:
Sugar (glucose or lactose) bouillon as in quantitative examination.

_Reagents Required_:
Hydrochloric acid, concentrated.
Hydrochloric acid, 25 per cent.
Sulphuric acid, concentrated (pure).
Phosphoric acid, concentrated solution.
Ammonia.
Ammonium sulphate.
Baryta water.
Sodium carbonate, saturated aqueous solution.
Absolute alcohol.
Ether.
Calcium chloride.
Calcium chloride solution.
Zinc carbonate.
Copper sulphate saturated aqueous solution.
Alcoholic thiophene solution (0.15 c.c. in 100 c.c.).
Animal charcoal.
Five per cent. sodium nitroprusside solution.
Potassium bichromate.
Schiff's reagent.
Arsenious oxide.
Ferric chloride, 4 per cent. aqueous solution.
Silver nitrate, 1 per cent. aqueous solution.
Lugol's iodine.
Ten per cent. caustic soda solution.
Hard paraffin wax (melting-point about 52 deg. C.).

METHOD.--

1. Prepare cultivation in bulk (500 c.c.) in a litre flask and add sterilised precipitated chalk, 10 grammes. Incubate at the optimum temperature.

2. After incubation throw a piece of paraffin wax (about a centimetre cube) into the cultivation and connect up the flask with a condenser.

The paraffin, which liquefies and forms a thin layer on the surface of the fluid, is necessary to prevent the cultivation frothing up and running unaltered through the condenser during the subsequent process of distillation.

3. Distill over 200 to 300 c.c.

Use a rose-top burner to minimise the danger of cracking the flask; and to the same end, well agitate the contents of the flask to prevent the chalk settling.

The distillate "A" will contain alcohol, etc. (_vide_ page 285); the residue "a" will contain the volatile and fixed acids.

4. Disconnect the flask and filter. The residue "a" then = filtrate B and residue b.

5. Residue b. Wash the residue from the filter paper, dissolve by heating with dilute hydrochloric acid, and add calcium chloride solution and ammonia until alkaline.

White precipitate insoluble in acetic acid = oxalic acid.

6. Make up filtrate B to 500 c.c. with distilled water and divide into two parts.

7. Acidify 250 c.c. with 20 c.c. concentrated phosphoric acid (this liberates the volatile acids) and distil to small bulk.

The distillate "B" may contain formic, acetic, propionic, butyric and benzoic acids.

DISTILLATE "B."
(Volatile Acids.)
|
|
1. Add baryta water till alkaline,
and evaporate to dryness.

2. Add 50 c.c. absolute alcohol and allow
to stand, with frequent stirring, for
two to three hours.

3. Filter and wash with alcohol.
|
|
|---------------------------------------|
| |
| |
FILTRATE RESIDUE
| |
| |
may contain barium propionate, may contain barium acetate,
barium butyrate. barium formate, barium benzoate.
| |
| |
1. Evaporate to dryness. 1. Evaporate off alcohol and
dissolve up the residue on
2. Dissolve residue in 150 the filter in hot water and
c.c. water. neutralise.

3. Acidify with phosphoric 2. Divide the solution into
acid and distil. four portions:

4. Saturate distillate with (a) Add ferric chloride solution.
calcium chloride and distill
over a few c.c. ~Brown~ colour = _acetic_ or
_formic_ acids.
5. Test distillate for butyric
acid: ~Buff ppt.~ = _benzoic_ acid
(see ether soluble acids).
Add 3 c.c. alcohol and 4 drops
concentrated sulphuric acid. (b) Add silver nitrate
solution; then add one drop
~Smell of pineapple~ = _butyric_ ammonia water, and boil.
acid.
~Black~ precipitate of metallic
Propionic acid in small silver = _formic_ acid.
quantities cannot be
distinguished from butyric (c) Evaporate to dryness; mix
acid by tests within the with equal quantity of
scope of the bacteriological arsenious oxide and heat
laboratory. on platinum foil.

Unpleasant ~smell of cacodyl~
= _acetic_ acid.

(d) Add a few drops of
mercuric chloride solution
in test-tube, and heat to
70 deg. C.

~Precipitate~ of mercurous
chloride which is slowly
reduced to mercury =
_formic_ acid.

8. If the distillation of "B" is continued as long as acid comes over (distilled water being occasionally added to the distilling flask) the distillate can be measured and 50 c.c. used for titration. This will give the amount of volatile acid formation.

9. The second part of the filtrate "B" (see page 282) should be examined for lactic, oxalic, succinic, benzoic, salicylic, gallic and tannic acids, as follows:

~Ether Soluble Acids.~--

1. Evaporate to a thin syrup, acidify strongly with phosphoric acid.

2. Extract with five times its volume of ether by agitation in a separatory funnel.

3. Evaporate the ethereal extract to a thin syrup.

4. Add 100 c.c. water and mix thoroughly.

5. To a small portion of this solution add slight excess of sodium carbonate, evaporate to dryness on the water-bath, dissolve in 5-10 c.c. pure sulphuric acid, add 2 drops saturated copper sulphate solution, place in a test-tube and heat in a boiling water-bath for 2 minutes, cool, add 2 or 3 drops of the alcoholic thiophene and warm gently.

Cherry red colour = lactic acid.

If a brown colour is produced on the addition of sulphuric acid, another sample should be taken and boiled with animal charcoal before evaporating.

6. If lactic acid is definitely present, prepare zinc lactate by boiling part of the solution of the ether extract with excess of zinc carbonate, filtering and evaporating to crystallise. The crystals so obtained have a characteristic form, and if dried at 110 deg. C, should contain 26.87 per cent. of zinc.

7. Test a portion of the rest of the solution of the ether extract for oxalic acid (page 282, step 5). Carefully neutralise the remainder and add ferric chloride solution.

Red brown gelatinous precipitate = succinic acid.

Buff precipitate = benzoic acid, and other acids related to benzoic acid.

Violet colour = salicylic acid.

Inky black colour or precipitate = gallic acid or tannic acid.

For further identification the melting-points of the crystalline acids, and the percentage of silver in their silver salts should be determined.

~3. Ammonia Production.~--

_Medium Required_:
Nutrient bouillon.

_Reagent Required_:
Nessler reagent.

METHOD.--

1. Prepare cultivation in bulk (100 c.c.) in a 250 c.c. flask and incubate together with a control flask.

Test the cultivation and the control for ammonia in the following manner:

2. To each flask add 2 grammes of calcined magnesia, then connect up with condensers and distil.

3. Collect 50 c.c. distillate, from each, in a Nessler glass.

4. Add 1 c.c. Nessler reagent to each glass by means of a clean pipette.

Yellow colour = ammonia.

The depth of colour is proportionate to the amount present.

~4. Alcohol, etc., Production.~--Divide the distillate "A" obtained in the 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:

_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.

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~.

_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.

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 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.

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.

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:

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.