Litmus milk.

METHOD.--

1. Prepare tube cultivations and incubate.

2. After incubation heat the cultivation to 55 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 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) _Quant.i.tative._--

_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 C.

for thirty minutes to sterilise.

3. Determine the _t.i.tre_ of the medium in "inoculated" and "control"

flasks as described in the preparation of nutrient media (_vide_ page 151).

4. The difference between the t.i.tre 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 t.i.tration.

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

_Medium Required_: Sugar (glucose or lactose) bouillon as in quant.i.tative 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.

Pota.s.sium bichromate.

Schiff's reagent.

a.r.s.enious 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 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.