The Elements of Bacteriological Technique Part 77

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

[Ill.u.s.tration: FIG. 152.--Arrangement of distillation apparatus for acids, etc.]

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.

quant.i.ties cannot be distinguished from butyric (c) Evaporate to dryness; mix acid by tests within the with equal quant.i.ty of scope of the bacteriological a.r.s.enious 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 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 t.i.tration. 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 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.