The Elements of Bacteriological Technique Part 21

1. Prepare and fix the film in the manner described above.

2. Boil the mixture and immerse the cover-slip in it, whilst still hot, for one minute.

3. Wash in water.

4. Examine in water; if satisfactory, dry and mount in Canada balsam.

~3. MacCrorrie's Method.~--

_Mordant-Stain._--

Measure out and mix.

Night blue, saturated alcoholic solution 10 c.c.

Potash alum, saturated aqueous solution 10 c.c.

Tannin, 10 per cent. aqueous solution 10 c.c.

NOTE.--The addition of gallic acid, 0.1 to 0.2 gramme, may improve the solution, but is not necessary.

METHOD.--

1. Prepare and fix the films as above.

2. Pour some of the mordant-stain on the film and warm gently, high above the flame, for two minutes (or place in the "hot" incubator for a like period).

3. Wash thoroughly in water.

4. Dry and mount.

~4. Loeffler's Method.~--

(a) _Mordant._--

Tannic acid, 20 per cent. aqueous solution 10 c.c.

Ferrous sulphate, saturated aqueous solution 5 c.c.

Haematoxylin solution 3 c.c.

Carbolic acid, 1 per cent. aqueous solution 4 c.c.

This solution must be freshly prepared.

_Haematoxylin solution_ is prepared by boiling 1 gramme logwood

with 8 c.c. distilled water, filtering and replacing the loss from evaporation.

_Alternative Mordant_ (Bunge's Mordant).--

Tannic acid, 20 per cent. aqueous solution 10 c.c.

Ferrous sulphate, saturated aqueous solution 5 c.c.

Fuchsin, saturated alcoholic solution 1 c.c.

(b) _Stain._--

Weigh out Methylene-blue } Or methylene-violet } 4 grammes Or fuchsin }

and dissolve in

Aniline water, freshly saturated and filtered 100 c.c.

METHOD.--

1. Prepare and fix films as above.

2. Pour the mordant on to the film and warm cautiously over the flame till steam rises; keep the mordant gently steaming for one minute.

3. Wash well in distilled water till no more colour is discharged; if necessary, wash carefully with absolute alcohol.

4. Filter a few drops of the stain on to the film, warm as before, and allow the steaming stain to act for one minute.

5. Wash well in distilled water.

6. Dry and mount.

NOTE.--The flagella of some organisms can be demonstrated better by means of an alkaline stain or an acid stain--a point to be determined for each. Speaking generally, those bacilli which give rise to an acid reaction in the culture medium require an alkali; those which form alkali in cultivation require an acid. According to requirements, therefore, Loeffler recommends the addition of sodium hydrate, 1 per cent. aqueous solution, 1 c.c.; or an equal quant.i.ty of an exactly comparable solution of sulphuric acid.

~5. Van Ermengem's Method.~--This method, being merely a precipitation of a silver salt on the micro-organisms and not a true stain, creates a false impression as to the relative proportions of bacteria and flagella.

(a) _Fixing Fluid._--

Osmic acid, 2 per cent. aqueous solution 10 c.c.

Tannic acid, 20 per cent. aqueous solution 20 c.c.

Acetic acid, glacial 1 c.c.

The fixing fluid should be prepared some days before use and filtered as required. In colour it should be distinctly violet.

(b) _Sensitising Solution._--

Silver nitrate, 0.5 per cent. aqueous solution.

This solution must be kept in a dark blue gla.s.s bottle or in a dark cupboard.

Filter immediately before use.

(c) _Reducing Solution._--

Weigh out

Gallic acid 5 grammes Tannic acid 3 grammes Pota.s.sium acetate, fused 10 grammes