The Elements of Bacteriological Technique Part 19

4. Weigh out 0.15 gramme of the dried precipitate; rub up in a mortar with 5 c.c. of methylic alcohol (Merck's puriss, for a.n.a.lysis).

Allow undissolved powder to settle, then decant the supernatant fluid to a clean 100 c.c. measuring cylinder.

5. Add further 5 c.c. alcohol to sediment in mortar and repeat the process, and so on until all the sediment has been dissolved.

6. Now make up the fluid in the measuring cylinder to 100 c.c. by the addition of more methylic alcohol.

METHOD.--

1. Prepare film, dry, but _do not fix_.

2. Flood the unfixed film with stain, allow it to act 30 seconds.

3. Add double the volume of distilled water to the stain on the film, and mix with gla.s.s rod or platinum loop.

4. Allow this diluted stain to act five minutes.

5. Wash off with distilled water.

6. Leave some water on film for thirty seconds to intensify the colour contrasts.

7. Dry and mount.

METHODS OF DEMONSTRATING STRUCTURE OF BACTERIA, ETC.

~To Demonstrate Capsules.~

~1. MacConkey.~--

_Stain._--

Weigh out

Dahlia 0.5 gramme Methyl green (00 crystals) 1.5 grammes

rub up in a mortar with

Distilled water 100.0 c.c.

Add

Fuchsin, saturated alcoholic solution 10.0 c.c.

and make up to 200 c.c. by the addition of

Distilled water 90.0 c.c.

Filter.

Allow the stain to stand for two weeks before use; keep in a dark place or in an amber gla.s.s bottle. Owing to the unstable character of the methyl green, this stain deteriorates after about six months.

METHOD.--

1. Prepare and fix film in the usual manner.

2. Flood the cover-slip with the stain and allow it to act for five to ten minutes.

3. Wash very thoroughly in water; if necessary, direct a powerful stream of water on the film from a wash-bottle.

4. Dry and mount.

~2. Muir's Method.~--

1. Prepare, dry and fix film in the ordinary manner.

2. Flood the film with carbolic fuchsin, warm until steam begins to rise. Allow the stain to act for thirty seconds.

3. Wash quickly with methylated spirit.

4. Wash thoroughly with water.

5. Subject the film to the action of the following mordant for five seconds:

Corrosive sublimate, saturated aqueous solution 2 c.c.

Tannic acid, 20 per cent. aqueous solution 2 c.c.

Potash alum saturated aqueous solution 5 c.c.

6. Wash thoroughly in water.

7. Treat with methylated spirit for about sixty seconds.

(The preparation should now be pale red.)

8. Wash thoroughly in water.

9. Counterstain in methylene blue, aqueous solution thirty seconds.

10. Wash in water.

11. Dehydrate in alcohol.

12. Clear in xylol and mount in xylol balsam.

~3. Welch's Method.~--

1. Prepare and fix film in the usual manner.

2. Flood the slide with acetic acid 2 per cent.; allow the acid to remain in contact with the film for two minutes.