The Project Gutenberg eBook of The Elements of Bacteriological Technique, by John William Henry Eyre

Allow the water to remain in contact with the dye for two weeks, shaking the contents of the bottle vigourously for a few moments every day. Filter.

Allow the alcohol to remain in contact with the dye for two hours, shaking vigourously every few minutes. Filter.

Add anilin oil drop by drop (shaking well after the addition of each drop) until the solution is opaque.

Note.—This solution will not keep longer than 14 days.

and proceed as in preparing the corresponding solution of methylene-blue (q. v.).

CONTRAST STAINS.

Eosin.—There are several commercial varieties of eosin, which, from the bacteriological point of view, possess very different values. Gruebler lists four varieties, of which two only are useful for bacteriological work:

TISSUE STAINS.

5. Allow the stain to stand for one month exposed to light; then filter again ready for use.

Prepare these two solutions in separate flasks. Take a clean flask of 250 c.c. capacity and insert a large funnel in its neck. Pour the solutions A and B simultaneously and slowly into the funnel to mix thoroughly. Store for future use.

Note.—If acid hæmatin is required, introduce glacial acetic acid (3 c.c.) into the mixing flask before adding the solutions A and B.

Place the beaker on a sand-bath, add the water in successive small quantities, and keep the mixture boiling for twenty minutes. Measure the solution and make up to 100 c.c. by the addition of distilled water. Filter.

BLOOD STAINS

When watery solutions of medicinal methylene blue and water soluble eosins are mixed a precipitate is formed which is soluble only in alcohol, and solutions of this precipitate impart a peculiar reddish-purple colour to chromatin. This compound was first used by Romanowsky to demonstrate malarial parasites, but various modifications are now employed for staining blood films generally, and also for bacteria and protozoa. The best modifications of the original Romanowsky are those of Jenner and Leishman—Jenner being most suitable for the histological study of the blood, and Leishman for the demonstration of protozoa.

1. Add B to A very slowly, stirring all the time. A viscous precipitate forms which frequently loses its viscosity when heat is applied. (This explains the necessity of mixing slowly).

2. Evaporate slowly in a porcelain basin, stirring occasionally, on a water bath at 55° C. When a paste begins to form scrape and break up occasionally. (On no account must the paste be allowed to fuse.)

2. Flood the unfixed film with the stain, allow it to act for 3 minutes (the methylic alcohol of the stain fixes the film).

3. Pour off the stain and wash in distilled water until the film presents a pink colour.

Keep at 65° C. for 12 hours in either a hot incubator or a water-bath; then stand in dark place at room temperature (20°C.) for ten days.

1. Mix the two solutions A and B in equal volumes, and allow the mixture to stand for 12 hours with occasional stirring.

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 analysis).

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.

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

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

METHODS OF DEMONSTRATING STRUCTURE OF BACTERIA, ETC.

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

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.

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

7. Treat with methylated spirit for about sixty seconds. (The preparation should now be pale red.)

2. Flood the slide with acetic acid 2 per cent.; allow the acid to remain in contact with the film for two minutes. This swells up and fixes the capsule and enables it to take the stain.

Warm to 36° C. (e. g., in the "hot" incubator) and saturate with dahlia. Filter.

2. Cover the film with the stain and allow it to act for one or two seconds only.

1. Muir's Modified Pitfield.—This is the best method and gives the most reliable results, for not only is the percentage of successful preparations higher than with any other, but the bacilli and flagella retain their relative proportions.

This solution is at its best some four or five days after manufacture; it keeps for about a couple of weeks, but must be re-centrifugalised each time, before use.

Method.—The cultivations employed should be smear agar cultures, twelve to eighteen hours old if incubated at 37°C, twenty-four to thirty hours if incubated at 22°C.

2. Emulsify it with a few cubic centimetres of distilled water in a watch-glass, by gently moving the spatula to and fro in the water. Do not rub up the growth on the side of the watch-glass. Some workers prefer to use tap water, others employ normal saline solution, but distilled water gives the best emulsion.

3. Spread a thin film of the emulsion on a newly flamed cover-slip, using no force, but rather leading the drop over the cover-slip with the platinum loop.

5. Fix by passing thrice through the Bunsen flame, holding the cover-slip whilst doing so by one corner between the finger and thumb.

6. Pour on the film as much of the mordant as the cover-glass will hold. Grasp the cover-slip with the forceps and hold it, high above the flame, until steam rises. Allow the steaming mordant to remain in contact with the film two minutes.

8. Pour on the film as much of the stain as the cover-glass will hold. Steam over the flame as before for two minutes.

2. "Pitfield" Original Method.—

(a) Mordant.—

Tannic acid	1 gramme
Water	10 c.c.

(b) Stain.—

Saturated aqueous solution of alum	10 c.c.
Saturated alcoholic solution of gentian violet	1 c.c.
Distilled water	5 c.c.

Mix equal parts of a and b before using.

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.
Hæmatoxylin solution	3 c.c.
Carbolic acid, 1 per cent. aqueous solution	4 c.c.

This solution must be freshly prepared.

Hæmatoxylin solution is prepared by boiling 1 gramme logwood

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.

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 quantity 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.

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

This solution will keep active for several days, but fresh solution must be used for each preparation.

1. Prepare emulsion, make and fix films as above in the preceding method, steps 1 to 4.

2. Pour on the film as much of the fixing solution as the cover-glass will hold, heat carefully over the flame till steam rises, and allow the steaming fixing fluid to act for five minutes.

6. Pour some of the sensitising solution on the film and allow it to act for from thirty seconds to one minute; blot off the excess of fluid with filter paper.

7. Without washing, transfer the film to a watch-glass containing the reducing solution and allow it to remain therein for from thirty seconds to one minute; blot off the excess of fluid with filter paper.

8. Without washing, again treat the film with the sensitising solution, this time until the film commences to turn black.

6. Differentiate in ferric ammonia sulphate solution for 1-1/2-2 minutes, examining wet under microscope during the process.

2. In fixing, pass the cover-slip film fifteen or thirty times through the flame instead of only three. This destroys the resisting power of the spore membrane and allows the stain to reach the interior.

1. Prepare and fix film in the usual way—i. e., pass three times through flame to fix.

2. Cover the film with hot carbol-fuchsin and hold in the forceps above a small flame until the fluid begins to steam. Set the cover-slip down and allow it to cool. Repeat the process when the stain ceases to steam and continue to repeat until the stain has been in contact with the film for twenty minutes. (This stains both spores and bacteria.)

4. Decolourise in alcohol, 2 parts; acetic acid, 1 per cent., 1 part. (This removes the stain from everything but the spores.)

6. Mount the cover-slip in water and examine microscopically with the 1/6-inch objective. (Spores should be red, and the rest of the film colourless or a very light pink.) If satisfactory, pass on to section 7; if unsatisfactory, repeat steps 2 to 5.

The spores of different bacilli differ greatly in their resistance to decolourising reagents; even the spores of the same species of organisms vary according to their age. Young spores are more easily decolourised than those more mature.

Sulphuric acid, 1 per cent. aqueous solution, and hydrochloric acid, 0.5 per cent. alcoholic (90 per cent.) solution, are useful decolourising reagents.

Methylene-blue	1.5 grammes
Carbolic acid	5.0 grammes

Methylene-blue, saturated alcoholic solution	30.0 c.c.
Caustic potash, 0.1 per cent. aqueous solution	100.0 c.c.

Gentian violet, saturated alcoholic solution	10.0 c.c.
Carbolic acid, 1 per cent. aqueous solution	100.0 c.c.

Absolute alcohol	10 c.c.
Saturated alcoholic solution gentian violet	11 c.c.

Thionine blue	1.0 gramme
Carbolic acid	2.5 grammes

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.

Acetic acid, glacial	12.5 c.c.
Alcohol, absolute	50.0 c.c.
Distilled water	100.0 c.c.

Tannic acid, 10 per cent. aqueous solution	10 c.c.
Corrosive sublimate, saturated aqueous solution	5 c.c.
Alum, saturated aqueous solution	5 c.c.
Carbolic fuchsin (Ziehl)	5 c.c.

Alum, saturated aqueous solution	25 c.c.
Gentian violet, saturated alcoholic solution	5 c.c.

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.

Gallic acid	5 grammes
Tannic acid	3 grammes
Potassium acetate, fused	10 grammes