In the following pages are collected the various "stock" stains in everyday use in the bacteriological laboratory, together with a selection of the most convenient and generally useful staining methods for demonstrating particular structures or differentiating groups of bacteria. The stains employed should either be those prepared by Gruebler, of Leipzig, or Merck, of Darmstadt. The methods printed in ordinary type are those which a long experience has shown to be the most reliable, and to give the best results—those relegated to small type comprise such as are not so generally useful, but give excellent results in the hands of the experienced worker.

BACTERIA STAINS.

Methylene-blue.—

1. Saturated Aqueous Solution.

Weigh out

Place in a stoppered bottle having a capacity of from 150 to 200 c.c. and add

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.

2. Saturated Alcoholic Solution.

Weigh out

Place in a stoppered bottle of 150 c.c. capacity and add

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

3. Carbolic Methylene-blue (Kuehne).

Weigh out

and dissolve in

and add

Filter.

4. Alkaline Methylene-blue (Loeffler).

Measure out and mix

Filter.

Gentian Violet.—

5. Saturated Aqueous Solution.

Weigh out

and proceed as in preparing the corresponding solution of methylene-blue.

6. Saturated Alcoholic Solution.

Weigh out

and proceed as in preparing the corresponding solution of methylene-blue.

7. Carbolic Gentian Violet (NicollÉ).

Measure out and mix

Filter.

8. Anilin Water Solution (Koch-Ehrlich).

Measure out

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

Filter until clear.

and add

Filter.

Thionine Blue (or Lauth's Violet).—

9. Carbolic Thionine Blue (NicollÉ).

Weigh out

and dissolve in

Filter.

Before use dilute with equal quantity of distilled water and again filter.

Fuchsin (Basic).—

10. Saturated Aqueous Solution.

Weigh out

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

11. Saturated Alcoholic Solution.

Weigh out

and proceed as in preparing the corresponding solution of methylene-blue.

12. Carbolic Fuchsin (Ziehl).

Weigh out

dissolve in

and add

Filter.

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:

13. Eosin Aqueous Solution (Yellowish or Bluish Shade), 1 per cent.

Weigh out

dissolve in

and add

Filter.

14. Eosin Alcoholic Solution, 0.5 per cent.

Weigh out

and dissolve in

Filter.

Safranine.—

15. Aqueous Solution.

Weigh out.

and dissolve in

Filter.

Neutral Red.—

16. Aqueous Solution.

Weigh out

and dissolve in

Filter.

Vesuvin (or Bismarck Brown).—

17. Saturated Aqueous Solution.

Weigh out

and dissolve in

Filter.

TISSUE STAINS.

Aniline Gentian Violet (For Weigert's Fibrin Stain).—

Weigh out

and dissolve in

then add

Shake well and filter before use.

HÆmatoxylin (Ehrlich).—

1. Weigh out

and dissolve in

2. Weigh out

and dissolve in

3. Mix 1 and 2, allow the mixture to stand forty-eight hours, then filter.

4. Add

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

HÆmatin (Mayer's).—

A. Weigh out

and dissolve in

B. Weigh out

and dissolve in

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.

Alum Carmine (Mayer).—

Weigh out

and place in a glass beaker.

Measure out in a measuring cylinder,

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.

Lithium Carmine (Orth).—

Weigh out

and dissolve in

Filter.

Picrocarmine.—

Weigh out

and dissolve in

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.

Jenner's Stain.—

A. Weigh out:

Dissolve in

This will make a thick solution.

B. Weigh out:

Dissolve in

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.)

3. Grind the resulting mass into an amorphous powder.

4. Weigh out:

Dissolve in

Allow time for true solution. (About three days is sufficient.)

Method.—

1. Prepare film, dry, but do not fix.

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.

4. Dry and mount.

Leishman's Stain.—

A. Weigh out:

Dissolve in

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.

B. Weigh out:

Dissolve in

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

2. Filter, and collect precipitate on filter paper.

3. Wash precipitate thoroughly with distilled water, and dry.

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.

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 glass 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

rub up in a mortar with

Add

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

Filter.

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.

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:

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. This swells up and fixes the capsule and enables it to take the stain.

3. Blow off the acetic acid by the aid of a pipette.

4. Immerse in aniline gentian violet, five to thirty seconds.

5. Wash in water.

6. Dry and mount.

4. Ribbert's Method.—

Stain.—

Measure out and mix:

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

Method.—

1. Prepare and fix films in the usual manner.

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

3. Wash thoroughly in water.

4. Dry and mount.

To Demonstrate Flagella.

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.

(a) Mordant.—

Mix thoroughly.

A precipitate forms which must be allowed to settle for a few hours.

Decant off the clear fluid into tubes and centrifugalise thoroughly.

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.

(b) Stain.—

Filter.

This stain must be freshly prepared.

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.

1. Remove a very small quantity of the growth by means of the platinum spatula.

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.

4. Allow the film to dry in the air, properly protected from falling dust.

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.

7. Wash well in water and dry carefully.

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.

9. Wash well in water.

10. Dry and mount.

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

Alternative Mordant (Bunge's Mordant).—

(b) Stain.—

Weigh out

and dissolve in

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 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.

(a) Fixing Fluid.—

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 glass bottle or in a dark cupboard.

Filter immediately before use.

(c) Reducing Solution.—

Weigh out

and dissolve in

Filter.

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

Method.—

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.

3. Wash well in water.

4. Wash in absolute alcohol.

5. Wash in distilled water.

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.

9. Wash in distilled water.

10. Dry and mount.

To Stain Nuclei of Yeast Cells.

1. Prepare and fix film in the usual manner.

2. Soak in ferric ammonia sulphate 3 per cent. aqueous solution for two hours.

3. Wash thoroughly in water.

4. Stain in hÆmatoxylin solution (see page 95) for thirty minutes.

5. Wash in water.

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

To Stain Spores.

1. Single Stain.—

1. Prepare cover-slip film in the usual way.

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.

3. Stain in the usual way with methylene-blue or fuchsin.

4. Wash in water.

5. Dry and mount.

2. Double Stain.—

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.)

3. Wash in water.

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

5. Wash in water.

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.

7. Counterstain in weak methylene-blue. (Now spores red, bacilli blue.)

8. Wash in water.

9. Dry and mount.

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.

DIFFERENTIAL METHODS OF STAINING.

Gram's Method.—This method depends upon the fact that the protoplasm of some bacteria permits aniline gentian violet and Lugol's iodine solution, when applied consecutively, to enter into a chemical combination which results in the formation of a new blue-black pigment, only very sparingly soluble in absolute alcohol. Such organisms are said to "stain by Gram," or to be "Gram positive."

1. Prepare a cover-slip film and fix in the usual way.

2. Stain in aniline gentian violet three to five minutes. Filter as much aniline water on to the cover-slip as it will hold; then add the smallest quantity of alcoholic solution of gentian violet which suffices to saturate the aniline water and form a "bronze scum" upon its surface—if too much of the alcoholic gentian violet is added the alcohol present redissolves this scum.

3. Wash in water.

4. Treat with Lugol's iodine solution until the film is black or dark brown.

To do this treat with iodine solution for a few seconds, wash in water, and examine the film over a piece of white filter paper. Note the colour. Repeat this process until the film ceases to darken with the fresh application of iodine solution.

Lugol's solution is prepared by dissolving

5. Wash in water.

6. Wash with alcohol until no more colour is discharged and the alcohol runs away clear and colourless.

The following mixture may be substituted for absolute alcohol as a decolouriser

7. Wash in water.

8. Counterstain very lightly with aqueous solution of Neutral Red. Other counterstains may be used such as dilute eosin, dilute fuchsin, or vesuvin.

9. Wash in water.

10. Dry and mount.

Gram-Claudius Method.—

1. Prepare a cover-slip film and fix in the usual way.

2. Stain in methyl violet, 1 per cent. aqueous solution for three to five minutes.

3. Treat with two lots picric acid, saturated aqueous solution.

4. Wash in water and dry.

5. Decolourise with clove oil.

6. Wash off clove oil with xylol.

7. Mount in xylol balsam.

Gram-Weigert Method.—

1-5. Proceed as for the corresponding sections of Gram's method (quod vide).

6. Dry in the air.

7. Wash in aniline oil, 1 part, xylol, 2 parts, until no more colour is discharged.

8. Wash in xylol.

9. Mount in xylol balsam.

Modified Gram-Weigert Method.—(To demonstrate trichophyta in hair.)

1. Soak the hairs in ether for ten minutes to remove the fat.

2. Stain thirty minutes in a tar-like solution of aniline gentian violet (prepared by adding 15 drops of the alcoholic solution of gentian violet to 3 drops of aniline water).

3. Dry the hairs between pieces of blotting paper.

4. Treat with perfectly fresh iodine solution.

5. Again dry between blotting paper.

6. Treat with aniline oil to remove excess of stain. (If necessary, add a drop or two of nitric acid to the oil.)

7. Again treat with aniline oil.

8. Treat with aniline oil and xylol, equal parts.

9. Clear with xylol.

10. Mount in xylol balsam.

To obtain the best differentiation the preparation should be repeatedly examined microscopically (with a 1/6-inch objective) between steps 5 and 9, as the actual time involved varies with different specimens.

Ziehl-Neelsen's Method.—(To demonstrate tubercle and other acid-fast bacilli.)

1. Smear a thin, even film of the specimen on the cover-slip by means of the platinum loop. (In the case of sputum, if it is a very watery specimen, allow the film to dry, then spread a second and even a third layer over the first.)

2. Fix by passing three times through the flame.

3. Stain in hot carbol-fuchsin (as in staining for spores) for five to ten minutes. (This stains everything on the film.) Avoid over-heating.

4. Decolourise by dipping in sulphuric acid, 25 per cent. (This removes stain from everything but acid-fast bacilli; e. g., tubercle, leprosy, and smegma bacilli and the film turns yellow.)

5. Wash in water. (A pale red colour returns to the film).

6. Wash in alcohol till no more colour is discharged. (This often, but not invariably, removes the stain from acid-fast bacilli other than tubercle; e. g., smegma bacillus.)

7. Wash in water.

8. Counterstain in weak methylene-blue. (Stains non-acid-fast bacilli, leucocytes, epithelial cells, etc.)

9. Wash in water, dry, and mount.

Pappenheim's Method.—

This method is supposed to differentiate between B. tuberculosis and other acid-fast micro-organisms.

1. Prepare and fix film in the usual way.

2. Stain in carbol-fuchsin without heat for three minutes.

3. Without previously washing in water treat the film with three or four successive applications of corallin (Rosolic acid) solution.

4. Wash in water.

5. Dry and mount.

Neisser's Method—Modified.—(To demonstrate diphtheroid bacilli.)

Stain I.—

Measure out and mix

Filter.

Stain II.—

Weigh out

and dissolve in

Filter.

Method.—

1. Prepare and fix films in the usual way.

2. Pour stain I on the film and allow it to act for two minutes.

3. Wash thoroughly in water.

4. Treat with Lugol's iodine for ten seconds.

5. Wash thoroughly in water.

6. Pour stain II on to the film and allow it to act for thirty seconds.

7. Wash thoroughly in water.

8. Dry and mount.

The bacilli are stained a light red by the neutral red, which contrasts well with the two or three black spots, situated at the poles and occasionally one in the centre representing protoplasmic aggregations (? metachromatic granules) stained by the acid methylene-blue.

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