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 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:

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. 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:

Acetic acid, glacial 12.5 c.c.

Alcohol, absolute 50.0 c.c.

Distilled water 100.0 c.c.

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.—

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.

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.—

Alum, saturated aqueous solution 25 c.c.

Gentian violet, saturated alcoholic solution 5 c.c.

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.

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

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 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.—

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 glass bottle or in a dark cupboard.

Filter immediately before use.

(c) Reducing Solution.—

Weigh out

Gallic acid 5 grammes

Tannic acid 3 grammes

Potassium acetate, fused 10 grammes

and dissolve in

Distilled water 350 c.c.

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.

3. Moeller's Method.—

1. Prepare and fix films in the usual manner.

2. Immerse in absolute alcohol for two minutes, then in chloroform for two minutes; wash in water. This dissolves out any fat or crystals that might otherwise retain the "spore" stain.

3. Immerse in chromic acid, 5 per cent. aqueous solution, for one minute; wash in water.

4. Pour Ziehl's carbolic fuchsin on the film, warm as in previous methods, and allow it to act for ten minutes.

5. Wash in water.

6. Decolourise in sulphuric acid, 5 per cent. aqueous solution, for five seconds.

7. Wash in water.

8. Counterstain with Kuehne's carbolic methylene-blue for one or two minutes.

9. Wash in water.

10. Dry and mount.

(Spores red, bacilli blue.)

4. Abbott's Method.—

1. Prepare and fix films in the usual manner.

2. Pour Loeffler's alkaline methylene-blue on the film; warm cautiously over the flame till steam rises and allow the hot steam to act for one to five minutes.

3. Wash thoroughly in water.

4. Decolourise in nitric acid, 2 per cent. alcoholic (alcohol 80 per cent.) solution.

5. Wash thoroughly in water.

6. Counterstain in eosin, 1 per cent. aqueous solution.

7. Wash.

8. Dry and mount.

(Spores blue, bacilli red.)