Although the greater part of the examination of microorganisms is done with stained specimens, yet they should always be examined in the unstained and living condition as well. The best way to do this is by means of the hanging drop. In this method a slide with a concavity is used. Around this depression a ring of vaselin is made. A drop of the material to be examined is placed in the center of a clean cover-glass, which is then inverted over the depression in the slide and is pressed down upon the vaselin. Care should be taken that the drop is not large enough to touch the slide. The edge of the drop should be examined, as the central portion is too thick. By this method can be determined the shape, size, grouping, division, sporulation, and motility of the organism.

Staining Bacteria. Staining Cover-Glass Preparations

A well-cleaned cover-glass or slide has a small portion of the material for examination spread out on it in a very thin layer by means of a sterilized platinum wire. The preparation is allowed to dry; it is best not to do it over a flame. When dry the cover-glass is passed rather slowly three times through the flame of a Bunsen burner. This coagulates the albumin and prevents the material being washed off during the process of staining. The cover-glass is covered with the stain and gently warmed for fifteen to twenty seconds over a small flame. The specimen is then washed in water, dried by blotting and by gently warming, and mounted in balsam.

Various of the anilin colors are the ones chiefly used in bacterial staining. They may be used alone or in combination with certain reagents employed to increase the staining power.

Saturated alcoholic solutions of the stains should be kept in stock, and from them the dilute aqueous solutions can be prepared. These latter, however, do not keep well, so various standard preparations are usually kept on hand.

Ldffler's Methylene-Blue

Sat. alc. sol. methylene-blue................ 30 c.c.

Caustic potash in water (1:10,000).......... 100 "

This keeps a long time and stains rapidly.

Neisser's Stain For Diphtheria

Solution No. 1

Methylene-blue.......................... 0.1 gm.

Alcohol................................. 2.0 cc.

Glacial acetic acid........................ 5.0 "

Distilled water........................... 95.0 "

Dissolve the methylene-blue in the alcohol and add it to the acetic-acid-water mixture. Filter.

Solution No. 2

Bismarck brown........................ 0.2 gm.

Water (boiling)......................... 100.0 cc.

Dissolve the stain in the boiling water. Filter. To stain: Fix the preparation. Pour on the acetic-acid-methylene-blue solution and allow to act from thirty to sixty seconds. Wash. Then pour on the Bismarck-brown solution, and after thirty seconds wash off with water. Dry and mount. The bodies of the bacilli are brown, with dark blue spots at either end. Best results are obtained with cultures from nine to eighteen hours old.

Carbol-Thionin

Sat. sol. thionin in 50 per cent, alcohol....... 10 cc.

Carbolic acid aq. sol. (2 per cent.)........... 100 "

Carbolfuchsin

Sat. ale sol. fuchsin........................ 10 cc.

Watery sol. carbolic acid (5 per cent.)........ 90 "

This stain is very permanent and is useful for many purposes. It is employed in the differential diagnosis of tubercle bacilli.

The following method, that of Ziehl-Nielson, is commonly employed for staining tubercle bacilli, particularly in sputum. After having made, dried, and fixed the smear, the cover-glass or slide is covered with carbolfuchsin and carefully heated, until steam rises, for some three or more minutes. Care must be taken not to boil the stain and to replace the solution as it evaporates. Then wash the smear thoroughly in water and decolorize with about a 10 to 15 per cent, solution of nitric acid in 95 per cent, alcohol. Will take about thirty seconds to a minute. Wash again in water and counterstain in Löfner's methylene-blue. Wash and examine. The tubercle bacilli will appear as minute red rods; all other organisms and cells will be blue.

With Gram's method the bacillus retains the stain.

Anilin Gentian-Violet

Sat. ale. sol. gentian-violet................... 16 c.c.

Anilin water............................... 84 "

Anilin water is made by taking:

Anilin oil.................................. 5 c.c.

Distilled water............................. 95 "

Shake thoroughly until a milky fluid is obtained; then filter. This stain should be freshly prepared when needed, as it does not last more than ten days.

Carbol Gentian-Violet

Sat. ale. sol. gentian-violet.................. 1 c.c.

Aq. sol. carbolic acid (5 per cent.)............ 10 "

This solution is permanent, but tends to overstain.

Gram's Method

After the cover-glass has been smeared and fixed it is stained in:

1. Anilin or carbol gentian-violet thirty seconds.

2. Washed in water two or three seconds.

3. Put in Gram's solution, as follows, for thirty seconds:

Iodin.................................... 1 gm.

Potassium iodid........................... 2 c.c.

Water................................... 300 "

4. Washed in 95 per cent, alcohol until the color ceases to come out of the preparation.

5. Dry by blotting and in air and mount in balsam. The value of this method lies in the fact that certain bacteria will retain the stain, while others give it up. In those organisms retaining the stain there has been a combination of mycoprotein, anilin dye, and the iodids that forms a compound insoluble in alcohol. The bacteria stain dark blue or black while the nuclei are only faintly colored. Nuclei that are undergoing division may stain rather deeply.

An organism is said to stain by Gram's method when it is not decolorized. This power is made use of to differentiate certain organisms that may resemble each other in size and shape.

The more important pathogenic bacteria are divided as follows, according to their reaction to Gram's:

Stained by Gram's Method.

Staphylococcus pyogenes.

Streptococcus pyogenes.

Streptococcus capsulatus.

Actinomycosis.

Bacillus anthracis.

Pneumococcus.

Bacillus diphtheriae.

Bacillus leprae.

Bacillus tuberculosis.

Bacillus tetanus.

Bacillus aërogenes capsulatus.

Decolorized by Gram's Method.

Gonococcus. Bacillus typhosus. Bacillus coli communis. Bacillus of malignant edema. Spirillum of Asiatic cholera. Diplococcus intracellularis meningitidis. Bacillus pyocyaneus. Bacillus of influenza. Bacillus of dysentery. Bacillus of bubonic plague. Bacillus of glanders. Spirochaeta of relapsing fever.