Microscopical Examination of Bacteria.
Bacteria in Liquids, Cultures, and Fresh Tissues.—In conducting bacteriological researches, the importance of absolute cleanliness cannot be too strongly insisted upon. All instruments, glass vessels, slides, and cover-glasses should be thoroughly cleansed before use. The same applies to the preparation and employment of culture media; any laxity in the processes of sterilisation, or insufficient attention to minute technical details, will be followed with disappointing results by contamination of the cultures, resulting in the loss of much time.
For the preparation of microscopical specimens it will be found convenient to use a platinum inoculating needle, sterilised, as before directed, in the sheet-iron box; in a few moments it will be cool enough not to destroy the bacteria with which it is brought into contact.
Unstained Bacteria.—The bacteria in liquids, such as blood and culture-fluids, can be investigated in the unstained condition by transferring a drop with a looped platinum needle, or a capillary pipette, to a slide, covering it with a clean cover-glass, and examining without further treatment. If it is desirable to keep the specimen under prolonged observation, a drop of sterilised water or salt solution must be run in at the margin of the cover-glass to counteract the tendency to dry.
Cultures on the solid media can be examined by transferring a small portion with a sterilised needle to a drop of sterilised water on a slide, thinning it out, and covering with cover-glass as already described. Tissues in the fresh state may be teased out with needles ([Fig. 249]) in sterilised salt solution, and pressed out into a sufficiently thin layer between the slide and cover-glass. Glycerine may in many cases be substituted for salt solution, especially for such as actinomyces and mould fungi.
Very small bacilli and micro-cocci are distinguished from granular matter or fat-crystals, or vice versâ, by the fact that the latter are altered or dispersed by the addition of acetic acid, and changed by solution of potash; ether dissolves out fatty particles, while micro-organisms remain unaffected. Baumgarten demonstrated tubercle bacilli in sections by treating them with potash, which clarified the tissues and brought the bacilli clearly into view. In examining unstained bacteria the iris-diaphragm should be used, and the sub-stage condenser carefully centred and focussed.
His’s Method of Staining.—A slide is prepared as for bacteria in the fresh state; the reagents are then applied by placing them with a pipette drop by drop at a margin of the cover-glass, and causing them to flow through the preparation by means of a strip of filter-paper placed at the opposite margin.
Babès’ Method is as follows: A little of the growth spread out on a cover-glass into as thin a film as possible; when almost dry, apply a drop or two of a weak aqueous solution of methyl-violet from a pipette to the film; any excess of the stain must be removed by gentle pressure with a strip of filter-paper.
Cover-glass Preparations.—A cover-glass is smeared with the substance to be examined spread out into a sufficiently thin layer; in the case of cultures on solid media, diffuse the bacteria in a little sterilised water. By means of another cover-glass the juice or fluid is squeezed out from between them into a thin layer, and on sliding them apart each cover-glass bears on it a thin film of the material. The cover-glass is then placed with its film side upwards and allowed to dry. After a few minutes it is passed from above downwards through the flame of a Bunsen burner three times. Apply two or three drops of an aqueous solution of fuchsine or methyl-violet to cover the film, wash away any surplus stain after a few minutes with distilled water. The cover-glass is then allowed to dry, when the preparation may be mounted in Canada balsam, or while still wet, turned over on a slide, and the excess of water removed with filter-paper.
If necessary to apply stain for a much larger period, pour staining solution into a watch glass and allow cover-glass to swim on surface with prepared side downwards.
Crookshank, instead of watery solutions of aniline dyes, prefers to use stronger solutions, and to reduce the staining by a momentary immersion in alcohol. The method is as follows: cover-glass preparations are stained with carbolised fuchsine (Neelsen’s solution) for about two minutes, rinsed in alcohol for a few seconds, and quickly washed in water. This method is specially valuable for sarcinæ and streptococci.
Gram’s Method.—The whole film is first stained violet with gentian-violet, fixed by a solution of iodine, in iodide of potassium in the bacilli, but not in any débris, pus cells, or tissue elements present. Transfer cover-glass to alcohol, the bacilli alone remain stained, the violet colour being changed to blue. By employing a contrast colour, such as eosin, a double staining is obtained.
For staining preparations with gentian-violet Crookshank employs the following useful method:—Place four or five drops of pure aniline in a test-tube, add distilled water to three-quarters full, close mouth with thumb, shake thoroughly. Filter the emulsion twice, pour filtrate into watch-glass. To the perfectly clear aniline water thus obtained, add, drop by drop, a concentrated alcoholic solution of gentian-violet till precipitation commences. Cover-glasses must be left in this solution ten minutes, transferred to iodine-potassic-iodide until the film becomes uniformly brown, then rinsed in alcohol. The decolourisation may be hastened by dipping the cover-glass in clove oil and returning to alcohol. Again immerse cover-glass in clove oil, dry by gently pressing between two layers of filter-paper, and mount in Canada balsam.
Double-staining of cover-glass preparations.—They can be treated by Ehrlich’s method for staining tubercular sputum, or by Neelsen’s modification, or by staining with eosin after treatment by the method of Gram.
Ehrlich’s Method is as follows: Five parts of aniline oil are shaken up with one hundred parts of distilled water, and the emulsion filtered through moistened filter-paper. A saturated alcoholic solution of fuchsine, methyl-violet, or gentian-violet, is added to filtrate in watch-glass, drop by drop, until precipitation commences. Cover-glass preparations are floated in this mixture for fifteen minutes to half an hour, then washed for a few seconds in dilute nitric acid (one part of nitric acid to two of water), then rinsed in distilled water.
Neelsen’s Solution and Methylene Blue.—Ziehl suggested the use of carbolic acid as a substitute for aniline blue. Neelsen recommended a solution of carbolic acid, absolute alcohol and fuchsine. (See Appendix.)
Gram’s Solution and Eosin.—After using Gram’s method as above and decolourising in alcohol, the cover-glass is placed in a weak solution of eosin for two or three minutes, washed in alcohol, immersed in clove oil, dried, and mounted in balsam.
Staining of Spores.—The cover-glass preparation must be heated to 210° C. for half an hour, or passed about twelve times through the flame of a Bunsen burner, or exposed to the action of strong sulphuric acid for several seconds, then a few drops of a watery solution of aniline dye applied in the usual way. To double-stain spore-bearing bacilli the cover-glass preparation must be floated from twenty minutes to an hour on Ehrlich’s fuchsine-aniline-water, or on the Ziehl-Neelsen solution. The stain must be heated until steam arises.