Bacteria: APPENDIX

Bacteria by George Newman is part of the HackerNoon Books Series. You can jump to any chapter in this book here. APPENDIX

It is proposed to add one or two notes on certain technical points in bacteriological work, with a view to assisting those medical men not able to obtain the advantages of a well-equipped laboratory, and yet desirous of occasionally attempting some practical bacteriology.

1. General Examination. All fluids may be examined for bacteria in two chief ways:

(a) A small quantity may be placed on a cover-glass or slide, dried over a lamp or bunsen flame, and stained with aniline dyes for a few minutes. It is then ready for microscopic examination. It is obvious that the result will generally be a mixture of bacteria, for which differentiating stains may be used (Gram, Ziehl-Neelsen, etc.).

(b) A minute drop of the suspected fluid may be added to various fluid media (broth, liquefied gelatine, etc.) and then plated out upon small sterilised sheets of glass. In the course of two or three days the contained bacteria will reveal themselves in characteristic colonies, which may be examined, and if possible sub-cultured, and carefully studied.

Double-Staining Methods. These are various, and are used when it is desired to stain the bacteria themselves one colour, and the matrix or ground substance in which they are situated another colour. Three of the commoner methods are those of Ehrlich, Neelsen, and Gram. They are as follows:

Ehrlich's Method. "Five parts of aniline oil are shaken up with 100 parts distilled water, and the emulsion filtered through moistened filter paper. A saturated alcoholic solution of fuchsine, methyl-violet, or gentian-violet is added to the filtrate in a watch-glass,338 drop by drop, until precipitation commences. Cover-glass preparations are floated in this mixture for fifteen to thirty minutes, then washed for a few seconds in dilute nitric acid (one part nitric acid to two of water), and then rinsed in distilled water. The stain is removed from everything except the bacilli; but the ground substance can be after-stained brown if the bacilli are violet, or blue if they have been stained red" (Crookshank, Bacteriology and Infective Diseases, p. 89).

Gram's Method. The primary stain in this method is a solution of aniline gentian-violet (saturated alcoholic solution of gentian-violet 30 cc., aniline water 100 cc.), which stains both ground substance and bacteria in purple. The preparation is next immersed in the following solution for half a minute or a little more:

In this short space of time the iodine solution acts as a mordant of the purple colour in the bacteria, but not in the ground substance. Hence, if the preparation be now (when it has assumed a brown colour) washed in alcohol (methylated spirit), the ground substance slowly loses its colour and becomes clear. But the bacteria retain their colour, and thus stand out in a well-defined manner. Cover-glass preparations decolourise more quickly than sections of hardened tissue, and they should only be left in the methylated spirit until no more colour comes away.

The preparation may now be washed in water, dried, and mounted for microscopic examination, or it may be double-stained, that is, immersed in some contrast colour which will lightly stain the ground substance. Eosin or Bismarck brown are commonly used for this purpose. The former is applied for a minute or two, the latter for five minutes, after which the specimen is passed through methylated spirit (and preferably xylol also) and mounted. The result is that the bacteria appear in a dark purple colour on a background of faint pink or brown. Carbol-thionine blue, picro-carmine, and other stains are occasionally used in place of the aniline gentian-violet, and there are other slight modifications of the method.

Ziehl-Neelsen Method. Here the primary stain is a solution of carbol-fuchsin:

It is best to heat the dye in a sand-bath, in order to distribute the heat evenly. The various stages in the staining process are as follows: (a) The cover-glass with the dried film upon it is immersed in the hot stain for one to three minutes.

(b) Remove the cover-glass from the carbol-fuchsin, and place it in a capsule containing a 25 per cent. solution of sulphuric acid to decolourise it. Here its redness is changed into a slate-grey colour.

(c) Wash in water, and alternately in the acid and water, until it is of a faint pink colour.

(d) Now place the cover-glass for a minute or two in a saturated aqueous solution of methylene-blue, which will counter-stain the decolourised ground substance blue.

(e) Wash in water.

(f) Dehydrate by rinsing in methylated spirit, dry, and mount. A pure culture of bacteria will not necessarily require the counter-stain (methylene-blue). Sections of tissue may require twenty to thirty minutes in the primary stain (carbol-fuchsin). This stain is used for tubercle and leprosy. With a little practice the staining of the bacillus of tubercle when present in pus or sputum becomes a very simple and accurate method of diagnosis. A small particle of sputum or pus is placed between two clean cover-glasses and thus pressed between the thumb and finger into a thin film. This is readily dried and stained as above, the bacillus of tubercle appearing as a delicately-beaded red rod with a background of blue.

Bacteriological Diagnosis. The following points must be ascertained in order to identify any particular micro-organism:

(1) Its morphology, bacillus, coccus, spirillum, etc.; the presence or absence of involution forms.

(2) Motility by the unstained cover-glass preparation ("hanging drop"); note presence of flagella.

(3) Presence of spores, their appearance and position.

(4) Whether or not the organism stains with Gram's method.

(5) The character of the growth upon various media (gelatine, agar, milk, potato, broth); the presence or absence of liquefaction in the gelatine culture; its power of producing acid, gas, or indol.

(6) Whether it is aërobic or anaërobic.

(7) Its colour in cultivation.

(8) If it is a disease-producing organism under examination, its effect upon the animal tissues and the course of the disease should be observed.

There are other points of importance, but the above are essential to a right conclusion.

(1) Diphtheria. This disease may be bacteriologically diagnosed with a minimum of apparatus and equipment. By means of a swab a rubbing from a suspected throat is readily obtained. This may be examined by the microscope, or sub-cultured on favourable medium. Blood serum is perhaps the best, but, as Hewlett remarks, "If no serum tubes can be had, an egg may be used. It is boiled hard, the shell chipped away from one end with a knife sterilised by heating, and the inoculation made on the exposed white surface; the egg is then placed, inoculated end down, in a wine-glass of such a size that it rests on the rim and does not touch the bottom.

A few drops of water may with advantage be put at the bottom of the glass to keep the egg-white moist. The preparation is kept in a warm place for twenty-four to forty-eight hours and then examined." The examination, of course, consists in staining and preparing for the microscope and observing the form, arrangement, and characters of the organism or organisms present. A small piece of the membrane may be detached, washed in water, and stained for the bacilli.

(2) Tubercle (Ziehl-Neelsen's stain, vide supra).

(3) Typhoid (Enteric Fever).

Widal's Reaction. This diagnostic test depends upon the effect which the blood of a person suffering from typhoid fever has upon the Bacillus typhosus. The effect is twofold. In the first place, the actively motile B. typhosus becomes immotile; and secondly, there is an agglutination, or grouping together in colonies, of the B. typhosus. Neither of these features occurs if healthy human blood is brought into contact with a culture of the typhoid bacillus. There are various ways in which this "serum diagnosis" can be carried out.

The simplest and quickest method is as follows: To ten drops of a twenty-four or forty-eight-hours-old neutral broth culture of the typhoid bacillus one drop of the blood serum to be tested is added. The serum and culture are rapidly mixed in the trough of a hollow ground slide (such as is used for the "hanging drop"), and a single drop is taken, placed upon an ordinary clean slide, and a cover-glass superimposed. The positive reaction of agglutination and immotility, if the blood comes from a case of typhoid fever, will probably appear within fifteen or twenty minutes. The fluid culture of typhoid may be taken from an agar culture as well as from broth. In both cases it may be desirable to filter through ordinary filter paper to remove any normally agglutinated masses of bacilli before commencing the test.

In his first experiments Widal used a test-tube in the following manner: The blood to be tested is diluted by one part of it being added to fifteen parts of broth in a test-tube. The mixture is inoculated with a drop of a typical Bacillus typhosus culture. The tube is then incubated at 37° C. for twenty-four hours, after which it is examined. If the reaction be positive, the broth appears comparatively clear, but at the bottom of the test-tube a more or less abundant sediment will be found. This is due to the clumps of bacilli having fallen owing to gravity. If, on the other hand, the reaction is negative, the broth will appear more or less uniformly turbid.

For the apparatus required to carry out the simpler methods of bacteriological work reference should be made to the standard laboratory text-books, which furnish all necessary details. A good microscope, with a 1/12 oil immersion lens, is, of course, essential. This can now be obtained for about £16 (Beck, Swift, Baker, Watson, etc.), and the other necessary apparatus is readily obtainable of Baird and Tatlock, Hatton Garden, E. C., and other makers.