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CHAPTER IV.

Methods of Observing Bacteria-Microscopical.

THE microscopic examination of bacteria is carried out in two ways: (1) observation of living organisms; (2) stained preparations. (1) Observations on Living Bacteria.-A small tin ring is cemented on to a glass slide with canada balsam, forming what is known as a hanging-drop slide (fig. 13). A clean coverglass (see

[graphic]

FIG. 12.-COVERGLASS JAR FOR KEEPING COVERSLIPS IN ALCOHOL.

appendix) is removed from the jar (fig. 12) and the alcohol burnt off; a drop of water placed in the centre by means of the platinum loop, and the drop inoculated with a minute amount of the culture to be examined; the ring on the hanging drop slide is smeared with a little vaseline by means of a small paint brush, and the coverslip placed upon it drop downwards; the vaseline prevents the coverslip falling off and keeps the preparation from evaporating. The preparation is now ready for examination, and is placed under the microscope and examined first with the . Motility, Brownian movement and spores should be looked for; the spores, if present, appear as highly refractile bright dots.

To watch the development of an organism under the microscope some sort of warm stage is required, a constant temperature being maintained by means of circulating water and a thermoregulator. The hanging drop is used, with a nutrient solution substituted for the water.

The hanging-drop method is used for the determination of the agglutinating power of serum, as in Durham's (Wedl's) typhoid reaction and spore germination, chemiotaxis, and many other experiments with living organisms. Every organism should be submitted to this process besides the methods of staining given below.

FIG. 13.-HANGING DROP SLIDE.

(2) Coverslip Preparations.-In coverslip preparations the bacteria are fixed on the coverslip, and stained by one or other of the various stains given below.

A coverslip is taken, and when the alcohol has been removed, a drop of sterile water is placed in the centre. With a sterilised platinum wire, sterilised by heating to redness in the flame, a small quantity of the culture to be examined is removed and added to the drop of water. Only a small amount is used, otherwise the preparation is too thick and the individual organisms massed together in clumps. A faint cloud throughout the drop is all that is required. The drop now containing the bacteria is carefully spread over the surface of the coverglass and allowed to dry.

When dry, but not before, the coverslip is "flamed." To do this take the coverslip between the finger and thumb and pass downwards through a bunsen flame, repeating the process three times. There is nothing mystic in the "three times through the flame," but it has been found by the experience of a large number of workers that this fixes the bacteria properly without damaging them for staining afterwards. After a little practice the student will be able to hold the coverslip in forceps, but it is far better to use the fingers at first until the method is mastered. After cooling the coverslip is flooded with stain. A good method is to use an

indiarubber coin pad such as is used for "change" in many shops; the slips can be easily manipulated in this way. In some processes it is better to immerse the coverslip in a watchglass full of stain.

When stained the preparation is well washed in water, dried between folds of blotting paper, and may be finally dried a safe distance above the flame (one foot). The preparation is then laid film upwards upon a piece of blotting paper, a drop of canada balsam dissolved in xylol placed in the centre, and a clean slide pressed upon it; the blotting paper absorbs any excess of balsam. The preparation is now ready for microscopic examination. A drop of cedar oil is placed on the coverslip and the oil immersion lens lowered until it touches the oil, and all but touches the glass; great care must be exercised to prevent the lens actually coming in contact with the glass, otherwise it may be irreparably damaged. To find the focus, rack upwards with the coarse adjustment until the film comes into view and then use the fine adjustment.

Films from Liquid Cultures.-The films made from broth or from the mouth direct contain a considerable amount of material which stains as well as the bacteria, forming an undesirable background. To prevent this the film must be "cleared" with some solution which does not interfere with the later processes of staining.

The films may be cleared in 1 per cent. acetic acid, or in absolute alcohol.

Another method of fixing film preparations suggested by Goulard is as follows: the coverslips, dried in air but not flamed, are immersed in a solution of absolute alcohol 25 ccm., pure ether 25 ccm., alcoholic solution of mercuric chloride 20 per cent. 0·5 ccm. The films are left in for five minutes or longer, washed well in water and stained.

Tissue Preparations. These preparations may be fresh, the tissue being cut with the freezing microtome, or fixed and hardened, and cut with the rocking or other microtome.

Fixation.—Small pieces of tissue may be hardened and fixed at the same time in absolute alcohol. Corrosive sublimate, a saturated solution in 0.75 per cent. sodium chloride solution is very useful; pieces in. in size or less are left in solution for twelve hours, larger pieces for a longer time. After fixing they are placed in a gauze bag in running water for twenty-four hours, and then passed through three percentages of spirit, 30 per cent., 60 per cent., 90 per cent.

Some iodine is added to the 60 per cent. to remove the last traces of the mercury salt.

Embedding. The various stages in the process are as follows:(1) Preliminary fixation and hardening as above.

(2) Absolute alcohol to complete dehydration.

(3) Absolute alcohol and xylol, equal parts, for twenty-four hours. (4) Xylol twenty-four hours.

(5) Xylol and paraffin twenty-four hours in paraffin bath. (6) Pure paraffin three or four hours.

(7) Melted paraffin poured into mould composed of two L-shaped pieces of brass, and just before it sets the tissue placed in it.

(8) Trim up when hard and cut on microtome.

Preparation of Sections of Teeth to show Bacteria in situ.— Owing to the leathery consistence of dentine when decalcified, the specimens cannot be cut in paraffin with any degree of success, and celloidin is generally employed.

Care must be taken in decalcifying, otherwise the bacteria do not stain well; the best agent is trichloracetic acid, suggested to me by Dr. Spriggs, which gives admirable results.

The tissue is fixed in Goulard's solution or other fixative, washed and transferred to the acid (5 per cent. solution) till soft.

When thoroughly softened the preparation is well washed, dehydrated and embedded in celloidin in the usual manner. The secțions are stained after cutting. Carious dentine, &c., may be embedded in gum while fresh and cut when frozen.

To prepare and stain the sections obtained by the paraffin method the embedding process is reversed.

Float the section in warm water on to a clean slide and dry. Remove the xylol with absolute alcohol, the alcohol with water.

The specimen is now stained, washed rapidly in water, then alcohol, and finally xylol, and mounted in Canada balsam dissolved in xylol.

Blood Films.-Method I.-Place a drop of the blood to be examined upon a clean slide near one end. Take a second slide and place the edge in the drop so that the whole of it becomes wetted, then push the slide along the surface of the first, keeping the second slide inclined.

Method II.-Moisten the edge of a cigarette paper with the blood to be examined and quickly smear the slide or coverslip. Two or more coverslips may be held in a clip or on a piece of blotting paper by means of a slide.

These films require special treatment if the corpuscles are to be preserved. The films are fixed by one of the following methods :— (1) In a hot air oven at 120° C. for an hour.

(2) In equal parts of alcohol and ether for half an hour.

(3) In saturated mercuric chloride solution for three to ten minutes.

After fixation the films are washed, stained, dried and mounted. For special stains for blood films the reader is referred to the large text-books.

METHODS OF STAINING BACTERIA.

General Principles.-The stains generally used in the laboratory for the staining of bacteria belong to the aniline series of basic nature, the bacterial plasm staining much in the same way as the nuclear chromatin of animal cells.

The aniline dyes are divisible into two series according to whether the acid or basic part of the dye is concerned in the process of staining. The basic stains are the ones that have the greatest affinity for the nuclear chromatin and bacteria, the acid for the protoplasm of the cell.

The following aniline dyes are among the ones commonly used in mycological work: Aniline gentian violet; dahlia (methyl-violet); methylene blue (phenylene blue); methyl green; thionin blue; Bismarck brown (vesuvin); fuchsin (basic rubin).

General Remarks on Stains.—The red and violet stains are the most intense in their action, and it is particularly easy to overstain with them; they are also liable to form "background." Specimens stained with gentian violet may often be cleared in absolute alcohol without decolourising the organisms.

The two blue stains are not so intense in their action but give more detail of structure; methylene blue particularly is useful in this respect, many appearances of the protoplasmic contents of the organisms being only demonstrable by its use. They are largely used for general routine work and for counterstaining for contrast.

Stock solutions of the above stains are conveniently kept in alcohol; a quantity of the stain is placed in a glass-stoppered bottle and alcohol poured in, as it is used from time to time the bottle is filled up with fresh alcohol.

Watery solutions of the stains are also used and may be kept made up, 1 per cent. being the usual percentage.

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