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The following list of antiseptics and disinfectants gives some of the more common ones in use with their relative strength as determined practically by laboratory experiments. These tests, however, are more favourable to the antiseptic used than the organism tested, which is growing artificially and not in its usual habitat. The use of spores and their death as determined by absence of germination is more reliable; the spores to be tested are dried upon sterile silk threads and immersed for various periods of time in the antiseptic to be tested, then washed with boiled distilled water to remove traces of antiseptic and transferred to a culture tube.

Another method is to add various quantities of the antiseptic under investigation to broth cultivations of the organisms experimented with; in this case care must be taken to avoid fallacies due to the neutralisation of the antiseptic by the medium used. The cultivations may be either fully developed ones, or one or more loop-fuls (öse) of culture may be inoculated into the nutrient medium containing the antiseptic to be tested. In making the subsequent sub-cultivations to test the destruction or inhibition of the organisms care must be taken to use a sufficiently large quantity of nutrient medium, otherwise the amount of antiseptic in the öse may invalidate the result. Control tubes should invariably be made.

Sternberg recommends mixing the standard culture and the diluted antiseptic in equal proportions; thus 10 cc. of sterile broth containing 1 in 200 carbolic is added to 10 cc. of a twenty-four hours' broth culture of the given organism (= 1 in 400); plate cultivations are then made at given intervals.

Many substances, such as concentrated solutions of sugar or common salt, prevent the development of bacteria but do not kill them.

Disinfection of Hands, &c.—The bacterial flora of the skin is of a varied nature, and owing to the cracks and fissures of the epidermis, particularly the hands, it is difficult to remove the bacteria ; moreover the bacteria actually live upon the dead epithelial cells, rarely however penetrating the true skin. The best method to adopt is first thorough scrubbing with a nail brush (boiled and kept in 2 per cent. lysol), soap and hot water, to remove as much of the dry epidermal scales as possible. The hand should be then soaked for two minutes in some antiseptic solution such as the one recommended by Lockwood', 1 of biniodide of mer

i Brit. Med. Jour., Jan. 11, 1896.

1 , 14,000

cury in 500 of methylated spirit, which is subsequently washed off in 1 in 3,000 biniodide solution. This treatment does not cause the same roughness that mercuric chloride or carbolic so often produces. It need hardly be added that unless a perfectly sterile towel is used the disinfection is of no avail, and in operative surgery they are discarded entirely, and should not be used by the dental surgeon to dry his hands before the operation of extraction.

The following short list of antiseptics and disinfectants gives the most useful; for other and extensive lists the reader is referred to Sternberg's “Bacteriology," Macfarland's “ Pathogenic Bacteria," and Hueppe's exhaustive article in “ Principles of Bacteriology."

Formalin (40 per cent, sol. of formic aldehyde gas in water) ... 1 in 40,000 Biniodide of Mercury ...

„, 35,000 Bichloride of Mercury Lysol ...

1, 1,000 Carbolic Acid ...

1, 133 These figures give the relative strengths of solutions which will restrain the growths of bacteria but will not always destroy them.

Salicylic acid and quinine are also powerful antiseptics, whilst iodoform, so commonly used, must be first changed into iodine—a somewhat rare thing—before it is effective. Its chief action is the neutralisation of the products of the organisms. The various mineral acids are strong disinfectants, and 5 per cent. HCl added to mercuric chloride greatly increases its efficiency. Lysol, consisting of coal tar oil, phenol and soap, is advantageous in that it is strongly alkaline and dissolves grease. It is used largely in general laboratory routine, especially for soaking used slides, the coverslips becoming detached by solution of the balsam as soap. A jar containing a 2 per cent. solution should be kept on the laboratory bench.

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 à 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.

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