The position and size of the spore are of considerable use in differential diagnosis. The terminal spore of Bacillus tetani is well known. It is rarely seen at both ends of the bacillus, and hence when poised only at one end causes the "drumstick" appearance. In the bacillus of Quarter Evil the spore is generally towards one end of the rod rather than in the middle; in Malignant Œdema the bacillus in the blood grows out into long threads, and when such a thread sporulates the spore is also near one end. The latter further illustrates the fact that in some species the spore is of greater diameter than the mother cell, and hence dilates the bacillary capsule. The spores of anthrax are typical oval endospores. When free in the field of the microscope, spores must be distinguished from fat cells, micrococci, starch cells, some kinds of ova, yeast cells, and other like objects. Spores are detected frequently by their resistance to ordinary stains and the necessity of colouring them by special staining methods. When, however, a spore has taken on the desired colour, it retains it with tenacity. In addition to their shape, size, thickened capsule, and staining characteristics, spores also resist desiccation and heat in a much higher degree than bacilli not bearing spores. Roux and some other eminent bacteriologists suggest that bacteria should be classified according to their method of spore formation.
THE INFLUENCE OF EXTERNAL CONDITIONS ON GROWTH OF BACTERIA
Nutritive Medium. In the very earliest days of the study of micro-organisms it was observed that they mostly congregate where there is pabulum for their nourishment. The reason why fluids such as milk, and dead animal matter such as a carcass, and living tissues such as a man's body contain so many microbes is because each of these three media is favourable to their growth. Milk affords almost an ideal food and environment for microbes. Its temperature and constitution frequently meet their requirements. Dead animal matter, too, yields a rich diet for some species (saprophytes). In the living tissues bacteria obtain not only nutriment, but a favourable temperature and moisture. Outside the human body it has been the endeavour of bacteriologists to provide media as like the above as possible, and containing many of the same elements of food. Thus the life-history may be carried on outside the body and under observation. By means of cover-glass preparations for the microscope we are able to study the form, size, motility, flagella, spore formation, and peculiarities of staining, all of which characters aid us in determining to what species the organism under examination belongs. By means of artificial nutrient media we may further learn the characters of the organism in "pure culture,"[7] its favourable temperature, its power or otherwise of liquefaction, the curdling milk, or of gas production, its behaviour towards oxygen, its power of producing indol, pigment, and chemical bodies, as well as its thermal death point and resistance to light and disinfectants. It is well known that under artificial cultivation an organism may be greatly modified in its morphology and physiology, and yet its conformity to type remains much more marked than any degeneration which may occur.
The basis of many of these artificial media is broth. This is made from good lean beef, free from fat and gristle, which is finely minced up and extracted in sterilised water (one pound of lean beef to every 1000 cc. of water). It is then filtered and sterilised. It will be understood that such an extract is acid. To provide peptone beef-broth, ten grains of peptone and five grains of common salt are added to every litre of acid beef-broth. It is rendered slightly alkaline by the addition of sodium carbonate, and is filtered and sterilised. Glycerine-broth indicates that 6 to 8 per cent. of glycerine has been added after filtration, glucose-broth 1 or 2 per cent. of grape-sugar. This latter is used for anaërobic organisms. The use of broth as a culture medium is of great value. It is undoubtedly our best fluid medium, and in it may not only be kept pure cultures of bacteria which it is desired to retain for a length of time, but in it also emulsions and mixtures may be placed preparatory to further operations. Gelatine is broth solidified by the addition of 100 grams of best French gelatine to the litre. Its advantage is twofold: it is transparent, and it allows manifestation of the power of liquefaction. When we speak of a liquefying organism we mean a germ having the power of producing a peptonising ferment which can at the temperature of the room break down solid gelatine into a liquid. Grape-sugar gelatine is made like grape-sugar broth. Agar was introduced as a medium which would not melt at 25° C., like gelatine, but remain solid at blood-heat (37·5° C.; 98·5° F.). It is a seaweed generally obtained in dried strips from the Japanese market. Ten to fifteen grams are added to every litre of peptone-broth. Filtration is slow and often difficult, and the result not as transparent as desirable. The former difficulty is avoided by filtering in the Koch's steamer or with a hot-water filter, the latter by the addition of the white of an egg. Glycerine and grape-sugar may be added as elsewhere. Blood agar is ordinary agar with fresh sterile blood smeared over its surface. Blood serum is drawn from a jar of coagulated horse-blood, in which the serum has risen to
Potato in a Roux Tube Prepared for Cultivation Glycerine is placed in the bulb of the tube the top. This is collected in sterilised tubes and coagulated in a special apparatus (the serum inspissator). Potato is prepared by scraping ordinary potatoes, washing in corrosive sublimate, and sterilising. They may then be cut into various shapes convenient for cultivation. Upon any of these forms of solid media the characteristic growth of the organism can be observed. Of the nutrient elements required, nitrogen is obtained from albumens and proteids, carbon from milk-sugar, cane-sugar, or the splitting up of proteids; salts (particularly phosphates and salts of potassium) are readily obtainable from those incorporated in the media; and the water which is required is obtainable from the moisture of the media.
There are two common forms of test-tube culture, viz.: on the surface and in the depth of the medium. In the former the medium is sloped, and the inoculating needle is drawn along its surface; in the latter the needle is thrust vertically downwards into the depth of the solid medium. Plate cultures and anaërobic cultures will be described at a later stage. When the medium has been inoculated the culture is placed at a temperature which will be favourable. Two standards of temperature are in use in bacteriological laboratories. The one is called room temperature, and varies from 18° C.-20° C.; the other is blood-heat, and varies from 35° C.-38° C. It is true, some species will grow below 18° C., and others above 38° C. The pathogenic (disease-producing) bacteria thrive best at 37° C., and the non-pathogenic at the ordinary temperature of the room. The different degrees of temperature are regulated by means of incubators. For the low temperatures gelatine is chosen; as a medium for the higher temperatures agar.
Staphylococcus Pyogenes Aureus
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