came to the conclusion that the streptococcus normally resident in the mouth was of a distinct species to the organism causing disease.

Other observers, however, have held that all streptococci, wherever obtained, are simply different races of the pathogenic streptococcus, and may be raised to the same standard of virulence by appropriate means. This question is considered more at length below, and I will only remark in passing that it cannot be definitely stated that all the streptococci found occurring in disease are of the same species.

Morphology.-Cocci, 0·4—1 μ in diameter, generally united in chains of four to twenty or more individuals, the longer chains are generally formed in liquid media. When rapid division is taking place the appearance of a chain of diplococci is produced. According to Sternberg, the cocci may occasionally occur as diplococci in culture media. Sometimes the cocci become so much elongated that the appearance of bacilli is produced.

In old cultures particularly, some of the elements of the chains appear enlarged and swollen and greatly exceed the size of the rest of the cocci in the chain; the bodies have been termed arthrospores by Hueppe and Du Bary, and are thought to be more resistant than the purely vegetative forms. Other bacteriologists consider them to be involution forms. No true endospores are formed, the cocci are not motile and do not possess flagella.

Staining Reactions.-Stains well with the ordinary aniline dyes and by Gram's method. Old cultures, and especially cultures on potato, show irregular staining.

Biological Characters.—An äerobic, facultative anäerobic, nonmotile pathogenic streptococcus. Spore formation not known, not motile, no flagella present, does not liquefy gelatin. Optimum temperature 37.5° C., but will grow at 16° to 18° C. Thermal death point 54° C. for ten minutes.

Gelatin Stab, 22° C.-Growth slow; in three days a line of small spherical, translucent, whitish colonies, giving a beaded appearance. Surface growth weak, flat, and edge entire. No liquefaction

occurs.

Gelatin Streak, 22° C.-A series of small, flat, whitish colonies, rarely forming a continuous streak.

Gelatin Shake, 22° C.-A cloud of minute spherical colonies are seen distributed throughout the medium in three to four days. No gas is formed.

Agar, 37.5° C.-A series of grey translucent flat colonies develop along the streak, in places becoming confluent. The edges of the colonies when observed with a lens are seen to be composed of loops of chains of cocci..

Blood Serum, 37·5° C.--Minute grey-white colonies similar to agar. Potato.-Little growth occurs, and in forty-eight hours at 37.5° most of the cocci have become swollen and involuted.

Broth.-Development somewhat slow at 37.5° C. The fluid remains clear, and numerous thin flat flocculi form, which tend to settle to the bottom or to the inclined side of the tube; when blood serum or ascitic fluid is added to the broth, development is more rapid. A faint acid reaction does not prevent the growth, although the organisms grow best in an alkaline or neutral medium. No indol formed.

Glucose Broth.—Acid, no gas.

Litmus Milk, 37.5° C.-Well marked acid reaction in two days, later the milk may be coagulated, but this is by no means constant. The vitality of the cultures is not great on liquid media. They are best kept on solid media, and withstand drying for some time.

Pathogenesis. The streptococcus varies considerably in the amount of pathogenic power possessed by various races, and is not, as generally obtained, very virulent for animals. Mice and rabbits, if inoculated with virulent culture, die in twenty-four hours to six days, of general septicemia, the organisms are present in large numbers in the heart blood and in the various organs. If the culture be less pathogenic, disseminated or metastatic abscesses are found distributed about the various organs.

Marmorek1 has shown that the initial virulence possessed by streptococci obtained from various pathological conditions arising in the human subject, may be raised to a great degree of virulence by the rapid passage through rabbits, one animal being inoculated with the blood of another just dead of streptococcal infection. The cultures were made upon a special medium, consisting of three parts of human blood serum to one of broth.

Bulloch also produced enormous exaltation of virulence by passage through rabbits. The culture on ascitic fluid-broth, which originally had a M.L.D.' of 0.1 ccm., was increased in virulence to

'Ann. de l'Inst. Pasteur, t. ix., No. 7, 593.

2 Lancet, April 11, 1896.

such an extent that 0.000001 ccm. was the M.L.D. Many other workers have confirmed these results. Widal and Besançon found that a streptococcus which originally possessed no virulence became pathogenic when inoculated with Bacillus coli communis, and that subsequent cultures could be raised in virulence by Marmorek's methods. These observers also found that the streptococci obtained from the mouth of a small-pox patient were nonvirulent, whereas those present in the blood exhibited a considerable degree of pathogenic power.

FIG. 35.-STREPTOCOCCUS PYOGENES.

From twenty-four hours' broth cultivation. Stained Gram.

× 1000.

Any one who has made cultivations from the mouth cannot but have been struck with the frequency of streptococci in the cultures. In all mouths, healthy or unhealthy, clean or dirty, I have never failed to obtain the streptococcus. Not only is it present in the mouth proper, but it exists in the antrum of Highmore, in the Eustachian tube, nose, and middle ear. In these situations it occurs typically as diplococci massed around the dead squamous

1 Minimal lethal dose.

Stained Gram.

× 1,000. (Washbourn and Goadby, Trans. Odont. Soc., 1896.)

× 1,000

epithelial cells (fig. 36). It is by no means confined to the human mouth, and I have observed it in the mouths of monkeys, dogs, rabbits, and guinea-pigs.

The diplococci form of this streptococcus can be easily seen in almost any coverslip preparation made from the mouth direct. A cover glass specimen is made by smearing saliva, scraped from the buccal sulcus with a platinum loop, on to the cover glass. The film is allowed to dry and then stained with carbolic-methylene blue or other stain. The diplococci will readily be recognised massed around and adhering to the squamous epithelial cells, some of the diplococci having the appearance of short bacilli owing to the elongated and somewhat pear-shaped form of the cocci. The question of the identity of these diplococci with the streptococcus may be proved in the following way: clean coverslips are smeared with melted agar to which a little saliva containing some epithelial cells has been added; when dry, the small coverslip-plate is cemented to a hanging drop slide (a slide with a glass ring cemented to it, see fig. 13) and fixed in place with a little canada balsam. The preparation is then placed on the stage of the microscope and an epithelial cell sought for with the diplococci attached. The microscope with the preparation in situ is then placed in the incubator at 37.5° C. for twenty-four hours. At the end of this time the preparation is examined, when the diplococci will be found to have developed into colonies which surround the cell and in which the streptococcal chains may be easily seen. With a little care one colony can be selected and marked, and cultures on broth and agar made from it as well as coverslip preparations. The culture tubes will show a good growth of streptococci.

This method of obtaining a pure culture of the mouth streptococcus is somewhat tedious, and the method adopted by Dr. Washbourn and myself is much less difficult. Broth cultures are made by adding a loopful of saliva to a tube of nutrient broth; the tube is then incubated for twenty-four hours, at the end of which time the tube will contain an impure culture of streptococci and other organisms. An agar tube is now inoculated from the broth tube and the agar tube incubated at 37° C. In eighteen to twentyfour hours the sloped surface of the agar tube will be found to be covered with a number of small grey-white colonies, which, transferred to another tube, will give a pure culture of the mouth streptoA little care must be exercised in picking out the colonies,

coccus.