we examine water, milk, or the intestinal contents in health or disease, we always find many varieties simultaneously present. This mixture certainly appears to us as a pure accident, but upon closer study it is found that also in the domain of bacteriology there are synergists (mutual or one-sided aid) and antagonists (mutually injurious, or one to the other). Nencki speaks of symbiosis and enantobiosis.

Experimentally, Garrè has demonstrated the antagonism by inoculating various bacteria simultaneously in streaks upon gelatin plates as parallel or intersecting lines. It then appears that many varieties thrive but slightly or not at all if another variety grows in the immediate neighborhood. The antagonism is very often only on one side; for example, the Bact. putidum grows very well if inoculated between closely placed, well-developed streaks of staphylococci; on the contrary, the Micr. pyogenes does not grow if inoculated between luxuriantly developed cultures of Bact. putidum, and if the two are simultaneously inoculated in alternating streaks, the former grows very slightly (Garrè, “Correspondenzbl. f. Schweizer Aerzte," 1887, 387).

Another way of showing antagonism is by preparing plates of gelatin or agar (for liquefying varieties), which, while liquid, are inoculated with equal quantities of two different varieties of bacteria; often only one variety will develop (Lewek, C. B. VII, 107).

A third way of carrying out the investigation is to inoculate simultaneously the same fluid nutrient medium with two varieties, and, later, determine microscopically or by plates which is triumphant in the battle. This is what is commonly observed when a cause of fermentation is abundantly introduced into a suitable nutrient medium; the contaminating bacteria are overgrown and sometimes perish.

From these observations the practical conclusion is reached that for determining the number of bacteria in a material the colonies in the plates must not be very thick, and also that for the isolation of definite varieties, thin plates are necessary; for example, if one wishes to isolate the Bact. Pflügeri from abundant Bact. putidum. In an area of several millimeters about each putidum colony no Bact. Pflügeri will grow (K. B. Lehmann).

Finally, in the animal body bacteria may counteract. each other as antagonists; as Emmerich has pointed out, animals infected with anthrax may be saved by subsequent infection with Streptococcus pyogenes. Literature by Mühlmann (C. B. xv, 895).

Symbiosis of bacteria appears to be of more practical

importance, and of this the following examples may be cited:

1. Some bacteria thrive much better together with others than when alone. Some anaerobes thrive even with the admission of oxygen, if only certain aerobic varieties are present. (Compare B. tetani.)

2. Certain chemical transformations-for example, the breaking up of nitrite with liberation of free nitrogen-are not accomplished by many bacteria alone, while two varieties jointly may do so. This fact is well worth considering when searching for the cause of certain decompositions. Always when the isolated varieties, acting singly, operate partially or not at all, the effects of combinations must be investigated.

3. In a similar manner it is observed that, for example, the single individuals of a series of soil bacteria are not pathogenic, while certain combinations, inoculated into animals, make them sick.1 (Liermann, C. B. vIII, 364.) This experience also demands that especial care be taken in searching for the cause of a new or puzzling disease-picture. Many authors believe that cholera has its origin in two germs, "diblastic theory" (Nägeli, Buchner).

4. Attenuated pathogenic varieties-for example, attenuated tetanus bacilli-may increase in virulence when cultivated together with other bacteria; for example, Bact. vulgare.

E. The Conditions of Spore-formation and Spore-germination.

The extent of the formation of endogenous spores appears hitherto to have been very insufficiently understood. Except in a large group of important varieties of bacilli, related to the B. anthracis and the B. tetani, undoubted

Not quite appropriate here is the experience that the metabolic products of one variety of bacterium, under some circumstances, may enhance the action of another variety; for example, the metabolic products of the Bact. vulgare the action of the tetanus bacillus.

endogenous spores are known only in Sarcina pulmonum, and the strange Spirillum endoparagogicum. 1

1

As H. Buchner (C. B. vi, 1) pointed out, sporulation occurs in suitable varieties when the nutrient medium begins to be exhausted, therefore most rapidly on nutrient media very poor in nutrient materials.

On the contrary, a good nutrient medium not only favors the growth of bacilli but also the formation of spores, in so far as the vigorously growing bacilli also luxuriantly and regularly sporulate (K. B. Lehmann and Osborne, A. H. XI, 51); see especially also Stephanidis (A. H. xxxv, 1). The crop of spores is exceedingly large. The quality (resistance) of spores which are grown upon various nutrient media was not found by Stephanidis to vary. For many details consult Schreiber (C. B. xx, 353).

For sporulation a higher temperature is sometimes (always?) required than for the vegetative growth. The anthrax bacillus, for example, thrives at 13° to 14°, but does not form spores below 18°.

All aerobic bacteria require, especially for spore-formation, the presence of oxygen; how the facultative anaerobes conduct themselves in this respect is still to be learned.

Obligate anaerobes only produce spores if oxygen is excluded or, with the admission of oxygen, in mixed cultures or in association with dead synergetic bacteria.

Spores never germinate in media in which they have developed when they have been exhausted or rendered detrimental by metabolic products. Only after transferring to fresh nutrient media does germination occur, appearing in one or more hours, and having the morphologic peculiarities described on page 26.

Against all injuries spores are substantially more re

1 As it is important for our classification, we have carefully sought, in a number of varieties generally considered as being free from spores, to obtain spores as had been done by Migula (Sys. 1, 207) by means of quince and marshmallow decoction. We never obtained a perfectly undoubted result. With Bacterium janthinum alone we saw detached pictures, which could be interpreted as spores, but we have not studied their germination. Upon the common nutrient media we have not once seen sporulation in a variety commonly known as not possessing spores.

sistant than the vegetative forms. They require no nourishment and no water in order to retain their ability to germinate after years and often decades. They are more indifferent to gases than the bacilli, the spores of anaerobic varieties usually bearing free oxygen well. 2 Spores are obtained by carefully removing sporulating agar streak cultures, and warming the emulsion, prepared with a little water, to 70° for five minutes.

Very important is the resistance of spores to dry and moist heat. Dry heat is especially well borne, a temperature of 100° being withstood by many spores for a long time. In a moist condition, a temperature of 70° kills the anthrax bacillus in one minute; on the contrary, anthrax spores withstand this temperature for hours; even in boiling water or live steam at 100° they die only after two to five, or at times after seven to twelve, minutes. The varying resistance of different anthrax spores (v. Esmarch, Z. H. v, p. 67; Stephanidis, A. H. xxxv, 1) appears to be partly a race peculiarity, but very probably also the nutrient medium, the temperature at which they were produced, the degree of maturity, etc., exert an influence upon the resistance. Very accurate investigations upon these points are almost entirely lacking. We only know from Percy Frankland that spores formed at 20° are more resistant to light than those originating at incubator temperature (C. B. xv, p. 101).

The resistance of spores is tested by hanging in the boiling steam-chamber little sacks of tulle containing fragments or little plates of glass upon which anthrax spores have been dried, and from minute to minute a sack is removed and the pieces of glass laid upon an agar plate, which is then kept at incubator temperature. A better way it seems to me is as follows: 1c.c. of an emulsion of spores is placed in 20 c.c. of water, and after shaking well five

1 According to an observation of v. Esmarch, if anthrax spores are kept a long time the virulence appears to be reduced before the power to vegetate is affected.

2 Spores of malignant edema in garden earth were well preserved in my institute for four years. On the contrary, very astonishingly, tetanus spores dried upon threads and kept in the room were still alive after two days, but dead after three days.

samples of 2 c. c. each are removed and placed in reagentglasses of equal thinness, while in a sixth one are placed 2 c.c. of water and a thermometer. All six glasses are now plunged in a large water-bath containing boiling water, and after two minutes the thermometer in the control tube reaches a maximum temperature (99° to 100°). Two minutes later one removes the first sample, four minutes later the second, etc., cools them rapidly in cold water, and utilizes 1 c.c. and c. c. of each sample in the preparation of plates. For further details, see Stephanidis, A. H. XXXV, 1.

The varying resistance of apparently identical anthrax spores is of great practical importance: (1) in disinfection experiments, which should be carried out with spores of known resistance; (2) in differential diagnosis, as it indicates how very careful one must be in placing dependence upon a slight difference in the resistance of spores in determining two species.

Very extraordinary is the resistance of many varieties occurring in hay and soil. Christen found, for example (C. B. XVII, p. 498), that with compressed steam the resisting spores in soil were killed—

At 100° in more than 16 hours.

The apparatus employed brought the objects to the desired elevation of temperature very quickly.

Also against chemical agents spores are very resistant; thus, anthrax spores (v. Esmarch, l. c.) resist 5% carbolic acid for at least two days, and in many cases as long as forty days. Very resistant anthrax spores withstood a 1% aqueous sublimate solution for three days, but the virulence is lost in twenty hours. Such experiments are best made with thin suspensions of spores in water, to which the disinfectant is added, just as was pointed out above for testing the action of antiseptics upon bacteria (p. 38).

In testing the resistance of spores to gases, they are best dried upon pieces of glass, and the gas allowed to operate in a dry and also in a moist chamber (compare p. 41).

Spores are also less injured by light than bacilli are. As