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

As H. Buchner (C. B. VIII, 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

1As 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. I, 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.

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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

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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 killedAt 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

with bacilli, an atmosphere containing oxygen is necessary in order for injury to occur. According to Dieudonné, anthrax spores upon agar plates were dead after an exposure to direct sunlight for three and a half hours (bacilli in one and a half hours); if oxygen was excluded, an exposure of nine hours produced no injury.

F. The Activities of Bacteria,

Especially in Regard to the Application of the Same to Diagnostic Purposes.

The activities of bacteria in the test-tube may be designated as: (1) mechanical, (2) optical, (3) thermal, (4) chemical. They will here be discussed in this order; a fifth section will show how the activities of the bacteria enable them to become the causes of disease (pathogenic action).

All the activities of a given variety of bacterium are especially dependent upon, (1) the momentary condition of the bacterium; (2) the nutrient substratum; (3) the entrance of air; (4) the temperature; (5) the illumination.

Since we have already stated what is most important regarding the influence of temperature and light, in the following I must especially discuss the influence of the nutrient medium and the admission of air on one side, and the composition of the final culture on the other. The latter point must always be made especially prominent, in order to show, in the largest possible range, how very much the activity of bacteria changes, according as they are examined when in a full zymogenic, chromogenic, or pathogenic condition, or in an attenuated state.

1 It is self-evident that to-day a division of bacteria into zymogenic, saprogenic, chromogenic, and pathogenic is not acceptable. Bact. coli causes, for example, in sugar-solutions powerful fermentation; on nutrient media rich in albumin it produces abundant indol and sulphuretted hydrogen; it forms upon potato very often a rather bright brownish-yellow colored layer, and is besides pathogenic for animals and man; it therefore combines the properties of all four groups.

1. MECHANICAL ACTIVITY.

Under the microscope we easily observe that many bacteria present pronounced inherent motion, and by study it is found that almost all the motile varieties 1 possess flagella by means of which they propel themselves. The character of the motion is exceedingly variable; for example, creeping (B. megatherium), wabbling (B. subtilis), rolling, snake-like (vibriones); sometimes it is very slow, and sometimes so rapid that any detailed observation can scarcely be made (B. typhi).

In many cases it is difficult to decide whether true active motion is present, or whether the micro-organisms present especially well-marked Brownian or molecular motion-i. e., the dancing and tremor exhibited also by finely divided inorganic particles. In such a case it is recommended to try to render the flagella visible by staining (Technical Appendix), and also to examine the organism in a drop of 5% carbolic acid or 1: 1000 sublimate solution, when, if the motion still persists, we have only to do with molecular motion. Many varieties appear on brief observation to be quiet, but on longer examination single individuals are observed to exhibit positive motion. It seems that the endowments with flagella and motility, when once present, are for the most part reasonably constant peculiarities. Many varieties do not always present motility, it being absent, especially, on many media. According to A. Fischer, with faultlessly developed flagella motion may be absent; for example, in Bac. subtilis on a nutrient medium containing 2% to 4% of ammonium chlorid. We have never observed spontaneous motion or flagella in two different cultures of the Micrococcus agilis AliCohen, obtained from reliable sources and grown upon all ordinary media. We have arrived at the conviction that the same variety may occur either with or without flagella. (Compare special part.)

Th. Smith has described a non-motile form of the mo

1 Upon the actively motile Spirochete Obermeieri and the slowly creeping Beggiatoa no flagella have thus far been demonstrated; therefore the motion is supposed to depend upon a narrow undulating membrane attached to the organism.