may be distilled after adding MgO and the distillate treated with Nessler's reagent. Yellow to reddish-brown color indicates ammonia. Control experiments must always be made.

7. Aromatic Metabolic Products.

Often, as the result of the action of very many varieties of bacteria, there arise from albumin aromatic bodies, of which indol, skatol, phenol, and tyrosin are best known. Methodical investigations are at hand regarding the occurrence of only indol and phenol, since these bodies are easily recognized.

Demonstration of Indol.-There is added to the bouillon culture, which is preferably not less than eight days old and prepared without the addition of sugar, about onehalf its volume of 10% sulphuric acid. If, now, on warming to about 80° a rose or bluish-red color at once appears, then both indol and nitrite are present. The nitroso-indol reaction just described requires both these bodies to be present for its success. With cholera and most other vibriones, at times also with diphtheria, the demonstration may be made (“cholera-red reaction”). Usually the addition of sulphuric acid is not sufficient, and it is necessary to add also a little nitrite. This may be added if, upon warming without the nitrite, no reaction, or only a doubtful one, is obtained. One adds 1 c.c. to 2 c.c. of a 0.05% solution of sodium nitrite until the maximum reaction is obtained. Addition of a stronger nitrite solution colors the fluid brownish-yellow, and entirely prevents the demonstration of indol.

Demonstration of Phenol.-The culture in non-saccharine bouillon receives the addition of about one-fifth its volume of hydrochloric acid and is then distilled. The distillate gives a flocculent precipitate when treated with bromin water. If carefully neutralized with calcium carbonate, the addition of neutral very dilute chlorid of iron gives a violet color.

In sixty varieties examined, we found (see Table I) indol production twenty-three times. Our findings accord well with the statements of Levandowsky (Deutsch. med.

Wochenschr., 1890, No. 51, 1186). The following are producers of indol: The colon group as a whole, glanders, diphtheria, proteus, and most vibriones. With the exception of the vibriones, according to Levandowsky, those mentioned as producers of indol also produce phenol. We have demonstrated phenol production only in Bacterium coli and vulgare, and have found only traces of phenol in five-days'-old cultures.

8. Decomposition of Fats.

Pure melted butter is no nutrient medium for bacteria. Rancid changes in butter depend upon (1) a pure chemical decomposition of butter through the action of oxygen under the influence of sunlight (Duclaux, Ritsert); (2) a lactic or butyric acid fermentation of the milk-sugar present in the butter. Compare v. Klecki (C. B. xv, 354). Finally, fat is also appropriated by bacteria with production of acids if it is mixed with gelatin as a nutrient medium. v. Sommaruga (Z. H. xvIII, 441).

9. Putrefaction. (Supplement to 1 to 7.)

By putrefaction the laity understand every decomposition brought about by bacteria, accompanied by the formation of foul-smelling substances.

The scientific view is, that the albuminous bodies and their relatives (glue, albuminoid substances) are the substratum for putrefaction, being often first peptonized and then further broken up.

Typical putrefaction occurs only with a deficient or limited supply of oxygen. Active passage of air through a putrefying culture of bacteria-an occurrence which never takes place in natural putrefaction-modifies the putrefactive powers most actively. This is accomplished (1) biologically, by killing or checking the growth of the anaerobic putrefactive bacteria, and (2) by the influence of oxygen upon the products or intermediate products of the aerobic and facultative anaerobic bacteria. Finally, it is probable that the same bacteria from the very start produce different putrefactive products when grown anaerobically from those produced under aerobic conditions.

As products of putrefaction we find those given in the preceding sections: Albumoses, ammonia and amine, leucin, tyrosin and other amido-bodies, oxyfatty acids, indol, skatol, phenol; then, sulphuretted hydrogen, mercaptan, carbonic acid, hydrogen, and finally marsh-gas.

In the decomposition of different nutrient media by various fungi, the metabolic products just enumerated are found, as a rule, only in part and in most variable combinations, so that putrefaction can scarcely be defined more exactly by chemical aids than is possible by the senses. I am, therefore, of the opinion that it is best to employ the expression "putrefaction" only in the general sense of the laity to indicate every foul-smelling decomposition of albuminous bodies. (Compare Kuhn, A. H. XIII, 40.)

10. Nitrification.

According to Heraeus (Z. H. 1, 193), the ability to form nitrite, at least in traces, from NH is widely distributed. 2 Most investigators, however, agree in stating that nitrite production from NH3 exclusively, or at least preponderantly, is dependent upon an organism, possessing slight morphologic characteristics, which Winogradsky, in his original work, designated nitrosomonas. For more detailed description see special part.

Winogradsky has described somewhat more completely the organism which forms nitrates from nitrites, and which he calls "nitrobacter" (compare special part). Both organisms are alike in that they grow only upon poor nutrient materials-on mixtures of inorganic salts or agar and mixtures of salts without peptone or sugar-and do not grow on any of our ordinary nutrient media. The contradictory statements of Stutzer and his pupils are generally considered incorrect. Both organisms are widely

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1 The assertion is often made that the albuminous bodies are first peptonized in every putrefaction, but since Bact. vulgare, B. Zenkeri, and Bact. putidum are generally recognized as causes of putrefaction," but never liquefy gelatin, it is not proper to speak of peptonization of albumin as being always present in putrefaction.

2 Rullmann calls attention to the nitrite present in laboratory air, which may easily cause mistakes (C. B. L. v, 212).

distributed in the soil, in meadows often the nitrite producers alone, in cultivated soil usually both. Both organisms possess the greatest theoretic interest, since out of inorganic nitrogen and carbonic acid (both free CO2 and Na,CO, or the presence of a bicarbonate is necessary) they are able to build up their body substances, i. e., albumin, without the aid of chlorophyll, which the higher plants require.

P. F. Richter (C. B. XVIII, p. 129) often observed marked nitrite reaction in urine freshly obtained with a catheter. From one specimen of urine he isolated a medium-sized coccus, which produced very intense nitrite reaction in fresh urine in twenty minutes. It also reduced nitrate to nitrite.

II. Transformation of Nitrites (and Nitrates) into Free Nitrogen (Denitrification).

An entire series of organisms, which are widely distributed in dung, straw, field-soil, and filthy water, set free gaseous nitrogen from nitrites (denitrification). Many are able simultaneously to transform nitrate into nitrite, also alone to set free gaseous nitrogen from nitrates (for example, Bacterium Stutzeri, B. pyocyaneum-compare special part), while others require synergetic bacteria to change the nitrate into nitrite (for example, Bact. denitrificans). Compare Burri and Stutzer (C. B. L. 1, 257, 350, 392, 422); Weissenberg (A. H. xxx, 274).

For the demonstration of the denitrifying action of bacteria, there is added to a liter of ordinary bouillon 2.5 gm. of sodium nitrate or, better,-since only thus are all denitrifying varieties recognized,-sodium nitrite.

As was first completely demonstrated in my institute by Weissenberg (Burri and Stutzer had made some similar observations), the reduction of nitrite to nitrogen is much promoted by the exclusion of oxygen, and is markedly or completely inhibited by very free entrance of oxygen (growth in shallow layers of fluids or with air passing through). The organisms thus break up the nitrite to obtain oxygen, and there thus originate considerable quantities of NaOH or Na2CO3, so that the fluid becomes strongly alkaline.

The test for denitrification is best made with fermentation tubes (p. 90), as the entrance of oxygen is here interfered with; still, usually test-tube cultures suffice. There occurs an abundant production of gas, which is not absorbed by KHO (not CO2), nor by KHO and pyrogallic acid (not O), and does not burn (not H or hydrocarbons), therefore is nitrogen.

According to Stoklasa, the denitrifying action of bacteria is most pronounced in nutrient materials which, like decayed vegetable matter, straw, and manure, contain abundantly the pentose, xylose (C5H1005). Recent literature upon denitrifying varieties: H. Jensen (C. B. L. IV, 401), Künnemann (C. B. L. IV, 906). Here are also described further denitrifying bacteria: Bact. agile Amp. and Gar., Bacillus Schirokikhi Jensen, Bact. filefaciens Jensen, Bact. centropunctatum Jensen, Bact. Hartlebii Jensen, Bact. nitrovarum Jensen, Vibrio denitrificans Sev.1 Most do not liquefy gelatin and are also able to break up nitrate without the aid of synergetic bacteria. The practical significance of the denitrifying organisms is very great. They rob the soil, manure, etc., of the nitrates and nitrites which are so necessary for the nourishment of plants, and so are powerful enemies of agricul

ture.

12. Assimilation of Nitrogen.

While, according to our present knowledge, none of the higher families of plants are able to assimilate nitrogen from the air, this property occurs in one variety of bacterium, Bacillus radicicola Beyerinck. This bacterium occurs in the small root-tubercles of various leguminous plants (peas, clover, etc.), and may be cultivated from them. It grows poorly or not at all upon the usual nutrient media, but well upon an infusion of pea leaves, to which is added 7% gelatin, 0.25% asparagin, and 0.5% canesugar. It does not liquefy gelatin, does not form spores

1 According to Severin (C. B. L. III, 504), there are yet many more denitrifying varieties; for example, Bacillus subtilis (or closely related varieties), Bacterium indicum, and a coccus.

2 Regarding the tubercles of the alder which assimilate nitrogen, and their inclusion of fungi, see Hiltner (C. B. L. II, 97)