bouillon, 0.1% of sugar sufficed to kill the cholera vibrio in a few days, 0.2% the Bact. typhi, and 0.3% most bacteria. In solutions rich in peptone the sugar produced less harm.

Since by many varieties the acid-production with the reduction of sugar is very rapid and intense, one designates this metabolism, brought about at the expense of the carbohydrates, as fermentation (compare p. 64). Because not rarely gas is produced in abundance, this designation also seems proper to the laity.

If, after the sugar is exhausted, the quantity of acids produced is not such as to kill the bacteria, then in the nutrient medium, now free of sugar, other metabolic processes occur and the acids are neutralized and the reaction becomes even alkaline.

Among the acids produced (besides the carbonic acid, to be spoken of under "gas-formation") the most important, so far as we know, is lactic acid; almost always there are at least traces of formic acid, acetic acid, propionic acid, butyric acid, and also not rarely some ethyl alcohol, aldehyd, or acetone. More rarely lactic acid is absent and only the other acids are produced.

For obtaining and separating the acids one proceeds somewhat as follows: One-half liter of peptone bouillon containing from 2% to 5% of grape- or milk-sugar is placed in liter flasks, and about 10 gm. of carbonate of calcium added to each. The acids produced unite with the calcium carbonate as soluble salts, and carbonic acid escapes. The reaction of the fluid remains neutral, and that is the principal thing; a strong acid reaction would prematurely hinder further growth of the bacteria.

When growth ceases (after eight to fourteen days), the insoluble carbonate is filtered off, and from the fluid, with neutral reaction, the alcohol, aldehyd, and acetone, etc., present are removed by distillation, thus reducing very much the amount of fluid. The three mentioned substances are tested for together by Lieben's iodoform reaction. To the slightly warm fluid in a test-tube are added five to six drops of a pure 10% aqueous solution of caustic potash; then drop by drop a weak solution of iodid of potassium is added until a brown color appears, and the

latter is again dissipated by a drop of potash. The presence of iodoform is proved by the characteristic odor and, microscopically, by the small six-sided iodoform plates. For the differentiation of alcohol, aldehyd, and acetone, consult Vortmann, Analyse organ. Stoffe, 1891. Then one acidifies strongly with phosphoric acid, and with the aid of a current of steam distils off the volatile acids. The distillation must be long continued, as the complete separation of the volatile acids is difficult. The non-volatile lactic acid (together with some succinic acid) remains behind and is separated by repeated shaking with pure ether, the ether then being distilled off.

The lactic acid obtained is always ethylidenlactic acid, CH3.CHOH.COOH, which occurs in two stereoisomeric forms: (1) dextrorotatory with levorotatory zinc salts; (2) levorotatory with dextrorotatory zinc salts. If, as is frequently the case, almost equal molecules of levorotatory and dextrorotatory lactic acid are present, then the mixture is optically inactive and is the so-called "fermentation lactic acid." I believe that often both lactic acids originate from sugar, but that many bacteria use up one acid exclusively or principally, while others appropriate the other acid. Thus may occur now a uniform mixture of both acids, now one acid exclusively or preponderantly.

Since Schardinger (Mitt. f. Chem. xI, 545) first discovered the previously unknown levorotatory lactic acid as a product of a short bacillus from water, many investigations have been made, especially by the pupils of Nencki and Rubner, regarding the lactic acids formed by different varieties of bacteria, with the hope of utilizing the results in differential diagnosis.

For the methods for determining which lactic acid is present, consult Nencki (C. B. IX, 305) and Gosio (A. H. XXI, 114). They have to do with the determination of polarization and the water-content of the zinc salt.

While at present these results are not of much value, yet a continuance of these theoretically interesting studies is desirable. (Compare special part, under Vibrio cholera and Bact. coli. )

Various bacteria-often, however, insufficiently studied morphologically or biologically-are able to produce butyric acid, butyl alcohol, or both from carbohydrates.

For a review of these varieties see Baier (C. B. L. 1, 17). Compare in special part: Bac. butyricus Hüppe, Bac. butyricus Botkin, Clostridium butyricum, etc.

In connection with the fermentation of sugar, decomposition of cellulose may be mentioned as caused by various bacteria. It occurs especially in the gastric and intestinal contents of herbivora, and also in quagmire, and forms marsh-gas as its striking product.

1 The statements regarding the coli group are from Nencki (C. B. IX, 305); regarding the typhoid, from Blachstein; regarding the cholera group, from Kuprianow (A. H. XIX, 283, 291) and Gosio (A. H. XXI, 114).

Unfortunately the fermentation of cellulose by bacteria is insufficiently studied. So much seems certain, that at least one anaerobic variety converts cellulose into marsh-gas and carbonic acid. Yet Van Senus maintains that the anaerobic "Bacillus amylobacter," isolated by him, attacks cellulose only in symbiosis with another small bacillus. (Compare the résumé by Herfeld, C. B. L. 1, 74, 114, and also the special part.)

Fig. 11.-Bacterium coli upon sugar-agar, after twelve, twentyfour, and forty-eight hours.

14. Gas-production from Carbohydrates and Other Fermentable Bodies of the Fatty Series.

The only gas eventually arising in visible quantity1 in nutrient media which contain no sugar is nitrogen (compare page 82).

1 Sulphuretted hydrogen and ammonia can scarcely occur in visible quantity.

If sugar is broken up energetically by bacteria, gasformation may be absent, only lactic or acetic acid being produced (for example, Bac. typhi on grapesugar), but very often an enormous production of gas occurs, especially if air is excluded. About one-third of the vigorous acid-forming varieties produce abundant gas, which consists always of carbonic acid, with a constant admixture, according to Smith (C. B. xviii, 1), of hydrogen. Marsh-gas appears to be rarely formed (aside from the bacteria causing fermentation of cellulose). Compare in special part: Bact. brassicæ acidæ.

To determine whether gas is formed, the shake-culture in 1% grape-sugar agar is very useful (Fig. 11). After

Fig. 12. Fermentation tube.

twenty-four hours (if incubator temperature is available, often after six to twelve hours) the agar is beset by gas bubbles or cleft by numerous deep holes and cracks. If it is desirable to collect and measure the gas, to investigate the curve of the intensity of gas production, or to analyze it, the gas is best collected after the method of Th. Smith in a fermentation tube, such as has been long employed in physiologic and pathologic chemistry (Fig. 12).

The tubes, which preferably have the same form, are filled with 1% grape-sugar peptone-bouillon (without airbubbles) and sterilized in the steam sterilizer.

After inoculating with a platinum loop, they are kept