Yet there is much greater difference in the dry substance of the cholera vibriones, if they are grown at one time upon soda bouillon rich in albumin, and again upon Uschinsky's medium, free of albumin. Cramer here found as follows (the figures are the average from observations upon five cholera cultures): He found also in the latter case a considerable quantity of non-nitrogenous bodies, a part of which may be thought to be hydrocarbons (or fats). For the analysis of the ash of bacteria, consult Cramer (A. H. XXVIII) and de Schweinitz and Marion Dorset (C. B. XXIII, 993). The latter found almost only phosphate in the ash of tubercle bacilli. Of importance for the classification, even though more in a critically negative sense, is the fact, discovered by Cramer, that closely related varieties, which, upon many nutrient media, present analogous slightly varying composition, suddenly upon a new medium conduct themselves differently. Most interesting in this respect is the behavior of five cultures of cholera, which in soda bouillon furnished vibriones of almost exactly the same composition, but upon Uschinsky's solution presented very variable composition. Cholera, Shanghai 64.25 33.87 98.12 47.50 11.64 59.14 Cholera, Hamburg II 63.94 29.81 93.75 34.37 14.74 49.11 This result shows again how dangerous it is to make a separation of two varieties because of any single chemical or biological reaction. In order to understand the astonishing differences, it is only necessary to recognize the ability of one of these varieties to form thick cell 1Compare p. 33. membranes from Uschinsky's solution. How easy it would be for an author to pronounce the cholera of Paris among this number, as a distinct species, since, upon Uschinsky's medium, it contains almost double the amount of albumin which the Hamburg cholera does. Bacterial spores have not so far, to my knowledge, been closely studied. One may naturally expect a decreased water-content, from the analogy to the spores of molds. C. Rapidity of Increase and Duration of the Life of Bacteria. Under favorable conditions (see below) bacteria multiply very rapidly; according to Buchner, the number of cholera vibriones, under most favorable conditions, is doubled in twenty minutes (C. B. II, 1). Compare also Ficker (C. B. XXIII, 1059). The duration of the life of bacteria is theoretically unlimited, since from each cell by division two new ones, with unlimited possibilities of division, are produced. Practically, however, in our cultures the case is quite different. As pointed out by Gotschlich and Weigang, in a cholera culture (agar-streak) at 37°, even after twenty-four hours the number of live germs is practically reduced, and after forty-eight hours many bacteria are injured by their own products (Z. H. xx, 376). D. Conditions of Life of Bacteria. 1. NUTRIENT MEDIA. While a number of bacteria have hitherto been met with only in the human or animal organism as parasites, and appear to us as obligate parasites (example, Spirochete Obermeieri), yet most of the parasites can be grown upon artificial nutrient media, either readily (example, Bacterium typhi) or with more difficulty (example, Micrococcus gonorrhea). Of the inhabitants of the inanimate surroundings of man, i. e., the so-called saprophytes, most are easily cultivated on artificial media, similar to those employed for parasites, while others-as, for example, saliva bacteria and certain water bacteria-offer great or in part insurmountable difficulties in their cultivation. All nutrient media for bacteria must be rich in water; the presence of salts, and sources for the supply of carbon and nitrogen are indispensable. Most varieties of practical importance and all pathogenic varieties prefer a medium containing albumin which is faintly alkaline in reaction. In some cases the demands of the bacteria as regards the composition of the nutrient medium are very different. As shown by Mead Bolton, a number of water bacteria (Bacillus aquatilis Flügge and B. erythrosporus Flügge) are contented with water which has been twice sterilized in glass vessels (Z. H. 1, 76). Here an increase of the bacteria must occur at the cost of traces of impurities, or of the ammonia and CO, of the atmosphere. 2 Almost simultaneously Heraeus (Z. H. 1, 193) observed a variety of bacterium, which thrived in water which contained ammonium carbonate as the only source of carbon and nitrogen, being free from every organic nutrient material. Here, then, there occurred the elaboration of living substance from simple materials, just as occurs in the higher plants which work with chlorophyll aided by sunlight. Hüppe and Winogradsky have demonstrated by extensive studies the truth and importance of this observation. It appears that the energy necessary for the synthesis of albumin is obtained by oxidation of ammonia into nitric acid. Among the practically important bacteria, such unparticular ones are very few. Many allow albumin to be absent from the medium and are content with very simply composed nutrient solutions. Cultures upon such fluids were formerly much employed, and more recently Uschinsky has again experimented with simple nutrient solutions. The solution of Uschinsky is as follows: Water, 1000. Glycerin, 30 to 40. Sodium chlorid, 5 to 7. Calcium chlorid, 0.1. Magnesium sulphate, 0.2 to 0.4. Instead of this complicated solution, one may employ many simpler ones; for example, such as is recommended by Voges and C. Fränkel (Hyg. Rundschau, 1894, No. 17, 769), which is as follows:" Upon this (although there is no sulphur in the nutrient medium) the following grow: Even with the addition of those substances recommended by Uschinsky, other varieties, as diphtheria1 and tetanus, did not grow luxuriantly, but, by adding 3% to 4% of glycerin, the medium can be used for cultivating many varieties, even the tubercle bacillus. While cultures upon the simple nutrient media just described possess a great theoretical interest, yet they have been but little employed for diagnostic purposes. Very much more use is found for meat-infusion, peptonegelatin, agar, and bouillon (each with or without the addition of grape- or milk-sugar), also glycerin-agar, milk, and slices of potato. (For preparation see Technical Appendix.) We must always keep these on hand, since without them 1 Recently Uschinsky has apparently obtained upon his non-albuminous nutrient medium a good growth with the production of toxin in the case of a certain culture of diphtheria. no differential diagnosis is possible, and no variety can be considered regularly described which is not tested in its relation to all these nutrient media (with the exception of glycerin-agar). More rarely the following nutrient media are employed: Potato water, veal bouillon, fluid and coagulated blood serum, serum-agar, ascites-agar, blood-smeared agar, meat, pieces of bread, potato-pap, rice-pap, cooked or raw eggs. (See Technical Appendix.) Uncooked, sterile organs of animals are actually poorer nutrient media for most bacteria than when cooked (Livingood, C. B. XXIII, 980 and 1002). Studies upon nutrient media containing liver, kidney, thymus, adrenal extract, etc., have been carried out, but without giving anything of practical importance. Literature: (Wroblewski, C. B. XX, 528). 2. REACTION OF THE NUTRIENT MEDIA. As stated above, the great majority of bacteria, especially the pathogenic, prefer a neutral or faintly alkaline nutrient medium, and formerly the advice was always given to neutralize the nutrient medium with soda solution, employing sensitive litmus paper as the indicator-i. e., to add alkali until red litmus paper was turned faintly blue. Every chemist knows that no accurate terminal reaction for the titration of nutrient media containing phosphates is obtained with litmus; that, further, various litmus papers influence the result; and, finally, that the titration is practically impossible with gaslight. As early as 1891, N. K. Schultz had therefore advocated phenolphthalein as an indicator in the titration of agar. He recommended that 8-10 c.c. less of normal sodium hydroxid be added than is required for complete neutralization with the indicator. Such a medium is found to be suited to many bacteria, yet there are others which demand a complete neutralization (C. B. x, 52). Without having noticed this proposal, I came upon the same idea in 1892, during my investigations upon breadacids. Often since then, and exclusively since the autumn of 1894, in my institute there has been employed as neutral |