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 diphtheria 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 gelatin (and agar) a medium which contains just so much sodium hydroxid as is required to produce a minimum reddening of phenolphthalein. All the plates in this atlas are prepared with the use of such nutrient media for such cultures. This was done after the investigation of five important bacteria had indicated to us that additions of acids or alkalis did not improve the growth. Since then I have had the great majority of the bacteria described in our atlas systematically studied as to their ability to grow on the following nutrient media, by Dr. Winkler (Dissert. Würzburg, 1896): 1. On "neutral" agar, neutralized with normal soda with the employment of phenolphthalein. 2. On "acid" agar-i. e., on neutral agar to which was added 10 and 20 c.c. of normal sulphuric acid per liter. 3. On a sort of alkaline agar-i. e., on neutral agar to which was added 10 c. c. of normal alkali solution per liter. The result, as indicated briefly in Table I (at end of book), is that almost all bacteria grow well on these three media. In every case the nutrient media neutralized by means of phenolphthalein as an indicator may be implicitly employed as universal nutrient media; moreover, the virulence of those varieties tested by us (Bac. anthracis, Bact. coli, Bact. of mouse septicemia and chicken cholera) is well preserved thereon. This method has the advantage over other methods in that it is easily carried out (compare Technical Appendix), and that it represents a very exact point, namely this, where all free acids and acid salts are changed into neutral salts (mono-sodium phosphate into di-sodium phosphate). Other recommendations-Timpe C. B. XIV, 845; Heim, Lehrbuch, p. 73; Deelemann (A. G. A. XIII, 374)-appear to have no advantage. If an acid nutrient medium is to be employed, we think it best to add 10-20 or 30 c.c. of normal acid to a medium previously neutralized with phenolphthalein. According to Winkler, the first degree of acidity is well borne by almost all bacteria. According to the certainly not super ficial reports of Schlüter (C. B. x1, 589), which were substantiated by subsequent publications, many bacteria bear much higher proportions of acid; according to observations in our own institute, as high as 100 c.c. of normal acid per liter. Nutrient media containing sugar usually favor the production of acid, which, according to Hellström, soon becomes so abundant that the micro-organisms are killed. Acid nutrient media are to be used for yeasts and molds and whenever it is wished to isolate a new bacterium from an acid nutrient substance. For counting the germs in air, soil, water, milk, etc., a neutral medium is always employed. 3. INJURY TO BACTERIA BY CHEMICAL SUBSTANCES. We have already learned that too large a proportion of either acid or alkali1 interferes with growth, or, if still stronger, produces death. Most varied chemicals, in certain concentrations, operate similarly. Those which are strongly active are called antiseptics or disinfectants. Usually, with Hüppe, the following grades of influence are distinguished: 1. Growth is not interfered with, 2 but the pathogenic or zymogenic functions are weakened: Weakening, attenuation. 2. The organisms are unable to increase, but are not killed Asepsis. : 3. The vegetative forms of the micro-organisms are destroyed, but not the resting forms: Antisepsis. 4. Vegetative forms and spores are both killed: Sterilization or Disinfection. 'Fermi (C. B. XXIII, 208) has published an extensive table regarding the sensitiveness of various micro-organisms to acids, alkalis, and various poisons. Unfortunately, he gives the number of drops of solutions of various percentages which inhibit the growth of bacteria in 5 c.c. of agar. At times there occurs a transitory or permanent interference with growth; in other cases briefly acting antiseptics, also heat, cold, etc., cause a retardation of subsequent growth without producing a weaken Since the diagnostic value of the test of the resistance. to chemicals plays only a modest rôle-various hopes in this direction remaining unfulfilled-this section must be very brief. To determine the minimal concentration of a chemical poison which produces asepsis—i. e., prevents growth—the following procedure is adopted: A solution of the disinfectant-for example, 1%-is employed; and of this, 1, 0.5, 0.3, 0.1 c.c. is added to 10 c.c. of liquefied gelatin. This nutrient medium, containing now 0.1, 0.05, 0.03, 0.01% of the disinfectant, is used for stab, streak, and plate cultures. Inoculations may also be made with material containing only spores (material freed from all bacilli by heating half an hour at 70°), and thus it may be determined whether the spores grow out in cultures. Behring has devised the following practical method of making these tests: A drop of fluid nutrient medium,for example, serum,-infected with the organism to be tested, is removed before the addition of the antiseptic. and suspended from the under side of a cover-glass on a hollow-ground slide, sealing it with a little vaselin (Technical Appendix). Then by degrees there is added to the tube of serum, increasing known quantities of the disinfectant. After each addition and thorough shaking a drop culture is prepared. The growth in each drop can be examined after being kept twenty-four or forty-eight hours in the incubator. If the concentration necessary for antisepsis is to be determined, the organisms to be examined are grown in bouillon, and to 10 c.c. of the bouillon, free from spores, and filtered through asbestos to remove any clumps of bacteria, various quantities of a solution of the disinfectant. of known strength are added. From each of the tubes after one, five, ten, fifteen, thirty minutes, one hour, etc., a platinum loopful of the material is removed, and placed in 10 c.c. of lukewarm liquefied gelatin, from which a plate culture is prepared. If it is suspected that a trace of the disinfectant carried in the drop renders the gelatin aseptic and so leads to an apparent death of the microorganisms, the result may be controlled by inoculating |