BACTERIOLOGICAL, PHYSIOLOGICAL, ETC. 351 BACTERIOLOGICAL, PHYSIOLOGICAL, ETC. Comparison of the Microscopical Method and the Plate Method of Counting Bacteria in Milk. J. D. Brew. (New York Agric. Exper. Stat., Bull. NO. 373, 1914, 1-38 ; through Monthly Bull. of Agric. Intelligence and Plant Diseases, 1915, 6, 624.) The number of bacteria was determined in 450 samples of milk by the usual plating method and also by counting the bacteria directly in microscopical preparations of the milk.I t was found that the microscope method almost invari- ably yielded higher figures than the plating method. The relative differences were veryirregular, but were generally greater when the bacteria were few in number. In samples of milk showing plate counts of less than 10,000 per C.C. of milk the count with the microscope showed approximately forty-four times as many indi- vidual bacteria, whilst those samples which gave a plate count of above 1,000,000 per C.C.only showed five or less times as many individual bacteria under the micro- scope. A large number of the bacteria counted under the microscope existed in clumps, and from the results obtained it would appear that each clump only gave rise to one colony.Beyond this, there was evidence that not all the bacteria were able to develop on the nutrient agar medium, and that this inability to develop tended to decrease and disappear as the number of organisms in the milk approached 1,000,000 per C.C. The microscope method, besides being more accurate when the number of organisms is low, has the further advantage of being rapid and relatively simple.w. P. s. Culture Media employed for the Bacteriological Examination of Water. I. : The Schardinger-Dunham Medium for Testing for the Presence of Hydrogen Sulphide forming Bacteria. E. M. Chamot and H. W. Redfield. (J. Amer. Chem. Soc., 1915, 37, 1606-1630.)-Three factors in the Schardinger medium were investigated-(1) Concentration of peptone, (2) that of the inorganic salt, and (3) the nature of the reaction of the medium.Irrespective of the inorganic salts present, and of the acidity of the medium, a concentration of between 3 and 4 per cent. peptone in the final inoculated and incubated medium appears to be best for the most rapid and energetic production of hydrogen sulphide. The addition of beef-broth to simple peptone media slightly increases its sensitiveness, but not in proportion to the increased trouble and labour involved ; it is therefore recommended that it be not included.If sodium chloride is used, the quantity must not be over 1.5 per cent. Cultures to which this salt was added showed an increased hydrogen sulphide production. In 3 per cent.peptone media the presence of from 0.5 to 1 per cent. of potassium chloride had a decidedly beneficial influence, and led to quicker and more uniform results than any other of the inorganic salts tried. Positive results of hydrogen sulphide formation by means of lead acetate paper may be obtained in eighteen hours. No hydrogen sulphide formation is obtainable in as long a period as seventy-two hours from natural waters which are truly ‘( clean,” while much is formed in from twelve to twenty-four hours with contaminated waters.The faeces of domestic animals contain bacteria which are capable of producing hydrogen352 ABSTRACTS OF CHEMICAL PAPERS sulphide from a simple peptone medium in as large amounts as in the case of the bacteria from human fmxs.The large amounts of hydrogen sulphide rapidly pro- duced by organisms of sewage appears to be not due primarily to members of the B. coli group. This group of' hydrogen sulphide producing bacteria do not actively ferment carbohydrates. Hence their presence is a supplementary test for gas- producers, and is of especial value in polluted waters in which the B. coli group is absent.Some evidence has been obtained which apparently indicates that hydrogen sulphide is more rapidly produced in waters containing a mixed bacterial flora than by the isolated pure cultures alone. H. F. E. H. Chemical Detection of Blood. D. Ganassini. (Boll. Chirn. Farm., 1914, 53, 777-781; through J. Chem. Soc., 1915, 108, ii., 295.)-The author has investi- gated Baecchi's test for blood (Arch.Int. Zdd. Ldgale, 1913, 4, 163), which is carried out as follows : Two C.C. of an aqueous alizarin-S-blue solution diluted to a mahogany yellow colour are mixed with about one-half the volume of 3 per cent. hydrogen peroxide solution. A little of the liquid to be tested is then poured slowly down the wall of the test-tube. If blood is present, gentle shaking rapidly renders the liquid intensely blue, the blue colour slowly fading and giving place to a red colour, which is moderately stable.This reaction is stated to give a positive result with 1 part of blood in 20,000, and to answer well with blood which has undergone alteration and become insoluble in water, the blood being then first dissolved in alcohol acidified with hydroohloric acid. Improved Hamin Test for Blood, with Notes on Some Recently Proposed Methods.W. Beam and G. A. Freak. (Bioclzem. J., 1915, 9, 161-170.)-The method described is a modification of that employed by Teichmann for the produc- tion of hemin crystals, and depends mainly on the precautions observed for insuring slow evaporation and crystallisation. A small quantity of the suspected material is placed at the bottom of a flat arsenic sublimation tube about 6 by 3 mm.and 35 mm. long. A few drops of acetic acid containing from 0.01 to 0.1 per cent. sodium chloride are added, and a, very fine cotton-thread adjusted so that its upper end is near the top of the tube and the lower end reaches to the bottom of the liquid. The thread should be everywhere in contact with the tube, to which it adheres readily by being moistened with the liquid.The adjustment is readily made by means of a glass rod, one end of which is drawn out for the purpose. The tube is now placed in a rack, and allowed to remain until crystallisation has taken place. Before placing the thread in position, the solution of the blood may be aided, if desired, by cautiously heating the tube over a small flame, loss of the liquid by violent ebullition being prevented by holding the finger tightly over the mouth of the tube.The clear liquid, filtered by its passage through and along the thread! is slowly drawn up by capillary attraction to the mouth of the tube. Under these conditions evaporation takes place so slowly that, even when the temperature of the atmosphere is above 38' C., it is complete (if the tube is held in an upright position) only after twelve to twenty-four hours or more.The rate ofBACTERIOLOGICAL, PHYSIOLOGICAL, ETC. 353 evaporation is readily controlled, within wide limits, by altering the dimensions of the tube and the angle at which it is supported. The crystals usually begin to appear on or near the upper half of the thread, and of sufficient size to be distin- guished with a magnification of 75 diameters, in about half an hour; and they ultimately become so large that, in place of requiring a magnification of 250 to 300 diameters, as usually recommended, they may readily be seen with one of 25 diameters, or even, in many cases, with a good hand-lens. The large size of the crystals is due in great part to the slow evaporation of the liquid ; but it is also a result of the removal, by the thread, of the interfering action of the blood-albumin on crystallisation.Examination of the thread at the end of the experiment shows that the soluble, non-crystallisable constituents of the blood are carried to its upper end, and that the maximum growth of haemin crystals is found lower down.If the solution is sufficiently weak, the first crystals form at the point of greatest concentra- tion-the upper end of the thread; but as the albuminous matter accumulates these cease to grow, and the largest and most numerous crystals are found later about midway down the tube, The following precautions should be observed : The tube should be thoroughly clean and dry, and the thread clean and as fine as can be obtained. The solution of the blood in acetic acid should be of such a degree of dilution that the liquid is faintly pink.If the method is to be applied experimentally to fresh blood, the latter must be completely dried before the addition of the acid, and care should be taken to work with very dilute solutions. Recent methods dealing with the hemin test by W.F. Whitney (Boston Medical and Surgical J., 1912) and C. T. Symons (Biochm. J., 1913, 7, 596) are criticised, and the general conclusions drawn are as follows : Of the reagents which have been recommended for use in Teichmann’s test for blood, acetic acid is the only one on which entire reliance may be placed if the method by evaporation is employed.Acetic acid, without salt of any kind, suffices for the test with blood-stains, fresh or old, provided the stains have not been extracted with water. As a precautionary measure it is advisable to use the reagent containing a minute proportion-about 0.01 per cent.-of sodium chloride. No advantage results from the substitution of other salts for sodium chloride in the acetic acid reagent.Iodides may not be used to substitute chlorides or bromides in the above. This does not apply to the reagent made with lactic acid. Where positive results have been obtained from the use of an acetic acid reagent containing iodide, the effect is apparently due to the chloride naturally present in the blood or as impurity in the reagents. The difficulties experienced with Teichmann’s test when applied to stains, both fresh and old, are due chiefly to the too rapid evaporation of the solvent, end to a less extent to interference of the albuminous matter of the blood with the crystal- lisation.Evaporation should be extremely slow, and when carried out in the manner detailed, which also eliminates the harmful effect of albumin, crystals are obtained with the greatest certainty and of remarkably large size, even though only a minute amount of blood be present.The test as described was found to be equally applicable to old blood-stains (twelve years), stains partially removed by soap and water, or heated to l l O o C., or354 ABSTRACTS OF CHEMICAL PAPERS mixed with earth, or to old stains on rusty iron which had been exposed to strong sunlight and atmospheric conditions for several days.H. F. E. H. Estimation of the Oxygen Demand by the Sodium Nitrate Method in Stoekyards, Tannery, and Corn Products Wastes. A. Lederer. ( J . Ind. and Eng. Chm., 1915, 7, 514-516.)--The method depends on the denitrification of sodium nitrate by the sewage bacteria; the amount of nitric oxygen absorbed during incubation is the same as in the usual method of incubating the sewage with the addition of water containing dissolved oxygen.Quantities of the sewage are treated with successively increasing amounts of nitrate, and, during the incubation period (ten days), the sediment, odour, and gas formation are observed. If the sediment becomes septic ” (ie.? black in colour), the bottle with the next higher nitrate content is selected for the estimation of the residual nitrate-nitrite oxygen.A large excess of added nitrate leads to an increased oxygen consumption, but the excess consumption is insignificant when compared with that obtained by the water- dilution method. The author discusses the application of the nitrate method to works effluent8 such as are met with in the district of Chicago. The effluent from packing-houses is usually mixed with domestic sewage, and the estimation of its oxygen requirement presents no difficulty; sulphurous acid is often present in the effluent from factories where corn-starch and glucose are made, but the injurious effect of this acid and the acidity produced by the fermentation of the carbohydrates may be eliminated by the addition of sodium bicarbonate. Effluents containing alkali hydroxides should be neutralised with hydrochloric acid before the estimation is commenced ; the same applies to lime effluents from tanneries. w, P. s.