ZOOLOGY AND BOTANY, MICROSCOPY, ETC. 833 



When developed the cohmies may be examined with low powers, and 

 even photographed. Individual colonies may also be taken out for further 

 examination. The estimation of the number of germs is made in the 

 ordinary way. The enumeiation of the colonies may be made by placing 

 a piece of paper divided into parts of a centimetre and multiplying tlie 

 number in a given square by the superficies, or a special ajiparatus devised 

 by the author may be used. 



If instead of gelatin agar be desired, it is advisable to add to each 10 cm. 

 of agar 2 or 3 drops of a neutral sterilized solution of gum arable or isinglass. 

 If anaerobic bacteria are to be studied, the central space must be filled with 

 gelatin while the tube is still in the ice-water. 



The advantages of this method over the ordinary plate cultivation are 

 its safety against impurities, the simplicity and rapidity of its execution, 

 the small amount of apparatus, and its facilit/ of transport. 



Bacteriological experiments with coloured nutrient media.* — It is 

 well known, says Dr. A. Spina, that indigo-blue turns white when acted on 

 by reducing agents, and recovers its former colour on exposure to the air. 

 It was this property which induced the author to make some experiments 

 in order to ascertain if it could not be made available for cultivation 

 research. 



A test-tube was half filled with the following solution : — 0-5 phosphate 

 of potash, 0*5 sulphate of magnesia, 1*0 tartrate of ammonia, and 100 dis- 

 tilled water ; and this stained with two or three drops of a watery solution 

 of sulphindigotate of soda. The coloured fluid was inoculated with some 

 drops of putrid blood, the test-tube plugged with cotton wool, and incubated 

 at 38°. After three or four days the fluid was decolorized, and the bacteria 

 much augmeoted in number. The nutrient medium acquired the appearance 

 of thin milk, and only on the surface was a blue layer evident. If the tube 

 was shaken the fluid became blue again, and white when put in the incu- 

 bator again. Methylen-blue behaves in a manner quite similar. 



The objection might be raised that the loss of oxygen was due, not to 

 the bacteria, but to the nutrient mediiim. That this is not the case the fol- 

 lowing exj)eriments prove : — (a) If a test-tube filled with the coloured 

 medium be inoculated, and after having been decolorized in the incubator, 

 and then sterilized, it rapidly becomes blue, but no further decoloi-ation 

 ensues, although it remains several clays in the incubator, (h) If a test-tube 

 filled with the coloured medium, and having been sterilized, be kept for 

 a week at a temperature of 38°, no decoloration of the fluid takes place. 

 Experiment also shows that the loss of oxygen was not produced by means 

 of the chemical products of the proliferating bacteria. It was remarked 

 before that sliaking or warming restored to the decolorized fluid its original 

 hue. This is explicable only on the assumption that the white methylen-blue 

 or indigo takes uj? oxygen, and the correctness of this view is shown by the 

 following experiment: — -A glass tube filled with the stained and inoculated 

 fluid is melted up at the open end after all air has been expelled, and de- 

 colorized in the incubator. In this case shaking will not bring back the 

 blue colour. 



From fluid the author passed to solid media, of which he employed twc — 

 (1) meat-peptoue-gelatin and (2) meat-peptone-agar. A weak solution of 

 the former, stained and inoculated, and kept at a temperature of 22°, became 

 decolorized below the colony in about three days. (The bacteria used were 

 developed on potato, and from the air, but no name is given.) In a few days 

 the decolorized column was quite large, but at the surface the layer in con- 



* Ceutralbl. f. Bacteriol. u. Parasituuk., ii. (1887) pp. 71-5. 



