408 APPENDIX. 



sealed in a flame, and a "tube plate" cultivation is made by distributing 

 the gelatine on the walls of the test tube. In this way excellent anaerobic 

 cultures may be obtained. The advantage of this method is that fresh inocu- 

 lations may be readily made from the growths that appear on the thin layer 

 of gelatine that covers the wall of the tube. Anaerobic cultures may also be 

 obtained by drawing the media, that have been de-oxygenated by means of 

 hydrogen or by means of the air pump, into capillary tubes, which are then 

 sealed so as to prevent the access of any fresh oxygen. Fluid media, of 

 course, may be used in exactly the same way. Where an air pump or 

 water exhaust apparatus is obtainable, an ordinary tube, with thick walls 

 and a long neck, may be used in which to make the culture ; but a com- 

 bination of these methods, as a rule, gives the best results. To such a tube 

 may be fitted a "T" piece with a stop-cock, through which a vacuum 

 is first created ; then hydrogen is allowed to come in, the process being 

 repeated several times until all the oxygen is removed. One very simple 

 method of obtaining a solid medium in which some anaerobic organisms 

 will grow is to add a small quantity of glucose (i to 2 per cent.) to agar- 

 agar or gelatine. For the more complicated methods we refer the reader 

 to special treatises. 



Various methods have been utilized for preserving anaerobic cultures or 

 of obtaining "needle" cultivations of such cultures in gelatine, &c. In 

 some of the earlier experiments on abiogenesis a layer of warm oil was 

 used to prevent the access of air to the nutrient medium, and this method 

 is still sometimes used when inoculating a solid medium with an anaerobic 

 organism. A puncture cultivation is made with a glass needle, and then 

 a layer of a couple of inches of boiled and cooled olive oil is carefully poured 

 over the surface. Roux used two methods one physical, the other biological 

 for the attainment of this object. For the former he draws out one end of 

 a pipette into a long capillary tube, then a short distance from the other 

 end a constriction is made ; the whole is carefully heated to 1 50 C. in the 

 hot air chamber, or by a Bunsen burner, the point of the pipette being 

 closed by fusion, and the other end plugged with sterilized cotton-wadding. 

 This pipette is then filled with sterilized nutrient gelatine that has been 

 brought to the boiling point in order that as much air as possible may 

 be driven off. To do this the capillary point is broken with sterilized 

 forceps ; the fluid is drawn up to the level of the constricted portion of 

 the tube by means of suction ; the capillary end is then fused, after which 

 the tube is also fused at the constricted part at the other end. Such tubes 

 may be kept for an indefinite period. To inoculate the gelatine in this 

 tube one end is broken, a fine glass needle on which is the material to be 

 inoculated, is carefully introduced and the end is again sealed. Under 

 these conditions no air can make its way to the organism, or to the medium 

 into which it is inoculated. 



Roux's second, or biological, method is to boil a quantity of nutrient 

 agar in a test tube, and then cool it as quickly as possible in cold 

 water. It is then inoculated with the anaerobic organism on a smooth 

 glass needle. A layer of melted nutrient gelatine is poured on the surface, 

 and when this is cooled a drop of a broth cultivation of " Bacillus subtilis " 

 is run on to the surface from a capillary pipette. The tube is then 

 fused, or the cotton-wadding plug is rendered impervious by being luted 

 with warm paraffin. As the Bacillus subtilis develops and grows it uses 

 up the oxygen at the surface. The organism below receives none, and 



