APPENDIX. 4oy 



because when it is used there is never any danger of drawing toxic fluids 

 into the mouth. 



Glass needles are especially useful when ansrobic organisms are being 

 dealt with, as the smooth surface of the glass does not allow of oxygen 

 being carried down with it along the track, which closes up as soon as the 

 needle is withdrawn. 



To Inoctilate Solid Culture Media. 



The test tube or flask is held inverted in the left hand, and the plug of 

 cotton wool is twisted once or twice in the mouth of the test tube to break 

 down any adhesions between it and the neck of the vessel. If the plug is at 

 all dusty, it is well to singe the surface by passing it rapidly through a 

 flame before removing it from its position. Wadding bums very rapidly, 

 and must be extinguished at once. The plug is removed and held between 

 two of the unoccupied fingers of the left hand, great care being taken that 

 no part of the plug that passes into the test tube shall come in contact 

 with any source of infection other than the air itself. At the same time 

 this portion of the plug is directed downwards, in order to avoid any falling 

 germs that may be present in the atmosphere. The platinum or glass 

 needle with its charge of seed material, is plunged straight into the gelatine 

 mass, then carefully withdrawn and the plug replaced. AATiere the seed 

 material is also in solid gelatine, the two tubes may be held inverted in the 

 left hand, one between the thumb and finger, the other between the first and 

 second, the plugs being held between the second and third and third and 

 fourth fingers. 



Methods of Cultivating Ancerobic Organisms. 



When it is necessary to cultivate such an organism as that of malignant 

 oedema or of tetanus the experimenter has to resort to the methods 

 used for keeping out the air. To isolate these organisms, Koch first 

 used the ordinary plate cultures, placing at irregular points on the 

 surface of the gelatine, thin sheets of mica that had previously been 

 sterilized (p. 172). Fraenkel, to render the exclusion of oxygen more 

 perfect, ran a little melted paraffin round the margins of these plates. By 

 filling a tube with gelatine or agar, Liborius succeeded in obtaining cultures 

 of anaerobic organisms at the lower part of his culture medium. If 

 the tube is filled with the solid medium close up to the plug, and the air is 

 carefully excluded by means of melted paraffin poured over the plug, this 

 method often succeeds exceedingly well. It is, however, difficult to obtain 

 pure inoculating material from such growths without removing the contents 

 of the tube or breaking it up altogether. 



One of the simplest methods of isolating ansrobic bacteria in gela- 

 tinized media is that described by Fraenkel, who uses a test tube fitted up like 

 a wash-bottle, with a long and a short glass tube passing through an india- 

 rubber cork. This, after being carefully sterilized, has about 10 cc. of 

 nutrient gelatine poured into it. An inoculation is made, and the india- 

 rubber cork, with its two tubes, is pushed home into the mouth of the test 

 tube, and the whole is luted down with paraffin, great care having been 

 taken to sterilize, by heating in a flame, those parts of the tubes that are 

 to go down into the test tube. A stream of hydrogen, purified by being 

 washed in an alkaline solution of pyrogallic acid, is passed through this 

 liquefied gelatine (kept at 35° C.) for four or five minutes. The tubes are then 



