760 DR JAMES BUCHANAN YOUNG ON THE 



proceeded with immediately on the arrival of the samples at the laboratory. In every 

 case cultivations were started within four hours from the time of taking the sample, and 

 in the majority of cases within two hours. This was important in order to minimise any 

 error arising from the rapid multiplication which takes place in the bacteria, as pointed 

 out by Fraenkel. 



Details of Bacteriological Examination. — For the cultivation of aerobic organisms 

 I found that plate cultivations in Foster's boxes were most suitable, for the reason that 

 many of the bacteria caused such rapid liquefaction of the nutrient medium that difficulty 

 was experienced in counting them in roll-culture, owing to the running together of 

 neighbouring liquefying colonies. Subcultures were also more easily made from the 

 surface of the nutrient jelly in the Foster's box than from the surface of a roll-cultivation. 

 For anaerobic forms roll-cultivation had, of course, to be resorted to. 



500 cubic centimetres of distilled water is put into a plugged and sterilised flask of 

 about one litre capacity, and sterilised in the autoclave by exposure to streaming steam 

 for ninety minutes, or by exposure to steam at fifteen pounds pressure for one hour. 

 The water having thoroughly cooled, have ready a plugged and sterilised test-tube. 

 Flame the lip and plug of the test-tube carefully, and with a sterilised metal spatula or 

 knife cut from the centre of the sample of soil a quantity (varying from about '5 gramme 

 at a depth of one foot from the surface to 5 grammes at a depth of eight or nine feet), and 

 add it with all precautions to the sterilised test-tube, and replug. 



The plugged test-tube containing the earth is now carefully weighed, and its 

 weight noted. The contents of this tube are now added, with all the usual precautions 

 to avoid contamination, to the flask containing the 500 cubic centimetres of sterile 

 distilled water. Now weigh the plugged test-tube. The difference between the first 

 and second weights is the weight of earth added to the water in the flask. The earth 

 so added to the water is thoroughly mixed with the water by shaking, so as to wash all 

 the bacteria out of the portion of soil added to the flask. When the earth is reduced 

 to the finest possible state of division, and is thoroughly mixed with the water, the 

 suspension is ready for inoculating the fluidified culture medium. 



With a sterilised pipette, each drop from which is equivalent to i^th part of a cubic 

 centimetre, a quantity of the suspension is sucked up, and the required number of drops 

 added to a test-tube containing about eight cubic centimetres of fluidified nutrient jelly, 

 all precautions being taken to avoid contamination. Having added the fixed number of 

 drops, the test-tube is replugged, and the suspension remaining in the pipette is returned 

 to the stock flask. This process is repeated until the desired number of inoculations has 

 been made. The stock flask containing the suspension is thoroughly shaken up every 

 time before introducing the pipette, in order to obtain a thorough mixture, and so ensure 

 that a fair sample of the whole is taken. 



The tubes containing the fluidified jelly, so inoculated, are now cautiously shaken 

 ii p to ensure thorough mixture, and the inoculated medium from each carefully poured 

 into a sterile Foster's box and allowed to solidify. When this jelly has set, the capsules 



