60 METHODS OF CULTIVATION OF BACTERIA 



of the mixture may be transferred to the medium. Shake a well, but 

 not so as to cause many fine air-bubbles to form. Transfer two loops of 

 medium from a, to b. Shake b and transfer five loops to c. The plugs of 

 the tubes are in each case replaced and the tubes returned to the beaker. 

 The contents of the three tubes are then poured out into three capsules. 

 In doing so the plug of each tube is removed and the mouth of the tube 

 passed two or three times through the Bunsen flame, the tube being 

 meantime rotated round a longitudinal axis. Any organisms on its rim 

 are thus killed. The capsules are labelled and set aside till growth 

 takes place. 



For accurate work it will be found convenient to carry out the dilutions 

 in definite proportions. The following is the procedure which we have 

 found very serviceable : In a number of small sterile test- tubes '95 c.c. 

 sterile water is put. To the first tube we add '05 c.c. of the bacterial 

 mixture. The contents of the tube are well shaken up, and the pipette 

 is sterilised by being washed out with boiling water. It is allowed to 

 cool, and "05 c.c. of fluid is transferred from the first tube to the second. 

 By a similar procedure '05 c. c. is transferred from the second to the third, 

 and so on. There is thus effected a twenty-fold dil ution in each successive 

 tube. After these steps have been carried out, a definite amount, say 

 '05 c.c, is transferred from each tube to a tube of melted medium, — the 

 medium being afterwards plated and the colonies counted when growth 

 occurs. The number of tubes required will vary according to the number 

 of bacteria in the original mixture, but usually four or five will be 

 sufficient. 



Enumeration of Colonies. — The dilution method just 

 described supplies the means of counting the number of living 

 bacteria in a fluid, the proviso being always made that they 

 are capable of growth in the medium used. For pathogenic 

 organisms one of the agar media is generally used, whilst in the 

 case of water, gelatine is most suitable. The dilutions are made 

 by the .quantitative method, and a given amount, say '1 c.c, is 

 taken from one of the dilutions and transferred to a tube of 

 melted medium, and, after gentle mixing, the medium is poured 

 in a Petri capsule. It is advisable to take samples in this way 

 from two or even three of the dilutions. To aid the counting of 

 the colonies which develop, various patterns of ruled glass plates 

 have been introduced. If the ruling is in the form of squares 

 of given size, the number of colonies in several squares is counted, 

 and as the area of the Petri dish can be got by multiplying the 

 square of its radius by 3-iJ-, the whole number can then be 

 calculated. Petri dishes are rarely flat, and unequal distribution 

 of the colonies has accordingly to be taken into account. The 

 dilution to be selected for taking the sample for plating will 

 depend upon the relative abundance of the organisms in the 

 original fluid. 



Separation of Pathogenic Bacteria by Inoculation of 

 Animals. — It is found difficult, and often impossible, to separate 



