MECHANICAL METHODS 



77 



Suppose it be required to isolate the organisms present in a drop of water. Add 

 the water to a tube A containing 10 c.c. of sterile broth. Thoroughly mix the water 

 with the broth by shaking the tube. The organisms present in the drop of water 

 are now diluted in 10 c.c. of broth, and since 1 c.c. corresponds to 20 drops, each 

 drop of broth contains 20 x 10, i.e. 200 times fewer organisms than the drop of 

 water under investigation. Now transfer one drop of the mixture from the tube 

 A to each of a series of tubes (B, B', B", ...) containing broth. If the original 

 drop of water contained 200 organisms, every drop of fluid in tube A will contain 

 igfl=l organism, so that every drop transferred from A to the series B, B', B", 

 etc., will carry one organism, and that organism will grow in the tube B, B', or B" 

 to which it has been transferred and will give rise to a pure culture. But if the 

 original drop of water contained only 50 organisms, then only one tube in four of 

 the series B, B', B", etc. will give rise to a pure culture. On the other hand, 

 suppose the drop of water contained a larger number than 200 organisms, it will then 

 be necessary to dilute further until in fact one drop contains not more than one 

 organism. Thus 10 drops from A will be transferred to a broth tube B, and a 

 series of sub-cultures C, C', C", etc., will be sown, each with one drop of broth 

 from B. 



2. Isolation by dissemination. 



The method of isolation by dissemination is due to Koch. 



For its application the use of solid media is necessary. It may be carried 

 out in one of two ways : either the medium may be liquefied and then sown, 

 or the organisms may be distributed directly over the surface of the medium. 



1. Dissemination in liquefied solid media. 



If it be required to isolate all the organisms present in a drop of water, 

 the method would be as follows : Transfer the drop of water to a tube of 

 sterile gelatin previously liquefied in the water bath, and mix the water 

 and gelatin thoroughly by rolling 

 the tube between the hands. The 

 organisms present in the water will 

 now be distributed through the gela- 

 tin. Pour the gelatin in a thin layer 

 on to a sterile glass plate, and cool 

 it rapidly. The organisms will be 

 scattered and held in the layer of 

 gelatin like the almonds in a piece 

 of nougat. If the plate be kept at 

 a suitable temperature, each organ- 

 ism will grow in an isolated position, 

 and will give rise to a colony com- 

 posed of a number of micro-organisms 

 all derived from the one organism 

 which originally settled in that 

 position, and therefore to a pure 

 culture (fig. 68). It will then be 

 easy to pick out each colony separ- 

 ately and sow it on a new medium. 



There are in practice several ways 

 of carrying this out, but the following 

 rules must always be observed : 



1. After liquefying the gelatin or 



agar and before sowing it, let it cool sufficiently (to 30-40 C.), to prevent 

 the organisms being killed by the temperature of the medium. 



2. Avoid contaminating the culture. 



3. Protect the plates from dust. 



FIG. 68. Isolated colonies of micro-organisms 

 on a gelatin plate (two-thirds natural size). 



