SEPARATION OF AEROBIC ORGANISMS 59 



making the smears. The characters of the colonies which appear 

 on the plates can be examined with a hand-lens, magnifying 

 about 6 diameters. In some cases examination under a low- 

 power of the microscope is an advantage ; the plate in the in- 

 verted position can be put on the stage of the microscope for this 

 purpose. For the culture of special organisms, as afterwards 

 detailed, the agar or other medium is smeared with sterile serum 

 or blood according to the growth requirements of the organism, 

 or the serum or agar is added before the medium is poured. 



The principle just described may be applied also to agar in 

 tubes, but the results generally are not so satisfactory, and the 

 characters of the colonies cannot be so readily studied. In this 

 case two or three agar tubes are taken, a platinum loop is 

 charged with the material to be examined, and a series of 

 undulating strokes is made from below upwards on the surface of 

 the agar, one tube after the other being used without recharging 

 the needle. The tubes after inoculation should be kept in the 

 upright position, so that the water of condensation is not allowed 

 to run over the surface. 



In the second method, which we have called the "dilution 

 method," the bacteria are added to the medium when liquid, and 

 mixed by careful shaking ; the inoculated medium is then poured 

 out into a capsule and allowed to solidify. As in this case the 

 organisms are diffused throughout the medium, some of the 

 colonies grow on the surface of the medium — " superficial 

 colonies " — others in its substance — " deep colonies." These often 

 show different appearances, which are sometimes used in the 

 systematic description of an organism. As the bacteria may 

 produce far too many colonies to allow separation, means must 

 also be used for making different dilutions, a separate plate 

 being prepared for each. If gelatine is used, the medium in 

 tubes is melted and kept in a beaker of water at about 28° C. 

 If agar is used, the medium is melted thoroughly by boiling in 

 a vessel of water and then allowed to cool to about 43° C, at 

 which temperature the inoculations are made. The following 

 are the details : — 



The contents of three tubes, marked a, b, c, 1 are liquefied as above 

 described. Inoculate a with the bacterial mixture. The amount of the 

 latter to be taken varies, and can only be regulated by experience. If 

 the microscope shows enormous numbers of different kinds of bacteria 

 present, just as much as adheres to the point of a straight platinum 

 needle is sufficient. If the number of bacilli is small, one to three loops 



1 For marking glass vessels it is convenient to use the red, blue, or yellow 

 oil pencils specially made for the purpose. 



