62 METHODS OF CULTIVATION OF BACTERIA. 



position under a cold tap till it solidifies as a uniformly 

 thin layer on the inside of the tube. Practically we deal 

 with a cylindrical plate of gelatine instead of a flat one. A 

 convenient form of tube for this method is one with a con- 

 striction a short distance below the plug of cotton wool 

 (Fig. 20). The great disadvantage of the method is, that if 

 organisms liquefying the gelatine be present, the liquefied 

 gelatine contaminates the rest of the plate. 



Separation by Agar Media i. Agar Plates. The only 

 difference between the technique here and that with gelatine, 

 depends on the difference in the melting points of the two 

 media. Agar, we have said, melts at 98 C., and becomes 

 again solid a little under 40 C. As it is dangerous to 

 expose organisms to a temperature above 42 C., it is neces- 

 sary in preparing tubes of agar to be used in plate cultures 

 to first melt them, by boiling in a vessel of water for a few 

 minutes, and then to cool them to about 42 C. before ino- 

 culating. The manipulation here must be rapidly carried 

 out, as the margin of time, before solidification occurs, is 

 narrow ; otherwise the details are the same as for gelatine. 

 Esmarch's tubes are not suitable for use here, as the agar 

 does not adhere well to the sides. If agar have 2 per 

 cent of a strong watery solution of pure gum arabic added, 

 Esmarch's tubes may, however, be used. 



2. Separation by Stroking Mixture on Surface of Agar 

 Media. -The bacterial mixture, instead of being mixed in 

 the medium, is spread out on its surface. The method 

 may be used both when the bacteria to be separated are 

 in a fluid, and when contained in a fairly solid tissue or 

 substance, such as a piece of diphtheritic membrane. In the 

 case of a tissue, for example, a small portion entangled in 

 the loop of a platinum needle is stroked in successive 

 parallel longitudinal strokes on sloped agar, the same 

 aspect being brought in contact with the agar in all the 

 strokes. Three strokes may be made on each tube, and 

 three tubes are usually sufficient. In this process the 

 organisms on the surface of the tissue are gradually rubbed 

 off, and when growth has taken place it will be found that 



