SEPARATION OF BACTERIA BY AGAR MEDIA. 57 



with a constriction a short distance below the plug of cotton 

 wool (Fig. 22). The great disadvantage of the method is, that 

 if organisms liquefying the gelatin be present, the liquefied 

 gelatin contaminates the rest of the gelatin. 



Separation by Agar Media. — i. Agar Plates. — The only 

 difference between the technique here and that with gelatin 

 depends on the difference in the melting-points of the two 

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

 soHd a little under 40° C. As it is dangerous to expose organ- 

 isms to a temperature above 42° C, it is necessary in preparing 

 tubes of agar to be used in plate-cultures to first melt the agar, 

 by boiling in a vessel of waterfor a few minutes, and then to 

 cool them to about 42° C. before inoculating. The manipulation 

 must be rapidly carried out, as the margin or time, before solidi- 

 fication occurs, is narrow ; otherwise the details are the same as 

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

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

 cent of a strong watery solution of pure gum arable is added, 

 Esmarch's tubes may, however, be used. 



2. Separation by Stroking Mixture 07i 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 proportion 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 in the later strokes 

 the colonies are less numerous than in the earlier, and sufficiently 

 far apart to enable parts of them to be picked off without the 

 needle touching any but one colony. When, as in the case of 

 diphtheritic membrane, putrefactive organisms are likely to be 

 present on the surface of the tissue, these can be in great part 

 removed by washing it well in cold water previously sterilised 

 {vide " Diphtheria "). In the case of liquids, the loop is charged 

 and similarly stroked. Tubes thus inoculated must be put in 



