CHAPTER IV 



STUDY AND IDENTIFICATION OF BACTERIA GENERAL 

 CONSIDERATIONS 



IN order to study bacteria it is necessary to isolate them in pure cul- 

 ture. This may be accomplished by taking one or more loopfuls of 

 the material and mixing it in a tube of melted agar or gelatin. From 

 this first tube one or more loopfuls are transferred to a second tube of 

 melted agar or gelatin, and from this a third transfer is made, thereby 

 giving us tubes in which the distribution of the bacteria is one or more 

 hundred times less in the second than in the first tube, and equally more 

 dilute in the third than in the second. When we pour the contents 

 of the tubes into Petri dishes we would have the bacterial colonies on 

 the first plate so thick that it would be impossible to pick up a single 

 colony with a platinum needle without touching an adjacent one. On 

 the second plate the distribution might be such that we should have 

 discrete, well separated colonies, material from which could be taken up 

 on the point of the needle or loop without touching any other colony. 

 If the second plate did not meet these requirements, the third would. 



In clinical bacteriology we work almost entirely with organisms preferring blood- 

 heat temperature, hence it is necessary to use agar or blood-serum as standard media 

 for the obtaining of isolated colonies. Gelatin is of little value for this purpose in 

 medical work. In using agar it will be remembered that it solidifies at a temperature 

 slightly below 4oC. and does not melt again until it is subjected to a temperature 

 practically that of boiling. Again, if the temperature of the media exceeds 44C. it 

 may affect injuriously the organisms we wish to study. Consequently it requires 

 careful attention and quick work to inoculate the tubes, mix, transfer and pour into 

 plates within the limits of a temperature which injures the organisms, and one which 

 brings about the solidification of the agar. 



Again, we not only have colonies developing from organisms which 

 have been fixed at the surface as the agar solidified in the plate, but 

 more numerous ones developing from bacteria caught in the depths of 

 the media. Therefore we have superficial and deep colonies. Except 

 to the person of great experience, all deep colonies look alike and there 

 is at times great difficulty in deciding whether a colony is deep or super- 

 ficial. It is in the matter of trying to obtain information from the 



48 



