248 LABORATORY EXERCISES IX BACTERIOLOGY. 



prehensive work it should not be neglected to arrange not one, but several plated 

 cultures of the same material upon agar as well as upon gelatine, with a view of placing 

 one culture at incubator temperature, one at room temperature, and a third in anaerobic 

 surroundings. Each should be prepared from as nearly equivalent original material 

 as possible, both in quantity and in kind, and the infected material thoroughly diffused 

 through the liquefied medium before plating. (One must not forget to have the 

 liquefied medium of as low a temperature as possible, lest harm be done to the bacteria 

 diffused in it ; and after diffusion, when about to pour the liquid upon a plate or in 

 a dish, let it be kept in mind to flame and cool the lip of the tube for fear of contam- 

 inating the medium passing over it.) In addition to these agar and gelatine prepara- 

 tions, smears are to be made upon solidified blood-serum and submitted to incubator 

 and room temperatures. 



After proper growth has been attained and one can distinguish one type of colony 

 from another, the sterile needle is to be used for transferring some of the organisms 

 from each to separate tubes of sterile medium, identical with that used in the plated 

 culture. Care is to be exercised in this step that the tip alone of the needle is touched 

 only to that colony from which transfer is to be made, and that all other colonies in 

 the preparation are avoided. It is not essential to have visible fragments or the 

 entire colony on the needle. It will be found of advantage, before attempting the 

 procedure, to bend the tip of the needle at right angles to its length, this shape lending 

 itself to the operation. After the needle with the adhering organisms is withdrawn 

 from the culture, it is introduced to the tube of fresh medium and a stroke or stab 

 inoculation made, after which the needle is to be at once flamed. Each tube thus 

 inoculated from the preliminary culture is for a time subjected to the same conditions 

 as those which proved successful for the plated culture, and after sufficient develop- 

 ment of the pure cultures the colonies in each are noted in the study of the gross and 

 minute features of the organisms composing them. 



The procedure is difficult only when the colonies to be separated are very close 

 to each other in the plated culture; and care in manipulation alone will then insure 

 success. Should one of the tubes inoculated prove to be mixed, the measure is to be 

 repeated until successful. 



Exercise 7 1 . Several loopfuls of Bacillus prodigiosus and Micrococcus 

 pyogenes are mixed in a test-tube containing a few cubic centimeters of 

 sterile water. Let each student diffuse a loopful of the fluid in liquefied 

 gelatine and plate in one or other manner, subsequently producing from the 

 plate a pure culture of each organism for inspection. 



2. Salomonsen's Capillary Tubes. Salomonsen suggested that after diffusion 

 of the infected material has been effected in a sterile liquefied medium, a small amount 

 of the latter be drawn into a sterile capillary glass tube instead of being spread out 

 over a surface. The principle is, of course, the same as in any of the plating methods, 

 the germs being disseminated, however, in a linear manner instead of over a plane. 

 After the tube has been filled with the inoculated medium, the ends may be sealed 

 in the flame or closed by wrapping a little sterilized cotton or paper closely about 

 them. It must be realized that in such tubes, even if closed in the latter manner, 

 but little air access is permitted to the organisms in the medium any distance from 

 the ends; and as a matter of fact it is especially in the isolation of anaerobic varieties 



