78 



ISOLATION OF AEROBIC MICRO-ORGANISMS 



A. Petri dishes. Method recommended. 

 (i) Using gelatin as the culture medium. 



Apparatus required. (a) Three Petri dishes (fig. 43) wrapped in filter 

 paper [or packed in a copper cylinder (Chap. I.)] and sterilized in the hot 

 air sterilizer (a number of these dishes should always be kept ready 

 sterilized). 



(b) Three sterile Pasteur pipettes. 



(c) Three tubes of sterile gelatin. 



Technique. 1. Melt the gelatin by standing the tubes in the water bath. 



Gelatin should never be liquefied by holding it in a gas flame, because the air 

 dissolved in the gelatin will appear as bubbles in the substance of the medium and 

 will interfere with the subsequent examination of the plates. 



2. Take up a drop of the liquid to be examined in a Pasteur pipette and 

 add it to one of the gelatin tubes (dilution 1), taking the necessary precau- 

 tions to prevent contamination. Replace the wool plug and mix thoroughly 

 by rolling the tube between the hands. 



Never shake tubes of media as is done in chemical investigations, because it 

 gives rise to frothing and this is highly inconvenient. 



3. With another pipette transfer three drops from the first tube to another 

 tube of gelatin (dilution 2). Mix as before. 



4. Transfer three drops of dilution 2 to the third gelatin tube (dilu- 

 tion 3). 



The three tubes of gelatin will now contain each a different number of organisms,, 

 and, according as to whether the original material contained many or few organisms, 

 dilution 3 or dilution 1 will give the best results. Thus if the number of organisms 

 be large, the colonies will be confluent in the plate poured with tube 1 and isola- 

 tion will be impracticable ; in that case dilutions 2 and 3 will be available. 



5. Take out a Petri dish from its envelope. Take the plug out of the first 

 gelatin tube, and flame the mouth. Then lift the cover of the Petri dish^ 

 pour the gelatin into it and cover the dish again as quickly as possible. 



Spread the gelatin in an uniform layer over the surface 

 of the dish by tilting it backwards and forwards, put it on a 

 cold and level surface and allow it to set. Then label it and 

 put it in the cool incubator (20 C.). 



6. Pour plates with the gelatin in tubes 2 and 3 in the 

 same way. 



7. Examine the plates every day, and without lifting the 

 cover note the appearance of the colonies and their charac- 

 teristics (both with the naked eye and with the aid of a 

 lens). Remove a portion of each colony for the purpose 

 of making sub-cultures and for microscopical examination. 



Roux bottles. It is often more convenient to use a flat flask,, 

 such as Roux's (fig. 69), instead of Petri dishes. The flasks are 

 perhaps better than the Petri dishes because they effectually 

 prevent contamination of the medium and evaporation is reduced 

 to a minimum. 



Note. The gelatin plate method has some disadvan- 

 tages, and is not available in all cases. Thus : 



(a) Some organisms rapidly liquefy gelatin, and if such are present in the 

 material under investigation the experiment is liable to be a failure. 



(b) It is only applicable in cases of organisms growing at tempera- 

 tures below 25 C. Above this temperature gelatin ceases to be a solid 

 medium. 



FIG. 69. Roux 

 bottle. 



