80 STUDY AND IDENTIFICATION OF BACTERIA 



anaerobic plates, either in a desiccator with the pyrogallic acid and caustic soda, or 

 less satisfactorily in the open air, is to sterilize the parts of the Petri dish inverted; 

 that is, the smaller part is put bottom downward in the inverted cover (as one would 

 set one tumbler in another). Then, in using, unwrap the Petri dish, lift up the inner 

 part, pour in the inoculated medium into the upturned cover. Then immediately 

 press down the inner dish, spreading out a thin film of the medium between the two 

 bottoms. 



Zinsser has originated a very satisfactory method for plate cultures of anaerobes, 

 which is shown in Fig. 8. 



Secure two small crystalh'zing dishes, about 3 and 4 inches in diameter by i inch 

 depth and sterilize as for Petri dishes. Pour the inoculated agar into the smaller 

 of the dishes or one can smear the surface of poured glucose agar with the material 

 to be plated out. In the bottom of the larger dish place the dry pyrogallic acid, 

 then invert the smaller dish with the agar surface over it. Quickly pour a 5% 

 solution of caustic soda into the space separating the sides of the inverted smaller 

 dish and the upright larger dish, to a depth of % inch and, as it is dissolving the 

 pyrogallic acid, very speedily superimpose paraffin oil on the soda solution to make 

 an air-tight seal. 



J. H. Wright's Method 



Make a deep stab culture in glucose agar or gelatin, preferably boiling the media 

 before inoculating. Then flame the cotton plug and press it down into the tube so 

 that the top lies about three-fourths of an inch below the mouth of the test-tube. 

 Next fill in about one-fourth of an inch with pyrogallic acid; then add 2 or 3 c.c. of 

 a 10% solution of caustic soda, and quickly. insert a rubber stopper. This method 

 is one of the most convenient and practical, and is to be strongly recommended. 



Method of Vignal 



In this a section of glass tubing (1/4 in.) is drawn out at either end, as in making a 

 bacteriological pipette, with a mouth-piece containing a cotton plug. The liquid 

 agar or gelatin is then inoculated and the medium drawn up into the tube by suction 

 with mouth or better with a rubber bulb. In a very small flame the capillary nar- 

 rowings are sealed off, and we have inside the tube very satisfactory anaerobic con- 

 ditions. To get at the colonies, file a place on the tube and break at this point. 



To obtain material for examination and isolation in pure culture from the deep 

 agar stab-tube, it is best to loosen the medium at the sides of the tube with a heated 

 platinum spud or a flattened copper wire. Then shake the mass out into a sterile 

 Petri dish. It is dangerous to break the tubes with a hammer as some do. 



With those anaerobes which produce gas in glucose agar the split in the column 

 of medium enables one to introduce a fine sterile capillary pipette to the site of a 

 colony and by releasing pressure on the rubber bulb to draw up into the tip of the 

 tube material for investigation. 



A Combination Method 



Recently as shown in the illustration in Fig. 7, I have been combining various 

 methods so that very satisfactory anaerobic conditions are obtained. First, a 



