1800 Journal of Applied Microscopy 



A Review of the Existing Methods of Cultivating Anaerobic 



Bacteria. 

 V. 



EXCLUSION OF ATMOSPHERIC OXYGEN BY MEANS OF VARIOUS PHYSI- 

 CAL PRINCIPLES AND MECHANICAL DEVICES. 



This category embraces all those methods and appliances that do not belong 

 to any of the preceding principles. They are arranged as follows : 

 The atmospheric oxygen is excluded : 



A. By deep layers of solid medium. 



B. By layer of oil. 



C. By layer of paraffin. 



D. By mica or glass plates. 



E. By boiling the medium to expel the air and sealing the appar- 



atus. 



F. By the use of the fermentation tube and its modifications. 



G. By inoculating into a hen's egg. 



A. DEEP LAYERS OF SOLID MEDIUM. 



Hesse (1885) and Liborius (188G) recommended the following method : Pour 

 into a test tube, Erlenmeyer flask, or deep Petri dish a sufiicient quantity of 

 nutrient gelatin, nutrient agar, or blood serum to form a layer of from 5 to 20 

 cm. deep. Boil the medium for at least five minutes to expel the air, cool down 

 to a temperature of about 40°C., inoculate the medium, distributing the inoculat- 

 ing material well through it. Care must be taken not to shake the tube or flask; 

 after inoculation cool rapidly by standing the apparatus in cold water until the 

 medium is set. In this way a large number of isolated colonies usually develop, 

 which are either distributed all through the medium, or appear only in the higher 

 zone, or inhabit exclusively the lower layers. The sharply marked distribution 

 of the colonies over the different zones, their size and other characteristics per- 

 mit a fairly accurate estimate of their relative want of oxygen. The inoculation 

 is made with a long, firm platinum wire, which has previously been brought into 

 contact with the culture material, or a long capillary pipette may be used, into 

 which the culture has been introduced by suction. This simple method, which is 

 very effective even in the case of the strictest anaerobes, is being used success- 

 fully in many laboratories. 



Often a layer of sterile medium is poured on top of the inoculated layer after 

 the latter has solidified. In order to gain access to the colonies the tube is 

 broken, Sanfelice, 1893, warms the bottom of the tube and shakes the column 

 of agar out into a sterile glass plate, where it can be cut into slices, the colonies 

 can be examined and subplanted. 



Esmarch recommends the following procedure : Make Esmarch roll cultures 

 in the ordinary way. Stand the tubes containing the roll cultures in ice-cold 

 water and pour liquified gelatin into the cavity of the tube. Upon cooling the 



