iii«-16 MANUAL OF METHODS FOR PURE CULTURE STUDY 



one week. C. perfringens (C. welchii) appears to be one of the most 

 difficult species in which to demonstrate spores microscopically 

 with regularity. If success is not attained using the above-men- 

 tioned media in cultures having the characteristics of this organism, 

 one may use the medium recommended by Svec and McCoy (See 

 Leaflet II). 



Since some taxonomic systems give considerable attention to the 

 size and position of the spore, these characteristics should be recorded 

 when the original laboratory examination is made. The characteris- 

 tic appearance of Clostridium tetani spores has been noted above; 

 these are round in shape and borne at the end of a slender vegetative 

 rod. This is almost the only instance in which the picture of the 

 spore and sporangium assumes importance in species diagnosis, 

 and this observation must be supported by cultural or pathologic 

 information as nontoxic organisms of similar microscopic characters 

 occur. 



GRANULOSE REACTION 



The cells of certain species, particularly during the early stages 

 of spore formation, store granulose. To test for this, add a drop 

 of Lugol's iodine to a wet mount preparation. Cells containing 

 granulose will stain blue or violet while others will appear yellow. 



Cultivation Technics^^ 



preliminary enrichment methods 



Ordinarily the best method to be followed in initiating growth of 

 an anaerobe from a sample is to inoculate one of the tubed media 

 rather than to proceed directly to plate culture. Certainly this 

 should be done if there is question concerning the possible success 

 of the preliminary culture, and it is advised that parallel tube cul- 

 tures be inoculated to serve as reserve cultures at the same time the 

 plating is done, if the plating technic is favored. The medium 

 to be used will be a matter of choice, as discussed in Leaflet II (9th 

 Edition), depending upon the nature of the sample. If aerobic 

 contamination is suspected and the anaerobe is thought to be in the 

 spore state, a duplicate primary culture should be heated briefly 

 (boil for one or two minutes, or hold at 80° C. for 20 minutes). This 

 should be a duplicate culture, however, in case the anaerobic form 

 is a non-spore-former or is a spore-former in the vegetative state. 

 Almost all types of tubed media should have the dissolved oxygen 

 driven off by boiling or heating in flowing steam. 



For the gas gangrene and tetanus group in infected wounds, Reed and Orr (IQll) 

 recommend a technic to those who work in clinical laboratories and examine such 

 material. The technic would appear to involve more cultures than is necessary but the 

 importance of the success of the preliminary culture, and the speed with which it is 

 attained, necessitate the routine suggested. Colonies which appear in the plates are 

 transferred to tubes of thioglycollate medium and species identification begun im- 

 mediately. It should be remembered that gas gangrene frequently is a polymicrobic 



i^The use of vaseline, mineral oil or other materials as a seal at the surface of liquid 

 media is not recommended. 



