132 MANUAL OF MICROBIOLOGICAL METHODS 



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 characteristic appear- 

 ance 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 LugoFs 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 cul- 

 ture, and it is advised that parallel tube cultures 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 dis- 

 cussed in Chap. Ill, 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 1 or 

 2 min, or hold at 80°C for 20 min). This should be a duplicate culture, 

 however, in case the anaerobic form is a nonsporeformer 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. 



The specimen of choice for bacteriological analysis of deep wounds 

 consists of bits of tissue taken at the time of debridement during the 

 initial surgery, or at the time of change of dressing. Alternate, though 

 less desirable, samples include swab samples or tissue exudate obtained by 

 aspiration. Such samples should be planted in heart-infusion broth with 

 heart particles (often called chopped-meat medium) and in addition, 

 streaked on blood or egg-yolk agar. (See Chap. Ill for details of prepar- 

 ation of these media.) Following incubation the heart-infusion cultures 



1 The use of petrolatum, mineral oil, or other materials as a seal at the surface of 

 liquid media is not recommended. 



