CHAPTER III 

 EXAMINATION OF FLUIDS AND SOLIDS 



Fluids other than milk are subjected to bacteriological examination for 

 several purposes: (i) to determine whether supposedly sterile fluids are so, and 

 if not the number, perhaps also the kind, of bacteria contained: (2) to determine 

 the character of bacteria present in fluids known to contain bacteria. 



TECHNIQUE 



Shake the sample thoroughly, take a large drop and spread it on a slide and 

 fix by gently heating until dry, stain with methylene blue and examine micro- 

 scopically. If no bacteria are observed or if only a few are seen in some fields 

 and none in others, plating will probably show less than 500,000 colonies per 

 cubic centimeter; if many bacteria are observed in most of the fields, plating 

 will show millions per cubic centimeter. Such a preliminary inspection, 

 together with other known facts, usually give one an idea as to the proper 

 amount of dilution required for plating. 



In order to make accurate counts, not more than 200 colonies should develop 

 on a plate, and to obtain this, the richer a substance is in bacteria the more it 

 must be diluted, the smaller the amount placed in the plate. 



To remove individual colonies from a plate containing various organisms, 

 in order to further study the organisms separately and to identify them, the 

 colonies must be discrete and sufficiently isolated from each other to permit the 

 removal of one colony with a loop, without touching any other colony. 



Having estimated the degree of dilution required, the fluid is diluted and 

 plated the same as when examining water or milk. 



Plates should be made on three different media, plain agar, litmus lactose 

 agar and gelatin. The gelatin plates are incubated at room temperature, and 

 the rest at 37C. 



Substances, both fluids and solids, contaminated with bacteria may contain 

 the tetanus bacillus. As this organism is an obligate anaerobe, several plates 

 should be incubated under anaerobic conditions. 



If in addition to a determination of the number of bacteria present, the 

 species must be determined, a representative plate is selected after making the 

 count, and one of each different kind of colonies observed is removed and plated 

 separately. If pure cultures develop on the second series of plates, further sub- 

 cultures are made on various media sufficient to establish the identity of the 

 organisms. If the second series of plates do not show pure cultures, colonies 

 are removed from them and again plated until a pure culture is obtained. 



Solids are examined for the same reasons and usually in the same way as 

 fluids, except that they are first dissolved or suspended in sterile distilled water. 



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