124 LABORATORY EXERCISES IN BACTERIOLOGY. 



operation, all combine to render the discovery of pathogenic germs a most difficult 

 part of bacteriologic investigation. 



Briefly stated, it is to be kept in mind, as of fundamental importance, that the 

 material collected should be obtained so as to represent its complete natural flora, that 

 it should be preserved from contamination and intrinsic changes before inoculation or 

 at once inoculated upon the nutrient medium, and that the infectious elements collected 

 be diluted or concentrated by artificial device as required for convenience of manipulation 

 and certainty of investigation. For these desiderata the application of method, care 

 of procedure, and a variety of needed apparatus must call forth the ingenuity and dex- 

 terity of the student ; and each case is to be approached individually for the application 

 of these principles, so as to provide eventual success. It is impossible to particularly 

 describe the technique of special procedures, and the consideration of a few general 

 examples, such as will presently be outlined (air, water, milk, soil, and disease material) , 

 must serve as illustration. 



THE DIFFERENT FORMS OF CULTURES. 



Tube Cultures. Growths of organisms inoculated upon media in test-tubes are 

 least liable to contamination from the various possible accidents of manipulation, 

 and are the most conveniently prepared and handled; they are therefore the type 

 usually employed unless some special purpose demands the use of others. Cultures 

 in tubes of liquid media are, of course, diffusion cultures, the organisms readily spreading 

 through the liquid in which they are grown. On solids, the cultures, from the mode 

 of inoculation and from peculiarities of the bacteria themselves, may be limited to 

 the surface (surface cultures), as smear or stroke cultures, or may grow in the interior 

 of the mass, as stab cultures or diffusion cultures. The same microorganism grown by 

 these different modes even upon the same medium is likely to present considerable 

 differences of appearances and of rate and profusion of development. Diffusions and 

 smears usually show the most characteristic appearances, although both punctures 

 and stroke cultures afford important information. Any of the media may be used 

 in tube cultures. 



Plate Cultures. These were introduced by Koch for use in the separation of 

 mixed into pure cultures, and although largely superseded by Petri dish cultures 

 and Esmarch's tubes, are still frequently employed. In preparing a plate culture, 

 the glass plates, supports, and culture dish already described (i>. Apparatus, p. 64), 

 sterilized as indicated, are to be provided, three plates and their platforms being usually 

 arranged as a set in a culture dish. Three diffusion inoculations are generally made 

 of the material to be examined ; the first from the original material, the second by the 

 transfer of a small amount (one or two loopfuls) of the first diffusion to the medium 

 in the second tube, and the third by like transfer from the second to the third tube. 

 (Sterilize the loop between inoculations.) A level surface is provided, usually by 

 means of a platform tripod with adjustable legs (Fig. 39), upon which is placed a 

 flat dish filled level full with ice-water or crushed ice. This dish is covered with a 

 large glass plate upon which rests a glass bell jar. The glass plate and bell jar should 

 have been disinfected and rinsed in boiled water before use, or otherwise sterilized. 

 One of the plates intended for the culture is placed upon the chilled surface of the 

 glass plate and the liquefied medium of the first diffusion inoculation poured over 

 it, the mouth of the tube having been flamed before the medium is poured out. The 

 bell jar is at once lowered, and by tilting the glass cover of the ice dish the liquid medium 

 can usually without difficulty be made to cover the culture plate in a fairly even film. 



