146 LABORATORY EXERCISES IN BACTERIOLOGY. 



of the various infectious diseases the first object of the investigator is the disco-very, 

 in the tissues or fluids of the diseased individual, of a probable microorganismal agent. 

 For the accomplishment of this, the first of the familiar postulates of Koch, two methods 

 of procedure are available and both should be pursued : the examination of the diseased 

 material by the microscope and the practice of inoculation of nutrient media with 

 such material. The examination by the microscope may be directed to the fresh, 

 unstained matter or, better, to stained films smeared and fixed upon a slide or cover- 

 glass (see Smear Preparations, Lesson VII). 



For successful culture of the pathogenic bacteria blood-serum probably offers 

 the greatest promise of any of the common nutrient media ; but, as in any other study 

 concerned with unknown forms of micro-organisms, one should at least in the pre- 

 liminary inoculations have recourse to the entire range of laboratory nutrients, several 

 inoculations being made upon each type to insure the fullest chance of avoiding failure. 

 Here may be recalled the method of McLaughlin, originally devised and employed 

 by him in the study of dengue fever, of employing as the artificial nutrient the natural 

 body material supposed to be the habitat of the organisms sought. This investigator 

 withdrew from veins into sterile glass tubes some of the blood of the infected subject 

 and with it the organisms he has described, thereafter keeping the blood in the collection 

 tube in the incubator for culture of the parasites. The method has wide application, 

 especially for infected blood and fluid exudates, excretions and secretions, but also 

 for infected solids, and has been utilized by a number of workers in a variety of ways. 

 Whatever media be employed, the various methods of inoculation possible for each 

 should be practised and the inoculated preparations subjected, as hereafter suggested, 

 to body temperature in the incubator as well as to room temperature, and to anaerobic 

 conditions as well as to the ordinary atmosphere. 



In case of exposed lesions, as the surface of an ulcer, or a diphtheritic throat, 

 either in the living or the dead subject, the wire loop or the swab may be well used 

 for taking up the infected matter for inoculation. In obtaining blood, it may be 

 removed from one of the large superficial veins of the arm of the living subject by 

 means of a sterile hypodermic syringe or from the pulp of the spleen, after thorough 

 disinfection of the skin and, in very, cautious operations, after a small superficial open- 

 ing has been made through the sterilized skin by a sterile knife. Other fluids, as spinal 

 fluid, peritoneal or pleural exudates, or the contents of an abscess, may be obtained 

 in the same way. When practicable for collection, Sternberg's bulbs, capillary tubes, 

 or pipettes may offer some advantages over syringes for subsequent convection, inocu- 

 lation, or culture. From the material thus procured smears and stabs are to be made 

 in solid media and diffusions in liquefied agar and gelatine, the latter being subse- 

 quently plated. When solid diseased tissues are removed by the surgeon and are to 

 be submitted to bacteriologic examination, they should at once be placed in sterile 

 jars and hermetically sealed for convection to the laboratory. After reception in 

 the laboratory, as soon as possible a knife-blade should be heated to red heat and 

 plunged into the mass, thus producing a sterile track into the midst of the tissue where 

 there cannot have taken place any air contamination after removal from the body. 

 Along this track a heavy platinum needle, heated to a red heat, is carried, plunged 

 through the eschar caused by the knife, and allowed to cool in the interior. When 

 cool, it is pushed beyond the zone influenced by its own heat and twisted about in 

 the structures until it has in all probability become infected. It is then withdrawn 

 and used in making the usual smear, stab, and diffusion inoculations. 



At autopsies it should be made a rule to obtain the material for inoculation as 



