142 MORPHOLOGY AND CULTURE OF MICROORGANISMS 



the details of sectioning and the staining of sections the reader is referred to Mallory 

 and Wright's Pathological Technic, W. B. Saunders and Co., and to Lee's Vade 

 mecum. 



The cultivation of free-living protozoa is usually accomplished by keeping a 

 supply of the medium in which they live on hand. Hay infusion prepared by boiling 

 a quantity of chopped hay in water is an easy and valuable method of preparing 

 culture media. For the cultivation of amoebae, the following media is widely em- 

 ployed. It should be noted, however, that the amoebae which have been cultivated 

 are regarded as free-living forms and the attempts to cultivate parasitic amoebae 

 have thus far been unsuccessful. 



MEDIUM OF MUSGRAVE AND CLEGG 



Agar 20 to 30 g. 



Liebig's extract of beef 3 to . 5 g. 



Common salt 3 to . 5 g. 



Water 1,000 c.c. 



This medium is designed to provide for slow bacterial growth in order to provide 

 food for amcebae. On a richer medium the latter are overwhelmed by the rapid 

 growth of bacteria. 



For the cultivation of trypanosomes, leishmania and other flagellates the so- 

 called triple N media is employed. This is prepared as follows: 



NlCOLLE, NOVY, MACNEAL MEDIUM 



Water 900 c.c. 



Salt 6 g. 



Agar 16 g. 



Dissolve, distribute in tubes, sterilize and add to the medium in each tube after 

 liquefying and cooling to 4o-5oC. one-third its volume of rabbit blood obtained by 

 cardiac puncture. Slope the tubes for twelve hours, incubate at 37 for five days 

 to prove the sterility of the medium and then keep them at the ordinary temperature 

 of the laboratory for a few days before sowing them. (The tubes should be sealed to 

 prevent evaporation.) 



The malaria organisms have been made to continue development outside the body 

 by the following method devised by Bass. 



Bass's Method. The blood in 10- to 2o-c.c. quantities is taken from the patient's 

 vein and received in a centrifuge tube which contains )-fo c.c. of 50 per cent, glucose 

 solution. A glass rod, or piece of tubing, extending to the bottom of the centrifuge 

 tube is used to defibrinate the blood. After centrifugalizing there should be at least 

 i inch of serum above the cell sediment. The parasites develop in the upper cell 

 layer about J^Q to %Q m ch from the top. All of the parasites contained in deeper 

 lying red cells die. To observe the development, red cells from this upper % 

 portion are drawn up with a capillary bulb pipette. 



