106 SUMMARY OF CURRENT RESEARCHES RELATING TO 



The fluid is then passed through a paper Alter, and afterwards through 

 a Chamberland filter. The reddish germ-free filtrate is preserved in 

 test-tubes or flasks, and if prevented from drying, the stock will keep 

 for weeks. For cultivation purposes it is mixed with solid or liquid 

 peptonised media. Thus, with 2 p.c. nutrient agar, the procedure is as 

 follows : test-tubes containing some 5 c.cm. of 2 p.c. nutrient agar are 

 liquefied and cooled down to 40-50° (."'., and then 1-2 c.cm. of the meat 

 extract are added ; in about a minute the medium is ready for use. 



Simple Method of Sterilising Blood for Cultural Purposes.* — 

 E. P. Bernstein and A. A. Epstein place 400 c.cm. of fresh ox-blood in 

 a sterile Erlenmayer's flask of 500 c.cm. capacity, in which have been 

 previously placed 30 c.cm. of 1 p.c. ammonium oxalate solution and 

 | c.cm. of 40 p.c. formalin. After shaking, and then allowing to stand 

 for i hour, an equal quantity of sterile physiological salt solution is 

 added to the blood. After 24 hours the blood may be used for cultural 

 purposes. One part of the diluted blood is added to 15 parts agar or 

 broth, so that the tubes contain about 1 : 3G000 formalin. 



Cultivation and Preparation of Myxomycetes.f — E. Pinoy culti- 

 vated Dictyostelium mucoroides on a medium composed of 20 grm. agar, 

 50 grm. linseed, and 1 litre of water. This was heated to 117° C, and 

 after having been distributed into glass vessels was sterilised at 115° C. 

 for \ hour. As the medium could not be filtered, the impurities were 

 got rid of by keeping the medium at 37° C. until the extraneous matters 

 had sedimented. When the agar had set, the clear portion was cut off 

 and was used. On this medium spores were sown, and cultures asso- 

 ciated with bacteria were obtained. The presence of one or more kinds 

 of bacteria seems to be indispensable for the nutrition of the fungi, 

 and all, with the exception of B. pyocyanms, were Gram-negative. 



For examining the cultures the condensation water was used, and 

 preparations made as hanging drops, or in Van Tieghem's cells. For 

 examination in vivo, neutral red was found to be the best stain, as it 

 ■colours not only the partially digested bacteria, but also has the property 

 of indicating the reaction of fluids, turning yellow if they be alkaline, 

 and red or blue purple if acid. Hence it indicates the acid or alkaline 

 reaction of the liquid in the vacuoles. Neutral red does not affect the 

 living organisms, but if in excess the myxamcebae are killed, and there- 

 fore stain. For fixed preparations Laveran's method was adopted. A 

 film is made in the usual way, and when dry is fixed with alcohol for 

 ten minutes. It is then stained with the following mixture : 4 c.cm. of 

 1 per thousand aqueous eosin, 6 c.cm. distilled water, 1 c.cm. Borrel's blue. 

 The stain is allowed to act for 15-20 minutes, and then the film is 

 differentiated with a 5 p.c. tannin solution. The results obtained by 

 the foregoing method were controlled by two other procedures, viz. 

 staining with Heidenhain's iron-hasmatoxylin after fixation in sublimate, 

 and by Borrel's method. This consists in fixing with the following 

 fluid : water 300 grm., acetic acid 20 grm., osmic acid 20 grm., 

 platinum chloride 2 grm., chromic acid 3 grm., then staining with 



^ * Journ. Infect. Diseases, iii. (1906) pp. 772. 

 ~ t Aim. Inst. Pasteur, xxi. (1907) pp. 622-56 (4 pis.). 



