PROTOZOA 567 



guaranteed salts. The diluted medium should be placed in sterile 

 test-tubes or small Erlenmayer flasks (100 c.c.), fitted with cotton- 

 wool plugs and sterilised by steaming for one hour on three 

 successive days as soon as possible after making up the con- 

 centrated mixture. 



It is best to have pyrex tubes, but others can be used. Tubes 

 of ordinary glass are apt to change thepH of the medium ; _pH 7-3 

 to 7-4 appears to be the optimum for those tested. 



All apparatus should be thoroughly cleaned with chromic acid, 

 finishing with glass-distilled water. Tubes and flasks for cultures 

 should, before filling with medium, be dried, plugged and heated 

 to 170° C. in an air oven for an hour to sterilise. 



Inoculation, using all precautions for maintaining sterility, is 

 performed most conveniently by using a small silica pipette. 

 Transfers to fresh tubes should be made every two to three weeks. 

 Experience has shown that 20° C. is the best temperature for 

 growth (Hearson's cold incubator is generally used for this purpose). 



Since 1931 Colpidium has been grown in the Biochemical Depart- 

 ment in a medium of approximately half the above strength containing 

 sterile yeast.* 



There is still considerable controversy as to whether ciliates such as 

 Colpidium, as well as other protozoa, can live for more than two or 

 three generations, if at all, in sterile media free from yeast or traces of 

 some accessory growth factors. 



1113. Cultures on Solid Media. Many coprozoic forms, as well 

 as small free-living amoebae and flagellates from soil, water, etc., 

 will grow well on a solid agar medium prepared as for bacterio- 

 logical use in test-tube slopes or Petri dishes (plate cultures). Of 

 the many formula; for making suitable media, that devised by 

 Musgrave and Clegg for cultivation of amcebae has been much 

 used : — 



Agar 2-0 grm. 



NaCl -03-05 „ 



Liebig's beef extract . . . •03--05 ,, 



Distilled water .... 100 c.c. 



Robertson {Quart. Journ. Micr. Sci., 1932, p. 540) use an 

 egg agar with Peters' medium very successfully for growing Bodo. 

 She also uses a 2 per cent, agar in which is incorporated about 

 18 per cent, of Peters' medium for Mastigamceba growing with a 

 suitable bacillus. By flooding this medium in Petri dishes with 

 more Peters' medium " pond " cultures may be obtained in 

 which Chlamydophrys or small flagellates, such as Bodo, flourish 

 exceedingly. 



* It would even appear that, when yeast is used, the glycero-phos- 

 phate is probably superfluous, since the Colpidium has grown well here 

 in the Zoology Department for a time in a medium consisting of sterile 

 yeast in Peters' saline solution only. — H. G. 



