748 THE ANIMAL PARASITES OF MAN 



film again dried. It is fixed in absolute alcohol and stained with 

 Romanowsky's solution. A cubic millimetre of blood divided into 

 quarters may be thus dehaemoglobinized and stained. The parasites 

 in such a cubic millimetre of blood may be counted. Such a proced- 

 ure was followed by R. Ross and D. Thomson, 1 in determining the 

 periodic variation of the numbers of trypanosomes in the blood of 

 a patient, as mentioned and figured on pp. 78 and 79. 



For cytologica details of various Protozoa, thin film preparations 

 on cover-slips or slides are often useful. Cover-slip preparations are 

 preferable, unless the organisms under investigation are extremely 

 scanty. The medium containing the organisms, such as blood, 

 lymph, intestinal contents, sputum, scrapings of ulcers, and urine, is 

 spread thinly, either alone or diluted with a little physiological salt 

 solution, on the cover-slip. Fixation while still wet is necessary. 

 Various methods are employed. 



Fixatives. A useful procedure is to fix the wet film by exposure 

 to 4 per cent, osmic acid vapour for ten to thirty seconds, then place 

 in absolute alcohol for five minutes to harden. Grade down from 

 absolute alcohol through 90 per cent., 70 per cent., 50 per cent., and 

 30 per cent, alcohols to water. Stain wet with a suitable stain such 

 as haematoxylin, and gradually dehydrate by grading through the 

 necessary strengths of alcohol, clear in xylol or other oily clearing 

 medium and mount in Canada balsam. 



Other fixatives may be employed, such as are also useful for fixing 

 pieces of tissue for sectioning. Films or smears on cover-slips while 

 still wet are floated on the surface of the fixative in a watch glass. 

 Some good fixatives of wide application are : 



Schaudinn's Fluid. This consists of a mixture of 



Saturated aqueous solution of corrosive sublimate ... ... 2 volumes 



Absolute alcohol ... ... ... ... ... I volume 



Two modifications of Schaudinn's formula may be found useful. 

 A saturated solution of corrosive sublimate in physiological salt 

 solution may be substituted for the aqueous one, and the addition of 

 a few drops of glacial acetic acid to either of the preceding mixtures 

 may be made. 



Some workers prefer to use hot fixatives, raised to a temperature 

 of about 50 C. 



Fixation by corrosive sublimate solutions must be followed by 

 thorough removal of the mercury salt by washing repeatedly in 

 30 per cent, alcohol or with iodine-alcohol. 



1 Proc. Roy. Soc., B, Ixxxii, p. 411. 



