STAINING BLOOD-FILMS 211 



triacid, hasmatoxylin and eosin, Smith's formol fuchsin, and with thionin, separate 

 fixation is necessary. For Giemsa and thionin, either absolute alcohol (ten to fifteen 

 minutes), or methyl alcohol (two to five minutes) answer well. 



Formalin vapor, for five to ten seconds, is also used for fixation. For Ehrlich's 

 triacid, haematoxylin and eosin and formol fuchsin, heat gives the best results. 

 The best method is to place the films in an oven provided with a thermometer. 

 Raise the temperature of the oven to i35C. and then remove the burner. After 

 the oven has cooled, take out the fixed slides or slips. 



Some prefer to place a crystal of urea on the slide, then hold it over the flame 

 until the urea melts. This shows that a temperature between 130 and i35C. has 

 been reached. 



One of the handiest methods is to drop a few drops of 95% alcohol on the slide 

 or cover-glass. Allow this to flow over the entire surface; then get rid of the exeess 

 of alcohol by touching the edge to a piece of filter-paper for a second or two. Then 

 light the remaining alcohol film from the flame and allow the burning alcohol to 

 burn itself out. A chemical fixation which gives good fixation for haematoxylin 

 and triacid stains (not equal to heat) is a modification of Zenker's fluid (Whitney). 

 To Miiller's fluid, which is potassium bichromate 2 grams, sodium sulphate i gram, 

 and water 100 c.c., add 5 grams of bichloride of mercury and 5 c.c. of nitric acid 

 (C. P.). Fixation is obtained in five seconds. 



When using corrosive sublimate fixation one should after thorough washing in 

 water treat the film with Gram's iodine solution for about two minutes and then wash 

 with 70% alcohol until the yellow color of the film disappears. Methyl alcohol for 

 two minutes is satisfactory. (See Staining of Protozoa.) 



Staining Blood-films. As separate staining with eosin and methyl- 

 ene blue rarely gives good preparations and as the modifications of the 

 Romanowsky stain recommended are easy to make and employ, and 

 give much greater information, the separate method of staining is not 

 recommended. The most satisfactory single stain is thionin. 



Rees' Thionin Solution Take of thionin 1.5 grams, alcohol 10 c.c., aqueous solution 

 of carbolic acid (5%) 100 c.c. Keep this as a stock solution. It should be at least 

 two weeks old before using. For use, filter off 5 c.c. and make up to 20 c.c. with 

 water. 



1. Fix films (a) by heat, (b) by alcohol and ether, or (c) preferably by i% 

 formalin in 95% alcohol for one minute. 



2. Stain for from ten to twenty minutes. Wash and mount. Malarial parasites 

 are stained purplish; nuclei of leukocytes, blue; red ceUs, faint greenish blue. 



Ehrlich's Triacid or Triple Stain. There are required: 



1. Sat. aq. sol. orange G. (Dissolve 3 grams in 50 c.c. water.) 



2. Sat. aq. sol. acid fuchsin. (Dissolve 10 grams in 50 c.c. water.) 



3. Sat. aq. sol. methyl green. (Dissolve 10 grams in 50 c.c. water.) 



These three solutions may be kept as stock solutions. They keep well in the dark. 

 To make the stain, add 9 c.c. of No. 2 (acid fuchsin) to 18 c.c. of No. i (orange G.). 

 After they are mixed thoroughly, add 20 c.c. of No. 3 (methyl green). Then after 

 the first three ingredients are well mixed, add 5 c.c. of glycerine. Mix, then add 15 



