COLLECTION, CULTIVATION, OBSERVATION 725 



Fixation for 10-50 minutes; wash for one hour or longer in running 

 water. 



The most commonly used stain is Heidenhain's iron haematoxy- 

 lin, as it is dependable and gives a clear nuclear picture, although it 

 is unsatisfactory for voluminous organisms or smears of uneven 

 thickness. It requires a mordant, ammonio-ferric sulphate (iron 

 alum) and a dye, haematoxylin. Crystals of iron alum become yellow 

 and opaque very easily. Select clear violet crystals and prepare 2% 

 aqueous solution. Haematoxylin solution must be well "ripe." The 

 most convenient way of preparing it is to make 10% absolute alcohol 

 solution. By diluting this stock solution with distilled water, pre- 

 pare 0.5 or 1% slightly alcoholic solution which will be ready for 

 immediate and repeated use. Smears are left in the mordant in a 

 jar for 1-3 hours or longer. Wash them with running water for 5 min- 

 utes and rinse in distilled water. Place the smears now in haematoxy- 

 lin for 1-3 hours or longer. After brief washing in water, the smears 

 are decolorized in Petri dish in a diluted iron alum, 0.5% HCl in wa- 

 ter or 50% alcohol, or saturated aqueous solution of picric acid under 

 the microscope. Upon completion, the smears are washed thor- 

 oughly in running water for about 30 minutes. Rinse them in dis- 

 tilled water. Transfer them through ascending series of alcohol (50 to 

 95%). If counter-staining with eosin is desired, dip the smears which 

 were taken out from 70%, alcohol, in 1% eosin in 95% alcohol for a 

 few seconds, and then in 95% plain alcohol. After two passages 

 through absolute alcohol and through xylol, the smears are mounted 

 one by one on a slide in a small drop of mixture of Canada balsam 

 and xylol. The finished preparations are placed in a drying oven at 

 about 60°C. for a few daj^s. 



Other stains that are often used are as follows : 



Delafield's haematoxylin. If the stock solution is diluted to 1:5- 

 10, a slow, but progrcvssive staining which requires no decolorization 

 may be made; but if stock solution is used, stain for 1-16 hours, and 

 decolorize in 0.5% HCl water or alcohol. If mounted in a neutral 

 mounting medium, the staining remains true for a long time. 



Mayer's paracarmine. In slightly acidified 70% alcohol solution, 

 it is excellent for staining large Protozoa. If over-stained, decolorize 

 with 0.5% HCl alcohol. 



Giemsa's stain. Shake the stock solution bottle well. By means of 

 a stopper-pipette dilute the stock with neutral distilled water (5-10 

 drops to 10 cc). Smears fixed in Schaudinn's fluid and washed in 

 neutral distilled water are stained in this solution for 10 minutes to 



