LABORATORY DIAGNOSIS OF PROTOZOAN INFECTIONS 



383 



11. Wash in distilled water. 



12. Place in a solution of 1% hydro- 

 quinone in 5% aqueous sodium 

 sulfite for 5 to 10 minutes to re- 

 duce the silver. 



13. Wash several times in distilled 

 water. 



14. Place in 1% (or more dilute) aque- 

 ous gold chloride for 4 to 5 min- 

 utes. 



15. Wash in distilled water. 



16. Place in 2% aqueous oxalic acid 

 for 2 to 5 minutes until a purplish 

 color appears. 



17. Wash several times in distilled 

 water. 



18. Place in 5% sodium thiosulfate for 

 5 to 10 minutes. 



19. Wash several times in distilled 

 water. 



20. Pass up thru a graded series of 

 alcohols to dehydrate, clear in 

 toluene or xylene, and mount in 

 per mount or balsam. 



GIEMSA STAIN FOR TISSUE SECTIONS 



The following technic is based on that 

 described by Hewitt (1940) for staining 

 tissue sections with Giemsa stain. 



1. Fix small pieces of tissue in for- 

 mol-Zenker's fluid for 18 to 24 

 hours. 



2. Wash in running tap water over- 

 night. 



3. Place in 30% alcohol, 50% alcohol 

 and 70% alcohol for 2 hours each. 



4. Treat overnight with 70%) alcohol 

 containing enough iodine to give it 

 a port-wine color. This removes 

 the excess mercuric chloride. 



5. Place in fresh 70% alcohol for 2 to 

 4 hours or longer to remove the 

 iodine. 



6. Finish dehydration, and infiltrate, 

 embed, section and mount in the 

 usual manner. 



7. Run the sections down thru xylene 

 and the alcohols into distilled 

 water, in the usual manner, 



8. Mordant in 2. 5% aqueous potassium 

 bichromate solution 1/2 to 1 hour. 



9. Wash quickly in distilled water. 



10. Stain for 24 hours in the following 

 solution: 



0. 5% aqueous sodium carbonate . 2 to 4 drops 



Methyl alcohol (CP) 3 ml 



Giemsa stain 2.5 ml 



Distilled water 100 ml 



11. Wash in distilled water colored 

 lemon yellow with 2. 5% potassium 

 bichromate to remove the excess 

 stain. 



12. Differentiate in 70% alcohol. This 

 is the most critical step in the 

 whole procedure. It usually takes 

 30 seconds to 2 minutes, but the 

 time varies with the type of tissue 

 and the thickness of the sections. 

 Liver usually takes less time than 

 enlarged, engorged spleen, which 

 takes less time than normal spleen. 

 Thick sections take longer than 

 thin. Stop differentiating as soon 

 as the stain is being removed in 

 noticeable quantities. Tissues 

 which contain a large amount of 

 blood will show sharply differen- 

 tiated red and blue areas macro- 

 scopically when they are properly 

 differentiated. 



13. Stop the differentiation by washing 

 quickly in distilled water. 



14. Dehydrate and mount. Alcohol 

 cannot be used for the dehydrating 

 process, since it will remove too 

 much dye. The simplest and best 

 method of dehydration is to pass 

 the sections thru 3 changes of aii- 

 Iiy (Irons tertiary butyl alcohol for 



5 to 10 minutes each (Levine, 1939). 

 (Ordinary samples of tertiary butyl 

 alcohol contain water and cannot be 

 used. A simple way of determining 

 whether a sample is anhydrous is 

 to place it in the refrigerator; its 

 melting point is 25° C, and it will 

 crystallize. ) 



Transfer the sections from the third 

 tertiary butyl alcohol to 2 changes of xylene 

 and then mount in permount or another res- 

 inous mounting medium. It is important 

 that the mounting medium be neutral; if it 

 is acid it will soon decolorize the prepara- 

 tions. 



