382 



LABORATORY DIAGNOSIS OF PROTOZOAN INFECTIONS 



Neutral xylene may be used in place 

 of toluene, altho it hardens the tissues 

 more. Neutral balsam or other resinous 

 mounting media may be used in place of 

 permount. Neutralize the xylene and bal- 

 sam by placing marble chips in their con- 

 tainers. If this is not done, the stains 

 will fade more or less after months to 

 years. 



FEULGEN STAIN 



The Feulgen nucleal stain, which is 

 used for the detection of deoxyribonucleic 

 acid (DNA), is essentially a modification 

 of the Schiff reaction for aldehydes. When 

 DNA is hydrolyzed by hydrochloric acid, 

 aldehyde-like substances are formed 

 which, when treated with colorless fuchsin 

 sulfite, stain a purplish red. Whether the 

 reaction is limited to DNA is doubtful, but 

 at any rate, when properly carried out, 

 the Feulgen technic produces a prepara- 

 tion in which only chromatin is stained. 



Not all samples of basic fuchsin are 

 satisfactory for the Feulgen stain. Hence, 

 care must be taken to use dye from a 

 batch which has been found satisfactory 

 and which has been certified as such by 

 the Biological Stain Commission. 



1. Fix material to be stained by this 

 method for 24 hours in a satur- 

 ated solution of mercuric chloride 

 containing 2% acetic acid. 



2. Wash in running water, and pass 

 thru 30%, 50%, and 70% alcohol. 

 Do not treat with iodine . 



3. Cut sections in the usual manner. 



4. Before staining, leave smears 

 and sections in 95% alcohol 48 

 hours to remove "plasmalogen" 

 substances which may take the 

 stain. 



5. To stain, run down thru the alco- 

 hols to distilled water, and place 

 the smears or sections in 1 N HCl 

 at 60" C for 4 minutes. 



6. Wash in cold 1 N HCl, then rinse 

 with distilled water. 



7. Transfer to the decolorized fuehsin 

 solution, and stain 1 to 3 hours. 



8. Wash thoroughly in water contain- 

 ing a little sodium bisulfite plus a 

 few drops of HCl. 



9. Wash in distilled water. 

 10. Dehydrate by passing up thru the 

 alcohols as described above, clear, 

 and mount in permount. 



BODIAN SILVER IMPREGNATION 

 TECHNIC 



This method is superior to ordinary 

 stains for demonstration of flagella and 

 other diagnostic structures of flagellates. 

 The technic given below is essentially that 

 described by Honigberg (1947). Not all 

 batches of protargol are equally good for 

 this stain, and care must be taken to use 

 a sample which has been tested and found 

 satisfactory. 



1. Fix in Hollande's or Bouin's solu- 

 tions for 10 minutes. 



2. Wash in 50% alcohol. 



3. Transfer to 30% alcohol and then 

 to distilled water. 



4. Bleach in 0. 5'c aqueous potassium 

 permanganate for 5 minutes. 



5. Wash in distilled water. 



6. Bleach in 5% aqueous oxalic acid 

 for 5 minutes. 



7. Wash several times in distilled 

 water. 



8. Place in freshly prepared 1% aque- 

 ous protargol solution. (To pre- 

 pare this solution, place the proper 

 amount of distilled water in a 

 beaker and scatter the protargol 

 powder on its surface; do not stir, 

 heat or disturb the vessel until the 

 protargol has dissolved. ) 



9. Keep copper wire or thin copper 

 sheeting in the vessel thruout the 

 staining process. Use 5 g copper 

 per 100 ml pi'otargol solution. 

 Columbia jars contain 10 ml of so- 

 lution. If they are used, it is con- 

 venient to place a coil of copper 

 wire weighing 0.5 g in the bottom of 

 each jar before adding the protargol. 



10. Stain for 1 to 2 days at room tem- 

 perature or 37" C in the protargol- 

 copper solution. The staining time 

 and temperature will depend on the 

 material being stained and the final 

 intensity desired. If staining is con- 

 tinued for more than a day, transfer 

 to fresh protargol solution contain- 

 ing fresh copper for the second day. 



