380 



LABORATORY DIAGNOSIS OF PROTOZOAN INFECTIONS 



PERMANENT FIXING AND 

 STAINING TECHNICS 



It is often desirable to make perma- 

 nent preparations of fecal smears or to 

 make hematoxylin-stained slides for de- 

 tailed study. For this purpose, smears 

 must first be fixed, i.e., the protozoa 

 must be killed by the action of a chemical 

 or mixture of chemicals which will pre- 

 serve their structures as nearly as pos- 

 sible in the same form as in life. 



Many different technics are used for 

 fixing and staining tissues, cells and 

 small organisms. Those given below are 

 especially suitable for protozoa. The 

 standard hematoxylin and eosin stain used 

 routinely for tissue sections is also valu- 

 able for protozoa in tissues, but it is so 

 well known that it is not described here. 

 For further information on fixing, sec- 

 tioning, staining and mounting technics, 

 any text or reference book on microscopic 

 technic may be consulted. 



FIXATION 



Schaudinn's fluid is probably the best 

 all-round fixative for intestinal protozoa, 

 and it also serves well for other forms. 

 Smears may be made on slides and stained 

 in Coplin jars, or they may be made on 

 coverslips and stained in Columbia jars. 

 The latter method has the advantages that 

 smaller amounts of reagents are necessary, 

 a neater preparation is obtained (since 

 there is no possibility of a portion of the 

 smear extending beyond the coverslip), 

 and in the completed slide the mounting 

 medium is beneath the smear rather than 

 above it, so that the microscope objective 

 can come closer to the smear. This fac- 

 tor may be of importance when the oil 

 immersion objective is used. Coverslips 

 are fragile, however, and greater care 

 must be exercised in handling them than 

 in handling slides. 



Clean the coverslips by dipping them 

 in 95% alcohol, and dry them with a clean 

 cloth before use. Be careful to handle 

 them only by the edges in order not to 

 leave fingerprints. 



Place a tiny drop of albumen fixative 

 in the center of the coverslip (or slide) and 

 smear it over the surface with the little 

 finger. (The finger should previously have 

 been cleaned and rid of its oil by dipping it 

 in 95% alcohol and wiping it with a clean 

 cloth. ) Albumen fixative is used to make 

 the feces adhere to the glass. 



Take up a small amount of feces on a 

 toothpick (preferably a round, smooth one) 

 and spread as evenly as possible in a very 

 thin layer over the surface of the coverslip. 

 Do not allow it to dry. Drop immediately 

 into a Columbia jar containing Schaudinn's 

 fluid at room temperature or 37° C. Allow 

 to remain about 10 minutes and then trans- 

 fer to 70% alcohol. 



In some cases it may be necessary to 

 mix the feces with a little physiological 

 salt solution in order to make it thin enough 

 to spread well. In other cases the feces 

 are so fluid that if the coverslips are drop- 

 ped edgewise into the fixative, all the ma- 

 terial will come off. To prevent this, place 

 the fixing solution in a small, flat vessel 

 such as a petri dish, and place the cover- 

 slip face down on its surface. After a few 

 seconds it can be transferred to a Colum- 

 bia jar. 



After fixation, wash the smear in 2 

 changes of 70% alcohol for at least 5 min- 

 utes each. Then transfer to 70% alcohol 

 containing enough iodine to give it a port 

 wine color. Allow to remain at least 10 

 minutes (preferably longer). This treat- 

 ment takes out the excess mercuric chlor- 

 ide which may otherwise form crystals in 

 the preparation. Then transfer to fresh 

 70% alcohol. Fixed material may be kept 

 in 70% alcohol indefinitely without injury. 



STAINING WITH HEIDENHAIN'S HEMA- 

 TOXYLIN 



In order to bring out many structures 

 of organisms it is necessary to color them 

 with a dye or dyes. The best and most 

 commonly used dye employed in parasitol- 

 ogic and histologic work is hematoxylin, 

 which is extracted from logwood. Hema- 

 toxylin alone has very poor staining 



