384 



lABORATORY DIAGNOSIS OF PROTOZOAN INFECTIONS 



(The following dehydration procedure, 

 recommended by Hewitt, can be used if 

 tertiary butyl alcohol is not available: 



MICROSCOPIC EXAMINATION 

 OF BLOOD 



In searching for blood protozoa, 

 thick or thin smears of the blood are pre- 

 pared and stained with one or another of 

 the Romanowsky (methylene blue-eosin 

 combination) stains. Thick smears are 

 preferable to thin ones for mammalian 

 blood because their use permits one to 

 examine a relatively large amount of 

 blood in a relatively short time. How- 

 ever, they cannot be used for avian blood 

 because of its nucleated erythrocytes. 

 The protozoa may be distorted in thick 

 smears enough so that some practice is 

 needed to differentiate species, especially 

 of the malaria parasites. 



Romanowsky stains may be either 

 rapid (such as Wright's and Field's stains) 

 or slow (such as Giemsa's stain). The 

 rapid stains are satisfactory if speed is 

 necessary, but they stain unevenly, par- 

 ticularly in thick smears, and they are 

 not as precise as the slow stains. Giemsa's 

 stain is best for most purposes. Mammal- 

 ian blood should be stained at pH 7.0 to 7.2, 

 and avian blood at pH 6. 75. These pH's 

 can be obtained by using Clark and Lubs 

 phosphate buffers. 



Trypanosomes, microfilariae and 

 most protozoa can be found in fresh, wet, 

 unstained smears, but for critical study 

 they must be stained. 



Preparation of Thin Blood Smears . 

 Clean 2 slides by rinsing in 95% alcohol 

 and wiping with a clean cloth. Handle the 

 slides only by their edges to avoid leaving 

 finger marks. Place a small drop of fresh 

 blood at the end of one slide, place the 

 other slide at a 30° angle to the first slide, 



touch the drop of blood with the end of the 

 slanted slide so that the blood runs into 

 the space beneath it, and then draw the 

 slanted slide rather quickly over the length 

 of the other slide. The blood should be 

 pulled behind the slide and not pushed ahead 

 of it as the smear is being made. A thin, 

 even film of blood should result. Wave the 

 slide in the air until it dries (a matter of a 

 few seconds if the smear is thin enough). 

 If the smear is to be stained in Giemsa's 

 stain, fix it by dipping in absolute methyl 

 alcohol (CP). If the smear is to be stained 

 in Wright's stain, fixation is not necessary, 

 since it will take place during the staining 

 process. If the smear is to be stored for 

 more than a day or so before staining, it 

 should be fixed. 



Preparation of Thick Blood Smears . 

 Prepare slides as for thin smears. Place 

 a medium-sized drop of blood or several 

 tiny ones on the slide, and mix with a 

 toothpick or the corner of another slide. 

 Allow to dry in air or in an incubator at 

 37° C. A hair dryer can be used to speed 

 up the drying process. Thick smears must 

 be laked (i.e. , the hemoglobin must be ex- 

 tracted) before being stained. This can be 

 done by placing them in water until the 

 color has disappeared. If Giemsa's stain 

 is used and the smears are fresh, laking 

 will take place during the staining process. 

 If the smears are to be stored for more 

 than a day or so before staining, they 

 should be laked and then fixed with absolute 

 methyl alcohol (CP) before storage, since 

 it is often extremely difficult to remove the 

 hemoglobin from smears which have been 

 stored for some time. 



While Leucocytozoon, microfilariae 

 and sometimes Trypanosoma can be found 

 with the low power of the microscope, the 

 stained blood smears should be examined 

 with the oil immersion objective for other 

 protozoa. The faster thin smears have 

 dried, the less distortion is produced. 

 Hence, the most natural appearing protozoa 

 will be found at the thin end and around the 

 edges of the smear. 



Cleaning Immersion Oil Off of Slides . 

 Stained blood smears are customarily not 

 covered with a coverslip, and immersion 



