VIROLOGICAL METHODS 249 



remove excess yolk. The membrane is then removed to be macerated 

 between two slides. Making two preparations at once allows for two 

 stains to be used, e.g., Giemsa's and Mcachiavello's. Other embryonic 

 membranes from the egg require blotting only before making the smear. 



While many stains and modifications could be recorded, it would be 

 better if the novice learns to use these two basic ones well. Two stains 

 which usually are found in almost every virologist's laboratory are the 

 two mentioned above, Giemsa's and Macchiavello's. 



Staining. Giemsa's stain and method are well known. The stain is 

 available commercially, and if the manufacturer's directions are followed, 

 good results can be obtained. This stain is used for both viruses and 

 rickettsiae. In addition to the infectious agent being demonstrated, the 

 host cell can be differentiated. Inasmuch as it is a polychrome stain, 

 inclusions may be classified with regard to the reaction, i.e., basophilic, 

 neutrophilic, or acidophihc. The position of the inclusion, whether 

 intranuclear or intracytoplasmic, should be noted. Macchiavello's 

 method is given in Chap. II, page 31. The stain is useful for locating 

 rickettsiae, which appear as red rods on a blue background. 



Staining for Negri bodies involves examining the proper area of the 

 brain as well as suitable staining technic. For this reason the reader is 

 referree to more extensive treatises dealing specifically with this problem 

 (Francis, 1948). 



Since viruses and rickettsiae are small and because they are observed 

 against a background of complex host material, one must always be 

 aware of artifacts. Examination of numerous preparations from normal 

 material followed by examination of numerous preparations known to 

 contain infectious agents is time well spent. Only in this fashion can one 

 become somewhat proficient in recognizing significant factors in a stained 

 preparation. 



Examination of tissue containing viruses by phase microscopy holds 

 some promise but will not be discussed here. 



CULTIVATION 



Viruses may be cultivated by a variety of methods all of which most 

 probably utilize living cells. Not uncommonly only certain cells will 

 support the increase of these rather fastidious organisms. Usually one 

 or more of three methods is employed : susceptible animals, embryonated 

 eggs, and tissue cultures. 



Cultivation in Animals 



Normal animals vary considerably as to susceptibility to various 

 viruses and rickettsiae, depending primarily on species and age. Corti- 



