T E C H N I Q U 1- 7 



released and combines with the stanung agent. At die same time the stainable 

 material ot the outer layers o{ the cell is more completely hydrolysed, so that 

 its masking eftect is reduced. This dift'erentiation is made possible by the 

 fact that the nuclear structures are composed largely of Fculgeii-positivc 

 deoxyribose nucleoproteins, whereas the cell membrane and surface layers 

 of the cytoplasm usually contain a higher proportion of ribose nucleoproteins. 



To pcrtorm the stain, smear preparations are made upon shdes or cover- 

 slips. They may be unfixed, although these tend to wash off, or they may 

 be fixed in osmic acid vapour. Most fixatives should be avoided as they may 

 completely alter the appearance of the nucleus. 



Hydrolysis in Normal HCl should be conducted at a temperature, approxi- 

 mately, of 60° C. Staining, in dilute Giemsa, is best performed at 37° C. 



The periods required tor hydrolysis and staining arc exceedingly variable 

 and may be different at different ages of the same culture. It is often necessary 

 to examine the preparation with the microscope, in order to determine 

 whether it is suitably stained, and for this purpose a water-immersion lens 

 is a great convenience. Most bacteria require from ten to twenty minutes 

 hydrolysis, and thirty minutes in the staining solution. Some require longer 

 periods or stronger solutions. 



A properly stained preparation is bright pink in colour, the nuclear 

 structures staining more intensely than the cytoplasm, which may stain bluish 

 or purple in some cases. Inadequate hydrolysis is indicated by a uniform 

 purple colour, and excessive hydrolysis by a pale pink colour and blurred 

 outline. Inadequate or excessive staining periods are self-evident in the 

 appearance of the preparation. 



It is important to use fresh reagents, and otherwise inexplicable failures 

 may be found to be due to neglect to do so. 



Other methods of staining give comparable results, and may be useful ill 

 the case ot bacteria which do not stain well by the classical method. Cold 

 perchloric or trichloracetic acid, or even weak alkalis, may be substituted for 

 hydrochloric acid. A variety of different dyes may be used instead of Giemsa. 

 Thionin gives good results and has been widely used, but is much less specific 

 than Giemsa, and stains the basophilic elements of the cell envelopes as well 

 as the nucleus, which is liable to cause confusion in interpretation. 



