112 Microbes and You 



faintly, if at all. This type of staining can be enhanced if the 

 organisms are grown or suspended in blood serum or in milk. 

 Many workers in the field are reluctant to accept the relatively 

 wide halo seen around such cells as representing the true size of 

 the capsule. Some shrinking occurs in the staining process and 

 capsules are exaggerated. By employing mordants and by ex- 

 amining undried preparations, more reliable results should be 

 obtained. 



A rather widely accepted technic for demonstrating capsules 

 is the Hiss Stain. This method recommends serum or ascitic 

 (body) fluid as background material in the preparation of the 

 smear. After allowing the slide to dry rather slowly at room tem- 

 perature, the smear is flooded with a crystal violet solution and 

 heated just enough to make steam visible. By washing off the 

 excess dye with a solution of copper sulfate (use no water any- 

 where in the process), draining away the excess fluid and blotting 

 gentlv until the slide is dry, the capsule will be seen as a faint 

 purplish halo about the more deeply stained underlying organism 

 when the oil immersion objective is employed. 



ACID-FAST STAIN 



As the name suggests, this stain remains fast even in the 

 presence of mineral acid. There are limits to this fastness, how- 

 ever, and just as timing is important in the various steps of the 

 gram stain, we must understand the limits of exposure of acid-fast 

 bacteria to acid. 



A relatively small group of organisms, principally members of 

 the genus Mycobacterium, possess chemical or physical properties 

 which make the cells difficult to stain, and once stained they are 

 difficult to de-stain by ordinary methods. Robert Koch's difficulty 

 in finding the causative agent in "consumption" ( tuberculosis ) may 

 be traced to this peculiarity in staining of the organisms. The 

 fatty-waxy nature of the capsule surrounding these organisms has 

 been the usual explanation of why the tuberculosis organism is 

 difficult to penetrate with dves. Once this tough barrier has been 

 breached, the reverse process of removing the dye is equally 



