30 MANUAL OF MICROBIOLOGICAL METHODS 



STAIN FOR FAT DROPLETS 



Burdon's Method 

 Bur don (194.6) 



Staining solution: 0.3 g of Sudan black B (commission certified) in 

 100 ml of 70 per cent ethyl alcohol. After the bulk is dissolved, shake at 

 intervals and allow to stand over night. 



Staining schedule: 



1. Prepare smears as usual from 18- to 24-hr cultures, and fix by heat. 



2. Flood the entire slide with the above staining solution and allow it to 

 stand undisturbed at room temperature for 5-15 min. (Exact time is 

 unimportant, as good results are often obtained after only 1 or 2 min; 

 on the other hand no harm results if the slides stain until the solution is 

 completely dry.) 



3. Drain, and blot slide completely dry. 



4. Cover with xylene by pouring from a dropping bottle or dipping 

 several times in a staining jar. Blot till dry. 



5. Counterstain 5-10 sec with 0.5 per cent aqueous safranin, taking care 

 not to overstain. 



Note: For acid-fast organisms, ZiehFs carbon fuchsin diluted 1:10 

 with distilled water may be applied for 1-3 min, instead of safranin. 



6. Wash in tap water, blot, and dry. 



Results: Fat droplets blue-black or blue-gray; rest of cell pink. 



ROBINOW'S STAIN FOR NUCLEAR APPARATUS 



Slightly modified from Eohinow {1944) 

 Among several methods given by Robinow for staining nuclear material 

 the following seems as generally applicable as any : 



Staining solution: Add 1 drop of Giemsa stain to 1 ml of Sorensen's 

 buffer of pH 6.9-7.0. Robinow specifies Gurr's R66. Giemsa stain as 

 certified by the Biological Stain Commission, however, is equally good 

 and requires less prolonged staining ; the staining time given below is that 

 called for by the American-type Giemsa. 

 Fixation and smearing: 



1. Incubate petri-dish cultures 2-5 hr. 



2. Remove a block of agar, and fix it from a few seconds to several hours 

 in the vapor of 2 per cent osmic acid. 



3. Make an impression smear on a cover slip or glass slide. 



4. Store in 70 per cent alcohol till needed. 

 Staining procedure: 



1. Remove preparation from alcohol, and wash in water. 



2. Place for 5-10 min in normal HCl at 60°C. 



