INTESTINAL FLAGELLATES OF RATS 125 



Intravitam staining of various cytoplasmic constituents may be 

 accomplished by using Janus green B, 1-5000 in physiological 

 salt solution (Hogue, 1926) ; Janus green, 1-1500 in 0.3% 

 NaCl ; brilliant cresyl blue, 1-50,000; neutral red, 1-50,000; or 

 methylene blue, 1-50,000 (Becker, 1926a). For further sugges- 

 tions see McClung (1929). 



Dark-field illumination is very useful for counting the flagella of 

 living flagellates and for the study of various cytoplasmic in- 

 clusions. 



Control of temperature may be accomphshed by either ( i ) mak- 

 ing the study in a warm room, (2) placing the microscope in a 

 warm box, or (3) using a warm stage. Several types of electric 

 warm stages which are reliable and convenient are on the market. 



Estimating the number of flagellates may be done roughly by 

 diluting the entire contents of the section of the digestive tract 

 to be dealt with in a measured quantity of salt solution and mixing 

 thoroughly ; then a measured quantity of this is placed on the slide 

 under a twenty-two millimeter square cover glass. Ten fields are 

 then counted and an average taken (Hegner, 1923a). Ratcliffe 

 (1928) made a i-io suspension of cecal contents and placed this 

 on a Leitz blood-counting slide. Counting the number of flagellates 

 in four groups of large squares gave the number in o.i cubic 

 centimeter of a i-io suspension. This figure times 100 gave 

 the number per cubic millimeter of the cecal contents. 



B. Temporary killing for counting flagella and recognizing other 

 details may be accomplished by diluting a small amount of in- 

 testinal or cecal contents with one of the following: (i) Lugol's 

 solution, diluted with water to a cherry red color, (2) saturated 

 alcoholic solution of iodine diluted in the same manner; (3) either 

 of the above with the addition of 0.5% eosin ; (4) 1% to 2% 

 osmic acid; (5) Janus green B, 1-2000; or (6) the flagellar stain 

 of Noland (1928).^ 



* Noland's flagellar stain : 



80 cc. of saturated solution of phenol in water 

 20 cc. of 40% solution of formaldehyde in water 

 4 cc. of glycerine 

 20 mgms. of gentian violet 



jMoisten the dye thoroughly with one cubic centimeter of water before 

 adding the other ingredients. Mix a drop of the reagent with a drop of the 

 culture or other material to be examined. 



