184 The Philippine Journal of Science isis 



The gastric vacuoles may be studied in the living organism 

 with very dilute solutions of neutral red or alizarin, added in 

 small quantity to the medium containing the organisms. In 

 connection with the use of intra vitam stains, it should not be 

 expected that they will stain living protoplasm. 



Occasionally, interesting results are obtained by adding an 

 opaque material to the culture medium, which will have the effect 

 of causing the organisms to stand out as bright, shining objects 

 on a dark field. This affords an excellent method of studying 

 the action of motile organs. Aniline black is one of a number 

 of the aniline dyes that may be used for this purpose; di- 

 phenylamin blue is another. India ink has been used by many 

 workers; it admits of observation of the discharge of the con- 

 tractile vacuole. Avoid the presence of acid in cultures so 

 treated. These substances may be used in combination with the 

 immobilizing media previously mentioned. 



Fixation and staining. — Though a study of the living animal 

 under normal conditions should always precede a study of fixed 

 and stained cells, it must be well borne in mind that staining 

 brings out many important details that cannot be observed in 

 the living cell. The nucleus, the form and finer structure of 

 which is subject to considerable variation in the Protozoa, is 

 seldom visible in any detail in the living cell. The technical 

 treatment of the protozoan cell is not a simple matter. There 

 are a multitude of methods that have been devised to meet 

 special conditions, and it is, of course, impossible to go into 

 this subject in any detail here. I shall be glad to correspond 

 with any investigator who desires aid in connection with any 

 special problem. A general method, which may be applied to 

 the usual run of free-living and parasitic species, is given below. 



To secure a good microscopical picture of the animal, the most 

 approved cytological methods must be employed. With the ex- 

 ception of studies of the hsematozoa, all staining must be done 

 by the "wet method;" that is to say, the preparation must never 

 be allowed to dry until it is sealed under the cover glass. Even 

 with the blood parasites special "wet methods" have been devised 

 that give infinitely better results from the cytological viewpoint 

 than the old Romanowsky methods. 



The best general fixative is sublimate-acetic fluid. It is made 

 up as follows : 



Saturated solution of mercuric chloride in sea water 



(per cent) 95 



Glacial acetic acid (per cent) 6 



