as needed and can be used repeatedly for at least 

 a month if kept cool: 



HgClo, saturated aqueous, 10 ml 



Glacial acetic acid, 2 ml 



Formalin, cone, 2 ml 



formol-nigrosin, 1-2 ml (depending upon 



intensity desired) 

 t-butanol, 10 ml. 



The formol-nigrosin is mixed as follows: 



Formalin, cone, 20 ml 

 nigrosin, soluble, 4 g 

 distilled water. 100 ml. 



The procedure for the NMF method is: Place 

 a drop of a concentrated suspension of organ- 

 isms on a clean slide and pipette onto it from 

 a height of 2-3 cm a drop of stain-fixative. After 

 a few seconds wash the culture fluid to the ends 

 of the slide by additional drops of the stain- 

 fixative. Practically all the specimens will be 

 fixed, stained, and attached to the slide, and after 

 about 15 sec the preparation may be dehydrated, 

 cleared, and covered. Ciliary organelles gener- 

 ally will appear black against a gray background, 

 cytopharyngeal rods, isolated dorsal cilia, gran- 

 ules between ciliary rows, compound buccal cilia, 

 surface ridges, and general surface morphology 

 are demonstrated (Borror, 1968a). Highly con- 

 tractile ciliates sometimes can be prepared by 

 this method following their relaxation in 8% 

 MgCl2; extremely fragile ciliates often can be 

 prepared by this method following preliminary 

 fixation in Champy's fluid (Borror, 1969). 



In addition, most ciliate specialists use one or 

 both of the following methods: the Chatton- 

 Lwoff' technique (sometimes called the French 

 silver method or the wet silver method) and the 

 protargol (or silver proteinate) method (Corliss, 

 1953; Tufl'rau, 1967a). The body form is par- 

 ticularly well preserved with the latter methods. 

 Both methods require several hours for prepa- 

 ration and careful attention to the variables of 

 time and temperature, and are part of the rep- 

 ertoire of the specialist. Although not neces- 

 sary for the use of this key generally, the Chat- 

 ton-Lwoflf technique is particularly desirable for 

 identification of members of the Hymenostoma- 

 tida, whose buccal structures are difficult to dis- 

 cern in life. This technique also demonstrates 

 basal bodies of cilia and other cortical features 

 such as cytostome, pore of the contractile vac- 

 uole, cell anus, and fiberlike "silver lines" that 

 apparently are places of contiguity of cortical 

 membrane systems. The protargol method stains 

 cilia and associated fibrillar systems as well as 

 nuclei and, in association with the Chatton-Lwoff 

 technique, is particularly useful in developmental 

 studies. 



Further information on the use of the micro- 

 scope for examination of ciliates, preparation of 

 slides with both fresh and permanent material, 

 as well as additional cytological techniques, can 

 be found in Kudo (1966). In the hands of a 

 nonspecialist, a small amount of lO^'r Methocel 

 in seawater greatly facilitates study of living 

 ciliates and does not prevent subsequent staining 

 with acidified methyl green. 



