FLAGELLA 



126 



FLOATATION TECHNIQUES 



the fixative of choice for mitochondria 

 because it penetrates so much better 

 than Osmic Acid containing fixatives. 

 No important new fixatives have recently 

 been devised. 



In making the selection one is natu- 

 rally guided by data concerning the 

 structures which it is desired to demon- 

 strate (see Nerve Endings, Mitochon- 

 dria, etc.) or the substances to be re- 

 vealed (Lead, Copper, Oxidases, Lipids, 

 etc.) or the techniques that seem best 

 adapted to the purpose in mind (Mal- 

 lory's Connective Tissue stain, Wei- 

 gert's Method, etc.). Some of the more 

 important fixatives are listed, further 

 data being given under each heading. 

 Flagella. 1. Of bacteria. Loeffler's stain. 

 Mordant in fresh 20% aq. tannic acid, 

 10 cc; sat. aq. ferrous sulphate, 5 cc; 

 3-5% basic fuchsin in 95% ale, 1 cc. 

 gently heated, 1 min. Rinse in water 

 stain with slight heat in Carbol Fuchsin 

 1 min. wash and dry. For other flagella 

 stains see discussion in McClung (pp. 

 143-145) and Shuuk, I. V., J. Bact., 

 1920, 5, 181 ; Galli-Valerio, B., Centralbl. 

 f. Bakt. Orig., 1915, 76, 233; Gray, P. H., 

 J. Bact., 1926, 12, 273. See technique 

 for darkfield study of flagella (Pi j per, 

 A., J. Path. & Bact., 1938, 47, 1-17). 



2. Of erythrocytes (Oliver, W. W., J. 

 Inf. Dis., 1934, 55, 266-270). Add 1 mg. 

 hirudin to 2-3 cc. sterile Ringer's solu- 

 tion in small, sterile test tube. Draw 

 up about 0.5 cc. into a sterile Pasteur 

 pipette fitted with rubber bulb. Apply 

 to drop fresh normal blood from finger. 

 Suck up quickly into pipette and expel 

 into test tube. Incubate at 37 °C. 40-50 

 min. which promotes flagella production. 

 Add small drop to clean slide held at 

 40° angle. After the drop has rundown 

 slide, let dry completely in horizontal 

 position at room temperature. Mor- 

 dant in fresh 10% aq. tannic acid, 50 cc. ; 

 sat. aq. ferrous sulphate, 25 cc. and sat. 

 ale. basic fuchsin, 5 cc. which is poured 

 on slide and warmed slightly 20 min. 

 Wash thoroughly in running tap water 

 and dry. Flood with fresh Ziehl-Neelsen 

 (1 gm. fuchsin, 10 cc. alcohol -j- 90 cc. 

 5%aq. phenol acid) 20 min. not warmed. 

 Wash carefully in running water, blot 

 dry and examine with oil immersion. 

 It will be helpful to examine Oliver's 

 illustrations. (Revised by Wade Oli- 

 ver, Dept. of Bacteriology, Long Island 

 Medical College, Brooklyn, N. Y., 

 1946). 



The interpretation of observations on 

 bacterial flagella offers many pitfalls. 

 Dubos, R. J., The Bacterial Cell. 

 Harvard Univ. Press, 1945, 460 pp. 

 calls attention to their fineness, the 

 slight affinity of their substance for 



stains, the use of mordants which ad- 

 here to their surface increasing their 

 apparent diameter when stained, and 

 the fact that mechanical agitation 

 alone is sufficient to detach them from 

 the cells. By thus releasing flagella 

 sufiicient flagellar material can be col- 

 lected for immunological study and the 

 action of flagellar antibody on mobile 

 flagella can be followed microscopically. 

 Dubos remarks that the amounts of 

 flagellar material available are too 

 small to permit chemical analysis but 

 we may hope that techniques both of 

 collecting material and of analysis will 

 be so improved as to make this feasible. 

 He refers to numerous papers on elec- 

 tron microscopic examination of flagella 

 as revealing structural details pre- 

 viously unknown. 



Details in the structure of flagellae 

 are revealed by Electronmicroscopy. 

 See Brown, H. P., Ohio J. Sci., 1945, 

 45, 247; and DeRobertis, E., and 

 Franchi, C. M., Exp. Cell Res., 1951, 

 2, 295-298. See also Cilia and Polysac- 

 charides. 



Flagellates, intestinal. Those commonly 

 found in man are, according to Craig, 

 p. 115, Giardia lamblia, Chilomastix 

 mesnili, and Trichomonas hominis; less 

 frequently seen are Embadomonas in- 

 testinalis and Enteromonis hominis. 

 Stains much the same as for Endameba 

 and Leishmania. See Craig for choice 

 of suitable culture medium. 



Flame Photometer. Use of this instrument 

 in the analysis of biological materials 

 is critically presented by Wallace, 

 W. M. et al., J. Lab. & Clin. Med., 

 1951, 37, 621-629. 



Flavins under fluorescence microscope show 

 green fluorescence in liver tissue. See 

 Riboflavin. 



Fleas, see method of double imbedding for 

 (Lee, p. 598). 



Flemming's Fluid. Weak: 0.25% chromic 

 acid, 0.1% osmic acid and 0.1% glacial 

 acetic acid in aq. dest. Strong: 1% 

 chromic acid, 15 cc; 2% osmic acid, 

 4 cc. ; glacial acetic acid, 1 cc. These are 

 classic fixatives now not much used. 

 The Bensleys (p. 45) advocate same 

 ingredients differently made up. A: 

 1% aq. chromic acid, 11 parts; glacial 

 acetic acid, 1 part; and aq. dest., 4 parts. 

 B : 2% osmic acid in 1% aq. chromic acid. 

 Immediately before use, mix 4 parts of A 

 with 1 part of B and employ a volume 

 ten times that of the tissue. Fix 2-72 

 hrs. and wash in water 24 hrs. See 

 Safranin-Gentian Violet and Orange G 

 method. Mitosis, Benda's Method. 



Floatation Techniques. Many methods are 

 available for separating helminth eggs 

 from feces for microscopic examination. 



