TRYPANOSOMES 



361 



TUMOR CELLS 



vital stains under the microscope but 

 in this case they are not permanent. 

 The determination of the chemical 

 nature of these inclusions by Ormerod, 

 W. E., Brit. J. Pharm. and Chemo- 

 therap., 1951, 6, 334-341 is a fine example 

 of the application of histochemical 

 methods to protozoa. They contain ri- 

 bonucleic acid and protein and resemble 

 Volutin granules. 



Trypanosomes. Media. Summarized from 

 Q. M. Geiman (Simmons and Gentzkow, 

 658, 661). 



Brutsaert and Henrard's (A) 6.50 gm. 

 NaCl., 0.14 gm. KCl, 0.12 gm. CaCU + 

 aq. dest. to make 1000 cc. (B) 8.0 gm. 

 NaCl, 0.2 gm. KCl, 0.2 gm. CaCls, 0.1 

 gm. MgClj, 0.05 gm. NaHP204, 1 gm. 

 NaHCOs, 1 gm. glucose + aq. dest. to 

 make 1000 cc. Sterilize both by filtra- 

 tion and distribute in culture tubes 

 2 cc. A + 2.5 cc. B. Add 2 cc. citrated 

 human blood (1% citrate) and incubate 

 at 37°C. 24 hrs. to prove sterility. 

 Keep in refrigerator useful up to 2 

 weeks. Into a syringe containing 1 cc. 

 1% aq. sodium polyanethol sulfonate 

 draw up 5 cc. patient's blood. Dis- 

 tribute 0.5 cc. to each of 10 culture 

 tubes, incubate 25-28°C. Examine mi- 

 croscopically for trypanosomes after 

 10-20 days. 



Kelser's. Dissolve 2.5 gm. Bacto- 

 beef (Difco) in 500 cc. aq. dest. on 

 water bath 55°C., 1 hr. Add 12.5 gm. 

 Bacto peptone (Difco) and 3.5 gm. 

 sodium chloride by placing flask in boil- 

 ing water 5 min. Clarify by filtering 

 through cotton and make pH 7 with 

 IN sodium hydroxide. Determine vol- 

 ume and add 1% Bacto-agar. Dissolve 

 and distribute 5 cc. per test tube or 

 10 cc. per small flask. Autoclave 12 

 lbs., 30 min. Store for latter addition 

 dextrose and blood or for immediate 

 use add 5% of 1% aq. dextrose (0.25 cc. 

 per tube or 0.5 cc. per flask) and 5% 

 fresh sterile defibrinated guinea pig 

 blood. After thorough mixing slant 

 with short slant or deep butt. Use 

 sterile rubber corks to prevent evapora- 

 tion. Prove sterility by incubation. 

 Inoculate by adding organisms to slant 

 or water of condensation. On incuba- 

 tion at room temperature (22-25°C.) 

 growth becomes apparent in approxi- 

 mately 1 week. Subculture at 6-8 

 week intervals. 



Trypsin, a gelatin plate method as described 

 under Pepsin but slightly modified is 

 recommended. 



Tryptagar, see Bacteria Media. 



Tryptophane Reaction. The procedure of 

 Scrra and Lopes is specified as follows 

 by Serra, J. A., Stain Techn., 1946, 21, 



5-18: Prepare tissue as described under 

 Ninhydrin Reaction. 



"1. Harden the fixed pieces in 10% 

 formaldehyde for at least 1-5 hours (an 

 unnecessary step if a fixative with for- 

 malin has been employed); then wash 

 well. 



"2. Immerse for 3-5 seconds in an 

 aqueous solution of sodium silicate 

 (a = 1.1). When the materials are 

 sufficiently hardened this step may also 

 be omitted; it is recommended, how- 

 ever, that the coloration should be tried 

 both with and without it. 



"3. Immediately afterwards, immerse 

 the pieces in the Voisenet reagent for 

 10-15 minutes, in a small glass stoppered 

 bottle. This reagent is composed of 

 10 ml. concentrated HCl to which is 

 added, with a thorough stirring, one 

 drop of 2% aqueous formol and one drop 

 of 0.5% aqueous NaNOj. The reagent 

 is prepared freshly every day and the 

 nitrite solution must also be freshly 

 made . 



"4. Mount directly in glycerin and 

 observe, with squeezing, if necessary. 

 As the coloration fades, it is necessary 

 to observe the preparations on the same 

 day. 



"The reaction is given by indolic 

 compounds, and in proteins it is specific 

 for tryptophane, which reacts even 

 when bound. The localization of the 

 reaction seems to be satisfactory and 

 the sensitivity is sufficient for it to be 

 used in cytophysiological work." See 

 Romieu Reaction. 

 Tubercle Bacilli. Stain by Carbol Fuchsin, 

 see Acid Fast Bacilli. See Concentra- 

 tion method for sputum. Fluorescence 

 with auramine has been described 

 (Hagemann, P. K. H., Miinch. med. 

 Woch., 1938, 85, 1066). Fix smears by 

 flame and stain 15 min. in 1:1000 aq. 

 auramine (Bayer) containing 5% pheno- 

 lum liquefactum (liquid carbolic acid). 

 Wash in tap water. Decolorize in 

 ethanol 100 cc. ; HCl cone, 4 cc. ; sodium 

 chloride, 4 gm. renewing solution after 

 li min. Wash thoroughly in tap water. 

 Examine without cover glass under 

 fluorescence microscope using apochro- 

 matic dry objective and 3 compensating 

 ocular (X about 180). For visible and 

 red rays employ 3.5 mm. "Uvet" lens 

 and 2% aq. copper sulphate. Bacilli, 

 golden yellow rods in violet fluorescent 

 background. Kaiserling, C. Deutsche 

 Med. Wochenschr., 1939, 64, 1354, has 

 described differences in fluorescence of 

 human bovine tubercle bacilli. See 

 Coproporphyrin and Sputum. 

 Tumor Cells. All of these are not cancer 

 cells but see Cancer for technique. 



