TRYPANOSOMES 



254 



TUNGSTIC ACID 



after being air dried should be stained 

 immediately. The methods of Giemsa 

 and Wright are preferred giving a little 

 more time for the stains to work. For 

 details of structure use iron hematoxy- 

 lin after Schaudinn's fluid (Craig p. 49). 

 The South American trypanosome, 

 T. cruzi, is more easily cultured than 

 either of the African forms, T. gam- 

 biense or T. rhodesiense. Kelser's 

 medium, described fully by Craig, seems 

 to be the best. See references supplied 

 by him (p. 199) to culture in chick 

 embryoes. 



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. CaClc, 0.1 

 gm. MgCla, 0.05 gm. NaUPjOj, 1 gm.. 

 NatlCOa, 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 appro.xi- 

 mately 1 week. Subculture at 6-8 

 week intervals. 



Trypsin, a gelatin plate method as described 

 under Pepsin but slightly modified is 

 recammended. 



Tryptagar, see Bacteria Media. 

 Tryptophane Reaction. The procedure of 



Serra 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 

 (d = 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 NaNOo. The reagent 

 is prepared freshly every day and the 

 nitrite solution must also be freshly 

 made. 



"4. Mount directlj^ 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 ISO). For visible and 

 red rays employ 3.5 mm. "Uvet" lens 

 and 2% aq. copper sulphate. Bacilli, 

 golden yellow rods in violet fluorescent 

 background. See Sputum. 

 Tungstic Acid, a stable solution (Abraham- 

 son, E. M., Tech. Bull., 1940, 1, 75). 



