LEISHMANIA 



134 



LEUCOCYTES 



fresh 10% aq. solution (Takasaki, S., 

 Lues, Tokyo, 1938, 16, 127). 

 Leishmania. Media. Direct, microscopic 

 examination of peripheral blood may 

 be negative while detection in culture 

 is feasible. Q. iNL Geiman (Simmons 

 and Gentzkow) recommends addition 

 of 10 cc. blood to sodium citrate in 

 physiological saline, centrifuge and in- 

 oculate few drops buffey coat into tubes 

 of NNN medium, incubate 22-28°C. and 

 examine microscopically 10-20th day 

 for motile forms. The following media 

 are abbreviated from Geiman 's ac- 

 count. 



1. Blood agar or NNN (Novy, Mac- 

 Neal and Nicolle, 1908). Agar, 14 gm., 

 sodium chloride, 6 gm., aq. dest. 1000 

 cc. Add I vol. sterile defibrinated 

 rabbit's blood cooled to 45°-50°C. Mix, 

 tube long slant. After agar sets, cap 

 with sterile rubber stoppers. Prove 

 sterility by incubation 37°C., 24 hrs. 

 Inoculate material to be cultivated on 

 slant and in water of condensation. 

 Incubate 20°-25°C. Transfer every 20- 

 30 davs to maintain. 



2. Leptospira (Noguchi, 1924). 0.9% 

 aq. sodium chloride, 800 parts; fresh 

 rabbit serum, 100 parts; 2% nutrient 

 agar pH 7.2, 100 parts, rabbit hemo- 

 globin solution 10-20 parts. (To make 

 this hemoglobin solution take 1 part 

 defibrinated rabbit's blood and 3 parts 

 aq. dest., centrifuge and use clear super- 

 natant fluid.) Tube, prove sterility by 

 incubation before using. Subculture 

 ever}' 30 days. An increase in hemo- 

 globin solution improves growth of 

 Leishmania. 



3. Adler's modification of above. 

 Agar, 1 part; Locke's solution contain- 

 ing 0.2% dextrose, 8 parts; fresh rabbit 

 serum, 1 part. For species of Leish- 

 mania and Trypanosoma cruzi. 



4. Modified, Salle and Schmidt 

 (Cleveland and Collier, 1930). Veal 

 infusion (50 gm. Bacto-veal, Difco + 

 1000 cc. aq. dest.), 250 cc; proteose, 

 peptone (Difco), 10 gm.; sodium chlo- 

 ride, 5 gm.; aq. dest., 550 cc. Dissolve 

 make pH 7.4 and autoclave. Add 20 cc. 

 50% aq. glucose (sterilized by filtration 

 or in autoclave 10 lbs., 10 min.) and 

 60 cc. horse red cells laked with 2 parts 

 aq. dest. Pour in medium flasks or 

 tubes. Vigorous long lived cultures. 



Length measurements : 



Millimeters to inches X 0.0394. Inches 

 tomm. X25.4. SeeMicron. 



Leprosy Bacilli. Stain by carbol-fuchsin in 

 smears. See Concentration method for 

 collecting bacilli from lesions. For 

 study in sections, see Acid Fast Bacilli. 



Leptospira Medium, Noguchi's, see Leish- 

 mania. 



Leptospiras, method for isolation from water 

 (Bauer, J. H., Am. J. Trop. Med., 1927, 

 7, 177-179. See Spirochetes. 



Leuco Basic Fuchsin. To make add to 200 

 cc. aq. sol. fuchsin, 2 gm. potassium 

 metabisulphite and 10 cc. N hydro- 

 chloric acid. After bleaching 24 hrs. 

 add 0.5 gm . Novit , shake 1 min . and filter 

 through coarse paper. Resulting clear 

 solution works nicely in Feulgen tech- 

 nique (Coleman, L. C, Stain Techn., 

 1938,13,123-124). 



Leuco-Dyes as vital stains. Make 0.01% 

 aq. solutions of methylene blue,azur A, 

 thionin toluidine blue and brilliant cresyl 

 blue. Add to 100 cc. 1-2.5 cc. N/10 

 NasSaOa and 1-4 cc. N/10 HCl. Mix 

 and store at room temperature in dark. 

 To stain, add 1-2 drops of leucobase to 

 the protozoa, blood cells, etc. in physio- 

 logical saline. Said to give good contrast 

 staining of nucleus and cytoplasm and 

 to be useful in oxidation-reduction 

 determinations (Roskin, G., Arch. Russ. 

 Anat. Hist. Embr., 1937, 16,107-109). 



Leucocytes. In the broad sense they in- 

 clude all white blood cells but the term 

 is generally restricted to the "granular" 

 leucocytes as compared with the "non- 

 granular" ones (Lymphocytes and Mon- 

 ocytes). In a still narrower sense the 

 leucocytes include only polymorphonu- 

 clear neutrophiles, eosinophiles and 

 basophiles which are easily found in 

 circulating blood as contrasted with less 

 differentiated leucocytes called Myelo- 

 cytes and Myeloblasts generally con- 

 fined to the bone marrow. 



For mitochondria within leucocytes 

 supravital staining with Janus green is 

 indicated. In smears Giemsa's stain 

 has a little advantage over Wright's in 

 the fact that it better demonstrates any 

 bacteria that may be present. The 

 May-Giemsa technique is most used in 

 Europe. It is, in effect,a double staining 

 because the air dried smears are first 

 treated with the May-Grunwald com- 

 bined fixative and stain and are later 

 colored by Giemsa's stain. It gives 

 satisfying deep colors. TheKardos- 

 Pappenheim modification is suggested 

 when a particularly intense coloration of 

 neutrophilic granules is desired. Ehr- 

 lich's triacid stain may likewise be use- 

 ful because it is said to stain the neutro- 

 philic granules leaving the azur granules 

 untouched. 



Leucocytes give strong Peroxidase 

 and Oxidase reactions, which are, how- 

 ever, not specific for them. The Golgi 

 Apparatus (reticular material) can be 

 demonstrated by long treatment with 

 osmic acid or by the Cajal uranium ni- 

 trate and silver method (Cowdry, E. V., 

 J. Exper. Med., 1921, 33, 1-11). The 



