COLORS 



87 



CONCENTRATION 



parts of the tissue with a hypodermic 

 syringe and long needle. Do not use 

 too much pressure and be careful not to 

 let any of the fixative spurt back into 

 one's face. Before the next step wash 

 in running water for about 12 hrs. 



2. For color restoration: Place the 

 tissue in 80% ethyl alcohol for 10-60 

 min. and watch for optimum coloration. 

 If left too long in the alcohol the colors 

 fade. Rinse in water and transfer to 

 No. 3. 



3. For final preservation: Change to 

 glycerin, 500 cc. ; 1% aq. arsenious acid, 

 200 cc; tap water, 2300 cc; potassium 

 acetate, 250 gms.; thymol, 2.5 gm. To 

 obviate difficulty of dissolving the 

 arsenious acid and to sterilize add 25 

 gms. arsenic trioxide to 2500 cc. water 

 + the thymol crystals first ground up 

 in a mortar and place in steam sterilizer 

 for 6 hrs. Then add other substances. 



There are 2 J ores solutions. 



1. For fixation: Chloral hydrate, 50 

 gms.; artificial Carlsbad salts (sodium 

 sulfate, 22 gm.; sodium bicarbonate, 20 

 gm. ; sodium chloride, 18 gm. ; potassium 

 nitrate, 38 gm.; potassium sulphate, 2 

 gm.), 50 gm.; formalin, 100 cc ; tap 

 water, 1000 cc Allow to act 2-14 days 

 depending on size, wash 12 hrs. in run- 

 ning water. 



2. For final preservation: Potassium 

 acetate, 300 gm., glycerin, 600 cc; aq. 

 dest., 1000 cc. 



Mai lory suggests fixation in Jores' 

 first solution and preservation in Kaiser- 

 ling's third solution. 



Colors, Interference, see Interference 

 Colors. 



Columbium, see Atomic Weights. 



Concentration. 1. Tubercle bacilli in spu- 

 tum. Nagy (A.H., J. Lab. & Clin. 

 Med., 1939, 25, 67-71) having critically 

 evaluated several techniques recom- 

 mends Pottenger's Dilution-Flotation 

 method. Shake equal parts sputum and 

 0.5% aq. sodium hydroxide for 10 min. 

 Digest in water bath at 56°C. for 30 

 min. Add 1 ml. (= 1 cc.) hydrocarbon 

 (gasoline or xylene), then 200 ml. aq. 

 dest. and shake 10 min. Allow hydro- 

 carbon to collect at top 15-20 min. 

 Take up hydrocarbon layer in rubber 

 bulbed pipette. Keep in vertical posi- 

 tion until supernatant fluid separates 

 from hydrocarbon, 5-10 min. Make 

 smears from hydrocarbon and dry. 

 Remove hydrocarbon by washing with 

 ether. Stain with carbol fuchsin 3 hrs. 

 or longer. Decolorize with acid alco- 

 hol 30 sec. or less. If further decolor- 

 ization is required employ 10% aq. 

 sodium sulphite. Counterstain with 1% 

 aq. picric acid or with methylene blue. 

 The concentration of bacilli is about 33 



times. Perhaps a modification of the 

 method could be used for leprosy or- 

 ganisms in emulsions of tissues. See 

 also Pottenger, J. E., Am. Rec Tuberc, 

 1939, 40, 581. Concentration of tuber- 

 cle bacilli in spinal fluids (Hanks, J. H. 

 and Feldman, H. A., J. Lab. & Clin. 

 Med., 1939, 25, 886-892). It is often 

 necessary to concentrate for micro- 

 scopic study objects which are not 

 present in abundance and which might 

 otherwise be overlooked. See exami- 

 nation of Feces for ova of parasites, of 

 Urine for sediment. 



2. Leprosy bacilli for chemical analy- 

 sis. Ra void's method for leprosy bacilli 

 can perhaps be used for others. Rela- 

 tively large masses of bacilli-laden cells 

 are dissected away from neighboring 

 uninvolved tissue and from necrotic 

 tissue when present in the centers of 

 the nodules. They are placed in a 

 Wueller press without addition of any 

 fluid. On exertion of pressure many 

 of the cells are ruptured and the tissue 

 fluid, together with cytoplasm, nucleo- 

 plasm and some entire cells, passes 

 through minute holes in the press and is 

 collected, leaving most of the fibrous 

 elements behind. Then a little saline 

 solution is added and the material is 

 ground up in sand and made up to a 

 volume of about 50 cc. The sand is 

 allowed to sediment out at the bottom 

 of a centrifuge tube. The supernatant 

 fluid is then centrifuged at low speed 

 (300 r.p.m.). This throws all the rest 

 of the debris to the bottom while the 

 bacilli remain in suspension. The 

 supernatant fluid, containing the bacilli, 

 is again decanted and centrifuged at high 

 speed (3500 r.p.m.) in an angle 

 centrifuge for 1 hr. The supernatant 

 fluid is discarded and the pasty material 

 at the bottom of the tube, made up of 

 bacilli, is diluted and washed by re- 

 peated centrifugation in some experi- 

 ments with saline solution and in others 

 with distilled water. 



Beginning with a large nodule or with 

 several small ones it is a simple matter 

 to collect in 4 or 5 hrs. billions of bacilli. 

 The pasty bacterial mass can be desic- 

 cated and weighed in grams. For our 

 experiments we used only the wet 

 bacilli. When viewed en masse they 

 appear dense white with a faint shade 

 of gray. They are not yellow or even 

 yellowish. Examination of a thick 

 smear, made after washing in saline, 

 shows myriads of bacilli without any 

 trace of cellular material. The bacilli 

 retain to a remarkable degree their 

 characteristic morphology, as seen in 

 sections and in smears of fresh tissue, 

 and their acid-fast properties are not 



