CAREY'S 



60 



CARMINE DUSTING 



knife cut the muscle quickly into pri- 

 mary pieces, 0.5 cm. long, and 0.5 cm. 

 thick, following the long axis of the 

 muscle fibers. Then cut the primary 

 pieces longitudinally into thin strips 

 1 to 2 mm. wide. 



2. Soak strips in freshly prepared 

 filtered lemon juice for 5 to 10 min. 

 until they become clear or translucent. 

 Rinse in cold tap water 4 to 5 times. 



3. Place strips in 1% aq. gold chloride 

 at 30°C. using at least 10 times the 

 volume of gold chloride solution to each 

 volume of muscle. While muscle is in 

 gold chloride solution, stir at least once 

 a min. The time for the optimum im- 

 pregnation of gold varies in the different 

 muscles of the same animal at a rela- 

 tively constant rate, for example, the 

 sternocleido-mastoid muscle of the 

 normal rat requires 16 min.; the pec- 

 toralis major, adductors of the thigh, 

 and biceps femoris, 13 min.; and the 

 gastrocnemius, tibialis anterior, and 

 the intercostal muscles 10 min. After 

 these muscles have been in the gold 

 chloride solution for the proper length 

 of time, they assume a yellowish-tan 

 color and have a firm consistency. It 

 is highly important that this variability 

 in the reaction of different muscles in 

 the same animal to gold impregnation 

 be realized. This may have been one 

 of the factors that led to the discarding 

 of the gold technique because it could 

 not be rigidly standardized. 



4. Pour off gold chloride solution and 

 rinse the tissue with tap water until the 

 water remains clear. Then place muscle 

 in 25% aq. formic acid in the dark 16 to 

 24 hrs. Too little time gives incomplete 

 reduction of the gold and too long time 

 excessive softening and maceration. 



5. Quickly rinse in tap water 5 or 6 

 times to remove as much of the formic 

 acid on the surface of the muscle as 

 possible. Even small amounts of for- 

 mic acid in the preserving fluid may 

 cause ultimate maceration of the tissue. 



6. Store the muscles until they are 

 teased in a mixture of § glycerine and 

 I5 70% alcohol. (The muscles have 

 been preserved in a good condition for 

 teasing in this mixture for 7 years.) 



7. To tease the muscle cut from one 

 edge with a flat bladed teasing needle a 

 piece 1 mm. thick and the full length of 

 the muscle fiber of short muscles. The 

 edge of the teasing needle may be flat- 

 tened by hammering the needle after it 

 has been placed in a Bunsen flame until 

 the needle is red hot. Orient this strip 

 of muscle in a drop of glycerine on a 

 clean, 1x3 slide. Gently add a clean 

 cover slip. Lightly press down with 

 the teasing needles, using a gentle 



lateral movement at right angles to the 

 long axis of the muscle fibers. The 

 muscle fibers, by this means, are gently 

 rolled out so that the preparation is one 

 muscle fiber thick. Check with micro- 

 scope. Such a preparation will keep 

 without any sealing of the cover slip 

 for at least 7 years. Any of the usual 

 cements, however, used for glycerine 

 mounts, may be used to make the prepa- 

 ration permanent. We have success- 

 fully used clarite. 



8. When cross or longitudinal sections 

 are desired reduce the gold by placing 

 muscle in a mixture of formalin 10% for 

 its hardening effect, and in formic acid 

 3% for the reduction of the gold. The 

 gold may, likewise, be reduced by strong 

 electric light for 16 to 24 hrs. The rou- 

 tine method for celloidin embedding is 

 then used. After the tissues have been 

 cut in sections, the nuclei can be coun- 

 terstained by various techniques. 



Carmalum (Mayer). Dissolve, if necessary 

 with heat, 1 gm. Carminic acid and 10 

 gms. ammonia alum in 200 cc. aq. dest. 

 Filter and to filter add 1 cc. formalin as 

 a preservative. The tissues stained 

 should not be alkaline (Lee, p. 141). 



Carmine has been very widely used as a 

 stain. Most of the formulae for stain- 

 ing of fixed tissues were proposed 40 or 

 more years ago chiefly by Ranvier and 

 Mayer. Now aniline dyes are more 

 popular but carmine is still of great use 

 for staining small animals m toto, for 

 staining tissues in bulk which are later 

 sectioned, as the best counterstain for 

 blue vital dyes like trypan blue, as the 

 most specific stain for Glycogen and for 

 Mucus in the form of mucicarmine, for 

 coloring gelatin used to inject blood 

 vessels and as a vital stain. Karsner, 

 H. T. and Swanbeck, C. E., J. Med. 

 Res., 1920, 42, 91-98 employed 15-25 

 cc. of fairly thick suspension for intra- 

 pleural injections in cats. At present 

 carminic acid is available and can be 

 employed instead of powdered carmine. 

 The only advantage is that the acid is of 

 more uniform composition. See Aceto- 

 carmine (Schneider), Alum Carmine 

 (Grenacher), Aluminum Chloride-Car- 

 mine (Mayer), Ammonia Carmine 

 (Ranvier), Best's Carmine for glycogen, 

 Borax Carmine (Grenacher), Carma- 

 lum (Mayer), Lithium Carmine (Orth), 

 Mucicarmine for mucus, Para-Carmine 

 (Mayer), Picro-Carmine (Ranvier). 

 Many more carmine combinations are 

 given by Lee (pp. 139-149). 



Carmine Dusting of the Lungs— Written 

 by C. C. Macklin, Dept. of Histological 

 Research, The University of Western 

 Ontario, London, Canada. November 

 28, 1951 — Mice or other mammals are 



