CAREY'S 



53 



CARMINE-GELATIN 



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 5 glycerine and 



I2 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 ^ 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 dj^es are more 

 popular but carmine is still of great use 

 for staining small animals in 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-Gelatin Injections of blood vessels. 

 Methods have been reviewed by Moore, 

 R. A., J. Tech. Methods, 1929, 12, 55- 

 58. He proposes a more accurate 

 technique for preparation of the gelatin 

 mass. Allow 80 gms. gelatin to take up 

 200 cc. cold water and heat to complete 

 the gel. Suspend 20 gms. carmine in 100 

 cc. water and add ammonia until dis- 

 solved. Mix the gelatin and carmine 

 solutions and add 15 gms. potassium 

 iocUde to reduce gelation point to less 

 than 25 °C. Place in water bath at 

 25 °C. and immerse a prepared platinum 

 electrode in it. Pass electrolytic hydro- 

 gen from a tank over the electrode and 

 agitate the gelatin with a motor stirrer. 

 Read electrical potential by balancing 

 against a standard cell. Add acetic acid 

 cautiously until reading of voltage corre- 

 sponds to pH 7.2. 



Two other techniques are listed by 

 Moore: 1. Dissolve 40 gms. carmine in 

 40 cc. strong ammonia and add water. 

 Allow to stand 12-24 hrs. and filter 

 through paper. Boil filtrate until it is 

 ammonia free. Precipitate the carmine 

 as a colloidal gel by adding 95% alcohol. 

 Filter, wash well with alcohol and dry 

 material collected. Dissolve 2 gm. in 

 5 cc. water and add 5 cc. 100 percent 

 gelatin in water thus making the product 



