DAVENPORT'S 



77 



DEAD CELLS 



protargoL 1946 modification written 

 by Dr. H. A. Davenport of original 

 (Davenport, H. A., Mc Arthur, J., and 

 Bruesch, S. R., Stain Tecbn., 1939, 14, 

 21-26). Fix for 1 to 3 days in: Form- 

 amide (Eastman Kodak Co.), 10 cc; 

 paranitroplienol, 5 gm.: 95% etiiyl alco- 

 hol, 45 cc; aq. dest., 45 cc. Transfer 

 thru graded alcohols to absolute, then 

 either n-butyl alcohol or xylene and 

 embed in paraffin. Sections are cut and 

 mounted in the usual manner, paraffin 

 removed and the slides run thru graded 

 alcohols to dist. water. Impregnate 

 for 1 hr. at 58-62°C. in a 5% aq. silver 

 nitrate. Rinse in 3 changes of aq. 

 dest. with 20-30 sec. allowed for each 

 change. The rinse water should cover 

 the slides completely, each slide sepa- 

 rate (not back to back) and the water 

 discarded with each change to prevent 

 carry-over of silver nitrate into the 

 protargoL Place the slides in 0.2% 

 protargol (Winthrop Chemical Co.) for 

 1 hour at room temperature. Rinse 

 quickly (2 sec.) in aq. dest. and reduce 

 for 1 to 2 min. in the following mixture : 

 Sodium sulfite, 5 gm. ; Kodalk (Eastman 

 K. Co.), 0.5 gm.; hydroquinone, 1 gm.; 

 aq. dest., 100 cc. Wash in running tap 

 water several minutes and rinse once in 

 dist. v/ater. Tone in 0.1% aq. gold 

 chloride for 5 to 10 min. Wash again 

 for about 1 min. and reduce in 1% aq. 

 oxalic acid for 10-20 sec. Rinse and 

 place in hypo (10% aq. sodium thio- 

 sulfate) for about 1 min. Wash in run- 

 ning water, dehydrate and cover. 



Notes: If the stain is too dark, try 

 any or all of the following modifications : 

 rinse longer after the protargol, use 

 0.1% protargol, omit the o.xalic acid re- 

 duction after gold toning. If too pale: 

 double the concentration of the pro- 

 targol, double the time of either or both 

 silver impregnations, omit rinsing after 

 protargol, double the concentration of 

 kodalk in the reducer, lengthen the 

 time of reduction in oxalic acid. The 

 technic is suitable for mammalian cen- 

 tral or peripheral nervous tissue, but 

 for sympathetic fibers in intestine and 

 uterus a moderate degree of success has 

 been obtained with material fixed in 

 Bouin's picric-fornialin-acetic (aq.). 

 Use clean glassware and fresh solutions! 

 Dead Bacteria. To distinguish from living 



try: 



1. Proca-Kayser stain (Gay, F. P. 

 and Clark, A. R., J. Bact., 1934, 27, 175- 

 189). Fix bacterial smear by drying 

 and flaming. Stain 3-5 min. in Loef- 

 fler's alkaline methylene blue. Wash 

 quickly and stain in Ziehl-Neelsen's 

 carbol fuchsin only 5-10 sec. Wash and 

 dry. Living bacteria blue, dead ones 

 purple to red^ 



2. Neutral red (Knaysi,G., J. Bact., 

 1935, 30, 193-206). Add a little neutral 

 red to the medium. Escherichia coli 

 and Schizosaccharoviyces pombi are 

 considered dead when tinged even 

 slightly by the sUxin. 



3. Decolor ization (Prudhomme, R. O., 

 Ann. Institut Pasteur, 1938, 61, 512- 

 518). Living bacilli separated from all 

 tissue decolorize solutions of 1-naphthol- 

 2-sodium sulphonate-indo-2-6-dibrom- 

 phenol, O-cresol-2-6 dichlorophenol 

 and 0-chlorophenol-indo-2-6-dichloro- 

 phenol. Bacilli killed by 100°C. for 15 

 min. do not decolorize them. 



The value of these methods is 

 questionable. 

 Dead Cells. Often it is very difl^icult to 

 say whether a particular cell was dead 

 or alive when the preparation was made. 

 The appearance of nuclei in Postmortem 

 Degeneration may be a clue. Evans 

 and Schulemann (H. M. and W. 

 Science, 1914, 39, 443-454) remarked 

 upon the extraordinary rapidity with 

 which dead cells take in vital benzidine 

 dyes and the diffuse, uniform coloration 

 that ensues. 



In cells supravitally stained with 

 neutral red Lewis and McCoy (W. H. 

 and C. C, Johns Hopkins Hosp. Bull., 



1922, 33, 284-293) employed the follow- 

 ing criteria for death : " (1 j loss of color 

 from the granules and vacuoles; (2) 

 diffuse pink staining of the cytoplasm 

 and nucleus; (3) the appearance of a 

 sharp and distinct nuclear membrane 

 and a change in texture of the cyto- 

 plasm and nucleus." Using dark-field 

 illumination W. H. Lewis (Anat. Rec, 



1923, 26, 15-29) observed the appear- 

 ance in dying cells of certain very small 

 brightly shining (white) bodies which 

 he called d or "death granules." These 

 were first in Brownian movement which 

 soon ceased. To quote Lewis: "During 

 the period when the cells were dying, 

 spherical blebs often appeared on both 

 the fiat and rounded cells. These were 

 pale grayish sacs with very thin walls 

 and fluid contents in which varying 

 numbers of small white granules in ac- 

 tive Brownian motion were seen. The 

 blebs varied in size and were occasion- 

 ally as large as a contracted cell. 

 Sometimes the blebs were so crowded 

 with granules that they were milky in 

 appearance. Frequently one would 

 burst, freeing its granular contents into 

 the surrounding fluid medium where 

 they showed Brownian motion until 

 they settled down on the slide." 



Luyet's (B., Science, 1937, So, 106) 

 method for the differential staining of 

 living and dead plant cells may prove 

 of value for animal cells also. He has 

 written the following account: A pieca 



