DEAD BACTERIA 



94 



DECALCIFICATION 



been obtained with material fixed in 



Bouin's picric-formalin-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 Schizosaccharomyces pombi are 

 considered dead when tinged even 

 slightly by the stain. 



3. Decolorization (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 difficult 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) 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 flat and rounded cells. These were 

 pale grayish sacs with very thin walls 

 and fluid contents in which va,rying 

 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, 85, 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 piece 

 of the lower epidermis of the scale of 

 the onion bulb is peeled off and placed, 

 cutin side down, on a sHde. A drop of 

 a .5 per cent, slightly alkaline, aqueous 

 solution of neutral red is deposited on 

 the piece of epidermis and left there for 

 2 minutes; then it is blotted off and re- 

 placed by a drop of a .4 per cent potas- 

 sium hydroxide solution, which is imme- 

 diately removed (also with a blotter) ; 

 then the preparation is washed with 

 tap water. The living cells take with 

 that treatment a bright cerise red color, 

 while the dead cells are of an intense 

 orange yellow. The contrasts are vio- 

 lent. There are intermediate tints 

 which correspond to the dying cells. 

 See Necrosis, Necrobiosis, Survival 

 of tissue. 

 Decalcification. The removal of calcium so 

 that bony tissues can be cut in sections. 

 There are many methods almost all of 

 which involve acid treatment. It is 

 generally better to apply the de- 

 calcifying agent after fixation, particu- 

 larily so when the agent is a poor fixative. 

 The volume of decalcifier should be 

 about 100 times that of the tissue. The 

 usual, crude, way of testing the progress 

 of decalcification is to stick a fine needle 

 into the bone being careful to avoid the 

 area that will be cut in sections; but 

 less objectionable methods can be used, 

 see Teeth, Decalcification. 



Saturated aq. sulphurous, 5% tri- 

 chlorlactic, 5% hydrochloric and equal 

 parts of 1% hydrochloric and 1% 

 chromic acids are all fairly good de- 

 calcifiers. Lactic, acetic, phosphoric 

 and picric acids are usually unsatis- 

 factory. Shipley (McClung, p. 347) 

 recommends slow decalcification by 

 long immersion in Muller's Fluid 

 through liberation of small amounts of 

 chromic acid from the bichromate. 

 The bones of an adult rat require 21-30 

 days. The process can be hurried 

 somewhat by using an incubator at 

 37°C. Adequate decalcification is de- 

 tected by slight bending of the bone or 



