134 



THE ART OF MAKING MICROSCOPE SLIDES 



Frog embryos 



ployed for a cluster of a dozen or two 

 amphibian eggs. The eggs are transferred 

 to the solution which is immediately 

 placed in a dark cupboard where it re- 

 mains for about 48 hours. The original 

 specifies that low temperature should be 

 employed, but the writer has been unable 

 to find the least difference in performance 

 between heavily yolked eggs fixed at room 

 temperature and those which have been 

 fixed in a refrigerator. The solution should 

 be changed once or twice during the 

 forty-eight hours, or at least as often as it 

 becomes dark green. It is inevitable that 

 it should become greenish, but by chang- 

 ing solutions before a dark-green color ap- 

 pears, the deposition of chromium oxides 

 on the surface of the egg and its mem- 

 branes may be avoided. At the end of 

 forty-eight hours the eggs are removed to 

 large volumes of 2% formaldehyde in the 

 dark; the solution must be changed as 

 often as it becomes discolored and wash- 

 ing must continue until no further color 

 comes away. After all possible color has 

 been removed by the 2% formaldehyde, 

 the jars may be taken from the cupboard 

 and stored indefinitely at room tempera- 

 ture in the light. Most of the eggs or 

 embryos will be found to have become de- 

 tached from their gelatinous membranes 

 in the course of this treatment, but the 

 vitelHne membrane is frequently left. It 

 becomes brittle, however, and may be re- 

 moved without difficulty with the aid of a 

 couple of needles. 



Whichever method of fixation has been 

 employed, we are now left with the eggs or 

 embryos in 2% formaldehyde. The process 

 of embedding is different according to the 

 technique to be used. By the technique of 

 Gregg and Puckett the eggs are dehy- 

 drated through graded alcohols, allowing 

 in the case of frog eggs two hours each at 

 35%, 50%, and 60%. It is also necessary 

 to treat them with iodine to remove the 

 mercuric chloride, and for this purpose 

 they are placed in 70% alcohol, to which 

 has been added al^out 5% of Lugol's iodine 

 (Chap. 22 ADS 12.2 Lugol (1905)). The 

 exact proportion of iodine is not impor- 

 tant and technical directions usually read 

 "add iodine until the fluid is the color of 

 port"; but the author sees no reason why 



this insult to a noble wine should be per- 

 petuated. It- requires a treatment of at 

 least 48 hours to make sure that the 

 mercuric residues are removed and the 

 eggs are then transferred to 80% alcohol 

 where they are washed until no further 

 color comes away. They are then dehy- 

 drated for one or two hours each in 95% 

 and absolute alcohol. Gregg and Puckett 

 specify clearing in xylene for 30 minutes 

 but the writer prefers cleaning in benzene, 

 which does not seem to render the eggs so 

 brittle. After complete clearing, they are 

 then transferred to a mixture of one part 

 of the hydrocarbon employed and two 

 parts of soft (48°C.) paraffin at room tem- 

 perature. They may remain in this mix- 

 ture until next required. When embedding 

 is finally to be completed this mixture is 

 placed in an oven at 58°C. and allowed to 

 remain until completely liquified. The 

 eggs are then transferred to 52° paraffin 

 for one hour and finally to whatever 

 medium is chosen for embedding (Gregg 

 and Puckett prefer 55° paraffin ; the writer 

 prefers rubber-paraffin) for another 3 or 4 

 hours. 



In the alternative technique of Smith, 

 the procedure is rather different. After the 

 eggs are taken from formaldehyde they 

 are passed through 35% and 50% alcohol 

 for two or three hours each and then 

 placed in Grenadier's alcoholic borax 

 carmine for about two days. The formula 

 for this fluid is given in Chapter 20 (DS 

 11.22 Grenadier 1879) and it must be 

 most strongly recommended that one use 

 the dry stock dissolved in 70% alcohol 

 rather than the usual solution prepared 

 direct. The eggs are removed from the 

 stain to one-quarter of 1 % hydrochloric 

 acid in 70% alcohol and left there for 

 about two hours or until the first rapid 

 color clouds have died down. It is not in- 

 tended to complete differentiation by this 

 process, but only to remove the excess 

 stain. They are then, exactly as in the pre- 

 vious technique, dehydrated through 95% 

 and absolute alcohol before being cleared 

 in whatever hydrocarbon is preferred. 

 Smith prefers embedding in 52° paraffin 

 but this, in the writer's experience, is too 

 soft and will only permit the tliickest sec- 

 tions. It is again recommended that one 



