DS 11.20 



DYE STAINS OF GENERAL APPLICATION 



299 



the preparation. The results leave nothing 

 to be desired, whether the mount is made 

 of the medusa, which has been taken as a 

 typical example, or of any other exceed- 

 ingly delicate material that is liable to 

 collapse in mounting. 



Preparation of a smear to show chromo- 

 somes in saUvary glands of Chi- 

 ronomus using the iron-aceto- 

 carmine stain of BelUng 1921 



Aceto-carmine preparations are easy to 

 make but their utility is limited by their 

 short life, which rarely exceeds, even in a 

 successful preparation, more than a week 

 or two. They are, however, excellent for 

 class demonstration, and the preparation 

 which follows should be placed in the 

 hands of every student of cytology who 

 wants to see for himself the really startling 

 detail which can be obtained in salivary 

 gland chromosomes. Though this tech- 

 nique is simpkst when the larva of the 

 dipteran Chironomus is employed, there 

 are many other small flies with which it 

 may be used. 



The larva of Chironomus which is recog- 

 nized by the rapidity of its movements 

 and its bright blood-red coloration, may 

 usually be collected from any fresh-water 

 stream. It is a waste of time to dissect out 

 the salivary glands since they may be ob- 

 tained more easily by pulling the animal 

 apart. Take the Chironomus larva and 

 strand it on a clean glass sUde on which the 

 final mount is to be prepared. If the 

 animal is living in ordinary pond water 

 containing much debris, it is desirable to 

 transfer it for an hour or two to filtered 

 water in order that the worst of the debris 

 may be removed. Two mounted needles 

 are all that are required to complete the 

 technique. The first needle is pressed 

 firmly upon the head of the larva which, 

 for a right-handed individual, may be 

 most conveniently directed toward the 

 left. With the point of the second needle a 

 very small hole should be torn in the upper 

 integument immediately behind the junc- 

 tion of the head with the thorax. Though 

 this hole is not absolutely essential it is an 

 insurance against the animal's breaking 

 in an undesirable place. The second needle 



is then pressed firmly and horizontally 

 across the abdomen and drawn with a 

 slow and steady motion to the right. Nine 

 times out of 10 the larva will break in 

 half at the junction of the head with the 

 tliorax, and there will be drawn out of the 

 thorax the anterior portion of the ahmen- 

 tary canal together with, of course, large 

 numbers of attendant structures, includ- 

 ing the salivary glands. The salivary 

 glands may be recognized as a pair of 

 flattened, transparent, pear-shaped struc- 

 tures in which the nuclei are so large that 

 they may be recognized with even the low 

 power of a microscope. Everything except 

 the salivary glands should now be dis- 

 sected away with the needles, the glands 

 themselves being severed from their at- 

 tachment either with the point of the 

 needle or with the edge of a minute scalpel. 

 A few drops of isotonic saline may now be 

 employed to wash away the remainder of 

 the adherent material, leaving the salivary 

 glands isolated on, and usually adherent 

 to, the surface of the slide. 



It is assumed that a supply of Belling's 

 aceto-carmine has already been secured, 

 made up according to the directions given 

 in the formula below (DS 11.23 Belling 

 1921). All that is now necessary is to place 

 a few drops of this on top of the salivary 

 glands and to add a coverslip. Care should 

 be taken that sufficient is added to pre- 

 vent undesirable squashing at this stage 

 of the proceedings. The slide is now placed 

 on one side for about five minutes, the 

 aceto-carmine being replaced as it evapo- 

 rates. Very little should normally be lost, 

 however, unless the atmosphere is unduly 

 dry. The specimen is then examined under 

 the low power of the microscope and, if it 

 appears to be too opaque, is flattened by 

 gentle pressure from a needle. The ejected 

 aceto-carmine is mopped up on a filter 

 paper until such time as it is sufficiently 

 thin. The specimen may then be exam- 

 ined under the high power of the micro- 

 scope and the remarkable structure of the 

 chromosomes noted. 



There is no really satisfactory method of 

 rendering permanent an aceto-carmine 

 preparation, though they will kooji for 

 some weeks if the edge of the coverslii) is 

 cemented (sec Chaptors 2 and '^) with 



