Section Cutting and Staining / A practical introduction to histological methods for students and practitioners

by Walter S. Colman

Section Mounting.

1. By flotation.

In this method the section whether stained or unstained is placed in a bowl of water, or normal salt solution (p. [53]). A clean slide is then introduced into the water at an angle of about 60°, a little more than half of its length being submerged. The section is then brought up by the needle and floated as far as possible into position on the slide. One corner is then fixed by the needle, and on gently withdrawing the slide the section should lie flat. If any folds are left no attempt should be made to smoothen them out with a needle, but the slide should be re-immersed until the folded part of the section is under water. It should then be gently withdrawn, when the fold will disappear. This manœuvre must be repeated in different directions until the section lies quite smoothly on the slide. Stained and unstained sections are floated out in this way before being mounted in Farrant’s medium, and unstained sections previous to staining in picrocarmine.

2. By transference with a section lifter.

This method is employed in mounting in Canada balsam in order to transfer the section from the clarifying agent (p. [63]) to the slide. The lifter is polished, and insinuated under the section. The section being held in position by the needle is now raised from the fluid, excess of which is removed by holding the section in position with a mounted needle, and tilting the lifter so as to allow it to drain off.

Removal of air bubbles from sections.—When sections contain many air bubbles, the best plan is to leave them in methylated spirit for a time. The bubbles then coalesce and escape from the section.

For delicate structures and for fresh sections the transference to spirit, and the subsequent flying out of the section when returned to water are risky, and the best method of treating these is to put the vessel containing them under the receiver of an air pump, if one is available, and slightly exhausting the air.

The most frequent cause of air bubbles in mounted specimens, however, is the employment of cover-glasses which have not been thoroughly cleansed. Proper cleansing is best effected by placing the covers when bought in a shallow wide mouthed stoppered bottle containing strong nitric acid, and leaving them in this fluid for twenty-four hours. The acid should then be drained off and water run through the vessel from a tap, until the washings no longer give an acid reaction with litmus paper. The water should then be drained off, and the glasses covered with absolute alcohol. They can be removed one by one and rapidly dried as required. With cover-glasses properly cleansed in this manner, not only will air bubbles be avoided, but the covers will be dried much more easily with the cloth, and fewer will be broken in the process.

Another very frequent cause is the transference of air bubbles with the mounting medium on the glass rod. This occurs especially if the rod be fused to the stopper. The proper bottles to use, both for Farrant’s medium and balsam are “balsam bottles” which have no stopper, but the mouth is closed by a glass cap which fits accurately (fig. 9). A short glass rod is attached to the cap, and is used to transfer the medium to the slide.

Fig. 9.—Balsam bottle.

Treatment of folded sections.—The folding may be due:—

(1.) To the section having creased through being cut with a knife whose surface was not perfectly smooth. This is best remedied by placing the section in methylated spirit for a minute, and then transferring it to a bowl of clean water, when the section will rapidly rise to the top, and spread itself out flat on the surface of the water, in consequence of the alcohol rapidly diffusing out at the edges into the surrounding water.

(2) To the section containing a large amount of fat, as in those of the skin and subcutaneous tissue. The fat may be removed from the fat cells without materially altering the appearance of the section. This is done by dehydrating the section in alcohol, and then transferring to a watch glass containing ether or chloroform to extract the fat. The tissue should be washed free from ether in the alcohol and then transferred to the bowl of water, and allowed to float out. This process does not interfere with subsequent staining operations.