In order to stain fluids, such as blood, pus, or sputum, for organisms, a very thin layer should be obtained by placing a little of the fluid between two clean cover-glasses and pressing them together. They are then separated and allowed to dry. The film is fixed by holding the cover-glass in a pair of forceps, and passing it slowly through the flame of a spirit lamp two or three times. Films of pus should be ‘cleared’ after fixing by placing them in a twenty per cent. solution of acetic acid for three minutes.
For clinical purposes it is often necessary to examine urine, fæces or vomited matter for bacilli. Films are prepared in the usual way and allowed to evaporate slowly, and then fixed by passing through the flame, and then washed in distilled water before staining. In the case of vomited matter and fæces this is usually done without difficulty. In the case of urine however it is often difficult to get the urine to evaporate completely. A syrupy layer remains, and if more heat be applied it decomposes and chars, and the products cause precipitation of aniline during subsequent staining processes. This may be partly avoided by gently washing the film in distilled water before staining.
Another plan is to mix the urinary deposit with a little gelatine free from organisms, such as that in unused culture tubes. The gelatin is liquefied by heat, and mixed with the deposit. Films are made from this mixture, and allowed to set, and then thoroughly washed in distilled water. The film is then dried thoroughly, and the cover-glass laid flat with the film uppermost, and a few drops of the staining fluid filtered on to it. After it has been stained sufficiently the stain is drained off, and the slip gently washed. The film may then be stained with some contrast stain in exactly the same way as sections, again washed, dried between folds of blotting paper, and mounted in balsam.
It is sometimes difficult to tell which is the side of the cover-glass which bears the film. This is readily done by holding the glass obliquely so that light from a window is reflected from its surface. The side which is coated appears dull; while the other is smooth and bright.
Methods of Examining Blood.
In all these methods blood is obtained by pricking the skin of one of the fingers, or the lobule of the ear, preferably the latter. The skin must previously be washed with soap and water or ether, to remove any grease or epithelial scales. The puncture should be made firmly so that blood may escape freely. The finger or ear must not be squeezed. Specimens must be made rapidly before red corpuscles have run into rouleaux. The slides and coverslips employed must be scrupulously clean, or it is impossible to get really good films. They should be cleaned with nitric acid and alcohol according to the directions on page [57].
Fresh specimens should be examined. The coverslip is made just to touch the drop of blood at one edge, so as to transfer a small quantity only, and is at once lowered on to the slide with the aid of a mounted needle. If slide and coverslip be perfectly clean the blood will spread out into a thin film, the corpuscles lying quite flat. If there be any delay, or if the cover-glass be not quite clean the red corpuscles will run into masses and the specimen will be useless for minute examination. Another specimen may be mixed with a little of Ferrier’s solution (p. [129]) before mounting. Permanent coverslip films may also be prepared.
Here again the use of absolutely clean coverslips is essential, and the blood must be taken immediately it escapes from the puncture. A little blood is taken on a cover-glass which is held horizontally. Another cover-glass is lowered on to this and by its weight and by capillary attraction, the drop of blood quickly becomes transformed into a thin film. The two covers are separated as soon as the film is formed by rapidly sliding them off one another. This manœuvre requires a little practice and dexterity. The movement of the slips must be in an exactly parallel direction otherwise the coating left will be uneven, just as when two pieces of bread and butter are pulled apart. Even with practice it is difficult to get more than one good film, the lower being usually best. There are four ways of fixing the film.
1. Exposure to osmic acid vapour.
The film while still moist is held over the mouth of a bottle containing at least one per cent. solution of osmic acid. In a minute or two the fixation will be complete, and the film becomes of a dirty brown colour. It is then left exposed to the air to get rid of all traces of osmic acid, and may afterwards be stained as described below.