First pour enough aniline into a test tube to cover the bottom and half fill with water, shake violently for two minutes, and filter through funnel, which has previously had wet filter paper fitted. It is essential that the filter paper be saturated with water, else the aniline oil will separate during filtration. Our next step is to deposit specimen of sputum in mortar (if very viscid, add a few drops of water), and triturate thoroughly in order to break up encapsulated colonies, and distribute evenly through the specimen.
Now remove an amount which will just cover end of toothpick, and deposit it on a previously cleaned cover glass, which should not be over 1
100 inch thick, and thinner if possible; immediately cover with another cover glass, allowing sputum to spread by capillarity or slight pressure, and separate by sliding apart, and put aside to dry without heat. I have found that specimens dried without heat (and consequent coagulation of albumen) will show a much larger number of bacilli than when heat is used. I believe this is due to the fact that the fuchsine penetrates more thoroughly through the albumen when not coagulated, or that when it is coagulated by heat it to a greater or less extent it protects them from the action of the stain. While the covers are drying we will pour out a sufficient quantity of the aniline water, which by this time has filtered into one of the staining glasses, and add one or two drops (not more) fuchsine solution. Now, placing one of the cover glasses on our cover holder, sputum side down, we lower it into the staining fluid and withdraw holder from the side, and repeat the operation for the other cover glass. It is my habit to allow the covers to remain in this solution for at least eight hours or over night. The time may be reduced to ten or fifteen minutes by heating the red stain to about 140 or 150 F., but the result is not so brilliant, nor is it sure, as I have frequently failed to find the bacilli by the short method, but have been able to demonstrate their presence by the long one.
At the end of either of the above periods of time, the cover glass is lifted out of the staining solution and, without washing, immersed in our five per cent. solution of nitric acid and alcohol. It is this part of the process, if any, which will give trouble, as the time of immersion is governed by the thickness and general character of the sputum. My custom is to hold the first cover immersed until the color has just disappeared, or say fifteen seconds, and the second five seconds longer; but a very little experience will remove any difficulty from over-decolorizing.
From the decolorizing solution they are immediately immersed in water and thoroughly washed, when they may be again floated in the contra-stain, which is prepared by filling the other staining glass with water to which a few drops (three or four) of our methyl blue has been added. They should remain here for from five to eight minutes, when they are again removed with the pincetts, and a few drops of alcohol poured over them to wash off the surplus stain. Again wash in clean water, and dry by gentle heat (which will now do no harm) over the alcohol lamp, and place sputum side up on table.
A very small drop of thin benzole balsam is now placed in the centre of each cover, and a cleansed slide gently lowered over one in such a position that both covers may be mounted on a single slide. As soon as the slide has been sufficiently lowered to come in contact with the drop of balsam, it spreads by capillarity, and draws the cover close to the slide without the slightest danger from air bubbles being engaged, and the slide may at once be inspected by a dry objective.
I have found it necessary to use an objective at least as high as one-fifth or one-sixth, with central illumination without diaphragm, as cases will frequently occur where the staining is so faint, that with a lower power they will escape observation, though a good, wide angle, four-tenths inch, will show them well when strongly stained.
I have endeavored to explain the method with perhaps too strict a regard to detail, but am sure that one who follows the various steps once or twice cannot fail to acquire the necessary technique without occupying more than fifteen minutes of working time; that is to say, five minutes to the first staining, and then the following morning to prepare and mount for observation.
171 Gates Ave., Brooklyn.
ADDRESS TO THE GRADUATES OF THE LONG ISLAND COLLEGE HOSPITAL TRAINING SCHOOL FOR NURSES, DELIVERED JUNE 12, 1888.
BY GEORGE G. HOPKINS, A.M., M.D.