| Saturated alcoholic solution of methylene blue | 3 parts |
| Aqueous solution of NaOH (or KOH), 1 to 10,000 | 10 parts |
| Mix and filter. | |
| Saturated alcoholic solution of fuchsin | 1 part |
| 5 per cent. aqueous solution of carbolic acid | 10 parts |
| Mix and filter. | |
| Dry methylene blue | 4 parts |
| Concentrated H2SO4 | 25 parts |
| Distilled water | 75 parts |
| Dissolve the dry dye in the acid and add the solution to the distilled water and filter. | |
Fig. 140.—Author’s staining set. Square bottles are set in square holes in the block. The capacity of each bottle is 30 cc.
Staining solutions are conveniently kept in square dropping bottles inserted in a block as shown in [Fig. 140]. This form of holder necessitates the use of one hand only in securing the stain and dropping it on the preparation.
The actual staining of bacteriological preparations can be learned only by repeated laboratory practice, yet the following methods have given such uniform results in class work that it is felt they are not out of place in a text-book.
Preparation of the “Film.”—The author learned to stain bacteria, on the “cover-glass” but does not recall having used this method in fifteen years and does not teach it to his students. All staining is done on the slide. To prepare a film from a solid culture medium the procedure is as follows:
First, be sure the slide is clean and free from grease. This is accomplished most readily by scouring a few minutes with finely ground pumice stone and a little water, then washing and drying with a grease-free cloth, handkerchief
, or piece of cheese-cloth. With the “loop” needle place in the middle of the slide a small loop of water. This is best done by filling the loop by dipping in water, then tapping it gently so that all that remains is the water that just fills the loop level full, and this amount is placed on the slide by touching the flat side of the loop to the glass. Then the straight needle is sterilized, dipped into the culture and just touched once into the small drop of water on the slide. The remainder of the culture on the straight needle is then burned off and the needle is used to spread the drop of water containing the bacteria into a thin even film, which will result, provided the slide is free from grease. This is dried and then “fixed” by passing three times through the Bunsen flame at intervals of about one second, passing through slowly for thick slides and a little more rapidly for thin ones. If the culture is in a liquid medium, the use of the loop of water is unnecessary; a loop of the fluid from the surface, middle or bottom as the culture indicates is spread out to a thin film, dried and fixed.