If we now examine the water from which we have taken our drop we shall probably find a scum floating on the surface; it consists of millions upon millions of hay bacilli, huddled together so to speak. It is the beginning of the end for them, nourishment is becoming scarce and important changes are about to take place. Let us continue our examination day by day, that we may discover what is happening. Before long, we shall find that within each cell wall, which is no longer jelly like, there is a darker cell contents than we saw before. The bacilli have, in fact, formed spores. Now we may plug our bottle containing the remainder of the water, with cotton wool and set it aside for some months if we wish. At the end of that time, by pouring a fresh supply of water upon the spores, we may start them all into a new vitality and the whole process will be repeated.
We have mentioned that bacteria should be stained, in order to make their presence more easily detected. This is hardly the place to enter into a lengthy discussion concerning the methods of staining but, for the benefit of our readers, who wish to pursue the subject further, we will state as concisely as possible how simple staining may be accomplished. Our requirements are, a pair of Cornet’s forceps, two small bottles of stain, say Carbol-Fuchsin and Methylene Blue and a larger bottle of 1/2 per cent. Acetic acid; these may be obtained from the firm who supplied our microscope and, for the beginner at anyrate, it is cheaper to buy the solutions ready made, than to attempt to make them up at home. Slides and cover slips, we require, of course, and they must be absolutely grease proof; it may be necessary to boil them in a strong solution of caustic soda to effect this result. A small bottle of Canada Balsam completes our requirements.
Should we wish to examine a drop of milk for bacteria, we proceed in this manner. With the aid of the Cornet’s forceps pick up two cover slips, place a drop of milk on one and cover with the other. With thumb and finger bring the glasses into close contact, so that the milk forms a thin film. Slide one glass from the other and set aside, milk side upwards, till dry. Next take each cover slip, separately, in the forceps and pass rapidly two or three times through the flame of a spirit lamp, this fixes the bacteria, if any be present, to the glass. Now having poured a little of the stain, say methylene blue, into a shallow vessel, a saucer will do, we place our cover slips therein for two minutes or so. Then, remove them with the forceps, wash in water till no more stain comes away and set aside to dry. When dry, take a clean slide, place a small drop of Canada Balsam at its centre and gently lower the cover slip thereon, stained side downwards. If we now examine our slide under a high magnification, we shall easily be able to see whether bacteria are present or not. Should our preparation be too deeply stained, a good slide will show the bacteria stained blue against an almost colourless background, we must immerse our second preparation for a few moments in a little of the 1/2 per cent. acetic acid which will have the effect of removing the excess of stain; then, after washing and drying, we proceed as before.
Beautiful double staining may be performed by the following method. In addition to the chemicals we already possess we shall require some 5 per cent. acetic acid. Double staining is especially useful for spore-forming bacteria, so we may take some of the Hay Bacillus at sporing time. Proceed exactly as described above substituting, of course, a drop of water known to contain the bacilli for the drop of milk. When the two cover slips are ready for staining, warm some of the Carbol-Fuchsin in a saucer and leave the cover slip therein for five minutes, then transfer to a 5 per cent. solution of Acetic Acid till all the stain appears to be removed, afterwards wash in water. The cover slips must next be immersed for a few minutes, two should be long enough, in Methylene blue solution, then washed and, when dry, mounted on a slide with Canada Balsam, as described above. If the staining has been properly carried out, we shall have a most beautiful preparation, showing spores stained red and the rest of the bacilli blue.
The work that may be done with bacteria is limitless but, to advance very far, we shall need facilities for obtaining what are known as “pure cultures.” Let us make the term clear. Suppose we take milk, water, butter, anything in fact upon which bacteria will grow and examine them carefully. If we have the requisite knowledge and recognise what we see, we shall find not one kind of bacterium but a number of different bacteria. Now by certain manipulations, which need not be described here, all the different kinds of bacteria may be sorted out, so that we have colonies consisting of one kind of bacterium only and such a colony is known as a “pure culture.”
In practice, bacteriologists do not use the rough and ready methods that we used in dealing with the Hay Bacillus. They prepare pure cultures and cultivate the bacteria on various substances, differing markedly from those on which they originally lived. For example, a jelly-like substance, mainly composed of beef broth and gelatine is one of the favourite substances on which to grow bacteria, milk is also used in some cases and also slices of potato. All this may seem to have little to do with the microscope, but indeed the bacteriologist relies as much on the behaviour of his pure cultures, growing on gelatine, etc., as on their appearance under the microscope. Some bacteria will not grow on the surface of the gelatine but only in the body of the substance, where air cannot reach them; others cause the gelatine to become liquid; others give off a characteristic smell or impart a well-marked colour to their food material; a few even cause the gelatine to become luminous. These easily seen characters are quite as typical of the various bacteria which bring them about as are the microscopic characters; in fact it sometimes happens that only by a combination of the two is it possible to be certain of what one has obtained.