There is probably no scientific work more wedded to the microscope than the study of bacteria. We may learn a great deal about birds or insects or rocks or minerals, without any instrument but we can learn little of the bacteria unless they are highly magnified.

There is such an extraordinary amount of misconception concerning bacteria that, it will be time well spent if we attempt to clear up all misunderstanding at the start. Bacteria, often called microbes or germs, are looked upon with considerable awe by most people, who associate them in some vague way with disease. There is no denying that many bacteria are responsible for certain diseases; many more are perfectly harmless and a goodly number are exceedingly useful.

To enumerate all the bacterial activities would require a large book but briefly, apart from the disease-causing bacteria, they enter into the manufacture of cheese and butter, of wine and vinegar; they are essential to brewing and tanning; they act as scavengers over the face of the earth, breaking up a mass of decaying animal and vegetable matter into simple chemical substances which can then be used again as food for plants; some of them also can take a gas called nitrogen, from the air, and pass it on to green plants.

What are these active little creatures? The question is a natural one. They are merely very minute, one-celled plants. Each one possesses a firm cell-wall, filled with living matter; in an earlier chapter, we described the one called protean animalcule and, although it was composed of but a single cell it had no definite wall. This is one of the essential differences between plants and animals, both of them are made up of one or more, maybe millions of cells, but each plant cell is surrounded by a well-defined wall, animal cells have no such walls. The exact position of the bacteria in the plant world is still open to doubt. Most scientists place them amongst the fungi; for, with very few exceptions they possess no chlorophyll. One or two of them, however, do possess a green colouring matter which, if not chlorophyll is very near to it; on this account other scientists are of the opinion that they are related to the seaweeds. It is a matter, however, that does not concern us very deeply, for our purpose it is sufficient to know that they are plants. When they were discovered, nearly three hundred and twenty-five years ago, they were looked upon as minute animals and it is curious that the belief has survived this long period of time in the popular mind. Long before the activities of bacteria were connected with various phenomena, such as infectious diseases, souring of milk, etc., it was thought that these changes were brought about by chemical action. Like many of the early theories, this one contained a half truth, for a great many of the changes brought about by bacteria are really due to chemical action initiated by the organisms. In other words, the bacteria set free certain substances which actually cause the changes to take place.

Let us make our statement clear by a simple experiment. To a little fresh milk we add a weak acid, the milk curdles at once and by dipping a piece of litmus paper (obtained at any chemist’s) into the mixture, it will turn red, showing the presence of acid. Litmus, by the way, is obtained from a lichen; in the presence of acid it is red, an alkali, the opposite of an acid, turns it blue. In a neutral solution, that is to say one that is neither acid nor alkaline, litmus is of a purplish hue.

To continue our experiment, we allow another sample of the same milk to stand for a day or two in a warm, dark place and again the milk will be curdled. A test with the litmus will show that the solution is acid. The bacteria themselves have not curdled the milk but they have liberated a substance, called a ferment, which has split up part of the milk into an acid, amongst other things and that acid has actually done the curdling. For this reason, weak alkalies are sometimes added to milk. Acids and alkalies, of equal strength form neutral solutions, so that, when the milk bacteria begin their activities which result in the formation of acid, it is at once made neutral by the alkali. By this means, curdling is postponed for a little while, though there comes a time, of course, when all the alkali is used up, then the acid gains the upper hand and curdling takes place. We could if we wished continue adding more and more alkali to keep pace with the formation of acid, but too much alkali would be as unpalatable as too much acid, so nothing would be gained.

Before we bring out our microscope to examine these lowly plants, we will first of all kill a myth which has survived, in the non-scientific mind, since the eighteenth century and then describe briefly the life history of a typical bacterium. Now for the myth. Bacteria are so minute, their activities so great and the results of their activities so far reaching, that it is hardly surprising to learn that, even at the present day, bacteria are supposed simply “to happen.” We talk of thunder turning milk sour, but thunder can no more sour milk than can a passing train or an air raid or a burst in a water main. True, milk turns sour more quickly in thundery weather than in frosty weather, because, when thunder threatens, the air is warm and the milk-souring bacteria increase more rapidly in warm weather than in cold. We must remember always that bacteria are living beings and in common with all other living things they must have parents. What probably took place at the beginning of the world we cannot consider here but one thing is certain that, at the present day, no living matter is produced from non-living matter; “life from life” is the only theory that will stand scientific tests and this has been the case ever since the simplest microscopes were thought of and thousands of years before that. Any substance, however liable to decay, if rendered germ free and kept germ free, will retain its fresh condition indefinitely. Could bacteria or germs, call them what you will, simply happen it would be useless attempting to fight against them.

Bacteria are everywhere. In the water we drink, in the milk, butter, cheese and in dust. We cannot avoid them, try as we will; it is fortunate, therefore, that the majority are harmless. You may be surprised that, with this ubiquity, you have never seen one. When, however, you learn that most of them are only about twenty-five thousandths of an inch long and that a thousand million of them could be packed comfortably into a little box, whose sides measured but a twenty-fifth of an inch in length, it is not really so surprising after all. Being so small, the activities of a single bacterium are insignificant; that “union is strength” was never better exemplified than amongst these lowly plants. There are no male and female bacteria, in the majority of cases they increase by splitting, in fact they are often called splitting plants. The change may be watched under the microscope. The plant elongates somewhat, it becomes narrower and narrower in the middle, it develops a waist in fact; finally the two halves part company and each one leads a separate existence as a bacterium. This splitting progresses at an extraordinary rate. A celebrated scientist once wrote: “Let us assume that a microbe divides into two within an hour, these two into four in the next hour, these again into eight in the third hour and so on. The number of microbes thus produced in 24 hours would exceed 16 1/2 millions; in two days they would increase to 47 trillions, and in a week the number expressing them would be made up of 51 figures. At the end of a day (24 hours) the microbes descended from a single individual would occupy one fortieth of a hollow cube with edges one twenty-fifth of an inch long, but at the end of the following day would fill a space of twenty-seven cubic inches, and in less than five days their volume would equal that of the ocean.” It is hardly necessary to add that these alarming figures represent what would happen if no accident befell the bacteria, they show the enormous vitality possessed by the smallest of all plants. Even allowing for misadventure their increase is alarming; actual tests, with a sample of milk containing originally 153,000 bacteria per cubic inch, show that the cubic inch contained after one hour, 539,750; after two hours, 616,250; after seven hours, 1,020,000; after nine hours 2,040,000 and after 25 hours 85,000,000 individuals.

The writer whom we have just quoted calculated that a single bacterium weighs about 0.000,000,000,024,243,672 of a grain, that forty thousand millions weigh one grain and that two hundred and eighty-nine billions weigh a pound. The descendants of one bacterium weigh 1/2666 of a grain, after twenty-four hours; more than a pound after two days, and sixteen and a half million pounds after three days. The assumption in this case, also, is that no harm comes to any of them; the mortality amongst bacteria is, clearly, very great.

Sometimes, owing to external conditions, such as lack of food certain bacteria produce spores. The power of spore formation is not possessed by all bacteria and those which are able to bring it about are difficult to kill for the spores, which contain the living material of the bacterium are surrounded with walls which will resist boiling, drying, freezing and all manner of ill treatment. The spore formation of bacteria is very simple, all or part of the living contents of the bacterium becomes surrounded by a tough wall and remains so surrounded till circumstances are favourable, when the wall bursts, its contents escapes and becomes a bacterium, capable of founding a new colony by the method of splitting we have already described.

Now let us try to find out what sort of plants we are to look for, when we are searching for bacteria, under our microscope. They exist in many forms, to which special names have been applied, and it is unfortunate that, very often, their external form varies according to their state, thus a bacterium may be spherical when young and rod shaped when older. Some bacteria are spherical and are known as Cocci or Micrococci, from Greek words meaning a berry or a little berry respectively; sometimes these spherical bacteria occur in pairs, then they are called Diplococci (double berries); or in chains, Streptococci (chain berries); or in bunches, Staphylococci (grape berries). They may resemble short rods, when they are called Bacteria, a name, by the way, which is also applied generally to all microbes; they may, on the other hand, have the appearance of longer rods and then they are called Bacilli. Some of these longer rods may be curved or even corkscrew shaped when they are known by the name of Spirilla. Rather fearsome names some of these we fear and we wished to avoid long names, but they appear over and over again in books and papers relating to bacteria so we are compelled to introduce them to our pages. Many bacteria possess no power of movement, others swim rapidly, by the aid of the lashing movement of little whip-like structures with which they are furnished.

After all this preamble, which we hope has cleared up certain misconceptions regarding bacteria and has given the reader some insight into their habits, we may proceed to the examination of some of the plants themselves. At the outset we have a confession to make. Bacteria can only be studied seriously, by those who possess very expensive and elaborate apparatus; considerable technical skill is required to prepare the plants for examination—many of them indeed can only be seen after they have been stained and lastly, to trifle with the disease-causing members of the family may lead to dangerous if not fatal results.

Having issued our warning let us see what we can do in the way of microscopic investigation. The easiest subject with which to make a start is the Hay Bacillus, Bacillus Subtilis, not because it is the largest of the bacteria by any means, but because it is very easily obtained. Each plant measures about five thousandths of an inch in length, so we shall require a high magnification to examine it. Having obtained a small quantity of hay, we must boil it in water for about three-quarters of an hour and then set it aside for some hours. In due course the water will contain hundreds upon hundreds of bacteria or, speaking more correctly, of bacilli. For our work, we shall require a special kind of microscope slide; instead of the piece of plain glass we have been accustomed to use we must obtain one with a circular portion, hollowed out from the centre. Having done so, we take a clean glass rod and, with it, transfer a drop of the water, containing the bacilli, to the centre of a clean coverslip. Invert the coverslip so that the drop is on the lower surface and place it over the hollow portion of the slide, in such a manner that the drop still remains suspended from the coverslip; this is known as the hanging-drop method and requires some little skill to accomplish satisfactorily. When our slide is prepared, with a magnification of at least one thousand diameters, we may reasonably hope that our trouble will be rewarded.

At first we shall probably see nothing. We recall that we had some difficulty in examining starch grains, on account of the fact that they were colourless. This time we are dealing with a far more difficult subject. When our eyes become accustomed to the light, however, we shall be conscious that there is something moving in our drop of water. The Hay Bacillus is one of the moving forms, each individual is furnished with a number of little whips whose lashings enable it to travel through the water. The whips cannot be seen in unstained bacilli; experience, however, tells us that they are there, for all these lowly plants which show movement are seen when stained, to possess the little whips. The process of staining kills the plants so that we cannot see the little whips in action.

Having detected that movement is taking place, a little adjustment of focus and a further search will reveal the bacilli to us, as little rod-like, colourless individuals. We shall see their cell contents if they are sufficiently highly magnified and also their cell walls. We may even observe them splitting, each one into two individuals. We must keep our sample of water for later examination. In fact, we may examine drops from day to day, in exactly the same manner. After a short lapse of time we shall notice that the bacteria have increased to an alarming extent and also that they no longer swim about. At this period they tend to arrange themselves in chains lengthwise, their cell walls also lose their clear cut appearance and become jelly like, yet withal they may still continue to split up.

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.