BACTERIA, WITH A METHOD OF STAINING FOR DIAGNOSTIC PURPOSES.
BY JOSEPH KETCHUM, ESQ.
Read and Demonstrated before the Section on Microscopy of the Brooklyn Institute.
In presenting the subject of Bacteria, I wish to disclaim any originality for the matter offered. I have endeavored to collect from such sources of information as I have access to the important dates, names and facts which have marked the progress of bacteriology up to the present time.
So far as we know, the first observer of bacteria and the so-called infusoria was Leeuwenhoek, who, with a simple magnifying glass, noticed in a drop of putrid water the multitude of little granules moving about in it. This was in 1675, and his observations were communicated to the Royal Society of Sciences in the same year. In the following year he recognized bacteria in the tartar from the teeth, and though he did not name them, his description of their forms and his drawings enable us to identify them as vibrios. There appears to have been no important investigations carried on until nearly one hundred years later, or in 1773, when Müller, a Dane, attempted to classify the organisms then known. He called them all infusoria, from the fact that they were the product of infusions, and divided them into two genera—the monas and vibrio. The monas he subdivided into ten forms and the vibrio into thirty-five; but his descriptions of them are so faulty that it is at present impossible to identify them from his writings. During the following century the study of bacteriology attracted more or less attention, and in 1829 Eherenberg, who is the Humboldt of the science, commenced his investigations, which for fifty years he pursued with an ardor and enthusiasm second to not even Darwin himself. He, in 1838, classified the family of vibrioniens, and with the additions made by Dujardin in 1841, placed them in a scientific category. Of course during this period many were the disputes and discussions as to specie, genera or family, each newly discovered member belonged to. And we have to come to the period of Hallier, Hoffmann and Cohn, and many others, before the questions, which had up to that time been in dispute, were settled. Ehrenberg’s original classification was into:
1. Bacterium, or rod-like—three species.
2. Vibrio, snake-like and flexible—nine species.
3. Spirillum, or spiral, but inflexible—three species.
4. Spirochœte, spiral, but flexible—one species.
Dujardin, in 1841, in his Natural History of the Zoophytes, accepted the classification of Eherenberg, except that he unites the spirillum and spirochœte, calling them all spirillum. Up to this time all bacteria had been considered animals, but a close study of their life history and habitat by those who followed declared them to belong to the vegetable kingdom, and as such they are accepted to-day.
In 1853, M. Chas. Robin pointed out the relationship of bacteria to Leptothrix, a form of fungi closely allied to that of mildew; and M. Davaine, in 1868, clearly demonstrated their relationship to the vegetable world. From this time the progress of bacteriological investigation has made rapid strides. Prof. Pasteur in the organisms of fermentation and the role they play therein; Davaine and Hallier in demonstrating the specific relationship of bacteria with charbon or anthrax; and the work of Koch, Nageli, Kohn, Bilroth, Miguel, Burdon, Sanderson, Klein, Weigert, Klebs, Ehrlich, Sternberg, and many others, are too recent to require special mention.
Few have more than the faintest conception of the minuteness of these organisms. Prof. Cohn, justifying himself for the unscientific method of comparison which he uses in class instruction by Prof. Tyndall’s argument on the scientific use of the imagination, says he compares man to the cheese mite, as the Strasburg cathedral to a sparrow. Of the animalcules which Leeuwenhoek discovered, they are to man as the bee is to the horse. As improvements have been made in microscopes, just so fast have we penetrated into the world of micro-organisms, until now the proportion between the smallest we can see and man, is as man is to Mont Blanc.
Of course, with these exceedingly minute structures, nothing can be made out except points. Among some of the larger forms, a few have been able to see cellia, and in some cases the growth of the spores; but in the present state of microscopical optics the work is slow, and progress in this direction is waiting an advance in the science of optics.
Like all living organisms, bacteria propagate themselves. The most usual method is by fission or by partition, though Magnin and Cohn have recorded their observations on the formation of spores and sporangia, and I have myself witnessed the last named method. It is of importance to note that while the bacterium is killed by continued exposure to temperatures of freezing or 176° F., the spores will germinate after protracted exposure to temperature as high as 205° F. or as low as °123 F. These spores will also withstand complete desiccation, and it is in this form, mixed with the air we breathe and move in, that present the conditions from which all zymotic diseases originate. Miguel has shown that, while the air contains very few adult bacteria, it contains myriads of their spores. To the researches of Koch, Pasteur, and others, we are indebted for the certain information that, while these omnipresent germs withstand such vicissitudes of temperature, they require certain food for their maintenance; and though we cannot as yet tell what that food is, we know that when nutrient material is submitted to their action they thrive for a time, and when the particular principle which supports them is exhausted they die. This is particularly true of pathogenic germs, and the accepted theory of the bacillus tuberculosis, or the germ of consumption, is a good illustration. It has been demonstrated by Koch, Klein, Pasteur, Frankell, Sternberg, and others, that they require some product of inflammatory action for their support within the body of their victim. This is also true of cholera, at least so far as their dietary requirements are concerned. The animal cannot be infected with tuberculosis by merely introducing the germ-laden material into the stomach or upon any of the mucous membranes; but if an inflammatory condition be present, either due to the puncture of the introducing needle or scalpel, or to extraneous causes, such as a catarrhal condition of the lungs, tuberculosis is as sure to follow as the sun is to rise again.
The human mind can scarcely comprehend the enormous numbers of these omnipresent atoms without a resort again to the legitimate use of the imagination. A computation of the increase from a parent germ shows as follows: We know that the parent grows until it reaches double its original size, when it constricts itself in the middle like a figure eight and breaks into two individuals. Each of these divides again, and, on account of the rapidity with which this is done, we find them usually in chains or squares. The warmer the air, the faster this proceeds, and at the temperature of the body the entire life history of a germ, from the time of fission of the parent to the time of his own subdivision into two new individuals occupies less than one hour. This gives us a known quantity for our problem. Let us look at the result. From a single germ increasing by the power of two each hour, we have at the end of twenty-four hours 16,777,220; at the end of two days the number has increased to 281 billions, and in three days to the enormous number of 48 trillions, and in one week the number can only be expressed by figures of fifty-places. In order to make this number comprehensible, let us figure the mass and weight of this, the result of a single bacterium. A single Bacterium Termo has an average width of 1
1,000 mm. A cubic mm. would therefore contain six hundred and thirty-three millions, and in one day would be one-fortieth full. At the end of the following day there would be required 444,570 such cubes to contain the product of the parent, or say half a litre. Suppose the seas of the earth cover two-thirds of its surface with a mean depth of one mile, the aqueous product would be 929 million miles. Now, our parent germ and its product would in five days completely fill this space. More wonderful still is a gravimetric estimation. Suppose we call the specific weight of the parent germ the same as water, which cannot be far from right, it would appear that the parent weighs, or his equal bulk of water weighs, 136 millionths of a gramme; in forty-eight hours, 442 grammes; in three days, nearly 7½ million kilograms; and, inside of thirty days, the weight of the earth itself.
Prof. Cohn, in offering these figures, says: “I don’t consider this idle play; without it we can form no conception of not only the enormous increase, but the tremendous destruction of these germs which is going on around us. Food is lacking to support more than a comparatively small proportion of the product of the parent, and, as it is demonstrated that they feed from their environment, one can readily understand that without a constant supply a given infectious germ will with its followers soon destroy its nidus or perish from starvation.”
Our breweries demonstrate the truth of this hypothesis; for, in twenty-four hours, a single yeast cell, which is 8
1,000 mm. in diameter, will yield one hundred-weight of yeast.
I have endeavored to present the subject in a condensed but general way without burdening you with technical details of species, genera or life history. The subject is a vast one and to which the best minds of the scientific world are devoting themselves. To those who are or may become interested in bacteriology and particularly to those who study the relation of these germs to disease, is held forth the reward which is sure to come to those who work persistently and intelligently.
The method which I shall employ to-night is eclectic. Doubtless each investigator will find fault with some parts of the process and perhaps suggest a better one. The following, however, has in my hands worked well and given entire satisfaction, so far as I know, to those who were and are most interested.
The apparatus necessary is as follows:
One two-inch glass funnel.
One package filter papers to fit same.
Four medium size test tubes.
Two glass or porcelain staining glasses.
One glass or agate mortar and pestle.
One cover holder.
One pair pincetts.
One alcohol lamp.
Package of wooden toothpicks.
The cover holder may be easily made by taking a piece of thin platinum, two inches long and one-eighth wide, splitting one end for half an inch up and bending into a Y shape, then lashing to a small handle (I use a match). This little tool is most convenient for floating cover glasses in staining fluids.
The reagents necessary are as follows:
A five per cent. solution of nitric acid in alcohol (95 per cent.).
Saturated alcoholic solution of fuchsine.
Saturated alcoholic solution of methyl blue.
Small quantity of alcohol, 80 to 95 per cent.
Pure colorless aniline oil (anilin).
The method is as follows:
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.