« EelmineJätka »
THE DIFFERENTIATION OF THE BACILLUS OF
For several years it was 'believed that the most marked and sufficiently diagnostic characteristic of the typhoid fever bacillus was its growth on sterilized potato. In two days at blood temperature, or in three to four days at room temperature, the inoculated potato gives a luxuriant, but to the unaided eye completely invisible, growth. The entire surface assumes a moist, shining appearance, but is not otherwise changed. If a small portion of the surface is removed with a sterilized platinum needle, and examined in a drop of water under the microscope, a large number of bacilli are to be seen moving about very energetically. It was thought for some time that this was a means of differentiation from all other species of bacteria. The results of many months' work in this laboratory, however, confirm the conclusion of several investigators abroad which have indicated that this is not the case, and for two reasons : first, because the typhoid bacillus does not invariably give this invisible growth ; and, second, because there are several species very similar to the typhoid bacillus in their growth on the usual culture media, which under some circumstances present the same appearance when grown on sterilized potato. Of these so-called “pseudo-typhoid” bacilli there are at least five species in the water of the Merrimack River at Lawrence. Like other observers, I have found that this characteristic of the typhoid bacillus is obtained only when the potato is slightly acid; slightly alkaline potato giving a gray or yellowish growth along the inoculation line. Moreover, as different specimens of potato appear to vary in their acidity or alkalinity, and as the same variety changes in this respect during the different
* This paper appeared in the “ Boston Medical and Surgical Journal," sept. 1, 1892.
seasons of the year, the potato test is not only to be no longer considered diagnostic, it is often contradictory.
At the Lawrence Experiment Station of the State Board of Health, under the direction of Mr. Hiram F. Mills, many experiments have been made upon the life-history of the typhoid fever bacillus in milk and in water, and upon the possibility of its passage through sand filters. Realizing the uncertainty of the differentiation of this micro-organism by means of the potato test, a comparative study of the different species of bacteria in the water of the Merrimack River at Lawrence, of which there are more than thirty, was systematically undertaken side by side with cultures of the typhoid bacillus. To this end, it became necessary to make a thorough investigation of the typhoid bacillus itself. The numerous special methods and media proposed for its differentiation were studied, and their diagnostic value ascertained. The observations of its characteristics and its behavior upon the various culture media now employed in this laboratory are as follows:Morphology. — When
grown on agar in tubes for one week at 200 C., it appears to be a plump bacillus, with rounded ends, about lu long and 0.6-0.8u in diameter. When grown in bouillon at blood temperature for two days it is a bacillus 1.5-2.54 long and 0.5-0.644 in diameter. Spore formation and involution forms under the above conditions have never been observed. Hanging drop preparations from bouillon cultures, at blood temperature, show that the bacilli possess very lively movements both of rotation and translation. The motility of the bacilli, when taken from agar cultures grown at blood temperature, is very constant but somewhat less marked ; but from agar cultures grown at 20° C. it is not only less marked but it is not uniformly present.
While the motility of the typhoid bacillus is one of its most marked characteristics and is an essential number in the series of characteristics which enables the bacteriologist to identify this micro-organism, yet in water, sewage and other sources are found species of bacteria which possess this characteristic to a very similar degree. Moreover, it has been learned from the repeated determinations of the species of bacteria in numerous samples of water that an element of variability in the morphological appearances of bacteria arises when cultures are grown on media of different composition, of different degrees of alkalinity, for different periods of time, at different temperatures. Accordingly, in order to make microscopical observations of value, a standard method of procedure should be adopted, making all conditions as nearly parallel as possible.
Relation to Temperature. — The typhoid bacillus can be grown in culture media at temperatures varying from 10° C. to 45.5° C. Its optimum temperature lies between 37° C. and 39° C. It has been found that at the extreme limits of temperature the bacillus grows better in liquid than in solid culture media.
Description of Growth on the Different Culture Media. - On the gelatine plate the bacillus grows and forms colonies in the usual time, namely, forty-eight to seventy-two hours, at 20° C. The colonies lying deep in the gelatine are small, white, spherical or spindle-shaped and sharply outlined. Those on the surface are larger, bluish-white, with slightly irregular outline ; under the microscope the colonies often appear to have ridges or folds. In some cases a centre is visible, but it is the exception. The gelatine is never liquefied. It was thought at one time that the typhoid colony was very characteristic, with its yellowish centre and bluish veil of irregular outline with ridges like the veining of a leaf. It is now known that the colony does not always present this characteristic appearance, and, further, that there are several species of bacteria in water which cannot be distinguished from the typhoid bacillus by their growth on the gelatine plate.
When grown in a gelatine tube, a moderately conspicuous, gray growth appears along the inoculation line, while at the surface there is a thin, gray skin of irregular outline which spreads toward the wall of the tube.
On the agar plate, incubated at 38° C., the surface colonies are slightly irregular, bluish-white, sometimes having a white centre. Those below the surface appear yellowish-white and are usually oval. On the inclined agar tube the typhoid bacillus gives a welldefined, smooth, moist, grayish growth.
When grown in sterilized milk the typhoid bacillus produces no coagulation and but a very slight amount of acid. It is not possible to detect the acid with litmus, but with phenolphthalein, which is a more delicate indicator than litmus for organic acids, it can be shown that a slight acid fermentation probably takes place. The milk test is manipulated as follows: The milk, drawn fresh from the cow and as nearly sterile as possible, is taken at once to the laboratory, tubed and sterilized, five cubic centimeters being carefully measured into each test-tube. The tubes are sterilized for three-quarters of an hour on three successive days. From time to time the apparatus and doubtless the atmosphere of the laboratory contain sporeforming bacteria, which are killed with great difficulty at steam temperature. Notwithstanding the precautions mentioned above, I have frequently found that some of the tubes of milk were not sterile. It was also observed that the species which were not killed at steam temperature produced coagulation. Accordingly, after the tubes have been sterilized three times they are placed in a thermostat at 38° C. for twenty-four hours. After another sterilization the tubes which had a growth in them are found to be coagulated and are discarded. Although many weeks may go by without having difficulty in this direction, the satisfaction of having sterile milk, and of knowing that it is such, well repays the additional labor.
The tubes are inoculated and the cultures allowed to grow for two days at 38° C., or for four days at 20° C. At the end of this time they are examined for coagulation, after which they are placed in boiling water for five minutes, and the coagulation, if any, again noted. The second observation gives uniformly constant results for the same species, whereas, before boiling, the coagulation by many species is sometimes positive and sometimes negative even after they have grown in the milk for a much longer time. The great advantage of this procedure is well illustrated in the case of the Bacillus coli communis, which ordinarily takes from two to seven days to coagulate milk. If, however, a culture is allowed to grow for twelve hours at 38° C., and is then placed in boiling water, coagulation at once follows. In explanation of this fact it is to be remembered that the caseine of milk is precipitated by acid (and redissolved by alkali) and that it is coagulated by heat in the presence of acid; therefore, in some cases, probably, the acid, although unable to precipitate the caseine, is sufficient to coagulate it in the presence of heat. The facts, however, that in some cases a small amount of acid effects precipitation before heating, while in other cases a comparatively large amount of acid produces no coagulation after prolonged heating, point to further chemical changes.
After the coagulation has been tested, the milk is placed in an evaporating dish and the amount of acid determined by titration against a twentieth-normal solution of caustic potash, using phenolphthalein as the indicator. As is well known, normal milk is acid with this indicator, the five cubic centimeters of milk requiring about two cubic centimeters of a twentieth-normal solution of caustic
potash for neutralization. This blank, which is determined for each lot of milk, is of course deducted from the amount of caustic potash required to neutralize each tube of milk after the bacteria have grown in it. It will be readily seen that there is a great advantage in using this indicator, inasmuch as the blank is sufficiently acid to enable alkali, if present to a moderate extent, to be estimated as well as if the fermentation had been an acid one.
When grown in a peptone solution containing potassium nitrate, the typhoid bacillus reduces the nitrate to nitrite. The explanation of this fact seems to be that, among the decomposition products of the peptone, some reducing agent or agents are formed, the nitrate being used simply as an indicator. It is not clearly known what the reducing agents are, but the test is of great value to the bacteriologist, as the statements of Heraus, Frankland and Jordan have suggested. After many hundreds of trials upon the typhoid bacillus, the test has never been known to fail if made with proper manipulation. This method is also of great service in the differentiation of other species, as from the single test three possibilities may result:
(1) The nitrate may remain as such. (2) It may be reduced to nitrite. (3) It may be reduced completely; that is, to ammonia or to free nitrogen.
The solution is prepared by dissolving one gramme of peptone and two-tenths of a gramme of potassium nitrate in one litre of city water. The reason that this water is used rather than distilled water is because it contains the phosphates and other inorganic salts which appear to be necessary for the growth of the bacteria. The solution is rendered alkaline to the same degree as ordinary nutrient gelatine, tubed, sterilized, inoculated, and the bacteria are allowed to grow for one week at 20° C. To determine in what condition the nitrogen is present, after the growth of the bacteria, a test for nitrites is first made. A cubic centimeter of the solution is removed to a clean test-tube, containing about ten cubic centimeters of distilled water free from nitrites. One cubic centimeter each of strongly acid sulphonilic acid and of napthylamine hydrochlorate solution are added ; and nitrites, if present, are indicated by a pink color. If nitrites are present, then the determination is sufficient; but, if they are absent, a determination of nitrates must be made, in order to learn, by their absence or presence, whether there has been a complete reduction, or none at all. To make the nitrate determination, the tube containing the solution is inverted and the small