others since then brought out may be very briefly reviewed here. In 1895 Spronck* called attention to the fact that the variable amount of sugar present in beef was responsible for the great fluctuations observed in the toxicity of diphtheria cultures. The writer had observed this independently of Spronck, by studying the relation between the amount of toxin in cultures and the amount of sugar as determined by the fermentation test. Sugar is present in all beef, but in perhaps 10 per cent. the amount is very small. In bouillon made from such 'beef the writer obtained very strong toxin. In bouillon from beef containing over 0.1 per cent. sugar the toxin was very feeble.

The cause for this difference lies in the acid or acids formed from the dextrose by the diphtheria bacillus, which inhibit the multiplication in a direct ratio to the amount formed. In sufficient quantity the growth may be entirely checked, and finally, when the acidity has reached a certain degree, the bacilli and the toxin are destroyed. Whether there are other causes at work besides mere inhibition of multiplication remains undetermined.

A small amount of dextrose, up to 0.05 per cent., is not inimical to toxin production; in fact, it seems to be more favorable than none at all, probably because a certain minimum amount is necessary for the cell life of the diphtheria bacilli. Bearing these facts in mind, we are better able to comprehend the various changes going on in cultures. The life of the culture begins with a rapid multiplication of the bacilli introduced and the formation of a surface membrane

usually within twenty-four hours. At the same time, any sugar present is acted upon at once, with the result that the reaction becomes more acid. If the acidity increases beyond 2 per cent. of a normal acid solution,† the culture is likely to become languid, the surface membrane rifted and settle to the bottom. Some bacilli, by a vigorous surface growth which probably oxidizes the acid products formed, may subdue a larger amount of acid, even to 3.5 per cent., and cause a rapid return towards the alkaline level. The toxin appears in greatest concentration when the alkaline level has been reached, usually within eight to twelve days when sugar is present in small amount only. When sugar is more abundant the acid period is prolonged, during which little growth is evident. After

"Annal. de l'Institut Pasteur," 1895, page 758.

I. e., each 100 cubic centimeters of the culture fluid requires 2 cubic centimeters of a normal solution of alkali to bring the whole to the neutral point as determined by phenolphthallein.

several weeks a slow alkalizing tendency brings the culture to a more vigorous growth and to an alkaline reaction, but without much accumulation of toxin.

Without going into more detail on this subject, we may summarize the conditions under which diphtheria bacilli produce maximum amounts of toxin in the ordinary 1 per cent. (Witte) peptone bouillon as follows:

1. Muscle sugar in the fluid from 0 to 0.05 per cent.

2. Initial reaction from 0.8 to 1.5 per cent. normal acid, the lower figure pertaining to bouillon containing the largest amount of sugar, the higher to bouillon containing none.

3. A thin layer of bouillon freely exposed to the air through one or more cotton-plugged openings in the vessel, and quiescent because the surface membrane which forms within twenty-four hours must not be disturbed.

4. The accumulation of toxin should be permitted to go on until the growth is checked by the alkaline reaction. This appears in from eight to twelve days, according to the initial reaction and amount of sugar present, and the growth ceases when the reaction is equivalent to 0.2 to 0.3 per cent. normal alkali.

The main difficulty before us, therefore, is to get beef containing only traces of dextrose. The writer's original plan, to select the bouillon in accordance with the fermentation test, is not feasible, because so little can be used. Spronck's suggestion, to allow the beef to lie for several days, in order that a partial decomposition by bacteria may transform the sugar, is better, but suffers from certain difficulties. The kind of bacteria cannot be controlled, and frequently the sugar is found but partially removed. Latterly, the writer has given up this method for one more rapid and certain in its action. The beef infusion is prepared either by extracting the chopped beef at 60° C. for several hours, or over night in the refrigerator. After removal of the beef the infusion is inoculated with a culture of some bacterium which rapidly acts upon dextrose, and placed in the thermostat over night. The writer has tried only B. coli, and found a complete transformation of carbohydrates taking place over night.

In the case of bouillon designed for diphtheria toxin the incubation should be as short as possible, so as to leave a trace of sugar in the fluid. This can be accomplished by placing the inoculated infusion in the thermostat at 10 P.M. and removing early next morn

ing (8 A.M.). The infusion is then made up in the usual way, with 1 per cent. peptone, per cent. sodium chloride. The final reaction should range, according to the amount of sugar left as stated above, between 0.8 and 1.5 per cent. normal acid, phenolphthallein being used as indicator. It can easily be brought to any desired point by adding from sterile solutions the calculated amount of normal acid or alkali (HCL or NaHO). The whole procedure is very simple after it has been put into routine practice. At any rate, the bacteriologist must make up his mind to give up the early slovenly methods of preparing culture media, or else be prepared for constant reverses and failures.

The bouillon must be sterilized finally in the autoclave, since the ordinary steaming frequently fails to destroy certain spore-bearing anaërobes, which begin to multiply after the diphtheria bacilli have formed a membrane and deoxidized the culture medium. These anaërobes inhibit the production of toxin.*

Park and Williams claim † that the amount of dextrose in beef purchased in New York City is not sufficient to interfere with the maximum accumulation of toxin if the culture be made sufficiently alkaline to begin with. This claim I cannot support by my experience with beef bought in the Boston markets. It may be that these authors had under observation bacilli which had acquired, through surface cultivation, a greater power to promptly oxidize acid products. This power is not possessed, as a rule, by bacilli recently isolated from the throat, with which this article deals.

A number of observers have published studies of the relative virulence of diphtheria bacilli from various sources, and those persisting in the throat after recovery for a variable length of time. It is not the object of this article to re-examine these writings and review the results obtained. For a summary of the literature the reader is referred to the article by J. H. Wright in the "Boston Medical and Surgical Journal," Vol. 131 (1894), page 329, and "Scientific Bulletin" No. 1 of the health department, city of New York (1895). A perusal of the various articles will show that the method of testing the virulence of the diphtheria bacilli was not adapted to give uniform or quantitative results. Thus, Park and Beebe, on page 23 of the

Since writing this, it has been observed that high temperatures in the autoclave may modify the bouillon in such a manner that only little toxin is formed subsequently. This matter is now under investigation.

+"Journal of Experimental Medicine," I. (1896), page 164.

bulletin referred to, recommend alkaline glucose bouillon as a culture medium, and the injection of cultures forty-eight hours old. Wright used sugar bouillon very largely. From what we now know of the inhibitory and destructive action of the acids formed from dextrose by diphtheria bacilli, the use of more than 0.1 per cent. dextrose in bouillon must be considered as at least unsafe. However, the authors followed general usage at that time, for even Escherich, in his work on diphtheria issued in 1894 (page 91), states that dextrose is not decomposed in appreciable manner by diphtheria bacilli, and therefore has no influence on growth.

Authors have not, so far as the writer knows, reported comparative tests of toxin production under conditions as nearly uniform as possible. It was mainly to fill this gap, if possible, that the series of cultures to be described were subjected to a comparative examination from the point of view of toxin production. Table I. gives a condensed account of the work done upon which the calculation of toxin production rests. In this table will be found: (1) the amount of acid produced in dextrose bouillon; (2) the condition of the bouillon used for the cultures; and (3) the test of the filtrate on guinea-pigs. The acid production will be dealt with farther on. The facts relative to the bouillon used need some explanation.

The beef used for bouillon, with one exception, was allowed to decompose according to Spronck's suggestion, but the results were not uniform, as stated above. In some of the bouillon the dextrose was absent, in some present in traces, in some in more appreciable amount, according to tests made with the fermentation tube and B. coli. In none was it present in the amount usually found in bouillon made from fresh beef. It is not probable that this slight fluctuation in the amount of dextrose had any appreciable influence on the culture. Where a doubtful result was obtained it was usually supplemented later on with a second test.

The question might be asked, Why not use the same bouillon for all bacilli studied, in place of the many lots actually employed? This would seem the simplest procedure, provided the bouillon did not change with time under the influence of light and air. A diminution in the amount of toxin produced in bouillon which had been standing for some time in a closet not absolutely dark had been casually observed. It is probable that bouillon in vacuo and kept in a dark place might meet the conditions of the problem, but bouillon kept under ordinary conditions would not. Further investigations

are now in progress to determine more precisely the degree of change produced in bouillon by age.

It might be claimed that different bacilli isolated from the throat would have different rates of growth in bouillon, and that the accumulation of toxin was simply a factor of the rate of multiplication, rather than of any inherent differences in the physiology of the bacilli. To answer this claim a determination of the number of diphtheria bacilli in cultures is not trustworthy, for the reason that diphtheria bacilli clump together, and the number of colonies in plate cultures may not indicate the number of bacilli used in preparing the plate. Again, bacilli may die in the course of growth, and others take their places. The writer has therefore endeavored to estimate the vigor of growth by the amount of change in the reaction produced. Cultures which in a given time in the same bouillon produce nearly the same amount of alkali may be regarded as having performed the same amount of work and grown with equal vigor. The uniformity of reaction in the various groups of bacilli studied together, after ten or twelve days, was such as to leave little doubt that the growth had been equally vigorous. When any culture lagged perceptibly behind, it was usually repeated with other bouillon.

The extent of the alkali production varies with the initial reaction of the bouillon and the presence of dextrose. Cultures containing the latter became at first more acid before swinging back to alkalinity. In Table I., therefore, it was deemed best to give both the initial reaction of the bouillon, the approximate amount of dextrose and the final reaction. Some idea may thus be gained of the amplitude of change which the fluid underwent during the period of growth permitted.

The culture vessel used at first was a large test tube placed in an inclined position after inoculation. This was soon given up for the Erlenmeyer flask, in which the depth of the bouillon was about 1.5 centimeters.

The toxin formed after ten to twelve days was tested upon guineapigs. The fluid was passed through filter paper until clear, then diluted with sterile salt solution, so that the quantity of toxin injected was contained in 1 cubic centimeter. Usually 0.1 cubic centimeter of toxin was injected. The place of injection chosen was the left side of the abdomen. Great care was exercised to deposit the fluid in the subcutis, and not to prick the muscles of the abdominal