(a) In the majority of cases microscopic examination is sufficient for the detection of the presence of the comma bacillus in the intestinal evacuations of cholera patients.

(b) Even in the most acute cases, running a very rapid course, the comma bacillus can always be found in the evacuations.

(c) In general the number of cholera spirilla present is greater the earlier death occurs; when death is postponed and the disease continues for a longer period, their number is diminished.

(d) Should the patient not die of cholera, but from some other disease, such as typhoid fever, that may be engrafted upon it, the comma bacilli may disappear entirely from the intestines.

2. With another slimy flake prepare a set of gelatin plates. Place them at a temperature of from 20° to 22° C., and at sixteen, twenty-two, and thirty-six hours observe the appearance of the colonies. Usually at about twenty-two hours the colonies of this organism can easily be identified by one familiar with them. 3. With another slimy flake start a culture in a tube of pepton solution either the solution of Dunham or, as Koch proposes, a solution of double strength of that of Dunham (Witte's pepton is to be used, as it gives the best and most constant results). Place this at 37° to 38° C., and at the end of from six to eight hours prepare cover-slips from the upper layers (without shaking) and examine them microscopically. If comma bacilli are present and capable of multiplication, they will be found in this locality in almost pure culture. After doing this prepare a second pepton culture from the upper layer, also a set of gelatin plates, and with what remains make the test for indol by the addition of 10 drops of concentrated sulphuric acid for each 10 c.cm. of fluid contained in the tube. If comma bacilli are growing in the tube. the rose color characteristic of the presence of indol should appear.

By following this plan "a bacteriologist who is familiar with the morphological and biological peculiarities of this organism. should make a more than probable diagnosis at once by microscopic examination alone, and a positive diagnosis in from twenty to, at the most, twenty-four hours after beginning the examination" (Koch).

There are certain doubtful cases in which the organisms are present in the intestinal canal in very small numbers, and microscopic examination is not, therefore, of so much assistance. In these cases plates of agar-agar, of gelatin, and cultures in the pepton solution should be made.

The plates of agar-agar should not be prepared in the usual way, but the agar-agar should be poured into Petri dishes and allowed to solidify, after which one of the slimy particles may be smeared over its surface. The comma bacillus being markedly aerobic, develops very much more readily when its colonies are located upon the surface than when they are in the depths of the medium. A point to which Koch calls attention in connection

with this step in the manipulation is the necessity for having the surface of the agar-agar free from the water that is squeezed from it when it solidifies, as the presence of the water interferes with the development of the colonies as isolated points and causes them to become confluent. To obviate this, he recommends that the agar-agar be poured into the plates and the water allowed to separate from the surface at the temperature of the incubator before they are used. It is wise, therefore, when one is liable to be called on for such work as this to keep a number of sterilized plates of agar-agar in the incubator ready for use, just as sterilized tubes of media are always ready and at hand. The advantage of using the agar plates is the higher temperature at which they can be kept, and consequently a more favorable condition for the development of the colonies. As soon as isolated colonies appear they should be examined microscopically for the presence of organisms having the morphology of the one for which we are seeking, and as soon as such is detected gelatin plates and cultures in pepton solution (for the indol reaction) should be made. The pepton cultures started from the original material should be examined microscopically from hour to hour after the sixth hour that they have been in the incubator. The material taken for examination should always come from near the surface of the fluid, and care should be taken not to shake the tube. As soon as comma bacilli are detected in anything like considerable numbers in the upper layers of the fluid, agar-agar plates and fresh pepton cultures should be made from them. The colonies will develop on the agar-agar plates at 37° C. in from ten to twelve hours to a size sufficient for recognition by microscopic examination, and from this examination an opinion can usually be given. This opinion should always be controlled by cultures in the pepton solution made from each of several single colonies, and finally the test for the presence or absence of indol in these cultures.

In all doubtful cases in which only a few curved bacilli are present or in which irregularities in either the rate or mode of their development occurs, pure cultures should be obtained by the agar-plate method and the method of cultivation in pepton solution as soon as possible, and their virulence tested upon animals. For this purpose cultures upon agar-agar from single colonies must be made. From the surface of one of such cultures a goodsized wire-loopful should be scraped, and this broken up in about I c.c. of bouillon, and the suspension thus made injected by means of a hypodermic syringe directly into the peritoneal cavity of a guinea-pig of about 350 to 400 grams weight. For larger animals more material should be used. If the material injected is from a fresh culture of the cholera organism, toxic symptoms at once begin to appear; these have their most pronounced expression in the lowering of temperature, and if one follows this decline in temperature from time to time with the thermometer, it will be

seen to be gradual and continuous from the time of injection to the death of the animal, which occurs in from eighteen to twentyfour hours after the operation.'

In general, this is the procedure employed at Berlin in the Institute for Infectious Disease under Koch's direction.

Typhoid Fever.-The bacteriological diagnosis of this disease may be made by two methods. One of these consists in the isolation of the typhoid bacillus from the feces by means of special culture-media; the other depends upon a peculiar effect which the blood-serum of a typhoid patient. exerts upon a suspension of typhoid bacilli when a mixture of the two is made.

Isolation of the Typhoid Bacillus from the Feces.— This may be accomplished in a certain proportion of cases by making Petri plate cultures from the feces with either of the following special culture-media. The material for cultures may be very conveniently secured by means of a "swab," and brought to the laboratory in the sterile testtube. A few grams of fecal material should be obtained, as this quantity will not readily dry up.

We are indebted to Dr. Mark W. Richardson of Boston for the following directions for preparing the special culturemedium of Elsner, and for the description of the method of applying it to the isolation of the typhoid bacillus from the stools, as practised by him:

kilogram

1. Pare carefully and cut up into small pieces of potatoes. The potatoes should be old rather than new, and not too large. The best size it that of an egg or slightly larger. New or large potatoes are too acid in reaction,

hence are not to be used.

2. Add 1 liter of water and boil for one and a half hours. 3. Mash potatoes thoroughly.

4. Measure and make up to 1 liter the loss of water by evaporation.

5. Strain through a cloth.

6. Make up amount once more to

liter.

7. Boil with 15 per cent. gelatin for five or ten minutes.

1 Pfeiffer: loc. cit.

8. Cool down to 60° C., and add a well-beaten egg to clear.

Boil.

9. Filter through cotton.

10. Filter through paper.

II. Test acidity with decinormal (4 grams to 1000 c.c.) sodium hydrate solution. The acidity should be such, according to Elsner, that 10 c.c. of gelatin are neutralized by 2.5 to 3 c.c. of the solution of alkali. The acidity may be somewhat lower, however (1.7 c.c.), with no harmful results. An acidity above 3 is to be corrected with normal sodium hydrate solution (40 grams to 1000 c.c.).

12. Add potassium iodid I per cent. This may be added(a) Before tubing the medium, in which case add to I liter of media 10 c.c. of a solution in which I c.c. is equivalent to I gram of potassium iodid, and mix thoroughly; or,

(b) Just before using the medium, in which case we add to 10 c.c. of medium c.c. of the above solution.

13. Tube, and sterilize three times.

In the examination of a suspected typhoid stool the first dilution is made with one "loop" of stool into bouillon (to economize on the medium) or into the medium itself.

Three or four Petri plates are generally sufficient, the dilutions being made as follows:

=

Plate II. I loop from plate I. or from the bouillon tube. Plate III. 1 loop from plate II.

Plate IV. 6 loops from plate III.

The plates are now kept at room-temperature (as near 20°-22° C. as possible) for forty-eight hours. The colonies. of the bacillus coli communis appear in twenty-four hours, but it is rare to see the typhoid colonies before the end of forty-eight hours. Naked-eye appearances have little or no value. The plates are best examined under the microscope with a Zeiss AA or Leitz No. 3 lens. Seen under these circumstances, at the end of forty-eight hours the colon colonies appear as larger, rounder, more well defined, coarsely granular, distinctly brown-colored colonies, while the typhoid colonies are much smaller, paler, less sharply defined, very finely granular, and are more often oval than round in shape.

It is, however, very important that the plates should not be too crowded, otherwise the small colonies of the bacillus coli communis may give rise to considerable confusion. The suspicious colonies are now picked up (always under the microscope) and transferred to bouillon and glucose agaragar tubes.

If, after twenty-four hours in the incubator, there has been no formation of gas on the glucose agar-agar, and the bouillon culture shows, in the hanging drop, an organism resembling in morphology and motility the typhoid bacillus, then the other tests for the typhoid bacillus (see Bacteriological Diagnosis) are indicated; for the appearances alone of the colony upon the potato-gelatin is certainly no more proof of the identity of the bacillus than any other one of the differential tests, unless it be the serum-reaction of Pfeiffer.

Make a solution of this composition and filter. To the filtrate add 20 grams of agar-agar and 10 c.cm. of a normal solution of sodium hydrate (40 grams to 1000 c.c.).

With this medium Petri plate-cultures are to be made. from the stools by first pouring the medium into the plates, allowing it to become solid, and then inoculating the surface by gently rubbing the infected platinum loop over it. The loop may be infected either by plunging it into the stool or into a bouillon suspension of the stool. In consequence of this manner of inoculating the plates the colonies all grow on the surface, and thus show more clearly anything characteristic in their appearance than if they developed in the depths of the medium. The colonies of the typhoid bacillus, when grown upon this medium, are shining, transparent,