fever. It is strikingly motile, always in the blood, and grows anaerobically. Both in the blood and in cultures many, but not all, of the individuals present one or two very long, motile, spiral appendages, which take the stain for flagella. Lewaschoff calls this form of remarkable spirochete, exanthematica. According to his opinion, there is perfect agreement between his findings and the newer investigations of Ljubimoff (cocci), Calmette and Thoinot (A. P., 1892, 39) (egg-shaped bodies and spirilla), von Dubief and Bruhl (C. B. XIV, 17), Curtis, and Combemale (diplococci).

APPENDIX IV.

Essentials of Bacteriologic Technic.

The following directions and short explanations include about all the technical material which is given in a thorough bacteriologic course. We have introduced only those things which are essential and, according to our experience, practical, without referring to the literature. More details will be found in the books mentioned in the preface.

I. Microscopic Examination of Bacteria.

1. Hints upon Microscopic Technic.

For bacteriologic examination we use almost exclusively the modern microscope with Abbé's illuminating apparatus, iris diaphragm, and a low-power and an oil-immersion objective.

(A) Low magnification (60 to 100 times) with a narrow diaphragm is used in the minute examination of plate cultures. In this examination either the cover is removed and the colony examined from above, or, if one does not wish to contaminate the plate by exposing it, the dish is laid upon the cover and the colony examined from below, but this does not give such characteristic pictures in all cases. (B) High Magnification. Oil-immersion Objective (700 to 1200 times).-This finds its use in the examination of single individuals. A drop of cedar oil is placed upon the preparation (slide, cover-glass) and the tube is lowered by means of the coarse adjust1 Our plate cultures are always poured into dishes.

ment until the lens just touches the oil; then it is accurately adjusted upon the preparation with the micrometer screw.

(a) Unstained preparations. Narrow diaphragm! They are examined in two ways:

1. A drop of pure culture in a fluid medium or a little drop of water with a trace of pure culture mixed in it is placed between the slide and cover-glass; or, better,

2. In a hanging drop. A drop of a pure culture in a fluid medium or a drop of bouillon in which is mixed a minute quantity of a pure culture is placed upon a cover-glass; the cover-glass is then turned over and placed upon a hollow ground slide so that the drop is suspended within the hollow. The cover-glass is now fixed to the slide by applying a very little water to each corner of the cover, or, if the observation is to be more prolonged, by means of vaselin.

(b) Stained preparations. Open diaphragm! Abbé's illuminating apparatus. In the examination of sections with a double stain, the wide diaphragm is required for the bacteria, the narrow opening for the tissues.

(C) Cleaning the Preparations and the Microscope.-The immersion oil is always gently brushed off, and now and then quickly cleaned with xylol and chamois skin; the setting of the lens is loosened by prolonged action of the xylol. Also immersion oil dried upon the cover-glasses of old preparations is readily removed by xylol.

2. The most Important Solutions for Use in Making Preparations.

(A) Staining Solutions.

1. Aqueous Alcoholic Solutions of Fuchsin and Methyleneblue. A concentrated "stock-solution" is prepared by pouring absolute alcohol upon the pulverized dyes (fuchsin, methylene-blue) in bottles, and after shaking and allowing them to stand a few hours, they are filtered. Of this saturated solution 1 part is mixed with 4 parts of distilled water, and before using is filtered. In order to obtain good preparations it is better to stain a longer time with weaker solutions than for a short time with strong solutions. 2. Carbol-fuchsin (Ziehl's Solution).—

3. Anilin Fuchsin.-Four parts of anilin oil (anilin. pur.) are well shaken several minutes with 100 parts of distilled water, and then filtered until all the water has passed through clear (then the funnel is removed, since otherwise oil will also pass through). In this anilin water 4.0 gm. fuchsin are dissolved and it is again filtered.

4. Anilin Gentian-violet (Ehrlich's Solution).-To 100 c.c. of anilin water, 11 c.c. of a concentrated alcoholic solution of gentianviolet (stock solution) is added. This solution does not keep long.

Czapiewski and E. Fränkel recommend that instead of anilin water, 25% carbole send water be employed. This has the advantage that the socation does not decompose so soon as anilin mixtures.

5. Loffer's Methylene-blue-To 100 e.e. of water is added 1 c.c. of 15 solution of potassium hydroxid, and 30 e.c. of concentrated alcobele solution of methylene-blue. The staining property of the dye is intensited by the addition of the alkali.

6. Acetic acid methylene-blue, according to M. Neisser, for the staining of grannies:

4 19 g of methylene-blue is dissolved in 20 c.c. of 90% alcohol, and to this is added 950 e.e, of distilled water and 50 c.e. of glacial acetie seid.

3.2.0 gm of Bismarck brown are dissolved in 1 liter of boiling distilled water filter!.

7. Bismarck Brown.-Prepared like No. 1 (stains tissues, but stains bacteria poorly.

8. Alum Carmin.-In 100 e.e. of a 5% solution of alum 2 gm. of carmine are placed... It is boiled for an hour and filtered. It stains nuclei and tissues, but not hacteria.

9. Eosin.-For staining the tissues about the bacteria. Two gm. of eosin are dissolved in 100 e.e. of distilled water.

10. Safranin.-Three gm. of safranin are dissolved in 100 c.c. of hot distilled water.

(B) Differentiating Agents.

1. Distilled water.

2. Absolute alcohol.

3. Iodin, iodid of potassium solution (Gram's solution):

C) Mordants for Staining Flagella.

mordant: 10 c.c alcoholic solution of fuchsin; 50 c.c. solution of ferrosulphate; 100 c.c. 20 solution of

mordant: 25 c.c. of officinal solution of ferric chlorid, times; 75 c.c. of a saturated aqueous solution of tannic ore using, enough of a 3% solution of hydrogen peroxid e a reddish-brown color, and it is then filtered. (We ispensed with this last addition.)

1. Xylol.

(D) Clearing and Mounting Agents.

2. Canada balsam.

3. Dammar varnish.

3. Preparations of Stained Specimens of Bacteria.

(A) Smear Preparations.

1. Simple Stain with Fuchsin or Methylene-blue. This may be used for all bacteria except the tubercle bacillus.

A drop of distilled water is placed upon a cover-glass (cover-glass preparation) or upon a slide (slide preparation), and in it is mixed a trace of pure culture (preferably from a solid nutrient medium), and the drop is then spread out in a very thin layer. After the fluid has evaporated, the preparation is passed quickly through the flame three times with the film up in order to fix the bacteria to the glass (avoid burning!). The film of bacteria is then covered with the staining solution. After a short time (a few seconds to a minute), perhaps with gentle heat, the preparation is washed with water and allowed to dry (perhaps again with gentle heat). Finally the dry cover-glass is fixed upon a slide by means of a drop of Canada balsam, the bacterial side being down.

It may here be remarked that slides and cover-glasses upon which the drop of water does not adhere or does not spread out evenly are best cleaned with soap. Alcohol and ether, which are much used for cleaning purposes, do not remove the particles of fat so completely. 2. Gram's Stain.

1. The smear preparation is made as above.

2. Stain with Ehrlich's solution three minutes.

3. Wash with water.

4. Differentiate with Gram's iodin solution one minute.

5. Decolorize with absolute alcohol until no more color is removed (usually one or two minutes).

If the alcohol does not decolorize rapidly enough, it can be accelerated by placing a drop of anilin-xylol upon the preparation and then again washing with alcohol.

6. Dry and mount.

According to our experience, the current idea that every variety of bacterium either stains well or not at all when treated by this method is false. Thus, we observed, in the case of the Bact. fluorescens, for example, which is usually said in the literature not to stain, that three out of twelve different cultures, of twenty-four hours' growth, stained very beautifully. According to Zimmermann, all the varieties of Bact. fluorescens stain in young cultures.

In like manner we found the bacillus of symptomatic anthrax from a culture to stain, although it has often been said not to stain. The contradictory results may be easily explained in part as due to varying age or youth of materials experimented with, and to variations in the differentiating process with alcohol. The Bacillus tenuis, which has been designated above as failing to stain by Gram's method, upon

a later test with the same culture and technic stained very well. In every case whenever the test is to be made, a fresh anthrax preparation should be simultaneously stained, and all preparations should be subjected to the action of alcohol for the same length of time (one or two minutes). Then we can judge very well whether a variety of bacterium retains or gives up the stain. A separation of the individual genera and varieties by Gram's stain now seems scarcely at all possible, since there are found within a single genus all stages, from those organisms which stain well to those that stain poorly.

The following may usually be stained by Gram's method:
Micrococci (not the Micr. gonorrhϾ).

Sporulating bacilli (symptomatic anthrax and malignant edema are uncertain).

Of the non-sporulating bacilli: Proteus, mouse septicemia, swine erysipelas, lactic acid bacterium. (Uncertain: fluorescens, some bacteria not unlike the coli Bact.)

Genus Corynebacterium, Mycobacterium, and Actinomyces.
The following usually do not stain:

Micr. gonorrhoeæ, Bact. influenza, Bact. coli and typhi, Friedländer's Bacillus pneumoniæ, bacteria of pest and glanders, the vibrios and spirilla.

3. Demonstration of Capsules.-The following is Johne's method: (1) Cover the preparation with 2% solution of gentian-violet and warm until it steams; (2) wash in water; (3) moisten with 2% acetic acid for six to ten seconds; (4) wash with water.

By this method also in varieties which are not regarded as "capsule-bacteria," a distinct membrane may often be demonstrated about the intensely stained bacterial cell. The capsule is seen most beautifully when examined in water.

4. Staining of Flagella.-The flagella, which are almost always invisible when unstained, are usually demonstrated according to Löffler's method:

1. Making the preparation (rubbing up a trace of young agar streak culture-not bouillon culture-in a very small drop of water, spreading well, and drying rapidly).

2. Heating the preparation with the mordant (p. 476) until it steams (not boils!) for one-half to one minute.

3. Wash with a vigorous stream of water.

4. Wash in alcohol to remove the remains of the mordant adhering at the edge.

5. Cover with staining solution (a few crystals are dissolved in 10 c.c. of anilin water, and to this is added, drop by drop, 0.1% sodium hydroxid solution until the clear fluid just begins to become opaque precipitation) and warm until it steams for one minute.

6. Wash with water, dry, and mount in Canada balsam. Extreme cleanliness is essential in the manipulations, especially very thorough cleaning of the cover-glasses with acid and alcohol, or, still better, with soap. Also the cultures must be young, although it is not essential, as some authors claim, to employ cultures which are only twenty-four hours old. We have often obtained very good prepa