2. The Employment of the Different Nutrient Media Depends upon the Following Points of View:

I. Fluids (Bouillon, Sugar-bouillon, Milk, Non-albuminous Nutrient Media)

are employed:

1. To produce culture en masse.

2. To obtain suspensions of bacteria in which the number can be accurately determined (counting by means of plates).

3. For the study of the formation of pellicles and sediments.

4. For the study of the metabolic products (compare p. 58 and what follows).

II. Solid Nutrient Media.

1. Gelatinous Nutrient Media.-The gelatinous, transparent nutrient media (agar and gelatin) are most extensively employed for the following reasons:

(a) They may be employed as fluid and solid nutrient media: as fluids, allowing a separation of the bacteria; and as solid substances, a fixing of the isolated germs and their separate growth into colonies.

(b) On account of their transparency they allow a macroscopic as well as microscopic observation of the cultures; they allow a thorough differential diagnosis of varieties and an early recognition of any contaminations.

They are especially used: (a) For plate cultures-i. e., for demonstration, for accurate separation and counting of the individuals and varieties.

(b) For obtaining characteristic, macroscopic cultures which serve in differential diagnosis.

(c) For permanent cultures, or collections of living bacteria. The special advantages of agar and gelatin are:

(a) Gelatin. - Advantages: Easily prepared, readily made into plates (at 25°); the property of being liquefied by many bacteria is of great diagnostic value. Disadvantages: Since it melts at 25° it cannot be used in hot weather nor at incubator temperature.

(b) Agar.-Advantages: It may be used at incubator temperature (i. e., for the rapid growth of bacteria-spores-and especially thermophilic bacteria). Disadvantages: Difficulty of preparation, more difficult to make plates from (the agar, melted at 80°, must be cooled to 40° before being inoculated). Colonies are often not characteristic. 2. Blood-serum, glycerin-agar, and glycerin-ascites-agar. — Employed especially for growing pathogenic varieties, which thrive poorly or not at all on other nutrient media. It is only possible to make plate cultures with glycerin-agar and mixtures of agar and serum. 3. Potato.-(a) To obtain macroscopically characteristic cultures of great durability and for differential diagnosis.

(b) Sometimes for spore-formation.

3. A Few Words Regarding the Technic of Ordinary

Cultures.

The platinum needle must be heated red-hot each time before it is used and before putting it down.

(a) Fluid culture media are inoculated with a loopful of pure

culture.

(b) Gelatin and agar stab cultures are made with a straight needle, only a single stab being made in each tube, but it should extend almost to the bottom of the tube.

(c) Agar and gelatin streak cultures and potato cultures are inoculated by a gentle superficial stroke over the surface with the platinum loop. It is sometimes necessary to rub the culture into the potato.

(d)_Gelatin plate cultures:

1. To isolate certain bacteria in pure culture: The gelatin in three tubes is melted, and after it is cooled down to 30°, a loopful of a fluid or a trace of a solid pure culture is introduced into one of them and well mixed. From this first tube one or two loopfuls of gelatin are carried to a second tube, and from this, after mixing, two or three loopfuls are again transferred to the third tube. After anything which may be upon the edge of the tubes has been burned off, the contents of each tube are poured into separate sterile plates, the cover being quickly raised for this purpose, and the plate inclined gently to and fro in order to distribute the gelatin as uniformly as possible. During the transferring from one tube to another it is recommended that they be held inclined, to prevent the falling into them of foreign germs. The plates thus prepared are then placed in a culture chamber with a constant temperature of 22° (or room temperature is used), and after two or three days the individual colonies which have developed are studied macroscopically and with slight (fifty times) magnification. Usually, of the three plates, only two are useful; at least one has been sown too thick or too thin.

2. If one wishes to ascertain the number of bacteria, for example, in water, 1 c.c., 0.5, and 0.1 c.c. of the water is placed in three tubes of liquefied gelatin, well mixed, and poured into plates. To ascertain the number of germs, if they are very numerous, the Wolfhügel counting plate is used; if only a few colonies appear, then the plate is inverted, the bottom divided into sextents with ink, and each visible colony marked with a dot. Plates made to determine the number of bacteria in drinking-water must be counted several times (on the second, third, and fifth days). In the case of fluids with very many germs (sour milk, canal-water, etc.), 1 c.c. is first placed in 100 c.c. of sterilized water, and this then treated as above. Solid bodies are first rubbed up in water. In the examination of air a definite quantity is drawn through a tube filled with sterilized sand, the sand then being washed in sterilized water and plates prepared from it.

(e) Agar plate-cultures are prepared in the same way. The agar must not be too cool when poured into the dish or it will solidify at once, forming an uneven surface. On the contrary, if it is too hot, the bacteria are killed by the temperature. Recently it has been much

advised that, in making agar (partly also gelatin) plates, the nutrient medium be first allowed to solidify in the dishes, and then the surface be superficially smeared over with the material to be examined by means of a platinum loop, strips of filter-paper, or a platinum brush. Only characteristic surface colonies are obtained in this way.

(f) Sugar-agar shake cultures: The contents of a tube are melted in the water-bath and cooled down to about 40°. A loopful of the pure culture is then introduced and thoroughly distributed, and after the medium solidifies the tube is placed in the incubator.

4. Anaerobic Cultures.

We have employed almost exclusively the method of H. Buchner: Absorption of oxygen by pyrogallic acid in the presence of potassium hydroxid.1

(a) For Stab Cultures.—At the bottom of a glass cylinder, which must be a little longer than the test-tube, is placed a heaping teaspoonful of pyrogallic acid and 20 c.c. of 3% potassium hydroxid solution. The inoculated stab culture is then placed in the cylinder, which is closed at one end with a soft rubber stopper or a ground-glass stopper which is sealed with paraffin. According to Kitasato, anaerobes which are less sensitive to oxygen may be cultivated in high stab cultures in sugar-agar without pyrogallic acid. A stab 8 to 10 cm. deep is made in sugar-agar with a small loop and the needle turned upon its long axis before being withdrawn.

(b) For Plate Cultures.-Instead of the glass cylinder, a wide exsiccator with a ground cover is used. The lower part is filled with sand and the pyrogallic acid mixture, and then the manipulation is as above (Arens).

If it is desirable to obtain the most perfect anaerobiosis, the pyrogallic acid method is combined with either the pumping out of the air with a water-pump or the displacing of the air with hydrogen, so that only a slight trace of oxygen remains to be taken up by the pyrogallic acid. We have employed the latter method many years. The cultures are placed in a roomy exsiccator with sufficient pyrogallic acid and potassium hydroxid, and then, by means of a double perforated rubber cork, hydrogen is allowed to flow through for one-half hour. closing the opening, we sink the whole apparatus, weighted with lead, in water.

After

Kabrhel recommends (C. B. xxv, 555), as a control for the absence of oxygen, that a tube be introduced which contains liquefied nutrient gelatin, to which is added, just before use, 0.3% to 1.0% grape-sugar, and which is rendered a transparent blue with a strong alcoholic solution of methylene-blue. Such an uninoculated tube is completely decolorized in twenty-four to thirty-six hours only in a chamber entirely free from oxygen. This indicator will also point out how essential it is to remove covers, corks, etc., in the case of anaerobic cultures.

1 Sensitive varieties are said to thrive better in an atmosphere of hydrogen.

III. Animal Experiments.

(A) Infection.

1. Subcutaneous Inoculation.-After the skin in some part has been washed with 1 : 1000 corrosive sublimate solution, a shallow incision is made with scissors, and inoculating material is introduced beneath the skin by means of a stout platinum wire with a loop. Mice are usually inoculated above the root of the tail, they being simply held by the tip of the tail and allowed to hang into a glass which is covered in great part by a piece of board. Guinea-pigs and rabbits are inoculated on the side of the thorax.

2. Subcutaneous injection is usually carried out with Koch's rubber-ball injection syringe or with Strohschein's syringe. A fold of skin is picked up upon some part of the body, and the needle introduced in the direction of the fold. If several cubic centimeters are to be injected, it may be simply done as follows: Upon a graduated pipet is fastened a short piece of rubber tubing provided with an injection needle, and the whole sterilized. The pipet is sucked full, and the fluid forced out with the mouth or a rubber bulb.

3. Intraperitoneal injection is made by perforating the abdominal wall at a single thrust with a sterile hollow needle; then, cautiously advancing the needle, the fluid is injected.

Regarding infection by feeding, inhalation, etc., consult more extensive works on technic.

(B) Observation.

Mice may be kept in sterile glass vessels provided with cotton and closed with wire gauze. Larger animals must be kept in sterilized cages or stalls.

(C) Autopsy and Disposition of the Body.

Autopsies must be made at once after death; at least, the animal must be placed on ice after death. The animal is placed upon a board on its back and nailed or tied by its four legs. The abdomen and chest are thoroughly moistened with sublimate solution and then the abdominal cavity first opened with a sterile knife. The abdominal walls are separated, and from the spleen, liver, and kidneys, some blood (or tissue juice) is obtained with a sterile platinum loop and smeared at once upon previously prepared agar plates. The organs are carefully cut out, avoiding contact with the intestines, and placed in absolute alcohol for further examination. Then the thorax is opened with scissors, and blood removed from the heart and also the lungs. These organs are also placed in alcohol. Before each operation the instruments must be carefully heated to a glow or thoroughly burned. It is better to have numerous sterilized instruments ready. The hands must be perfectly clean.

In interpreting the findings at the autopsy it is to be remembered that often very soon (sometimes during the death agony) micro-organ

isms migrate into the organs from the intestine. If living bacteria are injected into the abdominal cavity or trachea of cadavers, they can very often be found in the organs after a time (C. B. XXIII, 418).

After the autopsy the body is best burned. If this is not practicable, the body is wrapped in coverings wet with sublimate and buried at least 0.5 meter deep, and quicklime filled in about it.

APPENDIX V.

Brief Guide to the Recognition of Bacteria.

(Illustrated with an example.)

The case is one of eczematous conjunctivitis in which a number of the bacteria occurring in diseased eyes are present. Purulent or serous material removed from the conjunctival sac or edge of the lid with a platinum loop is made use of.

I. Microscopic Examination (Smear upon Slide or Cover-glass).

(a) Stained with fuchsin, we see:

1. Cocci, especially diplococci in heaps, usually distinctly "biscuit-shaped," many times within cells (perhaps gonococci).

2. Cocci, single or united in irregular clusters (probably Micro. pyogenes).

3. Short chains, of two or three links, of lance-shaped cocci, some with capsules (probably Streptoc. lanceolat.).

4. Rods, larger or smaller, often very irregular in form, staining in segments, ends rounded or pointed, often of the size of cocci (true diphtheria, pseudodiphtheria, or xerosis bacillus).

5. Rods regular, rather thick, but small (perhaps coli group).

6. Rods, often in pairs, quite large, the ends not rounded (perhaps, although at the time without spores, a bacillus or Bacterium duplex).