Gentian violet, saturated alcoholic solution.

Lugol's (Gram's) iodine.

Absolute alcohol.

Methylene-blue, } Fuchsin, basic, } saturated alcoholic solution.

Neutral red, 1 per cent. aqueous solution.

Leishman's modified Romanowsky stain.

Carbolic acid, 5 per cent. aqueous solution.

Acetic acid, 1 per cent. solution.

Sulphuric acid, 25 per cent. solution.

Xylol.

[Ill.u.s.tration: FIG. 65.--Staining rack, rubber change mat and lysol pot.]

[Ill.u.s.tration: FIG. 66.--Drop bottle.]

[Ill.u.s.tration: FIG. 67.--Canada balsam pot.]

And two pots with air-tight gla.s.s caps (Fig. 67), each provided with a piece of gla.s.s rod and filled respectively with Canada balsam dissolved in xylol, and sterile vaseline.

METHODS OF EXAMINATION.

Bacteria, etc., are examined microscopically.

1. In the living state, unstained, or stained.

2. In the "fixed" condition (i. e., fixed, killed, and stained by suitable methods).

The preparation of a specimen from a tube cultivation for examination by these methods may be described as follows:

~1. Living, Unstained.~--(a) _"Fresh" Preparation._--

1. Clean and dry a 3 by 1 gla.s.s slip and place it on one of the squares of filter paper. Deposit a drop of water (preferably distilled) or a drop of 1 per cent. solution of caustic potash, on the centre of the slip, by means of the platinum loop.

[Ill.u.s.tration: FIG. 68.--Holding tubes for removing bacterial growth, as seen from the front.]

TECHNIQUE OF OPENING AND CLOSING A CULTURE TUBE.

2. Remove the tube cultivation from its rack or jar with the left hand and ignite the cotton-wool plug by holding it to the flame of the Bunsen burner. Extinguish the flame by blowing on the plug, whilst rotating the tube on its long axis, its mouth directed vertically upward, between the thumb and fingers. (This operation is termed "flaming the plug," and is intended to destroy any micro-organisms that may have become entangled in the loose fibres of the cotton-wool, and which, if not thus destroyed, might fall into the tube when the plug is removed and so accidentally contaminate the cultivation.)

3. Hold the tube at or near its centre between the ends of the thumb and first two fingers of the left hand, and allow the sealed end to rest upon the back of the hand between the thumb and forefinger, the plug pointing to the right. Keep the tube as nearly in the horizontal position as is consistent with safety, to diminish the risk of the accidental entry of organisms (Fig. 68).

4. Take the handle of the loop between the thumb and forefinger of the right hand, holding the instrument in a position similar to that occupied by a pen or a paint-brush, and sterilise the platinum portion by holding it in the flame of a Bunsen burner until it is red hot. Sterilise the adjacent portion of the aluminium handle by pa.s.sing it rapidly twice or thrice through the flame. After sterilising it, the loop must not be allowed to leave the hand or to touch against anything but the material it is intended to examine, until it is finished with and has been again sterilised.

5. Grasp the cotton-wool plug of the test-tube between the little finger and the palm of the right hand (whilst still holding the loop as directed in step 4), and remove it from the mouth of the tube by a "s.c.r.e.w.i.n.g" motion of the right hand.

6. Introduce the platinum loop into the tube and hold it in this position until satisfied that it is quite cool. (The cooling may be hastened by touching the loop on one of the drops of moisture which are usually to be found condensed on the interior of the gla.s.s tube, or by dipping it into the condensation water at the bottom; at the same time care must be taken in the case of cultures on solid media to avoid touching either the medium or the growth.)

7. Remove a small portion of the growth by taking up a drop of liquid, in the case of a fluid culture, in the loop; or by touching the loop on the surface of the growth when the culture is on solid medium; and withdraw the loop from the tube without again touching the medium or the gla.s.s sides of the tube.

8. Replace the cotton-wool plug in the mouth of the tube.

9. Replace the tube cultivation in its rack or jar.

10. Mix the contents of the loop thoroughly with the drop of water on the 3 by 1 slide.

11. Again sterilise the loop as directed in step 4, and replace it in its stand.

12. Remove a cover-slip from the gla.s.s capsule by means of the cover-slip forceps, rest it for a moment on its edge, on a piece of filter paper to remove the excess of alcohol, then pa.s.s it through the flame of the Bunsen burner. This burns off the remainder of the alcohol, and the cover-slip so "flamed" is now clean, dry, and sterile.

13. Lower the cover-slip, still held in the forceps, on to the surface of the drop of fluid on the 3 by 1 slip, carefully and gently, to avoid the inclusion of air bubbles.

14. Examine microscopically (_vide infra_).

During the microscopical examination, stains and other reagents may be run in under a cover-slip by the simple method of placing a drop of the reagent in contact with one edge of the cover-gla.s.s and applying the torn edge of a piece of blotting paper to the opposite side. The reagent may then be observed to flow across the field and come into contact with such of the micro-organisms as lie in its path.

The non-toxic basic dyes most generally employed for the intra-vitam staining of bacteria are

Neutral red, } Quinoleine blue } Methylene green } in 0.5 per cent. aqueous solutions.

Vesuvin, }

_Negative Stain_ (Burri).--By this method of demonstration the appearances presented by dark ground illumination (by means of a paraboloid condenser) are closely simulated, since minute particles, bacteria, blood or pus cells etc. stand out as brilliantly white or colourless bodies on a dark grey-brown background.

_Reagent required:_

Any one of the liquid waterproof black drawing inks (Chin-chin, Pelican, etc.). This is prepared for use as follows:

Measure out and mix:

Liquid black ink, 25 c.c.

Tincture of iodine 1 c.c.

Allow the mixture to stand 24 hours, centrifugalise thoroughly, pipette off the supernatant liquid to a clean bottle and then add a crystal of thymol or one drop of formalin as a preservative.

METHOD.--

1. With the sterilised loop deposit one drop of the liquid ink close to one end of a 3 by 1 slide.

2. With the sterilised loop deposit a drop of the fluid culture (or of an emulsion from a solid culture) by the side of the drop of ink (Fig.

69, a); mix the two drops thoroughly by the aid of the loop.