The Elements of Bacteriological Technique Part 27

2. Remove xylol by flushing the section with alcohol.

3. If the tissue was originally "fixed" in a corrosive sublimate solution, the section must now be treated with Lugol's iodine solution for two minutes and subsequently immersed in 90 per cent. alcohol to remove all traces of yellow staining.

4. Wash in water.

5. Stain deeply, if using a single stain, as the subsequent processes decolourise.

6. Wash in water, decolourise if necessary.

7. Flood with several changes of absolute alcohol to dehydrate the section.

8. Clear in xylol. (Oil of cloves is not usually employed, as it decolourises the section.)

9. Mount in xylol balsam.

SPECIAL STAINING METHODS FOR SECTIONS.

~Double-staining Carmine and Gram-Weigert.~--

1. Prepare the section for staining as above, sections 1 to 3.

2. Stain in lithium carmine (Orth's) or picrocarmine for ten to thirty minutes, in a porcelain staining pot (Fig. 76).

3. Wash in picric acid solution until yellow. At this stage cell nuclei are red, protoplasm is yellow, and bacteria are colourless.

Picric acid solution is prepared by mixing

Picric acid, saturated aqueous solution 40 c.c.

Hydrochloric acid 1 c.c.

Alcohol (90 per cent.) 160 c.c.

4. Wash in water.

5. Wash in alcohol.

6. Stain in aniline gentian violet.

7. Wash in iodine solution till dark brown or black.

8. Wash in water.

9. Dip in absolute alcohol for a second.

10. Decolourise with aniline oil till no more colour is discharged.

[Ill.u.s.tration: FIG. 76.--Staining pot.]

11. Wash with aniline oil, 2 parts, xylol, 1 part.

12. Clear with xylol.

13. Mount in xylol balsam.

~Alternative Gram-Weigert Method for Sections.~--

1. Fix paraffin section on slide and prepare for staining in the usual manner.

2. Stain in alum carmine for about fifteen minutes.

3. Wash thoroughly in water.

4. Filter aniline gentian violet solution on to the section on the slide and allow to stain about twenty-five minutes.

5. Wash thoroughly in water.

6. Treat with Lugol's iodine until section ceases to become any blacker.

7. Wash thoroughly in water.

8. Treat with a mixture of equal parts of aniline oil and xylol until no more colour comes away.

9. Wash thoroughly with xylol.

10. Decolourise and dehydrate rapidly with absolute alcohol until there remains only a very faint bluish tint.

11. Clear with xylol.

12. Mount in xylol balsam.

(Then fibrin and hyaline tissue are stained deep blue, whilst bacteria which "stain Gram" appear of a deep blue-violet colour.)

~Unna-Pappenheim Method.~--

Stain.--

Weigh out and mix

Methylene green 0.15 gramme Pyronin 0.25 gramme

and dissolve in

Carbolic acid 0.5 per cent. aqueous solution 78 c.c.

Measure out

Alcohol 2.5 c.c. } Glycerine 20.0 c.c. } and add to the stain.