Dear Marian,

> Would anyone have a procedure for Twort's gram stain?

The following modification works quite well:

GRAM TWORTS STAIN FOR BACTERIA

          FIXATION:      10% buffered formalin

          MICROTOMY:     5um paraffin sections
          SOLUTIONS:

          1.   Crystal Violet:

               Crystal violet (CI 42555)               2g
               Ethanol                                 	20ml
               1% aqueous ammonium oxalate             80ml

          2.   Lugols Iodine

               Postassium Iodide                       2g
               Iodine                                         1g
               Distilled water                         100ml

               Dissovle potassium iodide in 2-3ml water, add iodine, dissolve
	 and dilute to 100ml with distilled water.

          3.   Acetone/Alcohol:
               Acetone                                     20ml
               Alcohol (absolute)                      20ml

               Prepare prior to use.

          4.   Tworts counterstain:
               0.2% neutral red (CI 50040) in ethanol  	9ml
               0.2% fast green (CI 42053) in ethanol   	1ml
               Distilled water                       		30ml

               Mix immediately before use.

          5.   1% Acetic acid.

          CONTROL:

          Section containing gram positive cocci and gram negative bacilli is
          preferred.

          STAINING PROCEDURE:

          1.   Dewax and hydrate sections.

          2.   Crystal violet, 5 minutes.

          3.   Rinse in tap water.

          4.   Lugols iodine, 5 minutes.

          5.   Rinse in tap water, blot section on filter paper.

          6.   Differentiate in acetone/alcohol till colour stops streaming from
	   the section.

          7.   Rinse in tap water.

          8.   Tworts counterstain, 10 minutes.

          9.   Differentiate in 1% acetic acid until stain stops streaming from
	   the section  (One dip).

          10.  Dehydrate rapidly, clear and mount.

          RESULTS:

               Gram positive bacteria                       blue
               Gram negative bacteri, nuclei            red
               Surrounding connective tissue           green

          REFERENCES:

          1.   Preston, Morrell.  J. Path. Bact. (1962), 84:241-5.
          2.   Twort,       J. State. Medicine (1924), 32:351.
          3.   Cherukian, Schenk.  J. Histotech. (1982), 5(3):127-128.

 
Tony Henwood
Senior Scientist
Anatomical Pathology
Royal Prince Alfred Hospital
Sydney, AUSTRALIA

http://www2.one.net.au/~henwood
http://www.pathsearch.com/homepages/TonyHenwood/default.html