[Histonet] gram stain

[Tony Henwood]

Jennifer,

Here is the method from our manual:

Gram Stain
Principle:
Crystal violet stains up both Gram positive and Gram negative bacterial wall but only the Gram positive wall structure is capable of retaining the violet molecules that are locked in by iodine. Gram negative cell wall loses the violet colour when differentiated with acetone, and stains up red as a result of counterstaining.

Fixation:      10% buffered formalin.
Microtomy:     Paraffin sections at 5µm.

Controls:	  Placenta containing gram positive cocci and gram negative bacilli
Reagents: 		

1.	1% Crystal Violet - Warning: Flammable, Suspected Carcinogen - see MSDS
Crystal Violet     (Or Methyl Violet)   		10g
Ethanol                                           			100 ml
1% Ammonium Oxalate                  			400 ml

2.	Lugol's Iodine - Warning: Toxic - see MSDS

3.	Acetone - Warning: Flammable - see MSDS

4.	Twort's 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.

 

Procedure:

1.	Bring sections to distilled water.
2.	Cover sections with crystal violet          	30-60 seconds
3.	Rinse slides in water
4.	Stain with Lugol's iodine                          	30-60 seconds
5.	Tap water wash thoroughly
6.	Blot sections lightly to remove excess water
7.	Differentiate with acetone until dye stops running off the section.
8.	Wash thoroughly with water
9.	Counterstain Tworts          	10 minutes
10.	Wash, dehydrate quickly, clear and mount.


Results: 
Gram positive bacteria                 			violet blue
Gram negative bacteria                     			red
Surrounding connective tissue           			green



Reference:	 
          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.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Jennifer MacDonald
Sent: Tuesday, 25 January 2011 9:15 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] gram stain

Does anyone have a method for the Twort stain or a gram stain that does not use picric acid or ether?  We were using a Twort stain and I cannot find the original reference for it and we suspect we have one of the concentrations wrong.
Thank you,
Jennifer MacDonald
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