[Histonet] gram stain

Jennifer MacDonald JMacDonald <@t> mtsac.edu
Mon Jan 24 22:04:07 CST 2011

Thank you!!

Tony Henwood <AnthonyH <@t> chw.edu.au> 
Sent by: histonet-bounces <@t> lists.utsouthwestern.edu
01/24/2011 04:54 PM

"'Jennifer MacDonald'" <JMacDonald <@t> mtsac.edu>, 
"histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>

RE: [Histonet] gram stain


Here is the method from our manual:

Gram Stain
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

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.



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.

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

          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 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 
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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