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: [email protected]
[mailto:[email protected]] On Behalf Of Jennifer
MacDonald
Sent: Tuesday, 25 January 2011 9:15 AM
To: [email protected]
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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