Thank you!!
Tony Henwood <antho...@chw.edu.au> Sent by: histonet-boun...@lists.utsouthwestern.edu 01/24/2011 04:54 PM To "'Jennifer MacDonald'" <jmacdon...@mtsac.edu>, "histonet@lists.utsouthwestern.edu" <histonet@lists.utsouthwestern.edu> cc Subject RE: [Histonet] gram stain 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-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jennifer MacDonald Sent: Tuesday, 25 January 2011 9:15 AM To: histonet@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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ********************************************************************************* This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses. ********************************************************************************* _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet