Karnovsky's Fixative

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SURGICAL PATHOLOGY - HISTOLOGY STAINING MANUAL Subject: No. 1:7 Rev.2 Date: Page: 1 of 2 KARNOVSKY'S GLUTARALDEHYDE PURPOSE: A fixative for electron microscopy. REAGENTS: Stock Solution: Paraformaldehyde 1M Sodium hydroxide 50% glutaraldehyde 0.2M cacodylate buffer, pH7.4 2.0 gm 2 - 4 drops 5.0 ml (25% glut - 10 ml) 20.0 ml Mix the paraformaldehyde with 25 ml of distilled water in a 125 ml Erlenmyer flask. Heat to 60°C. on a stir plate. When moisture forms on the sides of flask, add sodium h
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  SURGICAL PATHOLOGY - HISTOLOGYNo. 1:7 Rev.2STAINING MANUALDate:Page: 1 of 2Subject:  KARNOVSKY'S GLUTARALDEHYDEPURPOSE: A fixative for electron microscopy. REAGENTS: Stock Solution:Paraformaldehyde2.0 gm1M Sodium hydroxide2 - 4 drops50% glutaraldehyde5.0 ml (25% glut - 10 ml)0.2M cacodylate buffer, pH7.420.0 mlMix the paraformaldehyde with 25 ml of distilled water in a 125 mlErlenmyer flask. Heat to 60°C. on a stir plate. When moisture forms on thesides of flask, add sodium hydroxide and stir until the solution clears. Coolsolution under the faucet. Filter, add glutaraldehyde and 0.2M buffer, pH range7.2 to 7.4.Sodium Cacodylate Buffers:0.1M:Sodium cacodylate4.28 gmCalcium chloride25.0 gm0.2N hydrochloric acid2.5 mlDilute to 200 ml with distilled water, pH 7.40.2M:Sodium cacodylate8.56 gmMix as above, pH to 7.4 with HCL.Working Solution:Dilute 1:4 with 0.1M sodium cacodylate buffer to obtain an osmolarity of over500 mOsm.Dilute 1:2 for osmolarity over 700 mOsm.Place in individual vials, label, date and freeze. CAUTION: POSSIBLE CARCINOGENSAFETY: Use caution, work in a well ventilated area, wear gloves, lab coat, andgoggles.Paraformaldehyde: severe eye and skin irritant. Sensitizer by skin andrespiratory contact. Toxic by ingestion and inhalation. Target organ effectson respiratory system. Corrosive. Carcinogen.Gluteraldehyde: Severe skin and eye irritant; toxic by ingestion. FIXATION TIME: 1 hour minimum, 2 to 3 hours preferred, can be left overnight  KARNOVSKY'S GLUTARALDEHYDE  No. 1:7 Rev. 3Page: 2 of 2 PROCEDURE: 1. Thaw vial of fixative.2. Place tissue sample in fixative within 15 minutes of removal from patient.3. Date and time tissue is placed in fixative. REFERENCE: Sheehan,D and Hrapchak,B: Theory and practice of Histotechnology, 2nd Ed, 1980, pp330-331, Battelle Press, OhioCrookham,J, Dapson,R, Hazardous Chemicals in the Histopathology Laboratory, 2nd ED, 1991, Anatech  Prepared: By:Approved: By:  KARNOVSKY FIXATIVE Stock Solution: Paraformaldehyde2.0 gm1M Sodium hydroxide2 - 4 drops50% glutaraldehyde5.0 ml(25% glut 10.0 ml)0.2M cacodylate buffer, pH7.420.0 mlMix the paraformaldehyde with 25 ml of distilled water in a 125 ml Erlenmyer flask. Heat to 60°C. on a stir plate.When moisture forms on the sides of flask, add sodium hydroxide and stir until the solution clears. Cool solutionunder the faucet. Filter, add glutaraldehyde and 0.2M buffer, pH range 7.2 to 7.4. Sodium Cacodylate Buffers: 0.1M:Sodium cacodylate4.28 gmCalcium chloride25.0 gm0.2N hydrochloric acid2.5 mlDilute to 200 ml with distilled water, pH 7.40.2M:Sodium cacodylate8.56 gmMix as above, pH to 7.4 with HCL. Working Solution: Dilute 1:4 with 0.1M sodium cacodylate buffer to obtain an osmolarity of over 500 mOsm.Dilute 1:2 for osmolarity over 700 mOsm.Place in individual vials, label, date and freeze. CAUTION: POSSIBLE CARCINOGEN Obtain through VAH EM Laboratory and LDS EM Laboratory. SAFETY/PPE: Use caution, work in a well ventilated area, wear gloves, lab coat, and goggles.
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