Feb 13, 2023

Karnovsky's Fixative

This protocol is a draft, published without a DOI.
Karnovsky's Fixative
  • 1University of Gothenburg
  • CCI_Gothenburg
Jens Berndtsson
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Protocol Citation: Jens Berndtsson 2023. Karnovsky's Fixative. protocols.io https://dx.doi.org/
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: December 15, 2022
Last Modified: February 13, 2023
Protocol Integer ID: 74024
Keywords: glutaraldehyde fixative of high osmolality, glutaraldehyde fixative, fixation of tissue, formaldehyde, fixation time, fixation, fixative, microscopy, tissue
Funders Acknowledgements:
National Microscopy Infrastructure
Grant ID: NMI (VR-RFI 2019-00217)
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Abstract
Karnovsky's fixative is a standard fixative devloped by M. J. Karnovsky and consist of: 2.5% glutaraldehyde, 2% formaldehyde, 0.02% sodium azide in 0.05 M Na-cacodylate buffer at pH 7.4.

FIXATION TIME:
  • 1 hour minimum, 2-3 hours preferred, can be left overnight at 4 °C.
  • For fixation of tissue, add to the fixative within 15 min of removal from patient.

References
Karnovsky, M.J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron-microscopy. Journal of Cell Biology, 27, 137-138A.
Guidelines
It is best to make 1L every time. Always two people – to check the measurements and so that one person can be closing tube lids while the other is pipetting to avoid the fixative evaporating.
Materials
Aqueous Glutaraldehyde EM Grade 25% 100MLEMSCatalog #16210
PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
Sodium azide (99.5%)BDH Chemicals Ltd Poole EnglandCatalog #9115010C

Protocol materials
Aqueous Glutaraldehyde EM Grade 25% 100MLEMSCatalog #16210
PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
Sodium azide (99.5%)BDH Chemicals Ltd Poole EnglandCatalog #9115010C
Milli-Q water
Safety warnings
Use caution, work in a fume hood, wear gloves, lab coat, and goggles.

Aqueous Glutaraldehyde EM Grade 25% 100MLEMSCatalog #16210
H330 Fatal if inhaled.
H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled.
H314 Causes severe skin burns and eye damage.
H318 Causes serious eye damage.
H302 Harmful if swallowed.
H317 May cause an allergic skin reaction.
H335 May cause respiratory irritation.

PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled
H315 Causes skin irritation.
H319 Causes serious eye irritation
H227 Combustible liquid.

Sodium azide (99.5%)BDH Chemicals Ltd Poole EnglandCatalog #9115010C
H300 + H310 + H330 Fatal if swallowed, in contact with skin or if inhaled.
H373 May cause damage to organs (Brain) through prolonged or repeated exposure if swallowed.
H410 Very toxic to aquatic life with long lasting effects.
Before start
Book a fume hood and label the aliquot tubes.
1000 ml
Mix the following in a 1000 ml beaker:
  • 100 mL of Aqueous Glutaraldehyde EM Grade 25% 100MLEMSCatalog #16210
  • 125 mL of PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
  • 0.2 g of Sodium azide (99.5%)BDH Chemicals Ltd Poole EnglandCatalog #9115010C
  • 250 mL of 200 millimolar (mM) Na-cacodylate buffer
  • 525 mL of Milli-Q water

Aliquote the mixture in:
  • 20 x 5 mL - 15 ml falcon tube labelled K5
  • 30 x 10 mL - 15 ml falcon tube labelled K10
  • 30 x 20 mL - 50 ml falcon tube labelled K20

Dispose of the pipettes used for aliquoting in a hazardous waste bin.

Storage
Store long term (6-12 months) at -20 °C or short term (1-2 weeks) at 4 °C

Clean up:
Rinse the used 1000 ml beaker twice with 100 ml of water, disposing it in a liquid hazardous waste-jar for fixatives before putting the beaker in the dishwasher. Treat spillage as dangeorus and discard any contaminated saftey paper in a hazerdous waste bin. Spills outside of saftey papers should be cleaned up with paper and water which should then be discarded in a hazerdous waste bin.
500 ml
Mix the following in a 500 ml beaker:
  • 50 mL of Aqueous Glutaraldehyde EM Grade 25% 100MLEMSCatalog #16210
  • 62.5 mL of PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
  • 0.1 g of Sodium azide (99.5%)BDH Chemicals Ltd Poole EnglandCatalog #9115010C
  • 125 mL of 200 millimolar (mM) Na-cacodylate buffer
  • 262.5 mL of Milli-Q water

Aliquote the mixture in:
  • 10 x 5 mL - 15 ml falcon tube labelled K5
  • 15 x 10 mL - 15 ml falcon tube labelled K10
  • 15 x 20 mL - 50 ml falcon tube labelled K20

Dispose of the pipettes used for aliquoting in a hazardous waste bin.

Storage
Store long term (6-12 months) at -20 °C or short term (1-2 weeks) at 4 °C

Clean up:
Rinse the used 500 ml beaker twice with 100 ml of water, disposing it in a liquid hazardous waste-jar for fixatives before putting the beaker in the dishwasher. Treat spillage as dangeorus and discard any contaminated saftey paper in a hazerdous waste bin. Spills outside of saftey papers should be cleaned up with paper and water which should then be discarded in a hazerdous waste bin.
200 ml
Mix the following in a 500 ml beaker:
  • 20 mL of Aqueous Glutaraldehyde EM Grade 25% 100MLEMSCatalog #16210
  • 25 mL of PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
  • 0.04 g of Sodium azide (99.5%)BDH Chemicals Ltd Poole EnglandCatalog #9115010C
  • 50 mL of 200 millimolar (mM) Na-cacodylate buffer
  • 105 mL of Milli-Q water

Aliquote the mixture in:
  • 4 x 5 mL - 15 ml falcon tube labelled K5
  • 6 x 10 mL - 15 ml falcon tube labelled K10
  • 6 x 20 mL - 50 ml falcon tube labelled K20

Dispose of the pipettes used for aliquoting in a hazardous waste bin.

Storage
Store long term (6-12 months) at -20 °C or short term (1-2 weeks) at 4 °C

Clean up:
Rinse the used 500 ml beaker twice with 100 ml of water, disposing it in a liquid hazardous waste-jar for fixatives before putting the beaker in the dishwasher. Treat spillage as dangeorus and discard any contaminated saftey paper in a hazerdous waste bin. Spills outside of saftey papers should be cleaned up with paper and water which should then be discarded in a hazerdous waste bin.
100 ml
Mix the following in a 100 ml beaker:
  • 10 mL of Aqueous Glutaraldehyde EM Grade 25% 100MLEMSCatalog #16210
  • 12.5 mL of PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
  • 0.02 g of Sodium azide (99.5%)BDH Chemicals Ltd Poole EnglandCatalog #9115010C
  • 25 mL of 200 millimolar (mM) Na-cacodylate buffer
  • 52.5 mL of Milli-Q water

Aliquote the mixture in:
  • 2 x 5 mL - 15 ml falcon tube labelled K5
  • 3 x 10 mL - 15 ml falcon tube labelled K10
  • 3 x 20 mL - 50 ml falcon tube labelled K20

Dispose of the pipettes used for aliquoting in a hazardous waste bin.

Storage
Store long term (6-12 months) at -20 °C or short term (1-2 weeks) at 4 °C

Clean up:
Rinse the used 100 ml beaker twice with 20 ml of water, disposing it in a liquid hazardous waste-jar for fixatives before putting the beaker in the dishwasher. Treat spillage as dangeorus and discard any contaminated saftey paper in a hazerdous waste bin. Spills outside of saftey papers should be cleaned up with paper and water which should then be discarded in a hazerdous waste bin.