TITLE: Karnovsky&#39;s Fixative
AUTHORS: Jens Berndtsson

[DESCRIPTION]
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.

[BEFORE_START]
Book a fume hood and label the aliquot tubes.

[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. 

[STEPS]
SECTION: 1000 ml
1. Mix the following in a 1000 ml beaker:
100 mL of   
125 mL of   
0.2 g of   
250 mL of 200 millimolar (mM) Na-cacodylate buffer
525 mL of   

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.
SECTION: 500 ml
2. Mix the following in a 500 ml beaker:
50 mL of   
62.5 mL of   
0.1 g of   
125 mL of 200 millimolar (mM) Na-cacodylate buffer
262.5 mL of   

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.
SECTION: 200 ml
3. Mix the following in a 500 ml beaker:
20 mL of   
25 mL of   
0.04 g of   
50 mL of 200 millimolar (mM) Na-cacodylate buffer
105 mL of   

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.
SECTION: 100 ml
4. Mix the following in a 100 ml beaker:
10 mL of   
12.5 mL of   
0.02 g of   
25 mL of 200 millimolar (mM) Na-cacodylate buffer
52.5 mL of   

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.