GROWTH MEDIUM (Not in routine use)
500 mL Eagles' MEM with Hanks' salt/without glutamine
10 mL Vitamins
5 mL L-glutamine (200 mM)
50 mL Inactivated fetal calf serum
Store at 4oC. Stable for 2 weeks.
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MAINTENANCE MEDIUM (Made in-house, not in routine use)
500 mL Eagles' MEM with Hanks' salt/without glutamine (stored at 4oC)
5 mL L-glutamine (200 mM, stored frozen at -20 oC in clean room freezer)
10 mL Inactivated fetal calf serum (stored frozen at -20 oC in clean room freezer)
5 mL Fungizone (250 μg/mL, stored frozen at -20 oC in clean room freezer)
*5 mL Gentamicin (1 mg/mL, stored frozen at -20 oC in clean room freezer)
**5 mL Vancomycin (10 mg/mL, stored frozen at -20 oC in clean room freezer)
Store at 4oC. Stable for 2 weeks.
* Gentamicin Solution (1 mg/mL):
Dilute entire contents (10 mL) of 10 mg/mL Gentamicin Sulphate vial into 90 mL of d. H2O to achieve 1 mg/mL. Filter sterilize, then dispense into 5 mL aliquots (stored frozen at -20 oC in clean room freezer).
** Vancomycin Solution (10 mg/mL):
Add contents of 1 gram vial of Vancomycin to 100 mL of distilled H2O. Filter sterilize, then dispense into 5 mL aliquots (stored frozen at -20 oC in clean room freezer.
MAINTENANCE MEDIUM (Diagnostic Hybrids Inc., ready to use)
Media Quality Control:
a. A 1.5 mL aliquot each of the corresponding maintenance media is to be placed into each of MRC-5, R-Mix and E-Mix shell vials for a check on sterility/toxicity before the media are used on patient samples.
b. MRC-5, R-Mix and E-Mix Maintenance Media are to be registered into the LIS (micqc) as “on receipt” with lot numbers and expiry dates.
c. Maintenance Media QC results are all entered into the LIS daily (R-Mix for 2 days, MRC-5 for 4 days and E-Mix for 5 days).
d. Activate and inactivate the media lots in the LIS as appropriate.
Failed Media QC:
a. Any media/cell lines showing microbial contamination or toxicity is considered abnormal.
b. Do not release media lot for use pending resolution of the QC failure.
c. Inform charge/senior technologist to discuss further actions including the following.
d. Record in Incident Report (if necessary).
e. Repeat QC/re-make media to evaluate if it is the cell media, the supplement(s) or other materials causing the problem.
CELL WASHING MEDIUM (Purchased)
Hanks' Balanced Salt solution without CaCL2, MgCL2 and MgSO4.7H2O
CELL FREEZING MEDIUM (SIGMA C6164) (Purchased)
MEM supplemented with a mixture of fetal bovine serum and calf serum containing DMSO.
VIRAL FREEZING MEDIUM (SIGMA C6039) (Purchased)
MEM supplemented with a mixture of fetal bovine serum and calf serum containing 10% glycerol.
TRANSPORT MEDIA:
BARTEL'S VIRAL TRANSPORT MEDIUM (B1029-36A) (Purchased)
Eagles' MEM with nonessential amino-acid and L-glutamine in HBSS.
2% FBS
10 μg/mL Gentamicin
50 μg/mL Streptomycin
50 μg/mL Penicillin
4 μg/mL Amphotericin B
15 mM HEPES
9.5 mM Sodium bicarbonate
CHLAMYDIA TRANSPORT MEDIUM (Not in routine use)
500 mL Eagles' MEM with Hank's salt/without glutamine.
50 mL Fetal Calf Serum (normal)
5 mL 3M Glucose
5 mL Gentamicin (1000 μg/mL)
5 mL Vancomycin (1000 μg/mL)
5 mL Fungizone (250 μg/mL)
Combine all components aseptically. Dispense 1.5 mL transport medium into sterile screw-cap vials containing 3 glass beads. Store at -20oC.
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