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BAM Media M195: SHIBAM Components and Instructions

Heart Infusion Agar Base
10 mM CaCl2
4% defibrinated sheep blood washed five times (detailed below)
0.5 µg/mL mitomycin C (make 1mg/mL in H2O)

 

Agar Preparation

Heart Infusion Agar Base48 g
Tyrptose6 g
1M CaCl212 ml
Distilled water1.14 liter

Heat gently to boil. Autoclave for 15min at 121°C. Final pH, 7.2 ± 0.2. Cool to 45°C water bath. Add washed blood and 600µg of mitomycin C (600µL of 1mg/mL) to tempered (cooled) agar, mix, and then pour 20 ml portions into sterile 15 x 100 mm petri dishes. Dry at room temperature with lids closed. Should be stored at 4°C, protected from light and used before three months.

Blood Preparation a

  • Separate 48ml of defibrinated sheep blood into 2 - 24mL aliquots in 50mL conical tubes

  • Centrifuge at 740 RCF for 6min 4°C (older blood may require longer spin time for separation)

  • Remove supernatant with pipette and resuspend in equal volume cold filter sterilized 1X Phosphate Buffered Solution (PBS)

  • Repeat spin and PBS wash 5 times

  • Resuspend in 48mL of PBS

a Alternatively, purchase defibrinated sheep's blood washed 5X in 1X PBS (http://www.quadfive.com: product number 643, must request washing 5X in PBS disclaimer icon).