5.2.1. Agar base
- 2.5 g of peptone (Oxoid L37)
- 10 g of glucose
- 2 g of soluble starch
- 2.5 g of yeast extract (Oxoid L21)
- 5 g of neopeptone (Difco 0119)
- 2 g of trypticase (BBL211921)
- 50 ml of 1 M phosphate buffer (pH 6.7) = 49.7ml of 1M K2HPO4 + 50.3 ml of 1M KH2PO4.
- Mix all ingredients and make up to 1000 ml with distilled water.
- Adjust to pH 7.2 with 5M KOH.
- Add agar (Difco or Oxoid No 1) to a 1.5 % final concentration. For 250 ml amounts of agar base, weigh 3.75 g of agar into the 250 ml Duran flasks, for 500 ml, 7.5 gram. Dispense the correct amount of made up and pH corrected broth into the Duran flask.
- Mix well before dispensing broth into Duran flasks with agar, the soluble starch settles out quite quickly.
- Sterilize by autoclaving at 115°C for 15 minutes. Higher temperature autoclaving will tend to caramelize the agar (make it darker).
- Add cysteine hydrochloride H2O to a final
concentration of 0.025 %.
NOTE: Cysteine hydrochloride reduces the oxygen content in the agar more efficiently if added just before the agar is to be used. If the agar is made up in 250 or 500 ml amounts, then autoclaved, this base can be kept for quite a long time (2-3 months or longer), but if the cysteine is added before autoclaving, the base will only keep a week at the most.