SOP: Preparation of Agars, Broths and Other Reagents — Media Glossary, Sections Q–R

GIFSQ — Global Institute for Food Safety & Quality

Principle

This document gives the laboratory formulations of the agars, broths and other reagents in the Media Glossary that fall under the letters Q–R. For each medium it lists the basal medium ingredients with their quantities, any supplements, and the complete-medium preparation steps, so that the medium can be prepared in the laboratory and made available to the analytical methods of the compendium.

Apparatus required

• Analytical balance
• Autoclave
• Heating equipment for boiling
• Sterile screw-cap tubes (13 x 100 mm)
• Sterile Petri dishes

Rapid H₂S Test Medium (for biotyping Campylobacter)

Basal medium

• Brucella broth — 2.9 g
• Sodium phosphate (dibasic), anhydrous (Na₂HPO₄) — 0.118 g
• Potassium phosphate (monobasic), anhydrous (KH₂PO₄) — 0.023 g
• Agar — 0.2 g
• Distilled water — 97.0 mL
• pH about 7.3

Supplements

• A: 10% ferrous sulphate heptahydrate (FeSO₄·7H₂O) — 1.0 mL
• B: 10% sodium metabisulphite — 1.0 mL
• C: 10% sodium pyruvate — 1.0 mL

Complete medium

• Suspend the basal medium ingredients in 97.0 mL of distilled water
  ↓
• Autoclave the basal medium at 121 °C for 15 min
  ↓
• Cool to 45 °C
  ↓
• Prepare the supplement solutions separately in distilled water shortly before use and filter sterilize
  ↓
• Aseptically add 1.0 mL of solution A to 1.0 mL of solution B, mix well and add to 1.0 mL of solution C
  ↓
• Add the whole mixture to the basal medium
  ↓
• Aseptically dispense 3.0 mL into sterile screw-capped tubes (13 x 100 mm)
• Note: prepare fresh medium every two weeks

Rappaport-Vassiliadis Soya Peptone Broth (RVS)

Basal medium

• Sodium chloride (NaCl) — 7.2 g
• Soya peptone — 4.5 g
• Potassium phosphate (monobasic) (KH₂PO₄) — 1.26 g
• Potassium phosphate (dibasic) (K₂HPO₄) — 0.18 g
• Magnesium chloride, anhydrous (MgCl₂) — 13.58 g
• Malachite green — 0.036 g
• Distilled water — 1.0 L
• pH 5.2 ± 0.2

Complete medium

• Suspend the basal medium ingredients in 1.0 L of distilled water
  ↓
• Heat gently to dissolve
  ↓
• Dispense into tubes
  ↓
• Autoclave at 115 °C for 15 min

Reinforced Clostridial Medium (RCM)

Basal medium

• Yeast extract — 3.0 g
• Beef extract — 10.0 g
• Peptone — 10.0 g
• Soluble starch — 1.0 g
• Dextrose — 5.0 g
• Cysteine hydrochloride — 0.5 g
• Sodium acetate, anhydrous — 3.0 g
• Sodium chloride — 5.0 g
• Agar (for solid medium) — 15.0 g
• Distilled water — 1.0 L
• pH 7.4 ± 0.2

Complete medium

• Suspend the basal medium ingredients in 1.0 L of distilled water
  ↓
• Heat to boiling to dissolve the ingredients
  ↓
• Adjust the pH
  ↓
• Autoclave at 121 °C for 15 min
  ↓
• Dispense into sterile Petri dishes

References

• Health Canada, Compendium of Analytical Methods, Volume 1, Appendix G — Glossary of Agars, Broths and Other Reagents, Sections Q–R (January 2006)