SOP: Preparation of Agars, Broths and Other Reagents — Media Glossary, Section W

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 begin with the letter W. For each medium it lists the basal medium ingredients with their quantities, any supplement, 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
• Steam bath
• Refrigerated centrifuge
• Sterile Petri dishes
• Tubes or flasks

Wagatsuma Agar (for Kanagawa Test)

Basal medium

• Peptone — 10.0 g
• Sodium chloride (NaCl) — 70.0 g
• Yeast extract — 3.0 g
• Potassium phosphate dibasic (K₂HPO₄) — 5.0 g
• Mannitol — 10.0 g
• Agar — 15.0 g
• Crystal violet — 0.001 g
• Distilled water — 1.0 L
• pH: 8.0 ± 0.2

Supplement

• Human or rabbit red blood cells, fresh (within 24 h of drawing), with anticoagulant — 50.0 mL
• Physiological saline — 200–250 mL

Complete medium

• Suspend the basal medium ingredients in 1.0 L of distilled water
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• Heat to boiling to dissolve the agar
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• Adjust the pH to 8.0 ± 0.2
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• Place in a steam bath for 30 min
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• Do not autoclave
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• Cool to 45–50 °C
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• Prepare the blood: mix the fresh (within 24 h of drawing) human or rabbit blood with the same or a larger volume of physiological saline
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• Centrifuge at 4000 × g at 4 °C for 15 min
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• Decant the saline and wash two more times
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• After the third washing, pour off the saline and resuspend the cells to the original volume with saline
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• Add 50 mL of the washed blood cells to the basal medium
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• Mix and dispense into sterile Petri dishes
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• Dry thoroughly and use promptly

Wilkins Chalgren Anaerobe Blood Agar

Basal medium

• Pancreatic digest of casein — 10.0 g
• Gelatin peptone — 10.0 g
• Sodium chloride (NaCl) — 5.0 g
• Yeast extract — 5.0 g
• Glucose — 1.0 g
• L-Arginine — 1.0 g
• Sodium pyruvate (C₃H₃O₃Na) — 1.0 g
• Hemin — 5.0 mg
• Menadione (vitamin K) — 0.5 mg
• Agar — 10.0 g
• Tween 80 — 1.0 mL
• Distilled water — 950 mL
• pH: 7.1 ± 0.2

Supplement

• Lysed horse blood — 50.0 mL

Complete medium

• Suspend the basal medium ingredients in distilled water and bring the volume to 950 mL
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• Mix thoroughly
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• Gently heat to boiling to dissolve the agar
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• Autoclave at 121 °C for 15 min
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• Cool to 50–55 °C
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• Aseptically add 50.0 mL of lysed horse blood
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• Mix thoroughly and dispense into sterile Petri dishes

Wilkins Chalgren Anaerobe Broth

Basal medium

• Pancreatic digest of casein — 10.0 g
• Gelatin peptone — 10.0 g
• Sodium chloride (NaCl) — 5.0 g
• Yeast extract — 5.0 g
• Glucose — 1.0 g
• L-Arginine — 1.0 g
• Sodium pyruvate (C₃H₃O₃Na) — 1.0 g
• Hemin — 5.0 mg
• Menadione (vitamin K) — 0.5 mg
• Distilled water — 1.0 L
• pH: 7.1 ± 0.2

Complete medium

• Suspend the basal medium ingredients in 1.0 L of distilled water
  ↓
• Mix thoroughly
  ↓
• Dispense into tubes or flasks
  ↓
• Autoclave at 121 °C for 15 min

References

• Health Canada, Compendium of Analytical Methods, Volume 1, Appendix G — Glossary of Agars, Broths and Other Reagents, Section W (January 2006)