SOP: Preparation of Agars and Reagents — Media Glossary, Sections J–K

GIFSQ — Global Institute for Food Safety & Quality

Principle

This document gives the laboratory formulations of the agars and reagents in the Media Glossary that begin with the letters J and K. 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
• Screw-cap tubes (13 x 100)
• Heating equipment for boiling
• Tubes or flasks
• Sterile Petri dishes

King’s Medium B

Basal medium

• Proteose peptone No. 3 — 20.0 g
• Potassium phosphate, dibasic, anhydrous (K₂HPO₄) — 1.5 g
• Magnesium sulphate 7 hydrate (MgSO₄·7H₂O) — 1.5 g
• Glycerol — 15.0 mL
• Agar — 20.0 g
• Distilled water — 1.0 L
• Final pH: 7.2 ± 0.2

Complete medium

• Add all components to the distilled water and bring the volume to 1.0 L
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• Mix thoroughly
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• Heat gently and bring to a boil
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• Dispense into tubes or flasks
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• Autoclave for 15 min at 121 °C
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• Pour into Petri dishes, or leave in tubes

Klebsiella Selective Agar

Basal medium

• Agar — 15.0 g
• DL-Phenylalanine — 10.0 g
• L-Ornithine-HCl — 10.0 g
• Raffinose — 7.0 g
• Pancreatic digest of casein — 2.5 g
• Yeast extract — 2.5 g
• Potassium phosphate, dibasic (K₂HPO₄) — 2.0 g
• Phenol red solution (see Supplement 1 below) — 10.0 mL
• Distilled water — 990 mL
• Final pH: 5.6 ± 0.2
• Note: additional agar can be added to inhibit the spread of mucoid colonies

Supplement 1 — Phenol red solution

• Phenol red — 0.5 g
• 50% ethanol — 10.0 mL

Supplement 2 — Carbenicillin solution

• Carbenicillin — 0.05 g
• Distilled water — 10.0 mL
• Note: the carbenicillin solution is filter sterilized

Complete medium

• Add all components except the carbenicillin solution to 990 mL of distilled water
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• Mix thoroughly
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• Heat gently and bring to a boil
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• Autoclave for 15 min
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• Cool to 45–50 °C
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• Aseptically add 10.0 mL of the filter-sterilized carbenicillin solution and mix thoroughly
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• Adjust the pH
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• Pour into sterile Petri dishes

Kligler Iron Agar (KIA)

Basal medium

• Pancreatic digest of casein — 10.0 g
• Peptic digest of animal tissue — 10.0 g
• Lactose — 20.0 g
• Glucose — 1.0 g
• Sodium chloride (NaCl) — 5.0 g
• Ferric ammonium citrate — 0.5 g
• Sodium thiosulfate (Na₂S₂O₃) — 0.5 g
• Agar — 15.0 g
• Phenol red — 0.025 g
• Distilled water — 1.0 L
• Final pH: 7.4 ± 0.2
• Note: when used for halophilic Vibrio species, add sodium chloride to a final concentration of 2–3%

Complete medium

• Mix all ingredients thoroughly
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• Heat with agitation to dissolve
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• Dispense into 13 x 100 screw-cap tubes
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• Autoclave at 121 °C for 15 min
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• Cool, then slant the tubes so the agar forms deep butts

References

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