SOP: Preparation of Microbiological Media and Reagents — Glossary, Section B
GIFSQ — Global Institute for Food Safety & Quality
1. Purpose
This glossary lists the agars, broths, and other reagents prepared for use in food microbiological methods, from Baird-Parker medium to Buffered Peptone Water. Each entry gives the basal composition (quantities per 1.0 L of water unless stated) and the steps to prepare the complete medium, including sterilization conditions and any supplements added after cooling.
2. Baird-Parker (BP) Medium
Basal medium
- Tryptone — 10.0 g
- Beef extract — 5.0 g
- Yeast extract — 1.0 g
- Glycine — 12.0 g
- Lithium chloride — 5.0 g
- Sodium pyruvate — 10.0 g
- Agar — 20.0 g
- Distilled water — 1.0 L
- Final pH 7.0 ± 0.2
Supplements
- Filter-sterilized 1% potassium tellurite solution
- Egg yolk emulsion (50%)
Complete medium
Add the basal components to the water
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Heat gently and bring to the boil
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Autoclave at 121°C for 15 min
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Cool to 45–50°C
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Add the supplements and dispense. Example: aseptically add 50.0 mL of pre-warmed (45–50°C) commercial egg yolk/tellurite emulsion to the basal medium, mix thoroughly, and dispense. (Egg yolk tellurite emulsion is commercially available and usually contains about 50% egg yolk — follow the manufacturer’s instructions.)
Storage and use notes
- Dry the agar surface before inoculation.
- Freshly prepared BP medium may be toxic to injured cells — store the plates at refrigeration temperature overnight before inoculation.
- The medium should be opaque; do not use non-opaque plates.
- Shelf life is one to several weeks under the specified storage conditions, but the selectivity and diagnostic characteristics of the complete medium may deteriorate with time, especially if the plates are not prepared from good-quality commercial products. Each laboratory is responsible for validating the appropriate storage time for its BP plates based on selectivity, recoverability, and expected performance.
3. Bismuth Sulfite Agar (BS)
Basal medium
- Peptone — 10.0 g
- Beef extract — 5.0 g
- Dextrose — 5.0 g
- Disodium phosphate (Na₂HPO₄) — 4.0 g
- Ferrous sulfate (FeSO₄) — 0.3 g
- Bismuth sulfite indicator — 8.0 g
- Brilliant green — 0.025 g
- Agar — 20.0 g
- Distilled water — 1.0 L
- Final pH 7.6 ± 0.2
Complete medium
Suspend the ingredients in one litre of distilled water and mix thoroughly
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Heat uniformly to incipient boiling on a hotplate (not an open flame), swirling frequently; do not autoclave and do not overheat
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Cool to 45–50°C and swirl to evenly disperse the heavy precipitate just before pouring into plates
Storage and use notes
- Before use, store prepared plates overnight in a darkened refrigerator. Plates can be stored for up to 4 days. Discard uninoculated plates that begin to turn a darker green — the colour change indicates oxidation, which increases with storage time and light exposure and may make the plates inhibitory to some salmonellae.
4. Blood Agar — Trypticase Blood Agar Base
Basal medium
- Trypticase — 10.0 g
- Beef extract — 3.0 g
- Sodium chloride (NaCl) — 5.0 g
- Agar — 15.0 g
- Distilled water — 1.0 L
- Final pH 7.2 ± 0.1
Supplement
- Sterile defibrinated blood — 50.0 mL
Complete medium
Suspend the basal ingredients in 1.0 L of distilled water
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Heat to boiling to dissolve
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Sterilize at 121°C for 15 min
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Cool to 45–50°C and aseptically add the sterile defibrinated blood
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Mix thoroughly without incorporating air bubbles
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Dispense
Note
- The solidified, complete medium cannot be re-liquefied.
5. Blood Agar — Listeria
Prepare blood agar plates as soon as possible after receiving fresh blood, using blood agar base or preferably Trypticase Soy agar with 7% defibrinated horse blood. Rehydrate and sterilize as recommended by the manufacturer. Bring the agar and the blood both to 45–50°C before combining and pouring plates. Plates can be stored at 4°C for 1 month. Sheep blood plates for the CAMP test are prepared and stored in a similar way.
6. Brain Heart Infusion (BHI) Broth
Basal medium
- Calf brain, infusion from — 200.0 g
- Beef heart, infusion from — 250.0 g
- Proteose peptone — 10.0 g
- Dextrose — 2.0 g
- Sodium chloride (NaCl) — 5.0 g
- Sodium phosphate (Na₂HPO₄) — 2.5 g
- Distilled water — 1.0 L
- Final pH 7.4 ± 0.2
Complete medium
Dissolve the ingredients in 1.0 L of distilled water
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Distribute as required (tubes or flasks)
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Sterilize at 121°C for 15 min
7. Brain Heart Infusion Broth with 0.6% Yeast Extract (BHI-YE)
Basal medium
- Calf brain, infusion from — 200.0 g
- Beef heart, infusion from — 250.0 g
- Proteose peptone — 10.0 g
- Dextrose — 2.0 g
- Sodium chloride (NaCl) — 5.0 g
- Sodium phosphate (Na₂HPO₄) — 2.5 g
- Yeast extract — 6.0 g
- Distilled water — 1.0 L
- Final pH 7.4 ± 0.2
Complete medium
Dissolve the ingredients in 1.0 L of distilled water, adding the yeast extract to the BHI before autoclaving
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Distribute as required (tubes or flasks)
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Sterilize at 121°C for 15 min
8. Brilliant Green-lactose Bile (2%) (BGLB) Broth
Basal medium
- Peptone — 10.0 g
- Lactose — 10.0 g
- Ox-gall — 20.0 g
- Brilliant green — 0.0133 g
- Final pH 7.2 ± 0.2
Complete medium
Dissolve the peptone and lactose in 500 mL of water
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Add the ox-gall dissolved in 200 mL of water
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Bring the volume to approximately 975 mL with water and adjust the pH
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Add 13.3 mL of a 1% aqueous solution of brilliant green
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Bring the total volume to 1.0 L, stir, and filter through cotton if necessary to clarify
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Dispense in 10.0 mL volumes into tubes containing gas vials
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Sterilize at 121°C for 12 min
9. Brilliant Green Sulfa Agar (BGS)
Basal medium
- Yeast extract — 3.0 g
- Peptone — 10.0 g
- Sodium chloride (NaCl) — 5.0 g
- Lactose — 10.0 g
- Sucrose — 10.0 g
- Phenol red — 0.08 g
- Brilliant green — 0.0125 g
- Sodium sulfapyridine — 1.0 g
- Agar — 20.0 g
- Distilled water — 1.0 L
- Final pH 6.9 ± 0.2
Complete medium
Suspend the ingredients in 1 L of distilled water
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Heat to boiling with frequent swirling
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Autoclave at 121°C for 15 min
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Cool to 45–50°C
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Dispense into plates and allow to dry with the lids partially removed
10. Brilliant Green Water (0.002% w/v)
Basal medium
- Brilliant green dye — 1.0 g
- Sterile distilled water — 100 mL
Complete medium
Dissolve 1.0 g of brilliant green dye in 100 mL of sterile water (1% w/v solution)
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Add 2.0 mL of the 1% brilliant green solution to 1.0 L of sterile water (0.002% w/v)
11. Brucella Broth
Basal medium
- Tryptone — 10.0 g
- Peptic digest of animal tissues — 10.0 g
- Dextrose — 1.0 g
- Yeast extract — 2.0 g
- Sodium chloride (NaCl) — 5.0 g
- Sodium bisulfite (NaHSO₃) — 0.1 g
- Distilled water — 1.0 L
- Final pH 7.0 ± 0.2
Complete medium
Suspend the ingredients in 1.0 L of distilled water
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Boil to dissolve completely
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Autoclave at 121°C for 15 min
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Dispense
12. Brucella Semi-solid Medium
Basal medium
- Tryptone — 10.0 g
- Peptic digest of animal tissues — 10.0 g
- Dextrose — 1.0 g
- Yeast extract — 2.0 g
- Sodium chloride (NaCl) — 5.0 g
- Sodium bisulfite (NaHSO₃) — 0.1 g
- Neutral red — 0.02 g
- Agar — 1.6 g
- Distilled water — 1.0 L
- Final pH 7.0 ± 0.2
Complete medium
Mix the ingredients in distilled water
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Heat with frequent agitation and boil for about 1 min
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Dispense 5.0–7.0 mL into screw-cap tubes (16 × 125 mm)
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Autoclave at 121°C for 15 min
13. Buffered Glucose Broth (MR-VP medium)
Basal medium
- Peptone — 5.0 g
- Glucose — 5.0 g
- Dipotassium phosphate (K₂HPO₄) — 5.0 g
- Distilled water — 1.0 L
- Final pH 7.5 ± 0.2
Complete medium
Add the ingredients to 1.0 L of distilled water
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Heat gently with stirring to obtain complete solution
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Dispense into test tubes
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Sterilize at 121°C for 15 min
14. Buffered Glucose Salt Broth (MR-VP medium)
Basal medium
- Peptone — 5.0 g
- Glucose — 5.0 g
- Dipotassium phosphate (K₂HPO₄) — 5.0 g
- Sodium chloride (NaCl) — 2.0–3.0 g
- Distilled water — 1.0 L
- Final pH 7.5 ± 0.2
Complete medium
Add the ingredients to 1.0 L of distilled water
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Heat gently with stirring to obtain complete solution
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Dispense into test tubes
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Sterilize at 121°C for 15 min
Note
- For halophilic Vibrio species.
15. Buffered Peptone Water (BPW)
Basal medium
- Peptone — 10.0 g
- Sodium chloride (NaCl) — 5.0 g
- Disodium phosphate — 3.5 g
- Potassium dihydrogen phosphate — 1.5 g
- Distilled water — 1.0 L
- Final pH 7.2 ± 0.2
Complete medium
Dissolve the ingredients in 1.0 L of distilled water
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Dispense into appropriate glassware
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Autoclave at 121°C for 15 min
References
Compendium of Analytical Methods, Volume 1 — Glossary of Agars, Broths and Other Reagents, Section B, January 2006, Health Canada Microbiological Methods Committee.