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

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 M. 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
• Petri dishes
• Screw-cap culture tubes (20 x 150 mm)
• Flasks
• Sterile membrane filters and syringe
• Laminar flow hood

M Broth

Basal medium

• Yeast extract — 5.0 g
• Tryptone — 12.5 g
• D-mannose — 2.0 g
• Sodium citrate — 5.0 g
• Sodium chloride (NaCl) — 5.0 g
• Dipotassium phosphate (K₂HPO₄) — 5.0 g
• Manganese chloride (MnCl₂) — 0.14 g
• Magnesium sulfate (MgSO₄) — 0.8 g
• Ferrous sulfate (FeSO₄) — 0.04 g
• Tween 80 — 0.75 g
• Distilled water — 1.0 L
• pH 7.0 ± 0.2

Complete medium

• Suspend the ingredients in the distilled water and heat to boiling for 1–2 min
  ↓
• Dispense and autoclave at 121 °C for 15 min

MacConkey Agar

Basal medium

• Peptone — 20.0 g
• Lactose — 10.0 g
• Bile salts No. 3 — 1.5 g
• Sodium chloride (NaCl) — 5.0 g
• Neutral red — 0.03 g
• Crystal violet — 0.001 g
• Agar — 13.5 g
• Distilled water — 1.0 L
• Final pH: 7.1 ± 0.2

Complete medium

• Suspend the ingredients in 1.0 L of distilled water and heat to boiling with frequent swirling
  ↓
• Autoclave at 121 °C for 15 min and cool to 45–50 °C
  ↓
• Dispense the medium into plates and allow to dry with the lids partially removed

Malachite Green (5%)

Basal medium

• Malachite green — 5.0 g
• Distilled water — 100 mL

Complete medium

• Dissolve 5.0 g of malachite green in distilled water to make 100 mL of solution
  ↓
• Stir

Malt Extract

Basal medium

• Malt extract — 30.0 g
• Mycological protein — 5.0 g
• Agar — 15.0 g
• Distilled water — 1.0 L
• pH 5.4 ± 0.2

Complete medium

• Suspend the ingredients in the distilled water
  ↓
• Boil to dissolve completely
  ↓
• Autoclave at 115 °C for 10 min
• Note: if the medium is required for osmophilic organisms, add 500 g of sucrose per litre before autoclaving

mA-Mannitol (MAM) Agar

Basal medium

• Tryptone — 5.0 g
• Mannitol — 5.0 g
• Yeast extract — 2.0 g
• Sodium chloride (NaCl) — 3.0 g
• Potassium chloride (KCl) — 2.0 g
• Magnesium sulfate heptahydrate (MgSO₄·7H₂O) — 0.2 g
• Ferric chloride hexahydrate (FeCl₃·6H₂O) — 0.1 g
• Bromothymol blue — 0.08 g
• Agar — 15.0 g
• Distilled water — 1.0 L
• pH 8.0 ± 0.2

Supplement

• Sodium deoxycholate — 100 mg

Complete medium

• Add the basal ingredients, except the agar, to 1.0 L of water
  ↓
• Dissolve at room temperature and adjust to pH 8.0
  ↓
• Add the agar and autoclave at 121 °C for 15 min
  ↓
• Temper and add 100 mg of sodium deoxycholate
  ↓
• Mix well and dispense into Petri dishes
• Note: this medium can be stored for 2 months at 4 °C

Mannitol Maltose Agar

Basal medium

• PhytoneTM (papaic digest of soybean meal) — 5.0 g
• PolypeptoneTM (equal parts pancreatic digest of casein and peptic digest of animal tissue) — 5.0 g
• Beef extract — 5.0 g
• D-Mannitol — 10.0 g
• Maltose — 10.0 g
• Sodium chloride (NaCl) — 20.0 g
• Agar — 13.0 g
• 1000X dye stock solution (see below) — 1.0 mL
• Distilled water — 1.0 L
• pH 7.8 ± 0.2

Supplement: 1000X dye stock solution

• Bromthymol blue — 4.0 g
• Cresol red — 4.0 g
• Ethanol, 95% — 100 mL

Complete medium

• Suspend the ingredients and boil to dissolve
  ↓
• Adjust the pH and autoclave for 15 min at 121 °C

mA-Trehalose (MAT) Agar

Basal medium

• Tryptone — 5.0 g
• Trehalose — 5.0 g
• Yeast extract — 2.0 g
• Sodium chloride (NaCl) — 3.0 g
• Potassium chloride (KCl) — 2.0 g
• Magnesium sulfate heptahydrate (MgSO₄·7H₂O) — 0.2 g
• Ferric chloride hexahydrate (FeCl₃·6H₂O) — 0.1 g
• Bromothymol blue — 0.04 g
• Agar — 15.0 g
• Distilled water — 1.0 L
• pH 8.0 ± 0.2

Supplements

• Ethanol — 1.0 mL
• Ampicillin — 20.0 mg
• Sodium deoxycholate — 100 mg

Complete medium

• Add the basal ingredients, except the agar, to 1.0 L of water
  ↓
• Dissolve at room temperature and adjust to pH 8.0
  ↓
• Add the agar and autoclave at 121 °C for 15 min
  ↓
• Temper and add the additives
  ↓
• Mix well and dispense into Petri dishes
• Note: this medium can be stored for 2 months at 4 °C

Methyl Red Test Reagent

Basal medium

• Methyl red — 0.10 g
• Ethanol, 95% — 300 mL
• Distilled water — 200 mL

Complete medium

• Dissolve the methyl red in 300 mL of ethanol and bring the volume to 500 mL with distilled water
  ↓
• Store under refrigeration

Microbial Content Test Agar

(Also known as Tryptic Soy Agar with Lecithin and Polysorbate 80 [TSALT], and Casein Soy Peptone with Polysorbate 80 and Lecithin.)

Basal medium

• Pancreatic digest of casein — 15.0 g
• Sodium chloride (NaCl) — 5.0 g
• Tween 80 (polysorbate 80) — 5.0 g
• Enzymatic hydrolysate of soybean meal — 5.0 g
• Lecithin — 0.7 g
• Agar — 15.0 g
• Distilled water — 1.0 L
• pH 7.3 ± 0.2

Complete medium

• Add the components to the distilled water
  ↓
• Mix thoroughly
  ↓
• Gently heat and bring to a boil; boil for 1–2 min
  ↓
• Dispense into flasks or tubes and autoclave for 15 min at 121 °C
  ↓
• Pour into sterile Petri dishes or leave in the tubes

m-FC Agar (without rosolic acid)

Basal medium

• Tryptose — 10.0 g
• Proteose peptone No. 3 — 5.0 g
• Yeast extract — 3.0 g
• Lactose — 12.5 g
• Bile salts No. 3 — 1.5 g
• Sodium chloride (NaCl) — 5.0 g
• Aniline blue — 0.1 g
• Agar — 15.0 g
• Distilled water — 1.0 L
• pH 7.4 ± 0.2

Complete medium

• Heat the mixture to boiling, temper to 50–55 °C, and adjust the pH
  ↓
• Do not autoclave
  ↓
• Pour 15–20 mL into Petri plates
• Note: plates keep for 4 weeks at 4 °C
• Note: the formulation without rosolic acid was found to work better with the HGMF method for coliforms. If rosolic acid is added to the medium, use 10 mL of a 1% solution in 0.2 N NaOH. Add it after the other ingredients have been dissolved by heating, and continue to boil for an additional minute. This supplement is also commercially available; follow the manufacturer’s instructions.

Milk Agar

(There are various media under the names Milk Agar, Skim Milk Agar and Milk Plate Count Agar. The following formulation is specified for use with MFLP-61, an HGMF method for Pseudomonas aeruginosa.)

Basal medium: Mixture A

• Instant nonfat milk — 100 g
• Distilled water — 500 mL

Basal medium: Mixture B

• Nutrient broth — 12.5 g
• Sodium chloride (NaCl) — 2.5 g
• Agar — 15.0 g
• Distilled water — 500 mL

Complete medium

• Autoclave the two mixtures separately, and temper to 50 °C
  ↓
• Aseptically combine the mixtures and pour 20.0 mL per plate

Modified CCDA Preston

• See Campylobacter Agar with Charcoal and Deoxycholate (CCDA).

Modified Cellobiose-Polymyxin B-Colistin Agar (mCPC)

Basal medium: Solution 1

• Peptone — 10.0 g
• Beef extract — 5.0 g
• Sodium chloride (NaCl) — 20.0 g
• 1000X dye stock solution (see below) — 1.0 mL
• Agar — 15.0 g
• Distilled water — 900 mL
• pH 7.6

Basal medium: Solution 2

• Cellobiose — 10.0 g
• Colistin — 400,000 units
• Polymyxin B — 100,000 units
• Distilled water — 100 mL

Supplement: 1000X dye stock solution

• Bromthymol blue — 4.0 g
• Cresol red — 4.0 g
• Ethanol, 95% — 100 mL
• Note: for consistent medium colour, use the dye solution rather than repeatedly weighing out the dry dyes. Dissolve the dyes in ethanol for a 4% (w/v) stock solution. Using 1 mL of this solution per litre of mCPC agar gives 40 mg of bromothymol blue and 40 mg of cresol red per litre.

Complete medium

• Solution 1: adjust the pH to 7.6, boil to dissolve the agar, and cool to 48–55 °C
  ↓
• Solution 2: dissolve the cellobiose in the distilled water by heating gently, cool, and add the antibiotics
  ↓
• Add Solution 2 to the cooled Solution 1, mix, and dispense into Petri dishes
• Note: the final colour is dark green to green-brown. This medium, like TCBS, is very inhibitory and does not require autoclaving. The medium may be stored 2 weeks at refrigeration temperatures.

Modified E. coli Broth with Novobiocin (mEC-n)

Basal medium

• Tryptone — 20.0 g
• Bile salts No. 3 — 1.5 g
• Lactose — 5.0 g
• Dipotassium phosphate (K₂HPO₄) — 4.0 g
• Monopotassium phosphate (KH₂PO₄) — 1.5 g
• Sodium chloride (NaCl) — 5.0 g
• Distilled water — 1.0 L

Supplements

• Novobiocin — 100 mg
• Distilled water — 1.0 mL

Complete medium

• Add the components, except the novobiocin, to the distilled water
  ↓
• Mix thoroughly and dispense into 225 mL aliquots
  ↓
• Autoclave at 121 °C for 15 min
  ↓
• Let cool
  ↓
• Dispense in 225 mL aliquots
  ↓
• Add 50.0 µL of novobiocin solution (filter sterilized through a 0.45 µm filter) to each sample tube just before addition of the test sample

Modified Fraser Broth (MFB)

• See Fraser Broth.

Modified HC Agar (mHC)

Basal medium

• Tryptone — 20.0 g
• Bile salts No. 3 — 1.12 g
• Sodium chloride (NaCl) — 5.0 g
• Sorbitol — 20.0 g
• 4-Methylumbelliferyl-β-D-glucuronide (MUG), optional — 0.10 g
• Bromocresol purple, 1.6% — 0.94 mL
• Agar — 15.0 g
• Distilled water — 1.0 L
• pH 7.4 ± 0.2

Supplements

• Cefsulodin, 1% (1.0 g/100 mL) — 1.0 mL
• Potassium tellurite, 0.1% (0.1 g/100 mL) — 2.5 mL

Complete medium

• Suspend the basal ingredients in 1.0 L of distilled water, with stirring to dissolve all ingredients (except the agar), and adjust the pH
  ↓
• Heat to boiling to dissolve the agar
  ↓
• Autoclave for 15 min at 121 °C
  ↓
• Temper to 45–50 °C
  ↓
• Filter sterilize the supplements and add them aseptically to the medium
  ↓
• Gently swirl the medium (without adding air bubbles) to dispense the supplements thoroughly
  ↓
• Aseptically dispense the agar into Petri dishes and allow to solidify
  ↓
• Dry the plates in a laminar flow hood to remove excess moisture
• Note: the supplements are commercially available. If purchasing a reconstituted product (liquid form), ensure that the final concentration in the medium is correct. For mHC agar, potassium tellurite is 2.5 mg/L.
• Note: cefsulodin is not needed if the enrichment broth EEB is used.

Modified Oxford Agar (MOX)

Basal medium

• Columbia blood agar base (depending on the brand) — 39.0–44.0 g
• Agar — 2.0 g
• Esculin — 1.0 g
• Ferric ammonium citrate — 0.5 g
• Lithium chloride — 15.0 g
• Distilled water — 1.0 L

Supplements

• Moxalactam — 0.015 g
• Colistin sulfate — 0.01 g
• Distilled water — 10.0 mL

Complete medium

• Note: MOX agar is a slight modification of the Listeria selective agar (Oxford formulation).
  ↓
• Add the ingredients, except the supplement, to the distilled water
  ↓
• Boil gently to dissolve
  ↓
• Autoclave at 121 °C for 15 min
  ↓
• Cool rapidly to 46 °C in a water bath
  ↓
• Add the filter-sterilized supplement to the basal medium, mix well and dispense

Modified PE-2 Medium (PE2)

Basal medium

• Peptone — 20.0 g
• Yeast extract — 3.0 g
• Alcoholic solution of bromocresol purple, 2% — 2.0 mL
• Distilled water — 1.0 L

Supplement

• Alaska seed peas — 8–10 per tube

Complete medium

• Dissolve the ingredients with gentle heating, if necessary
  ↓
• Dispense 19.0 mL portions into 20 x 150 mm screw-cap culture tubes containing 8–10 untreated Alaska seed peas
  ↓
• Sterilize for 30 min at 121 °C or an equivalent
• Note: normally the prepared sterilized medium can be maintained unaffected under refrigeration (0–4 °C) for up to 4 weeks. Prior to use, the refrigerated medium should be deaerated at 100 °C for 10 min. Cool to ambient temperature prior to use.

Modified Semi-Solid Rappaport Vassiliadis (MSRV) Medium

Basal medium

• Tryptose — 4.59 g
• Casein hydrolysate — 4.59 g
• Sodium chloride (NaCl) — 7.34 g
• Monopotassium phosphate (KH₂PO₄) — 1.47 g
• Agar — 2.7 g
• Magnesium chloride (MgCl₂) — 10.93 g
• Malachite green oxalate — 0.037 g
• Distilled water — 1.0 L
• pH 5.2 ± 0.2

Supplements

• Novobiocin — 20.0 mg
• Distilled water — 10.0 mL
• Note: to prepare the supplement, dissolve the novobiocin in water and filter sterilize. Dispense in appropriate amounts and store frozen.

Complete medium

• Add the basal ingredients to the distilled water
  ↓
• Heat to boiling to dissolve; do not autoclave
  ↓
• Cool to 50 °C and aseptically add the 10 mL of novobiocin solution
  ↓
• Mix well and dispense 18.0 mL into sterile Petri dishes
  ↓
• Leave the plates completely dry (1–3 h) before replacing the lids; they can be left overnight at room temperature before being refrigerated
• Note: prepared MSRV plates must be stored in an upright position. They can be stored for up to 2 weeks at refrigeration temperatures in the dark. Examine the plates as soon as possible after removing them from the incubator, as false-positive motility reactions can develop at room temperature.

Modified Sorbitol MacConkey Agar (TCCSMAC)

Basal medium (Sorbitol MacConkey Agar — see the note below)

• Peptone — Difco: 15.5 g / Oxoid: 20 g
• Proteose peptone — Difco: 3.0 g / Oxoid: 0 g
• Sorbitol — 10.0 g
• Bile salts No. 3 — 1.5 g
• Sodium chloride (NaCl) — 5.0 g
• Neutral red — 0.03 g
• Crystal violet — 0.001 g
• Agar — 15.0 g
• Distilled water — 1.0 L
• pH 7.1 ± 0.2

Supplements

• Cefsulodin, 1% (1.0 g/100 mL) — 1.0 mL
• Cefixime (1.0 mg/mL) — 50.0 µL
• Potassium tellurite, 0.1% (0.1 g/100 mL) — 2.5 mL

Complete medium

• Suspend the basal ingredients in 1.0 L of distilled water, with stirring to dissolve all ingredients (except the agar), and adjust the pH
  ↓
• Heat to boiling to dissolve the agar
  ↓
• Autoclave for 15 min at 121 °C
  ↓
• Temper to 45–50 °C
  ↓
• Filter sterilize the supplements and add them aseptically to the medium
  ↓
• Gently swirl the medium (without adding air bubbles) to dispense the supplements thoroughly
  ↓
• Aseptically dispense the agar into Petri dishes and allow to solidify
  ↓
• Dry the plates in a laminar flow hood to remove excess moisture
• Note: the supplements are commercially available. If purchasing a reconstituted product (liquid form), ensure that the final concentration in the medium is correct. For TCCSMAC agar, potassium tellurite is 2.5 mg/L and cefixime is 0.050 mg/L.
• Note: cefsulodin is not needed if the enrichment broth EEB is used.
• Note: the basal medium (Sorbitol MacConkey Agar) is commercially available from a variety of manufacturers, for example BD-Difco and Oxoid. The formula of the basal medium may vary slightly (as above) and the variants can be used interchangeably. If basal medium from other sources is used, it is the responsibility of the laboratory supervisor to ensure equivalency.

Modified TSB with Novobiocin (mTSB-n)

Basal medium

• Tryptic Soy Broth or Tryptone Soya Broth — 30.0 g
• Bile salts No. 3 — 1.5 g
• Dipotassium phosphate (K₂HPO₄) — 1.5 g
• Distilled water — 1.0 L
• pH 7.4 ± 0.2

Supplement: novobiocin solution (100 mg/mL)

• Novobiocin (sodium salt) — 100 mg
• Distilled water — 1.0 mL
• Note: dissolve the novobiocin, filter sterilize using a 0.2 µm filter and syringe, and store. It may be stored several months in a dark bottle at 4 °C.

Complete medium

• Dissolve the ingredients, except the novobiocin, in the distilled water
  ↓
• Heat to boiling and dispense
  ↓
• Autoclave for 15 min at 121 °C
  ↓
• After cooling, store at 4 °C
  ↓
• Add 0.2 mL of novobiocin solution before use

Motility Test Medium

Basal medium

• Beef extract — 3.0 g
• Pancreatic digest of gelatin — 10.0 g
• NaCl — 5.0 g
• Agar — 4.0 g
• Distilled water — 1.0 L
• pH 7.2 ± 0.2

Complete medium

• Suspend the ingredients in water and mix thoroughly
  ↓
• Heat with frequent agitation and boil for 1 min to dissolve completely
  ↓
• Dispense and sterilize at 121 °C for 15 min

Motility Test Medium with 2,3,5-Triphenyltetrazolium Chloride (TTC)

Basal medium

• Pancreatic digest of gelatin — 10.0 g
• Sodium chloride (NaCl) — 5.0 g
• Beef extract — 3.0 g
• Agar — 3.5 g
• TTC — 0.05 g
• Distilled water — 1.0 L
• pH 7.2 ± 0.2

Complete medium

• Suspend the ingredients in water and mix thoroughly
  ↓
• Heat with frequent agitation and boil for 1 min to dissolve completely
  ↓
• Dispense and sterilize at 121 °C for 15 min
• Note: this medium should be stored in an upright position at 4 °C and protected from the light. Under these conditions the medium has a shelf life of 26 weeks from the date of manufacture.
• Note: this medium can be prepared by adding TTC to the regular commercially available motility test medium.

mPAC

Basal medium

• Agar — 12.0 g
• L-Lysine HCl — 5.0 g
• Sodium chloride (NaCl) — 5.0 g
• Sodium thiosulfate — 5.0 g
• Yeast extract — 2.0 g
• Magnesium sulfate heptahydrate (MgSO₄·7H₂O) — 1.5 g
• Lactose — 1.25 g
• Sucrose — 1.25 g
• Xylose — 1.25 g
• Ferric ammonium citrate — 0.80 g
• Phenol red — 0.08 g
• Nalidixic acid — 0.037 g
• Kanamycin — 8.0 mg
• Distilled water — 1.0 L
• pH 7.2 ± 0.2

Complete medium

• Add the components to distilled water and bring the volume to 1.0 L
  ↓
• Mix thoroughly
  ↓
• Gently heat and bring to a boil
  ↓
• Autoclave for 15 min at 121 °C and dispense

MR-VP Medium

• See Buffered Glucose Broth and Buffered Glucose Salt Broth (Vibrio).

Mueller-Hinton Agar (MH)

Basal medium

• Beef, infusion from — 300 g
• Casamino acids, technical — 17.5 g
• Soluble starch — 1.5 g
• Agar — 17.0 g
• Distilled water — 1.0 L
• pH 7.3 ± 0.2

Complete medium

• Suspend the ingredients in distilled water
  ↓
• Heat to boiling to dissolve completely
  ↓
• Autoclave at 121 °C for 15 min
  ↓
• Cool and dispense
• Note: when used for halophilic Vibrio spp., add NaCl to a final concentration of 2–3%.

Mueller-Hinton Agar with Blood

(commercially available)

Complete medium

• Add 50.0 mL of sterile defibrinated horse blood per 950 mL of Mueller-Hinton agar (final 5% blood concentration)

References

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