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

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 T. 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
• pH meter with electrode
• Membrane filter unit (0.45 µm)
• Screw-cap tubes (16 × 125 mm; 20 × 150 mm; 13 × 100 mm)
• Petri dishes
• Flasks or bottles with tight seals
• Refrigerator

T1N0, T1N1, T1N2, T1N3 Medium

• See the entry for Tryptone Salt Agar (below) for the formulation

Tetracycline, 0.2% stock solution

Basal medium

• Tetracycline — 50.0 mg
• Gelatin phosphate diluent (see formula) — 25.0 mL

Complete medium

• Dissolve the tetracycline in the gelatin phosphate diluent
  ↓
• Store at 4 °C for up to 4 days
• For longer storage, freeze in small portions
• Avoid re-freezing

Tetrathionate Brilliant Green Broth (TBG)

Basal medium

• Peptone/proteose peptone — 5.0 g
• Bile salts — 1.0 g
• Calcium carbonate (CaCO₃) — 10.0 g
• Sodium thiosulphate (Na₂S₂O₃) — 30.0 g
• Distilled water — 1.0 L
• pH 8.4 ± 0.2

Supplements

• Brilliant green dye — 1 g
• Distilled water — 100 mL
• Potassium iodide (KI) — 25.0 g
• Iodine (I₂) — 30.0 g
• Distilled water — 100 mL

Complete medium

• Suspend the basal ingredients in 1.0 L of distilled water and heat to boiling
  ↓
• Cool to 45–50 °C
  ↓
• Prepare the complete medium on the day of use
  ↓
• Aseptically add 1.0 mL of 1% brilliant green solution and 20.0 mL of iodine solution to 1 L of the basal medium
  ↓
• Mix thoroughly and aseptically dispense
• Do not heat the medium after the addition of iodine
• Brilliant green solution (1% w/v): dissolve the brilliant green dye in 100 mL of distilled water
• Iodine solution: suspend the potassium iodide and iodine in 100 mL of distilled water, without heating; prepare well in advance, because iodine is slow to dissolve

Thermoacidurans Agar (TA)

Basal medium

• Yeast extract — 5.0 g
• Proteose peptone — 5.0 g
• Dextrose — 5.0 g
• Dipotassium phosphate (K₂HPO₄) — 4.0 g
• Agar — 20.0 g
• Distilled water — 1.0 L
• pH 5.0 ± 0.2

Complete medium

• Suspend the ingredients in 1 L of distilled water
  ↓
• Heat to boiling to dissolve completely
  ↓
• Autoclave at 121 °C for 15 min

Thiosulphate Citrate Bile Salts Agar (TCBS) (Oxoid designation is Cholera Medium TCBS)

Basal medium

• Peptone — 10.0 g
• Yeast extract — 5.0 g
• Sodium citrate — 10.0 g
• Sodium thiosulphate · 5 H₂O (Na₂S₂O₃·5H₂O) — 10.0 g
• Sodium chloride (NaCl) — 10.0 g
• Ox-gall powder — 5.0 g
• Ferric citrate (FeC₆H₅O₇) — 1.0 g
• Sodium cholate — 3.0 g
• Sucrose — 20.0 g
• Thymol blue solution (0.2%) — 20.0 mL
• Bromothymol blue solution (0.2%) — 20.0 mL
• Agar — 15.0 g
• Distilled water — 1.0 L
• pH 8.6 ± 0.2

Complete medium

• Suspend the basal ingredients in 1 L of distilled water
  ↓
• Heat to boil to dissolve completely
  ↓
• DO NOT AUTOCLAVE
  ↓
• Cool to 45–50 °C and dispense

Toluidine Blue DNA Agar

Basal medium

• Deoxyribonucleic acid (DNA) — 0.3 g
• Calcium chloride (CaCl₂) — 1.1 mg
• Sodium chloride (NaCl) — 10.0 g
• Toluidine blue O — 0.08 g
• Agar — 10.0 g
• 0.05 M Tris, pH 9.0 (see below) — 1.0 L

Complete medium

• Add all basal ingredients, except toluidine blue O, to the Tris buffer
  ↓
• Boil to dissolve the DNA and agar
  ↓
• Add the toluidine blue O to the solution
  ↓
• Dispense in 50.0 to 100 mL volumes in flasks or bottles
  ↓
• Seal tightly
• Sterilization is not necessary
• Store at room temperature in the dark; the medium is stable at room temperature for up to 4 months and remains satisfactory even after several melting cycles
• Note: check all DNA sources against a boiled supernatant of a 24 h BHI broth culture of TNase-producing Staphylococcus aureus; the DNA is suitable if a DNA hydrolysis zone of at least 8.0 mm is observed with a 5.0 mL aliquot of the boiled supernatant after 4 h incubation at 35 °C
• Note: also check toluidine blue O against a suitable standard before use with unknowns, because the efficacy of the dye varies with the manufacturer

Trimethylamine N-oxide (TMAO) Medium

Basal medium

• Peptone — 10.0 g
• Beef extract — 10.0 g
• Sodium chloride (NaCl) — 5.0 g
• Yeast extract — 1.0 g
• Trimethylamine N-oxide (TMAO) dihydrate — 1.0 g
• Distilled water — 1.0 L
• Agar — 2.0 g
• pH 7.5 ± 0.2

Complete medium

• Dissolve all ingredients, except the agar, in 1 L of distilled water
  ↓
• Add the agar and heat to dissolve
  ↓
• Dispense 4.0 mL into 13 × 100 mm screw-cap tubes
  ↓
• Autoclave at 121 °C for 15 min
  ↓
• Cool with the tubes upright

2,3,5, Triphenyltetrazolium Chloride (TTC) Solution, 1%

Basal medium

• 2,3,5, Triphenyltetrazolium Chloride (TTC) — 1.0 g
• Distilled water — 100.0 mL

Complete medium

• Suspend the TTC in 100 mL of distilled water
  ↓
• Sterilize by filtration
  ↓
• Aseptically dispense into containers
  ↓
• Store refrigerated
• For TSA-TCC, aseptically add 10 mL to 1 L of sterilized and cooled TSA and mix thoroughly; dispense into petri plates
• For 0.1% TTC staining solution, add 10 mL of 1% TTC to 90 mL of distilled water and mix thoroughly

Triple Sugar Iron Agar (TSI)

Basal medium

• Beef extract — 3.0 g
• Yeast extract — 3.0 g
• Peptone — 15.0 g
• Proteose peptone — 5.0 g
• Lactose — 10.0 g
• Sucrose — 10.0 g
• Dextrose — 1.0 g
• Ferrous sulfate (FeSO₄·7H₂O) — 0.2 g
• Sodium chloride (NaCl) — 5.0 g
• Sodium thiosulfate (Na₂S₂O₃) — 0.3 g
• Agar — 12.0 g
• Phenol red — 0.024 g
• Distilled water — 1.0 L
• pH 7.4 ± 0.2

Complete medium

• Suspend the basal ingredients in 1.0 L of distilled water
  ↓
• Heat to boiling to dissolve completely
  ↓
• Dispense 8 mL into tubes
  ↓
• Autoclave at 121 °C for 15 min
  ↓
• Cool the tubes in a slanted position to form deep butts

Triple Sugar Iron NaCl Agar

Basal medium

• TSI agar dry basal ingredients — 65.0 g
• Sodium chloride (NaCl) — 25.0 g
• Distilled water — 1.0 L
• pH 7.4 ± 0.2

Complete medium

• Suspend the basal ingredients in 1.0 L of distilled water
  ↓
• Heat to boiling to dissolve completely
  ↓
• Dispense 8 mL into tubes
  ↓
• Autoclave at 121 °C for 15 min
  ↓
• Cool the tubes in a slanted position to form deep butts

Tris Buffer, 0.05 M, pH 9.0

Basal medium

• Tris (Hydroxymethyl) Aminomethane (F.W. 121.14) — 6.06 g
• Distilled water — 1.0 L
• pH 9.0

Complete medium

• Dissolve the Tris (Hydroxymethyl) Aminomethane in 1.0 L of distilled water
  ↓
• Adjust the pH to 9.0 with 0.2 N HCl, using an electrode

Tris-buffered Saline (TBS) (pH 7.5)

Basal medium

• Tris (Hydroxymethyl) Aminomethane — 2.42 g
• Sodium chloride (NaCl) — 29.24 g
• Distilled water — 1.0 L
• pH 7.5

Complete medium

• Dissolve the Tris (Hydroxymethyl) Aminomethane in 1.0 L of distilled water
  ↓
• Adjust the pH to 9.0 with 0.2 N HCl, using an electrode

Tris-buffered Saline (TBS) with 1% Gelatin

Basal medium

• Gelatin — 1.0 g
• Tris-Buffered Saline, pH 7.5 (see above) — 100 mL

Complete medium

• Heat the TBS to 40 °C
  ↓
• Add the gelatin and stir to dissolve
  ↓
• Cool to room temperature before use

Tris-buffered Saline (TSB) with 3% Gelatin

Basal medium

• Gelatin — 3.0 g
• Tris-Buffered Saline, pH 7.5 (see above) — 100 mL

Complete medium

• Heat the TBS to 40 °C
  ↓
• Add the gelatin and stir to dissolve
  ↓
• Cool to room temperature before use

Tris-buffered Saline (TBS) – Tween

Basal medium

• Tween 20 — 50 µL
• Tris-Buffered Saline, pH 7.5 (see above) — 100 mL

Complete medium

• Suspend the Tween 20 in 100 mL of TBS
  ↓
• Stir to dissolve

Trypsin-EDTA Solution, 1X

Basal medium

• Trypsin (1:250) — 0.05 g
• EDTA, disodium salt — 0.02 g
• NaCl, 0.9% — 100 mL

Complete medium

• Dissolve the trypsin and EDTA in the NaCl diluent
  ↓
• Filter sterilize
• Note: 1 g of trypsin (1:250) digests 250 g of casein substrate under standard conditions

Trypsin Solutions

Basal medium

• 1% trypsin solution:
• Trypsin — 200 mg
• Distilled water — 20.0 mL
• 1.4% trypsin solution:
• Trypsin — 280 mg
• Gelatin phosphate — 20.0 mL

Complete medium

• 1% solution: dissolve 200 mg of trypsin in 20.0 mL of distilled water; do not store
  ↓
• 1.4% solution: suspend 280 mg of trypsin in 20.0 mL of gelatin phosphate; filter through a 0.45 µm membrane; use vacuum for filtration to effect partial deaeration of the filtrate
• Note: in the following media formulations, the words tryptone, tryptic or trypticase peptone refer to a pancreatic digest of casein, and the words soya peptone, soy or phyto peptone refer to a papaic digest of soybean meal

Trypticase Peptone Glucose Yeast Extract (TPGY) Broth

Basal medium

• Trypticase — 50.0 g
• Peptone — 5.0 g
• Glucose — 4.0 g
• Yeast extract — 20.0 g
• Sodium thioglycolate (C₂H₃O₂SNa) — 1.0 g
• Distilled water — 1.0 L
• pH 7.2 ± 0.2

Complete medium

• Suspend the basal medium ingredients in 1.0 L of hot distilled water
  ↓
• Cool and adjust the pH
  ↓
• Dispense in 21.0 mL volumes into 20 × 150 mm screw-cap tubes
  ↓
• Autoclave at 121 °C for 10 min
• If stored in excess of 12 h, the medium must be deaerated (at 100 °C for 10 min) and cooled before use
• Note: Trypticase is manufactured by Oxoid as Special Peptone L72

Tryptic Soy Agar (TSA) (Soybean Casein Digest Agar) (ATCC Medium 77)

Basal medium

• Tryptone — 15.0 g
• Soya peptone — 5.0 g
• Sodium chloride (NaCl) — 5.0 g
• Agar — 15.0 g
• Distilled water — 1.0 L
• pH 7.3 ± 0.2

Complete medium

• Suspend the basal medium ingredients in 1.0 L of distilled water
  ↓
• Heat to boiling with constant stirring to dissolve all ingredients; do not overheat
  ↓
• Autoclave at 121 °C for 15 min

Trypticase Soy Broth (TSB) (Soybean Casein Digest Broth)

Basal medium

• Tryptone — 17.0 g
• Soya peptone — 3.0 g
• Dextrose — 2.5 g
• Sodium chloride (NaCl) — 5.0 g
• Dipotassium phosphate (K₂HPO₄) — 2.5 g
• Distilled water — 1.0 L
• pH 7.3 ± 0.2

Complete medium

• Suspend the basal medium ingredients in 1.0 L of distilled water
  ↓
• Heat to boiling with constant stirring to dissolve all ingredients
  ↓
• Dispense
  ↓
• Autoclave at 121 °C for 15 min

Tryptic Soy Broth (TSB) with Glycerol (extra NaCl and without dextrose)

Basal medium

• Tryptone — 17.0 g
• Soya peptone — 3.0 g
• Sodium chloride (NaCl) — 15.0 g
• Dipotassium phosphate (K₂HPO₄) — 2.5 g
• Glycerol — 240 mL
• Distilled water — 760 mL
• pH 7.3 ± 0.2

Complete medium

• Suspend the basal medium ingredients in 1.0 L of distilled water
  ↓
• Heat to boiling with constant stirring to dissolve all ingredients
  ↓
• Dispense
  ↓
• Autoclave at 121 °C for 15 min

Tryptic Soy-Fast Green Agar (TSFA)

Basal medium

• Tryptone — 17.0 g
• Soya peptone — 3.0 g
• Sodium chloride (NaCl) — 5.0 g
• Fast green FCF — 0.25 g
• Agar — 15.0 g
• Dipotassium phosphate (K₂HPO₄) — 2.5 g
• Distilled water — 1.0 L
• pH 7.3 ± 0.2

Complete medium

• Suspend the basal medium ingredients in 1.0 L of distilled water
  ↓
• Heat to boiling with constant stirring to dissolve all ingredients
  ↓
• Autoclave at 121 °C for 15 min

Tryptic Soy-Magnesium Sulfate Agar (TSAM)

Basal medium

• Tryptone — 15.0 g
• Soya peptone — 5.0 g
• Sodium chloride (NaCl) — 5.0 g
• Magnesium sulfate heptahydrate (MgSO₄·7H₂O) — 1.5 g
• Agar — 15.0 g
• Distilled water — 1.0 L
• pH 7.3 ± 0.2

Complete medium

• Suspend the basal medium ingredients in 1.0 L of distilled water
  ↓
• Heat to boiling with constant stirring to dissolve all ingredients
  ↓
• Autoclave at 121 °C for 15 min
• Note: tryptic soy agar is commercially available, to which MgSO₄·7H₂O (1.5 g/L) can be added

Trypticase Soy Agar with 0.01% TTC

Basal medium

• Trypticase soy agar (see above) — 40.0 g
• 1% TTC solution (2,3,5-Triphenyltetrazolium Chloride) — 10 mL
• Distilled water — 990 mL
• pH 7.3 ± 0.2

Complete medium

• Suspend the basal medium ingredients in 990 mL of distilled water
  ↓
• Heat to boiling with constant stirring to dissolve all ingredients
  ↓
• Autoclave at 121 °C for 15 min
  ↓
• Aseptically add the 1% TTC solution and mix thoroughly
  ↓
• Pour into plates

Trypticase Soy Agar with 0.6% Yeast Extract (TSA-YE)

Basal medium

• Trypticase soy agar (see above) — 40.0 g
• Yeast extract — 6.0 g
• Distilled water — 1.0 L
• pH 7.3 ± 0.2

Complete medium

• Suspend the basal medium ingredients in 1.0 L of distilled water
  ↓
• Heat to boiling with constant stirring to dissolve all ingredients
  ↓
• Dispense into tubes or flasks
  ↓
• Autoclave at 121 °C for 15 min
  ↓
• Leave in tubes or pour into sterile Petri dishes

Trypticase Soy Broth with 0.6% Yeast Extract (TSB-YE)

Basal medium

• Trypticase soy broth (see above) — 30.0 g
• Yeast extract — 6.0 g
• Distilled water — 1.0 L
• pH 7.3 ± 0.2

Complete medium

• Suspend the basal medium ingredients in 1.0 L of distilled water
  ↓
• Heat to boiling with constant stirring to dissolve all ingredients
  ↓
• Dispense
  ↓
• Autoclave at 121 °C for 15 min

Tryptone Broth (1%)

Basal medium

• Tryptone — 10.0 g
• Distilled water — 1.0 L
• pH to 7.0 ± 0.2

Complete medium

• Dissolve the tryptone in 1.0 L of distilled water
  ↓
• Dispense in 5.0 mL portions
  ↓
• Autoclave at 121 °C for 15 min

Tryptone Bile Agar (TBA)

Basal medium

• Tryptone — 20.0 g
• Bile salts no. 3 — 1.5 g
• Agar — 15 g
• Distilled water — 1.0 L
• pH to 7.2 ± 0.2

Complete medium

• Suspend the basal medium ingredients in 1.0 L of distilled water
  ↓
• Heat to boiling with constant stirring to dissolve all ingredients
  ↓
• Autoclave at 121 °C for 15 min
  ↓
• Pour 15–20 mL per plate on a flat surface

Tryptone Broth and Tryptone Salt Broths (T1N0, T1N1, T1N3, T1N6, T1N8, T1N10)

Basal medium

• Tryptone — 10.0 g
• Sodium chloride (NaCl) — 0.0, 10.0, 30.0, 60.0, 80.0 or 100.0 g
• Distilled water — 1.0 L
• pH 7.2 ± 0.2

Complete medium

• Suspend the basal medium ingredients in 1.0 L of distilled water
  ↓
• For T1N0 add no NaCl; for T1N1 add 10 g NaCl (1% w/v NaCl); for T1N3 add 30 g NaCl (3% w/v NaCl), and so on
  ↓
• Dispense into 16 × 125 mm screw-cap tubes; tighten the tubes to maintain the correct concentration in the tube
  ↓
• Autoclave at 121 °C for 15 min

Tryptone Salt (T1N1) Agar (also T1N0, T1N2, T1N3)

Basal medium

• Tryptone — 10.0 g
• Sodium chloride (NaCl) — 10.0 g
• Agar — 20.0 g
• Distilled water — 1.0 L
• pH 7.1 ± 0.2

Complete medium

• Suspend the basal medium ingredients in 1.0 L of distilled water
  ↓
• Heat to boiling with constant stirring to dissolve all ingredients
  ↓
• Autoclave at 121 °C for 15 min, cool and dispense
• Note: for T1N0, T1N2 and T1N3, use 10.0 g of tryptone and 0.0 g, 20.0 g and 30.0 g of NaCl respectively

Tryptose Broth and Agar for Confirmation Tests and Serology

Basal medium

• Tryptose — 20.0 g
• Sodium chloride (NaCl) — 5.0 g
• Dextrose — 1.0 g
• Agar (leave out of the broth formula) — 15.0 g
• Distilled water — 1.0 L
• pH 7.2 ± 0.2

Complete medium

• Suspend the basal medium ingredients in 1.0 L of distilled water
  ↓
• Heat to boiling with constant stirring to dissolve all ingredients
  ↓
• Autoclave at 121 °C for 15 min, cool and dispense
• For the agar, make generous slants

References

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