Modified Chopped Meat Broth Medium (ATCC Medium 1490, ATCC Medium 2124) | For 5L

Modified Chopped Meat Broth Medium (ATCC Medium 1490), for 5L

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Applications

Modified Chopped Meat Broth Medium (ATCC Medium 1490) is suitable for:

• Pre-reduced, anaerobically sterilised (PRAS) media applications
• Cultivation, maintenance, and metabolic profiling of spore-forming and non-spore-forming obligate anaerobes
• Use as a holding medium for anaerobic cultures
• Promoting sporulation and toxin production in Clostridium species
• Gas-liquid chromatography (GLC) analysis of bacterial metabolic end products

Components

[1]   Optional alternatives:

• Vitamin K₃ Stock
• Viscous liquid state of 98% Vitamin K₁ that has a longer shelf life at RT is also available, reagent pre-aliquoted into centrifuge tubes, each containing a sufficient amount to prepare a 10 mL stock solution at a final concentration of 0.5 mg/mL. See below the "Preparation of Vitamin K₁ Ethanol Stock Solution".

[2]   0.22µm filter sterilised. Adding Clindamycin Hydrochloride Stock will achieve ATCC medium 2124, Modified chopped meat medium with clindamycin. Select the corresponding option to purchase.

[3]   Adding 10g/L of Iron Filings, Dextrose 5.0g/L, and Sodium Chloride 5.0g/L will achieve Pre-reduced Chopped Meat medium, used specifically for culturing Clostridium species like Clostridium perfringens (ATCC® 13124) and Bacteroides fragilis (ATCC® 25285); Streptococcus pyogenes (ATCC® 19615). Select the corresponding option to purchase.

[4]   Replacing Defatted Ground Beef with Defatted Beef Extract will achieve ATCC medium 2210, Enriched Anaerobe medium.

Preparation

1. Add 80 g defatted ground beef granules and 40 g of Mixture A powder in 980 mL of Milli-Q water and mix well. [Optional: Supplement with the desired type and amount of sugar as required.]
2. Boil the broth in a water bath or microwave for 15 minutes, then cool the mixture under an anaerobic gas atmosphere (e.g.,  N₂ -80% / H₂ -10% / CO₂ -10%, or N₂ -100%).
3. Add 2.5 g of Mixture B powder and mix well. (If condition does not allow for step 7, add 2~10ml of stock C to the both at this step.  Please be aware that some Vitamin K₁ may degrade during the autoclaving process.)
4. Adjust the pH to 7.0 ± 0.2.
5. Dispense 7 mL into Hungate tubes or 1/5–1/4 volume of anaerobic culture bottles containing meat particles (1 part meat to 4–5 parts fluid) under the same anaerobic gas phase.
6. Autoclave at 121 °C for 20 minutes.
7. After cool down, under a sterile environment, add 14 µL of Stock C to each Hungate tube using microsyringe. This results in a final concentration of 1 mg Vitamin K₁ per Litre of broth.

For a solid medium, add 15.0g Agar. For a semi-solid medium, add 0.5g Agar. Dissolve together with Mixture A, to make 1000 mL of medium.

Storage

• Unopened Granules: Store at –20 °C in a dry, light-protected environment. For extended shelf life, –65 °C is recommended. Shelf life: 2~3 years.
• Opened Granules: Immerse in 25 mM NaOH solution and store at –20 °C. Shelf life: 3 months.
• Stock C: Store at 2~8 °C, protected from light. Shelf life: 6 months. After opening: 4 weeks.

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Preparation of Vitamin K₁ Ethanol Stock Solution (Aseptic)

Prepare the following materials in advance:

­ 95% ethanol, 10 mL

­ One 10‑mL centrifuge tube

­ One 2‑mL microcentrifuge tube pre‑loaded with vitamin K₁

­ One 20‑mL syringe

­ One 0.22‑µm syringe filter (single‑use)

Initial Dissolution

Add 0.5 mL of 95% ethanol into the 2‑mL tube containing vitamin K₁. Cap tightly and vortex or shake thoroughly until the vitamin K₁ is fully dissolved. Carefully decant the ethanol solution into the 10‑mL centrifuge tube.

Sequential Rinsing

Add another 0.5 mL of ethanol to the same 2‑mL tube, shake again to dissolve any remaining residue, and transfer the solution to the 10‑mL tube.

Repeat this rinse four to five times, or until no visible yellow vitamin K₁ remains on the walls of the small tube.

Make Up to Volume

After the final rinse, top up the 10‑mL centrifuge tube with 95% ethanol to reach a total volume of 10 mL.

Sterile Filtration (Perform inside a clean cabinet)

Remove the plunger from the 20‑mL syringe and attach the 0.22‑µm sterile syringe filter to the syringe outlet.

Carefully pour the 10 mL vitamin K₁–ethanol solution into the syringe barrel.

Reinsert the plunger and apply gentle pressure to filter the solution into a pre‑sterilized collection vial.

Storage

Vitamin K₁ is light‑sensitive. After filtration, wrap the storage vial in aluminium foil and keep it refrigerated to maintain stability.