Chopped Meat Broth with Carbohydrates, GMExpression Modified | For 5L

Chopped Meat Broth with Carbohydrates, Modified by GMExpression, for 5L

An alternative to ATCC Medium 1490 and DSMZ Medium 110.

This GMExpression formulation simplifies the preparation by eliminating the need for beef particles and filtrates. The medium provides an anaerobic culturing environment comparable to standard Chopped Meat Broth with Carbohydrates formulations. It is enriched in disulfide bonds, exhibits sustained reducing capacity, and effectively maintains anaerobic conditions—functionally replicating the role of beef granules in supporting microbial growth.

Other Names

• Chopped Meat Carbohydrate Broth (CMC)
• Chopped Meat Broth with Carbohydrates

Applications

Chopped Meat Broth with Carbohydrates, Modified by GMExpression, 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]   After autoclave, adding 10 µL of Stock C to 1mL of the broth can achieve an increased concentration of 0.5 g/L L-Cys in 0.005 M phosphate buffer, pH 7.2.

[3]   Adding cysteine stock enhances the maintenance of the broth's anaerobic conditions, effectively replicating the role of beef granules in prolonging broth anaerobiosis. It also replenishes reducing equivalents lost during autoclaving, thereby improving the redox stability of the medium.

Preparation

1. Dissolve 57.5 g chopped meat broth powder in  980 mL of Milli-Q water.
2. Mix until fully dissolved and adjust pH to 7.0 ± 0.2.
3. [Optional] Remove dissolved oxygen by anaerobic gas flashing or vacuum deoxygen, and then water bath at 60~70 °C for 10~15 min.
4. Autoclave at 121 °C for 15 min.
5. Cool to room temperature. Under anaerobic atmosphere:
   1. aseptically add Vitamin K1 stock (0.5 mg/mL in 95% EtOH): 2µL or 10µL (10µL for high Vitamin K₁ metabolic requirements) per mL broth.
   2. [Optional] aseptically add Cysteine phosphate stock (Stock C) 5~20 µL per mL (recommend 5 µL/mL for general use).

Storage

• Mixture A: Store at RT. Store with Oxygen absorbers once opened. Shelf life: 18 months.
• Stock B: Store at 2~8 °C. Protected from light. Shelf life: 6 months. After opening: 4 weeks.
• Stock C: Store at 2~8 °C. Shelf life: 1 year.

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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.