Songül Gönel I can guide a seven step process where you can try at your laboratory and it is quite simple and here is the process:

Inoculating lyophilized anaerobic bacteria without an anaerobic chamber requires careful handling to maintain the anaerobic environment. Here's a step-by-step approach:

1. Prepare the culture medium: Before inoculation, ensure the culture medium is reduced, meaning it has been treated to remove oxygen. This can be done by:

- Boiling and then cooling the medium under a nitrogen or argon gas stream

- Adding reducing agents like cysteine, thioglycolate, or sodium sulfide

2. Rehydrate the lyophilized bacteria: Reconstitute the lyophilized bacteria in a small amount (e.g., 1-2 mL) of reduced medium or anaerobic diluent, like thioglycolate broth.

3. Use a nitrogen or argon gas stream: During inoculation, maintain a gentle stream of nitrogen or argon gas over the work area to minimize oxygen exposure.

4. Inoculate quickly and carefully: Open the lyophilized bacteria vial and quickly add the rehydration medium. Immediately close the vial and mix gently.

5. Transfer to the culture medium: Using a sterile syringe or pipette, transfer the rehydrated bacteria to the reduced culture medium. Minimize exposure to air during transfer.

6. Seal the culture vessel: Immediately seal the culture vessel, ensuring it is airtight to prevent oxygen ingress.

7. Incubate: Incubate the culture in a temperature-controlled environment, typically between 30°C to 37°C, depending on the bacteria's requirements.

Srinivas Kasulla

Thank you for your suggestion

How can I inoculate without nitrogen or argon gas stream?

Songül Gönel Inoculating anaerobic bacteria without a nitrogen or argon gas stream requires careful handling to maintain an oxygen-free environment. Here are some alternative methods:

1. Use a glove box: Inoculate in a glove box filled with oxygen-free gas, like nitrogen or argon.

2. Anaerobic chamber: Use a portable anaerobic chamber or a vinyl anaerobic bag.

3. Oxygen-free water: Use oxygen-free water or medium to rehydrate the bacteria and prepare the culture medium.

4. Aspirate and inject: Use a syringe to aspirate the inoculum and inject it into the culture medium, minimizing exposure to air.

5. Butyl rubber-stoppered tubes: Use tubes sealed with butyl rubber stoppers, which are impermeable to oxygen.

6. Hungate tubes: Use Hungate tubes, which have a specialized stopper and technique to maintain anaerobic conditions.

7. Anaerobic broth: Use anaerobic broth, which contains reducing agents to maintain an oxygen-free environment.