I am working on a probiotic that have anaerobic bacteria spp. This probiotic is available in capsules commercially from Seeking Health LLC USA.
I need to dissolve the capsule powder in anaerobic PBS. In resulting Bacteria suspension I want to check the viability of the suspended bacteria.
I did a literature survey and found that few authors are telling to handle it dry anaerobic conditions. Mix the powder in anaerobic PBS and homogenize it. After this I can check cell viability via flow cytometry.
Do the homogenization maintain the cell integrity and viability?
If yes, what are the homogenization conditions to maintain the cellular integrity and cell viability.
Can I use Trypan Blue dye exclusion method for the viability check?
Or only Fluorescent dyes (Flow Cytometry) are the option ?
Expert advises are needed, please help me in this.
Thanks
Regards
Anuj
hi..
It is generally recommended to handle anaerobic bacteria under anaerobic conditions to maintain their viability. Handling the bacteria in the presence of oxygen can lead to oxidative stress and damage to the cells.
To dissolve the probiotic capsule powder in anaerobic PBS, you should first ensure that the PBS is anaerobic. This can be achieved by preparing the PBS under anaerobic conditions or by adding anaerobic gas to the PBS before use.
Once the capsule powder has been dissolved in the anaerobic PBS, you can use a homogenizer to homogenize the suspension. Homogenization can help to evenly distribute the bacteria in the suspension and can also help to release any bacteria that may be attached to the capsule material. However, it is important to use a gentle homogenization method to avoid damaging the cells.
To maintain the cellular integrity and viability of the bacteria during homogenization, it is important to use a low-speed homogenizer and to minimize the number of passes through the homogenizer. You should also avoid using high pressures or extreme temperatures during the homogenization process.
After homogenization, you can use a variety of methods to assess the viability of the suspended bacteria. The Trypan Blue dye exclusion method is a commonly used method for determining the viability of bacterial cells. In this method, a small aliquot of the bacterial suspension is mixed with a solution of Trypan Blue dye, which is selectively taken up by dead cells. The number of live and dead cells can then be counted under a microscope to determine the viability of the bacteria.
Alternatively, you can use fluorescent dyes and flow cytometry to assess the viability
Regards.
Thanks Alzeyadi!
I will implement your suggestions and let you know it is working or not
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