I prepare a Mcfarland 1 solution of H37Ra in 1 x PBS containing 0.05 % v/v Tween 80. I add between 1 - 5 ml of the solution to 50 ml 7H9 enriched with OADC and 0.05 % v/v Tween 80. Cells are grown at 37 degrees Celsius at 125 RPMs. Growth is slow as usual and then picks up but then my OD at 600nm plateaus at 0.3 and never rises? I would like to grow the culture to 0.6 and then store at - 80 in glycerol. What could be going wrong?
I would like to have stock cultures stored and thaw them when I need to use them, purely for plating onto 7H10 agar and working form that media.
Is it ok if I grow the cells to McFarland 1 in this manner then store? Or will this inoculmn size be too low for my purposes?
Usually, I start a new mycobacterial culture with an inoculum of 0.4 to 0.5 (OD, which is equivalent to McFarland Standard 2). The volume of the inoculum varies between 5% and 10% of the final volume of the new culture.
The "secret" of a good culture, is to have a turbid suspension, after inoculation.
However, this is not enough. You might have a high optical density but many bacteria could be dead. That's why we should always use a inoculum from a mid-logarithmic growth phase culture.
When you start a new culture, if you use an inoculum from a previous culture in exponential growth phase, the new culture will continue in exponential growth phase. Hopefully, you will reach a higher plateau. Good luck!
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