I will be doing a number of assays with BCG vaccine strain, which comes freeze dried. The assays include phagocytosis assay and testing if various drugs have bacterial effects on the BCG. Can I just reconstitute the BCG in normal saline and use it for the assays, or do I have to culture it for a while first?
Hello Steven,
You should prepare a stock from your freeze-dried lot. Grow the bacteria 21 days and store at -70ºC in storage medium (7H9 supplemented with ADC and 6% glycerol). Determine the CFU numbers so that you know the bacteria concentration of each vial. You need to have the bacteria in a metabolically active state for your experiments and you may need to know the inoculum size. In addition, you won't be able to do all your experiments at once. The working lot is going to provide you fresh bacteria for each experiment and the possibility to generate a new lot. Make sure not to use bacteria after many passages.
Hope it helps.
Esmeralda.
I prefer a highly viable batch to denser suspensions. The more you grow in broth you have less viable bacteria: I recommend growing lyo-stock in 7H9 broth with shaking for not more than 7-10 days; when it reaches faint turbidity they have the best viability (>90%). Spin down and freeze in aliquots in 10% medium (not saline) : use aliquots as one-time batch. Note that you can grow to denser turbidity but I do not prefer that since they have more dead bacteria; clumps of CFUs exclude nutrients into some bacilli within. This consistently gives you the best uptake into macrophages or for that matter plating. Please note all BCG suspensions should be gently sonicated to infect. You want a better infection mode, this suspension should once again be spun down gently to infect: standardize --otherwise you will get multiple bacilli into on macrophage. hope this helps.
Thanks for your input, Chinnaswamy. After i culture the BCG and freeze the aliquots, I assume I will need them to be metabolicaly active again before I can use them in my assays. For how long do i need to keep the aliquotes at 37 degrees before I can use them in my assay?
such aliquots can adapted to medium for just 3 days: this is what I use sine they show the highest viability for macrophage infections. Also remember most batches of lyo have variable and large portions of dead BCG: this has been confirmed by WHO during vaccine trials. Thus when you culture lyo only those which are viable grow faster and you need to harvest the 10 days -12 max. Rapid freezing in N2 liquid and when thawing in waterbath at 37 C for immediate use.
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