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I'm going to repeat an experiment I did in September, testing growth with different levels of arsenate and phosphate in plastic and glass screw-cap tubes. That time I only followed growth by changes in turbidity of the cultures, but this time I'll also follow the changes in the numbers of viable cells by plating samples on agar medium. I'll start the cultures tomorrow morning, plating the cells at t=0, so I know how many viable cells I started with, and again at t=1 hr, to see if cells are immediately dying in the arsenate. Then I'll plate at about 8 hr (after a family dinner, it being Sunday), and again the next morning.
The first step is to clean up all the old cultures on my bench this evening, so I'm ready to go in the morning...
You've probably covered this already, but do you make up the arsenic-containing medium fresh each time, or is there a stock? And if the latter, is there any correlation between age of the stock and bacterial growth?
ReplyDeleteIs there a straightforward way to confirm the arsenic concentration in your bacterial media at the time the bacteria are added and/or after the growth period?
Dr Redfield, Have you considered pH as your unidentified variable? AML60 would appear to be relying on carbonate/bicarbonate for its buffer capacity at pH 9.8, but that system requires a constant CO2 incubator to perform properly (cf. growth of eukaryotic cells). The different phosphate and arsenate concentrations will also affect the buffer capacity of AML60, so the pHs of your different cultures may vary significantly during growth.
ReplyDeletePS. Thank you for your efforts to sort this out!
This cartoon is a helpful reminder on why people draw comic characters with 4 (sometimes 3) fingers.
ReplyDelete;)
Dr. Redfield, I've been following your frustrations with results for GFAJ-1 grown in different containers. I would urge some caution with using new sterile polypropylene containers when repeating the Sept work. Have you ever placed your nose inside a new sterile PP tube fresh out of the bag? There is an awful smell there resulting (I believe) from degradation products from stabilizers the manufacturers use so that the tubes survive the sterilization process. In fact, many different stabilizers (including phosphites) are used in plastics. I use sterile polystyrene tubes for maintaining algae stock cultures as I could detect no volatile odor from them using my "nose" test and this type of tube is used by the CCMP collection....Larger volume vented tissue culture flasks (for suspensions; no binders) are available from Greiner One or Corning so that you could harvest more biomass for DNA. However, I wonder if polystyrene might sequester arsenate? Glass is probably the better choice, but not without considering potential issues of boron/silicon leeching from the glass?.. and we have also found there to be differences (at least in fatty acid content as measured by GC) between different manufacturer's phenolic caps used for glass tubes....I don't envy you your frustrations...it "should" be simpler (someone's famous last words..I'm sure)
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