DNA analysis attempt, again

First, no new developments seen in static flasks
Second, the bio grad student helping I'm working with for the PCR informed me that the reason we've been having so many problems is that the GoTaq was expired.  :-/  So, a PCR is being run right now, with new GoTaq.  Hopefully there are results this time around.  Since we have Gram-negative bacteria, they do not have a thick layer to get through, so it should work.  It'll be a great relief to see some bands this time in the gel.  The next step will be to sequence what we have, then compare that to a data base of known bacteria.  Here's hoping...

Going Green

Thus far, only the static flasks that have shown the obvious change of a precipitate have been kept and transferred for iron-reducing bacteria.  Today, I happen to take a closer look at a static flask of which the contents were going to be tossed out.  The media had a slight green tint.  After comparing to a control flask, there was certainly a color difference.  !!!  Note that the media used for this experiment is a very dark red/brown color and difficult to detect slight color changes, even in 250 mL flasks.  A color change from red/brown to green is expected of the iron-reducing bacteria.  Even though the flask with the green tinted media is fairly old, an attempt to transfer any live bacteria will be made.  A larger flask will be kept, as well as capped test tubes in hopes of more readily seeing a color change occur.  Here's to hoping some bacteria are still alive...

Third time's the charm? Not so much.

A DNA analysis of the bacteria our group is attempting to use is important for several reasons: for comparing other iron reducers of the same genus; knowing what growing conditions are best, what products are likely to be produced; possibility of a new species; and (most importantly for me) to carry out further experiments on the plausibility of iron leaching. 

Today was the third attempt at a PCR reaction and a third fail.  All three trials were slightly different, in an attempt to adjust for the particular bacteria.  Our next step will therefore be to isolate the DNA separately, then run a PCR.  Hopefully that will assure DNA and we will be able to sequence after.

Well,  I am off to try to call the vet before they close for the day: a friend's puppy tore her nail  and she need to get it looked at.  It's a very nasty tare, too.  The tare does not go down (vertically) through the nail, but horizontally back into the nail.  *sigh* Poor girl.

Bacteria pic

Lovely, no?  A 1000x clear view of the rod shaped, Gram negative bacteria  that I am lucky enough to be dealing with.

Well...

Well, it's bee awhile.  "Important" picks up and "other" goes down.  Ukulele not going so well.  It's enjoyable enough, but will probably never sound as good as Grant does.  As for the rats: Snowball has did of a respiratory infection.  Went to the vet, got antibiotics, but he never got better.  The other two are doing fine.  Active as always.

Research is going well enough.  Most likely the isolates are not Geobacter, since the colonies lack defining characteristics such as pink coloring turning to brown or green.  Current colonies are smooth, rough, and white to clear when grown on ATCC Geobacter media.  While doing a literature review, a paper published in 2007 claimed a new genus and species of fermentative, iron-reducing bacteria.  Citrate is fermented, using iron (III) as an electron sink, to produce acetate (if there is any interest in the paper, let me know and I'll give you the citation).  Two new experiments are currently set up and running to test the bacteria in our lab for citrate fermentation.  Also, if the PCR reaction ever goes right, DNA results will be immensely helpful in understanding what type of bacteria  we are dealing with.