Posts Tagged ‘agar’

A quick and dirty microbiology attempt

July 3, 2012

I recently felt compelled to do some microbiology work.  My goal was to make a crude growth media for microorganisms, swab my mouth for these buggers, and spell my name across a few petri dishes.

edit:  Forgive the bright spot from my lamp, it was the only way to get the letters to show.

J

Above you can see the letter “J”.  If you look to the right and bottom of the J you can see clumpy looking gunk – that is egg chunks.

I started out by boiling 300 g of sliced potatoes and 1 raw egg  in ~500 mL distilled water for 30 minutes.  Next I filtered the solution through numerous coffee filters and then filtered the solution a second time through a 0.45 micron membrane.  10 g of sucrose and 10 g of agar-agar (hardening agent) were added and stirred for ~10 minutes.  Next I sterilized the media and some glass petri dishes (~$1.50 per dish) in a pressure cooker for 25 minutes at 15 psi.  After sterilization I poured the media into the dishes and allowed them to cool and harden.

My open dishes

My open dishes

After I finished sterilizing the media I noticed the solution had clumpy-chunky junk floating all about.  I assume this was leftover egg that got through the filters, though it could also have been impurities from the agar-agar (least likely of the two).

I used a cotton swab to collect bacteria from my teeth and gums and I spelled my name (a letter per dish) across the plates using the swab.  On one plate I put nothing and on the last plate I spit into it to cover the whole thing.

e

e

I was happy to see that everything worked perfectly.  The dish with no bacterial additions had no growth.  The dish I spit into had growth all over and the dishes I wrote letters on only grew in the shape of the letter itself!0

s

s

Unfortunately condensation leaked all over the plates when I flipped them and blocked some letters from being photographed.

For future experiments I bought a “fitness multivitamin” that contains all of the amino acids and I am going to try and use it as an egg/tryptone/peptone replacement.

Finally got around to testing some gel electrophoresis.

January 16, 2012

It took much longer for me to get a round to testing out my gel electrophoresis equipment than I thought it would.  For now I have merely got it to work.  Next I will try and fine tune it to increase the quality of the gels.  More on that below.

This isn’t the most informative post but I was kind of frustrated by a lack of information when I was troubleshooting so I figured I would throw some data out and hope it helps someone.

Note:  The cameras on my phone and iPad both captured more wavelengths of light than I could see, so these images look worse than the gel actually is.

Zoomed out gel.

Zoomed out gel.

Gel closeup

Gel closeup

Gel Parameters:

  • GEL = 1.5% food-grade agar-agar gel (not agarose)
  • DNA LADDER= New England Biolabs 2-log DNA ladder
  • STAIN = GelRed Stain (Vendor; Biotium) (Approx. Equiv. to Ethidium Bromide, except safe).  Stain was used in the precast gel (1x) context.
  • TRANSILLUMINATOR = Fotophoresis I (Fotodyne)
  • BUFFER = TAE (MB grade reagents)
Failed gel.

Failed gel.

The image above is what the first two gels looked like – no fluorescence at all.  I still do not know for sure why they failed but I narrowed it down to either the composition of the DNA ladder or the staining method.

My set-up worked when I used GelRed in the molten agar-agar and composed the DNA ladder per the manufacturers instructions.  I used 5-10uL of ladder and 1-2uL of loading buffer on the failed gels whereas I used 1uL of ladder, 4uL H2O, and 1uL loading buffer on the successful gel.  During the failed gels I tried to use the 3x post-electrophoresis stain procedure with staining times between 0.5-1.5 hours – all with no luck.  Obviously changing two variables at once confounds the results – but at least I have a baseline now.

The setup

The setup

This was the gel box I built and used.  You can read my instructions for how to build it here.

Muh lab.

Muh lab.

This is ~90% of my apartment lab.

My next goal is to work out how to fine tune the procedure.  I am going to compare the gel quality in cases where I use reagent grade vs. industrial and food grade chemicals.  I am hoping that borax (sodium tetraborate) and roach poison (boric acid) buffer will work as good as its reagent grade cousin.