Inside Out?

Naturally, when you look at an insect you think:

“I wonder if I can sterilize the outside of that thing, crush it up in sterile media and plate the resulting goo to see if any microbes from the inside will grow up”

No?  Well congratulations on your full life.  I have more free time** on my hands, so I give you:

The Flaming Bug* Control

  1. Use forceps to drag recently expired adult beetle along surface of nutrient agar plate
  2. Dip adult beetle into 70% ethanol
  3. Remove beetle with forceps and light on fire with bunsen burner
  4. Drag flambéed beetle along different surface of nutrient agar plate
  5. Incubate at 31 C.

Picture1

Notice the colonies growing on the right side of the plate.  Those could be anything… bacteria from outer space, fungi from the cous cous etc  Not interested in those.  The fungus growing on the bug after its swim in the sauce though, is kind of cool.  Especially when you let it grow:

Picture2

Ashes to ashes and dust to must

I can’t be sure if that fungus came from within or without. The goal was to test if I could sterilize the outside of the insect and that didn’t happen, but at the very least, treatment with ethanol changed what grew up.  Already thinking of how to optimize this, although perhaps its a lost cause – exoskeletons do not seal off the viscera of an insect.  Need to think about that.

The Crushed Flambéed Bug Experiment

Never one to let muddy controls get in the way of doing more 2-bit, 10 minute experiments involving lighting dead bugs on fire, I proceeded to repeat the “sterilization” of a second, recently departed beetle and take it one step further by crushing it up in 1ml of sterile 1.5% peptone broth in a sterile mortar & pestle. Science at a community college often looks like science on Gilligan’s Island, although I have not yet actually used a coconut for anything.

Spreading ~200ul on a 60mm dish gives this after incubation at 31C (the temp I have my Tribolium beetles at) over the weekend:

beetle microbiota from inside nutrient agar

Hmmm, looks like a transformation that got plated on an LB plate without antibiotic, so, growth.  Re-streaking from the plate and from the fungus gave:

combined restreak

Nerd heaven. Or a waste of time. Maybe both.

There was (and still is) absolutely no reason to think this would work.  Any self respecting microbe that is adapted to growing inside an insect would refuse to grow on a drafty, unprotected agar plate.  In the pro column: it took 15 minutes to do, cost the college ~1.5 cents, was a break from grading, and I was curious.

The re-streak plates provided a perfect example for my micro lab; Thursday they had to practice streaking for single colonies using tubes spiked with two different species.  When I showed them my ongoing bug experiment as an example of why you would want to do this in the real world, they immediately showed their compassion by asking if they could buy my wife a drink.

I really need to figure out if sterilizing the outside is truly possible.  Dare I dream that the fungi grew up on the control plate because the bacteria had been knocked back?  Dear Santa: Please give me competitive exclusion of fungi by bacteria on or in insects as an early christmas gift.

* – and yes, I know that I am using a beetle and not a bug here; bug is more fun to say.  Thinking that ‘Mericans will start using “bug” to describe only Hemipterans is lot like thinking they will start using the metric system.

9-28-13: After doing more reading on entomopathogenic fungi: compare the fungus growing out of the beetle on the control plate with the fungus growing after being streaked out.  Assuming they are the same species (medium leap), look at the yellowish/brown color it makes on the insect versus the all white on the streak plate.  Color in fungi often comes from the production of sporangia (so for instance, black bread mold is white until it makes sporangia, which are black).  This excellent key to fungi (PDF)  states that non-spore forming mold structures are useless for identification and that if you can’t find any you should try to induce them by infecting an insect with the fungus.  Does the color of the fungus on the beetle indicate a happy entomopathogenic fungus?  I wouldn’t be surprised if it wasn’t… after all I am just joking around with cous cous cultures here…. why would I expect there to be relevant pathogens in Walmart cous cous?  Are there commensalistic fungi on insects that can become opportunists if given the chance?  I sure hope so, but its always good to keep in mind the most simple explanation: this is a yellow/brown fungus contaminant, not relevant to insects.

** – free time – \ˈfrê- tîm\

noun

1.  time that is not occupied by compulsory activites

2.  period of time during which a teacher should be grading

see: avoidance, denial, self soothing

 

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About SubOptimist

I am an Associate Professor in the Science Department at Georgia Perimeter College, Clarkston. I teach introductory biology courses at both the majors and non-majors level in addition to microbiology. Previous to that I spent 7 years as a postdoctoral researcher on different viruses. While I don't miss being on the "grant treadmill", I think better when I write and miss writing up data for papers and grants; this blog helps me with that a little. And sometimes my kids' insanely funny and cute antics need to be shared with the world. Any view expressed in this blog is that of me personally and not Georgia Perimeter College or the GPC Clarkston Science Department.
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