Snip, Snip: It’s All About Sterility

Just a note:  Today’s post is a two-fer, because 1) we worked until about midnight last night and I failed to make a post D:, and 2) today was very similar to yesterday.

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Giving a face to what we want to save (or at the very least understand). This is actually inaccurate since the mouth is underneath…

And no, although the title’s pun was intended, this post has nothing to do with vasectomies.  It’s about careful planning and maintaining sterility when you’re collecting samples and doing other things in the lab.

As you may know from mine and other posts, we just received 54 Pisaster sea stars that were being held at Merrowstone for an experiment.  They were collected from Ruby Beach on two different dates and were assumed to be healthy – completely free of SSWD pathogen.  This would have allowed us to do experiments to help us determine SSWD’s pathogen, comparing those that were naive and those that were infected with a putative pathogen.  However, what we thought were naive stars turned out to be infected, preventing their use in the experiment.  We had to choose between disposing them (what a waste!) or sampling from them, and so they were brought to FHL from Marrowstone for us to sample.

For our tissue sampling, we decided to be very careful (even more so than usual) and to use a very well laid-out process.  Because we were interested in seeing which tissues are targeted by the putative pathogen, we maintained sterility between stars and tissues (within the same star) to the best of our abilities.  It can be described kind of in recipe form:

Tools:

  • Plastic tray
  • Hypodermic needle (for sampling fluid)
  • Scalpel handle and blades
  • Dissections scissors
  • Plastic rulers
  • Forceps
  • Gloves
  • Paper towels
  • Tubes and bags
  • The trifecta: 10% bleach solution, reverse osmosis (RO) water, 95% ethanol (EtOH)
  • Sterile seawater (SSW).  I would actually argue this is super-sterile seawater (SSSW) since we made our best effort to have it really clean:  It was filtered to 0.22 μm and autoclaved.  But anyway…
  • Well plates
  • Alcohol lamp
  • Dry ice

Between each star:

  1. NEW GLOVES
  2. Remove tissue and bleach tray
  3. Sanitize tools:  scalpel handle, dissection scissors, forceps (bleach → RO water → EtOH → flame), ruler (just bleach: you can’t flame plastic safely…)
  4. NEW NEEDLE
  5. NEW SCALPEL BLADE

Between each tissue:

  1. Rinse tissue in SSSW in well plate (different well for each tissue!)
  2. Again, sanitize tools:  scalpel handle, dissection scissors, forceps (bleach → RO water → EtOH → flame), ruler (just bleach).
  3. Store sample as appropriate.  In most cases it was quick freezing in dry ice.
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Rinsing tissue in a well plate of SSSW. We didn’t want any tissue other than what we wanted in the tubes, but some contamination is inevitable.

This may seem excessive (I certainly hope it’s not), but being extremely careful with our sampling gave us confident in the utility of our samples.  It could have been faster or less expensive (could have not used a fresh blade between each star).  However, this would have sacrificed our trust in our methods, and if our samples didn’t work out we could needed to sample more – a luxury we might not have in the future.  Just a bit more work now can save us much more in the future.

 

 

 

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