Friday, March 18, 2016

Isolation

Sorry for the late update but I've been so busy at work I've hardly had any time to do anything else.

You could say I've been... isolated.

We scientists know our priorities.

Of course, what people don't understand is that we're never really alone.

As Gregori is constantly reminding me.

Our bodies are teeming with bacteria, so much bacteria that if you counted your own cells and then your bacterial cells, you'd discover that you were pretty thoroughly outnumbered.  If that thought horrifies you, never fear!  Most of those bacteria are completely benign and, in many cases, symbiotic with you.  Your immune system isn't so much a defense system as it is a checks-and-balances-style control system, designed to balance the delicate ecology that is you.  Congratulations.  You were a park reserve all along and didn't know it!  Now you have a good excuse to get out of planting a tree next Arbor Day.

Anywho, I've mentioned before my work with gnotobiotics, which is the study of "known life," aka, the aforementioned trillions of bacteria teeming all over you right now.  Gnotobiotics is a tricky business because obviously, it's impractical to try to count bacteria that outnumber us so much, just like we couldn't expect to reasonably inventory every cell in our bodies.  If you think of a body like a reservation and the bacteria like the wildlife, then you can say that anyone who studies gnotobiotics is the jackass trying to count all the birds in the park.  It just doesn't work like that.

So how are we supposed to know what's in us, on us, and around us, and in what amounts, if we can't count it?  This is a riddle with a pretty good answer.  The answer is that counting to zero is really easy, and if you carpet-bomb a wildlife preserve, you will be able to accurately count all the wildlife, and also would probably be the subject of international outcry for a while.

#CecilTheLion  #NeverForget

For five months now, we've been eagerly awaiting our germ-free isolators, which are giant plastic bubbles that can be sterilized (remember my carpet bombing metaphor!) and be used to house germ-free mice.  It's a simple concept with a shockingly complex execution.  The isolators need to be built before anything, and they have to be built correctly to ensure that the air flow in and out of them is controlled and filtered.  Then they're sterilized chemically.  The result, if you've done it right, is an inflated bubble of filtered air, with contents that is completely devoid of any and all bacteria, viruses, or fungi, making it a germophobe's wet dream.  Once sterilized, the isolators have to be treated very carefully to maintain sterility; all items are moved in and out of the isolators using a port that mimics a space station air lock, and all items must either be sterilized chemically or mechanically in an autoclave.  All work is done using a large set of heavy white astronaut gloves.  Meanwhile, weekly testing is used to confirm the germ-free status: methods include PCRing mouse feces, swabbing and incubating bacterial plates, and Western blot.  If you've never collected mouse feces while wearing astronaut gloves, then you have no idea just how hard this is.

To be fair, though, all science is hard.

So over the last month, I have been working on setting up our facility, building it from the ground up.  It's a labor of love and I'm delighted to see it coming together.  Below are some pictures.  Enjoy!

First, we enter the lab...



Ignore that sign.  It's actually very safe.  We use goggles and everything.



The "lab" is only where we conduct actual research and process data (which may refer either to actual DNA or to Excel spreadsheets, depending on what day of the week it is).



One of the places I spend a lot of time in within the lab is an anaerobic chamber.  This bubble lacks oxygen and so you can grow gut bacteria within it.  Hence the biohazard sticker.

The chamber is one of the places I test for isolator contamination.  If I swab anything and put it on a plate of media, bacteria will grow.  Here, for example, is a plate I coughed on personally.


But a germ-free chamber, swabbed, will yield no growth.  You can swab the mouse, you can smear its feces around, you can have the mouse spit on the plate... but there's no bacteria.  Nothing grows.  To establish sterility initially can take up to 50 plates to confirm.  My phone is nothing but pictures of plates right now.

 

But let's start at the beginning.  How do the isolators get set up?  It starts with the delivery of the components... 


It's like Christmas morning in the lab any time we get a big delivery like this.


Next we start setting things up.  Shelves are entered into the isolator and built inside them.  The isolator, a large plastic button, is attached to a round, rigid port that is attached to a table.


I liked this part because I got to use a wrench.

The large isolators and the shelving units were so large I had to climb inside them to set everything up.




In no particular order, we attach gloves, air filters, and ports to the plastic bubbles.  At this point the bubble isn't inflated and everything is still swarming with bacteria.  If you have OCD, feel free to take a break at this time and wash your hands.  It doesn't matter.  You're still teeming with bacteria.  Go ahead and let your pet bird sit in your food.  It doesn't matter.



Attachment points are secured with every mechanism known to man.  Tape, wires, stretched polyurethane, rubber bands, chewing gum... anything to ensure a seal and prevent bacteria from entering.


Next, we start entering supplies.  Some are obvious, like tweezers.  Others aren't.  Things like knitting needles, towels from AutoZone, and colorful stickers have all been put inside the isolators for completely legitimate scientific purposes.





 (Above: note the unassembled shelves in the background!)

Once everything's ready to go, we seal and sterilize the chamber.  How, you ask?  Why, chemicals, of course.  Just look at that chloride-dioxide fog!





Once the decontamination time has been met, we flush the isolator by turning on the air.


All air passes through HEPA filters to ensure no airbourne pathogens get in.  You can make sure air is flowing using ribbons.  I use blue ribbons so that I feel like a winner or sometimes pink for breast cancer awareness.  Also that's just what I happened to have at the time.


Now the isolators, much like my ego, are inflated!  


 

Confirmation of a successful sterilization takes a week or longer, but from this point on, any items entered must be chemically sterilized or placed into a large, metal cylinder, sealed, autoclaved, and attached and entered.  It's like getting supplies into a space station.



Look how cool it is once set up!




"Why are the lights all weird and red?"  Because first of all, it looks badass, and all evil science lairs need red lights.  Duh.  But also, the red filters protect delicate baby mouse eyes from harsh flourescent lights and make nesting mothers more comfortable.  Within a few weeks of getting our germfree mice, we were greeted with this little jelly bean (on the left)...


A successful, albeit arduous process.  So far, all of my isolators have been sterile on the first try.  I'm six for six on set-up and sterility, and have a colony thirty mice strong, along with three litters of new, germ-free babies!

Now I just have to take care of them forever and maintain that sterility.  Everything is done with gloves and lots of swearing to ensure the best results.


 
Let it blow your mind for a second.  These isolators are like the void of space.  You could shrink down to any size and you would never encounter even a single virus or living bacteria.  When I ask, "How many organisms occupy this space?" the answer might literally be, "Four.  I observe four mice.  There are no other living organisms even on a microscopic level in that bubble."

Now who's mind freaking whom, Criss Angel?
 
Once the colonies seem stable, I'm hoping to get into some research this summer.  So forgive me if my blog posts become erratic and infrequent... I'm busy doing science.

That's all, folks!

2 comments:

  1. Stumbled across your blog from Reddit--are you at Caltech?? One of those isolators has "Mazmanian" written on it, but I thought his gnoto lab had been up and running for a long while now?

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    1. Good eye!

      No, I'm at UCLA.

      A couple of these photos (the cylinder and the exhaust filter with the ribbon) are from CalTech, where I spent several weeks learning about gnoto management from their excellent team. Any of the photos with the green floor are my own lab, though. :)

      Also, where on Reddit? /r/labrats by any chance?

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