Fond Feelings for Tiny Deadly Things

 This might be weird but...

I have favorite bacteria, or rather species that I'm fond of and kind of 'cheer' for.

Bacillus subtilis: It's my first born and the species I really first focused on in grad school. We spent long hours in the lab together. I sent it to space (ISS). It came back and survived. I sent it to (simulated) Mars. It survived.

Legionella pneumoniae: It's SO PRETTY when you see it on BCYE agar. It's crackled iridescence and deadly deadly beauty if you breathe it in.

Stenotrophomonas maltophilia: This sucker is PERSISTENT. It can survive in very low nutrient conditions (purified water) and endure some major chemical trauma and just bounce back. I also learned today that some guy had to have his fingers amputated after this bug infected his skin and was just resistant to all the antibiotics they tried to throw at it.

Moral: Be like bacteria. Beautiful, deadly, and resilient. Also, better supported when you're in a community (biofilm).

Hidden Costs of Science

There is a push in the scientific community that data should be freely accessible and not hidden behind a paywall. I believe in this strongly. Many scientists I know got into the field because they feel a strong affinity for learning and being able to share what they've learned with everyone. As I'm juggling several things in life regarding science and lab work one thing has increasingly become quite apparent: There is an invisible cost bar to citizen science and citizen self study. As I'm trying to start my own lab (whooo, big post I've been avoiding making for reasons), dealing with current lab's recent audit (can't say much due to privacy and NDA), I've really been digging into the nitty gritty details of what it takes to run a certified, private laboratory. The initial costs that you expect (equipment, consumables, space) is understandable to some extent and can be mitigated by hunting for deals on used equipment and government auction sites of equipment they no longer need. But then there's the protocols that are industry standards and accepted as the way to have consistent results from lab to lab and person to person. In my current field there are two standards agencies that we pull methods from: ASTM and ISO. A single pdf copy of an ASTM method can be purchased for a little less than $50 USD. (So far that I've seen, there might be price variants that I haven't discovered). That copy is authorized for ONE user. To technically be in compliance with the copyright, my lab should be purchasing the rights for every person who uses the SOP. But instead we just copy off the one and pass it around. To acquire every single ASTM standard, for the current year, would be just over $10,000 USD for a digital copy, single user copyright. When it comes to an ISO standard they get trickier. We are currently working on an audit for an agency that requires us to follow ISO 12345* *not the actual # Several of the deficiencies cite laboratory failure to follow protocols, including details on holding time between sampling and testing, and quality control of an in house component made for the test. If you look up in ISO 12345 the details regarding what is required, the SOP just states to refer to ISO 12333 and ISO 1223. I went to an online store that sells access to these digital copies and added them to my cart to see what the total would be if I purchased all six (12345 + the 5 referenced) and the total came out to $943 USD. So now, as a citizen trying to start a lab, I need to add in a budget to purchase my SOP sources because they are not available in any reasonable manner. No wonder science, as a business, is out of reach for the average person, even one with a relevant degree. Not to mention the costs associated with achieving certification. Before you can even apply to have a lab certified with some agencies, you have to pay them for access to their audit standards to be prepared. Then you pay their employees to come to your lab for the audit. So if I want to achieve certification for my lab, I can't even tell if it's worth doing and preparing from the start without being out money. Because I can't take clients on until I can offer them the guarantee that the certification provides.

Test Protocol for Legionella: Science Sunday

At our lab we are certified for two different methods for identifying and quantifying Legionella in samples. One is the general CDC method and the other is a method that is WAY more involved and only required by the State of New York.  Why the state of New York has to be extra special is beyond me, but we have been audited by them and found to do everything properly so we can do New York samples.

In the following post, I actually am doing both methods simultaneously because one client is from not New York, and one client is from New York. Many of the steps are similar. Here we go!

Here is nearly everything I need to test for both methods of Legionella. From left to right:  vacuum pump, 100ml sterilized tips, pipetter, erlenmeyer flask with rubber fitting, vortexer, sharpie, chlorine test strips, samples, skinny tubes, glass tubes, black agar plates, blue large tubes, Page's Saline, saline, NY acid, Legionella acid, cultures of Pseudomonas aeruginosa and Legionella pneumoniae, sterile water, 2um disposable filters.

Not pictured: 1 ml glass pipettes, pipette bulb, forceps, ethanol, lighter

First step, required for NY, optional for CDC.  Test the chlorine content of the water.

Good result! You can proceed with testing of the sample.

BAD RESULT.

Add sodium thiosulfate until result is good. In this case it was 100 ml of sample, so I added 100 ul at a time.

Fill large tubes with 10 ml of saline (Pages for NY, general for CDC)

Filter 100 ml of sample.

Dip the forceps into ethanol and SET THEM ON FIRE! (to sterilize) Once cool, use the forceps to transfer the filter paper to the blue eppendorf tubes.

VORTEX!  Just... generally for CDC, for 2 VERY LONG MINUTES for NY.

Yay!

Forceps and ethanol

For NY, you have to run positive and negative controls. Using the McFarland standards, you can guesstimate your concentration of bacteria in liquid by comparing the cloudiness.  Pseudomonas is the negative control, Legionella is duh, the positive.

To save time, vortex multiple tubes at once and cut vortexing time in half!

CDC:  Use bi plates with two similar agars on each side. Pipette 0.1 ml onto each side.

Spread that sample around. You can use the same spread from side 1 to 2, but not 2 to 1 and obviously not from one sample to the next.

NY samples go on a single type of agar (Side 2 but poured onto a whole plate). You plate 0.5 ml and spread.

Not pictured:  Each method has an acid treatment stage. This is pretty neat. The acid will kill amoebas that can hide Legionella. Yay! CDC has a 15 minute acid treatment.  NY has a 5 minute acid treatment in a different acid.

NY also has a 30 minute 50deg C heat treatment.

Each treatment also gets plated onto it's own plate.

In the end, CDC method uses 4 plates (regular and 3 acid) per sample, NY uses 3 plates per sample (regular, heat, acid) plus the controls.

All of those plates go into anaerobic jars.  You light the candle and tighten the lid, the candle burns all the oxygen. Those jars get incubated for 5 days (CDC) or up to 10 days (NY) at 35 deg C.

How we identify legionella coming up next!

BONUS SCIENCE

A picture of the product my company manufactures. It's a two sided dip stick. You dip it in a limited volume of water and bacteria or fungi stick to the agar. You can enumerate how much bacteria is in your volume of water.