From May 13-14 2015 we hosted a “Live/Dead Workshop” here at UC Davis where we basically discussed a number of issues related to the topic of figuring out which bacteria are alive/viable in a given microbial community. This is particularly important in the built environment where we suspect that many (most?) microbes are dead and where DNA might persist for a long time. The talks were mostly methodological but we did cover some interesting terrain related to semantics, Planetary Protection, quantification, and such. The agenda of the meeting and list of participants is here. On the second day we made good progress towards producing a paper summarizing the state of the field and some future directions we think this work should go.
Below I’ve listed the talks and given my brief thoughts on them. As people (hopefully) upload their talks to Slideshare, I will link to them in this post. And finally a huge thank you to everyone who came to this meeting and made it such a great success!
Parag Vaishampayan (JPL)
“New perspectives on viable microbial communities in low-biomass cleanroom environments”
This talk covered a lot of ground, focusing on Planetary Protection and the importance of live/dead determination in that context. I learned that using PMA for live/dead also means finding low abundance OTUs who were otherwise masked by the high-abundance dead stuff in a sample. Parag also discussed “Double-PMA” which basically involves treating PCR reagents with PMA in order to get rid of exogenous contaminating DNA. Gotta try that!
Ivan G. Paulino-Lima (NASA-Ames)
“Use of cell staining techniques to study radiation resistance of uncultivated microorganisms.”
The title pretty much says it all here… Ivan talked about looking “beyond Deinococcus” at radiation-resistant organisms, mostly in the context of Astrobiology and Planetary Protection. Covered a lot of really interesting material and mechanisms of radiation resistance.
Andreas Nocker (Cranfield University)
“Practical tips to improve viability PCR”
Andreas is the father of PMA and so his talk covered this pretty exhaustively, including the pros and cons and a lot of methodological tips. He made a point to emphasize that membrane integrity is just a proxy for viability and not the end of the story. He also mentioned that flow cytometry and PMA are applicable to different questions and should not be considered exclusive.
Nicolas Justice (LBNL)
“A high-throughput method for absolute quantification of microbial community dynamics using next generation sequencing”
This talk really focused on the importance of absolute quantification in microbial ecology. One interesting idea I’d not heard of was spiking in a known concentration of a lab bug before doing DNA extraction in a 16S survey. He discussed the many systematic biases in PCR and most depressingly showed data suggesting a PCR efficiency disparity with different barcodes in the primers.
Tiffany Hsu (Harvard School of Public Health)
“Using PMA assays and sequencing to differentiate live/dead bacteria on a mass transit system.”
Tiffany’s talk covered their experience of trying to get PMA to work in their subway study. She showed data suggesting that PMA bound DNA is not isolated in DNA extraction (as opposed to just not working in the PCR).
Erica Hartmann/Clarisse Betancourt Román (BioBE Center, University of Oregon)
“Live/dead determination in dust with flow cytometry and PMA”
Erica and Clarisse gave a tag-team talk that could probably be summed as as “dust is really hard”. They discussed their experience of trying to get both flow cytometry and PMA to work on dust samples… significant difficulties in both cases.
Joanne Emerson (University of Arizona)
“Pros and cons of using flow cytometry and qPCR to count bacteria and fungi in air samples”
Joanne talked about her experience with flow cytometry and qPCR for absolute quantification in air samples. Of the two, she expressed a strong preference for qPCR in this context.
Cinta Silvan (LBNL)
“RNA as a proxy for metabolic activity”
Cinta rounded out the day by talking about RNA. This is a project not yet started, but she presented evidence from other work about the utility of RNA to determine metabolic activity. This sparked a lot of discussion about the difference between “alive”, “viable”, and “metabolically active” which I think is important to think about.
Here is a Storify of the tweets from the meeting:
I just read a wonderful article about dead-dormant-active soil microbes: “Active microorganisms in soil: Critical review of estimation criteria and approaches”. The active microbes compose only about 0.1-2 % of the total microbial biomass, the fraction of potentially active up to 60%. This is in temperate soil zones, which is usually a moist environment. What are the % on dry building surfaces? They conclude that “because active mo. Are the solely microbial drivers of main biogeochemical processes, analysis of the active and potentially active fractions are necessary in studies focused on soil functions”
http://www.sciencedirect.com/science/article/pii/S0038071713002988