Trends in Microbiology

Q: What is the focus of your research?

A: The focus of our research is microbial ecology in the ocean. We also study cystic fibrosis and look at microbes in the lung and gut. Some people in the lab study bacteria, but many study viruses. My work is mainly involved with the environmental projects in the ocean, looking at bacteria associated with coral reefs, although I do study some viruses too.

Q: What do you have to do to conduct these types of studies in the ocean?

A: We go into the field a lot and collect water samples from different types of reefs. We look at both healthy reefs and degraded reefs and study the microorganisms that live on the bottom, the corals and the algae, the microbes that live on the surface, and also those that live in the water column overlying these ecosystems. We see that a lot of things change in the water quality in terms of the nutrients and the microbial community in different regions of the ocean.

Q: How do you collect the water samples?

A: We collect large quantities of water, about 100 liters, and concentrate it to small volumes using filters, and that is what we bring back with us. We also collect water to do all the water chemistry, and that we collect in Niskin bottles and filter into vials to bring it back to analyze. We bring back punches of corals and samples of different algae. On our other project, looking at cystic fibrosis samples from humans, we are collecting mostly sputum.

Q: How do you keep samples from cross-contaminating or getting contaminated?

A: We do a lot of genomic work on our samples, and when you amplify the DNA using PCR you have to keep the post-PCR amplified DNA in another building, as it can easily contaminate our unamplified samples. We always isolate the DNA and set up PCR assays in a clean lab. Thermocyclers used to complete the PCR and all amplified DNA are stored in another building. We never bring equipment, such as pipettors, back and forth between the two buildings. We have had a couple of contamination issues, and it’s very difficult to decontaminate when that happens. This is very important to convey when we are training new people in the lab.

Q: What common contaminants do you routinely test for?

A: We routinely separate viruses from the bacterial community using cesium chloride gradients and ultracentrifugation. It is important to reduce the amount of bacterial nucleic acids when sequencing a viral metagenomic library, since bacterial genomes are so much larger. We test for contamination of bacterial DNA in our viral libraries by amplifying the 16S rRNA genes using bacterial-specific primers. It’s always hard to get rid of everything, but you can certainly minimize cross-contamination.

Q: What types of instruments do you commonly use in your lab?

A: We have many gene sequencers, thermocyclers, microscopes, microarray systems, and other types of instrumentation for routine sample extraction and preparation. We have a lot of specialized equipment that can be used in the field. We have a field microscope that can be taken apart, and it is custom-built to carry for travel. We work all over the world, in the Caribbean, islands in the Central- Pacific, French Polynesia, and Australia. Our PI, Dr. Forest Rowher, worked in the Arctic region off the coast of Russia last year.

Q: What changes or trends do you see in the field of microbiology?

A: Sequencing has definitely become a lot cheaper. A few years ago we used to work with small libraries consisting of a few hundred sequence reads. Now we can have more than a million sequences per library, and therefore the computing power is often a limitation. As biologists, we are faced with having to deal with these massive sequence libraries. Most of the viral diversity is unknown, but now with all the sequencing power the unknown is becoming a little more known. A few years ago many of the bacterial sequences that were known were those that had clinical implications. So they were either pathogens or bacteria that could be grown in a laboratory. Now with a technology called single-cell amplified genomes (SAGs), you can sort bacterial or viral cells from their environment, isolate one cell, and then sequence the genome of that cell directly without having to grow it on a plate. We can culture only about 0.1% of all the microbes out there. So if we have to grow a cell in order to sequence it, then we can study only a very small portion of the microbial diversity that exists. With technologies like this we can study the microbes that occur naturally in an environment and not just the ones that can be grown in culture or are pathogens.

Q: Have you taken advantage of all the lab automation that’s now available?

A: We don’t do a lot of high-throughput work, and hence we do not have robotic systems in place. We do have one project where we have to collect thousands of samples, and we are thinking about ways to process the samples if we get about 1,200 samples every couple of weeks. We could use robotics, but the first step still has to be done manually. We are still starting with a sample in a bag that needs to be dissolved in a buffer and moved to a plate. So we are limited by the steps that can be automated.

Q: Have there been any improvements in the options for sample collection and storage?

A: Sample collection is still the same whether you are working on a ship or in the field. However, there have been some nice advances for organizing the samples in our freezers once they are collected and analyzed. We now have a database with bar codes for all our samples. It’s hard to go back and bar code all the samples that we have collected over the past decade. But having this system in place will help us find samples more easily going forward. We also have backup systems for our freezers. If the power is down, the alarms go off, we are notified by phone, and the backup systems start releasing liquid CO2 so the samples can stay cold for a few days.