Man vs. Machine: Counting microbes at the bottom of the Baltic

The microbiology component of IODP expedition 347 to the Baltic Sea is well and truly underway, with drilling of our first hole dedicated to mi sampling completed. We retrieved 405 microbiology samples from up to 82 metres below the Lille Bælt seafloor. We have moved on to our next site just south of Anholt and have begun drilling. While my own samples will stay in the freezer at -80ºC until I return to Aarhus to extract microbial DNA, other microbiologists in the science party have begun some of the analyses that can be performed on board the ship. In this blog post I will talk about the two microbiologists on board who are addressing one of the most basic questions about microbes below the seafloor: how many microbes are down there?

The Man: Barry Cragg counting cells with a microscope
One of these two microbiologists is Barry Cragg from Cardiff University in Wales, a legend in the field of deep biosphere cell counting. This is Barry's sixth IODP expedition, and he has been counting cells in marine sediment for decades. His procedure is as follows: when a sediment sample is first collected it is immediately preserved in formaldehyde. Barry then mixes the formaldehyde-treated sediment with a dye that binds to the DNA in microbes and fluoresces brightly when exposed to a certain colour of light. He then observes this sample underneath a fluorescence microscope and counts the cells one by one. This is a task that requires skill and patience - sometimes particles that are not cells appear to be cells, and it takes practice to tell the difference. In order to accurately compare two different sediment samples, especially from the same location, the same individual should count the samples. This is why Barry is such an asset to the deep biosphere field: he has counted microbes in so many samples from so many different sites that his results allow us to compare microbial cell densities in deep marine sediments all over the world. The only problem is that it is hard to scale up this approach - the patient work of a scientist with years of training and experience in a small but important niche is difficult to replicate. This is why microbiologists are also looking for ways to automate the cell counting process.
Picture: Barry counting cells. Copyright Carol Cotterill/IODP/ECORD

The Machine: A flow cytometer operated by Nan Xiao
Nan Xiao is a microbiologist from the JAMSTEC Kochi Core Repository in Japan. She has brought on board the most sophisticated piece of microbiology equipment of all the microbiologists: a flow cytometer. The flow cytometer takes formaldehyde-treated cells labeled with fluorescent dye (similar to the dye Barry uses for his microscope technique) and pumps them through a narrow channel one cell at a time. A laser shines across this channel, and whenever a microbial cell passes through the laser beam it emits a flash of fluorescent light. To count the cells, the machine simply counts the flashes of light.

Like any automated solution, there are some drawbacks to using the flow cytometer. For example, while Barry can tell from the shape of a blob of light whether it is likely to be a microbe or some other kind of particle, the flow cytometer mostly ignores the shapes of the objects that drift past its laser. If a cell is attached to a sediment particle Barry can still see it and count it, but the flow cytometer requires a pre-treatment step to separate cells from the sediment that is not 100% efficient. However, the flow cytometer is faster: it can analyse a sample automatically in just a few minutes, while Barry needs at least thirty minutes of his undivided attention to do the same work. Moreover, the flow cytometer generates equivalent results regardless of the scientist operating it, while microscope counts are notoriously difficult to reproduce.
Picture: flow cytometer. Copyright Ian Marshall/IODP/ECORD

The winner?
There is little doubt that microbiologists studying deep ocean sediments would prefer to count cells using a tool like the flow cytometer than to rely on the skill of rare humans peering down microscopes. We are just unsure that a flow cytometer can deliver the same quality of results that a human operator can. That's what makes this cell counts on board this expedition so exciting - we will be able to compare the results of microscope counting to the results of a shipboard flow cytometer. If the flow cytometer can be shown to deliver results that match the microscopy technique we have come to rely on over the years then we could see a greater role for the use of these devices in counting cells in sediment in the future.