Sterile techniques on a dirty drillship

The deep subsurface is an energy-limited environment for microorganisms, which makes for a very low density of microbial cells. 100 m below the seafloor there are up to 100,000X fewer cells than in sediment on the seafloor.

This low cell density means that microbes from energy-rich sources may outnumber the deep subsurface microbes if they come into contact with the samples, leading to contamination.  Such potential sources of contamination include drilling mud, seawater, the ship, and the humans doing the sampling. In this post I will describe some of the measures that we are taking during IODP Expedition 347 on board the Greatship Manisha (currently drilling a few kilometres off the coast of Lyø near Fåborg) to prevent this contamination.

A "hacked" medical tool

When most people think of sterile work they think of medicine, and it turns out that one of the most widely used tools in environmental microbiology sampling is a "hacked" medical tool we have all encountered during a visit to the doctor: the plastic syringe.

The syringes (sizes from 3 mL up to 20 mL) are purchased pre-sterilized, and a sterile blade is then used to cut off the syringe nozzle. This "cut-off syringe" can then be inserted into the sediment and removed with sediment filling the syringe's interior. This is then placed directly into a sterile bag, with little risk of contamination by bacteria from the scientist's hands, clothing, or sampling tools. The sample is later processed under sterile conditions in a land-based laboratory.

The only drawback to this method is that on this expedition we plan to use over a thousand cut-off syringes, and each of these has to be carefully cut by hand by the scientists. I am certain that if a manufacturer of plastic syringes produced sterile pre-cut syringes this would be an instant hit amongst environmental microbiologists.

Keeping the lab clean

There are some procedures that cannot wait until we get back to land, and for these we need to use some other standard microbiology lab techniques to clean our working space and lab tools to avoid contamination. Exposure to a solution of 70% ethanol is sufficient to kill most microbes (100% ethanol evaporates too quickly to be effective). We have about 10 litres of 70% ethanol on board that we dispense using squeeze bottles. Unfortunately this ethanol is unsafe to drink, so it's not an easy way around the ship's ban on alcoholic beverages.

Fire is an even more effective means of killing microbes, and we have small blowtorches (the kind that come in that creme brulee kit) and a bunsen burner to flame-sterilize metal tools. Finally, we have a plastic working box fitted with a powerful blacklight that can be used to sterilize working surfaces and equipment before working with sediments. Sufficient exposure to ultraviolet light damages microbes' DNA (just as it damages humans' DNA in bright sunlight or a solarium) enough to prevent their further growth.

Chemicals trace contamination

While as scientists we have significant control over our working environment and resulting contamination risks, we have little control over the procedures used to drill into the seafloor and the potential for introducing contamination during drilling and coring.

Although drilling techniques are designed to produce cores as cleanly as possible, there is still a risk that the drilling mud or seawater may penetrate the cores, thus contaminating them with microbes from non-subsurface environments. We monitor this contamination by mixing a tracer chemical into the drilling mud - the tracer is an inert compound that does not affect our analysis or harm the environment, but can be easily detected down to very low concentrations. At regular intervals along the retrieved cores we take cut-off syringe samples and test them for the tracer chemical. This way we can monitor how far the drilling mud penetrated the core and thus how far any unwelcome microbes may have penetrated the core. We then avoid analysing samples from depths shown to have high concentrations of the tracer chemical.

Photo: Copyright Ian Marshall/IODP/ECORD