A Set of In Situ Informed Simulated Medium Formats for Culturing Environmentally Acquired Anaerobic Microorganisms

Our research focuses on terrestrial surface and subsurface microorganisms, culturing them and investigating their genomes and phenotypes. Of particular focus are microorganisms and environments that are not well understood, like those in the deep subsurface and in certain wetlands. Most of the microorganisms we culture are anaerobic, which means the culturing process generally requires more time and attention than processes for more commonly used aerobic microorganisms.

We hope the audiovisual presentation of anaerobic medium preparation will help researchers new to the technique learn it relatively quickly and easily. This medium preparation is based on the modified Hungary technique of Miller and Roland Culturing fastidious microbes is tedious and problematic. Having more tools to grow them is very welcome to the scientific community.

Future research in general will be similar to our current research of understudied terrestrial microorganisms. There are still many new microbes to discover and study. To begin, measure and add the required media components to a one liter bottle.

Adjust the pH according to the microbial culture of interest and mix the components to homogeneity by swirling the bottle. Heat the liquid to boiling in a microwave for five to six minutes, opening the microwave often and swirling the bottle gently using a heat resistant glove. Next, attach a sterile 10 milliliter glass pipette to a nitrogen gas manifold set up.

Open the manifold to vent oxygen and allow nitrogen gas to flow out. Place the pipette tip into the liquid and adjust the gas flow so that the bubbles are moderately vigorous. Flush the liquid with nitrogen until the bottle is no longer too hot to touch.

While waiting for the bottle to cool, set out 10 sterile 100 milliliter culture bottles. Once the media bottle reaches bearable warmth, pull the pipette tip up into the headspace of the bottle. Add 0.125 grams of sodium bicarbonate and 0.3 grams of sodium sulfide nonahydrate and wait until resazurin turns colorless.

While waiting for resazurin to change color, attach metal canulas to the gas manifold setup. Adjust the nitrogen flow to a moderate rate and place the canula tips into culture bottles. Once resazurin is colorless, aliquot 50 milliliters of liquid into each culture bottle.

Cap each bottle with a matching sized rubber stopper immediately after removing the canulas. Then secure the stopper with an aluminum seal. Place the bottles in an autoclavable bin and fill the bin with tap water until the water levels match those in the bottles.

Autoclave the bottles at 121 degrees Celsius for 30 minutes. To prepare the carboy set up, open the valve of a 6.4 centimeter hose bar ball. Attach two centimeter lengths of silicone tubing onto either end of the hose bar ball valve.

Attach a 7.9 to 6.4 millimeter hose bar adapter fitting to one end of the assembly. Attach the other end of the assembly to the hose barb of an eight liter carboy. Use a zip tie to secure the connection between the carboy's hose barb and the tubing and the connection between the hose barb adapter fitting and the tubing.

Measure and add the required media components to the eight liter carboy. After adjusting the required pH, mix the components to homogeneity by stirring in a hot plate and allow the liquid to boil for 30 minutes. Next, attach a sterile 10 milliliter glass pipette to a nitrogen gas manifold setup.

Open the manifold to vent oxygen and allow nitrogen gas to flow out. After turning off the heat, place the pipette tip into the liquid and flush it with nitrogen gas for 30 minutes, moderately vigorous but not overflowing. Next, pull the pipette tip up into the headspace of the carboy.

Add two grams of sodium bicarbonate, and 4.8 grams of sodium sulfide nonahydrate and wait until resazurin turns colorless. Once resazurin is colorless, remove the glass pipette and immediately cap the carboy with a 10 stopper. Twist a wire over the stopper and around the lip of the carboy to secure the stopper.

To acquire an anaerobic soil mixed culture, push the corer tool into the field sediment until the top of the sample collection cylinder is flushed with the surface of the sediment. Twist the corer at 90 degrees to free the corer and pull upward until the sample is extracted from the environment. For loose sediments, cover the base of the corer with a new nitro gloved hand to prevent the sample from falling out or disseminating into the water on extraction.

Quickly approximate by eye, six to seven centimeters up from the base of the core, and slice it to separate the bottom. Immediately transfer the bottom of the core to a culture bottle, minimizing exposure to the aerobic atmosphere as much as possible. After transferring, immediately reposition the stopper and hold it in place with a screw cap.

Keep the sample cool and refrigerate the collection bottle at 4 degrees Celsius upon returning to the lab. To begin, set out a stopper and sterile bottle of nitrogen and the culture bottles and a sample collection bottle. Drip 100%ethanol onto the stoppers and set them on fire using a lighter.

Once the stoppers are no longer on fire, insert a one milliliter syringe fitted with a 23 gauge needle into the nitrogen bottle and draw up approximately one milliliter of nitrogen. Extract the needle and slowly press the plunger to release the nitrogen. Immediately insert the needle into the sample collection bottle and draw up 0.5 milliliters of the environmental sample.

Inoculate the drawn sample into the culture bottle containing media. Gently swirl the bottle and place it in an incubator at the desired temperature. The direct cell counts before inoculation were often below the detection limit.

After inoculation, the cell counts increased over 10 million cells per milliliter on days 30, 37 and 44. Although the cell counts were below the detection limit before adding the inoculum, NGS indicated a basal presence of preinoculum microorganisms in the sediment and the major genus geobacillus was stable on days 8, 15 and 22. After inoculation with the BLM-1 inoculum on day 23, geobacillus no longer dominated and a new major genera arose along with a multitude of minorly present genera.