In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

The overall goal of this procedure is to culture bacteria in the system for analysis at the liquid vacuum interface, or SALVI, for subsequent imagining of the biofilms in their natural hydrated state with vacuum-based techniques such as Time-of-Flight Secondary Ion Mass Spectrometry. We present a method that helps answer key questions in environmental microbiology by visualizing the chemical-spatial distribution of living biofilms using imaging mass spectrometry like SIMS. The main advantage of this technique is that we can introduce biofilms alive to a mass spectrometer and many other analytical platforms to obtain multiplex measurements.

Although Shewanella oneidensis is used in this video, this method can also be applied to anaerobic chambers for biofilm growth. The latter requires a more stringent growth environment. Visual demonstration of this method is critical as the tubing setup steps are difficult to learn.

Specific placement of tubing and care are necessary when transferring to the biosafety cabinet. Using a 22 gauge needle, carefully punch a hole through a serum bottle rubber stopper. Cut 20 inches of 1/32 inch PTFE tubing with a razor, such that one end of the tubing is pointed.

Using the pointed end, thread the PTFE tubing through the hole in the top portion of the rubber stopper. Push the tube through until roughly two centimeters is through the stopper and cut the pointed end of the PTFE tube with a razor to have a flat end. Remove the plunger of a five milliliter syringe and fit the rubber stopper into the open end of the syringe.

The two centimeter end of PTFE tubing is within the barrel of the syringe. This is the bubble trap. Wrap the bubble trap with aluminum foil and secure it with autoclave tape.

Then autoclave the foil package to sterilize the tubing with a gravity cycle at 121 degrees celsius for 30 minutes. Store the sealed foil package at room temperature until it is ready for use. This procedure specifically covers culturing a Shewanella oneidensis MR-1 biofilm at room temperature after 24 hours of incubation.

However, different strains of bacteria require different growth conditions and lengths to achieve a desirable thickness for analysis. The SALVI tubing system is made up of the syringe containing the liquid reservoir, which is attached with a PEEK injector to the PTFE tubing. This is attached to the drip chamber, which has a PEEK injector fitting attached to the SALVI inlet tubing.

The drip chamber is also attached to the outlet container that the SALVI outlet tubing is sealed to. To promote sterility of the tubing system, detach the syringe from the syringe pump by unscrewing the metal syringe holder. Then bring the syringe with the attached tubing to a sterile biological safety cabinet.

Use a new SALVI device and attach a PEEk fitting and injector to one end of a PTFE tubing. SALVI devices are prepared fresh for each experiment following the device fabrication, which is detailed in previous research and patents. Next, aspirate two milliliters of 70%ethanol and deionized water into a syringe and connect the syringe to the PEEK fitting on the SALVI.

After connecting the syringe to a syringe pump and attaching the outlet of the SALVI to a waste bottle, allow the ethanol solution to run through the SALVI at 20 microliters per minute for one and a half hours. Then aspirate four milliliters of sterilized deionized water into a syringe and connect it to the inlet of the same SALVI. After connecting the syringe to a syringe pump, allow the water to run through the SALVI at 20 microliters per minute for at least three hours.

Within a biosafety cabinet, open the prepared foil packet and connect a sterile PEEK injector and fittings to the end of the PTFE tubing. Aspirate approximately three milliliters of sterile medium into a syringe and attach it to the PTFE tube. Invert the five milliliter drip chamber and using a sterile syringe inject the growth medium into the drip chamber until it reaches a total volume of one milliliter.

Next, connect the end of the five milliliter syringe drip chamber to the inlet of the SALVI. Aspirate 10 milliliters of growth medium into a sterile syringe and connect it to the inlet of the drip tubing. Allow the drip tubing to be vertical.

The syringe pump should be placed on an elevated surface with drip tubing secured by tape and with the SALVI secured on a flat surface below. Cover the outlet bottle with foil or plastic paraffin film to minimize the probability of medium contamination. Allow the medium to run through the SAVLI at 20 microliters per minute for 12 hours.

This ensures that all traces of ethanol have been removed from the microchannel and tubing of the SALVI before inoculating with bacteria. For added protection, keep the SALVI microchannel chamber within a sterilized petri dish with the sides cut to fit the inlet and outlet tubing. Additionally, keep tape always on the window to protect the silicone nitride membrane and channel prior to image analysis.

We move the whole tubing system from the syringe pump to bring it to a sterilized biosafety cabinet. Additionally, remove the serum bottle from the incubator that has been inoculated as described in the text protocol. Clean the surface of the serum bottle stopper with 70%ethanol to prevent any contamination.

Then use a sterile syringe with an attached sterile 22 gauge needle to extract four milliliters of bacteria from the bottle. Detach the SALVI from the five milliliter chamber of the drip tubing and connect the syringe with inoculated medium directly to the inlet of the SALVI. Leave the drip tubing within the sterile aluminum foil packet or within the biosafety cabinet.

Attach the syringe with the SALVI and outlet bottle to the syringe pump. Run at 20 microliters per minute for three hours to inoculate the channel of the SALVI and allow for a multiple volume changes of the liquid contained within the SALVI. Following inoculation, disconnect the syringe with the SALVI from the syringe pump and bring it to the biosafety cabinet.

Attach the inlet of the SALVI back to the five milliliter drip chamber. Then aspirate 20 milliliters of growth medium into a sterile syringe and attach it to the inlet of the drip tubing. The flow rate should never be increased or decreased, as changing the flow rate would create sheer stress within the channel and detach the biofilm.

To avoid flow rate changes ensure there are no bubbles in the tubing the day before SIMS. It is important to be cautious of bubbles within the bottom of the five milliliter drip tubing where it connects to the SALVI tubing. Fresh medium can be injected into the end of the PEEK injector to ensure that no air will be forced into the SALVI.

Bring the tubing attached to the SALVI from the biosafety cabinet to the syringe pump. Allow the medium to pass through the tubing at at rate of two microliters per minute for six to ten days. Refill the SALVI with fresh medium as it runs out within the biosafety cabinet by attaching a new sterile syringe filled with 10 milliliters of growth medium to the SALVI.

Using light microscopy or confocal fluorescence imaging, the presence of a biofilm can be detected on the window of the SALVI. Shown here is the negative mass spectrum of a hydrated Shewanella oneidensis MR-1 biofilm within the microfluidic channel. It's obtained by NC2 liquid Time-of-Flight Secondary Ion Mass Spectrometry.

The deionized water spectrum is also shown. A comparison of 2-D images of peaks of interest in the MR-1 biofilm to deionized water are shown. Once interesting m over z values can be identified from studying the mass spectra potential peak identification is performed through the software library, in lab reference spectra, and a literature survey.

Once mastered, this technique takes three days to set up, in addition to the specific time required for the biofilm to grow within the microchannel, which is about five to six days at room temperature for Shewanella. After watching this video, you should have a good understanding of how to grow insatiable biofilms in SALVI devices. Generally, individuals new to this method will struggle because bubbles may form within the growth medium due to bacteria growth.

It is necessary to get rid of bubbles before the SIMS analysis. While attempting this procedure, it's important to remember to never change the flow rate after inoculation of bacteria is completed. Even in fluorofil dying steps before optical imaging.

Following this procedure, other methods like confocal fluorescence imaging can be performed in order to answer additional questions, like how thick the biofilm was at the time the SIMS took place. After it's development, the technique paved the way for researchers in analytical chemistry and microbiology to explore the chemical profiles of biofilms in their naturally hydrated states.