Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electro spray ionization, mass spectrometry or CS EMS enables the analysis of bacterial volatiles without the requirement of any sample.Pre-treatment. The headspace of the bacterial culture is displaced by carbon dioxide into the cssi reaction chamber. As the volatiles traverse the SSI reaction chamber, they pass through the electro spray cloud and become ionized.

Once ionized, the volatiles are pulled into the mass spectrometer for analysis. Excess carrier gas and unreactive bacterial volatiles are passed through a 0.22 micron filter to remove any bacterial agents as an additional measure of protection vent to a chemical hood. Ultimately, this CSI MS protocol offers bacterial volatile fingerprints within minutes.

We anticipate that this technology could be used in many applications, including exhale, breath analysis for infectious diseases, or even applied to food systems where we might be able to rapidly detect pathogens that are on or N our food. There are several main advantages to this technique. Over existing methods like GCMS sift, MS and PT, RM S first, the volatiles don't require any pre-treatment prior to analysis.

Second, it's possible to fragment specific ions in the volatile mixture, which makes compound identification easier. And third, it's rapid. It typically takes less than five minutes.

To analyze each sample, Choose the appropriate vessel for growing your culture, considering the growth requirements of the species, as well as the efficient delivery of volatiles to the mass spectrometer. In this demonstration, we have selected standard 100 milliliter pyre media bottles. The bacterial volatiles are delivered to the mass spectrometer by a stream of carrier gas to assemble the carrier.

Gas lines fit the culture bottles with threaded caps that have at least two lure ports. Insert gas inlet and outlet lines through the lure ports, and plug any additional ports Prior to culturing your samples. Pressurize the vessels and submerge in water to check for leaks.

Gas Leaks are a primary cause of atypical results in the form of weak or absent. Volatile ion signals grow overnight. Cultures of e coli, K 12, and pseudomonas aerogen, PAO one in LB Lennox preparing two biological replicates for each species.

Prepare nine sterile culture bottles containing 50 milliliters of LB Lennox and one un inoculated bottle to be used as a blank inoculate each overnight culture into two sample bottles. Creating technical replicates, incubate the cultures and the blank for 24 hours at 37 degrees Celsius. Since the CEMS is specifically designed to sample volatiles, limit the use of fragrant personal care products before using the instrument tightly.

Cap all volatile chemicals in the lab and control air drafts as much as possible during testing to prevent contamination of your instrument and the gas transfer lines. With viable biological agents, install filters of the appropriate pore size into the carrier gas line. The filters will not interfere with the transfer of volatiles to the sesi reaction chamber, but may slightly impact the efficiency of aerosol transfer.

In order to verify that the voltage supply is turned off and that the system is discharged of electricity, check the indicator lights on the voltage supplier off. Ensure the voltage on the multimeter is zero and ground the electrical leads. At this time, install the electro spray solution for analysis.

In positive ion mode, we use an electro spray solution of 0.1%formic acid, 5%methanol, and 94.9%water In order to achieve optimal signal intensity and stability, turn on the carbon dioxide carrier gas and set the flow rate to two liters per minute. Apply pressure to the electro spray reservoir to initiate the delivery of the electro spray solution to the reaction chamber at a flow rate of five nanoliters per second. Next turn on the voltage supply and adjust the voltage to 2.5 kilovolts.

The applied voltage affects the signal ion intensity and the stability of the electro spray Tailor cone for our system voltages between two and five kilovolts. Provide the best results in positive ion mode exercise Caution as at this point the metal surfaces of the ionization source are capable of delivering a dangerous shock. Now set up a tuning method for monitoring the SEMS spectrum.

Clear the multiple channel acquisition or MCA checkbox. Set the acquisition time to 10 to 15 minutes and start data acquisition. Observe the spectra of the carrier gas background and make fine tuned adjustments to the applied voltage to obtain a stable total ion chromatogram and reproducible scans.

Continue to collect spectra and A TIC for five minutes to ensure the instrument is stabilized with instrument insured. Enter the acquisition parameters as appropriate for your experiment. Record a carrier gas background spectrum for Future experiments.

To collect a blank Spectrum. Direct the carrier gas flow through the bypass lines, and then attach the blank sample with the valves closed to the transfer lines of the instrument. Open the valves to the sample bottle and close the valves of the bypass lines.

For reproducible spectra. Allow the system to equilibrate for 30 seconds during which time the humidity in the reaction chamber is stabilizing. To ensure that the system is equilibrated, monitor the TIC, which will change during the equilibration period followed by stabilization.

Once the system is equilibrated, initiate spectrum collection. After the spectrum is collected, remove the sample bottle by first opening the carrier gas bypass lines. Then closing the sample valves.

And lastly, removing the sample bottle. Flush the system with the carrier gas for two to four minutes to remove the moisture and absorbed volatiles from the transfer lines and prevent sample to sample carryover. Collect volatile fingerprint data for each bacterial sample intermittently collecting additional blank spectra to ensure thorough blank Subtraction.

The Positive ion mode volatile fingerprints for e coli and p aerogen. Grown aerobically in LB Lennox for 24 hours at 37 degrees Celsius are distinct. The e coli volatile spectrum is dominated by indu at a mass to charge ratio of 118, whereas the spectrum of pseudomonas aerogen contains a larger variety of protein addable peaks.

While attempting This procedure, it's important that you record the instrumental parameters that you have optimized for your analysis, such as the applied voltage, the electro spray solution composition, and the carrier gas flow rate. It's also important that you maintain records of your representative spectra so that you can reproduce your analyses in the future. Don't forget that working with pathogen bacteria and high voltage could be extremely hazard.

Therefore, precaution such as limiting the access to css CMS during the experiment should always be taken. So now you should be able to reproducibly obtain a CSS, EMS spectra for the bacterial volatiles in your System. Good Luck.