Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

The overall goal of this set of consecutive methods is to visualize and monitor vitality parameters in DNA repair in reviving Bacillus subtilis spores after Low Pressure Plasma Treatment using time resolved confocal fluorescence microscopy and scanning electron microscopy. Our method can help answer key questions in the field of Microbial Inactivation and Sterilization. It helps us to analyze the detrimental effect of low pressure plasma treatment on biological indicators such as Bacillus subtilis spores.

The main advantage of our technique is that we combine several methods view determination, scanning electron microscopy and monitoring the DNA repair using time result fluorescence microscopy in order to verify the detrimental effect of low pressure plasma treatment. To carry out spore production, transfer a five milliliter overnight culture of the respective B.subtilis strain supplemented with appropriate antibiotics to 200 milliliters of double strength liquid shafer sporulation medium and cultivate it with vigorous aeration at 37 degrees Celsius for at least 72 hours or longer until greater than 95%of the culture has been sporulated. Harvest spores in 15-milliliter tubes by centrifugation at 3000 G for 15 minutes and purify the samples using sterile distilled H2O in repeated washing steps.

Check for purity and germination status by face contrast miscroscopy and ensure that spore suspensions consist of greater than 99%of phase bright spores and are free of vegetative cells, germinated spores and cell debris, otherwise further microscopy experiments can be disturbed. Determinate the spore titer by plating 50 microliters of ten-fold serial dilutions on LB agar to calculate the CFUs and incubate the plates at 37 degrees Celsius overnight. Following CFU determination, adjust the sample to 10 to the 9th spores per milliliter by concentrating or using sterile water to dilute the samples.

Place a sample carrier in the form of sterilized microscopic slides or round 25-millimeter cover slips inside the electrically operated aerosol spraying unit in alignment with the nozzle. Transfer the spore culture to the nozzle fluid inlet and initiate the spraying at 0.15 seconds with a pressure of 1.3 bar. The sprayed spore suspension forms a thin film on the microscopic slide that dries rapidly within seconds to form a uniformly distributed spore mono layer.

Store the treated sample carriers in a sterile container at room temperature. To clean and warm up all surfaces in the plasma system, initiate the system at five Pascals with Argon plasma at 500 watts for five minutes. The pretreatment reduces the sticking of molecules from ambient air such as nitrogen, oxygen, and water during venting of the chamber.

After venting the chamber, carefully place the samples on glass racks in the center of the reactor vessel. Close the chamber and evacuate it. Afterwards, use the desired process gas to fill the chamber and regulate the pressure in the system to five Pascals.

Then, start the plasma process. After the defined process time, turn off the power and gas supply and carefully vent the system to prevent blowing the samples from the sample holder. After ventilation, remove the samples and store them in a sterile container.

For non-plasma treated controls, expose samples to vacuum only in the presence of the process gas equivalent to the longest applied plasma time. Prepare a solution of autoclaved 10%polyvinyl acetate or PVA and use 500 microliters of it to carefully cover the sample carriers. After letting them air dry for four hours, use sterile forceps to strip off the dried PVA layer that now contains the spore sample and transfer it to a 2-milliliter reaction tube.

Add one milliliter of sterile water to the tube and vortex it to dissolve the PVA layer to recover greater than 95%of the spores capable of germination. Use sterile water to serially dilute the sample at one to 10 in a 96-well plate. After plating 50 microliters of each dilution on LB agar, and incubating at 37 degrees Celsius overnight, count the number of colonies and determine the CFUs per milliliter.

For germination experiments, prepare a one-millimeter thick 1.5 LB agar pad by boiling 700 microliters of medium and pipette it into a sterile microscopy petridish. After 10 minutes, use a sterile scalpel to cut out an eight millimeter by eight millimeter by one millimeter LB agar pad, and carefully transfer the agar on top of the spore monolayers which are resting on 25-millimeter glass cover slips already mounted in an imaging chamber. The agar put method combines two specific functions.

It stabilizes the samples in the optic plane during laser microscopy and it restricts lateral cell movement. Apart from using it for germination essays, this method can be applied to other microorganisms as well as to eukaryotic cells. After covering the sample with agar, quickly transfer the glass cover slip into an imaging chamber.

Keep the sample at 37 degrees Celsius on a heated stage during the entire imaging process. Use at least three biological replicates for each condition. Following the imaging of the samples by automated confocal laser scanning and bright field microscopy, record time lapse series with a laser power of 2.6%and set the confocal aperture to five aer units and a sample frequency of one frame per 30 seconds from zero to five hours depending on the experiment.

Applying high doses of monochromatic light, may completely inhibit spore germination during laser microscopy. Therefore, use the laser intensity and just longer intervals obtaining single frames. In case of spore aggregation, multi-layered spore distribution or contamination by dust particles, blocking of the plasma treatment or shadowing might occur and enable germination of shadowed spores.

To carry out scanning electron microscopy, once the spore monolayers have been sputter coded with gold palladium, image the samples with the field emission SEM operated at five kilovolts acceleration voltage including an inlan secondary electron detector to reveal topography contrast. As shown in this graph, plasma treatment of the B.subtilis spores results in a decrease in survival with increasing duration of the plasma treatment. These SEM images reveal characteristic longitudinal ridge-like structures, which are constantly visible in all treated and untreated spores.

Plasma treatment up to 30 seconds, induces no significant changes of the spore surface morphology in comparison to controls. Increasing duration of plasma treatment leads to a more granular surface and small cracks and fissures can be observed in spores treated for 120 or 240 seconds. In these time resolved confocal laser scanning microscopy experiments, almost all spores treated with vacuum as a control germinate and develop a rod-shape cell morphology with a rather uniform length of individual cells.

In contrast, spores treated for 15 seconds with plasma already show a reduced germination capacity of less than 75 plus or minus four percent. In addition, cells grow either into extensively long rods or remain small and stick in the spore coat. After 30 seconds of plasma treatment, only 25 plus or minus six percent of the spores germinate and vegetative cells are notably delayed in growth and vary in length.

While attempting this procedure, it's important to confirm the quality of the spore monolayers using phase contrast microscopy in order to ensure that there are no overlapping or germinating spores. After its development, the agar pad method for image stabilization, paved the way for researchers in plasma sterilization and live cell microscopy. After watching this video, you should have a better understanding of how to prepare spore monolayers on glass slides, determinacy of use using plasma treatments for spore inactivation and conduct live cell and scanning electron microscopy.

In contrast to other decontamination methods, for example ethylene oxide or gamma radiation, plasma sterilization is an efficient and safe approach to reduce a number of unwanted microorganisms on almost any kind of surface.