## 3 Results and Discussion
### Morphology and Cell Wall Integrity of the Plasma-Treated C. pruinosa Spores  
The results described in Section 3.1 prompted an investigation into what kind of cellular damage from the soft plasma jet could lead to the death of the spores. Because the fungal spore surface is covered with a protective layer of the cell wall [26], we examined the possibility of cell wall damage. Figure 5 shows SEM images of the _C. pruinosa_ spores before and after soft plasma jet treatments for 3 min and 10 min. Cell morphology of the plasma-treated spores showed remarkable changes as compared with that of control spores. The plasma-treated spores were shrunken, ruptured, and flattened. The deformation can be explained by the fact that the intracellular space had been at least partially emptied of its contents and/or cell rigidity was diminished. These explanations imply that spore cell wall was deformed by the plasma treatment. Because cell membranes control traffic into and out of cellular components, damaged membranes could allow intracellular components, such as nucleic acids, proteins, and other metabolites, to possibly leak out and/or make the cell vulnerable to attack by soft plasma jet-triggered radicals and/or reactive species that could induce cell membrane pore-opening or breakage.  
Figure 4: Fluorescence intensity of \(H\)2_DCF-DA_-stained _C. pruinosa_ spores for intracellular reactive oxygen species generated by a soft plasma jet. C, 1, 2, 3, 6, and 10 represent intracellular ROS for the control, 1 min, 2 min, 3 min, 6 min, and 10 min plasma-treated _C. pruinosa_ spores, respectively.  
To determine the level of structural damage in the membranes of spore cell walls, we stained plasma-treated and untreated spores with PI dye, which is impermeable through a normal membrane in living cells but permeable through the damaged membranes in dead cells. As shown in Figure 6, the fluorescence intensity of PI-stained _C. pruinosa_ spores is clearly more intense in plasma-treated spores than in untreated spores, indicating that cell membranes are damaged and that cell wall integrity has decreased in the plasma-treated spores.  
Figure 5: Typical SEM images of _C. pruinosa_ spores treated by a soft plasma jet. C, 3, and 10 represent SEM images of the control, 3 min, and 10 min plasma-treated spores, respectively.  
Figure 7 exhibits the CD spectra of the _C. pruinosa_ spores treated by the soft plasma jet. The control CD spectrum of the plasma non-treated spores exhibited a negative dip at about 230 nm and 285 nm. The CD dip at 230 nm might be attributed to the spore cell wall and/or membrane proteins [27], and the CD dip at 285 nm is attributed to ergosterol in the spore cell wall. The CD dip at 230 nm of the plasma-treated _C. pruinosa_ spores was lower than that of the control. A decrease in the spore chirality was thought to be originated from the degradation of the spore cell wall and/or membrane proteins by the soft-plasma jet.  
Figure 6: Fluorescence intensity of PI-stained _C. pruinosa_ spores treated by the soft plasma jet. C, 1, and 3 represent the fluorescence intensity of PI-stained _C. pruinosa_ spores for the control, 1 min, and 3 min plasma-treated spores, respectively. Cell wall integrity was investigated by measuring the fluorescence intensity of _C. pruinosa_ spores stained with a membrane-impermeable fluorophore, PI.  
Figure 7: Circular dichroism spectra of the _C. pruinosa_ spores treated by the soft plasma jet. C, 1, and 3 represent CD spectra of the control, 1 min, and 3 min plasma-treated _C. pruinosa_ spores, respectively.