## 3 Results
### TEM Analysis  
Changes in the mycelium ultrastructure were observed in the samples supplemented with hexamethylene-1,6-bis-(\(N\),\(N\)-dimethyl-\(N\)-dodecylammonium bromide) C6. In an earlier study [16], the authors observed large variations in the morphology of _A. brasiliensis_ mycelia treated with this compound compared with other gemini surfactants (C5, A5, and A6).  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Type of Sample** & **Biocide Concentration** & **Dice Index of Similarity (\%)** \\ \hline \multirow{3}{*}{C5} & \% MIC & 92.3 \\  & MIC & 62.5 \\  & 2 MIC & 54.5 \\ \hline \multirow{3}{*}{C6} & \% MIC & 92.3 \\  & MIC & 66.7 \\  & 2 MIC & 54.5 \\ \hline \multirow{2}{*}{A5} & MIC & 72.7 \\ \cline{1-1}  & \% MIC & 72.7 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Similarity of protein profiles of _A. brasiliensis_ ATCC 16404 after treatment with gemini surfactants.  
Figure 1: (**A**) Effect of neutral (A5 and A6) and cationic (C5 and C6) gemini surfactants on extracellular protein profiles of _A. brasiliensis_ ATCC 16404 at MIC; changes in protein profiles after (**B**) C5 and (**C**) C6 treatment at \(\%\) MIC, MIC, and 2 MIC in comparison with the sample without biocide (K). The sizes of the bubbles are related to pixel intensities in the protein profile patterns.  
Transmission electron microscopy (TEM) analysis revealed that the control cells of _A. brasiliensis_ were well organized (Figure 2A,B). In cells surrounded by well-defined cell walls (CW), regular nucleoli (N), mitochondria (M) with numerous cristae and electron-dense matrix, and vacuoles (V) with fibrillal material (Figure 2A) or electron transparent vacuoles (Figure 2A,B) could be distinguished. The hyphae in _A. brasiliensis_ treated with hexamethylene-1,6-bis-(_N_,_N_-dimethyl-_N_-dodecylammonium bromide) at 0.5 MIC exhibited different degrees of disorganization (Figure 2C-E). Some cells showed severe ultrastructural changes, with only remnants of organelles in the form of membranous structures (MS) and multivesicular bodies (MB). Even the cell walls exhibited loose structures (* Figure 2C). However, after treatment at 0.5 MIC, some cells were much less affected (Figure 2D). Organelles, such as nucleoli, mitochondria, and vacuoles, did not show disorganization but the cytoplasm was condensed and symptoms of membrane destruction were visible in the form of membranous structures and large lipid droplets (L). Some electron-dense material, similar to that which stuck to the cell walls in the form of more or less large deposits (arrow Figure 2E), was also visible in the small vacuoles (arrow Figure 2D,E). More severe effects of cationic gemini surfactant C6 were observed in the growing part of the hyphae where the cell wall structure was distended (** Figure 2E). Higher concentrations of hexamethylene-1,6-bis-(_N_,_N_-dimethyl-_N_-dodecylammonium bromide),at MIC (Figure 2F,G) and 2 MIC (Figure 2H,I) caused the death of _A. brasiliensis_ hyphae. Inside the cells, only membranous structures, multivesicular bodies, and electron-dense granules were observed (arrows). All three were numerous in cells treated at MIC (Figure 2F,G) and random in cells treated at 2 MIC (Figure 2H,I). Regardless of the concentration of cationic gemini surfactant C6, the cell wall maintained its integrity, although it was distended (* Figure 2G), especially at the hyphae tips (** Figure 2F).  
Figure 2: **(A,E,F,H)** TEM micrographs of longitudinal-sections and (**B–D,G,I**) cross-sections of _Aspergillus brasiliensis_: (**A,B**) control hyphae; (**C–E**) hyphae treated with hexamethylene-1,6-bis-(N,N-dimethyl-N-dodecylammonium bromide) C6 for 48 h at 0.5 MIC; (**F,G**) hyphae treated with C6 for 48 h at MIC and (**H,I**) hyphae treated with C6 for 48 h at 2 MIC. CW—cell wall; L—lipid; M—mitochondrion; MB—multivesicular body; MS—membranous structure; N—nucleus; V—vacuole; arrows-electron-dense deposit.