## 3 Results and Discussion
### TEM for AuNPs Samples with pH Adjusted Prior to Production  
TEM analysis was performed for the samples at pH 2, 6, and 10. The results are shown in Figure 4.  
As shown in Figure 4, the gold nanoparticles were not agglomerated at pH 2 before washing (Figure 4A), but they agglomerated afterwards (Figure 4C,D), indicating that they were unstable at the corresponding pH. The insert in Figure 4A shows some closely connected AuNPs without distinct boundary lines. This is in accordance with the observation from spectrophotometry as well as the results of [PERSON] et al. [31] (see Figure 2). However, we did not perform TEM for the sample with an initial pH of 4, and thus we cannot conclude that the AuNPs at pH 4 were rod-shaped (according to [PERSON] et al.), but the size distribution assay (see Figure 14) showed that that they were polydispersed and their sizes were over 100 nm.  
After washing, the nanoparticles were in a stable form at pH 6 and 10 and did not agglomerate (Figure 4B,E,F). The inset in Figure 4B shows that some AuNPs in close contact were separated by distinct boundaries. Image analysis confirmed this, and the thresholding procedure for particles easily separates them. The particle size analysis for the sample with an initial pH of 6 is shown in Table 1. Statistical analysis of the measured AuNP diameters confirmed the Gaussian distribution of the measured values (Figure 5). The mean diameter of AuNPs for the sample with an initial pH of 6 was \(16\pm 5\) nm. The value of the mean diameter of the AuNPs differed from the value of the z-average determined by DLS (Figure 14). A similar discrepancy in the size of AuNPs determined by TEM and DLS was described by [PERSON] et al. for AuNPs with a nominal diameter of 15 nm [32]. According to [PERSON] et al., the digital images from TEM differ from the DLS results because DLS measures the hydrodynamic diameter, unlike TEM, which only measures the diameter of the nanoparticles [33]. DLS results usually give larger sizes than TEM, and a combination of methods is needed to properly understand the size and distribution of the AuNPs [32]. Although TEM can show precise AuNP diameters, it cannot not show the dispersity and stability of the nanoparticles, which can be distinguished using other techniques such as DLS and spectrophotometry. For example, it was shown that for silica nanoparticles, DLS showed their solution behavior, while this could not be detected by TEM [32]. Analysis of the average size of the samples with the Zetasizer revealed that some samples of the AuNPs discussed in this article were approximately in a normal distribution.  
Figure 3: Samples with different pH levels before and after the washing process exhibited different behavior: (**A**) control (mycoilium-free supernatant); (**B**) gold nanoparticles prepared at pH 10 before washing; (**C**) gold nanoparticles prepared at pH 10 after washing; (**D**) gold nanoparticles prepared at pH 4 before washing; (**E**) gold nanoparticles prepared at pH 4 after washing.  
Figure 4: TEM images obtained from different samples: (**A**) unwashed sample at pH 2; (**B**) washed sample at pH 6; (**C**,**D**) washed sample at pH 2; (**E**,**F**) washed sample at pH 10. Scale bars: (**A**) = 200 nm; (**B**) = 200 nm; (**C**) = 200 nm; (**D**) = 1 \(\mu\)m; (**E**,**F**) = 100 nm; the inserts in (**A**,**B**) = 100 nm.