## 5 Results and Discussion
### Microbiological Analysis
#### 5.3.2 Cultural Analysis  
Culture techniques confirmed what was seen under microscopy. In fact, fungi and photosynthetic microorganisms were the main microorganisms isolated and involved in the biological patinas observed on the _tesserae_ surfaces. Results are summarized in Figures 6-8 and Tables 4 and 5. Fungi were isolated  
Figure 5: Adhesive tape samples stained with Acridine Orange (AO), seen under an epifluorescent microscope (EFM). Photosynthetic microorganisms are colored in orange/red being autofluorescenceent; bacteria and fungi are colored in green by the effect of fluorochrome AO. (**a**) Sample MRC1: mixed microbial community with cyanobacteria, algae, bacteria and fungi; (**b**) sample MRC3: mixed population mainly eukaryotic _Chlorella_ like type algae and bacteria; (**c**) sample MRC4: mixed population of eukaryotic algae (_Chlorella_-like and _Desmodes_-like and bacteria embedded in a polysaccharide matrix, EPS; (**d**) sample MRC6: massive presence of fungi, mainly black fungi as was seen by the thick dark cell wall. Bars are 10 \(\upmu\)m.  
from all samples both from the adhesive tape streaked on DRBC (with the exception of the treated samples MRC2 and MRC5) (Figure 7) and from the paper disc inoculated in BG11 liquid medium (except sample MRC2) (Figures 6 and 8). Fungal growth was observed in the DRBC plates mainly in strict contact with the adhesive tape; only sample MRC4 showed abundant and composite fungal growth (Figure 7). Black fungi were isolated with the technique of a wet paper disc placed in BG11 liquid medium (Figure 6 and Table 5). Figure 6: Main microorganisms growing in BG11 liquid. MRC1, MRC3 and MRC4 both fungi and algae were isolated. Scarce growth of algae in MRC2. Only fungi were isolated from MRC5 and MRC6. Figure 7: Growth of fungi in Dichlorane Rose Bengal Chloramphenicol (DRBC) medium. **(a)** Sample MRC1 with evident fungal colonies around and below the adhesive tape; **(b)** sample MRC2 with only yeasts; **(c)** sample MRC3 with fungi around the adhesive tape; **(d)** sample MRC4 with composite fungal growth; **(e)** MRC5 no fungal growth; **(f)** sample MRC6 with fungal growth around the adhesive tape. Algae and/or cyanobacteria were isolated from samples MRC1, MRC3 and MRC4 as shown in Figure 6. In addition, a slight growth was visible after three months for the disc sample MRC2 inoculated in BG11. No growth was observed for MRC5 and MRC6. Considering the abundant evidence of photosynthetic species in the MRC1-4 samples, an aliquot of culture grown in the flasks was taken and re-isolated in BG11 agar plates, and the type of photosynthetic microorganisms were observed under an EFM microscope as shown in Figure 8.  
In general, the mosaic was affected by different alteration forms, caused by a massive colonization of several microbial populations varying in relation to the sampling areas and to the bioreceptivity of the material, also considering portions that underwent previous restoration interventions. In particular, the MRC4 sample showed a greater and massive colonization than the other 5 specimens (MRC1-3, 5-6), displaying that mortar, as also clearly visible to the naked eye (Figure 3), had the higher bioreceptivity, allowing the colonization of both fungi and photosynthetic organisms (Tables 4 and 5). According to literature [29], the area in which MRC4 was sampled, underwent previous restoration works. The thesis was further confirmed by POM investigations, showing that in the same area the mosaic _tesserae_ were made of different stone materials (i.e., sedimentary and volcanic rocks), some of which were replaced during the restoration intervention. Additionally, some of the _tesserae_ sampled in the same area of sample MRC4 (Figure 2, such as the limestone ones) showed numerous micro-cracks, becoming suitable for the colonization of endolithic microorganisms.