Materials and Reagents

Petri dish 100 mm diameter (SYM Laboratorios, catalog number: 7730SYM)
Pipette tips (Thermo Scientific, catalog numbers: 94300210 [1,000 μl]; 94300110 [100 μl] and 101-96R-Q [10 μl])
5 mm diameter sterile stainless steel punch (Avantor, catalog number: 10194-928)
0.22 μm sterile filters (Millipore, catalog number: SLGVV255F)
5 ml syringe for filter sterilization (JTC, catalog number: 05LSA-NPRO)
15 ml tubes (Axygen, catalog number: SCT-15ML-500)
Caliper (MIYUTOYO, catalog number: 531)
Erlenmeyer flask (Pyrex, catalog number: CLS44501L-1EA)
Fungal isolates to test
Culture media Potato dextrose agar (PDA) (Bioxon, catalog number: 210700)
Distilled water (Meyer, catalog number: HDS 0345)
Selected heavy metals, used as salts:
Chromium Cr(III) (CrCl₃) (Sigma-Aldrich, catalog number: 450790)
Mercury Hg(II) (HgCl₂) (Sigma-Aldrich, catalog number: 215465)
Lead Pb(II) (Pb(C₂H₃O₂)₂) (Meyer, catalog number: HDS 1955)

Equipment

Analytical balance (Ohaus Adventurer, catalog number: H-5276)
Pipettes (Thermo Scientific, catalog numbers: 4651270N [1,000 μl]; 4651230N [100 μl] and 4651280N [10 μl])
Autoclave (Evar, catalog number: EV-24)
Microbiological incubator (Thermo Fisher Scientific, catalog number: 50129111)
Microbiological safety cabinet (Thermo Fisher Scientific, catalog number: 1310TS1)

Software

T-test package ggpubr (R package version 0.1.7. https://CRAN.R-project.org/package=ggpubr)
R Core Team (https://www.Rproject.org/)

Procedure

Heavy metal stocks and culture media
1. Prepare stocks under sterile conditions in six different concentrations: 2.5 mM, 5 mM, 7.5 mM, 10 mM, 15 mM, and 20 mM of Cr(III), Hg(II), and Pb(II).
2. Dissolve the meal salts for each concentration in distilled water, filter sterilize using 0.22 µm filters, and store at room temperature until use (up to 3 months).
3. Prepare PDA solid media in Erlenmeyer flasks using distilled water and sterilize by autoclaving (121°C and 15 psi pressure for 20 min). One flask of agar per heavy metal concentration and one without heavy metal solutions, to use as a control for each fungal isolate, are needed.
4. After sterilization, let the agar cool at 50°C and add the heavy metal solutions under sterile conditions. Pour 20 ml of supplemented agar per Petri dish. Store at 4°C until use (up to 3 months).

Cultivation methods
1. Under sterile conditions, inoculate fungal isolates (1 × 10⁶ conidia/ml) on PDA without heavy metal supplementation and incubate for 14 days at 28°C to induce conidiation (pre-grown state).
2. Punch out agar blocks of fresh mycelium from the edge of the colony using a 5 mm diameter sterile stainless steel punch after the first incubation time (pre-grown state). This step should be performed in the microbiological safety cabinet. Several plugs per strain are necessary depending on the number of plates with different heavy metal concentrations.
3. Inoculate the plates with different concentrations of heavy metals and the control in the center with one plug per plate and incubate at 28°C for 14 days.
4. Six replicates should be done for each fungus and heavy metal concentration and for control cultures. Representative fungal growth of two isolates is displayed in Figure 1.
  
  Figure 1. Fungal heavy metal resistance at four different concentrations after 14 days of incubation on PDA. The figure displays Periconia citlaltepetlensis (top) and Alternaria sp. growth (bottom) without heavy metals (A, F) and in the presence of Cr(III) at 2.5 mM (B, G), 5 mM (C, H), 7.5 mM (D, I), or 10 mM (E, J).
Minimum inhibitory concentrations and tolerance index
1. After inoculation, perform daily measurements of colony size with caliper.
2. Calculate the radial growth based on five measurements (in millimeters) during 14 days of growth by subtracting the initial diameter (5 mm) of the plug (Figure 2 and Table 1).

Data analysis

MIC is defined as the minimum concentration that inhibited the visible growth of isolates after 14 days (Joo and Hussein, 2012).
Results are graphed and statistically evaluated with the unpaired two-samples Student's t-test using T-test package ggpubr (Kassambara, 2018) in R (R Core Team, 2018). Statistics and graphs will help to understand the capacity to tolerate metals of each fungus tested (Figure 2).

Figure 2. Effect of different concentrations of lead on Alternaria sp. growth. The fungus was cultured at 28°C for 14 days on PDA agar at six different concentrations of Pb(II) (2.5 mM, 5 mM, 7.5 mM 10 mM, 15 mM, and 20 mM). Error bars represent the standard error of the mean of three technical replicates in at least two independent measurements.of each incubation period. n = 5. ***, P-value < 0.001 for fungal growth on lead compared with the control.

The TI calculation, which is an indication of how an organism tolerates stress (Mohammadian-Fazli et al., 2015), is determinate for each metal and fungal strain with the daily measurements of colonies (Figure 3). TI should be calculated based on measurements of heavy metal fungal growth (HMFG) at different concentrations divided by control culture fungal growth (CFG).
A TI close to 1 indicates that the fungus is resistant to the tested heavy metal concentration (Figure 3).

Figure 3. Measurements of Curvularia sp. at two different concentrations of chromium. The figure shows control fungal growth (CFG) (A) and on 2.5 mM (B) or 5 mM (C) of CrCl3 (HMFG). Red arrows represent daily measurements to monitor fungal growth.

To select the most tolerant fungi, the values of TI and MIC assays must be assessed. Representative data of MIC and TI values of three isolates are displayed in Table 1.

Table 1. MIC and TI of three extremophilic fungi at different concentrations of Cr(III), Hg(II), and Pb(VI) growth on media for 14 days

MIC: Minimum inhibitory concentrations in millimolar.
TI: Tolerance index calculation at 7.5 mM and 10 mM of heavy metals.
Notes
1. This is a simple, easier, and cheaper method to generate relevant data about metallo-tolerance and a possible bioremediation process.
2. The media (PDA) proposed here is useful with a broad range of fungal species, even those which are difficult to culture.
3. Fastidious isolates (e.g., extremophilic and psychrotolerant fungi) were successfully tested with this protocol.
Acknowledgments
The author thanks CONACYT Postdoctoral scholarship (005352) and appreciates the collaboration of Fernández-Tellez IE in Figure 1. This work is dedicated to empowered women who climb glaciers, fight against misogyny, and for equality in Mexico. This protocol is based on the original research paper (Calvillo-Medina et al., 2020).
Competing interests
The author declares that there are no conflicts of interest.
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