A Modified Slide-Embedded Scanning Electron Microscopy Preparation Method to Visualize Antagonistic Interactions Between Trichoderma viride and Fusarium sp.

Materials and reagents

Biological materials

1. Trichoderma viride TVI (Elamala Biotech Lab, Chellarcovil, Kerala)

2. Fusarium sp. isolate STSP (Elamala Biotech Lab, Chellarcovil, Kerala)

Reagents

1. Potato dextrose agar (PDA) (HiMedia, catalog number: MH096)

2. Acetone 99% (Hyma, catalog number: ASA2019)

3. Absolute ethanol 99.9% (MSB Chemical Limited, catalog number: 5268-39); ethanol solutions (10%, 20%, 40%, 60%, and 80%) were prepared in Milli-Q water and syringe-filtered (0.22 μm)

3. Glutaraldehyde 25% aq. solution (SRL, catalog number: 92577)

4. Sodium phosphate dibasic dihydrate (SRL, catalog number: 87258)

6. Sodium phosphate monobasic dihydrate (SRL, catalog number: 40597)

7. Milli-Q water

Solutions

1. 0.1 M phosphate buffer (pH 7.4) (see Recipes)

2. Glutaraldehyde fixative (see Recipes)

Recipes

1. 0.1 M phosphate buffer (pH 7.4)

Dissolve both components in 80 mL of Milli-Q water. Adjust the pH to 7.4 using 1 N HCl or 1 N NaOH and make up the final volume to 100 mL with Milli-Q water. Store at room temperature and chill the required volume to 4 °C before use. This solution can be stored at room temperature for approximately 1–2 weeks if prepared and handled aseptically. Without sterilization (e.g., filtration or autoclaving), it should be used within 24–48 h. When properly sterilized and stored in tightly sealed containers, it remains stable for up to 2 months. Because phosphate buffers are prone to microbial contamination, discard the buffer if cloudiness, particulates, or visible microbial growth is observed. For maximum stability and extended shelf life, store the buffer at 4 °C.

Note: Avoid using KOH to adjust the pH, as excess potassium may alter the buffer composition, disturb the Na⁺/K⁺ balance, and affect cellular integrity.

2. Glutaraldehyde fixative

Freshly prepare this solution and then syringe-filter it through a 0.22 μm filter before use. Chill all components to 4 °C prior to preparation (see Troubleshooting 1).

Caution: Toxic if inhaled, ingested, or in contact with skin.

Laboratory supplies

1. Petri dish 100 mm (Borosil Scientific, catalog number: 3160077)

2. Conical flask 500 mL (Borosil Scientific, catalog number: 4980024)

3. Kimtech Science^® Kimwipes^TM (Kimberly-Clark Professional, catalog number: 34155)

4. Inoculation loop (HiMedia, catalog number: LA019)

5. Syringe filter 0.22 μm (Sartorius, catalog number: 16532)

6. Disposable Pasteur pipettes (Genaxy, catalog number: GEN-153)

7. Parafilm M (Amcor, catalog number: PM992)

8. Microtips, 200–1,000 μL (Tarsons, catalog number: 521020)

9. Sterile tissue culture dish 60 mm (Tarsons, catalog number: 960020)

10. Sterile single-use syringe 10 mL

11. Forceps

12. Microscopic glass slide

13. Double-sided adhesive tape

14. Permanent marker

15. Rubber band

16. Newspaper

17. Non-absorbent cotton

Equipment

1. Scanning electron microscope (Carl Zeiss, model: EVO 18 Research)

2. Glass cutter (generic)

3. pH meter (HANNA instruments, model: HI98107)

4. Autoclave (EPS, model: EPS/VA-22)

5. Test tube rack (generic)

6. Laminar air flow (Clean Air Products, model: CAP412)

7. Microbiological incubator (Orbitek, model: LE)

8. Brightfield microscope (Carl Zeiss, model: Axioscope 5)

9. Bunsen burner (generic)

10. Refrigerator (2–8 °C) (generic)

11. Micropipettes 100–1,000 μL (Eppendorf, model: Research Plus Manual Single-channel pipette)

Procedure

A. Revival of fungal cultures from glycerol stocks

1. Prepare PDA according to the manufacturer’s instructions by dissolving the required quantity of medium in distilled water. Sterilize the PDA medium by autoclaving at 121 °C for 15 min.

2. Allow the PDA medium to cool to approximately 40–60 °C.

3. Aseptically pour the molten medium into sterile Petri dishes inside a laminar airflow chamber and allow it to solidify.

4. Inoculate on separate PDA plates with a loopful of conidial suspension (4 × 10⁸ spores mL^-1) of Fusarium sp. and Trichoderma viride preserved in 50% glycerol stocks at -80 °C.

5. Incubate the inoculated plates at 25 °C for 7 days to obtain actively growing cultures (Figure 1).

Figure 1. Fully grown fungal culture plates. (A) Seven-day-old culture dish of Trichoderma viride. (B) Seven-day-old culture plate of Fusarium sp.

B. Preparation of glass slide pieces

1. Sterilize a grease-free microscopy glass slide and mark ~1 × 1 cm squares using a permanent marker.

2. Cut the slides into small square pieces using a glass cutter along the markings made in the previous step.

3. Make a blunt cut at one corner of each glass piece to serve as an orientation marker and facilitate the localization of the interaction zone during SEM observation (Figure 2).

Critical: Make the blunt cut in a consistent orientation to enable easy identification of the glass piece and localization of the fungal interaction zone.

Figure 2. Slide piece with blunt end cut

4. Remove the glass debris generated during cutting and reclean the slide pieces with absolute ethanol to eliminate marker residues.

5. Wipe the cleaned slide pieces using lint-free tissue paper, wrap them, and sterilize them by autoclaving.

C. Slide embedding technique for fungal interaction studies

1. Prepare and sterilize PDA medium as described in steps A1–2.

2. Place sterile Petri dishes inside the laminar airflow chamber and mark a square corresponding to the dimensions of the glass slide piece on the bottom surface of each plate (Figure 3).

Figure 3. Petri plate marking. Marked regions indicate the positions for slide placement and fungal inoculation.

3. Pour molten PDA medium into the plates to a depth equal to the thickness of the slide piece (approximately 10 mL) and allow it to solidify.

4. Heat a sterile inoculation loop until red hot and aseptically remove a square section of agar corresponding to the marked dimensions from the solidified medium (Video 1).

5. Handle the autoclaved slide pieces using sterile forceps. Briefly dip the slide pieces in acetone, flame-sterilize them, and allow them to cool for 2 min.

Note: Acetone is used to remove residual grease and dust from the glass surface and serves as a combustion aid for subsequent flame sterilization.

Caution: Acetone is highly flammable. Keep it away from open flames except during the intended flame sterilization step. Handle acetone-soaked glass pieces with extreme care and follow appropriate fire safety precautions.

6. Carefully place the slide piece into the agar cavity created in the PDA plate (Figure 4A).

Figure 4. Slide piece placement and inoculation. (A) Slide piece placed on the agar surface. (B) Inoculation zones labeled for Fusarium sp. (F) and Trichoderma viride (T).

7. Inoculate the junction between the PDA medium and the embedded slide piece with a loopful of actively growing 7-day-old culture of Fusarium sp. (Video 2).

8. Seal the plates with parafilm and incubate them at 25 °C for 48 h.

Note: To obtain reproducible fungus–fungus interactions, the inoculation sequence, incubation period, and incubation temperature should be optimized according to the growth characteristics of the fungal species. The pathogen should be inoculated first and incubated under its optimal to sub-optimal growth conditions (temperature and duration) until it establishes sufficient growth along the PDA–glass interface, spreading bidirectionally from the inoculated edge, with the advancing mycelial front reaching approximately the midpoint of the two adjacent sides of the embedded slide piece. The fungal parasite should then be inoculated adjacent to the advancing hyphal margin of the pathogen (Figure 5). If the two fungi have different optimum growth temperatures, the incubation conditions should be adjusted accordingly following the second inoculation. In the present study, both the pathogen (Fusarium sp.) and the fungal parasite (Trichoderma viride) exhibited optimum growth at 25 °C; therefore, all incubations were performed at 25 °C. This optimization strategy ensures that the pathogen is well established without overgrowth while allowing the fungal parasite to grow toward the pathogen, thereby producing consistent and reproducible mycoparasitic interactions suitable for downstream SEM analysis. Because incubation temperature can influence fungal growth, host–parasite interactions, and the extent of mycoparasitism, it should be selected carefully according to the biological requirements of the interacting fungal species. After visible hyphal growth spreads across the slide surface, inoculate Trichoderma viride from a 7-day-old culture at the advancing margin of the Fusarium growth (Figure 4B).

Figure 5. Schematic diagram of a 48-h-incubated Petri plate illustrating Fusarium sp. growth and the inoculation point of Trichoderma viride

9. Incubate the plates for an additional 48 h (Figure 6).

Figure 6. Fully grown fungal cultures around the glass slides

10. Examine the interaction zone on the slide piece under light to confirm fungal growth and interaction (Figure 7).

Figure 7. Slide pieces with fungal interaction

11. Carefully remove the slide pieces using sterile forceps and mount them onto clean, sterile glass slides for microscopic examination.

12. Identify the precise location of fungal interaction under a brightfield microscope and document it for subsequent SEM observation.

D. Fixation and dehydration of fungal interaction samples

1. Immediately after identifying the interaction zone, transfer the slide pieces along with the glass slide into a sterile Petri dish.

Critical: Transfer the slide pieces using forceps by firmly holding only the two opposite edges. Avoid bringing the forceps into contact with the interacting surface, as this may damage the specimen. Follow this precaution in all subsequent steps involving slide-piece transfer (Figure 2).

2. Using a Pasteur pipette, add 2–3 drops of glutaraldehyde fixative solution to completely cover the fungal interaction area on the slide pieces (Figure 8A).

Figure 8. Fixation and dehydration. (A) Glutaraldehyde fixative covering the slide piece. (B) Washing and dehydration of the slide piece.

Critical: Due to the low volume of fixative, ensure that the solution does not dry during the subsequent incubation step. Alternatively, immerse the slide pieces completely in the fixative or add additional fixative to prevent drying.

3. Cover the Petri dish and incubate overnight (~14 h) at 4 °C in a refrigerator.

Critical: Do not incubate for more than 24 h.

Note: The fixation time and temperature may be adjusted depending on the sample and experimental requirements. While fixation for approximately 4 h at room temperature is generally sufficient, overnight fixation at 4 °C is recommended to achieve optimal preservation of fungal morphology and consistent SEM imaging outcomes.

4. Carefully discard the fixative solution without disturbing the slide pieces. Wash the slide pieces three times for 5 min each using 2–3 drops of sterile Milli-Q water to completely cover the surface.

Note: Perform washing by alternately transferring the slide pieces between two identical Petri dish setups containing glass slides. Transfer the slide pieces from the first setup to the second setup, add the respective reagent to the second setup, and then discard and renew the reagent in the first setup for the subsequent wash or treatment. Follow the same approach in the subsequent steps.

5. Perform dehydration sequentially using graded ethanol concentrations of 10%, 20%, 40%, 60%, 80%, and 100%.

6. Treat the samples thrice for 5 min each at every ethanol concentration by completely covering the slide surface with ethanol. Carry out the dehydration process in ascending order of ethanol concentration (Figure 8B).

Critical: Immediately proceed to the subsequent steps after 100% ethanol dehydration to minimize moisture exposure. Time is critical, but ensure that the ethanol has completely evaporated from the slide surface before proceeding to the next step.

7. Mount the dehydrated slide pieces carefully onto double-sided adhesive tape fixed inside a sterile 60 mm Petri dish to minimize disturbance during handling (Figure 9).

Figure 9. A 60-mm tissue dish with a slide piece attached to the double-sided adhesive tape

8. Seal the Petri dishes tightly with parafilm to prevent further moisture exposure.

9. Store the prepared samples in an incubator at 37 °C until SEM analysis or for up to 1 month.

E. Preparation and observation of samples under SEM

1. Mount the dehydrated slide pieces containing the fungal interaction zone onto SEM stubs using double-sided conductive adhesive tape prior to SEM analysis.

2. Sputter-coat the mounted samples with a thin (0.2–0.5 nm) gold-palladium conductive metal layer to enhance conductivity and improve image quality.

3. Observe the samples under a SEM using appropriate accelerating voltage (10–12 kV), working distance (10.5–19.5 mm), and magnification settings (1,500–4,000×) to visualize the interaction between Fusarium sp. and Trichoderma viride (Figure 10) (see Troubleshooting 2).

Figure 10. Scanning electron microscopy (SEM) images of interaction. Visible interaction between Trichoderma viride and Fusarium sp. Blue arrows indicate mycoparasitic interactions; green arrows indicate Trichoderma viride; orange arrows indicate Fusarium sp. (A) Coiling. (B) Cellular distortion. (C, D) Looping and cellular distortion.

Data analysis

During SEM acquisition, the blunt-end cut is used to locate the interaction zone between Trichoderma and Fusarium. The region is then appropriately magnified to identify specific interaction sites and acquire high-resolution images. Fusarium hyphae appear broader and larger (~2.98 ± 0.78 μm), whereas Trichoderma is observed as thin filamentous hyphae (~1.46 ± 0.53 μm). Hyphal coiling and looping of Trichoderma around Fusarium and cellular distortion of Fusarium hyphae are observed in Figure 9.

Validation of protocol

1. The protocol was validated using eight independent biological replicates to assess its reproducibility for visualizing mycoparasitic interactions between Fusarium sp. and Trichoderma viride by SEM.

2. In all replicates, Fusarium sp. was inoculated first and incubated for 48 h at 25 °C, followed by inoculation of T. viride and a further 48-h incubation at 25 °C. This sequential inoculation consistently produced intact pathogen hyphae and reproducible fungus–fungus interactions without pathogen overgrowth.

3. Some of the characteristic mycoparasitic interactions, including hyphal coiling, distortion, and degradation, were successfully observed in all eight biological replicates, demonstrating the reproducibility of the protocol.

4. An incubation period of 48 h for these fungi was found to be optimal. Longer incubation periods resulted in excessive mycelial growth, making the interaction zone overcrowded and more difficult to identify during SEM analysis.

5. To facilitate rapid localization of the interaction site during SEM, the interaction zone was first identified under a light microscope before fixation, and its position was recorded relative to the blunt-cut edge of the slide piece. This significantly reduced the time required to locate the interaction during SEM imaging.

6. The blunt-cut edge also served as a permanent orientation marker during fixation, dehydration, storage, and transportation, preventing accidental inversion of the slide pieces and ensuring that the interaction surface remained correctly oriented.

7. Throughout all these biological replicates, the prepared slide pieces remained intact during handling, storage, and transportation, enabling successful SEM imaging without damage or loss of the fungal interaction region.

General notes and troubleshooting

General notes

1. Maintain a dust-free working environment throughout the experiment to minimize contamination and artifact formation.

2. All fungal culture handling should preferably be performed inside a laminar airflow chamber to prevent spore dispersal and ensure aseptic conditions.

3. This technique can be used for fungal morphology visualization under SEM, other than interaction studies.

4. During incubation, fungal cultures should be monitored carefully to prevent overgrowth, which may result in dense filamentous structures and a crowded slide appearance that interferes with microscopic observation.

Troubleshooting

Problem 1: Glutaraldehyde fixative precipitation.

Possible causes: Improper temperature or pH.

Solution: Glutaraldehyde prepared in phosphate buffer may precipitate at room temperature (approximately 25–30 °C). To avoid precipitation, the glutaraldehyde fixative solution should be freshly prepared and used immediately.

Problem 2: Bright patches on the SEM field.

Possible causes: Exposure to moisture or improper drying during storage.

Solution: Excessive white appearance or glare in filamentous fungal samples may indicate incomplete drying after the dehydration process or exposure of the sample to moisture/humidity. To prevent such artifacts, ensure that the smears are completely dried before SEM analysis. Additionally, seal the SEM slides tightly with parafilm during storage and transportation, and preferably store them under warm conditions (≥37 °C) to minimize exposure to atmospheric moisture and humidity.

Problem 3: Excessive or insufficient fungal growth before interaction.

Possible cause: Incubation period not optimized according to the growth rate of the fungal isolates.

Solution: Adequate pathogen growth is achieved when the advancing mycelial front reaches the adjacent margins of the embedded slide piece. If the pathogen has not reached these margins, extend the incubation period before inoculating the fungal parasite. If the pathogen extends beyond the intended interaction zone or the slide surface becomes overcrowded with mycelia, reduce the incubation period before the second inoculation. The fungal parasite should be inoculated immediately adjacent to the advancing margin of the pathogen to ensure reproducible interactions and facilitate SEM visualization.

Acknowledgments

Conceptualization, S.P., S.T.; Investigation, S.T.; Writing—Original Draft, S.T.; Writing—Review & Editing, S.P.; Funding acquisition, S.P.; Supervision, S.P. The authors are grateful to the Vellore Institute of Technology, Vellore, Tamil Nadu, India, for providing the facilities, support, and encouragement. This work was supported by the seed grant provided by Vellore Institute of Technology, Vellore, India (sanction order no. SG20230036). S.P. acknowledges RV University, Mysuru, for infrastructural support and encouragement. The authors also acknowledge Elamala Biotech Lab for kindly providing fungal cultures and infrastructural support. The authors acknowledge the use of AI-assisted tools (Figurelabs) to generate the Graphical overview and Figure 5 in this manuscript. All images were critically evaluated and verified for scientific accuracy by the authors, who take full responsibility for the final content.

Competing interests

The authors declare no competing interests.

References

1. Viterbo, A. and Horwitz, B. A. (2014). Mycoparasitism. In K. A. Borkovich and D. J. Ebbole (Eds.), Cellular and Molecular Biology of Filamentous Fungi (pp. 676–693). Washington, DC, USA: ASM Press. https://doi.org/10.1128/9781555816636.ch42
2. Jeffries, P. (1995). Biology and ecology of mycoparasitism. Can J Bot. 73: 1284–1290. https://doi.org/10.1139/b95-389
3. Sun, J. Z., Liu, X. Z., McKenzie, E. H. C., Jeewon, R., Liu, J. K., Zhang, X. L., Zhao, Q. and Hyde, K. D. (2019). Fungicolous fungi: terminology, diversity, distribution, evolution, and species checklist. Fungal Diversity. 95(1): 337–430. https://doi.org/10.1007/s13225-019-00422-9
4. Atanasova, L., Crom, S. L., Gruber, S., Coulpier, F., Seidl-Seiboth, V., Kubicek, C. P. and Druzhinina, I. S. (2013). Comparative transcriptomics reveals different strategies of Trichodermamycoparasitism. BMC Genomics. 14(1): e1186/1471–2164–14–121. https://doi.org/10.1186/1471-2164-14-121
5. Mukherjee, P. K., Mendoza-Mendoza, A., Zeilinger, S. and Horwitz, B. A. (2022). Mycoparasitism as a mechanism of Trichoderma-mediated suppression of plant diseases. Fungal Biol Rev. 39: 15–33. https://doi.org/10.1016/j.fbr.2021.11.004
6. Bozzola, J. J. and Russell, L. D. (2006). Electron microscopy: principles and techniques for biologists (2. ed., [Nachdr.]). Boston: Jones and Bartlett.
7. Brundrett, M., Bougher, N., Dell, B., Grove, T. and Malajczuk, N. (1996). Working with Mycorrhizas in Forestry and Agriculture. Australian Centre for International Agricultural Research. https://doi.org/10.13140/2.1.4880.5444
8. Riddell, R. W. (1950). Permanent Stained Mycological Preparations Obtained by Slide Culture. Mycologia. 42(2): 265–270. https://doi.org/10.1080/00275514.1950.12017830
9. Bhat, K. A. (2017). A New Agar Plate Assisted Slide Culture Technique to Study Mycoparasitism of Trichoderma sp. on Rhizoctonia solani and Fusarium oxysporium. Int J Curr Microbiol Appl Sci. 6(8): 3176–3180. https://doi.org/10.20546/ijcmas.2017.608.378