TITLE: Viral Enumeration of EPS Samples Using Wet Mount Epifluorescence Microscopy
AUTHORS: Madeline Bellanger, Pieter Visscher, Richard Allen White III

[DESCRIPTION]
Epifluorescence microscopy (EFM) has been the gold standard method for environmental viral enumeration for over 25 years. Currently, standard EFM methods using the Anodisc filters are no longer cost-effective (>$15 per slide). We present a cost-effective method for environmental viral enumeration from aquatic samples, microbial mats, and exopolymeric substances (EPSs) within modern microbialites using EFM. Our integrated approach, which includes filtration, differential centrifugation, chloroform treatment, glutaraldehyde fixation, benzonase nuclease treatment, probe sonication (EPS and mat only), SYBR Gold staining, wet mounting, and imaging, provides a robust method for modern microbialites and aquatic samples. Our method provides a robust and cost-effective (~$0.75 per sample) viral enumeration within modern microbialites and aquatic ecosystems.

[GUIDELINES]
This method is used to enumerate viruses in EPS samples collected from the environment. See our protocols for water viral enumeration, microbial mat viral enumeration, and soil viral enumeration!

[STEPS]
SECTION: Sample Collection and Cleaning
1. Tare a 1.5 mL low protein binding nuclease free tube on a top loading balance.
SECTION: Sample Collection and Cleaning
2. Collect approximately 100 mg of mat using a scoopula or similar tool cleaned with 70% ethanol and transfer to the tube. Record the mass of the mat sample.
SECTION: Sample Collection and Cleaning
3. Add 900 µL ultrafiltrate from the same location to the tube.
SECTION: Sample Collection and Cleaning
4. Vortex at medium-high speed for 10 s.
SECTION: Sample Collection and Cleaning
5. Add 500 µL of   to the sample.
 

 

SECTION: Sample Collection and Cleaning
6. Centrifuge the samples at 14.000 rpm, 5 min (standard mini-fuge speed).

SECTION: Sample Collection and Cleaning
7. Carefully pipette 92 µL of the top layer of supernatant into a fresh 1.5 mL low protein binding nuclease free microcentrifuge tube, proceed to the next section: Fixation ( ).

SECTION: Fixation
8. Add 8 μL of EM grade 25% glutaraldehyde to each of the 92 μL samples (final concentration of 2%). Pipette to mix.
 

SECTION: Fixation
9. Allow samples to fix in the dark at 4 °C for 30 min.

SECTION: Fixation
10. OPTIONAL: Flash freeze samples using liquid nitrogen. If liquid nitrogen is not available, putting samples directly in a -80ºC freezer can be done. Samples can be stored in the -80ºC freezer until use.
 

SECTION: Preparation for EFM and Imaging
11. Thaw fixed samples at room temperature (if applicable). During this time, start heating a heat block to 37 °C. Start heating another heat block to 80 °C.
SECTION: Preparation for EFM and Imaging
12. Aliquot 20 µL of sample into a new 1.5 mL low protein binding nuclease free microcentrifuge tube.
SECTION: Preparation for EFM and Imaging
17. Add 1 µL of   to each sample. Pipette to mix.
SECTION: Preparation for EFM and Imaging
18. Incubate the samples in a heat block at 37 °C for 30 min.
SECTION: Preparation for EFM and Imaging
19. Add 4 µL SYBR Gold working stock to the sample in the dark and pipette to mix.
 

SECTION: Preparation for EFM and Imaging
20. Incubate the sample in a heat block covered in aluminum foil at 80 °C for 15 min.
SECTION: Preparation for EFM and Imaging
21. While the sample is incubating, prepare a 10% ascorbic acid antifade solution.
 

SECTION: Preparation for EFM and Imaging
22. Remove the sample from the heat block and add 2 µL of ascorbic acid antifade solution. Pipette to mix.
SECTION: Preparation for EFM and Imaging
23. Pipette 5 µL onto a clean, labeled, poly-L-lysine treated slide.
 

SECTION: Preparation for EFM and Imaging
24. Gently cover with a cover slide, avoiding creating air bubbles.
SECTION: Preparation for EFM and Imaging
25. Image on a fluorescence microscope under FITC blue excitation light (495 nm) with a 100x oil stage.
SECTION: Preparation for EFM and Imaging
16. Aliquot 19 µL of each sample into a new 1.5 mL low protein binding nuclease free microcentrifuge tube.

SECTION: Preparation for EFM and Imaging
15. Probe sonicate the samples in three 10 s intervals with 10 s between each interval at 50 Hz/40 amp while in an ice bath.
 

SECTION: Preparation for EFM and Imaging
13. Add 0.1 millimolar (mM) EDTA to the sample with the tube on ice (4 µL of 0.5 millimolar (mM) EDTA).
SECTION: Preparation for EFM and Imaging
14. Incubate the samples on ice for 15 min in the dark.