TITLE: Viral Enumeration of Water 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 water samples collected from the environment. See our protocols for microbialite viral enumeration, EPS viral enumeration, and soil viral enumeration!

[STEPS]
SECTION: Filtration and Cleaning
1. Record the starting volume of water, this will be used for calculations later.
SECTION: Filtration and Cleaning
2. Filter water through a glass fiber pre filter (Whatman Grade 1 qualitative filter paper).
SECTION: Filtration and Cleaning
3. Collect filtrate and filter through a 0.65 μm PVDF filter (Durapore PVDF membrane filter; hydrophilic).
SECTION: Filtration and Cleaning
4. Collect filtrate again and filter two times through 0.22 μm PVDF filters (Durapore PVDF membrane filter; hydrophilic).
SECTION: Filtration and Cleaning
5. If only filtering samples,  . Continue through this section for optional concentration.
SECTION: Filtration and Cleaning
6. Collect filtrate and add to the top portion of 30 kDa MWCO Centricon-70 plus centrifuge filters (Millipore UFC703008).
 

SECTION: Filtration and Cleaning
7. Centrifuge at 3500 rpm, 12 min (if using smaller volume filters adjust speed and time according to manufacturer instructions).
SECTION: Filtration and Cleaning
8. After centrifugation, collect the ultrafiltrate from the collection cup below the filter and add more 0.22 μm filtered water to the top. Continue to centrifuge as explained in step 7 until all water has passed through the filter.
 

SECTION: Filtration and Cleaning
9. After concentrating all the water, attach the retrieval cup to the top of the filter and flip upside down. Centrifuge at 3500 rpm, 5 min.
SECTION: Filtration and Cleaning
10. Collect the concentrate from the retrieval cup and pipette into a 1.5 mL low protein binding nuclease free microcentrifuge tube. Record the volume of concentrate recovered.
SECTION: Filtration and Cleaning
11. Dilute concentrate ~3:10 with ultrafiltrate.
SECTION: Filtration and Cleaning
12. Add 500 µL of   to the sample.
 

 

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

SECTION: Filtration and Cleaning
14. Use glass pasteur pipette to carefully pipette the sample portion of the supernatant (top part) into a fresh 1.5 mL low protein binding nuclease free microcentrifuge tube, making sure to not get any chloroform.
SECTION: Filtration and Cleaning
15. Aliquot 92 µL of the diluted concentrate into a fresh 1.5 mL low protein binding nuclease free microcentrifuge tube, proceed to the next section: Water Enumeration - Fixation ( ).

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

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

SECTION: Fixation
18. 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
19. 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
20. Aliquot 19 µL of sample into a new 1.5 mL low protein binding nuclease free microcentrifuge tube.
SECTION: Preparation for EFM and Imaging
21. Add 1 µL of   to each sample. Pipette to mix.
SECTION: Preparation for EFM and Imaging
22. Incubate the samples in a heat block at 37 °C for 30 min.
SECTION: Preparation for EFM and Imaging
23. Add 4 µL SYBR Gold working stock to the sample in the dark and pipette to mix.
 

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

SECTION: Preparation for EFM and Imaging
26. 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
27. Pipette 5 µL onto a clean, labeled, poly-L-lysine treated slide.
 

SECTION: Preparation for EFM and Imaging
28. Gently cover with a cover slide, avoiding creating air bubbles.
SECTION: Preparation for EFM and Imaging
29. Image on a fluorescence microscope under FITC blue excitation light (495 nm) with a 100x oil stage.