Virus B la sauce Yo-Pro: Microwave-enhanced staining for counting viruses by epifluorescence microscopy

A recently developed method for enumerating aquatic viruses (Yo-Pro stained) was modified to reduce staining time and allow counting of fixed samples. Marine and freshwater virus samples to be enumerated were stained with the cyanine-based dye Yo-Pro-l by using microwave irradiation, which reduced incubation time from 48 h to 4 min. The modified method and the original protocol yielded similar viral estimates. Easier sample handling and the addition of fixedsample capability should allow epifluorescence counting of viruses to become a routine part of field studies of viral ecology. Viruses are now considered to be an important component of aquatic microbial communities. Their extremely small size, however, makes them very difficult to study by direct enumeration. Nevertheless, reliable, uncomplicated estimation of viral abundance will be necessary for rapid progress in understanding the role of viral infection in the dynamics of protozoa, algae and bacteria in aquatic environments. Viruses currently enumerated with transmission electron microscopy (TEM) are either sedimented without preconcentration onto electron-microscopy grids by ultracentrifugation (Bergh et al. 1989; Borsheim et al. 1990; Mar-anger et al. 1994) or, in oligotrophic environments, they are preconcentrated by ultrafiltration before the sedimentation step (Proctor and Fuhrman 1990; Paul et al. 1991). These approaches are time-consuming, require expensive equipment, and yet may underestimate true viral abundance (Borsheim et al. 1990; Hennes and Suttle 1995; Maranger and Bird 1995). Recently, Hennes and Suttle (1995) established a method for counting viruses by epifluorescence microscopy using the cyanine-based nucleic dye Yo-Pro-l (4-[3-methyl-2,3-dihydro-(benzo-l,3-oxazole)-2-methylmethyledene]-l-(3’-t~methyl-amonium-propyl)-quinolinium diiodide, Molecular Probes, Oregon). Use of the Yo-Pro method offers numerous advantages such as moderately priced equipment, simplicity, and greater precision. Unfortunately, the Yo-Pro method requires long incubation times (24-48 h) for adequate staining. Also in our experience, bacteria can multiply during the long incubation period, hence making viral enumeration difficult. Furthermore, water samples fixed with formalin or glutaraldehyde resist staining with Yo-Pro so that only fresh samples can be counted with this method. Because certain circumstances, including field sampling, require fixation of water samples, conserving them with glutaraldehyde or formaldehyde is crucial for an accurate determination of viral abundance. We found a modification of the Yo-Pro method that reduces incubation time to minutes. The new method also permits staining of fixed samples. These improvements result from the enhanced diffusion that is produced by brief treatment with microwave irradiation. Microwave techniques are common in histopathology and histochemistry for staining and fixing samples (Moorlag et al. 1987; Suurmeijer et al. 1990). The use of microwaves is advantageous because it reduces staining and fixation time of biological tissues, and it can improve the quality of the microscopic preparation. In the present case, microwave irradiation seems to facilitate the diffusion of stain across the viral capsid, greatly increasing the rate of its bonding with DNA and RNA. Microwave irradiation increases internal temperature, thus enhancing diffusion and chemical reaction rates (Kok and Boon 1990). The modified Yo-Pro method was performed as follows. All manipulations were carried out in subdued light. The stain was prepared as described in Hennes and Suttle (1995). Yo-Pro was diluted to 50 PM in an aqueous solution of 2 mM NaCN. Prior to filtering samples, 80-~1 drops of the stain were placed in the bottom of a Pyrex Petri dish. Water samples were fixed with unbuffered formaldehyde or glutaraldehyde at a final concentration of 2%. Prior to filtration, fixed or unfixed freshwater samples (100 ~1) were diluted with 700 ~1 of prefiltered (pore size 0.02 pm) deionized distilled water (DDW). Marine water samples were diluted at a ratio of 1 : 4. We found it was more convenient to dilute 200 ,ul of marine sample with 800 ~1 of DDW because viral abundance is generally lower in marine environments (Maranger and Bird 1995). In ultra-oligotrophic environments, we dilute 1 ml of sample with 4 ml of DDW. Samples were filtered (400 mm Hg) on a 0.02-pm pore size A&O, Anodisc membrane filter (Whatman) with a premoistened backing filter (pore size 0.45 pm). Marine samples were additionally rinsed three times by filtering with 500 ~1 of prefiltered DDW. These rinses are critical to the final quality of the preparation and cannot be skipped.