The overall goal of this procedure is to determine the rate of which viruses are produced in aquatic microbial communities. This is accomplished by first reducing the abundance of free viruses in the sample of interest while keeping the native microbial community. The second step of the procedure is to incubate samples that incite you conditions and to collect subsamples every two to three hours for up to 10 hours.
The third step of the procedure is to enumerate the viruses within the collected subsamples from each time point. The final step of the procedure is to determine the rate at which virus particle abundance increased within the sample and determine virus production rates. Ultimately, results can be obtained that show the rates at which viruses are being produced within the microbial community from the enumerations, allowing researchers to infer rates of microbial mortality due to virus activity.
Hi everyone. My name is Audrey Madison, and I work in the laboratory of Dr.Steven Milham and the Department of Microbiology at the University of Tennessee. Today with the help of Charles Budoff and Claire Campbell, we'll be showing you a procedure for analyzing the rates that viruses are produced and aquatic systems.
To do this, we reduce the number of free viruses in samples and enumerate the rate in which they reoccur to the lysis of virally infected microorganisms. We currently use this procedure in our laboratory to study the ecology of viruses and how they may shape microbial communities. So now that we've introduced, let's go ahead and get started.
Before this procedure can begin, collect approximately 20 liters of sea water as aseptically as possible. Once the sample is collected, pre-filter the water through a 142 millimeter diameter 0.8 micron filter. For biologically productive systems, one may preempt this with filtration through a larger pore size or glass fiber filter.
To obtain ultra filtered water, set up an ultra filtration system like the AmCon M 12 system with a spiral wound membrane cartridge to exclude all viruses. The viruses in the sample have then removed and concentrated at approximately 25%of maximum speed with 15 to 16 kilo pascals of back pressure. The Tate, about 500 milliliters contains the now concentrated virus community, which may be saved for other studies while the virus free permeate will be used for viral production assays.
After each day of use, clean the Omicron M 12 system to prevent damage to the membrane of the filter cartridge. When working with sea water, rinse the membrane out with at least six liters of Milli Q water, followed by a wash with 0.1 molar sodium hydroxide solution for 30 to 45 minutes. Then rinse the cartridge with at least six more liters of Milli Q water.
When finished, the spiral cartridge can be stored in a 0.05 molar phosphoric acid solution at four degrees Celsius. To begin the viral reduction method, 500 milliliters of sea water sample from the same location we made our virus free water is placed in a stereo filter unit with a 0.2 micro nominal pore size low protein binding filter. At this point, also collect a subsample to determine the total abundance of viruses and bacteria in the starting water.
Gently vacuum pressurize the sample at less than 200 millimeters of mercury while continuously resus suspending the sample using a sterile transfer pipette to inhibit bacterial cells from concentrating on the filter slowly add three volumes of 500 milliliters of the ultra filtrate to the bacterial suspension to reduce the abundance of free viruses in the sample while maintaining the bacterial populations at a constant density. Finally dilute the bacterial fraction back to 500 milliliters with virus free ultra-filtered water. Next, divide it into three replicates of 150 milliliters and place the replicates in clear 250 milliliter polycarbonate bottles.
This represents your time zero sample set. Take samples for bacterial and viral abundance estimates from your time zero samples. Transfer the samples into cryo vials with a final concentration of 2.0 to 2.5%sterile glutaraldehyde.
As a fixative, immediately flash, freeze these samples with liquid nitrogen and store them at minus 80 degrees Celsius until processed. Alternatively, a tangential flow system can be used to concentrate the marine microbes. To use the TFF method, obtain approximately 500 milliliters of the natural sample.
Concentrate this sample using a 0.2 micron nominal pore size tangential flow filtration system when the bacterial fraction is reduced to approximately 10 to 15 milliliters at ultra-filtered virus free water as described for the virus reduction method, incubate the resulting replicate bottles at incite you conditions. Using environmental chambers at sea, we use flowing sea water incubators hooked up to a supply of surface water that maintains ambient temperatures. Light levels can be matched to surface conditions by using blue tinted acrylic or a neutral density screening net to decrease light intensity.
When working from a field station or lab, or when flowing, sea water is not available, a lighted growth incubator can be used. Continue to collect subsamples every 2.5 hours for at least 10 hours by the method performed for the sample at time zero. In addition to microscopy samples, some researchers will want to collect water for quantitative PCR analysis of specific viruses.
For QPCR samples, add five milliliters of the sample to a cryo vial with no fixative agent and immediately flash freeze in liquid nitrogen in preparation for viral production microscopy thaw frozen samples on ice and freshly prepare solutions as described in the written protocol. Then place a 25 millimeter 0.02 micron pore size, and a disc filter on top of a 0.45 micron cellulosic backing filter. Then add 850 microliters of the fixed sample to the top of the anadi filter and vacuum at less than 20 kilo Pascals until completely dried.
Once the ano disc filter is dry, pipette 100 microliters of cyber green working solution into a sterile petri dish. And with the vacuum still on, carefully remove the ano disk from the filter tower and place it onto the cyber green. Incubate the samples in the dark at room temperature for 20 minutes.
Carefully remove the filter from the cyber green solution and wick the back of the filter with a Kim wipe to remove all residual dye. Then add a small drop of Antifa solution to a microscope slide and place a cover slip on top. Remove the cover slip and add the dried filter to the microscope slide.
Again, add a small amount of Antifa solution to the cover slip and slowly place it on top of the filter, making sure to remove any bubbles that may form enumerate viruses using fluorescence microscopy with a wide blue filter count at least 20 fields of view for each filter, making sure to quantify total viruses from each field grid to ensure even distribution of viruses across the filter membrane. Calculate averaged rates of virus recurrence from the three independent replicate and determine a standard deviation from the production rates. Finally correct these rates for the dilution of microorganisms from the starting abundance to the diluted abundance in the virus reduced sample.
The primary data set resulting from this study is the recurrence rates of virus abundance in the subsamples from the incubations. These results form independent regressions of virus abundance versus time for each of the samples. The production of virus-like particles was monitored over a 10 hour incubation at insight due conditions using epi fluorescence microscopy.
These samples were collected during a phytoplankton bloom off the coast of New Zealand in September of 2008. We've just shown you how to estimate virus production rates. This includes using different ultra filtration systems and carrying out experiments to measure the virus production rates.
Also, we showed you how to enumerate these viruses by epi fluorescence microscopy. When doing this procedure, it's important to remember to use clean equipment, handle those stained viruses, and reduce light, process your samples quickly and to properly clean your ultra filtration cartridges after every use. So that's it.
Thanks for watching and good luck with your experiments.
