A Small Volume Procedure for Viral Concentration from Water

The overall goal of the following experiment is to optimize virus recovery from water. Using a small scale technique originally designed to optimize virus concentrations. A glass cellulose filter was found to be the best electropositive filter to concentrate adenovirus from water for the secondary elution.

A sea light secondary concentration technique proved to be best for eluding adenovirus from the filter surfaces. The result in technique recovers the most adenovirus from tap water based on having the highest percent recoveries observed between all tested filter and elution combinations. The main advantage of this technique over existing methods like large scale volume concentration, is that it utilizes smaller sample volumes, enabling a multitude of concentration variables to be evaluated quickly and easily.

When preparing for this protocol, have all the required solutions at the ready, including the point 22 micron filtered viral solution at 10 to 100, 000 MPN per milliliter. Begin by measuring out a liter of sterile tap water. Transfer it to a two liter beaker with a magnetic stir bar while stirring the water.

Add 0.7 grams of sodium thio sulfate and allow it to dissolve completely. Then adjust a pH to between seven and 7.5 by adding one molar acid in bay stock solutions dropwise. Next, spike the water with virus.

Add a full milliliter of the prepared virus solution. Cover the flask with a sterile wrap and let the virus mix into the water for 10 minutes. Meanwhile, prepare the millipore filter first, unwrap it from its packaging, and make sure it's a tight fit between the upper bowl and lower screen.

Next, check that the cork material on the filter fits snugly to the lip of a one liter sidearm flask. Now remove the top bowl of the filter and with forceps, place a 47 millimeter electropositive filter disc squarely onto the filter screen. Then reattach the bowl and lock it into place with a counterclockwise twist.

Measure out 100 milliliters of the spiked tap water using a sterile cylinder. Then transfer the water into the prepared filter housing on the two liter flask. The electropositive disc in the filter may be pleated glass cellulose or nano Illumina glass fiber gentle vacuum can be used to pass the residual water through the filter.

Now, move the filter housing to a 250 milliliter sidearm flask. Measure out 100 milliliters of beef extract and slowly pour it onto the filter. Allow the extract to pass through slowly and use a gentle vacuum to pull through the residual.

Then proceed with either of the two secondary concentration options to concentrate the virus using sea light. Transfer the 250 milliliter flask content, which contains the virus to a stir plate. Using a stir bar, create a small vortex.

Next, add 0.1 grams of cite powder to 100 milliliters of beef extract. Let the C light disperse and prepare to adjust the solutions pH using dropwise additions of 0.1 molar hydrochloric acid. Lower the pH to 4.0.

Then let the solution mix for 10 minutes while mixing. Attach your 47 milliliter pre-filter onto a two liter flask. Now pour 100 milliliters of the beef extract sea light mixture through the pre-filter.

A slight vacuum may be used to finish. Next place a metal tube clip on the end of a filter housing spout to the clip. Attach a sterile 15 milliliter polypropylene collection tube and then reassemble the filter on the flask.

Now elute the virus from the sea Light particles in five milliliters of one XPBS at pH nine. Allow most of the PBS to drip through into the 15 milliliter tube by gravity and pull the residual through with a light vacuum to concentrate the virus using organic flocculation. Transfer the 250 milliliter flask content containing the virus to a stir plate.

Using a stir bar, create a small vortex, adjust a pH of the solution to 3.5 using dropwise additions of one molar hydrochloric acid. Then let the mixture stir for a half an hour later. Transfer the solution to a sterile 250 milliliter conical centrifuge tube.

Spin the solution down at 2, 500 G for 15 minutes at four degrees Celsius after the spin. Be careful to not disrupt the pellet when handling the tubes carefully. To can the snat and resuspend the pellet in five milliliters of PBS pH nine.

Make sure the pallet is dissolved. Then centrifuge the Resus at 4, 000 G for 10 minutes at four degrees Celsius. Now collect the supernatant which contains the concentrated virus.

The pellet can be discarded. In optimizing this protocol, three types of sea light were compared larger. Sea light resulted in lower recoveries of both a DV 40 and a DV 41.

The filter elution was tested at PHS between 7.5 and 12 to determine which would be the most effective at collecting a DV particles from the filters. The highest recovery was obtained from a glass cellulose filter coupled with beef extract at pH 10 and with sea light. The result was the same with a DV 40 and a DV 41.

The beef extract at pH 10 proved superior to organic flocculation. The optimized method was compared with an addition of point 25%trypsin and compared with supplementing the beef extract with 0.1%sodium polyphosphate, neither. Treatment significantly improved.

A DV recovery Once mastered this technique can be done in 45 minutes if performed properly.