An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

The overall goal of this procedure is to isolate arthrobacter bacteria phases from soil samples using an enrichment protocol. This is accomplished by first collecting soil samples and isolating viable phage particles from contaminating soil microorganisms. The second step is to infect host arthrobacter bacterial cells with the isolated phage particles.

Next infected bacterial cells are plated on auger plates to identify phage as evidenced by plaque formation. The final step is to purify desired phages from clearly isolated plaques using the streak plate method. Ultimately, this enrichment technique is optimized for the relatively easy isolation of arthrobacter phages from arthrobacter species KY 3 9 0 1.

The main advantage of this technique over existing enrichment methods is the addition of a finely filtered soil sample to a pure culture of host bacteria to avoid the growth of competing bacteria. Though this method has been proven to be effective for the isolation of arthrobacter phages, it should be applicable for the isolation of phages from other bacteria hosts. We first had the idea for this method when we learned other researchers were having difficulty finding arthrobacter phages.

With traditional enrichment techniques. My students will be demonstrating the techniques used to successfully isolate arthrobacter phages. Arthrobacter cells are cultured from colonies streaked on an LB auger plate, incubated at 30 degrees Celsius for two to three days.

Use a sterile loop to pick a colony and add it to 50 milliliters of lb broth. In a baffled culture, flask incubate the flask in a shaking incubator at 225 RPM at 30 degrees Celsius for a approximately 24 hours. To obtain exponential or early stationary phase cells for phage infection experiments.

Monitor the state of bacterial growth closely to prevent cells from entering the middle to late stationary growth.State. The ideal OD 600 for phage isolation is between 0.5 and 0.7. Since bacteria in the arthrobacter genus are common soil bacteria, they and the phages that infect them can be found in most types of soil.

Gather 200 to 400 grams of soil from the desired location. Add at least 400 milliliters of phage buffer to the soil in a large enough beakers so that at least 200 milliliters of phage buffer is in the supernatant and is able to be extracted mixed by gently stirring or swirling until the soil is suspended and allow the soil sediment to settle typically for 30 minutes. Next, remove soil sediment debris by passing the soil extract through a filter paper by vacuum filtration.

Add four grams of LB powder to 200 milliliters of the filtered soil extract and mix to make a 2%solution. Pass the solution through a 0.22 micron filter by vacuum filtration to obtain a sterile filtrate. If possible, continue on to the next step immediately.

Nevertheless, if need be, the filtered samples can be stored at four degrees Celsius for up to a week before proceeding. However, the number of viable phage particles will be reduced using as septic techniques aliquot multiple 50 milliliter portions of the filter sterilized LB soil extract mixture into individual 250 milliliters. Sterilized shaking, baffled culture flasks add sterile 2.0 molar calcium chloride solution to the final desired concentrations.

Since different arthrobacter phages grow optimally under different calcium chloride conditions, begin this procedure by adding one to 2.5 milliliters of late exponential or early stationary phase bacteria culture to each of the flasks containing filter sterilized LB soil extract mixture. If the OD 600 of the culture is 0.5 to 0.7, use one milliliter. If the OD 600 is higher than 0.7, use 2.5 milliliters.

Shake the flasks at 250 RPM at 30 degrees Celsius for approximately 24 hours in a shaking incubator. After the 24 hour incubation period, remove the enrichment flasks from the shaking incubator. Dilute the enrichment samples tenfold in phage buffer.

Set up culture tubes with 0.5 milliliters of late exponential or early stationary. Phase bacteria culture. Add various amounts from five microliters to 500 microliters of the diluted enrichment culture to the culture tubes.

Add calcium chloride to the culture tubes to make a final concentration in five milliliters equal to the concentration present in the original enrichment flask. Use a range of different calcium chloride concentrations to select for many phages with varying calcium chloride dependencies. Add 4.5 milliliters of LB top auger to each culture tube, and pour the mixture onto an LB auger plate.

Swirl gently to distribute evenly across the plate and allow the top auger to solidify for approximately 15 minutes. Invert the plates and incubate at 30 degrees Celsius overnight to 48 hours. Vary the incubation times to optimize for the phages present after a 24 hour incubation check for plaque formation on the plates.

Most isolated phages come from plates incubated at 30 degrees Celsius at one to 2.5 millimolar calcium chloride concentration after a 24 hour incubation period, Purifying desired phages from clearly isolated plaques using a wooden stick as the most challenging part of this procedure. Technical expertise is required to gently spread the plaques across the auger plate to produce a uniform streak plate. Sterilize a wooden stick by running it through a flame and allowing it to cool.

Briefly touch a plaque with the sterile wooden stick and rub the stick on the auger plate in the gentle motion as shown. Repeat this using a clean stick for each pass. Once the LB auger plate is streaked at least three times, mark the area with the lowest concentration of putative phage particles.

Gently apply 0.5 milliliters of arthrobacter and 4.5 milliliters of molten LB top auger to the marked area, and allow it to spread evenly across the plate. Let the top auger solidify for at least an hour when the plate has solidified invert and incubate it overnight. Repeat the streak plaque technique for at least three iterations to ensure a pure phage species is isolated.

Plates can be stored at four degrees Celsius for up to a week as needed between streaks without significant loss of phage viability. Once desired plaques have been grown and isolated on the final streak plate, touch one well isolated plaque with a micro pipette tip and resuspend in 100 microliters of phage buffer serially Dilute this solution out to the 10 to the minus eighth dilution by passing 20 microliters of the sample into 180 microliters of fresh phage buffer. Add 10 microliters of each dilution to 0.5 milliliters of grown arthrobacter in a culture tube for five to 10 minutes at room temperature.

Then plate by mixing the infected bacteria with 4.5 milliliters of molten LB top auger containing the same concentration of calcium chloride used to isolate the phage from the original soil sample. Incubate the plates overnight. Save all dilution made at four degrees Celsius until the next day.

Determine the phage cereal dilution needed to make a web pattern using the traditional plaque titer assay. Identify a web pattern plate as mostly devoid of bacteria but containing remnants of bacteria that have not yet been laced by phages. Add five milliliters of late exponential or early stationary arthrobacter culture to a sterile 50 milliliter culture flask.

Add 100 microliters of the dilution that gave a web pattern to the arthrobacter culture and allow the phages to infect the bacteria cells for five to 10 minutes. At room temperature, add to the flask molten lb top auger containing the same concentration of calcium chloride as used to initially identify the phage plate. Five milliliters of this mixture onto each of 10 fresh LB plates.

After allowing the top auger to solidify, invert, and incubate the plates at 30 degrees Celsius overnight on the following day, flood the web pattern plates with five milliliters of phage buffer per plate and store overnight at four degrees Celsius. Filter the phage lysate through a 0.22 micron syringe filter. Store this final phage stock at four degrees Celsius for long-term storage.

Add an equal amount of the phage lysate to sterile glycerol in small vials for stock archiving at minus 80 degrees Celsius in the enrichment procedure demonstrated in this video, the plaque streak method to isolate pure phage populations after initial enrichment and plating on LB auger shown are representative results from the isolation of plaques for phage DI at room temperature, 30 degrees Celsius and 37 degrees Celsius. The red circles on two of the plates surround clearly isolated plaques. Phage Dylan produces similar plaques at three different temperatures.

Alternatively, a traditional standard plaque titer assay may be used to isolate a pure phage isolate. This assay was used here to empirically determine phage titer numbers and the number of plaque forming units to form a webbing pattern on a plate. A set of serial dilution for phage banana is shown with an example of how a good web pattern should look.

These are electron micrographic images of 20 arthrobacter phages isolated using the enrichment technique. Of the 25 phages currently isolated, 23 of them are cifo viruses with rather long tails. Two of the isolated phages are myo viruses containing head diameters similar in length to that of their tails.

Once mastered, the enrichment plating should allow for the successful isolation of arthrobacter phages from a variety of soil samples. After watching this video, you should have a good understanding of how to purify Phage is used in the streak paint method from an environmental soil sample.