Purpose:

Using collected samples, perform direct and enriched isolation to find bacteriaphage within your sample.

GPS Coordinates and Information regarding my phage: 

Collected by: Esperanza Sandoval

Date:  9/2/17

Type: Soil from Ranch

GPS: N 32, 40′ 47.81746″; W 97, 15′ 11.14521′

Depth: 1 in.

Temperature: 87 F

Environmental Sample Data Sheet:

Sample	1	2	3
Collector Name	Jessica Doty	Jessica Doty	Jessica Doty
Date of Collection	2-Aug-17	4-Aug-17	4-Aug-17
Sample Type	Soil	Soil	Soil
Location Description	Field	Flowerbed	Ground
General Location	Rural	Subdivision	Subdivision
Specific location	30.36, -96.83	32.217, -98.213	32.317, -98.213
Sample depth	3 in.	2 in.	3 in.
Ambient Temperature	82˚ F	71˚ F	71˚ F

 

Materials and Methods: 

-Collection of Environmental Samples

• 3- 50 mL conical tubes
• Tool for digging
• Permanent marker
• Device to tell GPS coordinates and temperature

-Direct Isolation

• Environmental sample
•  Enrichment broth (5 ml/sample)
•  Sterile 3 ml or 5 ml syringe
• 0.22 μm syringe filter
•  5 ml serological pipettes
•  Microcentrifuge tubes
•  15 ml conical tube

-Plaque Assay

• Phage samples for isolation, purification, or titering
•  Host bacteria (250 μl/plate)
•  Agar plates
•  Phage buffer
•  Top agar
•  Microcentrifuge tubes
•  5 ml sterile pipettes

-Enriched Isolation

• Solid environmental sample
•  0.22 µm Corning Tube-Top Vacuum Filter Systems or syringe filters
•  Enrichment broth
•  10X Enrichment Broth (if using liquid environmental samples)
• 50 ml sterile conical tubes
•  Sterile 5 ml syringes
•  0.22 μm syringe filters
• Microcentrifuge tubes
•  Host bacteria (500 μl)

–Spot Test

•  Liquid phage sample (either a picked plaque from a direct isolation, or an enriched isolation)
• Agar plates
• Host bacteria (250 μl/plate)
•  Top agar
• Phage buffer
• 5 ml sterile pipette

–Plaque Assay for Purification

• Phage samples for purification
• Phage buffer
• Microcentrifuge tubes
• Host bacteria
• Agar plates
• Top Agar
• 5 ml sterile pipettes

Procedure

Direct Isolation

• Prepare bench for aseptic work.
• Extract phage from solid environmental samples. Using a 15 ml conical tube, fill it approximately 1/3 full with soil. Add enrichment broth to conical tube. Incubate the tube for 1-2 hours. Allow the sample to sit until particulate matter settles.
• Prepare a phage filtrate. Open the package of .22 micron filter. Using a syringe remove 2ml of liquid from the flooded sample.
• Dispense a minimum of .5 ml of filtrate into a microcentrifuge tube. Proceed to Plaque Assay.

Plaque Assay

• Prepare bench for aseptic work.
• Inoculate the host bacteria with phage sample.
• Use a micropipettor and aseptic technique and dispense each phage sample into the appropriate culture tube.
• Mix each inoculated host culture by gently tapping the tube. Let the sample sit undisturbed.
• Plate the samples with top agar with 3 ml of molten top agar.
• Using a sterile 5 ml pipette, transfer 3 ml of top agar to an inoculated host tube. Aspirate the mixture back into the pipette and transfer it to the appropriate plate. Let the plates sit undisturbed for 20 minutes until the top agar solidifies.
• Incubate the inverted plates and place at the specified temperature for 24-48 hours.
• Check the plates for plaques.

Results of Direct Isolation and Plaque Assay

I did not find any plaques on my agar plates. My plates contained a lot of contamination from the air, table, and touch. To fix this, I must work closer to the flame at all times.

Procedure for Enriched Isolation

• Extract phages from a soil sample. Fill a 50 ml conical tube with your sample to the 15 ml mark. Add enrichment broth to 35 ml mark. Shake the sample for 1-2 hours.
• Balance the tubes and centrifuge for 10 minutes to pellet.
• Filter the supernatant through a .22 micron filter. Collect the flow in a 50 ml sterile conical tube.
• Add .5 ml of bacterial host to conical tube. Incubate the flask for 2- 5 days.
• Filter the enriched culture. Using a micropipette, transfer 1.4 ml of your culture to a microcentrifuge tube. Repeat this procedure so you have two microcentrifuge tubes.
• Spin the tubes for 1 minute in the microcentrifuge.
• Filter the supernatant through a .22 micron filter. Transfer the supernatant into a clean microcentrifuge tube.

Results for Enriched Isolation

Everything went great during my enriched Isolation purification. Everything went as planned and performed as shown in the Protocol.

Procedure for Spot Test

• Collect the liquid phage samples that need testing.
• Label the bottom of an agar plate. Divide the plate into as many sections as samples.
• Obtain host bacteria culture.
• Obtain 3 ml of molten top agar and dispense the top agar-bacteria mixture onto an agar plate.
• Allow the plate to sit undisturbed for 20 minutes or until the top agar solidifies.
• Aseptically transfer 10 microliters of each sample onto the proper location on the agar plate.
• Spot 10 microliters of sterile phage buffer on the plate as a negative control.
• Incubate the plates for 24-48 hours.

Results from my spot test

My spot test did not show any plaques that contained phage. I completed the procedure for enriched isolation and the spot test twice to try more than once to find phage, but did not have success. Since I did not find phage, for purification I used/adopted one from a fellow lab mate.

Procedure for Plaque Assay Purification

• Place a sterile tip onto a p200 micropipettor.
• Gently stab the agar in the center of the plaque. Place the end of the tip into the phage buffer. Vortex.
• Arrange microcentrifuge tubes into a 10-fold serial dilutions.
• Add 90 microliters of phage buffer to each tube.
• Add 10 microliters of your undiluted phage sample to 10^-1 tube and vortex.
• Continue each dilution until you get to your last tube.
• Inoculate the host bacteria with phage sample.
• Use a micropipettor and aseptic technique and dispense each phage sample into the appropriate culture tube.
• Mix each inoculated host culture by gently tapping the tube. Let the sample sit undisturbed.
• Plate the samples with top agar with 3 ml of molten top agar.
• Using a sterile 5 ml pipette, transfer 3 ml of top agar to an inoculated host tube. Aspirate the mixture back into the pipette and transfer it to the appropriate plate. Let the plates sit undisturbed for 20 minutes until the top agar solidifies.
• Incubate the inverted plates and place at the specified temperature for 24-48 hours.
• Check the plates for plaques.

Results for Plaque Assay Purification

After my first set of serial dilutions, I did not have any plaques.