Discovery of Aislinn

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by Matthew Bristerpostma, Shey Andrews and Abbigal Ramirez

Phage Isolation Notes

THE DISCOVERY OF AISLINN

Shey Andrews, Abbigal Ramirez, and Matthew Postma

Virus Isolation Class

Fall 2018

Dr.Edwards



INTRODUCTION

Bacteriophages have recently become one of the forefronts of modern research. Globally, their population amounts to over 10^31 particles and are highly dynamic in metabolism and genetic diversity With the resistance of bacterial superbugs becoming common clinical issue, the use of bacteriophages for antibacterial use against deadly infections has drawn the focus of the science community. Howard Hughes Medical Institutes created the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES). This program aides to increase undergraduates to explore the world of bacteriophages while developing -proper scientific laboratory skills. “The Howard Hughes Medical Institute SEA-PHAGES program takes advantage of the huge size and diversity of the bacteriophage population to engage students in discovery of new viruses, genome annotation, and comparative genomics.”

The following notes represent the combined efforts of the Astrid team, Shey Andrews and Abbigale Ramirez and the Aislinn team, Matthew Bristerpostma. Due to deficient results, the Astrid team joined the Aislinn team on October 15th, 2018. In an effort to provide thorough documentation, both sets of notes have been maintained with appropriate identifiers.

BACTERIAL HOST

Aislinn/Astrid: September 4th 2018: Protocol 5.1 Collecting Samples from the Environment

Objective: To extract bacteriophage from an environmental sample from three different locations.

Materials:

15mL conical tube
small spade
phone
pen and paper

Procedure:

A desired spot was located and the GPS coordinates, temperature of the air, and general type of environment in which the soil was collected in was recorded.
Using a spade, a 6-10 inch hole was made and a sample of the soil was transferred to a 15 mL conical tube to about two-thirds full.
The conical tube was labeled and stored immediately in the fridge until the next day when direct and enriched isolation were performed.

Abbigal Ramirez Collection

A total of three soil samples were collected in various locations in Cleburne, and Stephenville, TX.

Latitude: 32.3016 : Longitude: -97.4197
Latitude: 32.3373 : Longitude: -97.3943
Latitude: 32.2248 : Longitude: -98.2290

The first two locations are in Cleburne, TX. The first location was at the shore of Lake Pat in Cleburne, TX recovered at a temperature of 34.4 degrees Celsius, location two was a grave of a dog, located at a man’s home, recovered at a temperature of 33.9 degrees Celsius. The last location was in Stephenville, TX outside of an apartment complex, recovered at a temperature of 23.3 degrees Celsius. All three of the soil samples were rather dry, and easily accessed.

Shey Andrews Collection

A total of three soil samples were collected on September 4th, 2018 from various locations in Stephenville, TX.

Latitude: 32.21506276343063: Longitude: -98.20294943400457
Latitude: 32.21114128379872: Longitude: -98.22142983175213
Latitude: 32.2115830033962: Longitude: -98.2210702195454

The temperature recorded that night was 20 degrees Celsius. Location one was collected at the Stephenville City Park in a field next to a large body of water. The soil collected was a dark moist substance and was clumpy. The second location of collected soil was across from Tarleton State University, Stephenville, TX. The field was about a mile off campus and was next to a parking lot. The dirt collected here was a little more dry than the first location and was more sedimentary. The third location was about a half mile away from location two behind a storage shed. The dirt collected resembled the dirt from location one and was found near a running water supply.

Matthew Bristerpostma Collection

Collected September 4th, 2018 from several locations in Erath County, TX.

Latitude: 32°12’58” N: Longitude: 98°13’00” W
Latitude: 32°19’35” N: Longitude: 98°11’23” W
Latitude: 32°19’37” N Longitude: 98°11’22” W

The temperature in all cases was 27.7°C. Sample 1 was taken from an anthill on the Tarleton campus, samples 2 and 3 were taken from a moist pond bed, 2 from cow dung, and 3 from the soil. Both samples were quite moist.

Aislinn/Astrid: September 5th 2018: Protocol 5.2 Direct Isolation of Environmental Sample

Objective: The purpose of performing a direct isolation is to successfully extract bacteriophages from the collect environment samples. In order to do this, you have to use a liquid media and filter to make sure you only get potential viral particles. Then you inoculate the host bacteria in which you want to study and plate it using Plaque Assay Protocol 5.3.

Materials:

Environmental Sample
Liquid Media
Sterile 5mL syringe
0.22 um syringe filter
5ml serological pipette
Microcentrifuge tubes
15ml conical tube

Procedure:

The work bench was prepared using a decontaminate and working close to an open flame.
The sample and material were then gathered and the experiment was ready to be performed.
Extracting bacteriophage from solid environmental sample.

A 15mL conical tube was filled about one-third full of soil.
Liquid Media was added until the soil was submerged beneath about 2-3 mL of media.
The tube was then capped and inverted several times to insure that the soil was mixed thoroughly.
The tube was then shaken in a shaking incubator set at 250 rpm for 1-2 hours.
After incubating the soil was allowed to settle in the tube for about 15 minutes.

Preparing the Phage Filtrate.

A 0.22 microliter syringe filter was used to filter out any bacteria/contaminant so that only bacteriophages go through. The package containing the filter was opened and placed on the bench with the filter still in the package. This was an important step because the filter is easily contaminated.
Approximately 2 ml of liquid from the flooded sample was carefully removed to avoid withdrawing solid material and clogging the filter.
The syringe was attached to the filter and was removed from the packaging.
0.5 ml of filtrate was dispensed in a microcentrifuge tube and labeled accordingly.
The tube was capped immediately and stored in the fridge at 4 degrees Celsius.
The filter and syringe were discarded and the filtrate was ready to proceed to Plaque Assay Protocol 5.3.

Aislinn: September 5th 2018: Protocol 5.5 Enriched Isolation on Phage from anthill

Objective: The objective of doing an enriched isolation is to amplify the amount of phage present in the environmental sample. The result of doing the incubation of the soil sample is a culture with an exponentially larger in concentration of phages that is specific to the bacterial host.

Materials:

Solid Environmental Sample
0.22 microliter syringe filter
Liquid Media
Microcentrifuge tubes
500 microliters of host bacteria
50 mL sterile conical tube

Procedure:

The solid environmental sample was gathers along with the materials.

Extracting the Phage from the soil sample

A 50 mL conical tube was filled with the soil to the 15 mL mark.
Liquid media was added to the 35 mL mark and the tube was vortexed.
The sample was then placed in the shaking incubator for 1-2 hours at 250 rpm.
After settling, the tube was placed and balanced inside the centrifuge machine and was centrifuged at 2,000xg for 10 minutes in order to pellet the soil.

Preparing Bench and Seeding the Culture with Host Bacteria

The supernatant was filtered through a 0.22 microliter filter to remove unwanted bacteria and soil particles.
The volume of supernatant collected was about 15 mL.
0.5 mL of bacterial host culture was added to the conical tube.
The cap was placed on the conical tube. Care was taken to make sure that the cap was properly aerated. This was done by loosely placing the cap and securing it with a piece of tape.
The conical tube was incubated in the shaking incubator (upright to avoid spillage) for 24 hours at 220 rpm.

After the enriched culture incubated for 24 hours, the next step was done.

The plunger from the syringe was removed
The sterile filter was opened and attached to the barrel of the syringe
1 mL of supernatant was pipetted from each of the microcentrifuge tubes into the syringe barrel for a total of about 2 mL.
The tip of the syringe (that was attached to the filter) was placed over the microcentrifuge tube and the plunger was inserted to the syringe and depressed.
1.4 mL of the enriched culture was transferred using a pipette into a microcentrifuge tube. This step was repeated twice so that there are two microcentrifuge tubes with 1.4 mL in each.
The tubes were spun at high speed in a microcentrifuge for 1 minute to pellet the bacteria.
The supernatant was not clear so the following was performed:
The microcentrifuge tube was centrifuged for at high speed for 1 minute to ensure that the bacteria is pelleted.
The supernatant what transferred into a clean microcentrifuge tube and capped immediately.

Materials:

Environmental Sample
Liquid Media
Sterile 5mL syringe
0.22 um syringe filter
5ml serological pipette
Microcentrifuge tubes
15ml conical tube

Procedure:

The work bench was prepared using a decontaminate and working close to an open flame.
The sample and material were then gathered and the experiment was ready to be performed.
Extracting bacteriophage from solid environmental sample.

A 15mL conical tube was filled about one-third full of soil.
Liquid Media was added until the soil was submerged beneath about 2-3 mL of media.
The tube was then capped and inverted several times to insure that the soil was mixed thoroughly.
The tube was then shaken in a shaking incubator set at 250 rpm for 1-2 hours.
After incubating the soil was allowed to settle in the tube for about 15 minutes.

Preparing the Phage Filtrate.

A 0.22 microliter syringe filter was used to filter out any bacteria/contaminant so that only bacteriophages go through. The package containing the filter was opened and placed on the bench with the filter still in the package. This was an important step because the filter is easily contaminated.
Approximately 2 ml of liquid from the flooded sample was carefully removed to avoid withdrawing solid material and clogging the filter.
The syringe was attached to the filter and was removed from the packaging.
0.5 ml of filtrate was dispensed in a microcentrifuge tube and labeled accordingly.
The tube was capped immediately and stored in the fridge at 4 degrees Celsius.
The filter and syringe were discarded and the filtrate was ready to proceed to Plaque Assay Protocol 5.3.

Materials:

Environmental Sample
Liquid Media
Sterile 5mL syringe
0.22 um syringe filter
5ml serological pipette
Microcentrifuge tubes
15ml conical tube

Procedure:

The work bench was prepared using a decontaminate and working close to an open flame.
The sample and material were then gathered and the experiment was ready to be performed.
Extracting bacteriophage from solid environmental sample.

A 15mL conical tube was filled about one-third full of soil.
Liquid Media was added until the soil was submerged beneath about 2-3 mL of media.
The tube was then capped and inverted several times to insure that the soil was mixed thoroughly.
The tube was then shaken in a shaking incubator set at 250 rpm for 1-2 hours.
After incubating the soil was allowed to settle in the tube for about 15 minutes.

Preparing the Phage Filtrate.

A 0.22 microliter syringe filter was used to filter out any bacteria/contaminant so that only bacteriophages go through. The package containing the filter was opened and placed on the bench with the filter still in the package. This was an important step because the filter is easily contaminated.
Approximately 2 ml of liquid from the flooded sample was carefully removed to avoid withdrawing solid material and clogging the filter.
The syringe was attached to the filter and was removed from the packaging.
0.5 ml of filtrate was dispensed in a microcentrifuge tube and labeled accordingly.
The tube was capped immediately and stored in the fridge at 4 degrees Celsius.
The filter and syringe were discarded and the filtrate was ready to proceed to Plaque Assay Protocol 5.3.Aislinn Observational Notes:

Both isolation procedures were redone using the same procedure due to deficient liquid. Direct isolation set to incubate at 1830 20180904, Enriched isolation set to incubate at 1801.

Aislinn/Astrid: September 5th 2018: Protocol 5.3 Plaque Assay

Objective: Using bacterial lawns of a selected host to detect the presence of phages. When phages are present on the bacterial lawn, there will be areas of “clearing” called plaques. Plaques let you know that viruses have replicated and lysis the bacterial in the area (area of clearing are areas that no longer have bacteria present). Each plaque may be a different phage since they arise from a single phage particle.

Materials:

Phage sample
Host Bacteria (250 ul/plate)
Agar plates
Phage Buffer
Top Agar
Microcentrifuge tubes
5 ml serological pipettes

Procedure:

The workbench was prepared using aseptic technique and an open flame
The phages from the direct isolation were gathered to be used for the plaque assay.

Inoculating the host bacteria with the phage sample

Since there was only one sample to test, one aliquot of 250 ul of host bacterial was collected and the tube was labeled accordingly.
A micropipette was used to dispense the phage sample volume, according to the table below, into a culture tube containing 250 ul of host bacteria.
The tube was tapped gently to mix the inoculated host culture.
The inoculated host culture then sat undisturbed for 5-10 minutes to allow for attachment.

After 5-10 minutes of letting the sample sit, the samples were plated using top molten agar that was provided by the instructor

An agar plate was collected and labeled Shey Andrews; 9/05/2018:3:19PM @23C.
A bottle of top agar was removed from the 55C bath and brought to the workbench.
Using a 5 ml pipette, 3 ml of molten top agar was aseptically transferred into the inoculated host tube.

Sample Type	Sample Volume
Direct Isolation sample	500 ul
Enriched culture	10 ul
Serial Dilutions of picked plaques	10 ul
Lysates for titering	10 ul
Negative Control	10 ul of phage buffer
Positive control	10 ul provided phage sample

The molten top agar/inoculated host mix was then immediately aspirated back into the pipette and was transferred to the agar plate.
The agar plate was quickly and gently tilted so that the top agar mixture evenly coated the plate.

Incubating the plates

The plates were incubated to allow for bacterial growth and phage infection.
The top agar was allowed to solidify by letting the plates sit for approximately 20 minutes undisturbed.
The plates were inverted and placed in the incubator for 24-48 hours at 23-30C

Aislinn: September 6th 2018: Protocol 5.3 Results from 09/06/2018 Plaque Assay

The plates that were put in the incubator on 09/06/2018 shows numerous plaques. Though the direct isolation agar did not set properly before incubation. 3 plaques were selected from the enriched isolation plate following incubation. The below photo shows the numberous plaques found on the direct and enriched isolation plates first incubated following the initial isolation protocols:

Astrid: September 7th 2018: Protocol 5.3 Results from 09/06/2018 Plaque Assay

The plate that was put in the incubator on 09/06/2018 shows no plaques. This means that there was no phage particles successfully transfered to the host and plate. Also the top agar did not set properly. The supernate from the direct isolation used in this plaque assay was used as a duplicate and the sample was tested again for the presence of phage.

Astrid: September 7th 2018: Protocol 5.3 Plaque Assay Duplicate

Objective: To extract bacteriophage from the direct isolation distillate.

Materials: same as Protocol 5.3 Plaque Assay on September 5th.

Procedure:

Modifications

When pipetting the extracted phage from the centrifuge tube to the bacterial host test tube, extra care was taken to make sure that the phage sample made it completely into the host by avoiding the sides of the test tube.
Before inoculating the host, the tube was flicked and observed to make sure that the culture was as homogenous as it could get and after adding the phage it was mixed again by flicking it.
The attachment time was also physically timed to 20 minutes to make sure the phage was properly attached. The inoculated culture was then plated using the same steps in the previous run of protocol 5.3.
The procedure was the same as the Plaque Assay performed on September 5th with a few modifications.

Aislinn: September 10th 2018: Protocol 5.6 Spot Test on Enriched Isolation

Objective: To test a sample for the presence of phage on a bacterial lawn by “spotting” the plate.
Materials:

Agar plates
250 ul of host bacteria
To agar
Phage Buffer 5mL serological pipette

Procedure:

The bench was prepared for aseptic work and the supplies and liquid phage from the enriched isolation was gathered.

Preparing Bacterial Lawn Using Aseptic Technique

The agar plate was labeled into three sections (AR1, AR2, SA1).
3 mL of molten top agar (provided by the instructor) was transferred into a 250 ul culture of host bacteria using a sterile 5 mL pipette.
The solution was immediately drawn from the tube and dispensed onto an agar plate.
The agar plate was gently and quickly tilted in multiple directions until the agar evenly coated the plate.
The plate sat undisturbed for 20 minutes and the top agar solidified completely.

Spotting the Liquid Phage on the prepared bacterial lawn.

10 ul of each sample was used to spot the bacterial lawn one at a time.
The tip was held slightly above the agar and expelled so that the drop did not splatter.
The spots were placed in a triangle pattern on each of the three sections of the plate.
Without inverting the plate, it was incubated at _________ for 24-48 hours.

The below photo shows the results of the spot test, completed on 20180910 following approximately 48 hours of incubation at a 10^-1 dilution:

xA was selection to move forward with serial dilutions.

Aislinn: September 10th 2018: Protocol 5.2 Picking a Plaque and Performing Serial Dilutions

Using the above shown xA plaque, serial dilutions were performed:

Materials:

Phage Buffer
Samples requiring diluting
Microcentrifuge tubes

Objective: To prepare the liquid phage samples of decreasing concentrations. The goal is to use the method of serial dilutions to manipulate the number of phages in a sample. This can allow for easy mathematical calculations

Procedure:

The bench was prepared using aseptic technique and the material were gathered.
A 10-fold serial dilution was set up.

The microcentrifuge tubes were arranged in a tube rack and labeled according to their dilution.
90 microliters of phage buffer to each of the test tube.

Performing the 10-Fold Serial Dilutions

10 microliters of the undiluted phage was added to the first microcentrifuge tube labeled 10^-1 and the tube was vortexed.
Using a different clean pipette tip for each transfer, 10 microliters of the 10^-1 sample was transferred to the centrifuge labeled 10^-2 and was then vortexed.

This process was continued until all the dilutions were made.
Performing Spot test using the liquid phage from the Serial Dilution Protocol.

The plate were sectioned in rows and labeled accordingly to their dilution. Since three plaques were picked, a total of three dilutions were made, thus two plates per sample.
3 mL of molten top agar was added to a 250 microliter aliquot of bacteria and the mixture was transferred to the plates and left undisturbed for 15 minutes to allow it to settle.
Once the top agar on the plate solidified, 10 microliters of each microcentrifuge tube were plated carefully to avoid splashing or spilling over to other sections.
The plates were incubated agar side down in the incubator for 48 hours.

Astrid: September 10th 2018: Protocol 5.3 Results from Plaque Assay on 09/07/2018

Results: This photo was taken on September 10th 2018. This is the plate that was incubated from 09-07-2018 and was from the same sample of direct isolation. There were no plaques present on this plate which meant that there were no phages present. Our next step was to perform an enriched isolation on another soil sample.

Astrid: September 11th 2018: Protocol 5.5 Enriched Isolation of Soil Sample from Collection Site Two

Materials:

Solid Environmental Sample
0.22 microliter syringe filter
Liquid Media
Microcentrifuge tubes
500 microliters of host bacteria
50 mL sterile conical tube

Procedure:

The solid environmental sample was gathers along with the materials.

Extracting the Phage from the soil sample

A 50 mL conical tube was filled with the soil to the 15 mL mark.
Liquid media was added to the 35 mL mark and the tube was vortexed.
The sample was then placed in the shaking incubator for 1-2 hours at 250 rpm.
After settling, the tube was placed and balanced inside the centrifuge machine and was centrifuged at 2,000xg for 10 minutes in order to pellet the soil.

Preparing Bench and Seeding the Culture with Host Bacteria

The supernatant was filtered through a 0.22 microliter filter to remove unwanted bacteria and soil particles.
The volume of supernatant collected was about 15 mL.
0.5 mL of bacterial host culture was added to the conical tube.
The cap was placed on the conical tube. Care was taken to make sure that the cap was properly aerated. This was done by loosely placing the cap and securing it with a piece of tape.
The conical tube was incubated in the shaking incubator (upright to avoid spillage) for 2-5 days at 220 rpm.

After the enriched culture incubated for two days, the next step was done.

The plunger from the syringe was removed
The sterile filter was opened and attached to the barrel of the syringe
1 mL of supernatant was pipetted from each of the microcentrifuge tubes into the syringe barrel for a total of about 2 mL.
The tip of the syringe (that was attached to the filter) was placed over the microcentrifuge tube and the plunger was inserted to the syringe and depressed.
1.4 mL of the enriched culture was transferred using a pipette into a microcentrifuge tube. This step was repeated twice so that there are two microcentrifuge tubes with 1.4 mL in each.
The tubes were spun at high speed in a microcentrifuge for 1 minute to pellet the bacteria.
The supernatant was not clear so the following was performed:
The microcentrifuge tube was centrifuged for at high speed for 1 minute to ensure that the bacteria is pelleted.
The supernatant what transferred into a clean microcentrifuge tube and capped immediately.

The next step that was performed was the Spot Test Protocol 5.6

Astrid: September 10th 2018: Protocol 5.6 Spot Test on Enriched Isolation

Objective: To test a sample for the presence of phage on a bacterial lawn by “spotting” the plate.

Materials:

Liquid phage sample from enriched isolation
Agar plates
250 ul of host bacteria
To agar
Phage Buffer 5mL serological pipette

Procedure:

The bench was prepared for aseptic work and the supplies and liquid phage from the enriched isolation was gathered.

Preparing Bacterial Lawn Using Aseptic Technique

The agar plate was labeled into three sections (AR1, AR2, SA1).
3 mL of molten top agar (provided by the instructor) was transferred into a 250 ul culture of host bacteria using a sterile 5 mL pipette.
The solution was immediately drawn from the tube and dispensed onto an agar plate.
The agar plate was gently and quickly tilted in multiple directions until the agar evenly coated the plate.
The plate sat undisturbed for 20 minutes and the top agar solidified completely.

Spotting the Liquid Phage on the prepared bacterial lawn.

10 ul of each sample was used to spot the bacterial lawn one at a time.
The tip was held slightly above the agar and expelled so that the drop did not splatter.
The spots were placed in a triangle pattern on each of the three sections of the plate.
Without inverting the plate, it was incubated at _________ for 24-48 hours.

Astrid: September 12th 2018: Results from Spot Test of Enriched Isolation

This image was taken on 09/12/2018. The results show that there are no plaques on the plate so none of the samples from the enriched isolation contained phage. There is evidence that there was some bacterial contamination (yellow areas on the plate). This most likely resulted from a cross contamination between the bacterial host and the phage during transfer. A second spot test was performed using the same liquid phage sample from the enriched isolation. During the second spot test, a phage buffer was added as a negative control.

Aislinn: September 12th 2018: Results from September 10th: First Round of Serial Dilutions

Serial dilutions failed. Brownish-white turbidity in plates indicated contamination on 10^-5 and 10^-6 samples. Re-performing serial dilution procedure as given above.

Astrid: September 15th 2018: Protocol 5.6 Spot Test on Enriched Isolation

Objective: Test sample for the presence of phage.

Materials: Same as before with Protocol 5.6.

Procedure:

Modifications

The only modification that was performed was to the labeling of the agar plate. The agar plate this run was sectioned into four different sections (Phage Buffer, AR1, AR2, SA1). A phage buffer was added as a negative control. The samples that were collected from the enriched isolation used in the previous spot test was also used during this run of the experiment.

Aislinn: September 17th 2018: Results from First Round of Serial Dilutions:

Serial dilutions were successful. No contamination immediately evident.The second round of serial dilutions was started using the above protocol.

The photo below is of the completed first round of serial dilution plates. An approximately 10-fold decrease of plaques was observed, tapering off at 10^-6.

Astrid: September 17th 2018: Protocol 5.6 Results from Spot Test 2

The plate was incubated for two days. There were no plaques observed on 09/17/2018 when this picture was taken which means that there are no phage present in the sample.

Astrid: September 19th 2018: Protocol 5.2 Picking a Plaque and Performing Serial Dilutions

Note: This represents the last step attained by the Astrid team prior to joining the Aislinn team, therefore all results and procedural notes are contained below, irrespective of date.

Objective: The objective was to pick a plaque off a donated phage.

Materials:

Agar plates with plaques of interest
Phage Buffer
Microcentrifuge Tubes

Procedure:

The bench was prepared using aseptic technique and the agar plate with the plaques were collected.
Using a sharpie, three plaques on the plate were circled to indicate which ones we picked. Since the plate that was given had already be serially diluted, the plaques that were picked were from the original phage concentration section of the plate. The morphology of the plaques were really turbid and very small.
We labeled three microcentrifuge tubes 1, 2, and 3.
Using aseptic technique, 100 microliters of phage buffer were translated to each of the three centrifuge tubes.
Picking the Plaque

A sterile tip was placed on a p200 micropipette.
The pipette was held perpendicular to the plate and the tip was lowered carefully inside the center of the plaque. Care was taken to avoid touching the outer bacterial part of the plate.
The end of the tip was placed into the phage buffer in the corresponding microcentrifuge tube. The tip was tapped into the sidewall of the tube and the phage buffer mixture was pipetted up and down to dislodge phage particles.
The tubes were vortexed.
Steps a-d were repeated for each microcentrifuge tube.

Serial Dilutions

Materials:

Phage Buffer
Samples requiring diluting
Microcentrifuge tubes

Procedure:

The bench was prepared using aseptic technique and the material were gathered.
A 10-fold serial dilution was set up.

The microcentrifuge tubes were arranged in a tube rack and labeled according to their dilution.
90 microliters of phage buffer to each of the test tube.

Performing the 10-Fold Serial Dilutions

10 microliters of the undiluted phage was added to the first microcentrifuge tube labeled 10^-1 and the tube was vortexed.
Using a different clean pipette tip for each transfer, 10 microliters of the 10^-1 sample was transferred to the centrifuge labeled 10^-2 and was then vortexed.

This process was continued until all the dilutions were made.
Performing Spot test using the liquid phage from the Serial Dilution Protocol.

The plate were sectioned in rows and labeled accordingly to their dilution. Since three plaques were picked, a total of three dilutions were made, thus two plates per sample.
3 mL of molten top agar was added to a 250 microliter aliquot of bacteria and the mixture was transferred to the plates and left undisturbed for 15 minutes to allow it to settle.
Once the top agar on the plate solidified, 10 microliters of each microcentrifuge tube were plated carefully to avoid splashing or spilling over to other sections.
The plates were incubated agar side down in the incubator for 48 hours.
After diluting the phage sample from the picked plaque, the Spot Test Protocol 5.6 was performed.
The plates were set up in the following manner:

Results:

All the slides were negative for phage. There was one slide that has a contamination problem and there was a bacterial growth on the plate.

September 26, 2018: Results of picking a plaque and serial dilutions.

The only real result we obtained was also in a plate that had massive contamination, although we used it because we believed it could be salvaged.

We repeated the process of picking plaque and performed serial dilutions yet again, except this time we used one plate per dilution.

October 1, 2018: Results of picking the plaque and serial dilutions part 2.

Our samples were in the incubator for too long of a period of time, therefore the result was almost all the same.

However, one sample was quite odd compared to the rest. It had contamination, but unlike our previous samples that had experience this, it was so developed it completely ate the intierty of the agar leaving only the bacteria.

Because of this failed attempt, we tried again using a new sample from our phage plate with another failed result, due to what seemed to be the same mistake of it cooking too long in the incubator. This was taken October 8, 2018.

October 8,2018: Repeated the process yet again.

Due to another failed attempt I learned that not only had i plated it incorrectly, but I hadn’t correctly added my dilutions to the bacteria before applying it to the molten agar. So i switched up the procedure a bit, adding the steps

Use 10ul and add it to the bacteria
Allow bacteria and dilutions to settle for approximately 20 minutes.
Mix bacteria/ dilution mixture with molten agar\
Apply to agar plate
Incubate for 24-48 hours.

Result of updated process

The result of this procedure went better than the previous, however there was no longer a trace of phage.

October 10, 2018: One last time.

Due to our many attempts, we again tried to repeat this procedure, had an observer to see if we were doing anything wrong, or if we could do anything in a better way to avoid the issues we continued to have. We used our first and best result from the spot test, which was OG 1, and gave it our best efforts. Then we waited the perfect amount of time to check our plaques.

With yet again NO result, we felt defeated ultimately, and did what we believed would be the most rational option. We adopted into another phage, and have abandoned our own.

October 15, 2018

It was this day we officially abandoned our phage and adopted another. We took the last picture we needed for this journal, and have officially put the rest to rest.

Aislinn: September 19th 2018: Results of second round of Serial Dilutions

Plates 10^-3 and 10^-5 were found to have been flipped to early. No plaques on 10^-4 or 10^-6. Serial dilution procedure was redone. The images below show the failure of plates 10^-4 and 10^-6.

Aislinn: September 24th 2018: Results of second round of Serial Dilutions

Serial dilutions failed. Contamination was evident in whitish turbidity on all plates. Plaques were only evident on 10^-5 and 10^-6, likely because of low contamination concentration.

Note: An incident of human error was identified. I had been removing the pipette nozzels from their paper prior to using them, thereby opening them up to contamination.

Aislinn: September 27th 2018: Results of second round of Serial Dilutions and collecting plate lysate using Protocol 6.3: Collecting Plate Lysates

Serial dilutions were successful.

A webbed plate was formed on the 10^-4 dilution plate. Plates 10^-1 through 10^-3 were completely lysed.

Protocol 6.3: Collecting Plate Lysates

Objective: To generate a highly concentrated liquid phage sample

Materials:

Webbed plate(s) with clonal phage population (10^-4 plate mentioned above)
Phage buffer (8 ml/plate)
0.22 μm filter
5 ml syringe
15 ml sterile conical tube for lysate storage

Procedure:

The bench was prepared for aseptic work.

The webbed 10^-4 serial dilution plate was used and 8mL of sterile phage buffer was applied using a sterile pipette. This was left to sit after being covered at 1045 for 3 hours, allowing the phage to diffuse into the buffer. At 1340 the lysate was harvested using a sterile 5mL syringe and filtered through a sterile 0.22 μm filter into a conical tube.

Aislinn: September 28th 2018: Protocol 6.4: Spot Titer

Using the lysate harvested according to the previously listed procedure, Protocol 6.4 was executed.

Objective: To determine the concentration of phage particles in a lysate using a spot test.

Materials:

Lysate for titering
Agar plate
Host bacterial culture
Top agar
Phage buffer
Microcentrifuge tubes
5 ml serological pipettes

Procedure: Using aseptic techniques, a single agar plate was prepared. Molten agar inoculated with M. Foliorum host bacteria was plated and allowed to set. Meanwhile, a 10µL aliquot of lysate was diluted down according to the serial dilutions protocol. These dilutions were then spotted on to the set plate and incubated over the weekend, approximately 48 hours.

Aislinn: October 1, 2018: Spot Titer results

Spot Titer success. The below picture represents the successful spot titer.

Note: A mistake was made in labeling the above plate. The lysate was take from a 10^-4 dilution plate, not a 10^-7. Titer of lysate calculated at 2.3×10^9 using 10^-6 spot.

Aislinn: October 2, 2018: Protocol 6.5: Full plate titer

Objective: To determine the concentration of phage particles in a lysate more accurately than a spot titer by using a plaque assay.

Materials:

Liquid phage sample requiring titering
Phage buffer
Agar plates for plating dilutions
Top agar
Host bacteria
Microcentrifuge tubes
5 ml serological pipettes

Procedure: Using aseptic techniques, the lysate was diluted according to the serial dilution protocol down to 10^-8 and plated on prepared agar by inoculating host M. Foliorum. This was left over night to incubate.

Aislinn: October 3, 2018: Results of Full plate titer

Plate titer failed. Unknown mass contamination event had taken place in multiple student samples, indicated by the presence of white turbidity and spots, foul odor, and resistance to phage lysis.

The below photograph shows the results of the plate titer and the contamination issue:

Aislinn: October 9, 2018: Protocol 6.5: Full Plate Titer:

Following the identification and elimination of the contamination issue the plate titer protocol was re-performed and incubated overnight.

Aislinn: October 10, 2018: Results of Full plate titer:

Plate titer was a success. Counted 48pfu on 10^-3 plate, and a 10-fold increase on 10^-2. Titer calculated at 4.8×10^9.

Note: Instability of phage particles attributed to a lack of notable results after the 10^-5 concentration.

Aislinn: October 10, 2018: Protocol 7.1: Making webbed plates from lysate of known titer

Objective: To create a plate with a very high density of plaques from a lysate of known titer

Materials:

Lysate of known titer
Agar plates
Host bacterial cultures
Phage buffer
Top agar
Microcentrifuge tubes
5 ml serological pipettes

Procedure: Using aseptic techniques, the lysate was diluted down to the calculated concentration required to yield webbed plates: 3.125×10^-6 mL or 3.125×10^-3µL.

Aislinn: October 15, 2018: Results of webbed plates

Webbed plates failed. Plates were neglected and completely lysed following 5 days of incubation. Contamination was present on 3 of the plates. Procedure was repeated according to the above protocol on 3 sets of bracketed plates at dilutions 10^-4, 10^-5, and 10^-6, for a total of 9 plates.

Aislinn: October 17, 2018: Results of webbed plates

Webbed plates partially successful. Contamination evident on 10^-5 plates. 2 of the 10^-4 plates were webbed. These plates were flooded and harvested using the collecting lysate protocol. 3 additional plates were made using this collected lysate at the appropriate concentration (3.125*10^-3) and incubated.

Note: A change in plaque morphology was observed at this step. Lytic plaques were no longer visible on 10^-5 or 10^-6 plates, replaced by less numerous lysogenic and turbid ones.

Aislinn: October 18, 2018: Results of webbed plates

Plates incubated on October 17^th showed no plaques. These were placed back in the incubator.

Aislinn: October 22, 2018: Results of webbed plates/Collecting Plate Lysates

Webbed plates partially successful. All 3 were webbed. These plates were flooded and harvested after diffusing for 3 hours according to protocol 6.3.

Note: The incubation time of these plates was significantly longer than expected.

Aislinn: October 22, 2018: Calculating the titer of the lysate collected using the plate titer protocol

The plate titer protocol was performed to the 10^-6 dilution using 10 of the lysate collected. These plates were incubated.

Aislinn: October 27, 2018: Results of lysate plate titer

Plate titer incubation took 5 days. The growth of the plates was checked every day during this period to ensure viability. Titer calculated using 10^-3 (webbed) plate, equaling 1.6×10^10.

16mL of lysate was collected overall.

Aislinn: October 30, 2018: Protocol 8.1b Microscopy

Objective: To prepare your phage sample for viewing with a transmission electron microscope

Materials: High-titer lysate
EM forceps
Pelco tabs
200–400 mesh carbon–formvar-coated copper grids
Wedges of Watman filter paper for wicking
Sterile filteredwater
Phage buffer
1 % uranyl acetate

Procedure: Using aseptic techniques, 1mL of HVL (High volume lysate) was transferred to a 1.5 mL microcentrifuge tube and centrifuged at top speed at 4°C for 1 hour to pellet. A micropipettor was used to remove .90 mL of supernate, leaving behind the pelleted phage and .10mL of fluid. 100µL of phage buffer was then added and left standing for 1 hour at 4°C to resuspend the pellets.

A work space under the fume hood was prepared, using a PELCO tab, grid, and petri dish with parafilm stuck to the inside. The phage was then mounted on the grid by micropipetting 10µL of phage directly on to the grid. This was allowed to settle for two minutes, after which, using sterile filter paper, excess moisture was wicked off the grid.

The grid was then rinsed twice by the use of sterile filtered water, each time wicking the excess moisture off with filter paper.

After this, 10µL of uranyl acetate was used to stain the phage. This was allowed to settle for 2 minutes before the excess was wicked away using filter paper and then allowed to air dry.

After the grid was dried, it was placed into its transportation slot.

Note: The grid was slightly bent during storage.

Aislinn: October 31, 2018: Baylor DNA extraction protocol:

Goal: The goal of this procedure is to obtain a high quality DNA from the concentrated phage lysate.

Materials:

50 mL conical tube
Filter-sterilized phage lysate
Nuclease Mix
Precipitant solution PEG8000/NaCl
Microcentrifuge tube
Sterile water
DNA Clean Up Resin

Procedure:

Degrading the Bacterial DNA

10 mL of filter-sterilized phage lysate was transferred into a 50 mL conical vial.
40 microliters of Nuclease Mix (DNase I and RNase A) was adding to the conical vial and inverted several times to gently mix the solution. It should be noted that the proper use of gloves are important when handling nuclease solutions.

Precipitating the Phage Particles

4 mL of phage precipitant solution (PEG8000/NaCl) was added to the nuclease-treated lysate and the tube was inverted several times to gently mix.
The tube containing the phage precipitant solution and the nuclease-treated lysate was incubated at 37 C for 30 minutes followed by 45 minutes of incubation at room temperature.
The tube was placed into a swinging bucket centrifuge and was spun at 4000 rpm for 30 minutes.

Results: There was no pellet present in the tube after centrifugation.

On October 31st, 2018, the tube containing lysate that did not have a pellet after centrifuging was centrifuged again at 4300 rpm for 40 minutes.

Results: There was still no pellet present in tube. Began using the same lysate sample to perform another protocol on DNA isolation.

To conserve remaining lysate, this protocol was abandoned in favor of the tertiary protocol.

Aislinn: October 31, 2018: Tertiary DNA extraction protocol Day 1:

-Pippetted 100 microliters of lysate for backup into sterile microcentrifuge tube for a possible back up to plate up to ten plates

-Alternative DNA Isolation protocol using ZnCl2

Objective: Isolate phage DNA from collected lysate.

Materials

5 mL of lysate
Nuclease mix
0.5M EDTA
2M ZnCl2 (filtered sterilized)
TES Buffer:

0.1M Tris-HCl, pH 8
0.1M EDTA
0.5% SDS

10 mg/ml Proteinase K
3M potassium acetate, pH 5.2
Isopropanol
70% ethanol
Nuclease-free water
Microcentrifuge tubes
37C heat block
60C heat block
Ice
Nanodrop

Procedure:

The HVL was gently mixed and then an aliquot of 5 mL of the lysate was transferred into a 15 mL conical tube.

20 microliters of nuclease was added to the mix

The tube was then inverted and incubated at 37 C for 10 minutes
1 mL of lysate was aliquoted into a centrifuge tube. This was repeated until there were 5 microcentrifuge tubes with 1 mL of lysate in each.
20 mL of ZnCl2 was added to each of the centrifuge tubes that were then mixed gently by inverting and then incubated at 37 C for five minutes to precipitate the phage.
The five tubes were then placed into the centrifuge and ran at 10000 rpm for one minute.
The supernatant was aspirated carefully to avoid disturbing the pellet. The supernatant was transferred into a new sterile 50 mL conical tube.
500 microliters of TES buffer were added to each tube and incubated at 60 C for 15 minutes.
1 microliter of Proteinase K were added to each microcentrifuge tube and mix gently. The tubes were then incubated at 37 C for 10 minutes to allow for the elimination of any residual nuclease activity.
60 microliters of potassium acetate were added to each tube and mixed gently. This was then placed on ice for 15 minutes. A white residue formed at the bottom of the tube representing the capsid of the bacteriophage.
The tubes were centrifuged at 4 C for one minute at 12,000 rpm to pellet the capsids.
The supernatant from the tubes were transferred into five new microcentrifuge tubes to discard the capsids. The tubes with the pellets (the phage capsids) were discarded.
500 microliters of isopropanol were added to each of the tubes with the supernatant mix and left on ice until the next day.

Aislinn: November 1, 2018: Tertiary DNA extraction protocol Day 2:

Procedure: The supernate-isopropanol mix was centrifuged at top speed for 10 minutes, pelleting DNA. The supernate was conserved in a 15mL conical tube. 250µL of 70% ethanol was added to each microcentrifuge tube and spun again to wash at top speed for 1 minute. The superate was collected and conserved in a 15mL conical tube. The DNA pellets were inverted and allowed to dry for 18 hours.

Aislinn: November 2, 2018: Tertiary DNA extraction protocol Day 3:

Procedure: The first dry DNA pellet was resuspended in 50µL of nuclease-free water, then the solution was transferred to the second to allow resuspension. This process was repeated until the 5^th microcentrifuge tube, which contained approximately 50µL of highly concentrated DNA suspended in nuclease-free water.

This concentrated sample was used to check DNA concentration using the Nanodrop device twice. The results are shown below:

INTRODUCTION

BACTERIAL HOST

Aislinn/Astrid: September 4th 2018: Protocol 5.1 Collecting Samples from the Environment

Objective: To extract bacteriophage from an environmental sample from three different locations.

Materials:

15mL conical tube
small spade
phone
pen and paper

Procedure:

A desired spot was located and the GPS coordinates, temperature of the air, and general type of environment in which the soil was collected in was recorded.
Using a spade, a 6-10 inch hole was made and a sample of the soil was transferred to a 15 mL conical tube to about two-thirds full.
The conical tube was labeled and stored immediately in the fridge until the next day when direct and enriched isolation were performed.

Abbigal Ramirez Collection

A total of three soil samples were collected in various locations in Cleburne, and Stephenville, TX.

Latitude: 32.3016 : Longitude: -97.4197
Latitude: 32.3373 : Longitude: -97.3943
Latitude: 32.2248 : Longitude: -98.2290

Shey Andrews Collection

A total of three soil samples were collected on September 4th, 2018 from various locations in Stephenville, TX.

Latitude: 32.21506276343063: Longitude: -98.20294943400457
Latitude: 32.21114128379872: Longitude: -98.22142983175213
Latitude: 32.2115830033962: Longitude: -98.2210702195454

Matthew Bristerpostma Collection

Collected September 4th, 2018 from several locations in Erath County, TX.

Latitude: 32°12’58” N: Longitude: 98°13’00” W
Latitude: 32°19’35” N: Longitude: 98°11’23” W
Latitude: 32°19’37” N Longitude: 98°11’22” W

Aislinn/Astrid: September 5th 2018: Protocol 5.2 Direct Isolation of Environmental Sample

Materials:

Environmental Sample
Liquid Media
Sterile 5mL syringe
0.22 um syringe filter
5ml serological pipette
Microcentrifuge tubes
15ml conical tube

Procedure:

The work bench was prepared using a decontaminate and working close to an open flame.
The sample and material were then gathered and the experiment was ready to be performed.
Extracting bacteriophage from solid environmental sample.

A 15mL conical tube was filled about one-third full of soil.
Liquid Media was added until the soil was submerged beneath about 2-3 mL of media.
The tube was then capped and inverted several times to insure that the soil was mixed thoroughly.
The tube was then shaken in a shaking incubator set at 250 rpm for 1-2 hours.
After incubating the soil was allowed to settle in the tube for about 15 minutes.

Preparing the Phage Filtrate.

A 0.22 microliter syringe filter was used to filter out any bacteria/contaminant so that only bacteriophages go through. The package containing the filter was opened and placed on the bench with the filter still in the package. This was an important step because the filter is easily contaminated.
Approximately 2 ml of liquid from the flooded sample was carefully removed to avoid withdrawing solid material and clogging the filter.
The syringe was attached to the filter and was removed from the packaging.
0.5 ml of filtrate was dispensed in a microcentrifuge tube and labeled accordingly.
The tube was capped immediately and stored in the fridge at 4 degrees Celsius.
The filter and syringe were discarded and the filtrate was ready to proceed to Plaque Assay Protocol 5.3.

Aislinn: September 5th 2018: Protocol 5.5 Enriched Isolation on Phage from anthill

Materials:

Solid Environmental Sample
0.22 microliter syringe filter
Liquid Media
Microcentrifuge tubes
500 microliters of host bacteria
50 mL sterile conical tube

Procedure:

The solid environmental sample was gathers along with the materials.

Extracting the Phage from the soil sample

A 50 mL conical tube was filled with the soil to the 15 mL mark.
Liquid media was added to the 35 mL mark and the tube was vortexed.
The sample was then placed in the shaking incubator for 1-2 hours at 250 rpm.
After settling, the tube was placed and balanced inside the centrifuge machine and was centrifuged at 2,000xg for 10 minutes in order to pellet the soil.

Preparing Bench and Seeding the Culture with Host Bacteria

The supernatant was filtered through a 0.22 microliter filter to remove unwanted bacteria and soil particles.
The volume of supernatant collected was about 15 mL.
0.5 mL of bacterial host culture was added to the conical tube.
The cap was placed on the conical tube. Care was taken to make sure that the cap was properly aerated. This was done by loosely placing the cap and securing it with a piece of tape.
The conical tube was incubated in the shaking incubator (upright to avoid spillage) for 24 hours at 220 rpm.

After the enriched culture incubated for 24 hours, the next step was done.

The plunger from the syringe was removed
The sterile filter was opened and attached to the barrel of the syringe
1 mL of supernatant was pipetted from each of the microcentrifuge tubes into the syringe barrel for a total of about 2 mL.
The tip of the syringe (that was attached to the filter) was placed over the microcentrifuge tube and the plunger was inserted to the syringe and depressed.
1.4 mL of the enriched culture was transferred using a pipette into a microcentrifuge tube. This step was repeated twice so that there are two microcentrifuge tubes with 1.4 mL in each.
The tubes were spun at high speed in a microcentrifuge for 1 minute to pellet the bacteria.
The supernatant was not clear so the following was performed:
The microcentrifuge tube was centrifuged for at high speed for 1 minute to ensure that the bacteria is pelleted.
The supernatant what transferred into a clean microcentrifuge tube and capped immediately.

Materials:

Environmental Sample
Liquid Media
Sterile 5mL syringe
0.22 um syringe filter
5ml serological pipette
Microcentrifuge tubes
15ml conical tube

Procedure:

The work bench was prepared using a decontaminate and working close to an open flame.
The sample and material were then gathered and the experiment was ready to be performed.
Extracting bacteriophage from solid environmental sample.

A 15mL conical tube was filled about one-third full of soil.
Liquid Media was added until the soil was submerged beneath about 2-3 mL of media.
The tube was then capped and inverted several times to insure that the soil was mixed thoroughly.
The tube was then shaken in a shaking incubator set at 250 rpm for 1-2 hours.
After incubating the soil was allowed to settle in the tube for about 15 minutes.

Preparing the Phage Filtrate.

A 0.22 microliter syringe filter was used to filter out any bacteria/contaminant so that only bacteriophages go through. The package containing the filter was opened and placed on the bench with the filter still in the package. This was an important step because the filter is easily contaminated.
Approximately 2 ml of liquid from the flooded sample was carefully removed to avoid withdrawing solid material and clogging the filter.
The syringe was attached to the filter and was removed from the packaging.
0.5 ml of filtrate was dispensed in a microcentrifuge tube and labeled accordingly.
The tube was capped immediately and stored in the fridge at 4 degrees Celsius.
The filter and syringe were discarded and the filtrate was ready to proceed to Plaque Assay Protocol 5.3.

Materials:

Environmental Sample
Liquid Media
Sterile 5mL syringe
0.22 um syringe filter
5ml serological pipette
Microcentrifuge tubes
15ml conical tube

Procedure:

The work bench was prepared using a decontaminate and working close to an open flame.
The sample and material were then gathered and the experiment was ready to be performed.
Extracting bacteriophage from solid environmental sample.

A 15mL conical tube was filled about one-third full of soil.
Liquid Media was added until the soil was submerged beneath about 2-3 mL of media.
The tube was then capped and inverted several times to insure that the soil was mixed thoroughly.
The tube was then shaken in a shaking incubator set at 250 rpm for 1-2 hours.
After incubating the soil was allowed to settle in the tube for about 15 minutes.

Preparing the Phage Filtrate.

A 0.22 microliter syringe filter was used to filter out any bacteria/contaminant so that only bacteriophages go through. The package containing the filter was opened and placed on the bench with the filter still in the package. This was an important step because the filter is easily contaminated.
Approximately 2 ml of liquid from the flooded sample was carefully removed to avoid withdrawing solid material and clogging the filter.
The syringe was attached to the filter and was removed from the packaging.
0.5 ml of filtrate was dispensed in a microcentrifuge tube and labeled accordingly.
The tube was capped immediately and stored in the fridge at 4 degrees Celsius.
The filter and syringe were discarded and the filtrate was ready to proceed to Plaque Assay Protocol 5.3.Aislinn Observational Notes:

Both isolation procedures were redone using the same procedure due to deficient liquid. Direct isolation set to incubate at 1830 20180904, Enriched isolation set to incubate at 1801.

Aislinn/Astrid: September 5th 2018: Protocol 5.3 Plaque Assay

Materials:

Phage sample
Host Bacteria (250 ul/plate)
Agar plates
Phage Buffer
Top Agar
Microcentrifuge tubes
5 ml serological pipettes

Procedure:

The workbench was prepared using aseptic technique and an open flame
The phages from the direct isolation were gathered to be used for the plaque assay.

Inoculating the host bacteria with the phage sample

Since there was only one sample to test, one aliquot of 250 ul of host bacterial was collected and the tube was labeled accordingly.
A micropipette was used to dispense the phage sample volume, according to the table below, into a culture tube containing 250 ul of host bacteria.
The tube was tapped gently to mix the inoculated host culture.
The inoculated host culture then sat undisturbed for 5-10 minutes to allow for attachment.

After 5-10 minutes of letting the sample sit, the samples were plated using top molten agar that was provided by the instructor

An agar plate was collected and labeled Shey Andrews; 9/05/2018:3:19PM @23C.
A bottle of top agar was removed from the 55C bath and brought to the workbench.
Using a 5 ml pipette, 3 ml of molten top agar was aseptically transferred into the inoculated host tube.

Sample Type	Sample Volume
Direct Isolation sample	500 ul
Enriched culture	10 ul
Serial Dilutions of picked plaques	10 ul
Lysates for titering	10 ul
Negative Control	10 ul of phage buffer
Positive control	10 ul provided phage sample

The molten top agar/inoculated host mix was then immediately aspirated back into the pipette and was transferred to the agar plate.
The agar plate was quickly and gently tilted so that the top agar mixture evenly coated the plate.

Incubating the plates

The plates were incubated to allow for bacterial growth and phage infection.
The top agar was allowed to solidify by letting the plates sit for approximately 20 minutes undisturbed.
The plates were inverted and placed in the incubator for 24-48 hours at 23-30C

Aislinn: September 6th 2018: Protocol 5.3 Results from 09/06/2018 Plaque Assay

Astrid: September 7th 2018: Protocol 5.3 Results from 09/06/2018 Plaque Assay

Astrid: September 7th 2018: Protocol 5.3 Plaque Assay Duplicate

Objective: To extract bacteriophage from the direct isolation distillate.

Materials: same as Protocol 5.3 Plaque Assay on September 5th.

Procedure:

Modifications

When pipetting the extracted phage from the centrifuge tube to the bacterial host test tube, extra care was taken to make sure that the phage sample made it completely into the host by avoiding the sides of the test tube.
Before inoculating the host, the tube was flicked and observed to make sure that the culture was as homogenous as it could get and after adding the phage it was mixed again by flicking it.
The attachment time was also physically timed to 20 minutes to make sure the phage was properly attached. The inoculated culture was then plated using the same steps in the previous run of protocol 5.3.
The procedure was the same as the Plaque Assay performed on September 5th with a few modifications.

Aislinn: September 10th 2018: Protocol 5.6 Spot Test on Enriched Isolation

Objective: To test a sample for the presence of phage on a bacterial lawn by “spotting” the plate.
Materials:

Agar plates
250 ul of host bacteria
To agar
Phage Buffer 5mL serological pipette

Procedure:

The bench was prepared for aseptic work and the supplies and liquid phage from the enriched isolation was gathered.

Preparing Bacterial Lawn Using Aseptic Technique

The agar plate was labeled into three sections (AR1, AR2, SA1).
3 mL of molten top agar (provided by the instructor) was transferred into a 250 ul culture of host bacteria using a sterile 5 mL pipette.
The solution was immediately drawn from the tube and dispensed onto an agar plate.
The agar plate was gently and quickly tilted in multiple directions until the agar evenly coated the plate.
The plate sat undisturbed for 20 minutes and the top agar solidified completely.

Spotting the Liquid Phage on the prepared bacterial lawn.

10 ul of each sample was used to spot the bacterial lawn one at a time.
The tip was held slightly above the agar and expelled so that the drop did not splatter.
The spots were placed in a triangle pattern on each of the three sections of the plate.
Without inverting the plate, it was incubated at _________ for 24-48 hours.

The below photo shows the results of the spot test, completed on 20180910 following approximately 48 hours of incubation at a 10^-1 dilution:

xA was selection to move forward with serial dilutions.

Aislinn: September 10th 2018: Protocol 5.2 Picking a Plaque and Performing Serial Dilutions

Using the above shown xA plaque, serial dilutions were performed:

Materials:

Phage Buffer
Samples requiring diluting
Microcentrifuge tubes

Procedure:

The bench was prepared using aseptic technique and the material were gathered.
A 10-fold serial dilution was set up.

The microcentrifuge tubes were arranged in a tube rack and labeled according to their dilution.
90 microliters of phage buffer to each of the test tube.

Performing the 10-Fold Serial Dilutions

10 microliters of the undiluted phage was added to the first microcentrifuge tube labeled 10^-1 and the tube was vortexed.
Using a different clean pipette tip for each transfer, 10 microliters of the 10^-1 sample was transferred to the centrifuge labeled 10^-2 and was then vortexed.

This process was continued until all the dilutions were made.
Performing Spot test using the liquid phage from the Serial Dilution Protocol.

The plate were sectioned in rows and labeled accordingly to their dilution. Since three plaques were picked, a total of three dilutions were made, thus two plates per sample.
3 mL of molten top agar was added to a 250 microliter aliquot of bacteria and the mixture was transferred to the plates and left undisturbed for 15 minutes to allow it to settle.
Once the top agar on the plate solidified, 10 microliters of each microcentrifuge tube were plated carefully to avoid splashing or spilling over to other sections.
The plates were incubated agar side down in the incubator for 48 hours.

Astrid: September 10th 2018: Protocol 5.3 Results from Plaque Assay on 09/07/2018

Astrid: September 11th 2018: Protocol 5.5 Enriched Isolation of Soil Sample from Collection Site Two

Materials:

Solid Environmental Sample
0.22 microliter syringe filter
Liquid Media
Microcentrifuge tubes
500 microliters of host bacteria
50 mL sterile conical tube

Procedure:

The solid environmental sample was gathers along with the materials.

Extracting the Phage from the soil sample

A 50 mL conical tube was filled with the soil to the 15 mL mark.
Liquid media was added to the 35 mL mark and the tube was vortexed.
The sample was then placed in the shaking incubator for 1-2 hours at 250 rpm.
After settling, the tube was placed and balanced inside the centrifuge machine and was centrifuged at 2,000xg for 10 minutes in order to pellet the soil.

Preparing Bench and Seeding the Culture with Host Bacteria

The supernatant was filtered through a 0.22 microliter filter to remove unwanted bacteria and soil particles.
The volume of supernatant collected was about 15 mL.
0.5 mL of bacterial host culture was added to the conical tube.
The cap was placed on the conical tube. Care was taken to make sure that the cap was properly aerated. This was done by loosely placing the cap and securing it with a piece of tape.
The conical tube was incubated in the shaking incubator (upright to avoid spillage) for 2-5 days at 220 rpm.

After the enriched culture incubated for two days, the next step was done.

The plunger from the syringe was removed
The sterile filter was opened and attached to the barrel of the syringe
1 mL of supernatant was pipetted from each of the microcentrifuge tubes into the syringe barrel for a total of about 2 mL.
The tip of the syringe (that was attached to the filter) was placed over the microcentrifuge tube and the plunger was inserted to the syringe and depressed.
1.4 mL of the enriched culture was transferred using a pipette into a microcentrifuge tube. This step was repeated twice so that there are two microcentrifuge tubes with 1.4 mL in each.
The tubes were spun at high speed in a microcentrifuge for 1 minute to pellet the bacteria.
The supernatant was not clear so the following was performed:
The microcentrifuge tube was centrifuged for at high speed for 1 minute to ensure that the bacteria is pelleted.
The supernatant what transferred into a clean microcentrifuge tube and capped immediately.

The next step that was performed was the Spot Test Protocol 5.6

Astrid: September 10th 2018: Protocol 5.6 Spot Test on Enriched Isolation

Objective: To test a sample for the presence of phage on a bacterial lawn by “spotting” the plate.

Materials:

Liquid phage sample from enriched isolation
Agar plates
250 ul of host bacteria
To agar
Phage Buffer 5mL serological pipette

Procedure:

The bench was prepared for aseptic work and the supplies and liquid phage from the enriched isolation was gathered.

Preparing Bacterial Lawn Using Aseptic Technique

The agar plate was labeled into three sections (AR1, AR2, SA1).
3 mL of molten top agar (provided by the instructor) was transferred into a 250 ul culture of host bacteria using a sterile 5 mL pipette.
The solution was immediately drawn from the tube and dispensed onto an agar plate.
The agar plate was gently and quickly tilted in multiple directions until the agar evenly coated the plate.
The plate sat undisturbed for 20 minutes and the top agar solidified completely.

Spotting the Liquid Phage on the prepared bacterial lawn.

10 ul of each sample was used to spot the bacterial lawn one at a time.
The tip was held slightly above the agar and expelled so that the drop did not splatter.
The spots were placed in a triangle pattern on each of the three sections of the plate.
Without inverting the plate, it was incubated at _________ for 24-48 hours.

Astrid: September 12th 2018: Results from Spot Test of Enriched Isolation

Aislinn: September 12th 2018: Results from September 10th: First Round of Serial Dilutions

Serial dilutions failed. Brownish-white turbidity in plates indicated contamination on 10^-5 and 10^-6 samples. Re-performing serial dilution procedure as given above.

Astrid: September 15th 2018: Protocol 5.6 Spot Test on Enriched Isolation

Objective: Test sample for the presence of phage.

Materials: Same as before with Protocol 5.6.

Procedure:

Modifications

The only modification that was performed was to the labeling of the agar plate. The agar plate this run was sectioned into four different sections (Phage Buffer, AR1, AR2, SA1). A phage buffer was added as a negative control. The samples that were collected from the enriched isolation used in the previous spot test was also used during this run of the experiment.