Bacteriophage found September 4, 2018 in a dog hole in Stephenville, Texas 32.3166 x -98.2810.

 

 

 

September 5th

 

• Extracted the sample found in horse pen at coordinates at 32.3166 x -98.2825 in Stephenville plated for direct isolation.
  1. Added liquid media to scat sample sample in the 15 mL conical tube until the sample was submerged about 2 mL.
  2. Inverted several times until it was thought sample was mixed thoroughly, then sample was put in shaking incubator for an hour.
  3. When sample was observed after an hour it had little precipitate and many particles of grass at the top of tube. Sample was then left in the incubator for another hour.
  4. Sample was taken out after additional hour, but showed no signs of debris moving down. The sample was then transferred to a 50 mL conical tube to decant.
  5. Syringe was shoved into conical tube and 2 mL of liquid was taken up.
  6. The 2 mL of sample was then transferred to a 1.5 mL centrifuge tube using a .22 um filter. The filter continuously clogged and when final amount of sample was observed it was just at the recommended.5 mL. The sample was observed to have a unusual green color thought to be from the filter breaking.
• A plaque assay was then preformed to observe the presence of any phages. The protocol was misunderstood and done incorrectly. (Other two samples were put in fridge for use as backup or as an enriched sample.)
  1.  A sample of host bacteria was obtained and vortexed. Top agar was then added to the sample and transferred to the agar plate.
  2. The plate was then divided into two sections a negative control section and a section where 500 uL of direct isolation was plated.
  3. The plate was never inverted and placed into the incubator at 29.5 C. Other two samples put in fridge for use as backup or as enriched sample.

 

 

 

 

 

September 6th

 

• Direct isolation samples were checked on after 24 hrs and no growth was observed.
• Enriched sample isolation was preformed.
  1. Sample was collected in 15 mL conical tube and was then transferred to a 50 mL conical tube to follow enriched protocol.
  2. Less than 15 mL of soil sample was collected, so around 25 mL of liquid media was added.  It was then vortexed and left to shake at 250 rpm for 1 hour. The tube was had little to no debris with an evident supernatant and was a yellow clear color that seemed standard.
  3. The tube was then centrifuged at 2,000 for 10 mins
  4.  Around 20 mL of supernatant was collected and filtered through a vacuum flow conical tube with .22 um filter paper.
  5. Around 250 uL of host bacteria was then added to the conical tube. The tube was then placed strategically one quarter turn so that it was properly aerated. Masking tape was placed on the conical tube lid so the lid did not fall off in the shaking incubator.  The tube was then placed upright in the shaking incubator at 220 rpm for 2 days.

 

September 8th 

 

• Day 2 of enriched isolation protocol was continued.
  1.  1.4 mL of enriched sample was transferred from 50 mL conical tube to 2 different 1.5 mL microcentrifuge tubes.
  2. Tubes were spun for around 1 minute in centrifuge to pellet the bacteria. The pellets were extremely noticeable when taking tubes out of the centrifuge.
  3. Supernatant was clear but extra precautions were taken and the supernatant was filtered through a .22 um filter to be sure there was not any non-host bacterium. The filtration was done by pipetting 1 mL from each tube into syringe barrel, then depressing the plunger with the .22 um filter into new microcentrifuge tube.
  4.    The culture was then returned to the incubator with the lid loosely taped on.
• A spot Test was then done using the liquid phage sample just collected.
  1.  The plate was divided into 3 sections with one being the negative control side.
  2. 3 mL of top agar was added to the  250 uL of host sample bacteria then placed onto the plate.  The plate was then allowed 20 minutes then inverted to check for solidity. The plate flipped and the agar fell and crumbled. It was thought to be a result of the plate being too cold to add the top agar onto. The plate was discarded of and another plate was used but not inverted.
  3. 2 10 uL of sample were then placed into each section of the bacterial lawn excluding the negative control side. The plates were left undisturbed for 10 minutes then moved to the incubator without inverting at 29.5 C.

 

September 10th 

 

• Plates were checked for clearing. Clearing in both bacterial lawn were observed. The negative control was observed to have no growth.
•  Picking a plaque protocol was then preformed
  1.  3 different plaques were isolated that seemed to have the morphology of a lytic phages and were far away enough away from other clearings to indicate it was a singular bacteriophage. The plaques were circled and labeled A,B,C.
  2. Each plaque was picked using a sterile pipet tip then placed into a microcentrifuge tube containing 100 uL of phage buffer. The plaque was mixed into buffer by pipetting up and down then vortexing after. The technique was preformed on A, B, and C into each of their according tubes.
• Serial Dilutions were then done for purification
  1.  A 10-fold dilution down to 10 E-5 as recommended when working with enriched samples. This was done with each tube containing 90 uL of phage buffer and transferring 10 uL from the original to the -1 then 10 uL of E-1 to E-2 and so on.
  2.  All 5 dilutions and the original were plated on the same plate but in divided sections. 10 uL of each dilution and the original samples were pipetted to each section. This was done for A, B, and C, which resulted in three plates total.

 

September 11th

 

• After 24hrs growth was observed on all three plates.
• 1A was the least concentrated and stopped growth at E-5. There was noticeably smears on the plate that led to the belief that there was error or bad technique when pipetting out the dilutions. The morphology was thought to be constant throughout the dilutions.
• 1B was observed to have the same morphology though out but did not drop off at E-5 dilution. Serial dilutions were repeated and E-9.
• 1C was observed to have the same morphology though out the dilutions but also did not drop off at -5 dilution.
• It was noted that all 10-fold serial dilutions needed to be repeated but to the  E-8 instead of just to the E-6.

 

 

 

September 12th

 

• 10-fold serial dilutions were repeated to the E-8 for all three plaques picked and plated (by inoculating provided bacteria and adding 3mL of top agar to each plate, as previous protocol recalls).

 

 

 

September 14th

 

• A spot Titer for 1B and 1C was  done to check concentration which seemed to mock the exact results the initial dilutions for purification did due to the misunderstanding when following the protocol for how the serial dilutions should first be plated. This was done by making a bacterial lawn and pipetting 3 uL into each section as previously done.
• When calculating the math, there was confusion on how if calculating plaques correctly.
• The full plate titer protocol was then followed for 1A because of the plating low number of plaques. The full plate titer protocol was done wrong and the phage was not added to the host bacteria, then mixed with top agar and dispensed. Instead, a bacterial lawn was made and the phage sample was dispensed directly in the middle of the plate.
  1.  It was estimated that the correct concentration of the most countable number would be in the plates E-4, E-5,E-6.

 

September 17th 

 

•  Plates for 1B and 1C had observed growth in their spot test. An estimated titer was calculated of the correct concentration of a countable number of plaques.
  1.  For B it was estimated a serial dilution of E-5,E-6,E-7 would yield the most countable number.
  2. For C it was estimated a serial dilution of E-5,E-6,E-7,E-8 would yield the most countable number.
• Full plate titer was preformed again with same dilutions on 1A due to inaccurate inoculation of bacteria.

 

September 18th

 

• Full Plate Titer growth was observed for 1C E-6 but not any of the other plates. No growth for 1B found.

 

 

 

September 19th 

 

• Full plate titer was preformed again on 1A, 1B, 1C  due to no growth being observed.
  1. The serial dilutions for 1A of E-4,E-5,E-6 were plated.
  2. The serial dilutions for 1B of E-5,E-6,E-7 were plated.

 

September 21st

 

• Growth was observed for 1A and a conformational round of full plate titers were done E-4,E-5,E-6.
• The other two plaques 1B and 1C were offered out for donation to anyone that did not have any growth.

 

September 24th

 

• All Full Plate Titers had observed growth and plaques 1B and 1C were put in the fridge and further pursues were discontinued.
• 1A conformational titers had plaques and did confirm that the morphology was the same and that E-2 was the concentration that would be used to make webbed plates.
  • 3 more full titer plates were made in the dilution of E-2 to make webbed plates. When adding top agar and inoculated bacteria to the plate it was suspected one of the plates had contamination.

 

 

 

September 26th

 

• The possible contaminated plate had a small yellow colony growing in the middle of the plate. All plates were put back in the incubator for one more day to let grow even further. It is suspected that the bacteriophage “Hamburger” is at its optimum growth after 3-4 days of incubation.

 

September 27th

 

• Plate lysate from 1A E-2 was collected

 

1.  8mL of sterile phage buffer was added to each plate and the plates were left for 2 hours at room temperature undisturbed.
2.  After 2 hours the lysate was harvested using a 5mL syringe to aspirate.
3.  The syringe was then attached to a .22 um filter while still remaining in the package.
4. The filter (with the syringe still attached) was put on top of a 15mL conical tube aseptically. The syringe was then depressed into the tube. The filter remained on the 15mL tube and the syringe was just switched out when it got covered in lysate. The filter needed to be changed for the last plate due to resistance thought to be from clogging.

 

• Around 8mL of high titer lysate was collected from the plates. It was recommended that a stock of around 30mL would be needed for the sample to go through a DNA extraction and used for TEM.

 

September 28th

 

• A spot Titer was done with the lysate to determine the concentration of phage particles. A spot test was done twice on two different plates.

 

1.  Both plates were labeled with 9 sections to contain a sample of the original and all of the dilutions up to the E-8. The plates were then left out to get to room temperature.
2.  8 serial dilutions were done with the lysate in phage buffer.
3.  Once plates were at room temperature a bacterial lawn was made by adding 3mL of top agar to bacteria provided then dispensing that mixture to the plate. The plate was then dest undisturbed for 10 minutes.  This was done for each plate.
4.  3uL of each samples was transferred to their corresponding place on each plate. The spots were allowed to sink into the agar for 30 mins then placed in the incubator. (Plates were NOT inverted)

 

October 1st

 

• Plates had observed growth but there was contamination on one plate in E-1, E-3, E-4, and E-8. The other plate had contamination also in 1A original spot. It suspected that since the contamination is not consistent that it could possibly be due to dirty pipet tips because the lab is shared. Designated pipet tips for the phage research were not being used.
• Three more webbed plates were made in the concentration of 1A E-2.

 

October 5th 

 

• Webbed plates had different growth than normal and the plate looked like sand. It was suspected that there was mass contamination with the bacterial stock. The plates were discarded of due to their contamination.

 

 

 

October 10th

 

• Used 10mL of collected lysate for DNA extraction
  1.  10mL of lysate was transferred to 50mL conical tube
  2. 40uL of Nuclease mix was added and inverted multiple times.
  3.  4mL of phage precipitant solution was added to to tube and then inverted multiple times.
  4.  Sample was incubated for 30 mins at 37C  then left at room temperature for an hour.
  5.  Sample was then put in fridge at 4C.

 

October 12th

 

• Webbed plates from a known  titer were made. (Step 2-3 were done 3 different times)
  1.  The number of math was calculated on what concentration to get a webbed plate. It was estimated to be around 10uL of phage needed to inoculate the bacteria provided. 
  2. Bacteria was inoculated and then left to set for 10 minutes.  
  3.  3mL of top agar was then added to the inoculated bacteria and plated.  Plates were then placed in the incubator. 

 

 

October 15th

 

• Baylor DNA extraction protocol was attempted to be resumed
  1.  Centrifuged for 25 mins at highest setting 4000 rpm but no pellet was formed
  2.  Centrifuged again for 10 minutes at highest setting of 4000 rpm until it was noted that nuclease mix was obviously left in the solution for too long and degraded all DNA that could form pellet.
• Plates were flooded with 8mL of phage buffer and high titer lysate was collected from plates that were previously plated on 10.12.18.

 

October 16th

 

• Four more webbed plates were made with an old sample of 1A (that were then diluted down to E-2 as previous entries recall) in phage buffer from 3 weeks ago. It was unknown if there would be growth or if the phage would of completely degraded in the buffer.

 

 

 

October 17th

 

• Plates were checked on and no growth was seen. Plates were put back in there but it is suspected that the sample used for making serial dilutions could possibly not of had any of the “Hamburger”  bacteriophage.

 

October 19th

 

• Growth was observed on webbed plates. They were parafilmed and put into the fridge until I had time to work on them. 

 

October 22nd

 

• Plates were flooded for lysate
  1. Plates were taken out of the fridge and flooded with 8mL of phage buffer each. 
  2.  Plates were left undisturbed for around 2 hours.
  3.  Using a syringe and filter lysate was collected and added to 15mL conical tube with lysate. 

 

October 27th

 

• Microscope mounting for TEM done:
  1.  2mL of lysate was transferred into two 1.5mL microcentrifuge tubes with each one containing 1 mL of lysate.  Tubes were then centrifuged at 4C, 12000 rpm for 1 hour.
  2.  Supernatant was removed without disrupting the pellet and 100 uL of phage buffer was added to the pellet. It was resuspended and stuck in the fridge at 4C for 1 hour. 
  3. The fume hood was set up with a petri dish lid that contained Parafilm and a Pelco tab. A grid was removed from the grid box and left to touch the very edge of the Pelco tab. 
  4. 10 uL of lysate was pipetted onto the grid without touching the tip to the grid. It was left on for 5 minutes.  The excess fluid was then wicked off.
  5. The grid was then washed by adding 10 uL of sterile water and allowing it to sit for 2 minutes. The water was wicked after two minutes. Washing was repeated twice.
  6. 10 μl of 1 % uranyl acetate was added to the grid and let sit for 2 minutes.  The grid was then wicked until it looked like an oil slick rainbow. The grid was left to air dry. 
  7. Once grid was dry it was put in the transportation grid box and from there put into a vacuum sealed chamber so that no moisture made contact with the grids. 

 

October 29th 

 

• Baylor DNA extraction protocol was performed again.
  1.  10mL of sterile phage lysate was transfered to a 50 mL conical tube. 
  2. 40uL of nuclease mix was added then inverted. 
  3. 4mL of PEG8000 was added then inverted.
  4. Sample was put in incubator at 37C for 30 mins
  5. Sample was then incubated for 1 hr at room temperature.
  6.  Tube was then place in the swinging bucket centrifuge at 4000 rpm for 35 mins
  7. No pellet was observed. The sample was separated into multiple 1.5mL microcentrifuge tubes then put into a different centrifuge at 12000 rpm, 20C, for 1 hr. No pellet was observed.  Protocol was stopped. 

 

October 30th

 

• Alternative DNA isolation protocol using ZNCl2 was performed
  1.  5mL of HVL was transferred into a 15mL conical tube. 20uL of nuclease mix was added then inverted.
  2.  The tube was incubated at 37C for 10 minutes
  3. 5 mL of sample was transferred to 5 different 1.5 mL tubes that each contained 1mL. 
  4. 20uL of ZnCl2 was added to each tube and inverted. The tube was then incubated at 37C for five minutes to precipitate. 
  5. Samples were centrifuged for 1 min  at 10000 rpm at 20C to pellet. 
  6.  Supernatant  around the pellet was removed without disturbing the pellet.
  7. Pellets were resuspended in 500uL of TES buffer per each tube and placed on the heat block at 60C for 15 mins. 
  8. 1uL of Proteinase K was added to each and mixed gently. Tubes were then placed in the incubator at 60C for 15 min.
  9. 60uL of potassium acetate was added to each tube then mixed well through inversion. Sample was left on ice for 15min.
  10. Sample was centrifuged at 4C for 1 min at 12000 rpm to pellet. Supernatant was extracted out and placed into new microcentrifuge tubes.
  11.  500 uL of pure isopropanol was added to each tube. All tubes were then left on ice overnight. 

 

October 31st

 

• Day two was continued
  1.  Samples were taken out of ice and centrifuged at top speed for 10 min, the waste was then put in a waste tube. 
  2.  250 uL of 70% ethanol was added to each tube and centrifuged at max for 1 min. This was repeated a second time due to the assumption that the DNA could have residual salt left over. 
  3. Supernatant around pellets was discarded of into waste tube and pellets were left on their side to dry in the hood.  Pellets did not observably become white so they were left in the hood overnight. 

 

November 1st

 

• 50uL of nuclease-free water was added to first tube then transferred to second tube.   Each tube had a white and viscous mix in it and seemed to absorb the water immediately. only about 20uL of water/DNA mix was left when transferring it to the third tube.  At this point it was determined it was more salt than DNA and there was residual salt left over from purifying the DNA.  The protocol was stopped here and no reading of concentration of DNA was taken due to the appearance of the “DNA”. 

 

November 6th

 

• Alternative DNA isolation protocol using ZNCl2 was performed again
  1. 5mL of HVL was transferred into a 15mL conical tube. 20uL of nuclease mix was added then inverted.
  2.  The tube was incubated at 37C for 10 minutes
  3. 5 mL of sample was transferred to 5 different 1.5 mL tubes that each contained 1mL. 
  4. 20uL of ZnCl2 was added to each tube and inverted. The tube was then incubated at 37C for five minutes to precipitate. 
  5. Samples were centrifuged for 1 min  at 10000 rpm at 20C to pellet. 
  6.  Supernatant  around the pellet was removed without disturbing the pellet.
  7. Pellets were resuspended in 500uL of TES buffer per each tube and placed on the heat block at 60C for 15 mins. 
  8. 1uL of Proteinase K was added to each and mixed gently. Tubes were then placed in the incubator at 60C for 15 min.
  9. 60uL of potassium acetate was added to each tube then mixed well through inversion. Sample was left on ice for 15min.
  10. Sample was centrifuged at 4C for 1 min at 12000 rpm to pellet. Supernatant was extracted out and placed into new microcentrifuge tubes. This was twice and tubes were spun for 3 mins on the second time to make sure no salt had gotten over. 
  11.  500 uL of pure isopropanol was added to each tube. All tubes were then left on ice overnight. 

 

November 7th

 

• Day two of protocol was continued:
  1. Samples were taken out of ice and centrifuged at top speed for 10 min, the waste was then put in a waste tube. 
  2.  250 uL of 70% ethanol was added to each tube and centrifuged at max for 1 min. 
  3. Supernatant around pellets was discarded of into waste tube and pellets were left on their side to dry in the incubator at 37C for 20 mins.  Pellets did not observably become white, but this time they were the same size as the original pellets so it was assumed they would actually be DNA this time.
  4. 50uL of nuclease-free water was added to the  first pellet and it began to soak up some water. The 50uL had turned into 45uL and the solution became noticeably thicker and more resistant to being mixed well. Once contents of the first tube had reached the third tube there was almost no water in the tube at all and the white pellet had oversaturated the water. 10uL more of water was added to the final tube, the final “DNA” was ran on the nanodrop to be 100 ng/uL but the solution was white and viscous when pipetting leading to the belief that the solution was full of salt contamination.

 

November 10th

 

• A PCR prep was began on the DNA sample but when pipetting, it became obvious it was not DNA and the procedure was stopped. 
• Three more plates were made from lysate to make more high titer lysate.
  1. 9.6 uL of lysate was added to 90 uL of phage buffer. 
  2. 10uL of the first dilution was added to another 1.5 mL tube with 90 uL of phage buffer. 
  3. 3 tubes of bacteria were inoculated with 9.6 uL of the second dilution of phage that was performed. The inoculated bacteria was left undisturbed for 10 minutes to allow for attachment.
  4.  3mL of top agar was added to inoculated bacteria and placed on agar. This was repeated 3 times.
  5.  Plates were left un-inverted for 20 minutes,  until solidified. After solidified plates were inverted and placed into the incubator.  

November 13th

• Checked plates in incubator and 2 out of the 3 plates plated had contamination. The plates that were contaminated were from the high titer lysate stock, so it was determined that all lysate needed to be re-filtered to get out all bacterial contamination.
• 2 more plates were re-plated for lysate to be obtained so that another DNA could be performed.
  1.  9.5 uL of lysate diluted twice was inoculated into bacteria that was provided. The bacteria and virus was left to sit for 10 minutes to allow for attachment. 
  2.  3mL of top agar was added the the phage-bacteria mix then placed on the plate. 
  3. Plates were left undisturbed for 20 minutes then placed into the incubator. ( This was repeated for the stock solution of lysate I had the was in the fridge in another area to see if the contamination only from the large conical tube I had been working with. 

November 14th

•  Lysate was re-filtered
  1. Lysate was aspirated from 2 15mL conical tubes using a 5mL syringe.
  2. The syringe was then attached to a filter that was on top of the conical tube and gently pushed through. There was less than 5mL left after re-filtering the lysate.  

• 1 plate was taken out of the incubator and flooded to collect more lysate. Other two plates were not usable due to no growth from bad plating technique and the other from too high contamination so no phage growth.
  1. 8mL of phage buffer was added to each plate and left to sit undisturbed for 2 hours.
  2. Lysate was then collected by aspirating all liquid from the tube into a 5mL syringe. 
  3. The syringe was then carefully attached to filter that was on top of the conical tube and lysate was pushed through into the pool of 5mL lysate that I currently had. 

November 15th

• Entered phage into phagesdb.com
  1. If you did not use the SEA-PHAGES App when you collected your soil sample, you can add phages directly from PhagesDB by selecting the “Data” dropdown menu and clicking on “Add Phage.”
     • If you have not logged in to PhagesDB, you will be redirected back to the login page. You can log in if you already have an account, or you can create an account using the green “Sign in or Register” button on the left, at the bottom of the page.
  2. Fill in as many fields as possible, paying special attention to the following:
     • GPS coordinates: If your GPS fields were not prepopulated by the SEA-PHAGES App, you can find the GPS coordinates by using a web mapping service application. One such service can be found via this link: http://www.heywhatsthat.com/profiler.html OR http://bit.ly/yqKB. Simply drag the map to the location where the environmental sample was found, zoom in as much as possible, and click on the map to obtain the GPS coordinates of that location. The output should be to the right of the map and in the N/W Decimal Degree format.
       • Program
       • Institution
       • Bacterial host
       • Name

November 16th

•  Bacteriophage “Hamburger” was archived
  1.  3 tubes were obtained and labeled  with barcodes provided from Dr. Edwards
  2. Sterile beads were aseptically transferred into each tube 1.5cm from the top.
  3.  DMSO/lysate mixture was made by adding 2.8mL of lysate with 200 uL of DMSO into a 15 mL conical tube. Mixture was vortexed.
  4.   Mixture was dispensed into tubes equally with liquid just covering beads.
  5.  Samples were placed in the fridge. 
• Plates were made for more high titer lysate so that another DNA extraction could be performed. 
  1.  Webbed plates for high titer lysate were made using 9.5uL of lysate.
  2. The lysate was added to the bacteria and the mixture was left undisturbed for 10 minutes. 
  3.  3mL of top agar was added to the inoculated bacteria and then plated. The plate was left undisturbed for 20 minutes and then placed in the incubator. This was repeated twice for both plates.

November 17th

•  Plates were checked on and it was obvious I had incorrectly done the protocol when plating. 1 plate had been added twice, while the other had not had top agar and inoculated bacteria on it. Only one plate was replated for webbed plate because I had just under 10mL of lysate, which was the least available needed for another DNA extraction. 
  1.  Webbed plate for high titer lysate was made using 9.5uL of lysate.
  2. The lysate was added to the bacteria and the mixture was left undisturbed for 10 minutes. 
  3.  3mL of top agar was added to the inoculated bacteria and then plated. The plate was left undisturbed for 20 minutes and then placed in the incubator. 
• A plate of the dilution of E-4 was made to exhibit plaque morphology for pictures on the phagesdb.com and for further reference in notebook to note characteristics.
  1.  An old tube that was already diluted was obtained from the fridge and 10 uL was added to the bacteria. Tube was left undisturbed for 10 minutes.
  2. 3 mL of top agar was added to the inoculated bacteria  and then plated. Plate was left undisturbed for 20 minutes. 
  3. Plate was put into the incubator