Date: August 22nd 2018 

Title : Soil Sample Washing  

Rationale : We washed soil samples that we collected from our designated area in hopes of isolating a possible bacteriophage that maybe present in the soil.  

Procedure:  

Preventing Contamination:  

• In hopes of preventing contamination we cleaned the table with Cidecon and wiped the table dry. Then we cleaned the table with 70% ethanol and let it air dry.  
• We created an aseptic zone with an ethanol flame, so that while transferring materials we can avoid further contamination 

Creating the Soil Sample:  

• I added the LB Broth until the 35 mL mark to the soil sample of 12 mL I had collected the previous day. Therefore, it was a total of 22.5 mL of LB broth  
• Made sure while transferring the broth we stayed near the aseptic zone.  
• One mistake that I made was placed the cap on the table while focusing on the LB Broth, so will have to see in the future if that has any effects on my results  
• After I added the LB broth to the soil we shook the tube for 20 minutes.  
• I weighed the mass of the entire tube and it turned out to be 53.23 grams.  
• After finding the mass of our soil and LB broth solution we found a partner who had a mass within the range of plus or minus 0.1 grams. This was important for when we were using the centrifuge. 
• In order for accurate results out of the centrifuge we had to make sure that there had to be a equal balance of weight on either side of the centrifuge.
• After the 20 minutes of shaking, we placed the test tubes into a centrifuge.  
• The use of the centrifuge is to separate the liquid and the heavier particles

Creating the Enriched and Direct Lysate Samples 

• After our test tubes came out of the centrifuge we extracted the supernatant, in other words the liquid part at the top half of the test tube, using a dropper.  
• We put the supernatant in another vacuum filter, which can filter out some of the smallest particles, but leaves bacteriophages behind.  
• While using the dropper, I avoided any big particles left behind such as small pieces of bark from my sample 
• I also left behind the pellet, which is the heavier material at the bottom of the test tube 
• After extracting most of the supernatant into the vacuum we waited as it filtered through  
• Since the filter was taking a long time, I filtered 10 mL which I put in a separate test tube and labeled as my enriched sample  
• Then I poured the rest of the unfiltered sample into another test tube and labeled it as an unfiltered direct sample.  
• I stored both my enriched and direct sample in the fridge.  

Observations/ Results/ Data : 

• I observed that as I shook my sample for 20 minutes the color turned to be a chocolatey color and started foaming up
• I will have to keep an eye on whether any contamination occurred because I placed the cap on the table  
• There were no results or data that were collected this time, as we just washed our soil sample and tried to separate possible phages with the rest of the soil particles 

Conclusions/ Next Step  

• Next time we come to lab, I will need to filter my direct sample to use it for testing on the spot test.  
• I will also be performing a spot testing to check if our sample has phages, and if not I might have to retake a soil sample and retry hoping to find a phage in the new sample. 

Date: August 27nd 2018 

Title : Spot Testing of Soil Sample

Rationale :  We are performing a a spot test using the direct, enriched and control soil samples on a petri dish in hopes of finding a plaque which shows us that there are phages in our sample  

Procedure:  

First we followed the same cleaning procedure as last time for lowering the chances of contamination, and also set up a ethanol flame.  

Creating the Top Agar Solution for Control  

• I brought the enriched sample and direct sample from the fridge and placed on my lab table  
• We also got syringes, four 50 mL test tubes, and four petri dishes  
• I took 2 mL of the enriched sample using a syringe and put it into micro test tubes to use for the lab.  
• I did this in an aseptic zone  
• After using the syringe, I disposed the syringe  
• I then labeled my petri dish. I divided it into 3 different sections on the bottom and labeled them control, enriched, and direct.  
• My lab group and I decided to do the control first, so we worked on that first  
• We first added 4.5 mL of the LB broth using pipettes into a larger test tube (50 mL)  
• Using the formula C1V1 = C2V2 we found the required amount of CaCl2 is 42.75 ul.  
• So, then we added 42.75 ul of CaCl2 into the control test tube.  
• We used the pipette marked 20-200 ul.  
• We also made sure to change the pipette tips between uses.  
• Next, we added the Top Agar into the LB broth and CaCl2 solution using a pipette, since Top Agar sets quickly and therefore would have needed to work fast.  
• After adding the top Agar our solution was a total of 10 mL.  
• We then shook the solution a few times to make sure it was an even mixture.  
• Lastly, we poured the solution into a petri dish and let is sit to solidify.  
• While adding and mixing the solutions we made sure that we were near an aseptic zone.  

Creating Individual Spot Testing  

• My lab group decided to create a big batch, so we tripled all the measurements  
• We first obtained a new pipette tip and used the leftover LB Broth at our tables to pipette 4.5 mL of LB broth into each of our tubes  
• After changing pipettes, we added 45 ul of CaCl2 in each tube as well.  
• After adding these two ingredients to make our individual plates, we obtained 0.5 mL of Arthro from the hood, almost completing our solution. 
• We then finally added the 5.0 mL of Top Agar using a glass pipette and pipette bulb. 
• Also while adding all the solutions we made sure that we were in the aseptic zone. 
• I ended up with 10.0 mL of my Top Agar solution and let it sit for 10 minutes so that it will solidify  
• An additional step I did was to filter the unfiltered direct solution which I did while waiting for my Top Agar to set.  
  • Since the solution was untouched for a few days, the solution had separated the heavier parts and the liquid remained at the top  
  • Using a syringe I extracted 2mL of the liquid part of the direct solution and put it into a mini test tube basically filtering it a little more  
• Using a pipette I added 5 microliters of the enriched, direct and buffer, which was provided by the professor, into its respective parts on the petri plate.  
• I closed it and let it sit for about 10 minutes so that there was enough time for the agar to soak up each of the solutions. 
• Then lastly I placed the petri dish in the incubator.  

Observations/ Results/ Data: 

• Some observations were as I was pouring in the Top Agar I realized there was this bubble on the side of the plate, which did not pop.  
• Some of the other observations in this experiment was making sure that we made the correct mixture for the solution .  
• Another observation we had to make was which pipette to use for what and in general the max and min pipetting values for each 
• Hopefully the result will be that there is a plaque, showing to us that there is a phage in the sample.  
• There was no data or results for this part of the experiment   

Conclusions/ Next Step  

• The next step is that we will look at our spot test and see if we found a plaque or not. Depending on the results we will proceed with a plaque assay  
• If there is plaque on the spot test, then we will perform an enriched and direct petri dish for the plaque assay  
• If there is no plaque on the spot test, then we will only perform an enriched plaque assay  
• Some conclusions that we can get from this test is whether we found a phage or not.  
• The control can show us if there is any source of contamination from the Top Agar solution

Date: August 29nd 2018 

Title : Plaque Assay  of Soil Sample

Rationale :  We are performing a plaque assay, to either confirm that we have a phage or to retry to see if the phage was missed in the spot test.  

Procedure:  

Making the Top Agar solution for Plaque Assay:  

• After cleaning up the table with Cidecon and 70% ethanol, we set up an ethanol flame for a aseptic zone.  
• Obtained the spot test from the incubator and inspected the results  
• Decided to perform an enriched plaque assay since I think that my spot test had plaque in the enriched portion of the petri dish  
• I obtained the enriched lysate from the fridge.
• I then obtained the 0.5 ul Arthro and labeled it with name and information. 
• Then I used a 0-10 ul pipette to add our lysate of 10 ul to our 0.5 ul Arthro near the aseptic zone  
• Then we were instructed to waited for 15 minutes, but in the meantime, we decided to make our Top Agar solution  
• I took the 8 ml of the LB broth and added it to our 50 ml tube using the pipette  
• We took 90 ul of the CaCl2, which was passed out in class, and pipetted it into our solution in the 50 mL tube.  
• My lab group and I decided to wait some more time before we added the Top Agar into the solution so that the solution doesn’t solidify fast, since by this time only 7 minutes had passed.  
• We proceeded with our experiment at the 14 minutes mark  
• Lastly, we added 10 mL of Top Agar to our solution using our glass pipette and bulb  
• We then stirred the solution to make sure that its evenly mixed and proceeded by pouring 5 mL of the entire solution into each of our plates with the Top Agar and the Arthro  
• There was one bubble which showed up which could later be mistaken for a plaque, so I noted it. 
• I waited 10 minutes before placing the solution into the incubator upside down.  
• In the meantime, I wrapped up my spot test and kept it in a different place to keep the test for plaque in case one doesn’t show up in the plaque assay  

Observations/ Results/ Data: 

• Some observations from the results of the spot test:  
• Enriched had a spot which looks like a plaque.  
• Direct – sort of had a clearing but not completely sure if plaque  
• Buffer – had very little clearing kind of looked hazy but probably not plaque 
• In my plaque testing plate there was one bubble while pouring the Top Agar 
  

  Spot Test – Can see a possible plaque on the enriched portion

• 

  Plaque Essay – noticed a bubble

Possible Question:  

• Does different tree species affect the Arthrobacter phage we are trying to find?  

Conclusions/ Next Step  

• There seems to be phage in my soil sample from the spot test in the enriched section of the plate.
• Just to confirm it we performed a plaque assay, which we will observe next time.
• If there is some clearing in the plaque assay, then we can use the plaque to continue with the next portion of our experiment.  
• If there is no plaque on the plaque assay plate, then we will use the plaque that we found on the spot testing to continue with our experiment.