September 14

Lab Day 6: Plaque Assay + Sand, Silt, Clay + Metadata Results

Rationale

Make plaque assay and record results from Lab day 5

Detailed Procedure

  1. Since I was the only one in my group without a direct lysate, I had to make a plaque assay while the rest did a spot test. (therefore I had to make my own control)
  2. Add water to help balance my partner’s weight= final enriched weight= 22.38 g
  3. Filtered enough enriched lysate into a 15 mL vial.
  4. Took 10 microliters of enriched lysate and added to 0.5 m Arthro vial
  5. Mixed it well and let sit for 10 mins
  6. During that time, took 2.0 mL LB Broth, 23 microliters of 1M CaCl2, and 2.5 mL 2X TA and mix well through pipetting in a separate 50 mL vial
  7. Took enriched lysate + Arthro mixture and added to the 50 mL vial
  8. Mixed one more time through pipetting
  9. Poured mixture onto a plate label “Plaque Assay B”
  10. Repeat step 6 for control onto a new plate labeled “Plaque Assay Control”
  11. Let both plates sit for 15 mins

Detailed Procedure: Sand, Silt, Clay+ Metadata results

  1. Record total mL in tube
  2. Record how many mL of sand, silt, clay, and calculate the %

Observations/Results

  • Sand= 50%
  • Silt= 43.75%
  • Clay= 0.0625%
  • Soil + petri dish= 5.75 g

Next Steps

  • Wait for results from plaque assay and see if there’s any presence of plaque.
  • Results and comparison to rest of class will help resolve/eliminate any variables or questions we are trying to solve

enriched lysate w water

control vs exp

after 48 hours

Category: Soo-Un Jeong | LEAVE A COMMENT
September 14

Plaque Assay Soil B

9/12/18

Rational:

To do a plaque assay to see if there’s any arthrobacter phage in soil B. I will also be calculating the sample’s water percentage and percent of sand, silt, and clay based on the results from lab on 9/10.

Procedure:

  • Weight of the dry soil in the weigh boat- 5.33 g (weight of dry soil- 2.9)
  • Weight of enriched lysate 21.6 g
  • Percent water- 16.18%
  • The 10 mL soil sample contained 4 mL sand (40%), 3 mL silt (30%), and 3 mL clay (30%)
  • Spun the enriched lysate at 3,000 g for 5 minutes
  • Filtered the enriched lysate
  • Put 10 ML FS lysate into .5 mL Arthro
  • Put 2 mL LB broth into the TA mixture
  • Put 2 mL LB broth into vial for the control mixture
  • Added 22.5 ML 1M CaCl2 to the TA mixture and 22.5 ML 1 M CaCl2 to the control TA
  • Added .5 Arthro to my TA mixture
  • Added 2.5 TA to the TA mixture and control TA
  • Poured my mixture onto my plate and lets set for 10 minutes
  • Poured control TA onto the control plate and let set for 10 minutes
  • Placed in the incubator at 26 degrees Celcius at 3:45 9/12
  • Cleaned lab dab desk with CiDecon and ethanol at the beginning and the end of lab

                                                        

Fig.3.B – Shows the plate for the plaque assay for soil B             Fig.4.B – Shows the control plate for soil B after the TA after the TA was added. The yellow circle shows where              was added. The yellow circles indicate where bubbles  a bubble is on the plate.                                                                      were observed on the plate.

 

Category: Emily Balint | LEAVE A COMMENT
September 14

Analyzing Soil B

9/10/18

Rational:

To analyze the soil sample (type of soil, percent water, and pH). This will give metadata that could give further insight into the results that our group finds.

Procedure:

  • Cleaned the lab desk with CiDecon and 70% ethanol
  • Placed 2 mL of soil in a 15 mL conical vial
  • Used aseptic technique to add 10 mL LB broth using a serological pipette
  • Started 15 minute timer
  • Shook vial for 15 minutes
  • Weighed vial at 18.99g then centrifuged at 10,000 G for 5 minutes
  • Added 10 mL soil to a falcon tube
  • Added DI water up to 30 mL
  • Added soil dispersion (3 drops)
  • Shook for 30 seconds
  • Let sit for 30 seconds
  • Poured off supernatent into a conical tube (later poured supernatent back into the falcon tube)
  • Weighed empty weigh boat- 2.43 g
  • Weighed weigh boat with wet soil- 5.89 g (wet soil- 3.46 g)
  • Placed weigh boat  under hood to dehydrate
  • Put some soil into a pH vial and filled the rest of the tube with DI water
  • Shook the tube for 10 seconds and then let it sit for 2 minutes
  • Placed pH paper in the vial for 45 seconds and checked the pH (pH- 6)
  • Filtered out centrifuged soil sample with a top filter
  • About 7.5 mL was filtered out
  • Added Arthrobacter into the tube using the aseptic technique (0.5 g Arthrobacter)
  • Then placed tube into the shaking incubator at 3:50 pm 9/10 at 26 degrees Celcius (until 2:30 9/12)
  • Cleaned lab desk with CiDecon and ethanol

Fig.2.B – shows the pH of soil sample B to be 6

Category: Emily Balint | LEAVE A COMMENT
September 14

9-10-18 — Soil Filtration and Metadata

Date: Monday, September 10th, 2018

Title: Soil Filtration and Metadata

Rationale: The purpose of today’s lab was to filter out our soil, get an enriched and direct sample, and record soil metadata.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

  1. We began by creating an aseptic zone spreading CiDecon over the workplace then letting Ethanol (70%) evaporate off the table, dehydrating and therefore killing any organisms that could contaminate the equipment or samples.
  2. We lit a burner in the middle of the table in order to keep falling particles from contaminating the equipment or samples from above since the flame creates a circulating air current.
  3. Next I filled a 50 mL conical with my soil up to the 2 mL mark.
  4. I filled the same conical up to the 12 mL mark with LB broth and shook for 14 minutes.
  5. I massed my conical vial and recorded it to weigh 19.79 g. I spun this for 5 minutes at 10,000 g.
  6. I filtered the supernatant with a 22 micrometer syringe filter to a new 50 mL conical.
  7. I added .5 mL ATC 21022 to my lysate, labelled it BJR and E for “enriched,” and set it in the incubator at 3:50 pm.
  8. I then filtered 1 mL of my supernatant to a 15 mL vial to use as my direct isolation, storing it in the fridge.
  9. While waiting for my soil to soin, I added soil to a Falcon tube to the 10 mL mark.
  10. I added DI water to the 30 mL mark, added 2 drops of dispersion fluid, shook it for 30 seconds, and left it to disperse.
  11. I set some soil on a plastic boat to let it dehydrate to find the percent water. The boat weighed 2.479 grams, weighing 9.738 grams after adding soils. This means I had 7.259 grams of wet soil in the boat.

Observations:

  • The soil in the Falcon tube already began to separate by the time I left, showing different layers of different sized particles.

Results:

  • This project yielded me a direct isolation, an enriched isolation, soil metadata, and a dispersed Falcon tube allowing me to record soil composition.

Next Step

  • My next step is to use my enriched lysate, direct isolation, and a phage buffer to make a spot test. I will also finish recording metadata.
Category: Brandon Reider | LEAVE A COMMENT
September 14

Lab Day 5: Soil Enrichment, Metadata, pH

Rationale:

  • Make enriched lysate of soil B, find pH of soil and start soil metadata to find future calculations of sand, silt, and clay.

Detailed Procedure: Enriched Lysate

  1. Took vial from Lab day 4 and added LB Broth up to 12 mL mark
  2. Shook vial for 10 mins
  3. Weigh vial= 19.04 g
  4. Centrifuge for 5 mins
  5. Syringe filter 7.5 mL of supernatant (did not have enough for direct)
  6. Add 0.5 mL Arthro to vial

Detailed Procedure: Metadata

  1. Add ~10 m of soil in a 50 mL vial.
  2. Add water up to 30 mL mark
  3. Shook vial for 30 secs
  4. Add soil disperse solution to it and mix for a bit
  5. Let sit for 48 hours
  6. Took some soil and weigh it with petri dish
  7. Let sit for 48 hours to dry

Detailed Procedure: pH

  1. Took pinch of soil in a small pH vial and filled the rest with water
  2. Shook for a bit and rip off 1 inch of pH paper
  3. Dip paper for 45 secs and compare color
  4. Recorded ph was green= 7.0

Observation/Results

  • petri dish weight= 2.35 g
  • petri dish weight + soil= 6.26 g
  • after this, I ran out of all of my soil, so I have no more left to test if needed in the future.

Next steps

  • calculate sand, silt, clay
  • weigh petri dish when soil dries
  • plaque assay with enriched because I have no direct lysate
Category: Soo-Un Jeong | LEAVE A COMMENT
September 14

Soil Metadata, Enriched Lysate, Spot Test

Soil Metadata and Enrichment (9/10/18) 

Rationale: Find the metadata of our soil, such as the PH, the % water, silt, sand ,and clay, and to create a enriched and direct lysate for our spot or plaque assay testing.  

Procedure: 

  1. We first cleaned our table with Cidecon 70% ethanol and then we used a ethanol flame to create an aseptic zone for us to work in.
  2. Got a 15mL test tube and with the soil we had collected from the prior week we added 2mL of soil into it.  
  3. Added 9mL of LB broth using a glass pipette into the 15 mL test tube, for a total closer to 11mL of solution.  
  4. Shook the test tube for the next 15 minutes, since the solutions were going to be centrifuged 
  5. In the meantime, we started to obtain the metadata results.  
  6. Started with percent water and measured the dish without the soil to find a value of 2.33 grams.  
  7. Added about 5 grams of soil onto the petri dish and found the total of the petri dish and the soil was 7.91 grams.  
  8. In order to find the percent water, let the soil and the petri dish sit for 48 hours in the fume hood.  
  9. After 10 minutes of shaking we took the masses of our solution and found that to be 18.231 grams.  
  10. We then found a partner to centrifuge with, since there must be a balance of weight in the machine while the solutions are being centrifuged  
  11. After the test tubes came back after being centrifuged, we used a syringe to get our enriched and direct lysate.  
  12. After getting 5 mL of filtered solution, we realized that the liquid was too low and the syringe would not go in more.  
  13. We then transferred the liquid into a 50 mL test tube and used a syringe to filter out the solution into another 50mL test tube, which made it easier to reach the liquid.  
  14. Only obtained 8mL of solution for lysate, so decided to not have a direct sample.  
  15. We then obtained Arthro and added it to the 50mL conical tube, and then shook up the tube to make sure that it was well mixed.  
  16. Started with soil PH testing by obtaining a small PH vial and adding a little bit of soil to it, and then filled up the rest of the tube with DI water. 
  17. We shook the small vial for 10 seconds and let it settle for the next 2 minutes.  
  18. We waited 45 seconds and then put a 1 inch PH testing strip inside the small vial, and took it out as the color started changing, and quickly recorded our results by comparing the colors.  

Observations/  Results : 

We found that the petri dish weight was 2.33 grams, and with the soil the total of grams was 7.91. 

We found out that the Ph of the soil was 6.0

Next Step:  

Since there were limited test tubes for performing the salt, silt, clay percentage tests for our metadata, we will have to set up and perform the data next time. Also next time we will set up our testing, either a spot test or a plaque assay, to test for the presence of plaques and phages.  

 

Metadata and Spot Testing (9/12/18) 

Rationale: Procedure: Continuing to find soil metadata and setting up the spot test, using the enriched lysate we created from last week, to find phages in our soil sample.  

Procedure: 

  1. Clean tables with Cidecon and 70% ethanol and create an aseptic zone with an ethanol burner.  
  2. Mass the enriched sample and found its weight to be 21.53g 
  3. Found a partner with a similar mass and let the enrichment sample centrifuge for five minutes at 3,000 g to pellet the Arthro.  
  4. While waiting for centrifuged sample, take the mass of the soil left in the hood to find the % water in the soil.  
  5. Recorded the results and found the total mass to be 7.34 grams.  
  6. Obtain the enriched sample after being centrifuged and filtered 2.5 mL of the enriched sample into a 15mL conical tube.  
  7. Next start on the process for creating the top agar solution for a spot test.  
  8. First pipette 2.0 mL of Lb Broth into a 50 mL conical tube for the top agar.  
  9. Add 22.5 mL of 1 M CaCl2 into 50 mL conical tube using a micropipette  
  10. Next add 2.5 mL of 2x Top Agar using a pipette.  
  11. Mixed all the materials for creating a top Agar solution and poured it into a plate and let it sit for 10 minutes  
  12. Created a 5mL solution for our entire plate for spot test.  
  13. Divided and labeled our plate into 4 sections: control, SS, EAG, and LIP.  
  14. While waiting for the top agar to set, we started on our soil metadata for percent sand silt and clay.  
  15. Obtain a 50 mL vial and added about 9.9 mL of soil to the conical tube, and added DI water until the 30 mL mark on the tube.  
  16. Add 3 drops of soil dispersion liquid into my 50 mL vial and shook the solution for 30 seconds, and let it sit in the hood for 48 hours.  
  17. 10 minutes passed, so we returned back to our spot testing.  
  18. Added 5 uL of enriched lysate, using a pipette, to each of the three sections of the plate labeled SS, EAG, and LIP.  
  19. Add 5 uL of PH buffer to the control part of our plate, and let the plate sit for 10 minutes. 
  20. Placed the top spot test plate in the incubator upside down.  

Observations/  Results : 

Found the percent water in my soil was 10.22% 

Before centrifuging today, realized that my enriched lysate still had some pellet in it.  

While setting the top agar, realized that there were 2 bubbles. Also noted that the bubbles were around the control section of our plate.  

Next Step:  

Next time we will view the results of our spot test and check the plates for plaques. We will also observe and take data on our soil metadata for percent silt, sand, and clay.  

Category: Sona Subramanian | LEAVE A COMMENT
September 14

Spotting Test, Plaque Assay & Soil Metadata for Soil Sample (9.5.18) 9/12/18

Research Question:

To find out how the presence of bacteriophages in the soil around red or white oak trees has a correlation with the health condition of oak trees.

Rationale:

A Plaque Assay helps us determine if there is the presence of bacteriophages by adding Arthrobacter directly to the lysate. We can tell the existence of bacteriophages by checking the presence of plaques on the agar plate. A spotting test can help determine if bacteriophages are present in the lysate or not. And Soil Metadata can help us to learn more about the soil sample’s characters.

Experimental Procedure for Spotting Test for Soil 9.5.18:

……………………. 1. Set up an Aseptic zone(Sprayed with Ci-Decon, wiped dry, then sprayed 70% …………………………Ethanol and let it evaporate)  on the workbench,  prepare:

…………………………………………………….(1) Direct Isolation & Enrichment Lysate

…………………………………………………….(2) LB Broth

…………………………………………………….(3) 2x TA

…………………………………………………….(4) 1M CaCl2

…………………………………………………….(5) 0.5 ml Arthrobacter

…………………………………………………….(6) 50ml conical tubes

…………………………………………………….(7) Micropipettes & Serological Tubes

…………………………………………………….(8) Phage Buffer

 …………………….2. Centrifuge the enrichment Lysate at 3000x g for 5 min

……………………..3. Add 2 ml LB Broth, 0.5 ml Arthrobacter & 22.5 ul CaCl2 (aq) to a 50 ml conical …………………………tube.

……………………..4. Section a new Agar Plate into 3 sections, each label  PB (Phage Buffer as control), ………………………..(D) Direct Isolation & (E) Enrichment. (Plate labeled: JY5 Spotting test 9.5.18 …………………………9/12/18)

……………………..5. Add 2.5 ml of 2x TA to 50 ml conical tube and pipette with serological tube …………………………before adding the solution on to a new Agar plate, slightly swirl the plate to let it …………………………cover the whole plate evenly and wait for 10 min.

……………………..6. Filter 2 ml Soil 9.5.18 Enrichment Lysate with filter paper (0.22um) to filter out ………………………….bacteria into an Eppendorf. (labeled: JY5 9.5.18)

……………………..7. Use micropipette to drop 1o ul of Phage Buffer, Direct Isolation Lysate & ………………………….Enrichment Lysate to the middle of each section and set still for 10 min before ………………………….placing in the incubator.

Experimental Procedure for Plaque Assay for Soil 9.5.18:

…………………….1. Add 0.5 ml Arthrobacter and 10 ul Filtered Enriched Lysate to an Eppendorf 10

……………………….min for infection.

…………………….2. Add 8 ml of LB Broth, 90 ul Calcium Chloride (aq) to a 50 ml conical tube

…………………….3. Add 10 ml of 2x Top Agar to the 50 ml conical tube, quickly distribute among four

………………………..new 15 ml conical tubes for the whole group.

…………………….4. Take one tube and add the infected lysate, pipette and carefully pour on to a new

………………………..agar plate

…………………….5. Wait for 10 min to solidify (slightly shooked during) and place into the incubator.

Soil Metadata:

…………………….1. Remove the supernatant in the falcon tube

…………………….2. Record the results and discard the soil in the biohazard bin, wash up the tube and

………………………..place on rack

…………………….3. Weigh the petri dish, record the mass, discard the petri dish in the biohazard bin

…………………….4. Calculate the Soil Composition and % Water

Observations, Results & Data:

.Soil Metadata for Soil Sample 9.5.18:

…………………………….(1) % Water: 21.6% ( (12.24 – 11.16) / (12.24 – 7.24 ) = 0.216 )

 ……………………………(2) Sand: 57.1% (4ml / 7 ml)

 ……………………………(3) Slit: 28.6% (2 ml / 7 ml)

 ……………………………(4) Clay: 14.3% (1 ml / 7 ml)

The raw data from the falcon tube is extremely hard to identify since the differentiation of layers isn’t obvious.

From the data collected, the soil type seems to be sandy loam, which is generally the soil type for gardening.

Spotting Test for Soil Sample 9.5.18:

The plate appears to have an unknown white dot on the plate, its effects on the experiment are yet to be seen.

Interpretations & Conclusions:

By spinning down the Enrichment lysate, the bacteria are mostly at the bottom of the 50 ml conical tube, which made the syringe filter step extremely easy due to the lack of blockage on the filter paper.

When mixing the top agar for Plaque Assay, the time passed since the 2x Top Agar was added to the mixed solution to the mixture was poured onto the plate could potentially affect the quality of the test, since the agar starts to solidify as soon as it was added to the mixture.

The white dot is a giant variable in the spotting test, if the dot is a contamination on the agar plate the test would lose its accuracy and reliability,  after further investigation the dot seemed to be embedded in the bottom agar so it was not removable.

The result of the soil composition metadata corresponds to the environment it was sampled in since the sample was originated from an artificial garden, it is not surprising that the soil analysis indicated that it was sandy loam. However, the high percentage of water contained in the soil was astounding and the effects of the % water in the soil needs more background research.

Next Step:

I will be checking the results of both the Spotting Test and Plaque Assay on the Soil Sample 9.5.18

Category: Yang-En Yu | LEAVE A COMMENT
September 14

Soil Washing, Enrichment and Metadata for Soil Sample (9.5.18) 9/10/18

Research Question:

To find out how the presence of bacteriophages in the soil around red or white oak trees has a correlation with the health condition of oak trees.

Rationale:

To search for possible bacteriophages in the soil, this experiment takes Soil Sample 9.5.18 and go through the Washing process to collect potential bacteriophage-containing lysate for further analysis. By using LB Broth, centrifuge and syringe filters, potential bacteriophage lysate can be collected. And by adding Arthrobacter to some of the lysate, Enrichment protocol can help with the confirmation of bacteriophage’s existence. And Soil Metadata can help us to learn more about the soil sample’s characters.

Experimental Procedure for Soil Washing, Enrichment of sample Soil 9.5.18:

Soil Washing:

1. Set up an Aseptic zone(Sprayed with Ci-Decon, wiped dry, then sprayed 70% Ethanol and let it evaporate)  on the workbench,  prepare:

  1. Sample Soil 9.5.18
  2. Two 15 ml Conical Tube
  3. One 50 ml Conical Tube
  4. LB Broth
  5. Syringe Filter

2. Place sample Soil A to the 2 ml mark in the 15ml Conical Tube.

3. Pour LB Broth to the 12 ml mark in the 15 ml Conical Tube Soil 9.5.18

(1) 15 ml Conical Tube Soil 9.5.18 weight: 20.15 g

4. Vortex(5 min), Shake(5 min) & Vortex(5 min) for 15 min to mobilize the soil.

5. Centrifuge at 3000 G for 10 min.

6. Filter the supernatant from the 15 ml Conical Tube Soil 9.5.18 with syringe filter to a new 50 ml conical tube and the rest in a new 15 ml conical tube.

7. Add 0.5 ml Arthrobacter into the 50 ml conical tube with the filtered supernatant and place on shaker.

10. Stored the Supernatant filtered from the filter paper into a new 15 ml Conical Tube(Direct Isolation) and store in fridge.

Soil Metadata:

11. Soil Composition:

…..(1) Add soil to the 10 ml mark on the falcon tube.

…..(2) Add water to the 30 ml mark.

…..(3) Add 3 drops of Soil Dispersion Liquid

…..(4) Wait for 48 hrs to see the results

12. % Water:

…..(1) Weigh the empty petri dish: 7.24 g

…..(2) Weigh the petri dish with wet soil: 12.24 g

…..(3) Wait for 48 Hrs for Soil to dry and check results

13. Soil pH:

…..(1) Add a small amount of soil to the pH vial

…..(2) Add water to 80% full

…..(3) Shake the vial for 30 sec and wait for 2 min

…..(4) put in a pH paper(should not exceed 1 inch)

…..(5) Wait for 45 sec and compare the color with the pH scale to determine the pH of the soil

…..(6) The pH level for Soil Sample 9.5.18 is  pH 7.5

14. Workbench was sterilized with Ci-Decon and 70% Ethanol for clean up.

Observations, Results & Data:

During the syringe filter stage, the bubbles in the syringe cause the filter process harder due to the compressible characteristic of air.

The metadata of Soil Sample 9.5.18: pH 7.5,

other data would have to wait for 48 Hrs to get the results.

Interpretations & Conclusions:

Even with a pH scale, the results can be hard to interpret and can be subjective due to the nature of the naked eye.

It would be ideal if the lab reports next time can feature a picture of the pH paper and the scale together,

Next Step:

I will be doing spotting test and Plaque Assay on Soil Sample 9.5.18, and finishing up the data collection of the soil metadata.

In the Next Lab:

I will need (1) LB Broth

……………….(2) Arthrobacter

……………….(3) Calcium Chloride

……………….(4) Centrifuge

……………….(5) Agar Plate

……………….(6) Syringe Filter

……………….(7) Micropipette and Serological Pipette

Category: Yang-En Yu | LEAVE A COMMENT
September 14

Soil Metadata (continued) and Spot Test for Soil B (09/12/18)

Rationale:

Today, the percent water, percent sand, percent silt, and percent clay will be calculated for later used to determine whether there is any correlation between the present of phage and the metadata. The spot test will be run only with the enrich lysate (since no isolated lysate was collected) will reveal whether or not the sample soil collected has any phages that target Arthrobacter specifically.

Procedure:

  1. Cleaned the counter area with CiDecan and wiped it dry. Then, cleaned with EtOH (70%) and allowed it to evaporate.
  2. Weighed the mass of “KEA 9/10/18 Soil B enrich” conical vial.
  3. Added a few drops of de-ionized (DI) water with disposable pipette to have a similar mass to a peer’s enriched lysate conical vial mass.
  4. Centrifuged “KEA 9/10/18 Soil B enrich” conical vial at 3,000g for 5 minutes.
  5. Finished collecting data for the percent water by weighing the mass of “KEA 9/10/18 Soil B” petri dish with the dry soil.
  6. Finished collecting data for the percent sand, percent silt, and percent clay by analyzing and calculating from “KEA 9/10/18 Soil B” falcon tube.
  7. Cleaned “KEA 9/10/18 Soil B” falcon tube with DI water.
  8. Wiped a 0.22 μL tip with EtOH (70%) to prevent contamination.
  9. Through aseptic technique (over an EtOH (100%) flame), used a 3 mL syringe with a 0.22 μL tip to filtered out “KEA 9/10/18 Soil B enrich” conical vial into a microcentrifuge tube.
    • Labeled the microcentrifuge tube as “KEA 9/12/18 enrich filtered lysate.”
  10. Obtained two plates for a control TA, which was labeled “LEF KEA 9/12/18 Spot Test Control” and one for self, which was labeled “KEA 9//12/18 Spot Test.” Circles were also drawn to indicate where the enrich lysate and the phage buffer were placed.
  11. Tripled the original recipe for performing a spot test.
  12. Used a serological pipette with bulb with 10 mL tip to transfer 6.0 mL of LB Broth into a 50 mL conical vial.
    • Labeled this 50 mL conical vial “Team 2 9/12/18 Soil B TA.”
  13. Transferred over with a serological pipette with bulb with 10 mL tip 7.5 mL 2X TA into “Team 2 9/12/18 Soil B TA” conical vial.
  14. Used a P200 micropipette to add 67.5 μL CaCl2into “Team 2 9/12/18 Soil B TA” conical vial.
  15. Added 1.5 mL of Arthrobacter into “Team 2 9/12/18 Soil B TA” conical vial.
  16. Used a serological pipette with bulb with 10 mL tip to transfer 5 mL of “Team 2 9/12/18 Soil B TA” mixture onto both “LEF KEA 9/12/18 Spot Test Control” and “KEA 9//12/18 Spot Test” plates and swirled to even out the TA.
  17. Allowed 15 minutes for the TA to solidify.
  18. Used a P10 micropipette to add 10 μL of phage buffer in the phage buffer circle on “KEA 9/12/18 Spot Test” plate.
  19. Used a P10 micropipette to add 10 μL of the enriched lysate from the “KEA 9/12/18 enrich filtered lysate” microcentrifuge tube in the enrichedcircle on “KEA 9//12/18 Spot Test” plate.
  20. Placed plates in incubator for the next 48 hours.
  21. Cleaned lab counter with CiDecan and EtOH (70%).

Calculations and Metadata Soil Data:

Percent Water

Data Table

mass of empty petri dish with lid (mi)

7.58 grams

mass of petri dish with wet soil sample (mt)

 11.00 grams

mass of wet soil sample (mwet soil)

mt – mi= mwet soil

3.42 grams

mass of petri dish with dry soil (mf)

10.67 grams

mass of dry soil sample (mdry soil)

mf – mi= mdry soil

3.09 grams
mass of water in the soil sample (mwater)

mwet soil – mdry soil = mwater

0.33 grams

Equation for Percent Water

Percent Water = 9.65%

Percent Sand, Percent Silt, and Percent Clay

Data and Calculation Table

Texture Approximate Amount Calculations Percent
Sand 4.3 mL 4.3 mL / 7 mL 61.43%
Silt 1.2 mL 1.2 mL / 7 mL 17.14%
Clay 1.5 mL 1.5 mL / 7 mL 21.43%
Total 7 mL 7 mL / 7 mL 100.00%

Percent Sand = 61.43%

Percent Silt = 17.14%

Percent Clay = 21.43%

Classifying Soil Texture

Based off the percent sand, percent silt, and percent clay, calculations using a soil texture triangle classifies the soil sample’s texture to be sandy clay loam. The point is plotted on the soil texture triangle below.

This image was retrieved from the Natural Resources Conservation Service website at https://www.nrcs.usda.gov/wps/portal/nrcs/detail/soils/survey/?cid=nrcs142p2_054167.

Calculations for the Amount of Materials Needed

Original Recipe

X3

2 mL LB Broth

6 mL LB Broth

0.5 mL Arthro

1.5 mL Arthro

2.5 mL 2X TA

7.5 mL 2X TA

22.5 μL CaCl2

67.5 μL CaCl2

Observations:

  • “KEA 9/10/18 Soil B enrich” conical vial had a mass of 22.28 grams before DI water was added. After DI water was added, “KEA 9/10/18 Soil B enrich” conical vial had a mass of 22.32 grams.
  • In the “KEA 9/10/18 Soil B” falcon tube, the levels of sand, silt, and clay were not straight across. The following photos illustrate how the levels tended to shift towards one side of the falcon tube than the other causing there not to be straight define lines of where exactly the silt starts and stops.

  • As shown above, the sand had a light brown color. The silt had a dark gray tint. The clay had a dark cinnamon-brown color.

Next Steps:

Next time, check the spot test to see if there is any contamination, positive, or negative results. If contaminated, I will run another spot test. If not, I will run a plaque assay with the “KEA 9/10/18 Soil B enrich” lysate. The results from both the spot test and plaque assay will ultimately determine whether or not I have to collect more soil.

 

Category: Kathryn Adkins | LEAVE A COMMENT
September 14

09/10/18 Soil Enrichment and Metadata

Rationale:

Today’s lab goal was to set up the lysate for a plaque assay and spot test as well as begin the process of gathering soil metadata.

Materials Used:

  • 15-mL Conical Vial
  • Weigh Plate for soil
  • Serological Pipette
  • DI Water
  • Soil Dispersion Liquid
  • LB Broth
  • 0.5 mL of Artho
  • pH Paper

Procedure:

  • The experiment began with gathering the soil samples previously collected the week prior .
  • After gathering soil and a 15-mL conical vial, 2-mL of soil was added to the bottom of the conical vial along with 10 mL of LB broth.
  • After the LB Broth was added, one of my lab partners put my sample through the vortex for 15 minutes, while this was occurring I added 10-mL of soil to a 50-mL tube along with 20-mL of DI water and soil dispersion liquid to gather soil composition metadata.
  • I shook the soil for 30 seconds and let it sit for 30 seconds, I left the soil to sit for 48 hours for the particles to separate.
  • Once the soil composition experiment was finished, my sample was finished with its vortex my vial lid broke and I had to transfer my liquid to another conical vial before sending it off to centrifuge at 10,000 g for 5 minutes.
  • While my soil was in the centrifuge, I started the experiment to calculate my percent water of the soil sample.
  • To begin, I weighed my empty petri dish to get a weight of 6.90 g.
  • I added my wet soil to the empty Petri dish and got a mass of 11.60 g.
  • Then, using subtraction, I found the mass of the wet soil which is 4.7 g.
  • After calculating the mass of the wet soil, I left the petri dish out for 48 hours as well to let the water evaporate from the soil.
  • After my vial was retrieved from the centrifuge, my lysate was still extremely cloudy. To try and make up for this, I split my lysate and put it in the centrifuge again for another 5 minutes at 10,000 g.
  • After removing the vial from the centrifuge, my lysate was significantly clearer. After examining it, I filtered the enrichment through a syringe filter.
  • After filtration, I split the lysate in 2 vials. One was my direct isolation which had 2-mL and the other was my enriched with 10-mL with 0.5-mL of Arthobacter.
  • After splitting the two, I put the vials up for 48 hours to use for the next lab.
  • At the end of the lab, I added a small amount of soil to a vial, and filled it up with D.I water and shook vigorously for 10 seconds.
  • After shaking, I waited for 2 minutes for the soil to set and inserted a piece of pH paper no longer than an inch into the vial for 45 seconds. After 45 seconds I removed the paper to examine the pH.

Data/Observations

  • The lysate after the second centrifuge was still slightly cloudy despite the second centrifuge.
  • The soil separated very well for the metadata, there appears to be a lot of sand with very little to no silt or clay present in the soil.
  • pH paper came from the soil a blue, the closest color to match was a navy blue

Conclusions:

  • The soil pH was approximately 7.5
  • Soil appears to have a large amount of sand and little silt and clay present.
  • Lysate appeared slightly dark, not entirely clear throughout, despite the second centrifuge. Must be due to the switching the tubes after the lid cracking. Hopefully that does not affect the phage present in the plaque assay and spot test.

Next Steps:

  • The next steps will be to test for the presence of phage in the lysate with a spot test and a plaque assay. Also I will need to examine the soil composition and calculate the percent of water present in the soil.
Category: Gabriel Andino | LEAVE A COMMENT