September 14

Metadata + Plaque Assay + Spot Test

Title: Metadata, Spot Test, and Plaque Assay

Date: September 12, 2018

Rationale: The sand-silt-clay results and the % water results are ready, and the lysate is done incubating, therefore metadata can be recorded, and tests be done to check for plaques.

Procedure: Aseptic zone created by the following procedure:

  • Counter washed and wiped with CiDecon
  • Counter sprayed with 70% EtOH and allowed to evaporate completely (to dehydrate and kill any bacteria on the counter and avoid contamination)
  • Ethanol lamp lit to create rising heat and a current that protests samples from falling contamination.

% Water: The dirt weighed 5.37 grams before evaporation and weighed 5.066 after evaporation. The following calculation yields the % water of the collected soil:

1-(5.066/5.37)= .057 = 5.7% water

Sand – Silt – Clay: 3.25 mL of soil was layered:

1 mL of sand      1.75 mL of silt   .5 mL of clay

= 30.77% sand     =53.85% silt    = 15.38% clay

  1. The enriched lysate from Monday was pulled from the incubator, massed at 21.47g, and spun in a centrifuge to separate cells.
  2. A spot test and a plaque assay were done to assure the best chances of a phage discovery.

 

Spot Test                                                                              Plaque Assay (All multiplied by 4 for 3 plates + negative control)

– 2.0 mL LB Broth                                                                            – 2.0 mL LB Broth

– 0.5 mL Arthrobacter                                                                    – 0.5 mL Arthrobacter

– 2.5 mL 2X Top Agar                                                                      – 2.5 mL 2X Top Agar

– 22.5 microliters CaCl2                                                                  – 22.5 microliters CaCl2

—————————————————————————————————————-

~ 10 microliters spotted onto plate –> put into incubator                       Set 15 min –> put into incubator

Category: Uncategorized | LEAVE A COMMENT
September 14

New Soil Enrichment + Metadata

Title: New Soil Enrichment + Metadata

Date: September 10, 2018

Rationale: The new soil collected in the previous week is to be washed and enriched for further testing to make progress on our research question

Procedure: Aseptic zone created by the following procedure:

  • Counter washed and wiped with CiDecon
  • Counter sprayed with 70% EtOH and allowed to evaporate completely (to dehydrate and kill any bacteria on the counter and avoid contamination)
  • Ethanol lamp lit to create rising heat and a current that protests samples from falling contamination.

 

  1. ~2mL of soil was added to a 15mL vial
  2. ~10mL of LB Broth was added to bring the vial up to the 12mL mark
  3. Vial was shaken for 10 minutes

Meanwhile, soil metadata was taken

% Water: An empty weigh boat was massed at 2.39 grams, soil was added to bring the mass of the weigh boat to 7.76 grams. The weigh boat was left to evaporate for 48 hours. The mass of the dry dirt will then be used to calculate the % water of the soil.

Sand, Silt, Clay: ~4mL of dirt added to Falcon tube, DI water was added to cover the dirt, and soil dispersion liquid was added to separate the soil. The entire mixture was shaken for 30 seconds and left to disperse over a 48-hour period.

pH: A small amount of dirt was added to a pH testing vial and was filled the rest of the way with DI water. The mixture was shaken for 10 seconds and the pH was recorded on a pH testing strip. The recorded pH was 6.0

A 22 micrometer Top filter was then used to generate ~7.5 mL of lysate. 0.5mL of Arthrobacter was added to enrich the lysate and incubated at 28 degrees Celsius for 48 hours.

Category: Cooper Johnson | LEAVE A COMMENT
September 14

9/12/18 – Soil B Spot Test and Plaque Assay + Results of % Water & % Sand/Silt/Clay

Rational:

Today, we will be setting up and performing both a plaque assay and spot test for the enriched lysate of Soil B, as well as checking the results of the % water and % sand/silt/clay

Procedure:

  • We first created an aseptic zone on our table using CiDecon and 70% ethanol, and lit an ethanol burner to create the aseptic zone
  • I then massed my enriched lysate (21.59g) to find a partner for centrifusion. The enriched lysate was centrifuged at 3000 g’s for five minutes to pellet the arthrobacter
  • While the lysate was being centrifuged, I checked the results of percent water and percent sand/silt/clay on my soil
  • For percent water, I re-massed my soil+dish and got 11.72g, which meant the soil was 9.33g (Total dry weight – weight of dish)
    • I then took my dry soil weight and divided it by the wet soil weight (9.33/12.064), and got 77. I then subtracted 100 from this number to get 23% water
  • For percent sand/silt/clay, I looked at my vial and determined where all the clay ended (Total 2 mL)
    • For sand, I determined it was 25% of the soil
    • For silt, I determined it was 50% of the soil
    • For clay, I determined it was 25% of the soil
  • My table group decided that we will all be performing our spot test on one plate (1/4 per test), with the left over 1/4 being for our negative control (Phage buffer), as well as performing a plaque assay separately, while sharing one TA (Top agar) control plate
  • For the spot test, we mixed together 2 mL LB Broth, 0.5 mL Arthrobacter, and 22.5 μL 1M CaCl2 into a 50 mL conical vial
    • The LB Broth was added with a cartwheel pipette with a 5mL tip, while the CaCl2 was added with the P200 pipette
  • We then added in 2.5 mL 2xTA, and then immediately poured it onto our plate, and then let it solidify for 10 minutes
    • 2xTA added with the P200 pipette
  • While waiting for the TA to solidify, we filtered our enriched and centrifuged lysate using a syringe and a 22μL filter
  • We then spotted 10 μL of each of our enriched lysate on our separate sections of the dish, as well as spotted 10μL of phage buffer into our negative control 1/4
  • We then moved the plate to an incubator
  • For our plaque assay, we multiplied all our materials by four; we added 8 mL of LB Broth and 90 μL of CaCl2 into a 50 mL conical vial
  • We then took our separate enriched lysates and added .5 μL arthrobacter into each one and let it sit for 10 minutes to infect
  • We then added 10 mL 2xTA to our mixture, and immediately poured 5mL into our TA control dish (The TA will start solidifying after being added into the mixture)
  • We then separately added 5mL of our TA mixture to each of our enriched lysate + arthrobacter mixture, and immediately added it to our plaque assay plates
  • We let our plates sit for 15 minutes, and then moved the plates to incubation

Observations:

You can clearly see the separations of the dirt

My dried dirt, used to calculate % water

Our spot test dish; three of the quadrants are for the separate three members in our group, as well as 1/4 for our negative control

My Plaque Assay dish prior to anything added into it

My Plaque Assay dish with the procedure completed

  • There was a significant color difference between the wet dirt and dry dirt
  • The procedure for the spot test and plaque assay was performed more quickly than our first time

Next Steps:

Next time we come into lab, we will see if the spot test and plaque assay turn out negative or positive, and see if our controls worked

Category: Justin Yu | LEAVE A COMMENT
September 13

9/10/18 – Soil B Washing and Enrichment

Rationale:

We will be washing and filtering our Soil B to created an enriched lysate. This is so we will be able to perform a spot test and plaque assay for the Soil B lysate.

Procedure:

  • First, we created an aseptic zone on our table using CiDecon and 70% Ethanol. We also lit a ethanol burner to create the aseptic zone
  • I then put 2mL of Soil B into a 15 mL conical vial, and added 10 mL of LB Broth
  • I then shook the vial for ten minutes, with both my hand as well as the vortex machine
  • My vial massed at 17.488 g and was then centrifuged at 10,000 g’s for five minutes
  • While my vial was being centrifuged, I needed to begin the preparations for finding the percentage water,  percentage sand/silt/clay in my soil sample, and my soil pH
  • To calculate the percentage water, I first obtained a weighing dish and massed it. It weighed 2.39 g
    • I then added soil into the dish, and massed it again. The dish + soil weighed 14.454 g, so the weight of the soil was 12.064 g
    • The dish with my soil sample in it was then placed into a hood to evaporate for 48 hours (Results in next post)
  • To calculate percentage sand/silt/clay, I first obtained a 50 mL measurement vial, and added 4mL of soil into it
    • I then added water to the 10mL mark, as well as SDL (Soil Dispersion Liquid) to the vial, and shook for 30 seconds
    • After the 30 seconds, I placed the vial in the hood in a rack to let sit for 48 hours (Results in next post)
  • To find my soil pH, i First took a pH vial and added a small amount of Soil B into it
    • I filled the rest of the vial with DI (Deionized) water, and then shook for 10 seconds
    • After letting the soil sit, I placed a strip of pH paper into the vial, and let it react for 45 seconds
    • The recorded pH of my soil was 6.5
  • After my soil was centrifuged, I ran my liquid through a top filter (22 μL filter) to get my enriched lysate
  • I had around 10 mL of enriched lysate in a 50 mL conical vial; I labeled my vial with my initials and the date, as well as “Soil B”
  • I then added .5 mL of arthrobacter and incubated my tube

Data/Observations

My Enriched Lysate prior ro incubation

The soil-water solution with SDL in it; results in 48 hours

The pH vial and pH paper after measuring. My weighing dish can also be seen in the background

  • My pH indicated that my soil is slightly acidic; it would be interesting to compare results to others in the lab
  • The SDL had already started to work in the 50 mL measuring vial; I could see some distinct separation of materials in the vial

Next Steps:

My next steps would be to perform a spot test or plaque assay with the enriched lysate of Soil B. I will also need to check on the results of the percentage water and percentage sand/silt/clay next lab

Category: Justin Yu | LEAVE A COMMENT
September 13

9.12.18 Soil Metadata and Spot Test

9.12.18 Soil Metadata and Spot Test

Rationale)

I will finish collecting metadata about Soil B and conduct a spot test with enriched lysate produced from Soil B, in the effort of determining if an isolatable phage is present in Soil B.

Procedure)

  1. Set up an aseptic zone by cleaning the work surface with CiDecon and 70% ethanol, and lighting an ethanol flame.
  2. Collect 50mL conical vial labeled “NMN 9.10.18 Soil B Enriched Lysate”  and add enough DI water using a pipette that mass of the vial registers 22.043g. Proceed to centrifuge the vial at 3000g for 5 minutes to pellet the arthrobacteria.
  3. While the vial is centrifuging, determine the percent water of Soil B by taking the mass of the weigh dish labeled “NMN %H2O” which holds now dried sample of Soil B from Monday. The new mass of the soil is 3.197g and comparing that to Monday’s weight of 3.72g that means a total of .523g of water evaporated. And dividing the grams of water over the original soil mass and multiplying by 100 gives us a percent of water that is equal to 14.059%.
  4. Observe the now settled middle-back falcon tube and use the markings to determine the total milliliters of the sand, silt, and clay in the sample and record. Sand: 2mL, Silt: .5mL, Clay: .5mL, dispose of the soil in the biohazard container and wash out the tube for later reuse.
  5. Collect LB broth, a petri dish with base agar, and a 50mL conical vial that is labeled “Top Agar for Spot Test, Group 3 9.12.18”.
  6. Label the plate by dividing it into four sections and write the initials of each Group 3 member in a section(NN, HB, SS), leaving one section blank to act as a control. On the rim of the plate label it “Spot Test 9.12.18”.
  7. Using a 10mL serological pipette transfer 2mL of LB broth to “Top Agar for Spot Test, Group 3 9.12.18” under aseptic conditions, add .5mL of arthrobacter to “Top Agar for Spot Test, Group 3 9.12.18” under aseptic conditions as well.
  8. Add 22.5 microliters of 1M CaCl2 using a 10-100 microliter pipette to “Top Agar for Spot Test, Group 3 9.12.18” under aseptic conditions.
  9. Add 2.5mL of 2xTop Agar to the 50mL conical vial labeled “Top Agar for Spot Test, Group 3 9.12.18”, cap the vial and swirl for 10 seconds to mix. Pour the contents of the vial onto the previously labeled plate, shaking the plate slightly in order to distribute the top agar evenly. Wait 5 minutes for it to solidify.
  10. Retrieve a .22 micron syringe filter and a 3mL syringe and under aseptic conditions filter enough of “NMN 9.10.18 Soil B Enriched Lysate” to fill a micro test tube, label the test tube “Filtered Lysate Enriched NMN 9.12.18”. Dispose of the vial and filter in the biohazard bag.
  11. Add 10 microliters of “Filtered Lysate Enriched NMN 9.12.18” to the section of the now solidified plate in the section labeled “NN”. Also, add 10 microliters of Phage Buffer to the control section of the plate. All of this is conducted under aseptic conditions using a 2-20 micropipette.
  12. Let the plate “Spot Test 9.12.18” sit for 15 minutes then store inverted in the incubator for 48 hours.
  13. Clean the work area with CiDecon and 70% ethanol, and place any remaining equipment in the biohazard bag or the appropriate autoclave container.

Data/Observations)

The mass of the dried soil is 3.197g. The mass of the pre-dried soil was 3.72g. The mass of water in the soil is therefore .523g. The percent of water in Soil B is therefore 14.059%.

The volumes of Sand, Silt, and Clay are Sand: 2mL, Silt: .5mL, Clay: .5mL. Therefore the soil composition is: Sand: 66.66%, Silt: 16.66%, Clay: 16.66%.

I also observed that the pellet of arthrobacter was rather large, and left the lysate much clearer than it was before the vial was centrifuged. Additionally, it was relatively difficult to use the syringe to extract lysate from the 50mL conical vial which could increase the potential for contamination.

Conclusions/Next Steps)

From the data gathered it can be determined that the soil from which Soil Sample B has an average water percentage of 14.059%, additionally the next steps for collecting soil metadata will be to use a soil composition chart to determine the soil type for Soil B based upon the gathered percentages of sand, silt, and clay. In regards to the spot test, the next step will be to check on Friday for the presence of plaques in my section of the plate, which if positive will require the need to conduct a plaque assay with the same enriched lysate used for the spot test. Additionally, we will also begin the process of picking and isolating the plaques in order to isolate the phages that caused them. If the test comes back negative we will have to proceed to collect another soil sample, in the effort to find a phage that can be isolated and eventually sequenced.

Category: Nathan Newton, Uncategorized | LEAVE A COMMENT
September 13

09/12/2018- Soil Metadata analysis and Spot test

09/12/2018

Soil Metadata Analysis and Spot test

Research Question: 

Does the presence of arthrobacter appear more dominant in the soil of one oak species than the others? Is there a correlation between the presence of Arthrobacter Phage and the presence of oak wilt fungus?

Objectives:

  • calculate % water, % sand, %silt and % clay.
  • make spot test using the lysate extracted from soil sample B

Materials required:

micropipettes, agar plates, micro centrifuge tubes, soil metadata collection samples prepared on 09/10/2018, 50 ml conical vials, serological pipettes, 1 M CaCl2 , phage buffer, enriched lysate, direct isolation lysate, 0.5 ml of arthrobacter, 2X top Agar, LB broth, syringe, syringe filters ( 22 microns).

Procedure:

Soil Metadata:

% Water:

  1.  collected the soil that was put in the weighing boat outside to dry.
  2. massed the dry soil and boat on the scale
  3. subtracted the mass of the boat from the total weight( boat and dry soil)
  4.  using % water = mass of dry soil / mass of wet soil x 100, the percent water was calculated

Calculations

mass of boat = 2.330 g

mass of wet soil and boat= 8.700g

mass of wet soil= 8.700-2.330= 6.370g

mass of dry soil and boat = 7.929g

mass of dry = 7.929-2.330= 5.599g

% water= mass of wet-mass of dry/ mass of wet x 100= [( 6.370-5.599)/6.370] x 100= 12.1%

% sand, silt and clay

  1. retrieve falcon tube with soil sample for collecting metadata on the type of soil
  2. record the level to which there sand on the bottom
  3. do the same for silt and then clay
  4. the total for all was 10 ml.
  5. calculate % sand silt and clay as done below.

Calculations :

amount of sand= 7ml

amount of silt= 0.5 ml

amount of clay= 2.5 ml

total = 10 ml

% sand = amount of sand / total x 100 = 7/10 x 100 = 70%

% silt= amount of silt / total x 100 = 0.5/ 10 x 100 =  5 %

% clay= amount of clay/ total x 100 = 2.5/10 x 100 = 25 %

Type of Soil :  loam

based on chart below

Spot test:

Calculations:

conversion factors:

1M= 1000mM

1 ml =1000 microliters

M1V1=M2V2

1M(V1)=(4.5mM)(10ml)

1000mM(V1)=(4.5mM)(10000 microliters)

V1=45 microliters

Procedure:

  1.  First the aseptic zone was set up: pour Cidecon on the desk and wiping it till the desk dry. then pour ethanol and wipe it all over the table and let it evaporate.
  2. Light the ethanol lamp to create an air current near which the samples can be opened to prevent things from getting into the samples.
  3. make on TA for the group. because four plates are made ( 1 per person and control), adjust amounts accordingly
  4. Retrieved the LB broth, a 50 ml conical tube and a serological pipette
  5. While in the aseptic zone, transfer 8 ml of LB broth to the 50 ml conical vial.
  6. now retrive the 1 M CaCl2 stock solution.
  7.  Using the micropipette, i transfered 90 microliters of the CaCl2 to the 50 ml conical tube with the LB broth.
  8. retrieved 0.5 ml of arthrobacter from Lathan ( Teaching Assistant)
  9. add the arthrobacter to the 50 ml conical tube.
  10. add 10 ml of top agar to the 50 ml conical tube.
  11. pour the contents of the 50 ml conical tube onto the agar plate.
  12. to let the top agar solidify, i waited for 10 minutes.
  13. collect the enriched sample tube, a syringe and a filter of 22 microns.
  14. take a sample from the enriched tube using the syringe.
  15. attach filter to the syringe and pour the lysate out slowly into a microcentrifuge tube
  16. collect the direct isolation sample from the fridge.
  17. collect a phage buffer from the instructor
  18. mark the agar plate with three spots , one for the enriched, one for the direct and one for the phage buffer.
  19. spot 10 microliters each of the phage buffer, enriched sample and the direct isolation sample.
  20. let the sample rest for 10 minutes
  21. i put the plate in the incubator, where it will remain for 48 hours.

Analysis:

there was no event that may have caused contamination to the sample. the aseptic zone was properly maintained. the procedure was properly followed.

Future notes:

take more pictures to have a proper record of all set ups.

Pictures:

 

 

Category: Dr. Adair, Uncategorized | LEAVE A COMMENT
September 13

9.12.18: Metadata Collection and Spot Test

9.12.18: Metadata Collection and Spot Test

Rationale: Metadata tests that needed additional time to settle had been given enough, so results of the tests could be processed and analyzed. Then, a Spot Test could be performed with Enriched Isolation that was obtained on Monday, 9/10/18.

Metadata Results/Data:

  • % Water: 2.390g measured after 48 hours of evaporation/3.145g initial soil added= 75.99% Soil. 1-0.7599= 24.01% Water
  • After observing tube that contained soil being analyzed for percentages of sand, silt, and clay, it was found to contain 3.25mL of layered soil. The bottom layer was 1.5mL of sand, the middle layer was 1mL of silt, and the top layer appeared to be 0.75mL of clay. The corresponding percentages were: 46.15% Sand, 30.77% Silt, and 23.08% Clay. The category of soil this fits under is Sandy Clay Loam.

Procedure:

  1. Lab bench was cleaned with Cidecon and 70% ethanol.
  2. Ethanol burner was lit to establish an aseptic zone surrounding the flame.
  3. Obtained mass of 50mL Conical tube labelled “HMB Enriched Lysate 9/10/18 S.S.B.”
  4. Paired tube with another of similar mass and sent for centrifuging at 3000g for 5 minutes in order to pellet out Arthrobacter.
  5. While the tube was being centrifuged, metadata results were collected.
    1. Weigh boat containing original soil sample B labelled “HMB Water” was re-massed and found to contain 2.390g of soil. The corresponding calculations resulted in discovering the % water of the soil was 24.01%. Weigh boat containing soil was disposed of under the instruction of Leo.
    2. The tube with soil in layers was taken up from lab bench and examined. After examination and documentation, the tube was cleaned and returned properly.        
  6. LB Broth, plate, and 50mL conical tube were obtained. Plate was labelled with each of Group Three’s initials on one section of the plate and “Spot Test 9.12.18” on the bottom.
  7. Placed 2mL of LB Broth in the 50mL conical tube.
  8. 0.5mL of Arthrobacter was added to same 50mL conical tube.
  9. 22.5 microliters of 1M CaCl2 was pipetted into same 50mL conical tube.
  10. Finally, 2.5mL of 2x Top Agar solution was added and swished in same 50mL conical tube. After mixing, solution was rapidly poured onto plate to avoid cooling and hardening in tube rather than on plate.
  11. Plate with freshly poured Top Agar was given 15 minutes to settle and harden.
  12. While plate was setting, a 3mL syringe was obtained along with the enriched lysate from Monday (9/10/18).
  13. 2mL of enriched lysate were drawn up into syringe. Filter was screwed onto end, and the 2mL were pushed through the filter and into a microcentrifuge tube.
  14. 10 microliters of filtered sterilized enriched lysate was added to the section of the plate labelled “HB”.
  15. Plate was allowed to set for 15 minutes before being placed into the incubator to be observed on Friday.

Observations:

  • Soil in tube was not evenly layered. For example, the silt on one side appeared to encompass over 1mL of the total sample, but on the other side it made up closer to 0.5mL. To get values, averages of these were taken, leading to possible inaccuracies in final values.
  • While the top agar was being formed, some residual bubbles remained on the outside of the agar. They were unable to be popped, so they remained around the ring. However, there was more than enough space on the plate to avoid them.
  • When samples were applied to the plate during the initial spot test, they seemed to sit on the surface momentarily before settling into the agar. However, the same phenomena was not observed during this Spot Test. It will be interesting to see whether or not the results are different, but any discrepancies would not be able to be formally attributed to that due to the number of variables changed.

Next Steps:

  • On Friday during open lab, the spot test sample will be analyzed. If there is a plaque, it will need to be picked and a Plaque Assay will be run to ensure that the results obtained were accurate and repeatable. If there is no plaque, another soil sample will need to be obtained and examined.
Category: Henry Burns | LEAVE A COMMENT
September 13

Spot Test and Metadata 9/12/18

Rationale

Today we will conduct a spot test to isolate bacteriophage and collect the remaining metadata on Soil Sample B.

Procedure

  1. Mass the weigh boat labeled “EAG 9/10/18 %H2O Metadata” and calculate the percent water lost.
  2. Use the aseptic technique to clean the area. With gloves on, spray CiDecon on the lab table and wipe dry. Then wipe 70% EtOH on the surface and let the excess evaporate.
  3. Obtain your enriched sample created on 9/10/18 and mass it.
  4. Spin the 50 mL conical vial labeled “EAG 9/10/18 Enriched Isolation” at 3000 g to pellet Arthro for 5 minutes.
  5. Using a syringe and a 0.22 μm filter attached to the end of the syringe to filter 2 mLs of the supernatant produced into a micro test tube.
  6. Label a petri dish by dividing the dish into 4 quadrants. Label one quadrant “EAG 9/12/18 Spot Test”, another quadrant “LIP 9/12/18 Spot Test”, another quadrant “SS 9/12/18 Spot Test”, and the last quadrant “9/12/18 Control.”
  7. In a 50 mL conical vial labeled “9/12/18 EAG,SS, LIP Spot Test,” pipet 2 mLs of LB Broth in the vial using a 10 mL pipet and a suction device.
  8. Micropipette 22.5 μL of CaCl2 into “9/12/18 EAG,SS, LIP Spot Test.”
  9. Add 0.5 mLs of Arthrobacter into the 50 mL conical vial and quickly add 2.5 mLs of 2X Top Agar using a 5 mL pipet and a suction device. Lightly mix the tube and then pour into the petri dish.
  10. Let the plate sit for 10 minutes.
  11. Using a 5 μL micropipette, pipet EAG, SS, LIP, and phage buffer into four different quadrants in the plate.
  12. Let the plate sit for 10 minutes and then place into the incubator.
  13. Begin collecting the remaining metadata by determining the percent sand, silt, and clay of Soil Sample B. Obtain a 50 mL falcon tube and label “EAG 9/12/18 Sand/Silt/Clay Metadata”
  14. Place 10 mLs of soil into the falcon tube and fill with DI water to the 30 mL line.
  15. Add 3 drops of soil dispersion liquid and shake vigorously for 30 seconds. Let the falcon tube sit in the fume hood for 48 hours.

Observations

  • Air bubbles occurred when pouring the Top Agar to the plate. Two air bubbles were present near the center of the control quadrant and one air bubble was present near the edge of quadrant “EAG 9/12/18 Spot Test.”
  • When collecting the metadata on percent sand, silt, and clay of Soil Sample B, soil clumped up at the bottom of the falcon tube and the was not able to be mixed properly.
  • The mass of the weigh boat is 7.192 g. The % water in the soil is 46.9%.

Conclusion and Next Steps

We will let the spot test and the sand/silt/clay tubes sit for 48 hours. The spot test results will be observed in hopes of plaque presence and the percent sand, silt, and clay will be determined.

Category: Emily Gaw | LEAVE A COMMENT
September 13

9/12/18 Spot Test/Plaque Assay/Continuation of Soil Metadata

Rationale: Isolate bacteriophage through both a Spot Test and Plaque Assay., and to collect more metadata soil sample B.

Procedures:

Before starting we had to create an aseptic zone to ensure that all bacteria were killed, and the working space would not contaminate our experiments.

  1. Cleaned off the workspace with CiDecon and applied the table with 70% ethanol solution.
  2. Wiped off the table after CiDecon was applied, same with the 70% ethanol solution, only, we let the ethanol solution evaporate.
  3. We then got an ethanol burner, and our aseptic zone was created.

Spot Test and Plaque Assay

Spot Test

  • The enriched lysate was made on 9/10/18.
  • Measured the mass of the enriched lysate. Mass of enriched lysate: 20.47 g.
  •  The enriched lysate was spun in the centrifuge for 5 minutes at 3000 x g.
  • The enriched lysate was filtered and put into a small cap.
  • Divided one plate into four quadrants
    • I – ML Spot Test
    • II – CJJ Spot Test
    • III – JLL Spot Test
    • Buffer
  • Added 2mL LB broth into a 50mL vial.
  • Added 22.5 microliters of M Calcium Chloride in the 50mL vial.
  • Added 0.5mL OF Artho PA Phage lysate
  • Added 2.5 mL TA into the 50mL vial.
  • Poured our solution onto the one plate that is divided into four quadrants.
  • Sat the petri dish aside to let TA solidify (1o minutes).
  • Added 0.5 microliters directed (filtered) lysate on top of the TA.
  • Added the buffer onto the last quadrant, our negative control.
  • After all the quadrants were filled with a spot, we placed the plate in the incubator.

Plaque Assay

  • Got four different petri dishes, one for each lab partner, and the fourth one being our control buffer.
  • In each dish we added:
    • Added 2mL LB broth into a 50mL vial.
    • Added 22.5 microliters of M Calcium Chloride in the 50mL vial.
    • Added 0.5mL OF Artho PA Phage lysate
    • Added 2.5 mL TA into the 50mL vial.\
      • Note this times 4 since we had to fill 4 petri dishes.
  • We then separated the solutions into 15mL vials and we added our 0.5 microliters of direct lysate into the 15mL vial.
  • Poured the Solution onto my petri dish labeled ML 9/12/18 Plaque Assay.
  • Let the solution sit for 15 min and placed in the incubator.
  • Cleaned off the workspace with CiDecon and applied the table with 70% ethanol solution.
  • Wiped off the table after CiDecon was applied, same with the 70% ethanol solution, only, we let the ethanol solution evaporate.

Soil Metadata

  • Percent water of the soil was calculated to be 11%.
    • Mass Dry= 10.8
    • Mass Wet= 12.09
    • 10.8/12.09=.89
    • 1-.89=.11=11%
      • Side note: Lots of debris which could have an effect on the % water.

  • The mass of the soil was recorded and left to sit under a vented hood in the lab (this was done 9/10/18). This allowed the procedure above to be done today in lab.
  • % Soil, Clay, and Silt was then calculated
  • 50mL vial was left under a vent hood from Monday to let the soil disperse.
  • The vial was observed and these are the results.
    • 1.5mL Sand
    • .5mL Clay
    • 2mL Silt

Observations:

  • The soil had a lot of debris in both the % water mass and the vial shown above which was used to measure % soil.
  • Results from the metadata:
    • 11% Water
    • 1.5mL Sand
    • .5mL Clay
    • 2mL Silt
    • 6.5pH

Conclusions/Next Steps:

The overall spot test and plaque assays procedures were easy since we knew what to expect. We were better prepared versus last time. Next steps: to see if our Bacteriophage has been isolated, and if so, we will try to amplify it.

Category: Michael Lum, Uncategorized | LEAVE A COMMENT
September 13

Soil B Enrichment and Metadata 9/10/2018

Rationale

Today’s goal is to wash Soil Sample B using a new technique to produce a direct and enriched isolation. We will also collect metadata on the soil sample, consisting of pH and percent water composition.

Procedure

  1. Use the aseptic technique to clean the area. With gloves on, spray CiDecon on the lab table and wipe dry. Then wipe 70% EtOH on the surface and let the excess evaporate.
  2. Place 2 mLs of the Soil Sample B into a 15 mL conical vial. Label the tube “EAG 9/10/18 Soil Sample B Isolation.”
  3. Add 10 mLs of LB broth to the vial and place the vial on the vortex on setting 10 for 10 minutes to mix.
  4. Mass the 15 mL conical vial and prepare to centrifuge by finding another vial with a similar mass.
  5. Place “EAG 9/10/18 Soil Sample B Isolation” into the centrifuge and spin for 5 minutes at 10,000 g.
  6. While the 15 mL conical vial is spinning, begin conducting percent water metadata on Soil Sample B. Weigh the mass of the weigh boat and then add some of Soil Sample B to the weigh boat and mass again.
  7. Label the weigh boat “EAG 9/10/18 %H2O Metadata” and place into the vacuum hood inside the lab. Let the weigh boat containing the sample sit for 48 hours.
  8. After the 15 mL conical vial labeled “EAG 9/10/18 Soil Sample B Isolation” has finished spinning, use a 2 mL syringe and a 0.22 μm filter attached to the end of the syringe to filter all of the supernatant available into another 50 mL conical tube labeled “EAG 9/10/18 Enriched Isolation.” (note: only 8.5 mLs of filtered enriched solution was produced, therefore no direct solution was made.)
  9. Add 0.5 mLs of Arthrobacter to the 50 mL conical vial and incubate the tube for 48 hours at 28 degrees Celsius.
  10. Next, measure the pH of Soil Sample B by placing a small amount of soil into a pH vial. Fill the rest of the vial with DI water. Shake vigorously for 10 seconds. Let the vial sit for 2 minutes.
  11. Place a 1 inch strip of pH paper into the vial for 45 seconds. Use the color of the pH paper to determine the pH of the soil sample.

Observations

  • The mass of the 15 mL conical vial before being centrifuged is 19.219 g.
  • The mass of the weigh boat is 2.39 g and the mass of the soil sample and weigh boat is 10.08 g.
  • The pH of the soil is 5.5.
  • The supernatant produced after centrifugation resembled a clear yet slightly yellow tint.

Conclusion and Next Steps

The sand, silt, and clay composition of Soil Sample B was not determined due to a lack of falcon tubes present. The composition metadata will be observed during the next lab. We will also conduct a spot test in hopes of producing plaques.

Category: Emily Gaw | LEAVE A COMMENT