Objectives:

• To analyze the plaque assays from 10/01/18
• To make a plaque assay with 50 μL of phage extract from 09/26/18
• To acquire a webbed plate for flooding

Pre Lab Observations:

The control plate for the plaque assays from 10/01/18 was not contaminated. There was one possible plaque on the plaque assay for the third passage of purification. To confirm the strength or lack there of due to the presence or absence of phages in the sample, a plaque assay will be made with 50 μl of phage extract from 09/26/18.

Procedure:

1. Cidecon was poured on the desk and wiped till the desk was dry. Then, 70% ethanol was poured and wiped until it was all over the table and then it was allowed to evaporate. After the ethanol had evaporated, an ethanol lamp was lit, setting up the aseptic zone.
2. 0.5 ml arthrobacter was retrieved from the lab instructor
3. Using the micropipette, 50 microliters of the 10^0 bacteriophage mixture was transferred to the arthrobacter vial.
4. The vial was then allowed to rest on the test tube rack for 15 minutes
5. While the vial was resting, one Top Agar mixture was made for the group.
6. The LB broth was retrieved from its storage bath, along with a 50 ml conical tube and a serological pipette
7. While in the aseptic zone, 8 ml of LB broth was transferred to the 50 ml conical vial.
8. Then, 1 M CaCl2 stock solution was retrieved from the lab instructor.
9.  Using the micropipette, 90 microliters of the CaCl2  was transferred to the 50 ml conical tube with the LB broth.
10. The vial was then set on the rack.
11. 10 ml of the 2X TA was added to the LB broth and Cacl2 after the sample was allowed to enrich the arthrobacter for 15 minutes.
12. Using another serological pipette, 4.5 ml of the top agar mixture was transferred to the test tube with the arthrobacter and the phage extract.
13. The contents of the test tube were then poured onto the agar plate.
14.  Part of the top agar mixture was poured into the top agar control plate for the group.
15. To let the top agar solidify, the plates were allowed to rest for 20 minutes.
16. The plates were placed upside down in the incubator, where they will remain for 48 hours

Analysis and Conclusion:

The top agar did not properly solidify and therefore the plates where not inverted to prevent movement of top agar. It seems that the through cleaning and bleaching of lab equipment may have resulted in positive results, implied from the lack of contamination of the control plate from 10/01/18. the procedure was properly performed in the aseptic zone. there were no apparent sources of contamination.

Objective: 

• Acquire a concentrated sample of bacteriophages
• Pick a plaque from the plaque assays formed after serial dilutions
• Make a plaque assay from the picked plaque

Pre-Lab Observations:

• Plaques were formed on all the plates ( 10^0, 10^-1, and 10^-2 were the dilutions)
• The control plate was contaminated.

Procedure:

1. Cidecon was poured on the desk and wiped till the desk was dry. Then, 70% ethanol was poured and wiped until it was all over the table and then it was allowed to evaporate. After the ethanol had evaporated, an ethanol lamp was lit, setting up the aseptic zone.
2. Phage buffer was acquired from the lab instructor and microcentrifuge tips.
3. Using the micropipette, 100μl of phage buffer was transferred to a microcentrifuge tube.
4. In the aseptic zone, at a 90° angle, a plaque on the 10^-1 plaque assay was stabbed using a micropipette tip ( attached to the micropipette) and the tip was then put into the microcentrifuge tube with 100μl of phage buffer and stirred to properly remove bacteriophages on the tip.
5. This microcentrifuge tube was then vortexed for 30 seconds and was labelled 10^0.
6. One Top Agar mixture was made for the group.
7. The LB broth was retrieved from its storage bath, along with a 50 ml conical tube and a serological pipette
8. While in the aseptic zone, 8 ml of LB broth was transferred to the 50 ml conical vial.
9. Then, 1 M CaCl2 stock solution was retrieved from the lab instructor.
10.  Using the micropipette, 90 microliters of the CaCl2  was transferred to the 50 ml conical tube with the LB broth.
11. The vial was then set on the rack.
12. 0.5 ml of arthrobacter was retrieved from the lab instructor
13. Using the micropipette, 10 microliters of the 10^0 bacteriophage mixture was transferred to the arthrobacter vial.
14. The vial was then allowed to rest on the test tube rack for 15 minutes
15. After the 10 minutes had ended, 25 ml of the 2X TA was added to the LB broth and Cacl2.
16. Using another serological pipette, 4.5 ml of the top agar mixture was transferred to the test tubes with the arthrobacter and the lysate.
17. The contents of the test tube were then poured onto the agar plate.
18.  Part of the top agar mixture was poured into the top agar control plate for the group.
19. To let the top agar solidify, the plates were allowed to rest for 12 minutes.
20. The plates were placed upside down in the incubator, where they will remain for 48 hours

Analysis and Interpretations:

The repeated contamination of the plate is perplexing. there may be another factor causing the contamination of the plates is that the agar plate could be contaminated plates before top agar was poured onto the plates. the purification process will not be repeated a few more times to acquire more concentrated and similar phages.

Future Notes 

Check plates properly before using them and test the broth and 2x agar for potential contamination.

09/17/2018

Plaque Assay and Soil Collection

Pre- lab Observations:

The spot test made for sample soil B on 09/12/18 was retrieved from the incubator and analyzed. The plate had no plaques. The Top Agar control plate did have peculiar features that seemed to indicate contamination, which was not reflected in the spot test plates. According to the Teaching Assistant, they seemed to be arthrobacter, which may have entered the plate due to contact of the bacteria with an instrument used during the procedure.

Objectives:

• For verification, make a plaque assay from the filtered enriched lysate to test the presence of arthrobacter
• Collect soil sample C

Procedure:

1. Cidecon was poured on the desk and wiped till the desk was dry. Then, 70% ethanol was poured and wiped until it was all over the table and then it was allowed to evaporate. After the ethanol had evaporated, an ethanol lamp was lit, setting up the aseptic zone.
2. The group decided to make 1 top agar for the group.
3. The LB broth was retrieved from its storage bath, along with a 50 ml conical tube and a serological pipette
4. While in the aseptic zone, 8 ml of LB broth was transferred to the 50 ml conical vial.
5. Then, 1 M CaCl2 stock solution was retrieved from the lab instructor.
6.  Using the micropipette, 90 microliters of the CaCl2  was transferred to the 50 ml conical tube with the LB broth.
7. The vial was then set on the rack.
8. 0.5 ml of arthrobacter was retrieved from the lab instructor ( 0.5 ml for each group member)
9. Using the micropipette, 10 microliters of filtered enriched lysate was transferred to the arthrobacter vial.
10. The vial was then allowed to rest on the test tube rack for 10 minutes
11. After the 10 minutes had ended, 10 ml of 2X TA was added to the conical vial.
12. Using another serological pipette, 4.5 ml of the top agar mixture was transferred to the test tube with the arthrobacter and the lysate.
13. The contents of the tube were then poured onto the agar.plate
14. a part of the top agar mixture was into the top agar control plate.
15. To let the top agar solidify, the plate was allowed to rest for 10 minutes.
16. The plates were placed up side down in the incubator, where they will remain for 48 hours.

Soil Collection

1. Due to the low probability of finding plaques, a new soil was acquired in case a new lysate was to be extracted for testing for phages again
2. The lab group went into the field ( went outside ) to find a live oak tree.
3. The group picked a live oak and metadata was collected as the survey required.
4. First, numerous pictures of the tree were taken.
5. The diameter of the tree trunk was measured 137 cm from the ground.
6. Average diameter of the canopy was measured by taking the average of the longest and shortest diameter of the tree canopy.
7. Using the known height of an individual, the length of the individuals shadow and trigonometry, the angle of elevation of the sun was calculated, which was later used with the length of the shadow of the tree and trigonometry to find the height of the tree.
8. A soil sample was collected a certain distance from the tree trunk, one sample per group member, and the sample was put into a Ziploc bag. A leaf sample was also collected.
9. Store the sample in the fridge.

Conclusion and Interpretation:

The analysis of the control plates indicates that there was contamination. It indicates that more caution is required to maintain the aseptic zone and the methods used at the time of this procedure require more care and attention. There is also a chance that the LB broth or Top Agar. Currently it is not clear which is more probable.

Other Notes:

1. Group 4 had plaques on their Plaque Assays. They group also did  spot tests in addition to plaque assays, but only one group member (Justin) had plaques on the spot test whereas the entire group had plaques on their plaque assays. The same group member also had the most well defined plaque on the plaque assay. One possible reason could be that the soil sample collected by Justin was more concentrated with bacteriophages than the samples of the rest of the group, yielding him the best results.
2. Calculations for Lathan’s (lab instructor) webbing plate

no. of plaques= 14

dilution of sample= 10^-3

titer of sample = (14/10 μl)x(1000ul/1μl)(10^3)= 1.4×10^6 pfu/ml

Diameter of plate= 8 cm

Diameter of the plaque= 1mm

Area plate=πr^2=6400π

Area plaque=πr^2=π

area plate/ area plaque=6400π/π=6400

volume of lysate required= 6400/1.4×10^6=4.57×10^-3

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:

09/10/2018

Washing And Enrichment of Soil B

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?

Objective:

• Prepare direct and enriched lysate for soil sample B
• Collect metadata for soil sample B ( %water, pH, and sand silt clay percentages)

Materials Required:

50 ml conical vials, 15 ml conical vials, pH vials, pH paper, serological pipettes, Cidecon, 70% ethanol, LB broth, Soil Sample B, Falcon tubes, DI water, soil dispersion liquid, scooper, syringe, syringe tip filter (22 microns), shaking incubator, weighing boat

Procedure

1.  set up an aseptic zone by cleaning your desk with Cidecon (wipe till dry) and ethanol ( 70%)( wipe on the desk and let it evaporate) after clearing the table.
2.  lit the ethanol lamp to set up an air current to help keep other microbes from getting into tube when it is open.

Washing and Enrichment:

1. retrieved Soil Sample B from the refrigerator.
2. retrieved the 15 ml conical vial with 2 ml of soil in it.
3. retrieved the LB broth
4. using a serological pipette, transferred LB broth to the 15 ml conical vial until it reached the 11 ml mark.
5. closed the 15  ml conical vial and shook it by hand for 12 minutes
6. after  12 minutes, find the mass of the vial ( 19.31g), and find another lab partner with a tube of mass within 0.05g of your tube mass.
7. take the vials to the centrifuge and put them in for 10 minutes at an acceleration  10000 times g.
8. take the vial back to the aseptic zone.
9. using a sterile syringe and a syringe tip filter (22 microns), transfer 10 ml of filtered lysate to a 50 ml conical vial. ( take care to use the aseptic zone to avoid contamination of lysate.
10. after you acquire 10 ml of filtered lysate, add 0.5 ml of arthrobacter to the vial. this is the enriched sample.
11. place this tube, loosening the cap a little bit , into the shaking incubator for 48 hours
12. filter the rest of the lysate into another 15 ml conical vial ( 1/2 ml for my sample). this will be your direct isolation sample
13. store direct isolate in the fridge

% Water

1.  weigh the weighing boat on the scale ( g)
2. pour some soil onto the boat and weigh the weight of the boat plus the wet soil
3. take the difference of the final weight ( boat and soil ) and initial weight (boat) to find the weight of the soil.
4. put the soil outside for 48 hours to allow the water to evaporate.
5. weigh the soil again.
6. take the difference of the weight of the wet soil and dry soil to find weight of the water in the soil ( the water that evaporated)
7.  calculate the percent of water in the soil by using     percent water= (weight of water/ weight of wet soil) x 100

Sand, Silt, Clay

1. take 50 ml conical vial and fill it with soil sample to the 10 ml mark
2. add DI water to the vial until it reaches the 30 ml mark.
3. add soil dispersion liquid to the vial.
4. shake vial vigorously for 30 seconds
5. let the vial rest for 48 hours.

pH

1. add a small amount of sand to the pH vial
2. add water so that the level reaches the top of the vial
3. shake vial for 10 seconds and then let is rest for 120 seconds
4.  after 120 seconds, dip 1 inch of the pH paper in the vial for 45 seconds
5. immediately compare the color of the strip to the scale to acquire pH

Analysis and Data:

there is no data that can yet be calculated. that will be done in pre lab calculation for the lab on 09/12/2018.

the following was collected for today

Mass of wet soil = 6.370 g

pH of soil= 6

the aseptic zone was properly maintained and there were no apparent events that may have caused contamination.

Future:

calculate % water, %sand, %silt, and % clay.