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: