Date: 8/29/2018

Plaque Assay Soil A

Objectives:

• perform the spot test

Materials Required: Filtered Enriched lysate, serological pipettes, micropipettes, micropipette tubes, LB broth, Top Agar 2X, 50 ml                                               conical vials, 1M CaCl2 stock solution

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. Retrieved the LB broth, a 50 ml conical tube and a serological pipette
4. While in the aseptic zone, transfer 4.5 ml of LB broth to the 50 ml conical vial.
5. now retrive the 1 M CaCl2 stock solution.
6.  Using the micropipette, i transfered 45 microliters of the CaCl2 to the 50 ml conical tube with the LB broth.
7. retrieved 0.5 ml of arthrobacter from Lathan ( Teaching Assistant)
8. add the arthrobacter to the 50 ml conical tube.
9. add 5 ml of top agar to the 50 ml conical tube.
10. pour the contents of the 50 ml conical tube onto the agar plate.
11. to let the top agar solidify, i waited for 10 minutes.
12. collect the enriched sample tube, a syringe and a filter of 22 microns.
13. take a sample from the enriched tube using the syringe.
14. attach filter to the syringe and pour the lysate out slowly into a microcentrifuge tube
15. collect the direct isolation sample from the fridge.
16. collect a phage buffer from the instructor
17. mark the agar plate with three spots , one for the enriched, one for the direct and one for the phage buffer.
18. spot 10 microliters each of the phage buffer, enriched sample and the direct isolation sample.
19. let the sample rest for 10 minutes
20. 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:

read the instructions carefully and work faster so as to finish on time and prevent mistakes.