User:Pakpoom Subsoontorn/Notebook/Shape Determination in Bacteria/2008/10/17: Difference between revisions

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==10/25/08==
==10/25/08==
*Start reading "Cell shape dynamics in E.coli" by Reshes G, et al 2007
*Start reading "Cell shape dynamics in E.coli" by Reshes G, et al 2007
==11/03/08==
*Skim through Rob's review paper of cell membrane and protein biophysic
==11/05/08==
==11/05/08==
*Goal:
*Goal:

Revision as of 02:20, 6 November 2008

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Lab note: 10/17/08-10/23/08

  • In summary,

10/17/08

  • Make an agar slide of E.coli (K-12 wild type) cell that has been cultured for about 24 hours at 37 C in 5 ml plain LB.
  • The slide is made from glass slide, cover slid, newly-made LB agar (20 ml LB+0.3gram of agar, heated up on hot plate with stirrer). About 1.5 ul of cultured E.coli is dropped on the agar patch directly without dilution. After the cultured solution is dried, put cover slid on and sealed with... (see image)
  • Take phase-contrast image at 4 different positions, looping every 10 seconds for 360 cycles. Exposure time is

10/21/08

  • Meet with Tony: neofilm sandwich and laser cutter for cell-flow template
  • Start Cultured E.coli K-12 in 5 ml LB 37 C at 10 am. Streak a new plate (plain LB) of E.coli K-12 from Steph's stock, grow at 37 C
  • Install AutoCAD 2008 in macbook

20/22/08

  • Start Cultured E. K-12 in 5 ml LB 37 C at 8.30 am from 10/21/08 stock. Note: no gas for burner!

20/24/08

  • dilute FM464 dye 1:200 from stock (1 mg/ml)
  • start drop cell 9 am : 1 ul of over night culture + 2 ul of dye
  • Take phase-contrast image at 4 different positions, looping every 10 seconds for 200 cycles. Exposure time is
  • emission filter, Nikon 59004 (C93105), 49002 (C91306), 49004 (C91530)
  • finish the first draft of the simple microfluidic design and gave it to KC

SimpleMask1

  • Skim through the first three chapters of AutoCAD bible

10/25/08

  • Start reading "Cell shape dynamics in E.coli" by Reshes G, et al 2007

11/03/08

  • Skim through Rob's review paper of cell membrane and protein biophysic

11/05/08

  • Goal:
    • We made a forzen stock of E.coli 4213 and a stock of E.coli+PZS21-GFP-Kan in 20% glycerol
    • We tried to estimate shape recovering time of E.coli-K-12 from the round shape in Stationery state to normal rod shape of the exponential phase by taking sample once in a while and image in microscope. The cells were also stained with dye XXX
  • Detail experiment:
    • PREP: Start growing E.coli from plate to 5 ml LB around 2 pm of 11/05/08. Note that E.coli+PZS21-GFP-Kan culture had 1:1000 Kan from stock (conc XXX). K-12 and 4213 were in plain LB. All were grown at 37 C in shaker.
    • STRAIN TEST:
      • Vancomysin was dissolved 10mg/1ml, dilute 10ul/100ul ..to 1mg/ul. Then, we add 20ul to 2 ml of diluted cultures (20ul overnight culture/2ml LB) of the three stains. They were grown in the shaker at 37 since 2.30 pm...this one is for testing 4213 strain
    • SHAPE RECOVERY:
      • sample of overnight K-12 were diluted 20ul/2ml LB and grow on a shaker at 37 C
      • Take sample and at put on agar patch at 2.30pm, 2.55pm, 3.45pm and 4.15pm. The first sample was taken directly from the overnight cultures and too concentrated for imagine. The fluorescence staining is weird: there are lots of bright lumps on the agar patch outside cell ares.
      • Between 3.45 and 4.15 pm I forget to put the culture back to 37 C



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