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

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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
      • I manually took 9-14 snapshots at each time points.. with phase-contrast 100X, exposure time 100ms
    • STOCK PREP
      • add 800 ul of each overnight sample to 200 ul of glycerol, mix, throw into liquid nitrogen kept item there for about 2 hours then put -80 freezer
  • Result
    • I clearly see shape transition between 2.55 pm to 3.45 pm sample..cells became more rod. Note that some small fraction of cells remain round and small. Not much different between 3.45 and 4.15
    • Images files are saved at my whatislife account folder 110508EcoliShapeReovery..file tf1, tf2 and tf3 for the three time points sample
  • Things to learn and be concerned next time:
    • sample preparation
      • murky and dry agar undermines image quality
      • As we took sample out every 20-30 min, the temperature of the cultured cells were not remained at 37 C all the time.
      • what should be "the time point"?: take a tube from incubating room (2min)/ pipet sample on agar patch and wait it to dry(5min), put in microscope and find the focus (3 mins), looking around to get good image field (~ 20-30 sec each).
    • Microscopy
      • How to automatically tune pixel-range display
      • What can explain unknown fluorescence grain we see today?
      • Light intensity from light lamp may be changed manually (even when the shutter time is the same), how can we standardized imaging protocol? How about fluorescence standardization?
      • We need at least strict protocol for scanning image field view
      • In the end, we do need autofocus
      • Any program for scanning and imaging various position automatically?
      • I should start making a whole list of dye and their profile.. absorption/emission, filter,etc****this also mean standardized tools, strain and other inventory...also ask pp at Theriot lab about microscope filter
      • Ask about turning off microscope...I saw a spark today! something might went wrong
    • logistic issue
      • how many time we should repeat, standardized extc


11/13/08

  • At Theriot Lab, inoculate E.coli K-12 and E.coli 4312 from the plates in the fridge to two tubes for 5 ml LB. Grow in shaker at 37 C from 4 pm

11/14/08

  • Spin down cells from the day before..maximum speed 14k for 2 and 1 mins, wash with PBS twice, resuspend in 1 ml PBS...spin down at 5000g and resuspend at about 100 ul
  • (need detailed protocol from KC), EM of the two E.coli strain from the day before..Hewlet bldg sub-basement..nano fabrication lab


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