User:Christian Niederauer/Notebook/RacingBacteriaMicrofluidic

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Contents

Description

Summer project at IISER Pune (India) about the biophysics of chemotaxis.
The aim is to build a microfluidic racing track and to quantify the speed of E.coli swimming along a nutritional gradient in the device.
The project is funded by the DAAD.

To Do

Microfluidics

  • make araldite duplicate from silicon wafer
  • create functioning H-Pattern device

Bacteria

Computation

  • install Comsol
  • simulate gradient
  • play around with bacteria recognition & tracking code

August 2014

September 2014

October 2014

Lab Meetings


Stock

Protocols

Bacteria

Streaking Bacteria onto Agar Plate

  • Petri dishes filled with 20ml of molten (80°C) 1.5% Agar, cool for 15 minutes
  • with inoculator loop: dip into liquid culture of revived bacteria and spread one line
  • after initial line, burn inoculator ring & let it cool
  • dip ring into initial line and spread bacteria diagonally
  • burn ring again
  • repeat ...

Reviving Bacteria from Glycerol Stock

  • Scrape the frozen surface of the culture with a sterile inoculating needle, and then immediately streak the bacteria that adhere to the needle onto the surface of an LB agar plate containing the appropriate antibiotics. Incubate the plate overnight at 37°C
  • Scrape the frozen surface of the culture with a sterile inoculating needle or tip, and immediately immerse it in 2ml growth media within a 2059 snap-cap tube. Grow the bacteria overnight in a 37°C shaker.
  • Return the frozen culture to storage at -70°C

Source: doi:10.1101/pdb.prot4452

Preparation of Glycerol Stock for Long Term Storage of Bacteria

  • sterilize glycerol by autoclaving
  • to 1.5 ml of bacterial culture, add 0.5 ml of the sterile glycerol
  • vortex the culture to ensure that the glycerol is evenly dispersed
  • transfer the culture to a labeled storage tube
  • freeze the culture directly to -70°C for long-term storage (up to 6 months)

Important

always work under hub (switch on Fan and Light), afterwards close hub and switch on UV
Stuff contaminated with bacteria should be given to autoclaving room

Microfluidics

How to Create a PDMS-Stamp from Silicone/Araldite Mold

Why PDMS? -> PDMS is optically clear (good for microscopy!), and, in general, inert, non-toxic, and non-flammable.

Preparation of PDMS mixture

  • with pipette boy and 5ml stripette pour ~4g of elastomer into a (clean) cup
  • with micropipette (1000µl) add one tenth of elastomer mass (~0.4g) of linker fluid to the cup
  • mix well with stripette
  • degass for 30min-60min

Preparation of PDMS membrane

  • pour degassed mixture onto wafer placed in petri dish with aluminium foil
  • pour just enough to cover the waver
  • use Leveling Petri Dish to ensure constant thickness
  • araldite wafer: cure it for 3-4 hours in oven at 60°C
  • silicon wafer: cure it for 2 hours in oven at 80°C for
  • peel off membrane from wafer surface with forceps
  • cut out the pattern but leave enough space around it

Final Processing

  • punch holes with 2mm coring tool at the desired connection points under dissection microscope (tip: target the hole with coring tool under microscope, then put the stamp onto one finger with alufoil between stamp and finger and push/turn until you feel the coring tool on your skin)
  • plasma clean the treated face of the stamp and a coverslip and press them together with alufoil in order to prevent contamination


If the stamp is too thin

BEFORE PUNCHING HOLES

  • take a slice of PDMS with bigger or same area than the stamp
  • plasma clean unshaped face of stamp and extra slice and stick them together
  • now punch desired holes with 2mm coring tool

How to clean the PDMS cup

  • scratch out all solid PDMS residues with forceps
  • use tissue to clean out crudely
  • pour pentane into the cup and gently sway the cup
  • clean out with water and let dry

How to clean dirty stamp

  • let the stamp soak in ethanol
  • use forceps and (with caution) submerge the stamp into a jet of water
  • let dry on clean alufoil


Plasma Cleaning Protocol

  • turn on the dry air outside the building to 50 kg*m^-1*s^-2 (first open gas bottle valve by turning ccw, then turn black knob cw)
  • turn on the dry air inside the building above the plasma cleaner to 30 (unit?) by turning cw
  • turn on plasma cleaner, close all vents (vent ctrl, gas1 & gas2 by turning cw)
  • settings: 70,30,10,1,restr,60,10,1,5,0
  • add whatever you want to clean
  • push start
  • after "bleeding champer with gas" open gas 1 quickly to ~ 10^0 mbar by turning it ccw
  • after "vent hold" open vent ctrl by turning it ccw
  • after finishing process, close gas1 and vent ctrl again
  • after end of lab work: close inside and outside gas valves


Why Plasma Cleaning?

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