Christian Niederauer/Notebook/RacingBacteria/Calendar

August 2014

06.08.2014 Revive Bacteria

• revive bacteria strain RP437 from glycerol Calendar (see Glycerol Protocol)
• Master Plate is incubated ON, then stored in fridge
• Bacteria have to be checked for motility

08.08.2014 PDMS Preparations

PDMS Duplicate Protocol
Plasma Cleaning Protocol
Problems encountered with creating PDMS stamps:

• oven is not entirely flat
• the "coring tool" is a cut glass pipette which continues to break and creates jagged holes
• device is prone to leakage if the holes are placed near the edge of the stamp
• devices are very thin and making it bigger by sticking additional pdms layer is tedious

-> build leveling device, order coring tool, try cutting stamp so that the device is not near an edge, make deep araldite molds (thick stamp is hard to achieve with silicon wafer, as ideally the surface tension of the PDMS should keep the fluid on the wafer. If you add to much fluid, it will break surface tension and spread over the whole petri dish. Silicon wafer is too delicate to experiment with some ring-shaped devices to keep PDMS on the wafer.)

11.08.2014 Checking for Motility

• make suspension of one big and one small colony from the Master Plate created on 06.08. in eppi with 1mL PBS by picking it up with a micropipette tip and suspend tip into eppi
• cut window in double sided tape and put onto slide
• add a drop of bacteria suspension from the eppi(3µL) and put coverslide onto slide

Result: bacteria from the big colony are motile, whereas bacteria from the small colony were not
-> reinoculate bacteria from one or more big colonies of the master plate into liquid LB medium over night

As a comparison: 1µm latex beads on same coverslip-slide arrangement in 1:1000 dilution
Analysis of Bead Movement

Data: E:\12-08-rp437\ and E:\12-08-beads\

12.08.2014 Reinoculate motile colony

• put 5ml LB broth into falcon tube
• pick up one big colony with pipette tip and discard the tip in falcon tube
• incubate ON
• dip sterile inoculate ring into liquid colony and streak on agar plate the next day
• incubate plate over night => this is the new master plate

13.08.2014 Tracking Beads

As a check, track beads based on microscopy images acquired with Epitome, using BergLab tracking code. Brownian Motion is observed and after binning the frame-to-frame displacements, a rayleigh distribution is fit to the data with the curve fitting tool.

• 

  tracked positions

Matlab code for binning:
botEdge=0; %first bin should start at 0 displacement
topEdge=25; %last bin should reach to 25
numBins=20; %number of bins
edges=linspace(botEdge,topEdge,numBins+1);
binned=histc(disp,edges);
figure, plot(edges,binned,'.'); %plots the number of displacements which lie in each bin

• 

  distance in µm

25.08.2014 Araldite Replica from PDMS & COMSOL

COMSOL Multiphysics
Downloading COMSOL Multiphysics 4.3b 64-bit at the fedora linux machine in the dry lab.

Araldite Replica
According to the Araldite Replica Protocol, 9g (5g of part A-resin and 4g of part B-hardener) are prepared, degassed and should have been poured after ~30 minutes. The break during the introduction course about the new microscope was after 1 hour. By then, the epoxy already was beginning to solidify. In addition to that, it seemed that the amount of epoxy mixed should be increased for easier pouring and handling (solidifies more slowly, more tolerance in terms of weight ratio).
Manufacturing of replicas is postponed to 26.08.2014.

Microscopy Course Notes

• make new optical configuration: Configuration-> New Optical Configuration
• basic setting windows: View -> Acquisition Controls -> Clara Settings -> TI PAD, Sensor
• for DIC: ANALY & Condensor DIC N1 (20x,40x)
• always: [D] in, [NCB (neutral color balance)] in
• enable mouse focusing: Device -> Enable Mouse Joystick
• to save current window configurations: Save Current Layout as..
• Fluorescence: turn on NTNSL & filter 1 (blank with paper because shutter is not installed yet)
• high frame rate: no delay, exposure 1 frame, small ROI, Binning

26.08.2014 ACTUAL Araldite Replica from PDMS & COMSOL

Araldite Replica
Using Araldite Protocol: Mixing 10g part A and 8g part B of Araldite and pouring onto the H-Pattern PDMS stamp from 20.08.
The replica will be called Araldite Replica I.
Another Araldite replica is made with a pre-cut PDMS stamp made on 22.08..
An extra layer of PDMS is added below the stamp to achieve a deeper mold in the end. It is called Araldite Replica II.

Araldite Replica II

Araldite Replica II

Comsol Multiphysics
Always change into usr folder with cd ../

Mount .iso with
sudo mount -r -t iso9660 -o loop COMSOL43b_dvd.iso /media
cd media/
sudo sh setup

Start License Server manually:
cd local/comsol43b/license/glnxa64/
./lmgrd -c ../license.dat -l ../comsol44.log

Run Comsol in local/comsol43b/bin/glnxa64/ -> comsol

Gives error:

• 

27.08.2014 PDMS from Araldite & Liquid Colony

• with PDMS-Protocol: create PDMS from Araldite Replica II and compare with original entire-pattern stamp from 20.08..
• Liquid Colony is created under hood with some left over sterile LB medium by discarding pipette tip into that tube after picking up a few colonys from the Master Plate.
• coring tools are too big (orange: 2.5mm, green: 3mm), have to wait for the right ones

Plan: punch holes into the PDMS from Araldite II, plasma clean with coverslip and attach tubings, then insert safranin. If it works properly, insert bacteria suspension

Stock

Bacteria

Master Plate

Created on 12/08/2014 from liquid culture, which again was created from one motile colony of the original master plate.
The original master plate again was created from glycerol stock of RP437 E. Coli on 06.08..

Liquid Colony

• Created on 19/08/2014 [disappeared]
  

Suspended a few colonies from Master Plate into 1ml of LB media.

• Created on 27/08/2014
  

Suspended a few colonies from Master Plate into ~5ml of LB media.

RP437 Strain

Datasheet of the RP437 Strain

After reviving this strain, test on soft agar plates and pick a colony that is motile and chemotactic because nonmotile variants arise in room temperature stab-cultures.

Microfluidics Devices

Leveling Petri Dish

Problem: Oven is not even, the PDMS stamps therefore will be to thin at one side or the fluid even leaks over the waver and the stamp will be to thin everywhere.
Solution: Filling up a Petri Dish with ~20ml PDMS and curing it in the oven (lower rack, placing dish at the very back on the right wall with the marker pointing perpendicular to the backwall). The fluid's plane will be arranged perpendicular to the gravitational force eventually. Future petri dishes with PDMS then can be placed on top of this device.

H-Pattern PDMS stamps

close up of 20.08. stamp

problem: jagged holes, glass residues in stamp

• 20.08. (entire pattern)
• 22.08. (pre-cut)
• 27.08. (Araldite Replica II)

Original Silicon Wafer with H-Pattern

• 

Original Pattern etched on the Silicon Wafer
Track width: 2µm
Track length: 362µm
Number of tracks: 32

Araldite Molds

Araldite Replica II in the making

Araldite Replica II

• Araldite Replica I, created on 26.08. from H-Pattern PDMS stamp (20.08.)
• Araldite Replica II, created on 26.08. from H-Pattern PDMS stamp (20.08.) with increased thickness

Material

• Coring Tool 2mm
• PDMS Chemicals (Sylgard, Silicone Elasteromer KIT 184)
• Araldite Chemicals (Resinova Bondtite)

Protocols

Bacteria

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

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 ...

• 

  source

Inoculating Bacteria into Liquid Colony

• take micropipette with sterile tips and pick up a few colonies from your plate
• discard the tip into liquid LB medium
• incubate ON

Reviving Bacteria from Glycerol Calendar

• 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 Calendar 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)

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

Additional Procedures

If the stamp is too thin
(DO 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

• 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?
PDMS has a hydrophobic surface because of a -CH_3 group present in its monomers. When oxidized with O_2 it reacts to Silanol (-SiOH) group which is hydrophilic. This group bonds with other hydrophilic groups (e.g. coverslip).

Paramters in Detail for Plasma Cleaning:

• Air inlet valve: 0.5kg/cm^2
• Power: 70W
• Ashing time: 20-30s
• Bleed delay time: 10s
• Gas 1 (left most valve): Set value to 1mbar
• Vent valve: restricted
• Restricted vent time: 120s
• Pump spin down time: 10s
• Vent hold time: 1s
• Gas shut time: 5s
• Turbo pumping No

Reference: Chattopadhyay et al., 2005

How to create a Araldite Mold of PDMS Stamp for PDMS Stamp

In order to minimize the use of the (expensive and delicate) silicon wafer, one can create a epoxy (Araldite) mold, which can be used in the same way as the silicon wafer, by the following steps:

• in discardable container, mix Bondtite Araldite by taking weight ratio of 10:8 for resin(typ A) : hardener(typ B)
• mix thoroughly with (outside) pipette tip
• degass for at least 30mins
• pour onto PDMS mold inside petri dish wrapped in alufoil
• cure 2 days at RT or 3 hours at 50-60°C
• let cool afterwards and recollect the stamp (it is still usable)