Bioflux machine

Bio Flux machine

Starting up

1)	Turn on hardware first
•	Bioflux box
•	MCU 2008
•	Powersupply
•	Microscope (button on back left of scope)

2)	Then turn on computer
Bioflux has two programs:
1)	Bioflux: controls pumps 
2)	Bioflux montage: microscope, analysis and imagery
Bioflux
a)	Enter plate serial number (last four digits)
b)	Select Manual and enter dines required for each
•	Columns 1 -4 operated by one pump and 5 – 8 by another so check each. 

Priming the plate 
i.e. wetting the pipes etc so they work properly. 
If you want to coat wells, then do so at this step.
•	Put media in the outlet wells -> going to be pumped into left well (see section below for how to put plate in bioflux)
•	Media will run from light green to dark green so click on ‘outlet’ to choose these as light green
•	Run at 1 dine for a couple of minutes. 

Putting plate into holder

•	Move little grey clip on right back of holder and put plate in
•	Put lid on with silver things on left of groove -> slide to right and then tighten the red clips. 
•	If red clips are tight enough then the pressure won’t be right and liquid won’t run properly.

Temperature
Turn on heating unit and control using little display box under computer screen
If using 37°C then heat media to 37 BEFORE placing in wells (or will make bubbles as it heats = problems with microfluidics) 

Seeding the plate

Inlet: Media
Outlet: Cells to seed
•	Place roughly same volume in inlet and outlet (or vol difference will result in irregular flow)
•	Choose 2 dines for 5 secs
•	Click lanes you want to run and then run
Check with m/scope that cells are in channel (but not past channel as you don’t want them going into your inlet and therefore contaminating media)

Microscope

Has bright field at top and fluorescent at the bottom
Bright field is sufficient for viewing development of biofilm -> fluorescent scope used if have dyes eg gfp (doesn’t pick up bioluminescence) but can be used for live/dead stain
Use Joystick to move light source to channel of interest
On little display use eye/camera icon to switch between eyepiece view and computer screen view. 
Use 10x to view biofilm – more zoomed in wont capture what’s happening enough 

Settings

Main – check first 3 boxes
Saving – choose folder for data to be put in -> put in your own directory 
Timing – Eg every 20 mins for 18 hours
Stage – here you give coordinates of each channel to the microscope so it can find them and take a picture at the relevant time. Each coordinate will be x, y and z with z being focus so you can change the focus between channels if necessary.
Once here, use joystick (or mouse?) to scroll til you find channel 1 (there will be a no 1 above it so you know). When you are focused and in correct position, click in ‘Enter Position’ tab and write 1. Then scroll to find channel 2 and continue. 
Wavelength – try 4 to start. 
Brightfield – 10millisec
FITC/Red etc 500 millisec
All closed -> on this tab choose ‘all closed’
Click ‘Aquire’ and wait -> don’t use computer during this time as could crash. 

Analysis of Data

NM Standards -> Apps -> Multidimensional data

Click little box on left of name of your data and will show all your frames
Either check the ones you want or to check them all right click over them so select all
Click load images = create a video

Getting data on biofilm size (for graphs etc)

1)	Telling computer what is background noise and what is not
While video is on screen, click on second from bottom icon (a little graph image) and click auto threshold for dark objects ¬-> hopefully this will be good but if not then manually change. 

2)	Getting actual data:
Click on NM Standards -> measure -> region measurements
Should bring up a graph that is equivalent to a growth curve with time on x axis and threshold on y
Can use this graph directly or export data to excel.

Done!