Basic Protocol

This is the best practice default way of performing a Scan-o-matic experiment.

The document is currently being written and may undergo major revisions

(0) Preparing Scan-o-matic for the first time

First thing you should do before starting your first experiment in a scanner is to setup a fixture. Once it is setup, it can be used over and over indefinitely so name the fixture wisely, e.g. Scanner 1, and write that name on the fixture too to avoid mix-ups in the future.

(1) Experimental design

Normalisation strain

(2) Preparation of media

Sterile filter

Typically 10x - 40x stock solutions of the defined media components are made so that they can be mixed into a 2x concentration stock mix to which a 2x concentration stressor/specific environment solution can be added.

20x - 40x Glucose

10x CSM

1. In milliPour H2O, add 10x the CSM nutrient powder and a clean magnet.
2. Put on stirrer until dissolved. Content may be heated to 40 °C to ease dissolving.
3. Working sterile, suction filter the mixture into an autoclaved bottle using X μM filter.

Casting of plates

If you have a plate pourer robot it is highly recommended that you use it.

After cast, let dry in room temperature until the surface is no longer visibly wet. Plates can be used after that, but can also be stored in fridge for a couple of days.

How well they store will of course depend on the exact composition of the plate in question so you must take stability of your compounds into account.

Plate pouring robot

Manual pouring

WORK STERILE BY FLAME (you may also need it for step 5 below)

1. Mix
2. Cool to __ C
3. Measure 50mL in Falcon tube
4. Pour contents slowly from tube.
5. If bubbles appear and media isn't heat sensitive, use Bunsen burner flame on area (a quick flaming) to remove bubble. If stressor or content in media is heat sensitive, discard plates that have more than a very few bubble directly at the edge of the plate.

(3) Pinning

• Ensure that the plates don't have a wet surface
• Each source colony should only be pinned from a maximum of __ times.
• If you are pinning to start an experiment, don't pin more than 4 plates at a time, else you will have substantial difference in time between pinning and first image between the plates.
• If scaling up a plate from lower to higher number of colonies per plate, you should never start an experiment with a scaled up plate. You must allow for one iteration of pre-culture so that the pinning to the experimental plate is done with one single pinning pad (e.g. 1536 to 1536). This implies that when using the 3:1 or other interleaving of control-surface pinning you should also allow for a pre-culture iteration before going to experiment
• Pre-cultures should be done on defined media.

Lawn to control surface

1. Inoculate 5mL of YNB or other defined media over night with normalisation strain (see (1) Experimental Design for discussion about suitable strain).
2. Pour inoculate onto YNB or other defined media plate and rake it carefully to cover the entire plate.
3. Put parafilm around plate and let grow for 1-2 days until sufficient amount of cells appear.
4. Pin using the target pinning format of the normalisation reference. Typically, this implies 384-format of references then to be used on 1536 experimental plates. You can use the same lawn to produce several 384-format plates, but in that case switch offset in pinning between the plates.
5. Allow for 384 plates to grow for 1-2 days.
6. Normalisation plates are now ready to use. PLEASE NOTE THAT THEY SHOULD BE STORED ONLY A LIMITED TIME BECAUSE IT IS CRUCIAL THAT THEY SHARE SIMILAR HISTORY AS THE EXPERIMENTAL COLONIES OR ELSE THERE'S A SUBSTANTIAL RISK THAT THEY WILL WORK POORLY AS NORMALISATION

Wet to Solid pinning

Solid to solid pinning

(4) Starting experiment

Preparing the scanner.

• NEVER USE EtOH ON THE FIXTURE & AVOID CLEANING IT AS FAR AS POSSIBLE. IF YOU MUST CLEAN IT USE LENS TISSUE, NOT REGULAR PAPER AND STAY AWAY FROM THE CALIBRATION STRIP*
• If you need to clean the scanner, first carefully remove the fixture and place it somewhere safe where it won't collect dust nor be exposed to scratching. Use lens tissues, not ordinary paper and EtOH. Let the scanner stay open until the surface is fully dried. If you trap liquid between your plates and the scanner you will ruin you experiment, so it is worth waiting a couple of extra seconds.
• Ensure that all plates are properly placed inside their slots and that the fixture is properly placed in the scanner.
• Remove the lids from the plates
• Carefully lower the scanner lid, to ensure that you don't move the plates while doing so, to ensure that you don't scratch the glass of the scanner lid, and to ensure that the lid rests perfectly even on all plates.

The user interface

• In your web browser go to localhost:5000 and click on experiment.
• Fill in all the information with sufficient detail. You will hate yourself for being sloppy.

For more information look at start the experiment.

• Press start. This will not only run the experiment for you (scanning at your set interval), but automatically run through all of the analysis too.

(5) When the experiment is done

REMOVE YOUR PLATES FROM THE SCANNER because we don't want to culture microbes outside our experiments in the scanner!

With the resulting phenotypes you then should do some quality control followed by spatial bias normalisation and then from these interfaces you can export your results.