MBEC Assay (28Sep17)

Aims and objectives: Grow and measure the activity of a C. albicans biofilm in different
substrata Hydroxyapatite and Polystyrene

1. Set up

Day 0 - Prepare inoculum of C. albicans in BHI for 18/24 hours (1 colony in 10 ml).
Day 1 - Prepare the media to be used artificial saliva (AS) add

Method
 On the following day (morning) harvest a loopfull of your bug and place it in 10ml of your
growing media and incubate under agitation for around 6 hours.

 Load the plates as shown in MBEC inoculation 150 ul AS in columns 1, 3-12

 Wash the suspension 2X (4000RPM, 5min) and ressuspend it in 10 ml AS then inoculate your
96 well plates with 150ul per well in column 2.

 Serially dilute from column 2 to 12

 Place the MBEC lid on top and incubate 100 RPM 37 °C for 24 hours

2. Treatment/ testing

No treatment.
Before start this step, to speed up the process, prepare and label all the plates that are
needed for one “go” 2 wash plates, 2 Alamar blue plates (note that the Alamar blue needs to
be prepared not longer than 10 minutes before use)
Note: Use 200ul per well instead of the 150 ul previously used to measure the biofilm that
grows above the liquid.

Prepare the Alamar Blue Solution in BHI, it needs to be prepared less than 10 minutes before
use and to be covered from light.

1st Wash the biofilm with PBS (30sec) (mild rotation)

2nd Incubate with the challenge plate 10 min

3rd Wash the biofilm with PBS (30sec) (mild rotation)

4th Alamar blue 10% incubate for 4 hours.

During this time prepare your treatment plate and set up the plate reader Fluorescence
(Excitation 530nm Emission 590).

5th Read the Alamar blue fluorescence.

 MBEC inoculation
1 2 3 4 5 6 7 8 9 10 11 12

A Ve-

B 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul
AS AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca
C 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul
AS AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca
D 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul
AS AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca
E 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul
AS AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca
F 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul
AS AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca
G 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul
AS AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca
H 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul 150ul
AS AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca AS + Ca

24 Well plate with PMMA disks

1 2 3 4 5 6

A Ve-

B 1750 uL As + 1750 uL As + 1750 uL As + 1750 uL AS

Ca Ca Ca
C 1750 uL As + 1750 uL As + 1750 uL As + 1750 uL AS
Ca Ca Ca
D
 MBEC Test Plate
1 2 3 4 5 6 7 8 9 10 11 12

24 Well Test plate with PMMA disks

1 2 3 4 5 6

A Ve-