Organic Acid Tolerance Response Assay
1. Inoculate 3 ml LBS cultures; grow at 28°C with shaking for 1-2 hours.
2. Inoculate 30 ml LBS cultures to an initial OD of ~0.02 and incubate at
28°C with shaking.
3. When cultures reach an OD of 0.2 - 0.4, harvest cells in sterile
centrifuge tubes (SS-34 rotor, 6 minutes @ 6500 rpm)
4. Remove supernatants and resuspend pellet in 1 ml LBS. Transfer 100
µl to one 1.5 ml Eppie tube and the other 900 µl to another.
Centrifuge @ 12,000g for 1 min.
5. Remove supernatants and resuspend the pellet from the 100 µl tube in
1 ml LBS; this will be the "Unadapted" cultures. Resuspend the pellet
from the 900 µl tube 1 ml LBS + 0.075x organic acid cocktail, pH 5.7
(see notes); this will be the "Adapted" culture.
6. Transfer both adapted and unadapted to sterile falcon tubes and
incubate for one hour at 28°C with shaking.
7. Remove half the volume from the adapted and unadapted cultures and
centrifuge @ 12,000g for 1 min. Save the remaining half volumes
8. Resuspend pellets in 1 ml LBS + 0.1x organic acid cocktail, pH 4.5.
9. Immediately serial dilute (see notes) and plate cultures; this will be
the 0 time point. Also plate dilutions of the "unshocked" half volumes
from step 7 (this is a control for cell survival)
10. Repeat step 9 at 30, 60, and 90 minutes post-exposure.
11. Incubate overnight and count CFUs on plates. Percent survival at 30,
60, and 90 minutes is calculated by dividing the CFUs per ml for a
given time point by CFU's per ml at time 0.
Notes:
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This assay is modified from the following reference: Merrell & Camilli. 1999.
The cadA gene of Vibrio cholerae is induced during infection and plays a role in
acid tolerance. Molec. Micro. 34: 836-849.
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1x Organic Acid Cocktail:
74 mM Propionic Acid
174 mM Acetic Acid
50 mM Butyric Acid
(Stench Advisory: For god sake, make this in the hood! Filter sterilize)
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To make the organic acid amended media, you'll need to make twice the amount
that you'll actually need. That is, if you'll need 10 ml LBS + 0.1x acids, pH 4.5,
you'll need to start out by making up 10ml LBS + acids and adjust the pH to 4.5,
monitoring the pH and keeping track of the volume HCl that you add. However,
this batch of media is no longer sterile, so you'll need to make up a second, equal
volume, adding the predetermined amount of HCl, and then sacrifice a small
volume to confirm the final pH. Unfortunately, batches of LBS tend to slightly
vary in buffering capacity, so you'll need to do this every time you do the assay.
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You'll need to work out the best way to do the dilution series by trial and error. In
my experiments, I've needed to plate directly from the shocked cultures for the
unadapted cells (they're killed very effectively). For the adapted cultures, the
dilutions between 103 and 107 tend to give countable plates most frequently, with
the lower end of those dilutions becoming most useful during the later time
points.