GVA FAQ

• Try tilting the plate while adding the agar.
• Place the pipette tips at the bottom of the tilted wells but not pressing down hard enough to clog tips.
• Mix a volume that is ~50µL less than the total volume in the well.
• If small bubbles form, let them rise to the top before placing the tip in ice.
• If bubbles are forming after the tips are ejected into the box, hold the tip in ice a few seconds longer.

Depending on the temperature of your environment, every person’s agar will gel at a different rate – if it is gelling particularly quickly here are a couple of tips:

1. Make sure you use a heat bath if one is available to keep the reservoir of agar in while performing GVA
2. If performing GVA with many plates, prepare 2 or more 100mL bottles of agar, using one at a time and keeping the other in a heat bath
3. Low melt agar may be used at the same concentration but the tips will need to be held in the ice longer to gel before ejecting into the tip box

• Double check that you are adding the proper stain at the right concentration to your agar after boiling – they may just be invisible!
• Make sure the agarose powder is completely dissolved in the LB after microwaving or it will not gel properly.
• Increase the intitial dilution of the sample.  Your colonies might be too dense.

We can’t distinguish between colonies that are perfectly overlapping each other in images, however due to the robustness of GVA calculations to missing colonies as well as the low likelihood of this occurring, we have not found this to skew results or make them unreliable.

Nope! We have tested the ability for water to diffuse up into the tips and have seen no indication that it can on the timescales of gelling.

We purposefully use a concentration of agarose (0.5%, pore size 600nm) to ensure that the bacteria are unable to move once it is solidified. Dipping the tips in ices causes the agarose to solidify almost immediately, trapping the bacteria in place through incubating and imaging. Increasing the concentration of agarose will decrease the pore size.

We don’t fully know! The leading theory is nutrient depletion, but if you figure it out, let us know!

• E. coli 
• B. sub
• S. Typhimurium
• P. aeruginosa
• Staph
• Yeast (Saccharomyces cerevisiae)

• MHB
• LB
• Blood agar
• YNB
• PMM
• YEPD

• Bacto agar
• Low melt agar

• GVA can easily be performed in a hood – one thing to note is the agarose will gel more quickly so be sure to have enough extra liquid agarose on hand
• In order to safely transport and image pipet tips with drug resistant strains we dip the tips in bleach for about 1 minute.  The colonies inside the tip remain countable but the exterior of the tip is disinfected.

Generally, yes! Your cells should still be somewhat visible upon closer look or under the magnification of a camera. Take pictures and if you can see and count the cells you are all set! If the cells are still indistinguishable in the images then it may need to be redone.

Our rule of thumb is 10 or once the CFU count at the top starts stabilizing upon clicking more colonies

• Unfortunately, they will not be able to be accurately analyzed if they are all too dense as the CFU count will be estimated as 10^9 for all of them
• We recommend redoing the experiment with an extra 10^2 dilution (or more depending how concentrated they seemed) of the cells before adding agarose on top
• For estimating how much to dilute prior to running the experiment, generally 10^2 is sufficient for lab strain cells that are being treated with a killing agent. Generally, 10^4 dilution is better for untreated cells or cells that are treated with a compound that promotes growth

If the bubbles are small and there is a contigous region of colonies that are countable, the bias will likely be small.  If the bubbles are large and substantially change the geometry of the agarose cone, we consider this a pipette error (hotkey e in software).