1. How to avoid condensation. I red advices above and agree with people recommending temperature gradients. Keeping high moisture does not help. Other alternatives: a) buy plate reader which has the mentioned option to keep the lid 1 oC warmer (say, Bioscreen), you can't make arrange it yourself; or b) shut down temperature control and run plates at RT; or c) remove cover and run experiment non-sterile, be aware of liquid evaporation; or d) do kinetic measurements manually, several times a day with new dry lid.

2. How to transfer cells from rock particles. Many techniques are available to remove/desorb bacterial cells. They are based on mechanical action (shaking, vortexing, ultrasound treatment), use of mild detergents and differential centrifugation, filtration, flotation or elutriation to separate desorbed cells from solid particles. But none of them gives 100% success (hundreds of papers published on the subject). With rocks, sediments or soils, the best way to record kinetics of growth on various C-substrates would be to use non-optical monitoring technique to avoid cell removal from solids. Examples: to record heat production with sensitive microcalorimeter, to measure gas exchange, e.g. CO2 production and O2 uptake. Not as popular as microtiter plates, but there are high throughput systems based on such principles.

My guess would be, incubate them at 100% RH chamber.

what I did, when I used BIOLOG, is to incubate the plate in a humidity chamber to avoid evaporation and drop formation.

These suggestions might be good for incubation with endpoint reading, but what to do when looking at the kinetics with (automatic) measurements every hour over 3 days?

We also used a humidity chamber for biolog incubation

The obvious solution is to raise the temperature of the lid to slightly higher than that of the wells but I never found a way to do that. If you are doing one plate at a timeand all wells are innoculated with the same sample than the answer would be leave the plate open (since you are not worried about cross contamination) and keep high humidity in the room to minimize evaporation.

we are using a plate reader from Tecan for continous measurements, in which the lid temperature is slightly higher then the the ambient temperature

Incubate the plate in a humidity chamber 100% RH to avoid evaporation and drop formation. But be sure to steralize the chamber.

It is possible you are getting condensation because of the humidity settings of your incubator. adjust the humidity in your incubator

1. How to avoid condensation. I red advices above and agree with people recommending temperature gradients. Keeping high moisture does not help. Other alternatives: a) buy plate reader which has the mentioned option to keep the lid 1 oC warmer (say, Bioscreen), you can't make arrange it yourself; or b) shut down temperature control and run plates at RT; or c) remove cover and run experiment non-sterile, be aware of liquid evaporation; or d) do kinetic measurements manually, several times a day with new dry lid.

2. How to transfer cells from rock particles. Many techniques are available to remove/desorb bacterial cells. They are based on mechanical action (shaking, vortexing, ultrasound treatment), use of mild detergents and differential centrifugation, filtration, flotation or elutriation to separate desorbed cells from solid particles. But none of them gives 100% success (hundreds of papers published on the subject). With rocks, sediments or soils, the best way to record kinetics of growth on various C-substrates would be to use non-optical monitoring technique to avoid cell removal from solids. Examples: to record heat production with sensitive microcalorimeter, to measure gas exchange, e.g. CO2 production and O2 uptake. Not as popular as microtiter plates, but there are high throughput systems based on such principles.

Good suggestion above. To add:

Condensation happens in these plates and I do not think it is easily preventable. If feasible for your design/sterility requirements, you can lift the lids by offsetting the lid on the plate itself (i.e. on the bench next to the plate reader). By offsetting the lid on the plate (for approximately 15 minutes before you take your readings), there should be enough airflow to remove the built up condensation in the lids. Then, simply place the lid firmly back onto the plate right before your 72 hr reading. Likewise, it may be more feasible to simply read the plates without the lid.

Q: And what is the best method to transfer microorganisms from rock particles to a low optical density supernatant?

A: Depending on the size of the rock: (small gravel) for example could be shaken or sonicated in a Nalgene bottle (with the supernate) to transfer microorganisms. One method to quantify the transfer success (i.e. colonies transferred) is using a serial dilution plate method...but many methods are available depending on the desired resolution of your research questions.

During kinetic measurements I always spin them in a plate centrifuge prior reading and to avoid mixing of the wells, I reduce the amount of inocula by 25 uL.

I don't think the humidity will help, that's a good solution against drying but not for this kind of evaporation. However, a warm-lid reader sounds like a great solution.

It is inevitable to get condensation free BIOLOG plate after incubation. To remove it before reading in microplate reader raise temperature of lid by rubbing warm piece of cloth on to the lid. To transfer of microorganisms from rock particles: soak the rock particle in phosphate buffer saline and shake it for 30 min. Filter it to get particle free filtrate and use it as inoculum for BIOLOG analysis.

Best of luck

You might spray the lid of the BIOLOG plate with an anti-fogging agent, known from scuba diving. We used "nanofog" from Technostar to prevent condensation.

I tend to have a spare sterile BIOLOG plate, and just before reading, under sterile conditions, I switch the plate lids. This way, if I need to measure at 24h, 48h, and 72h, I can without contaminating the samples.

The Tecan plate readers take this issue away as mentioned above. It really is a matter of trying to balance the humidity in the room and plate/lid temperature.

Commentary:

I just want to give my opinion on what this method is meant to provide - and that is a functional representation of the microbial community being sampled. This means minimum processing where possible (to reduce any biased loss of specific species) and incubation at a temperature more representative of the original environment from which the microbial community originated. Just remember it's a mixed microbial community.

In terms of detachment methods - we need to keep the above in mind. While ultrasonic detachment is ok for later DNA/RNA extraction etc. it is not appropriate for a situation where we are detaching culturable/viable communities. Praveen Rahi has given a good example of a method which has been widely accepted in this field. While not perfect (you likely won't detach everything from your sample), it doesn't bias the community which is detached.

While there have been a number of papers on the subject of detachment - many are not in the proper context of this type of method. One paper which does look specifically at this situation is:

Method for the detachment of culturable bacteria from wetland gravel. Journal of Microbiological Methods. Volume 80, Issue 3, March 2010, Pages 242–250

Here the authors compare a few different shaking methods. They found a small improvement in detachment (from a CLPP perspective) when using enzymes to breakdown biofilm components holding the microbial communities to the gravel - you just need to watch the lactose wells if using the same enzymes as studied here. This is for a thick highly active biofilm though - different situations may call for different methods.

I am happy to continue conversations via private messages.

If it's possible for you to waste some more sterile plates, I would just change the lids in the moment of reading the plate for a new sterile lid from a new plate that you can put aside just to make the quick change at that moment and keep all sterile and with no condensation... That's what I usually do :)

Other solution, for multi well trays, would be using these transparent films, or stickers which have the same dimentions than the plate and they're sterile; so, you would just lift the lid and change it for an sticker the very moment of reading the plate, so the plate could be read with no condensation.

the recently launched Spark 10M microplate reader from Tecan allows to lift off plate lids inside the reader for measurement, while keeping the microplate protected by the lid during incubation. This works with all available plate/ lid combinations... Isn't that cool? And I've invented it...

I would not celebrate a victory. What about contamination of plates while they are open? It is added cost and higher risk of malfunction. Finally, you do not have solution to evaporation of liquid over ling-term incubations.

Well, at least it gives a fresh spin to an old problem. In my opinion, the possibility to have microplate protection by a lid, while being able to measure without lid (or inject intermittently) opens up whole new ways of doing complex incubation and measurement sequences. Regarding evaporation, a new way of evaporation protection was also developed together with this new reader, which is again compatible with all types of microplates. I'm not the marketing guy but a developer, therefore I won't present marketing links or more advertising...

Just find out yourselves, if interested.