Dear Casper,

I am not familiar with this particular model, if I have found the correct model it looks like a flask based freeze dryer.

There are several keys to good freeze drying, first step is good freezing. Question - what does good look like? With a complex matrix like feaces, then the volume of your sample will be important. I assume that you have a small quantity in a vial or sample tube and do not seek to dry the whole sample from the patient / donor. [We had a zoo doing whole samples from Rhino ... whole other story!]

As you know there's lots of stuff in feaces, and you want to get the moisture out. Also, some components may make freezing hard. Therefore, I would generally recommend diluting the sample with little RO water or buffer, and mix well. This sounds counter intuitive but will help normalize the samples as every donor will be different. The extra water will help the sample to freeze more easily and when freezing, freeze slowly to allow large ice crystals to grow, do not freeze in a -85 or LN2 as you will get smaller ice crystals. Assuming I found the correct model dryer, then freeze samples in a -20 freezer before placing in the flask to dry, and cover each one with some film and put some holes in the top with a hypodermic needle to allow the moisture to escape. Depending on the volume of your sample, the time taken could be ... 24 to 48 hours at a best guess.

If you have to freeze dry a the sample taken directly from the donor, the challenge will be variability (dry to wet) and when drying, the dry sample can block the vapour escaping from the remaining ice - we see this in many types of sample. Addition of 10% or 20% RO water or buffer will not make the drying time longer but for an already relatively dry sample, may make the drying time shorter as the water can escape more easily from the complex core of the sample.

Kind regards

Rob

Dear Rob

Thanks for taking your time to provide such a thorough answer.

To provide a little bit more background:

Different top compartments can be applied to the freeze-dryer, we have a shelf system (the one in the middle picture https://www.labogene.com/CoolSafe-4-15L-Freeze-Dryers), where we can have multiple tubes in each tray.

We have home collected samples provided in prefrozen fecal tubes, it is not possible to add water at collection (Practical reasons) or in the lab (to avoid freeze-thawing).

Unfortunately the tubes are tall with a low surface to volume ratio.The amount of feces in each tube is also very different with some being stuffed while others only provide a pea sized amount.

We already have samples in a biobank and the reason why we want to proceed with freeze--drying is that our current method of drilling samples for further processing is too time consuming for our large cohorts.

Can you please elaborate on why we want large ice crystals. Is that specifically to allow efficient escape of water? In terms of DNA quality I have always been told to store samples at -80 °C. However, in my own experiments it did not seem to influence microbiome composition if samples where stored at -20 °C or -80 °C. I was also recommended to store samples at -80 °C, before freeze-drying to make sure not to have compartments in the sample that goes into liquid form. Would you recommend to initially freeze samples at -20 °C and then move samples to -80 °C prior to freeze-drying?

Do you have any input on specific freeze-drying settings in terms of pressure and temperature. I also saw some dividing the process into primary drying and secondary drying (http://www.pdxwildlife.com/freeze-drying-panda-feces/)?

Best,

Casper

Dear Casper,

Wow ... you have a challenge!

For the basics of freeze drying, including the difference beteween primary and secondary drying, look here:

https://www.spscientific.com/freeze-drying-lyophilization-basics/

Good freeze drying is all about suface area for water vapour to escape. Ice crystal size, sample suface area, sample depth, matrix composition all are factors. Faeces are a complex and highly variable matrix, intra and inter donor. Things like undigested fibrous material will be the hardest to dry, they trap moisture in their cellular structure and then can be "burried" in the sample itself.

Large ice crystals that run throughout the sample create a highway for water vapour to escape from. Lots of tiny ice crystals mean that you have a network of fine channels for moisture to escape from, making drying the middle of the sample much harder.

Given the wide variety of samples that you have, I fear that you cannot have a truly "optimal" method, therefore you have to play safe and go for a "low and slow" method. The pea size samples will dry and sit in the vacuum for longer than needed while the other samples dry, unless you can hand sort for sample weight / volume before loading samples into the dryer.

The shelves you have are not refrigerated, this is a basic dryer with a set of trays (can you heat them?). Set vacuum to 100mTorr (ice temperature is then -40 C) and then when the samples are under vacuum, if you have it, use some shelf heat to help speed the latter half of drying. It may help to place the taller tubes on their side for good heat input, but not necessary if you have a basic shelf with no heat.

I have no idea about time ... the small samples will be

Thanks Rob this was very helpful.

I'm still concerned regarding DNA quality, do you or anybody else have experience with freeze drying for the specific purpose of storing feces prior to DNA-extraction and 16S rRNA gene sequencing?