After writing for a month I’ve gotten my first reader question and I’m excited to share the answer with the rest of you. Here’s the question I got a week ago from a reader working in a tissue bank regarding their samples:

I work in a tissue bank and have some concerns in the practice we use for handling our tissue. When the tissue is ready for processing we receive it in a partially frozen state. To thaw, we have the tissue soak in sterile water for a period of 15 min. We process in rooms with air exchanges that are similar to a hospitals OR. We do have preventative measures after the process we take to protect the tissue. However, we recently have been trying to accommodate clients by processing aseptically. My question is, can the use of water create a harbor for bacterial growth considering the length of time the tissue will remain in the room throughout the duration of the process. (8 to 16 hours) and would there be any alternative means of a safer way in which to thaw the tissue?                                                                                                                -G

 This is a great question but a little bit outside of lab work that I’m personally familiar with. Thankfully though, aseptic technique works the same way everywhere, so here are some suggestions I came up with based on my experience.

In making suggestions, I’m going to make a few assumptions about your current process. Since you’re working in a tissue bank I assume that you’re soaking this tissue for the fifteen minutes and moving to another media after this time and that they don’t just sit in dishes for the full eight to sixteen hours, which would be bad for the tissues. I’m assuming that these tissues are not going back into people or being processed for extremely sensitive downstream analysis like RNA microarrays, which would change the protocol and is a topic for another day.

You’re already using sterile water, so that’s good. Be sure to use fully sterilized dishes for the thaw as well, using either disposable single-use sterile plastics or autoclaved glass or metal dishes. Just cleaning dishware in the sink does to effectively sterilize it. Dishwashers also don’t sterilize what is washed. In fact, it can be very difficult to fully sterilize certain items and even hospitals fail occasionally. There have been instances where hospital-grade sterilization breaks down and can result in the transmission of bacteria between patients¹. Keep all reagents and dishes used for thawing tissue samples sterile in whatever manner is most appropriate for you.

During the thawing process I would recommend that the whole thawing system be kept at 4 °C so the water does not get warm enough for bacteria to grow and multiply. Ice buckets are great for this and I used them extensively when thawing cochlea for tissue processing. There is the simple fact that any and all tissues you process will harbor endogenous bacteria that can grow once at a permissible temperature. If the surrounding water becomes warm it is possible for bacteria in the tissue to grow provided that there are enough nutrients. With that in mind, remember to keep it cold.

From your question it also sounds like the tissues are being thawed out in the open in a room with hospital-grade air exchange. This is good assuming that the samples are in an undisturbed area. However, if persons will be regularly walking by or activities taking place near the thawing samples that could disrupt the air currents I would recommend using a vessel with a lid to maintain sterility. Contamination can easily occur due to bacteria and fungi in the air and those being shed constantly from our hair, skin, clothes, etc. Therefore, if these samples are in a disturbed area I would recommend further precautions such as using lidded container or placing samples in a tissue culture hood.

It’s great that your group is processing these samples aseptically because this will give you more accurate, reproducible results without the confounding effects of microbial contamination. It sounds like the protocol in place will work for your purpose. Just keep in mind the three guidelines:

• Only use sterile materials
• Don’t let the material get above 4 °C
• Prevent or minimize any chances for downstream contamination

Clean, cold, and isolated. If you follow these basic outlines to aseptic technique I see no reason why 15 minutes thawing before processing would lead to problems with bacterial outgrowth.

I wish you the best of luck with your new aseptic protocol!

1.           Shimono, N. et al. An outbreak of Pseudomonas aeruginosa infections following thoracic surgeries occurring via the contamination of bronchoscopes and an automatic endoscope reprocessor. Journal of Infection and Chemotherapy 14, 418-423 (2008).