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Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity

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Cryoprotectant-free preservation of bacteria using semi-spherical drops.

Jian Wern Ong¹, Zhixiong Song¹, Hassan Ali Abid¹

¹Laboratory for Optics and Applied Mechanics, Department of Mechanical & Aerospace Engineering, Monash University, Clayton, Victoria, 3800, Australia.

|November 28, 2021

Summary

This study introduces a novel cryopreservation method using small, semi-spherical drops, eliminating the need for cryoprotectants. This technique enhances microbial survival rates and supports green laboratory practices by reducing plastic waste.

Keywords:

Bacteria Biobanking Cryopreservation Cryoprotectant Drops Staphylococcus dry ice

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Last Updated: Oct 11, 2025

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Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

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Published on: August 3, 2013

Area of Science:

Microbiology
Biotechnology
Cryobiology

Background:

Cryopreservation is vital for microbial preservation but faces challenges like freeze-thaw damage and osmotic stress.
Optimizing cryoprotectant selection and concentration is resource-intensive and complex.
Current methods require significant expertise and resources for effective microbial long-term storage.

Purpose of the Study:

To develop a cryopreservation method that bypasses the need for cryoprotectants.
To leverage unique thermal properties of semi-spherical drops for improved microbial viability.
To offer a high-throughput, cost-effective, and environmentally friendly preservation technique.

Main Methods:

Utilizing semi-spherical drops (10 μl) on a non-wetting substrate with holes, frozen at -70°C.
Employing an f(x) metric to quantify relative microbial viability.
Comparing survival rates of Staphylococcus epidermidis in drops versus standard cryovials.

Main Results:

Cryopreservation in drops without cryoprotectants improved Staphylococcus epidermidis survivability by 1.91 times compared to larger volumes in tubes.
This method's efficacy (1.91x improvement) approaches that of standard cryopreservation with cryoprotectants (2.33x improvement).
The drop method facilitates high-throughput aliquoting and reduces freeze-thaw cycles, minimizing cell damage.

Conclusions:

The novel drop-based cryopreservation method effectively preserves microbial viability without cryoprotectants.
This technique offers a scalable, efficient, and greener alternative to traditional cryopreservation.
The method reduces plastic waste and associated laboratory costs, promoting sustainable practices.