# Long-term preservation of bacteria in washed sterile sand: A 14-year study

## TL;DR

A simple and low-cost method of preserving bacteria in sterilized sand kept most strains viable and stable for over 14 years.

## Contribution

Demonstrates that washed and sterilized sand is a viable, low-cost alternative to cryopreservation for long-term bacterial storage.

## Key findings

- 13 out of 14 bacterial strains remained viable after 14 years in sterilized sand.
- Spore-forming Gram-positive bacteria showed the highest survival rates.
- Enterobacter hormaechei retained phenotypic stability with minimal viability loss over 14 years.

## Abstract

Cryopreservation and lyophilization are standard methods for preserving microbial cultures, but they are expensive and labor-intensive, creating a barrier for resource-constrained laboratories. This study evaluated the efficacy of a simple and inexpensive alternative: long-term storage in washed and sterilized sand.

Fourteen bacterial strains from ten genera were preserved in sealed tubes containing washed and sterilized sand at room temperature. Viability, assessed by colony-forming units (CFU/g), was quantified at inoculation and after 7 and 14 years. Phenotypic stability was evaluated by comparing the pre- and post-storage profiles for enzyme activity and stress tolerance.

After seven years, all 14 strains remained culturable with preserved phenotypic features. After 14 years, 13 strains (93%) were viable. The highest recovery rates were observed in spore-forming Gram-positive bacteria (e.g., Bacillus sp.), indicating taxon-specific survival. Notably, the Gram-negative strain Enterobacter hormaechei remained stable with only a approximate 1.58 log₁₀ reduction over 14 years (half-life = 2.67 years). All resuscitated strains maintained their pre-storage characteristics. The distinct survival patterns for each species indicate that contamination was not the primary driver of the outcomes.

Washed and sterile sand offers a practical, low-energy matrix for long-term preservation of a diverse range of bacteria, effectively maintaining cultivability and key phenotypic attributes for over a decade. This approach is an economical preservation strategy for resource-limited laboratories and field collections.