Development of a Protocol to Retrieve Microorganisms from Ancient Salt Crystals

Abstract

Previous research has indicated that halophilic microorganisms are associated with salt crystals from ancient formations. However,this research has generally failed to convincingly demonstrate that these organisms were originally trapped inside the crystals during their deposition and were not recent surface contaminants. This paper presents techniques for crystal selection; verifiable, noninvasive, surface sterilization of salt crystals; and sterile extraction of biological material from the crystals. Immersing salt crystals in 10 M NaOH for 5 min; rinsing with sterile, saturated brine before immersing for 5 min in 10 M HCl; and rinsing with saturated brine is an effective method for surface sterilization. No growth has resulted from contamination tests of 216 faces from 36 natural salt crystals exposed to these treatments. Pure culture experiments using the halotolerant eubacteria, Halomonas elongata and Bacillus sp. (2-9-3), and the archeon, Halogeometricium borinquense, showed that exposure to either 10 M HCl or NaOH reduced the population of these organisms by a factor of 107 to 108 colony-forming units/ml. The fluid is extracted from inclusions by drilling into surface-sterilized salt crystals with a variable-speed drill using sterilized 0.5-mm-diameter carbide drill bits and extracting the fluid with a sterilized microliter syringe. Drilling and extraction are performed in a Class II biosafety cabinet. All procedures are carried out in a biosafety level 3 facility. All personnel involved wear cleanroom coveralls, shoe covers, hair caps, and gloves. The above protocol has resulted in the isolation of a Bacillus sp. from a Permian-age salt crystal.