Isolation of Halophilic Archaea from ( 121-112 Million Year Old) Primary Salt Crystals
INTRO
In recent years, several studies have attempted to isolate living halophiles from within primary salt crystals. These salt crystals areevaporite rocks
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formed from the evaporation of ancient seas and the precipitation of the minerals left behind. Within these halite crystals reside what are known as inclusions, which house a small amount of residual fluids thought to be able to support microbial life.Fluid Inclusions in a single Chevron Halite crystalThe idea is that because these inclusions have been protected within the crystals for millions of years, the organisms occupying them upon formation are the same organisms occupying them now. In this particular study the authors obtained their samples from the Sergipe basin in Brazil.
The Sergipe Basin covers a total area of 20,000 km sq. and reaches a depth up to 800m. Within this 800 m researchers sampled Chevron halite,
Fluid Inclusions in a single Chevron Halite crystal
known to have fluid inclusions, at 768m in the GTP-8 core. Core sampleswere taken and crystals were subsequently removed, photographed and inspected at places other than those being used for molecular or microbiological extraction. The crystal samples were taken using a hand held electric drill and coring bit. The underground mining techniques used in this facility does not include blasting and the shaft was constructed using up-reaming Up-reaming
Up-reaming
This limits mechanical damage to the wall rock compared with blasting. Only crystals from primary sedimentary features were removed. Each crystal used for microbiological sampling was examined, and crystals were rejected as damaged if inclusions showed rounded edges, elongation or stress-related roughness along the walls of the inclusion.
After the crystals were selected they were placed into clean unused sample vials and transported to the microbiological laboratory. Various precautions were taken before and during isolation procedures so as to assure that no contamination would occur.
Preventing Contamination:
Halite crystals were sampled in a biological safety level three (BSL3) clean room. Prior to use the entire room was cleaned with anti-bacterial cleaners and sprayed with Cidex Plus liquid sterilizing agent. All personnel involved in sampling were gowned in clean, bleach washed coveralls, caps, boots, as well as sterile latex gloves. The class IIB laminar flow hood, where the crystals would be sampled, was first treated for two hours under a germicidal UV light.
Contamination Checks
Due to the need for high levels of contamination control, extensive contamination checks were conducted throughout all experiments.
Controls included:
1. autoclaving of all steam stable equipment, media and chemicals for at least 60 min.
2. pre-incubation of all sterilized media for at least 14 days prior to use, all medium batches were also prepared in groups of 50 flasks with the rule that all were discarded without use if even a single flask was contaminated.
3. sampling of all wash brines after treatment of six crystals, once again any contamination event resulted in discarding the 6 samples if any of these already produced positive growth.
4. during incubation the wash brines were incubated along with the samples and needed to remain negative for at least 14 days after the last positive crystal from that set of 6 samples.
5. presence of open control plates of solid media were scattered inside the laminar flow hood during the entire sampling operation. In this case, any contamination resulted in the rejection of all samples taken during that session.
6. Finally, all media used for contamination checks were selected at random from the flasks being used for actual isolation.
Dissolving the Crystals:
The individual halite crystals were surface sterilized using the five minute NaOH, 5 minute HCl procedure in groups of 6 using sterile 24 well tissue culture plates. At the end of thee surface sterilization, the crystals were placed individually, one per flask, into small 50 ml screw cap Erlenmeyer flasks containing 25 ml of CAS medium that had been autoclaved at 121 degrees Celsius for 60 minutes and incubated before use to check for contamination.
All media used in these experiments contained 20% NaCl so the crystals were allowed to dissolve slowly after they were placed in the medium. The inoculated medium was allowed to stand undisturbed for at least 2 hours after the crystals dissolved. The media were then placed into an incubated shaker at 30 degrees Celsius. The flasks were checked weekly for several weeks.
Analysis of Isolates:
For details on how the authors did their lipid analysis go to Torreblanca et al 1986.
For further details on Sequencing of 16srRna and to read this article go to Vreeland et al. 2007
INTRO
In recent years, several studies have attempted to isolate living halophiles from within primary salt crystals. These salt crystals areevaporite rocks
The Sergipe Basin covers a total area of 20,000 km sq. and reaches a depth up to 800m. Within this 800 m researchers sampled Chevron halite,
This limits mechanical damage to the wall rock compared with blasting. Only crystals from primary sedimentary features were removed. Each crystal used for microbiological sampling was examined, and crystals were rejected as damaged if inclusions showed rounded edges, elongation or stress-related roughness along the walls of the inclusion.
After the crystals were selected they were placed into clean unused sample vials and transported to the microbiological laboratory. Various precautions were taken before and during isolation procedures so as to assure that no contamination would occur.
Preventing Contamination:
Halite crystals were sampled in a biological safety level three (BSL3) clean room. Prior to use the entire room was cleaned with anti-bacterial cleaners and sprayed with Cidex Plus liquid sterilizing agent. All personnel involved in sampling were gowned in clean, bleach washed coveralls, caps, boots, as well as sterile latex gloves. The class IIB laminar flow hood, where the crystals would be sampled, was first treated for two hours under a germicidal UV light.
Contamination Checks
Due to the need for high levels of contamination control, extensive contamination checks were conducted throughout all experiments.
Controls included:
1. autoclaving of all steam stable equipment, media and chemicals for at least 60 min.
2. pre-incubation of all sterilized media for at least 14 days prior to use, all medium batches were also prepared in groups of 50 flasks with the rule that all were discarded without use if even a single flask was contaminated.
3. sampling of all wash brines after treatment of six crystals, once again any contamination event resulted in discarding the 6 samples if any of these already produced positive growth.
4. during incubation the wash brines were incubated along with the samples and needed to remain negative for at least 14 days after the last positive crystal from that set of 6 samples.
5. presence of open control plates of solid media were scattered inside the laminar flow hood during the entire sampling operation. In this case, any contamination resulted in the rejection of all samples taken during that session.
6. Finally, all media used for contamination checks were selected at random from the flasks being used for actual isolation.
Dissolving the Crystals:
The individual halite crystals were surface sterilized using the five minute NaOH, 5 minute HCl procedure in groups of 6 using sterile 24 well tissue culture plates. At the end of thee surface sterilization, the crystals were placed individually, one per flask, into small 50 ml screw cap Erlenmeyer flasks containing 25 ml of CAS medium that had been autoclaved at 121 degrees Celsius for 60 minutes and incubated before use to check for contamination.
All media used in these experiments contained 20% NaCl so the crystals were allowed to dissolve slowly after they were placed in the medium. The inoculated medium was allowed to stand undisturbed for at least 2 hours after the crystals dissolved. The media were then placed into an incubated shaker at 30 degrees Celsius. The flasks were checked weekly for several weeks.
Analysis of Isolates:
For details on how the authors did their lipid analysis go to Torreblanca et al 1986.
For further details on Sequencing of 16srRna and to read this article go to Vreeland et al. 2007