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Applied and Environmental Microbiology, April 2004, p. 2540-2544, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2540-2544.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Comparisons of Protocols for Decontamination of Environmental Ice Samples for Biological and Molecular Examinations

S. O. Rogers,1,2* V. Theraisnathan,1 L. J. Ma,2,{dagger} Y. Zhao,2,{ddagger} G. Zhang,1 S.-G. Shin,1 J. D. Castello,2 and W. T. Starmer3

Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403,1 Faculty of Environmental and Forest Biology, College of Environmental Science and Forestry, State University of New York, Syracuse, New York 13210,2 Department of Biology, Syracuse University, Syracuse, New York 132443

Received 5 September 2003/ Accepted 7 January 2004


   ABSTRACT
Top
 Abstract
Introduction
 Experiment set 1.
 Experiment set 2.
 Experiment set 3.
 References
 
Drilling and laboratory manipulations of glacial ice cores introduce contemporary microbes and biomolecules onto the cores. We report herein a systematic comparative study of several decontamination protocols. Only treatment with 5% sodium hypochlorite eliminated all external contaminating microbes and nucleic acids while maintaining the integrity of those within the cores.


   INTRODUCTION
Top
 Abstract
 Introduction
Experiment set 1.
 Experiment set 2.
 Experiment set 3.
 References
 
Glacial ice and permafrost sampled from ice cores to depths of nearly 4 km at many locations throughout the world have been assayed for ancient viable microbes and intact nucleic acids, some of which were deposited up to 700,000 years ago (1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; L. J. Ma, H. Fan, C. Catranis, S. O. Rogers, and W. T. Starmer, Abstr. Mycol. Soc. Am. Inoculum, abstr. 48:23, 1997; L. J. Ma, C. Catranis, W. T. Starmer, and S. O. Rogers, Abstr. Mycol. Soc. Am. Inoculum, abstr. 49:34, 1998). The major concern in all of these studies has been authentication because of the potential for contamination. We report herein a comparative study of the most commonly used decontamination protocols (1, 9, 10, 11, 12, 15, 16, 17).

"Sham" ice cores were produced in our laboratory by freezing (at –20°C) cell suspensions and/or nucleic acids in sterile beakers (12.5-cm diameter by 10-cm length). Cells and/or nucleic acids from species different from those inside the cores also were spread and frozen onto the core surfaces. The cores were then cut into eight equivalent sections by using a sterilized saw. The outer organisms were chosen for their hardiness, while the inner organisms were chosen for their susceptibility to sterilization methods. Concentrations of cells and nucleic acids were similar to those found in glaciers. For each treatment, the cores were warmed by exposing them to a temperature of 4°C for 30 min. Solutions and instruments were chilled to 4°C. Assaying was performed by culturing and/or PCR amplification. Culturing was on malt extract agar (2% malt extract, 1.5% agar [pH 7.5]) for fungi, Luria-Bertani agar (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar [pH 7.5]) for Escherichia coli, or nutrient agar (0.3% beef extract, 0.5% peptone, 1.5% agar [pH 6.8]) for Bacillus subtilis. The cultures were grown in 10-cm culture plates and were incubated at 22°C (for fungi and B. subtilis) or 37°C (for E. coli) for 2 to 7 days. Each plate was inoculated with 200 µl of ice meltwater. PCR amplifications were performed for fungi (15, 16), bacteria (20), and tomato mosaic tobamovirus (ToMV) (7, 8).


   Experiment set 1.
Top
 Abstract
 Introduction
 Experiment set 1.
Experiment set 2.
 Experiment set 3.
 References
 
Aspergillus terreus (103 spores/ml and/or 1 pg of DNA per µl), B. subtilis (300 or 107 spores/ml and/or 1 pg of DNA per µl), E. coli (107 cells/ml), and ToMV (50 fg of RNA per µl) were frozen (at –20°C) separately or in combination into sham ice cores. The cores were coated with 1 ml of a Ulocladium atrum spore suspension (1010 spores/ml) and 1 ml of a U. atrum DNA solution (1 pg/µl) and then stored at –20°C. Under a sterile laminar flow hood, the core sections were completely immersed in 800 ml of 5.25% sodium hypochlorite (undiluted Clorox bleach product) for 10 s and rinsed twice with 200 ml of sterile water (18.2 M{Omega}, <1 ppb total organic carbon). The core sections were then transferred into sterile funnels and melted at room temperature under the hood. Meltwater was collected in 20-ml aliquots, termed shells, where the outermost shell is designated shell 1 and the innermost shell is designated shell 5. Each shell then was assayed at least three times by culturing, PCR amplification, or reverse transcription-PCR amplification (for ToMV RNA).

Growth was never observed in shell 1 when treated with undiluted Clorox, but many U. atrum colonies grew on plates inoculated with shell 1 meltwater from untreated cores (Table 1) (Fig. 1). After Clorox treatment, growth was observed from the second shells inward at 103 cells/ml for fungi and at 107 cells/ml for bacteria or from the third shells inward at 300 cells/ml for bacteria (Table 1). Levels of growth similar to those of untreated controls were observed in the third shells (103 cells/ml for fungi and 107 cells/ml for bacteria) or in the fifth shells (300 cells/ml for bacteria). Robust PCR amplification bands were always present in the untreated samples (Table 1) (Fig. 1), whether at low or high nucleic acid concentration (50 fg/µl or 1 pg/µl, respectively). Amplification was not observed in the first shell of any core treated with undiluted Clorox. Faint amplification was observed in shell 2 of the high-concentration cores and in shell 3 of the low-concentration cores. Robust amplification bands were observed in high- and low-concentration sham cores for the third and fourth shells, respectively.


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  		TABLE 1. Results of Clorox decontamination tests of sham ice cores seeded with various organisms

 


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  		FIG. 1. Number of colonies of U. atrum (outer contaminating organism [upper graph]) and A. terreus (the inner sensitive organism [lower graph]) before (white bars) and after (black bars) treatment with 5.25% sodium hypochlorite. No colonies of U. atrum were observed on any plates from the sodium hypochlorite-treated sham ice core meltwater samples. In the treated ice cores, colonies of A. terreus were at untreated levels in shells 3 to 5. The difference in scale for number of colonies is due to the difference in the colony size for the two species. U. atrum forms large colonies, while A. terreus forms small colonies. The relatively low number of colonies for A. terreus in the first and second shells of the untreated sham core is due to competition for area on the culture plates, since the colonies of U. atrum occupied more space on those plates (both fungi were on the same plates). Colonies were counted in all five shells for all treatments and controls. Standard deviations, based on triplicate experiments, are indicated at the top of each bar.

 

   Experiment set 2.
Top
 Abstract
 Introduction
 Experiment set 1.
 Experiment set 2.
Experiment set 3.
 References
 
Aureobasidium pullulans (107 cells/ml) and E. coli (107 cells/ml) were frozen together into the ice cores (at –20°C). The exterior surfaces of the cores then were coated with U. atrum and B. subtilis cells (107 cells/ml). The cores were stored at –20°C. The following treatments were performed on separate core sections: treatment 1 (sodium hypochlorite), immersion in 800 ml of 5.25% sodium hypochlorite (undiluted Clorox) for 10 s, followed by three 200-ml rinses with sterile water; treatment 2 (ethanol), immersion in 800 ml of 95% ethanol for 60 s, followed by three 200-ml rinses with sterile water; treatment 3 (heated probe), after immersion of the core in 800 ml of 95% ethanol for 60 s, a heated glass rod (diameter, 5 mm; length, 150 mm) was pressed into the bottom of the core near the outer edge after each shell was removed (by melting) and the resulting meltwater was collected; treatment 4 (drilling), after immersion of the core in 800 ml of 95% ethanol for 60 s, a sterilized drill bit was used to circumscribe an inner core subsection that was removed with sterilized forceps; treatment 5 (sterile water ablation), three 300-ml rinses with sterile water; treatment 6 (mechanical ablation), a sterilized razor blade was used to scrape off 5 mm from all of the outer surfaces of the core; treatment 7 (UV irradiation), the outer surface of the middle of the ice core was covered in black paper and aluminum foil (in that order), followed by UV irradiation (27,540 J/m–2/s) for 10 min, and then the inner portion of the core was extracted by drilling and sawing from both ends with a sterile drill bit and saw blade, followed by removal with sterilized forceps; and treatment 8 (control), no treatment. After each treatment, the cores were melted in shells and meltwater was collected, as described for experiment set 1 (except as noted in the description of treatment 3, above).

The 5.25% sodium hypochlorite treatment killed all externally applied microorganisms, although some reduction in the number of internal organisms was observed. Treatments 2, 3, and 4 produced similar results (Fig. 2). Contaminating organisms were always present in the first and second shells. They were diluted or absent in the third shells. Since all three treatments employed 95% ethanol as the surface sterilant, it is likely that the reduction in the number of outer organisms was due primarily to the action of ethanol. Treatments 5 and 6 (ablation) were less effective than were other methods in removing outer contaminants, since outer contaminating microbes were found all the way into the fourth shell (Fig. 2). Viable U. atrum was detected in all shells of the UV-irradiated ice cores. Spores of this fungus are highly resistant to UV irradiation (data not shown). A dose of 27,540 J/m2/s of 254-nm-wavelength UV irradiation (5-min exposure) was sufficient to kill 100% of Penicillium commume spores, but a dose 12 times higher caused no reduction in the number of germinating spores of U. atrum.



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  		FIG. 2. Number of colonies of the outer organisms (upper graphs; U. atrum [white bars] and B. subtilis [black bars]) and inner organisms (lower graphs; A. pullulans [white bars] and E. coli [black bars]) after various treatments (described in the text). Shown are the results of experiment set 2. Colonies were counted in all five shells for all treatments and controls. Standard deviations, based on triplicate experiments, are indicated at the top of each bar. Results for untreated controls are shown at the far right.

 

   Experiment set 3.
Top
 Abstract
 Introduction
 Experiment set 1.
 Experiment set 2.
 Experiment set 3.
References
 
Cores were prepared as described for experiment set 2. The following treatments were performed separately on each core section: treatment 1 (sodium hypochlorite), immersion in 800 ml of sodium hypochlorite (1, 2, 3, 4, 5, or 6%) for 10 s, followed by three 200-ml rinses with sterile distilled water; treatment 2 (dilute base and acid), immersion in 800 ml of 1 N NaOH for 10 s, followed by two 200-ml rinses with sterile water and then immersion in 800 ml of 1 N HCl for 10 s and two 200-ml rinses with sterile water; treatment 3 (concentrated base and acid), same as treatment 2 (above), except that 10 N NaOH and 10 N HCl were used; treatment 4 (dilute hydrogen peroxide), immersion in 800 ml of 6% H2O2 for 10 s, followed by three 200-ml rinses with sterile water; and treatment 5 (concentrated hydrogen peroxide), same as treatment 4 (above), except that 35% H2O2 was used.

Only treatments of 5 and 6% sodium hypochlorite killed all externally applied contaminants (Fig. 3 and 4). However, only the 5% treatment yielded little or no reduction in the growth of interior organisms. The dilute NaOH-HCl treatment (1 N NaOH followed by 1 N HCl) eliminated all external fungi, but some external bacteria remained in shells 1 and 2, and inhibition of inner organisms was observed in some shells (Fig. 3). At high concentrations (10 N NaOH and 10 N HCl), external bacteria also survived and inhibition of inner organisms was observed in all shells (Fig. 4). Both concentrations of H2O2 failed to kill all outer organisms and reduced the number of inner organisms.



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  		FIG. 3. Number of colonies of the outer organisms (upper graphs; U. atrum [white bars] and B. subtilis [black bars]) and inner organisms (lower graphs; A. pullulans [white bars] and E. coli [black bars]) after various treatments (described in the text). Shown are the results of experiment set 3. Colonies were counted in all five shells for all treatments and controls. Standard deviations, based on triplicate experiments, are indicated at the top of each bar. Results for untreated controls are shown at the far right.

 


View larger version (36K):
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  		FIG. 4. Number of colonies of the outer organisms (upper graphs; U. atrum [white bars] and B. subtilis [black bars]) and inner organisms (lower graphs; A. pullulans [white bars] and E. coli [black bars]) after various treatments (described in the text). Shown are the results of experiment set 3. Colonies were counted in all five shells for all treatments and controls. Standard deviations, based on triplicate experiments, are indicated at the top of each bar. Untreated controls are shown paired with each of the treatments.

 
Researchers have been isolating viable microbes and their nucleic acids for a few decades by using a variety of decontamination, isolation, and detection protocols. These protocols have not been standardized or compared for effectiveness. The results reported here provide the basis upon which standardized decontamination protocols can be developed and used by all researchers working with ancient ice.


   ACKNOWLEDGMENTS
 
We acknowledge C. M. Catranis and J. Smith for their kind assistance with aspects of this project.

This work was partially supported by a grant from the National Science Foundation (grant no. 9808676) and by Bowling Green State University.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Sciences, Bowling Green State University, 217 Life Sciences Bldg., Bowling Green, OH 43403. Phone: (419) 372-2333. Fax: (419) 372-2024. E-mail: srogers{at}bgnet.bgsu.edu. Back

{dagger} Present address: Whitehead Institute, MIT Center for Genome Research, Cambridge, MA 02141. Back

{ddagger} Present address: School of Medicine, Wayne State University, Detroit, MI 48201. Back


   REFERENCES
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 Experiment set 3.
 References
 

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Applied and Environmental Microbiology, April 2004, p. 2540-2544, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2540-2544.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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