Applications of a Rapid Endospore Viability Assay for Monitoring UV Inactivation and Characterizing Arctic Ice Cores

Shafaat, Hannah S.; Ponce, Adrian

doi:10.1128/aem.00255-06

Cited by 47 publications

(39 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We note that enzymatic activity has been detected in actinomycete spores at 40°C (45), which may indicate some capability for damage repair. However, Shafaat and Ponce (46) have shown that spores are only Ϸ1% as abundant as vegetative cells in GISP2 ice; at subzero temperature, the total rate of damage repair of spores is thus negligible relative to the rates shown by the purple lines in Fig. 3.…”

Section: Discussionmentioning

confidence: 99%

In situ microbial metabolism as a cause of gas anomalies in ice

Rohde

Price

Bay

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Isolated spikes of anomalously high concentrations of N2O have been reported at depths in Greenland and Antarctic ice cores corresponding to narrow time intervals over the past Ϸ10 5 years. Now, using a calibrated spectrofluorimeter to map protein-bound Trp, a proxy for microbes, versus depth in the 3,053-m GISP2 ice core, we find six depths at which localized spikes of high cell concentrations coincide with N2O spikes. We show that the excess gases are consistent with accumulation of in situ metabolic wastes during residence times of the excess microbes in the ice. Because of sparseness of N2O measurements and our spectrofluorimetry versus depth, the total number of microbially produced N2O spikes in GISP2 is probably much larger than six. Spikes of excess methanogens coincident with CH4 spikes are found at three depths in the bottom 3% of GISP2, most likely because of methanogenic metabolism in the underlying silty ice, followed by turbulent flow of the lowest Ϸ90 m of ice. The apparent rates of in situ production of N2O and CH4 spikes by metabolism are observed to be consistent with a single activation energy, U, and maintain proportionality to exp(؊U/RT) over the entire temperature range down to ؊40°C. Fluorescence of nonmicrobial aerosols in GISP2 ice is distinguishable from microbial fluorescence by its different emission spectra. Our spectrofluorimetric scans throughout the GISP2 ice core lead us to conclude that both microbes and nonmicrobial aerosols are deposited in discontinuous bursts, which may provide a tool for studying wind storms in the distant past.climate ͉ ice cores ͉ proxy for dust storms ͉ scanning fluorimetry ͉ N2O

show abstract

Section: Discussionmentioning

confidence: 99%

In situ microbial metabolism as a cause of gas anomalies in ice

Rohde

Price

Bay

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…This communication shows that low concentrations of Tb 3ϩ and some other multivalent metal ions strongly inhibit the germination of coat-deficient spores. This finding is notable, as these same low Tb 3ϩ concentrations are used to assay viable spores by measuring DPA release in spore germination (26,28,29,31,32). Since coat-deficient spores have viability comparable to that of intact spores (8,15), Tb 3ϩ -based assays of spore viability could yield false-negative results with coat-deficient spores.…”

mentioning

confidence: 93%

“…DPA forms a strong 1:1 highly fluorescent complex with terbium (Tb 3ϩ ) ions, and spore quantification by Tb 3ϩ -DPA fluorescence takes only a few minutes (4,12). DPA is also released in the first minutes of spore germination (23), and since germination is essential for spore viability, Tb 3ϩ -DPA fluorescence has been used to detect spore germination, and there is a good correlation between DPA release and spore viability (4,26,28,29,31,32). However, we report a flaw in the latter assay of spore viability, as Tb 3ϩ and some other multivalent metal cations strongly inhibit germination of coat-deficient spores.…”

mentioning

confidence: 99%

Efficient Inhibition of Germination of Coat-Deficient Bacterial Spores by Multivalent Metal Cations, Including Terbium (Tb ³⁺ )

Bond

Sarker

et al. 2011

Appl Environ Microbiol

View full text Add to dashboard Cite

Release of dipicolinic acid (DPA) and its fluorescence with terbium (Tb 3؉ ) allow rapid measurement of the germination and viability of spores of Bacillus and Clostridium species. However, germination of coat-deficient Bacillus spores was strongly inhibited by Tb 3؉ and some other multivalent cations. Tb 3؉ also inhibited germination of coat-deficient Clostridium perfringens spores.Many Bacillus and Clostridium species can form dormant, resistant spores under conditions unfavorable for growth (24,25). These spores can resist extreme conditions and survive for years in the absence of nutrients. However, when nutrients return, these spores can germinate and become growing cells (16,23,25). Some spore-forming bacteria can also cause food spoilage, food poisoning, and disease, and spores of Bacillus anthracis are a potential bioterrorism agent (9, 13, 25). Therefore, rapid, sensitive assays for bacterial spores have applied importance.An assay has been developed to detect low spore concentrations, based on spores' high concentration (ϳ10% [dry weight]) of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) (9). DPA forms a strong 1:1 highly fluorescent complex with terbium (Tb 3ϩ ) ions, and spore quantification by Tb 3ϩ -DPA fluorescence takes only a few minutes (4, 12). DPA is also released in the first minutes of spore germination (23), and since germination is essential for spore viability, Tb 3ϩ -DPA fluorescence has been used to detect spore germination, and there is a good correlation between DPA release and spore viability (4,26,28,29,31,32). However, we report a flaw in the latter assay of spore viability, as Tb 3ϩ and some other multivalent metal cations strongly inhibit germination of coat-deficient spores. The coats comprise the outermost layer of spores of many species, including Bacillus subtilis, and protect spores against many agents, including enzymes and reactive chemicals (8,10,11,24).The B. subtilis strains used are isogenic derivatives of a laboratory 168 strain and are (i) PS533 (wild type), carrying plasmid pUB110, encoding resistance to kanamycin (22); (ii) PS3328 (⌬cotE mutant), in which much of the cotE coding sequence was replaced with a tetracycline resistance (Tc r ) cassette (15); and (iii) PS3738 (⌬safA mutant), which carries a Tc r cassette replacing much of the safA coding sequence (10). These strains were sporulated at 37°C on 2ϫ Schaeffer's-glucose (SG) agar plates without antibiotics, and spores were harvested and cleaned as described previously (14). The Bacillus cereus strain used was strain T obtained from H. O. Halvorson, and its spores were prepared at 30°C in a defined liquid medium and were purified as described previously (3). The Bacillus megaterium strain used was strain ATCC 12782, and its spores were prepared at 30°C in liquid supplemented nutrient broth and were harvested and cleaned as described previously (5). The Clostridium perfringens strain used was MRS101 (⌬cpe::catP mutant) in which the enterotoxin gene (cpe) in the C. perfringens food poisoning isolate S...

show abstract

“…Previously, we reported a related method where germinating endospores were enumerated in bulk suspension by luminescence spectroscopy (i.e., spectro-EVA), where Tb-DPA luminescence intensities were tabulated against a B. atrophaeus endospore calibration curve (28). Results obtained by a comparison of EVA to spectroEVA showed that the two methods are in good agreement (see Fig.…”

Section: Resultsmentioning

confidence: 53%

Fast Sterility Assessment by Germinable-Endospore Biodosimetry

Yung

Ponce

2008

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

The increased demand for sterile products has created the need for rapid technologies capable of validating the hygiene of industrial production processes. Bacillus endospores are in standard use as biological indicators for evaluating the effectiveness of sterilization processes. Currently, culture-based methods, requiring more than 2 days before results become available, are employed to verify endospore inactivation. We describe a rapid, microscopy-based endospore viability assay (EVA) capable of enumerating germinable endospores in less than 15 min. EVA employs time-gated luminescence microscopy to enumerate single germinable endospores via terbium-dipicolinate (Tb-DPA) luminescence, which is triggered under UV excitation as 10 8 DPA molecules are released during germination on agarose containing Tb 3؉ and a germinant (e.g., L-alanine). Inactivation of endospore populations to sterility was monitored with EVA as a function of thermal and UV dosage. A comparison of culturing results yielded nearly identical decimal reduction values, thus validating EVA as a rapid biodosimetry method for monitoring sterilization processes. The simple Tb-DPA chemical test for germinability is envisioned to enable fully automated instrumentation for in-line monitoring of hygiene in industrial production processes.The era of modern microbiology began in the 1870s when the life cycle of an endospore-forming pathogen, Bacillus anthracis, was elucidated using new methods for isolating pure cultures from single-cell clones on solid growth media. Bacterial endospores are dormant microbial structures that are highly resistant to chemical, physical, and radiation sterilization processes (2,6,19,26). In fact, Bacillus subtilis endospores have survived for 6 years in space while exposed to highvacuum conditions, temperature extremes, and intense solar and galactic radiation (12,18). Bacterial endospores are routinely employed as biological indicators (i.e., biodosimeters) to validate the effectiveness of sterilization methods (e.g., autoclaves) used in the medical device (15, 16), pharmaceutical, health care (5), food preparation, wastewater remediation (23), and biodefense (29) industries.The effectiveness of sterilization processes is measured and reported in terms of sterility assurance levels (SALs), which are defined as the expected probability that a product remains contaminated with viable microorganisms after exposure to a validated sterilization process. A sterilization process that yields predictable SALs is considered to be validated. Confidence in achieving a required SAL is obtained by the use of biological indicators that present a considerably greater population and resistance challenge than the expected bioburden (21), and the most effective way to test the efficiency of a sterilization process is to place biological indicators within and on test products of interest.Currently, endospore inactivation is quantified by measuring the log reduction in CFU. This method, however, requires several days of incubation, during which ...

show abstract

Applications of a Rapid Endospore Viability Assay for Monitoring UV Inactivation and Characterizing Arctic Ice Cores

Cited by 47 publications

References 49 publications

In situ microbial metabolism as a cause of gas anomalies in ice

In situ microbial metabolism as a cause of gas anomalies in ice

Efficient Inhibition of Germination of Coat-Deficient Bacterial Spores by Multivalent Metal Cations, Including Terbium (Tb ³⁺ )

Fast Sterility Assessment by Germinable-Endospore Biodosimetry

Contact Info

Product

Resources

About

Applications of a Rapid Endospore Viability Assay for Monitoring UV Inactivation and Characterizing Arctic Ice Cores

Cited by 47 publications

References 49 publications

In situ microbial metabolism as a cause of gas anomalies in ice

In situ microbial metabolism as a cause of gas anomalies in ice

Efficient Inhibition of Germination of Coat-Deficient Bacterial Spores by Multivalent Metal Cations, Including Terbium (Tb 3+ )

Fast Sterility Assessment by Germinable-Endospore Biodosimetry

Contact Info

Product

Resources

About

Efficient Inhibition of Germination of Coat-Deficient Bacterial Spores by Multivalent Metal Cations, Including Terbium (Tb ³⁺ )