Evaluation of microbial culture techniques for the isolation of Pythium insidiosum from equine tissues.
Abstract: The purpose of this study was to evaluate the effects of sample handling, storage, and culture techniques on the isolation of Pythium insidiosum from infected equine tissues. Tissue and kunker samples obtained immediately posteuthanasia from a horse with subcutaneous pythiosis were used to assess the effects of sample type (kunkers vs. tissues), media type (selective vs. nonselective), storage technique, and storage time on P. insidiosum isolation rate. Overall, isolation rates were higher from fresh kunkers (94.6%) and stored kunkers (76.4%) than from fresh tissues (8.3%) or stored tissues (4.6%). Isolation of P. insidiosum also occurred more often on antibiotic-containing media than on nonselective media for both fresh and stored samples. For samples that were stored for 1-3 days prior to culture, P. insidiosum isolation rates were highest for the following techniques: kunkers stored at room temperature and plated on selective media (100%), kunkers stored at 4 C and then plated on either nonselective (91.7%) or selective (95.8%) media, kunkers stored on cold packs and then plated on either nonselective (93.8%) or selective (100%) media, kunkers stored in ampicillin solution and plated on selective media (100%), and kunkers stored in ampicillin/gentocin solution and plated on selective media (87.5%). For samples stored for 4-5 days, P. insidiosum isolation rates were highest for kunkers stored at 4 C and then plated on either nonselective (81.3%) or selective (87.5%) media, kunkers stored in ampicillin solution and then plated on selective media (87.5%), and kunkers stored in ampicillin/gentocin solution and plated on selective media (87.5%). Results of this study suggest that optimal isolation rates of P. insidiosum from infected equine tissues are achieved by culturing fresh kunkers on selective media. For samples that cannot be processed immediately, acceptable handling techniques include storage at room temperature for up to 3 days, refrigeration for up to 5 days, shipping on cold packs, and storage in antibiotic solution, each combined with subsequent inoculation on selective media.
Publication Date: 2002-08-03 PubMed ID: 12152807DOI: 10.1177/104063870201400403Google Scholar: Lookup
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- Journal Article
Summary
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The study aims to understand the effects of storage, handling, and cultural methods on the isolation of Pythium insidiosum, a pathogen, from equine tissues. The researchers found that the most effective way was through culturing fresh kunkers on selective media; however, if immediate processing isn’t possible, storing samples at room temperature for a maximum of 3 days, refrigerating for up to 5 days, or using cold packs and antibiotic solution, accompanied by inoculation on selective media provided satisfactory results.
Research Methodology
- The study used tissue and kunker samples from a horse with subcutaneous pythiosis collected shortly after euthanasia.
- Several factors were examined in this study, including the type of sample (kunkers vs tissues), the kind of media used (selective vs non-selective), storage techniques, and time of storage for isolation of P. insidiosum.
Findings of the Study
- Fresh kunkers yielded the highest isolation rates at 94.6%, followed by stored kunkers at 76.4%.
- Fresh tissues had an isolation rate of 8.3%, with stored tissues trailing at 4.6%.
- P. insidiosum was more frequently isolated on antibiotic-containing selective media than on non-selective media for both fresh and stored samples.
- For samples kept for 1-3 days before culturing, the highest isolation rates were observed in kunkers stored at room temperature, refrigerated at 4C, stored on cold packs, or stored in ampicillin solutions and then plated on selective media.
- For longer storage periods of 4-5 days, highest isolation rates were seen in kunkers stored in refrigerated conditions at 4C or those stored in ampicillin solutions.
Recommendations
- The most optimal isolation rate of P. insidiosum from infected equine tissues can be achieved by culturing fresh kunkers on selective media.
- For samples that cannot be processed promptly, suggested handling techniques include storage at room temperature for up to 3 days, refrigeration for up to 5 days, shipping on cold packs, and storage in antibiotic solution.
- All these stored samples should be subsequently inoculated on selective media.
Cite This Article
APA
Grooters AM, Whittington A, Lopez MK, Boroughs MN, Roy AF.
(2002).
Evaluation of microbial culture techniques for the isolation of Pythium insidiosum from equine tissues.
J Vet Diagn Invest, 14(4), 288-294.
https://doi.org/10.1177/104063870201400403 Publication
Researcher Affiliations
- Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge 70803-8410, USA.
MeSH Terms
- Animals
- Culture Media
- Evaluation Studies as Topic
- Female
- Horse Diseases / diagnosis
- Horse Diseases / microbiology
- Horses / microbiology
- Microbiological Techniques / standards
- Pythium / isolation & purification
- Pythium / pathogenicity
- Specimen Handling
- Temperature
Citations
This article has been cited 18 times.- Yolanda H, Lohnoo T, Rujirawat T, Yingyong W, Kumsang Y, Sae-Chew P, Payattikul P, Krajaejun T. Selection of an Appropriate In Vitro Susceptibility Test for Assessing Anti-Pythium insidiosum Activity of Potassium Iodide, Triamcinolone Acetonide, Dimethyl Sulfoxide, and Ethanol. J Fungi (Basel) 2022 Oct 24;8(11).
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