Monitoring of active but non-culturable bacterial cells by flow cytometry
| Autor: | Karina Yew-Hoong Gin, Ramaiah Sachidanandham, Chit Laa Poh |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
DNA
Bacterial Time Factors Cell Survival Population Cell Count Bioengineering Biology medicine.disease_cause Applied Microbiology and Biotechnology Flow cytometry Microbiology Cell wall Freezing Escherichia coli medicine Seawater education education.field_of_study Staining and Labeling medicine.diagnostic_test Flow Cytometry biology.organism_classification Enterobacteriaceae Culture Media Staining Fecal coliform Microscopy Fluorescence Filtration Bacteria Biotechnology |
| Zdroj: | Biotechnology and Bioengineering. 89:24-31 |
| ISSN: | 1097-0290 0006-3592 |
| DOI: | 10.1002/bit.20304 |
| Popis: | Flow cytometric signatures (i.e., light scatter, red and green fluorescence) were obtained for the active but non-culturable (ABNC) cells of E. coli and a coliform isolate H03N1, in seawater microcosms using BacLight, a live-dead assay kit from Molecular Probes (Eugene/Portland, OR). Previous studies have reported that there are two major adaptations, which cells undergo during the formation of ABNC states: cell wall toughening and DNA condensation. Therefore, we hypothesized that the matured ABNC forms should be more resistant to extreme temperature treatments (i.e., by freezing in liquid nitrogen and thawing at room temperature) than the normal and transition populations. It was shown that the membrane-compromised cells (comprising of normal wild-type and dead cells which are less resistant to rapid freeze thaw) could be differentiated from the matured ABNC using BacLight staining and fluorescence detection by flow cytometry. The population of ABNC cells, which could not be cultured using m-FC media (for the enumeration of fecal coliforms), was resuscitated in phosphate buffer saline followed by growth in Luria broth. Flow cytometry was thus able to detect and differentiate the ABNC cells against a mixed population comprising of culturable cells, transition populations, and dead cells. The results also showed that the formation of ABNC is as early as 2 days in seawater microcosms. By directly comparing the coliform levels enumerated by the BacLight based flow cytometry assays and m-FC technique, it was shown that the presence of coliforms can be undetected by the membrane filtration method. |
| Databáze: | OpenAIRE |
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