Quantification of Bacterial Polyhydroxybutyrate Content by Flow Cytometry
流式细胞术定量分析细菌聚羟基丁酸酯含量
发布: 2017年12月05日第7卷第23期 DOI: 10.21769/BioProtoc.2638 浏览次数: 8049
评审: Valentine V TrotterPaula María TribelliAnonymous reviewer(s)
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The authors used this protocol in:
Mar 2017
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Abstract
We describe here a detailed protocol for the quantification of the intracellular content of polyhydroxybutyrate (PHB) in a population of bacterial cells by flow cytometry, which is based on a consensus of several previously reported works.
Keywords: Polyhydroxybutyrate (聚羟基丁酸酯)
PHB (PHB)
Bacteria (细菌)
Flow cytometry (流式细胞术)
Nile red (尼罗红)
Background
Upon nutrient exhaustion and in the presence of an available carbon source, several bacterial species (e.g., Cupriavidus necator, Sinorhizobium meliloti, species of Pseudomonas) switch on the accumulation of the neutral polymer of β-hydroxybutyrate, polyhydroxybutyrate (PHB), for storage of carbon and reducing power (Jendrossek and Pfeiffer, 2014). Classical methods for quantification of bacterial PHB content are based on its initial depolymerization, followed by the quantitative detection of specific chemical derivatives of β-hydroxybutyrate by UV spectrophotometry or gas chromatography (Law and Slepecky, 1961; Braunegg et al., 1978). More recently, the application of the fluorochrome Nile red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) (Greenspan and Fowler, 1985) to stain intracellular PHB granules (Müller et al., 1993) led to the development of novel rapid fluorometric methods to quantify the content of the polymer in bacterial samples. Subsequently, coupling of Nile red–based PHB staining to flow cytometry made possible the quantification of the polymer content at a single-cell level within bacterial populations (Gorenflo et al., 1999; Kacmar et al., 2006; Tyo et al., 2006; Alves et al., 2017; Lagares Jr. et al., 2017). We report here a detailed protocol that summarizes the consensus steps for the quantification of PHB in bacterial cells by Nile red staining coupled to flow cytometry.
Materials and Reagents
- Pipette tips (2-200 µl and 100-1,000 µl tips)
- 1.5 ml Eppendorf tubes
- 12 x 75-mm Falcon® 5 ml uncapped Round Bottom Polystyrene Test tubes (Corning, catalog number: 352052 )
- Bacterial samples for analysis
Note: The bacteria can be harvested from multiple sources (e.g., liquid cultures, colonies on semisolid agar plates, natural environments). However, it must be taken into account that adaptations in the steps for cell preparation should be considered, depending on the specific source of the cells. - Absolute ethanol (e.g., Sigma-Aldrich, catalog number: E7023 )
- Sterile deionized water (e.g., Sigma-Aldrich, catalog number: W3500 )
- Sodium chloride (NaCl) (e.g., Sigma-Aldrich, catalog number: S9888 )
- Potassium chloride (KCl) (e.g., Sigma-Aldrich, catalog number: P3911 )
- Sodium phosphate dibasic (Na2HPO4) (e.g., Sigma-Aldrich, catalog number: S9763 )
- Potassium phosphate monobasic (KH2PO4) (e.g., Sigma-Aldrich, catalog number: P0662 )
- Dimethyl sulfoxide (DMSO) (e.g., Sigma-Aldrich, catalog number: D8418 )
- Nile red stain (e.g., Sigma-Aldrich, catalog number: N3013 )
- 1 N HCl and 1 N NaOH solutions for pH adjustments
- Cytometer sheath fluid (e.g., BD FACSFlowTM solution, BD, catalog number: 342003 )
- Full-strength bleach (for waste disposal)
- Phosphate-buffered saline (PBS; see Recipes for 10x and 1x solution preparation)
- Permeabilization solution (see Recipes)
- Nile red stock solution (see Recipes)
Equipment
- Pipets (e.g., Gilson Pipetman Classic) (Gilson, models: P20 , P200 , P1000 )
- Centrifuge for 1.5 ml Eppendorf tubes (e.g., Eppendorf, model: 5424 , catalog number: 5424000010)
- pH meter
- Spectrophotometer suitable for measurement of optical density at 600 nm (OD600)
- FACSCalibur flow cytometer (BD, model: FACSCALIBUR ) equipped with an excitation laser of 488-nm-wavelength and an emission filter with bandwidth of 585 ± 45 nm
Note: The suitability of other flow cytometers than FACSCalibur (BD, USA) for this assay is subjected to the availability of a proper excitation laser source and of the filter for the emitted fluorescence, as indicated above.
Software
- Flow cytometer acquisition software
- FlowJo [FlowJo, USA]
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Lagares Jr., A. and Valverde, C. (2017). Quantification of Bacterial Polyhydroxybutyrate Content by Flow Cytometry. Bio-protocol 7(23): e2638. DOI: 10.21769/BioProtoc.2638.
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分类
微生物学 > 微生物新陈代谢 > 其它化合物
微生物学 > 微生物生物化学 > 其它化合物
细胞生物学 > 基于细胞的分析方法 > 流式细胞术
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