Minimal Bactericidal Concentration for Biofilms (MBC-B)
体外微量滴定测定法测定生物膜的最小杀菌浓度(MBC-B)
发布: 2014年05月05日第4卷第9期 DOI: 10.21769/BioProtoc.1115 浏览次数: 13591
评审: Fanglian HeAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2013
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Abstract
A biofilm is a multicellular consortium of surface associated microbes surrounded by a hydrated, extracellular polymer matrix. The biofilm matrix plays a critical role in preventing desiccation, acquiring nutrients, and provides community protection from environmental assaults. Importantly, biofilms are significantly more resistant to antimicrobials relative to their free-swimming counterparts. The level of antimicrobial tolerance is influenced by a number of factors, including genetic/adaptive resistance mechanisms, stage of biofilm development, and pharmacokinetics of the antibiotic. Here, we describe an in vitro microtiter-based assay to quantify the minimal bactericidal concentration for biofilms (MBC-B) for short exposure times (2 h). This exposure period is significantly shorter than standard over-night and 24-hour treatments described in traditional protocols. This assay was developed to approximate the time an antibiotic is available during a one-time treatment before it is metabolized, sequestered by host proteins, or digested.
Materials and Reagents
- Glycerol stock of Pseudomonas aeruginosa (P. aeruginosa) (acquired from a single colony, stored at -80 °C)
- Bacto-tryptone (BD Biosciences, catalog number: 211705 )
- Bacto-yeast extract (BD Biosciences, catalog number: 212750 )
- Bacto-agar (BD Biosciences, catalog number: 214010 )
- Sodium chloride (Sigma-Aldrich, catalog number: S7653 )
- 10x PBS stock (Life Technologies, Gibco®, catalog number: 70011-044 )
- Colistin sodium sulfate (Sigma-Aldrich, catalog number: C4461 )
- Polymyxin sulfate salt (Sigma-Aldrich, catalog number: P4932 )
- Tobramycin (Sigma-Aldrich, catalog number: T4014 )
- Ciprofloxacin (Sigma-Aldrich, catalog number: 17850 )
- LB medium for agar plates (see Recipes)
- 1% TB liquid medium (see Recipes)
Equipment
- Glass beads (425-600 µm, sterile) (Sigma-Aldrich, catalog number: G8772 )
- Aluminum film (sterile) (Sigma-Aldrich, catalog number: Z722642 )
- 15 ml polystyrene culture tubes (sterile) (VWR International, catalog number: 89497-782 )
- Falcon 96 well polystyrene plates (flat-bottom, sterile with lid, non-treated) (Corning Incorporated, catalog number: 351172 )
- Spectrophotometer to measure absorbance of cell culture (OD600)
- 37 °C shaking and static incubators
- Titer plate shaker (Thermo Fisher Scientific, catalog number: 4625-Q )
- Multichannel pipette (volume ranges 10 μl-200 μl)
- Sterile plastic loops (1 μl) (Santa Cruz, catalog number: SC-200266 )
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Billings, N. and Ribbeck, K. (2014). Minimal Bactericidal Concentration for Biofilms (MBC-B). Bio-protocol 4(9): e1115. DOI: 10.21769/BioProtoc.1115.
- Billings, N., Millan, M., Caldara, M., Rusconi, R., Tarasova, Y., Stocker, R. and Ribbeck, K. (2013). The extracellular matrix component Psl provides fast-acting antibiotic defense in Pseudomonas aeruginosa biofilms. PLoS Pathog 9(8): e1003526.
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分类
微生物学 > 微生物生物膜 > 杀伤试验
生物化学 > 其它化合物 > 抗菌剂
微生物学 > 微生物细胞生物学 > 细胞活力
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