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Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds
Published on: January 14, 2017
A comparative study between overlay method and selective-differential media for recovery of stressed Enterobacter
Murad A Al-Holy1, Mengshi Lin, Hamzah M Al-Qadiri
1Department of Clinical Nutrition and Dietetics, Faculty of Allied Health Sciences, Hashemite University, P.O. Box 150459, Zarqa-Jordan, Jordan. murad@hu.edu.jo
Abstract:
This study compares the performance of different selective-differential media with the overlay method for recovery of stressed cells of Enterobacter sakazakii from infant formula milk (IFM). Five different selective-differential media were used in this study: OK medium, violet red bile agar (VRBA), Druggan-Forsythe-Iversen agar (DFI), Enterobacteriaceae enrichment (EE) agar, and fecal coliform agar (FCA). Tryptic soy agar supplemented with 0.1% sodium pyruvate (TSAP) was used as a control. The overlay method involved applying a thin layer (8ml) of each of the selective media onto TSAP after spreading a sample onto TSAP. Reconstituted IFM was inoculated by ca 1x10(7)CFU/ml of a mixture of four strains of E. sakazakii and subjected to different stress conditions: heat (55 degrees C for 10min), a freeze-thaw cycle (-20 degrees C for 24h, thawed at room temperature, frozen again at -20 degrees C, and thawed), acidic pH (pH 3.56 for 15min), alkaline pH (pH 11.04 for 15min), and desiccation (E. sakazakii was inoculated onto powdered IFM at a level of ca 1x10(6)CFU/g, held at 21 degrees C, water activity of the inoculated product was 0.29 and examined at 0, 15, and 30d). No major differences were noticed between the control (TSAP) and the overlay methods. However, the overlay method recovered significantly higher numbers of stressed E. sakazakii cells compared to selective-differential media. Also, the selective-differential media exhibited some variability in terms of their capabilities to recover stressed cells of E. sakazakii. Among all the examined selective-differential media, DFI performed better for recovering stressed E. sakazakii cells. This study suggests that the overlay method may serve as a potential alternative to direct selective plating for best recovery of E. sakazakii from IFM.
Insights
The overlay method significantly improved recovery of stressed Enterobacter sakazakii from infant formula milk compared to direct plating. Druggan-Forsythe-Iversen agar showed the best performance among selective media for detecting these stressed bacterial cells.
Area of Science:
- Food Microbiology
- Bacteriology
- Analytical Methods
Background:
- Enterobacter sakazakii poses a risk in infant formula milk (IFM).
- Accurate detection of stressed Enterobacter sakazakii is crucial for food safety.
- Traditional selective media may have limitations in recovering stressed bacterial cells.
Purpose of the Study:
- To compare the efficacy of different selective-differential media and the overlay method for recovering stressed Enterobacter sakazakii from IFM.
- To identify the most effective method for detecting stressed Enterobacter sakazakii in infant formula.
Main Methods:
- Five selective-differential media (OK, VRBA, DFI, EE, FCA) and an overlay method were evaluated.
- Infant formula milk was artificially contaminated with stressed Enterobacter sakazakii (heat, freeze-thaw, acid, alkali, desiccation).
- Recovery rates were compared between direct plating on selective media and the overlay technique.
Main Results:
- The overlay method demonstrated significantly higher recovery of stressed Enterobacter sakazakii compared to direct plating on selective media.
- Variability was observed in the performance of different selective-differential media.
- Druggan-Forsythe-Iversen agar (DFI) exhibited superior performance among the tested selective media for stressed cell recovery.
Conclusions:
- The overlay method is a promising alternative to direct selective plating for enhanced recovery of stressed Enterobacter sakazakii from IFM.
- Optimized methods are needed to ensure accurate detection of potentially viable but stressed bacteria in infant formula.
