The four bacterial strains used in this study were the L. monocytogenes AAL20066 (ser. 1/2a), AAL20074 (ser. 4b), AAL20105 (ser. 1/2c), and AAL20107 (ser. 1/2b), all isolated from fresh mixed salads and kindly provided by Dr. Andritsos (Athens Analysis Laboratories S.A.; Metamorfosi, Attica, Greece). All strains were kept frozen (at −80 °C) in Tryptone Soy Broth (TSB; Lab M, Heywood, Lancashire, UK) containing 15% glycerol and were resuscitated through streaking on to the surface of Trypticase Soy Agar (TSA; Condalab, Torrejón de Ardoz, Madrid, Spain) and incubating at 37 °C for 24 h (precultures). Working cultures were prepared by inoculating a colony from each preculture into 10 mL of fresh TSB and further incubating at 37 °C for 18 h. Bacteria from those final working cultures were sedimented by centrifugation (4000× g for 10 min at room temperature), washed twice with quarter-strength Ringer’s solution (Lab M), and finally suspended in the same solution, so as to present an absorbance at 600 nm (A600 nm) equal to 0.1 (ca. 108 CFU/mL). Those adjusted saline suspensions of each strain were finally mixed together and used for the subsequent attachment experiments.
Tryptone soy broth tsb
Manufactured by Lab M
Sourced in United Kingdom
Tryptone Soy Broth (TSB) is a general-purpose nutrient-rich medium used for the cultivation of a wide range of microorganisms. It provides the necessary nutrients and growth factors to support the growth of bacteria, yeasts, and other microbes.
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3 protocols using tryptone soy broth tsb
1
Preparation of Listeria monocytogenes Strains
2
Reviving Bacterial Strains for Experiments
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The two bacterial strains used in this research were the S. aureus DFSN_B26, isolated in our lab from non-pasteurized milk cheese and the S. epidermidis DFSN_B4 (C5M6), originally isolated from fermenting grape juice and kindly provided by Professor G.-J. Nychas (Agricultural University of Athens, Greece). Before their use in the subsequent experiments, both strains were stored frozen (at −80 °C) in Tryptone Soy Broth (TSB; Lab M, Heywood, Lancashire, UK) containing 15% glycerol in cryovials and was then each one revivified by streaking a loopful of its frozen culture on to the surface of Tryptone Soy Agar (TSA; Lab M) and incubating at 37 °C for 24 h (precultures). Working cultures were prepared by inoculating, using a microbiological loop, cells of a district and well isolated colony from each preculture into 10 mL of fresh TSB and incubating at 37 °C for 18 h. Bacteria from each final working culture were collected by centrifugation (4000× g for 10 min at RT), washed twice with quarter-strength Ringer’s solution (Lab M), and finally suspended in the same solution, so as to display an absorbance at 600 nm (A600 nm) equal to 0.1 (ca. 107 CFU/mL).
3
Antimicrobial Evaluation of Honey Dilutions
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For each honey, two dilutions (25 and 12.5 % v/v) were prepared using quarterstrength Ringer's solution (Lab M, Heywood, Lancashire, UK) as the diluent. Then, 40 μL of each dilution were placed in duplicate in wells (of 5 mm diameter) prepared in soft Tryptone Soy Agar (TSA; i.e., Tryptone Soy Broth [TSB; LabM] also containing 0.7% w/v agar) in a petri dish (of 90 mm diameter). In each petri dish, eight wells had been created with the help of an inverted Pasteur glass pipette. Before the creation of the wells, each soft agar medium had also been inoculated with the target microorganism (ca. 10 6 CFU/mL) and left to solidify in the dishes. Following the addition of the diluted honey samples to the wells, dishes were left for 2 h at room temperature and were then placed at 37 °C for 24 h (except for B. cereus, which was incubated at 30 °C). Soft TSA also contained 3% (w/v) NaCl in the case of alophile V. parahaemolyticus. Following incubation, the growth inhibition zones around each well were measured with the help of a ruler. Ampicillin (50 μg/μL) and corn glucose syrup (82% v/v; Haitoglou Bros SA, Kalochori, Thessaloniki, Greece) were used as positive and negative antimicrobial controls, respectively. The last one was selected because it has the average sugar content of honey. The experiment was repeated three times using independently grown bacterial cultures.
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