A weighted amount (25 g) of cream sample was transferred aseptically to a sterile stomacher bag (Seward Medical, London, UK) containing 225 mL of sterilized quarter-strength Ringer’s solution (Lab M Limited, Lancashire, UK) and homogenized in a Stomacher device (Lab Blender 400, Seward Medical) for 1 min at room temperature. For the enumeration of total mesophiles (i.e., total viable counts (TVC)), appropriate serial decimal dilutions in Ringer’s solution were surface plated on tryptic glucose yeast agar (Biolife, Milan, Italy) and colonies were counted after incubation of the plates at 30 °C for 48 h. The obtained microbiological data were expressed as log (colony forming units) per gram of cream (log CFU/g).
Quarter strength ringer s solution
Manufactured by Lab M
Sourced in United Kingdom
Quarter-strength Ringer's solution is a sterile, isotonic electrolyte solution. It contains a balanced mix of sodium, potassium, and calcium ions, similar to body fluids. This solution is commonly used in laboratories to maintain the physiological environment for various applications.
Automatically generated - may contain errors
Lab products found in correlation
Tryptone soy broth tsb, by Lab M (3 mentions)
Tryptic glucose yeast agar, by Biolife (1 mentions)
Heraeus multifuge 1s r, by Thermo Fisher Scientific (1 mentions)
Plate count agar, by Biolife (1 mentions)
Trypticase soy agar, by Condalab (1 mentions)
Tryptone soy agar tsa, by Lab M (1 mentions)
Pseudomonas agar base, by Lab M (1 mentions)
8 protocols using quarter strength ringer s solution
1
Enumeration of Cream Microbiota
2
Characterization of Listeria monocytogenes Strains
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The two tested L. monocytogenes strains were the foodborne AAL20066 (ser. 1/2a) and AAL20074 (ser. 4b) isolates deposited in the microbial culture collection of the Microbiology Laboratory in Athens Analysis Laboratories S.A. (AAL). Both strains were previously recovered from mixed fresh salads and were kept frozen long-term (at −80 °C) in Trypticase Soya Broth (TSB; Condalab, Torrejón de Ardoz, Madrid, Spain) containing 15% (v/v) glycerol. When needed for the experiments, each strain was streaked on to the surface of Tryptone Soya Agar (TSA; Oxoid, Thermo Fisher Specialty Diagnostics Ltd., Hampshire, UK) and incubated at 37 °C for 24 h (preculture). 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 each of those final working cultures were collected by centrifugation (2000× g for 10 min at 4 °C), washed once with quarter-strength Ringer’s solution (Lab M, Heywood, Lancashire, UK), and finally suspended in 5 mL of the same solution (ca. 109 CFU/mL). The purity of each cellular working suspension was verified through streaking on TSA plates.
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.
4
Evaluating Bacterial Growth in Microbial Supernatant
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The growth behavior of the tested S. Enteritidis, S. Typhimurium 4/74 and St. aureus strains was evaluated in LBG broth (pH 6.5) containing: (i) no microbial supernatant (Control); (ii) 20% (v/v) microbial supernatant (MS); and (iii) 20% (v/v) microbial supernatant that was subjected to heat treatment (MS-HT), as described in Section 2.2. The 18-h culture of each one of the strains was centrifuged (Heraeus Multifuge 1S-R, Thermo Electron Corporation) at 5000 ×g for 10 min at 4 °C. The harvested cells were washed with quarter strength Ringer's solution (Lab M Limited) and centrifuged under the same conditions. The harvested cells of the washed cultures were finally resuspended in 10 ml of LBG of the above characteristics (Control, MS or MS-HT), and 300-μl aliquots of appropriate dilution(s) (conducted in the same medium) were transferred in 96-well polystyrene microplates (Nuova Aptaca S.r.l., Canelli, Italy). The growth kinetic parameters of the tested strains were estimated from optical density (OD) measurements, with the latter being taken at 600 nm using the Synergy HT automated spectrophotometer. The microplates were placed in the spectrophotometer at an incubation temperature of 25 °C, and OD measurements were Bassler et al., 1997 taken at 15-min intervals after agitation of the microplates for 10 s at medium amplitude.
5
Quantifying Microbial Populations in Marinated Chicken
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A portion of 25 g of marinated chicken souvlaki (chicken thigh fillet, sodium chloride, sodium acetate, sodium citrate, enzyme tenderizer, and ascorbic acid) was transferred aseptically to a Stomacher bag containing 225 mL of sterile quarter-strength Ringer’s solution (Lab M Limited, Lancashire, UK) and was homogenized by a Stomacher device (Lab Blender 400, Seward Medical, UK) for 60 s. From this 1:10 sample solution, serial decimal dilutions were prepared using the same diluent, and 0.1 mL of the appropriate dilution was spread to the following media: (a) tryptic glucose yeast agar (Plate Count Agar, Biolife, Milan, Italy) for the enumeration of total viable counts (TVCs), when incubated at 25 °C for 72 h; (b) Pseudomonas agar base (LAB108 supplemented with selective supplementation with Cetrimide Fucidin Cephaloridine, Modified C.F.C. X108, LABM) for the determination of the presumptive Pseudomonas spp. counts, when incubated at 25 °C for 48 h.
6
Preparation of Listeria monocytogenes Strains
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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.
7
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).
8
Quantifying Pseudomonas on Chicken Meat
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Four slices with about 2-mm thickness of chicken breast and thigh fillets were aseptically removed using a sterile stainless steel cork borer (diameter: 2.5 cm), scalpel, and forceps. The contaminated surface (external fillet surface) of each slice was approximately 5 (4.91) cm2. The four slices with a total contaminated surface of approximately 20 (19.64) cm2, were added to 100 mL of sterile quarter strength Ringer's solution (Lab M Limited, Lancashire, UK) and homogenized in a Stomacher device (Lab Blender 400, Seward Medical, UK) for 120s at room temperature. About 0.1 mL of the appropriate decimal dilution was spread on Pseudomonas Agar Base supplemented with cephalothin-fucidin-cetrimide (LabM Limited) and incubated at 25°C for 48 h. After incubation, typical colonies of presumptive Pseudomonas spp. were enumerated, and the populations were expressed as log cfu/cm2. The selectivity of the medium was checked routinely by Gram staining and microscopic examination of smears prepared from randomly selected colonies.
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