Bread samples (25 g) were transferred aseptically into individual stomacher bags (Seward Medical, Worthing, UK), containing 225 mL of sterile Peptone Water solution (0.1%) and homogenized in a stomacher (Lab Blender 400, Seward Medical, Worthing, UK) for 60 s. For each sample, appropriate serial decimal dilutions were prepared in Peptone Water solution (0.1%). The amount of 0.1 mL of these serial dilutions of bread homogenates was spread on the surface of dry media. The following groups of microflora were determined according to official protocols [26 ]: Total Viable Counts (TVC), yeasts/molds and Bacillus cereus. TVC were determined using Plate Count Agar (LABM, Heywood, UK) after incubation at 30 °C for 3 days. Yeasts and molds were enumerated using Rose Bengal Chloramphenicol Agar (LABM, Heywood, UK) after incubation at 25 °C for 3 and 5 days respectively in the dark. Finally, Bacillus cereus was enumerated using mannitol–egg yolk–polymyxin (MYP) agar (LABM, Heywood, UK) after incubation at 30 °C for 24 h. All plates were examined visually for typical colony types and morphological characteristics associated with each growth medium. In addition, the selectivity of each medium was checked routinely by Gram staining and microscopic examination of smears prepared from randomly selected colonies from all of the media.
Plate count agar
Manufactured by Lab M
Sourced in United Kingdom
Plate Count Agar is a culture medium used in microbiological testing to enumerate the number of viable bacteria in a sample. It provides a standardized environment for the growth and enumeration of bacteria, allowing for the quantification of microbial populations.
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Lab products found in correlation
Rose bengal chloramphenicol agar, by Lab M (2 mentions)
Palcam agar, by Merck Group (1 mentions)
Violet red bile glucose agar, by Thermo Fisher Scientific (1 mentions)
Palcam agar base, by Lab M (1 mentions)
Sterile quarter strength ringer s solution, by Lab M (1 mentions)
Pseudomonas agar base, by Lab M (1 mentions)
Eosin methylene blue agar, by Lab M (1 mentions)
Tryptic soy agar, by Lab M (1 mentions)
5 protocols using plate count agar
1
Bread Microbiological Analysis Protocol
2
Salmonella Typhimurium Sensitivity Test
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The sensitivity test was carried out using Salmonella Typhimurium ATCC 14028 (Oxoid Ltd, Basingstoke, UK). The strain was revitalized by incubation in 10 ml of Tryptone Soy Broth medium (Lab M Ltd, Heywood, UK) for 24 h at 37°C. Furthermore, the strain was standardized by determining the bacterial load with the method of serial dilutions and sowing by inclusion on Plate Count Agar (Lab M Ltd, Heywood, UK) that yielded about 108 CFU/ml. An aliquot (2 ml) of strain was centrifuged at 14 000 rpm for 10 minutes, the pellet was suspended in 1 ml of distilled water and centrifuged at 14 000 rpm for 10 min, and the pellet was suspended in 200 μl of 6% Chelex. The suspension was incubated for 8–10 min at 100°C, finally placed on ice for 1 minute. The suspension was then centrifuged (5 min at 14 000 rpm) to allow the Chelex, cell components, membrane residues and cell wall to settle to the bottom of the tube. 100 μl of the supernatant was carefully transferred to a new tube and used as a template in molecular methods.
3
Microbial Analysis of UV-C Treated Juices
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Microbial analyses were carried out for untreated and UV-C treated juices.
For total mesophylls and yeasts and molds enumeration, juice decimal dilutions were carried out in buffered peptone water. Total mesophylls were assessed in duplicate, using Plate Count Agar (Lab M, Lancashire, UK). Samples were incubated at 37 °C, during 48 hours. Yeasts and molds were determined also in duplicate using Rose Bengal Agar (Lab M, Lancashire, UK). Samples were incubated at 25 °C for 60 h.
A. acidoterrestris spores were enumerated according to Silva, Gibbs, and Silva (2000) , by spread plating the diluted samples onto Bacillus acidoterrestris agar (pH 4). The plates were incubated at 45 °C (Sanyo MIR-262) for 2-3 days. Microbial counts were performed in triplicate.
L. innocua was quantified in duplicate through decimal dilutions and using Palcam agar containing selective supplement (Merck, Darmstadt, Germany). Samples were incubated at 30°C for 3 days.
Enumerations were expressed as CFU per mL of juice.
For total mesophylls and yeasts and molds enumeration, juice decimal dilutions were carried out in buffered peptone water. Total mesophylls were assessed in duplicate, using Plate Count Agar (Lab M, Lancashire, UK). Samples were incubated at 37 °C, during 48 hours. Yeasts and molds were determined also in duplicate using Rose Bengal Agar (Lab M, Lancashire, UK). Samples were incubated at 25 °C for 60 h.
A. acidoterrestris spores were enumerated according to Silva, Gibbs, and Silva (2000) , by spread plating the diluted samples onto Bacillus acidoterrestris agar (pH 4). The plates were incubated at 45 °C (Sanyo MIR-262) for 2-3 days. Microbial counts were performed in triplicate.
L. innocua was quantified in duplicate through decimal dilutions and using Palcam agar containing selective supplement (Merck, Darmstadt, Germany). Samples were incubated at 30°C for 3 days.
Enumerations were expressed as CFU per mL of juice.
4
Enumeration of Microbial Flora in Ham Slices
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Samples (10 g) of ham slices were weighed aseptically, added to sterile quarter strength Ringer’s solution (LabM, Lancashire, UK) (90 mL), and homogenized in a stomacher (Stomacher 400, Circulator, Seward) for 60 s at room temperature. The resulting suspensions were serially diluted in the same diluent and 1 or 0.1 mL samples of the appropriate dilutions were poured or spread, respectively, on the following agar media: de Man–Rogosa–Sharp Agar (MRS, Oxoid, Hampshire, UK) for LAB, incubated at 30 °C for 72 h; Plate Count Agar (LabM, Lancashire, UK) for TVC, incubated at 30 °C for 48 h; STAA Agar Base (Oxoid, Hampshire, UK) for Brochothrix thermosphacta, incubated at 25 °C for 48 h; Rose Bengal Chloramphenicol Agar (LabM, Lancashire, UK) for yeasts/molds incubated at 25 °C for 5 days; Violet Red Bile Glucose Agar (Oxoid, Hampshire, UK) for Enterobacteriaceae, incubated at 37 °C for 24 h, Pseudomonas Agar Base (LabM, Lancashire, UK), for Pseudomonas spp. incubated at 25 °C for 48 h, as well as Palcam Agar Base (LabM, Lancashire, UK), for Listeria spp. incubated at 30 °C for 48 h.
5
Isolation of Lytic Phages from Wastewater
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Two wastewater samples of about one L were collected from two locations of the municipal sewer system of Trabzon, Turkey. For primary isolation of gram-negative bacteria, the wastewater samples were diluted and plated on eosin methylene blue agar, tryptic soy agar, and plate count agar (Lab M, Lancashire, UK). The cultures were incubated at 37°C for 24-48 hours. The bacterial colonies with distinct morphology were purified, gram stained, and used as hosts for lytic phage isolation. The same wastewaters were used for the isolation of lytic phages using the culture-enrichment method described by Lin et al8 (link) with some modifications. One hundred twenty-five milliliters of each wastewater was mixed with 125 mL of double-concentrated Luria-Bertani (LB) broth (Lab M) and incubated at 37°C for 24 hours with shaking at 150 rpm. The cultures were centrifuged at 8000 ×g for 10 minutes at 4°C, and the supernatants were filtered using 0.22 μm pore size filters. The phage lysates were stored at 4°C in the dark with one drop of chloroform until use.
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