The antimicrobial activity of the impregnated cotton disks was performed accordingly to the ISO 20743 standard [19 ]. Briefly, inocula were grown overnight, at 37 °C, in Tryptic Soy Broth (Biokar Diagnostics, Beauvais, France) after which the bacterial load was adjusted to a concentration between 1 × 105 to 3 × 105 CFU/mL. Following this, 200 µL of adjusted inoculum was added to 0.40 g (± 0.05 g) of cotton dyed with blue navy everzol NPs (25 mg/mL). Simultaneously, two controls were accessed; one with non-dyed cotton the other with cotton dyed with navy blue everzol at 25 mg/mL. Viable counts were determined at 0 and 24 h and plated by the drop method as previously described by Costa, Silva [20 (link)] in Plate Count Agar (Biokar Diagnostics, Beauvais, France). Plates were then incubated at 37 °C for 24 h and the results were given in log of CFU. All assays were done in sextuplicate.
Plate count agar
Manufactured by Solabia
Sourced in France
Plate Count Agar is a microbiology culture medium used for the enumeration of bacteria in samples. It provides a nutrient-rich environment for the growth and isolation of heterotrophic bacteria. The agar supports the development of bacterial colonies, which can then be counted to determine the total bacterial population.
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Lab products found in correlation
Tryptic soy broth (tsb), by Solabia (1 mentions)
Peptone water, by Thermo Fisher Scientific (1 mentions)
Chloramphenicol glucose agar, by Solabia (1 mentions)
Metaphosphoric acid, by Merck Group (1 mentions)
Peptone, by Solabia (1 mentions)
2 4 6 tris 2 pyridyl s triazine, by Merck Group (1 mentions)
Tryptone soy broth (tsb), by Solabia (1 mentions)
Palcam base agar, by Solabia (1 mentions)
Gallic acid, by Merck Group (1 mentions)
Sodium carbonate, by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
2 2 azino bis 3 ethylbenzothiazoline 6 sulfonic acid diammonium salt, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Violet red bile glucose agar, by Bio-Rad (1 mentions)
Baird parker medium, by Merck Group (1 mentions)
Man rogosa sharpe agar, by Merck Group (1 mentions)
Tryptone salt broth, by Solabia (1 mentions)
Mrs agar, by Solabia (1 mentions)
7 protocols using plate count agar
1
Antimicrobial Activity of Cotton Impregnated with Everzol NPs
2
Microbial Quality Assessment of Strawberries
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The microbiological quality of strawberry samples was determined by counting aerobic mesophilic bacteria, psychrotrophic bacteria [27 ], and yeasts and molds [28 ]. Ten grams of each sample was transferred to 90 mL of peptone water (Oxoid) and homogenized by a homogenizator classic/panoramic (IUL Instruments, Barcelona, Spain) and consequently diluted 10 times. To determine the counts of yeasts and molds, Chloramphenicol Glucose Agar (Biokar, Paris, France) was used, and to determine psychrotropic and aerobic mesophilic bacteria, Plate Count Agar (Biokar, Paris, France) was used. The incubation temperature of agar plates for yeasts and molds was 25 ± 1 °C for 48–72 h, 30 ± 1 °C for 24–72 h for aerobic mesophilic bacteria and 6.5 ± 1 °C for 5 to 10 days for psychrotrophic bacteria. Results were expressed as Log10 colony-forming units (CFU) per gram fresh weight [29 (link)].
3
Listeria Detection and Antimicrobial Analysis
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Tryptone soy broth (TSB), tryptone soy agar (TSA), Palcam base agar, Palcam selective supplement for Listeria, yeast extract, plate count agar (PCA), Dichloran Rose Bengali Chloramphenicol Agar (DRBC), and peptone were obtained from Biokar Diagnostics (Allonne, France), and Dew-Engley medium was obtained from Merck (Darmstadt, Germany).
Ascorbic acid, gallic acid, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), sodium carbonate, metaphosphoric acid, acetic acid, and 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was obtained from Sigma-Aldrich (Steinheim, Germany). Methanol, acetone, chlorhidric acid (37%), sodium acetate, sodium hydroxide, sodium chloride, potassium chloride, ferric chloride hexahydrate, and Folin–Ciocalteu’s reagent were procured by Panreac (Llinars del Valles, Spain).
Ascorbic acid, gallic acid, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), sodium carbonate, metaphosphoric acid, acetic acid, and 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was obtained from Sigma-Aldrich (Steinheim, Germany). Methanol, acetone, chlorhidric acid (37%), sodium acetate, sodium hydroxide, sodium chloride, potassium chloride, ferric chloride hexahydrate, and Folin–Ciocalteu’s reagent were procured by Panreac (Llinars del Valles, Spain).
4
Microbial Load Quantification Protocol
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Mesophilic aerobic counts were carried out according to AFNOR NF V08-051 on plate count agar (Biokar, France). Inoculation was performed by incorporating 1 mL of inoculum from each dilution. Coliforms were counted in accordance with AFNOR standard, NF ISO 4832 July 1991; inoculation was carried out by incorporation onto Violet Red Bile Glucose Agar (Bio-Rad, France). Sulfite-reducing bacteria were counted by incorporation into test tubes containing Tryptone Sulfite Neomycin Agar (Bio-Rad, France). Streptococci were enumerated on Bile-Esculin-Azide Agar (Bio-Rad, France). Inoculation was carried out by incorporating 1 mL of inoculum from each dilution. Dichloran Rose Bengal Chloramphenicol (Biokar, France) was used for yeast counts. Inoculation was carried out by spreading according to standard NF V08-059.
The various microbial loads, expressed in cfu/mL, were calculated using the formula of ISO 7218.
The various microbial loads, expressed in cfu/mL, were calculated using the formula of ISO 7218.
5
Comprehensive Microbiological Analysis Methods
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Microbiological analysis was performed according to the standard methods. The buffer solution (peptone water containing 0.03g/L of tween 80) was used to revive the bacteria. Plate Count Agar (Biokar Diagnostics, France) was used to enumerate the total Mesophilic Aerobic Microorganisms on Petri dish after incubation at 30°C for 72 h. Yeasts and molds were enumerated on Sabouraud Agar containing Chloramphenicol (Sigma Aldrich, India) following incubation at 25°C for 5 days. VRBL Agar was used to evaluate the Total coliforms (TC) and Fecal Coliforms (FC) following incubation at 30°C/24 h and 44°C/48 h, respectively, for total coliform and fecal coliform, respectively. Tryptone Sulfite Neomycin Agar (Sigma Aldrich, Switzerland) was used for determination of anaerobic sulfite reducing (ASR) bacteria after incubation under strict anaerobic conditions. Baird Parker medium (Sigma Aldrich, Switzerland) medium supplemented with egg yolk and potassium tellurite was used to enumerate Staphylococcus flora following incubation at 37°C for 48 h. The lactic acid flora were enumerated on Man Rogosa Sharpe Agar (Sigma Aldrich, Switzerland) medium following incubation at 37°C for 48 h.
6
Microbiological Analysis of Sausage Samples
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TVC (mesophilic and psychrotrophic) were performed according to ISO 4833-1:2013 (total mesophilic flora) and ISO 17410-1:2019 (psychrotrophic microorganisms) by plating in Plate Count Agar (BIOKAR Allonne, France) followed by incubation for 3 days at 30 ⁰C and 10 days at 6.5 ⁰C, respectively.
Slices of three sausages from the same package were aseptically taken and pooled until a 25 g portion was obtained. Then, to prepare the initial suspension, 10 g of this pooled test portions were aseptically weighted in a sterile bag and homogenised with 90 g of sterile Tryptone-Salt Broth (BIOKAR Allonne, France) for 60 seconds in a stomacher blender (Stomacher Star Blender LB 400, VWR, Leuven, Belgium). Decimal dilutions (up to 10 -3 ) were prepared in Tryptone-Salt Broth (BIOKAR Allonne, France).
Slices of three sausages from the same package were aseptically taken and pooled until a 25 g portion was obtained. Then, to prepare the initial suspension, 10 g of this pooled test portions were aseptically weighted in a sterile bag and homogenised with 90 g of sterile Tryptone-Salt Broth (BIOKAR Allonne, France) for 60 seconds in a stomacher blender (Stomacher Star Blender LB 400, VWR, Leuven, Belgium). Decimal dilutions (up to 10 -3 ) were prepared in Tryptone-Salt Broth (BIOKAR Allonne, France).
7
Quantifying Microbial Cell Culturability
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Cell viability was measured using the agar plate count method. Fresh or thawed cell suspensions were diluted in saline water, then plated into an agar growth medium: Plate Count Agar (Biokar Diagnostics, Paris, France) for C. maltaromaticum and MRS Agar (Biokar Diagnostics, Paris, France) for the L. bulgaricus strains. Incubation was carried out at the strain's respective optimal temperatures for growth (30 °C for C. maltaromaticum and 42 °C for L. bulgaricus), for 48 h. The cell plate counts were expressed in CFU.mL -1 and results were obtained in triplicate.
Culturability loss after each freezing cycle (FC) (in log[CFU.mL -1 ]) was calculated using the following equation (Eq. 1):
[𝐶𝐹𝑈 𝑚𝐿 -1 ] 𝑡ℎ𝑎𝑤𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 𝑎𝑓𝑡𝑒𝑟 𝐹𝐶(𝑖-1) (Eq. 1)
For the first freezing cycle (i=1), [CFU.mL -1 ]thawed cells after FC(i-1) corresponds to the cell concentration in the fresh sample.
Culturability loss after each freezing cycle (FC) (in log[CFU.mL -1 ]) was calculated using the following equation (Eq. 1):
[𝐶𝐹𝑈 𝑚𝐿 -1 ] 𝑡ℎ𝑎𝑤𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 𝑎𝑓𝑡𝑒𝑟 𝐹𝐶(𝑖-1) (Eq. 1)
For the first freezing cycle (i=1), [CFU.mL -1 ]thawed cells after FC(i-1) corresponds to the cell concentration in the fresh sample.
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