The juice was homogenized under aseptic conditions, and appropriate dilutions with 0.1% sterile peptone water were prepared for natural juice contamination analysis. Samples were pour-plated in Plate Count Agar (PCA, Himedia®, Mumbai, Maharashtra, India) for aerobic mesophilic bacteria enumeration and counted after 48 h at 35 ± 1 °C. Molds and yeasts were analyzed based on the spread-plated technique in Potato Dextrose Agar (PDA, Fluka Analytical®, São Paulo, São Paulo, Brazil), and plates were incubated at 25 ± 1 °C for 5–7 days [54 ]. Coliforms (at 35 °C) and Escherichia coli were analyzed in Petrifilm plates (3M®, Maplewood, NJ, USA) and incubated at 35 °C for 48 h. The analysis was carried out in three repetitions, and plating was performed in duplicate. Results were expressed in CFU/mL.
Plate count agar pca
Manufactured by HiMedia
Sourced in India
Plate Count Agar (PCA) is a culture medium used for the enumeration of aerobic mesophilic bacteria. It provides a balanced nutrient source to support the growth of a wide range of microorganisms. PCA is commonly used in standard plate count techniques to determine the total viable bacterial count in food, water, and other samples.
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Lab products found in correlation
Potato dextrose agar (pda), by HiMedia (3 mentions)
Petrifilm plate, by 3M (1 mentions)
Sodium carbonate, by Merck Group (1 mentions)
Peptone, by HiMedia (1 mentions)
Zinc acetate dihydrate, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
2 thiobarbituric acid, by HiMedia (1 mentions)
Di chloran rose bengal chloramphenicol drbc agar, by HiMedia (1 mentions)
Malt extract agar, by HiMedia (1 mentions)
Potassium chloride (kcl), by Vetec (1 mentions)
Hexane, by Tedia (1 mentions)
Acetonitrile, by Tedia (1 mentions)
Chloroform, by Tedia (1 mentions)
Whatman, by Cytiva (1 mentions)
Potato dextrose agar (pda), by Merck Group (1 mentions)
De man, by Merck Group (1 mentions)
Violet red bile glucose agar, by HiMedia (1 mentions)
Petrifilm e coli coliform count plate, by 3M (1 mentions)
11 protocols using plate count agar pca
1
Microbiological Analysis of Fruit Juice
2
Eggshell-Derived Silver Nanoparticles
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Chicken eggshells (ES) were obtained from household waste, and commercial calcium carbonate particles were obtained from Sand and Soil Industry Co., Ltd. Silver nitrate (≥99.0%, ACS reagent), sodium carbonate (≥99.5%, ACS reagent), sodium citrate tribasic dihydrate (trisodium citrate) (≥99.0%, ACS reagent) and poly (sodium 4-styrenesulphonate) (PSS, average Mw∼70 000 g mole−1) were obtained from Sigma Aldrich. Nitric acid (65%, AR grade) was purchased from ANaPURE. Plate count agar (PCA) and peptone were purchased from HiMedia Laboratories Pvt. Ltd and Sisco Research Laboratories Pvt. Ltd, respectively. Unpacked fresh beef and vacuum-packed fresh beef (MAX BEEF) were purchased from a local fresh market and a Home-Fresh Mart supermarket, respectively.
3
Sperm Tyrode's Albumin Lactate Pyruvate Media
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Reagents for preparing Sperm Tyrode’s albumin lactate pyruvate (Sp-TALP) media and MODENA extender, 2-thiobarbituric acid (TBA), trichloroacetic acid (TCA), hydrochloric acid (Finar Reagent), and Dulbecco’s phosphate-buffered saline (DPBS; 10X), were procured from HiMedia Laboratories Pvt. Ltd., Maharashtra, India, and Merck Ltd., Darmstadt, Germany. Fluorescent dyes such as 5(6)-carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) (≥94%) were purchased from Sigma-Aldrich Chemicals Private Limited, Bangalore, India, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1) was purchased from Morek Life Solanos Pvt. Ltd., Maharashtra, India, for the semen quality assays. ZnO-NPs and zinc acetate (ZA) were obtained from ICAR-Indian Institute of Agricultural Biotechnology, Ranchi, India. Zinc acetate dihydrate and sodium hydroxide for NP synthesis and reagents of analytical purity grade were commercially purchased from Merck Ltd., Darmstadt, Germany. For antimicrobial studies, Plate Count Agar (PCA) and sterile disposable Petri plates (90 mm × 15 mm) were purchased from HiMedia Laboratories Pvt. Ltd., Maharashtra, India.
4
Standardizing Aflatoxins Detection Workflow
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Standard aflatoxins (AFB1, AFB2, AFG1, and AFG2) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The reagents KCl and CuSO4 were ACS grade from Vetec (Rio de Janeiro, Rio de Janeiro, Brazil). Trifluoroacetic acid for HPLC was purchased from Tedia (São Paulo, São Paulo, Brazil). The organic solvents hexane, chloroform, acetonitrile, and methanol HPLC grade were acquired from Tedia (São Paulo, São Paulo, Brazil); ultrapure water was obtained using a Rios/Milli-Q® purification system (Millipore, Danvers, MA, USA). Qualitative filter papers grade 1 were purchased from Whatman (Maidstone, Kant, UK), and the HPLC 13 mm polypropylene filters with 0.45 µm PVDF membrane Durapore® (Millipore, Danvers, MA, USA) were purchased from Merck (São Paulo, São Paulo, Brazil). Plate count agar (PCA), used for total mesophilic count, Dichloran Rose Bengal Chloramphenicol (DRBC) agar, malt extract agar (MEA), and potato dextrose agar (PDA) used in fungal analyses were obtained from Himedia (Curitiba, Paraná, Brazil).
5
Evaluating Extracellular and Lipolytic Activities
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The extracellular proteolytic activity was evaluated following the protocol described by Randazzo and co-workers (2021) [18 (link)] and Franciosi and co-workers (2009) [19 (link)]. In detail, 2 µL of cells, standardized as previously described, were spotted on the surface of petri dishes containing Plate Count Agar (PCA, HiMedia, Mumbai, MH, India) supplemented with skim milk (10% w/v, Oxoid Ltd., Basingstoke, UK). After incubation at 30 °C for four days, plates were checked for the presence of a clear zone surrounding the colonies.
The lipolytic activity was tested by spotting 2 µL of standardized cells (about 3 × 108 CFU/mL) on the lipolytic medium composed of peptone, 2.5 g/L; casein peptone, 2.5 g/L; yeast extract, 3 g/L; Agar, 12 g/L; and 1% of tributyrin. After incubation at 30 °C for 72 h, the presence of a halo around the bacterial spot revealed lipolytic activity [20 (link)].
Each test was performed in triplicate.
The lipolytic activity was tested by spotting 2 µL of standardized cells (about 3 × 108 CFU/mL) on the lipolytic medium composed of peptone, 2.5 g/L; casein peptone, 2.5 g/L; yeast extract, 3 g/L; Agar, 12 g/L; and 1% of tributyrin. After incubation at 30 °C for 72 h, the presence of a halo around the bacterial spot revealed lipolytic activity [20 (link)].
Each test was performed in triplicate.
6
Fungal Growth in Brined Olives
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All the samples of brined table olives were prepared using fifteen green olives of the Arauco cultivar (graded-size 320/360 olives/kg), with 200 mL of acidified brine, to analyze the fungal growth under real conditions. The formulation was prepared according to the experimental design. The final pH of 3.5 was reached by the addition of lactic acid 85%.
Initially, the samples were pasteurized at 100 °C for 10 min, in order to eliminate the natural microbiota and thus be able to evaluate exclusively the deterioration resulting from contamination by M. ruber. Preliminary experiments showed that the time × temperature used were the most appropriate thermal treatment conditions. The experiments were carried out in quintuplicate where, it was not observed any growth after plating with 1 mL of samples in Potato Dextrose Agar (PDA) – (Fluka – Sigma–Aldrich®) and Plate count agar (PCA) – (Himedia®) after 3–5 days of incubation at 30–35 °C.
Pasteurized samples (100 °C/10 min) were inoculated with 0.2 mL of the ascospores suspension (“Preparation of ascospores suspension” section), to reach a concentration of around 102 ascospores/mL of brine. These were the samples incubated at temperatures set in the experimental design (30, 35, 40 °C). Inoculum concentrations between 102 and 103 CFU/mL are commonly used and recommended in studies involving microbial behavior.26
Initially, the samples were pasteurized at 100 °C for 10 min, in order to eliminate the natural microbiota and thus be able to evaluate exclusively the deterioration resulting from contamination by M. ruber. Preliminary experiments showed that the time × temperature used were the most appropriate thermal treatment conditions. The experiments were carried out in quintuplicate where, it was not observed any growth after plating with 1 mL of samples in Potato Dextrose Agar (PDA) – (Fluka – Sigma–Aldrich®) and Plate count agar (PCA) – (Himedia®) after 3–5 days of incubation at 30–35 °C.
Pasteurized samples (100 °C/10 min) were inoculated with 0.2 mL of the ascospores suspension (“Preparation of ascospores suspension” section), to reach a concentration of around 102 ascospores/mL of brine. These were the samples incubated at temperatures set in the experimental design (30, 35, 40 °C). Inoculum concentrations between 102 and 103 CFU/mL are commonly used and recommended in studies involving microbial behavior.26
7
Microbial Enumeration in Food Samples
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Ten (10) g of each sample was placed in a Stomacher filter bag, and 90 mL of sterile buffered peptone water (BPW, Oxoid CM1049, Thermo Fisher Scientific, Waltham, MA, USA) was added. The content of each bag was homogenized with a laboratory blender (Stomacher 400, Seward Ltd., Company, West Sussex, UK) for 2 min. Serial decimal dilutions were made with buffered peptone water, and then, the proper quantity of each dilution was plated. The aerobic plate counts (APCs) were enumerated on the Plate Count Agar (PCA, HiMedia, Mumbai, India) plates incubated at 30 °C for 48 h, and the lactic acid bacteria on MRS plates (De Man, Rogosa Sharpe, Merck, Darmstadt, Germany) were incubated at 37 °C for 48 h anaerobically (BBL GasPak jar anaerobiosis system). For yeast counting, 0.1 mL of the serial dilutions was spread plated on Potato Dextrose Agar (PDA, Merck, Germany) and incubated at 20 °C for 5 days. Counts of microorganisms were determined as log cfu/g.
8
Microbial Enumeration of Chewy Santol
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To measure the microbial populations of the chewy santol samples, a 25 g of samples were homogenized with 225 mL of 1% sterile peptone water using a Stomacher (Stomacher ® 400 Circulator, England) for 1 min. Ten-fold dilution series were made in sterile peptone water as required for plating on (1) Plate Count Agar, PCA (HiMedia) incubated at 37±2°C for 24±3 h for determining the total bacteria counts; (2) Potato dextrose agar (HiMedia) incubated at 28±2°C for 7 days for determining the yeast and mold counts. The microbial populations were expressed as log CFU/g sample (colony forming units per gram). All measurements were performed in triplicate.
9
Enumeration of Fish Microbiome
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Samples (10 g) for enumeration of the total viable count (TVC), Enterobacteriaceae, and psychrotrophic bacteria, were taken aseptically from the mackerel muscle. The initial dilution were prepared in accordance with specific rules for the preparation of fish samples (ISO 6887-3). Plate Count Agar (PCA; HiMedia, India) was used to enumerate TVC of microorganisms (ISO 4833). Violet Red Bile Glucose Agar (HiMedia, India) was used to determine Enterobacteriaceae (ISO 21528-2). The PCA was used also to determine the psychrotrophic microorganisms (ISO 17410). After incubation of inoculated Petri dishes, the numbers of characteristic colonies were enumerated, calculated and displayed in CFU/g.
10
Microbiological Assays for Cider Samples
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Cider samples were plated at regular intervals for microbiological counts. Plate count agar (PCA) (Himedia, Mumbai, India) was used to determine the counts for total aerobic microbes. From each sample bag, triplicate serial dilutions were made with peptone water. Samples (1 mL) from each dilution were aseptically placed in Petri dishes, to which 20 mL of sterilized agar was poured and mixed thoroughly. Upon agar solidification, the Petri dishes were inverted and incubated for 48 h at 37ºC. Yeast and mold counts were determined using acidified Potato Dextrose Agar (PDA) (Himedia, Mumbai, India). The pH of the sterilized agar was adjusted to 3.5 with sterilized 10% (w/v) tartaric acid. Sample (0.1 mL) of each dilution was spread on solidified agar, and incubated for 5 days at 25 ºC. Colonies were counted using a Quebec colony counter and are reported as log10 CFU/mL. The E. coli tests were evaluated using 3M Petrifilm E. coli/Coliform Count Plates (3M, MN, USA).
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