Microbiological analysis was performed by the traditional plate method [15], according to the following standards: ISO 15214:1998 [16] and ISO 6611:2004 [17] . The number of each type of bacteria was determined with three repetitions. The following microbiological media were used for the analyses: MRS (de Man, Rogosa, and Sharpe) agar (Merck, Darmstadt, Germany), for determining the number of lactobacilli cells; M17 agar (Merck, Darmstadt, Germany), for determining the number of lactococci cells; and YGC (Yeast Extract Glucose Chloramphenicol) agar (Merck, Darmstadt, Germany), for determining the number of yeasts. All media were incubated at 30 • C for 72 h. The Petri dishes with the MRS agar were inserted in the anaerobic jars (Merck, Darmstadt, Germany), those containing the M17 agar were grown aerobically, and Petri dishes with the YGC agar were incubated aerobically for up to 5 days at 25 • C. The results are expressed as mean values of colony-forming unit per mL of kefir sample (CFU/mL) in two parallel replicates, and then as decadic logarithm.
M17 agar
Manufactured by Merck Group
Sourced in Germany, United Kingdom, United States, Poland
M17 agar is a culture medium used for the growth and isolation of lactic acid bacteria. It is a nutritionally rich medium that supports the growth of a variety of Gram-positive and Gram-negative bacteria. The medium contains lactose, peptones, and yeast extract, providing essential nutrients for bacterial growth.
Automatically generated - may contain errors
Lab products found in correlation
Mrs agar, by Merck Group (24 mentions)
Ringer solution, by Merck Group (4 mentions)
Rogosa agar, by Merck Group (3 mentions)
Plate count agar pca, by Merck Group (3 mentions)
Anaerocult a, by Merck Group (3 mentions)
Potato dextrose agar pda, by Merck Group (2 mentions)
Vancomycin hydrochloride, by Merck Group (2 mentions)
Ygc agar, by Merck Group (2 mentions)
Yeast extract glucose chloramphenicol agar, by Merck Group (2 mentions)
De man rogosa sharpe agar, by Merck Group (2 mentions)
83920 rogosa agar, by Merck Group (2 mentions)
China blue lactose agar, by Merck Group (2 mentions)
Perkin elmer series 200, by PerkinElmer (1 mentions)
Pb 10, by Sartorius (1 mentions)
Tsk gel columns, by Tosoh (1 mentions)
G4000pwxl, by Tosoh (1 mentions)
Safranin, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
51 protocols using m17 agar
1
Microbiological Analysis of Kefir
2
Determination of Levan and Sugars in TSJM
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The levels of sucrose, glucose and fructose in the cultivated TSJM were determined by a high-performance liquid chromatography (HPLC)-based method described previously [24 (link)].
The Mw of the levan obtained was determined by high-performance size exclusion chromatography (HPSEC) using a Perkin-Elmer series 200 liquid chromatography (PerkinElmer, Waltham, MA) equipped with a Perkin-Elmer series 200 refractive index detector. Two TSK-GEL columns (G6000PWXL and G4000PWXL, Tosoh Bioscience Co., Japan) were maintained in series, utilizing 0.1 M NaNO3 as the eluent at a flow rate of 0.6 ml/min. The columns were maintained at 25 °C, and 5 mg/ml of BD1707 levan dissolved in the 0.1 M NaNO3 was filtered through a 0.22-μm filter before injection. Commercial pullulans with molecular mass ranging from 6000 to 2,560,000 Da (6000 Da, 12,000 Da, 110,000 Da, 800,000 Da, 2,560,000 Da) (Sigma, St. Louis, MO, USA) were used as standards (see Supplementary material, TableS2 ).
The viable cell counts of BD1707 in the cultivation broth were enumerated by plating the serially 10-fold diluted sample on M17 agar (Merck, USA) and incubating at 30 °C aerobically. The pH was measured by using a pH meter (PB − 10, Sartorius AG, Goettingen, GER) [19 (link)].
The Mw of the levan obtained was determined by high-performance size exclusion chromatography (HPSEC) using a Perkin-Elmer series 200 liquid chromatography (PerkinElmer, Waltham, MA) equipped with a Perkin-Elmer series 200 refractive index detector. Two TSK-GEL columns (G6000PWXL and G4000PWXL, Tosoh Bioscience Co., Japan) were maintained in series, utilizing 0.1 M NaNO3 as the eluent at a flow rate of 0.6 ml/min. The columns were maintained at 25 °C, and 5 mg/ml of BD1707 levan dissolved in the 0.1 M NaNO3 was filtered through a 0.22-μm filter before injection. Commercial pullulans with molecular mass ranging from 6000 to 2,560,000 Da (6000 Da, 12,000 Da, 110,000 Da, 800,000 Da, 2,560,000 Da) (Sigma, St. Louis, MO, USA) were used as standards (see Supplementary material, Table
The viable cell counts of BD1707 in the cultivation broth were enumerated by plating the serially 10-fold diluted sample on M17 agar (Merck, USA) and incubating at 30 °C aerobically. The pH was measured by using a pH meter (PB − 10, Sartorius AG, Goettingen, GER) [19 (link)].
3
Enumeration and Identification of Cheese Microbiota
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Ten grams of each cheese sample was transferred to 90 mL of sterile
quarter-strength Ringer’s solution (Merck). Cheese samples were
homogenized in a stomacher (AES Chemunex, Easy mix) for 2 minutes, and
serial dilutions were prepared by mixing 1 mL of the homogenized sample with
9 mL of sterile quarter-strength Ringer’s solution. NSLAB were
enumerated and identified by plating samples on M17 Agar (Merck) and DeMan,
Rogosa and Sharpe (MRS, Merck) Agar containing 20 μg
mL−1 vancomycin (V, SigmaTM) to isolate
lactococci and leuconostocs, respectively, after aerobic incubation at
30°C for 72 h. Rogosa Agar (Merck; anaerobic incubation at
30°C for five days) and Kanamycin Aesculin Azid Agar (KAA) (Merck;
aerobic incubation at 37°C for two days) were used to enumerate and
isolate lactobacilli and enterococci, respectively (Navidghasemizad et al., 2009 (link)). Total mesophilic aerobic
bacteria (TMAB) and yeast and molds (YM) were counted by plating serial
dilutions on Plate Count Agar (PCA) (Merck; aerobic incubation at
30°C for two days) and Yeast Extract Glucose Chloramphenicol Agar
(YGEC) (Merck; aerobic incubation at 25°C–28°C for five
days), respectively. For each microbial group, the experiments were repeated
in triplicate. Following the incubation, the plates with 10–300
colonies were selected for enumeration, and the number of colonies was
expressed as Log CFU/g.
quarter-strength Ringer’s solution (Merck). Cheese samples were
homogenized in a stomacher (AES Chemunex, Easy mix) for 2 minutes, and
serial dilutions were prepared by mixing 1 mL of the homogenized sample with
9 mL of sterile quarter-strength Ringer’s solution. NSLAB were
enumerated and identified by plating samples on M17 Agar (Merck) and DeMan,
Rogosa and Sharpe (MRS, Merck) Agar containing 20 μg
mL−1 vancomycin (V, SigmaTM) to isolate
lactococci and leuconostocs, respectively, after aerobic incubation at
30°C for 72 h. Rogosa Agar (Merck; anaerobic incubation at
30°C for five days) and Kanamycin Aesculin Azid Agar (KAA) (Merck;
aerobic incubation at 37°C for two days) were used to enumerate and
isolate lactobacilli and enterococci, respectively (Navidghasemizad et al., 2009 (link)). Total mesophilic aerobic
bacteria (TMAB) and yeast and molds (YM) were counted by plating serial
dilutions on Plate Count Agar (PCA) (Merck; aerobic incubation at
30°C for two days) and Yeast Extract Glucose Chloramphenicol Agar
(YGEC) (Merck; aerobic incubation at 25°C–28°C for five
days), respectively. For each microbial group, the experiments were repeated
in triplicate. Following the incubation, the plates with 10–300
colonies were selected for enumeration, and the number of colonies was
expressed as Log CFU/g.
4
Enumeration of Yogurt Bacteria
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Serial dilutions of ayran samples were prepared by using Ringer’s solution. S. thermophilus and L. bulgaricus colonies were counted on M17 agar (Merck Co., Kenilwoth, NJ, USA) and De Man, Rogosa and Sharpe (MRS) agar (Merck Co.), respectively. The M17 agar plates were incubated aerobically at 37 °C for 72 h and the MRS agar plates were incubated under microaerophilic conditions at 43 °C for 72 h (15 ). Microaerophilic conditions were achieved by means of an anaerobic kit (Anaerocult A®; Merck-Millipore, Burlington, MA, USA).
5
Dairy Ingredient Analysis Protocol
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Agar and d ‐glucose were purchased from Merck (Merck, Darmstadt, Germany). Skimmed milk powder, whole milk powder, and cream powder were produced by Pegah Fars Company (Shiraz, Iran). Sodium chloride, phenol red, crystal violet, safranin, acetone, M17 Agar, MRS Agar, phenolphthalein, trichloroacetic acid, ethanol 98%, o‐phthaldialdehyde, hydrochloric acid, sulfuric acid, ß‐mercaptoethanol, sodium tetraborate, sodium dodecyl sulfate, and sodium hydroxide were from purchased from Merck.
6
Quantitative Microbiological Analysis of Cheese
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Representative duplicate 10 g portions were retrieved from each cheese sample during various time intervals and blended with 90 mL of sterilized 2% w/v tri-sodium citrate solution. The suspension was then submitted to 10 decimal serial dilutions with ¼ strength Ringer’s solution. Viable counts of total aerobic bacterial counts, Lactococci, Lactobacilli, yeasts and fungi, and coliforms were determined in triplicate by pour plating of appropriate dilutions (0.1 mL or 1 mL) on the selective media for each species according to instructions of the manufacturer [26 (link)]. Specifically, viable counts of total aerobic bacterial counts were enumerated on plate count agar (PCA) (Merck) by suspended 0.1 mL of the sample after incubation at 30 °C for 72 h. Lactococci were enumerated on M-17 agar (Merck) by suspended 1 mL of the sample after incubation 30 °C for 48–72 h. Lactobacilli were enumerated on MRS agar (Merck) by suspended 1 mL of the sample after incubation at 37 °C for 48 h. Yeasts and fungi were determined by suspended 0.1 mL of the sample after incubation on Potato Dextrose agar (PDA) (Merck) at 30 °C for 72 h. Finally, coliforms were enumerated on Violet Red Bile agar (LabM, Heywood, U.K.) by suspended 1 mL of the sample after anaerobic incubation at 30 °C for 24 h. All cell counts were expressed as log of mean colony forming units (CFU).
7
Enumeration of Probiotic Bacteria
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The enumeration of Str. thermophilus and Lb. bulgaricus was determined by the method mentioned in the procedures outlined by the ISO [24 ]; by pour plating on M17 agar (Merck, Darmstadt, Germany) and MRS agar (Merck, Darmstadt, Germany), respectively, and incubated for 72 h at 37 °C in anaerobic jar AnaeroGen (Oxoid, Basingstoke, UK) of Lb. bulgaricus and aerobic condition of Str. thermophilus. Bifidobacteria counts were enumerated in RCA (pH 7.1) plus 1 µL mL−1 dicloxacillin (Oxoid, Basingstoke, UK) after incubation for 72 h at 37 °C in anaerobic jar [25 (link)]. The coliforms were determined as described by APHA [26 ]. Yeasts and molds counts were performed as described by IDF [27 ]. The Total aerobic mesophilic bacteria (TAMB) count was carried out in plate count agar (Oxoid) incubated at 30ºC for 48 hours; at 5ºC for 10 days for total psychrotrophic bacteria count. All bacterial counts were enumerated in duplicate. The numbers were tableted as logarithm colony forming units per gram (log10 CFU g−1).
8
Cheese Microbiological Analysis Protocol
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For the analysis of total bacteria count (TBC), Lactococcus spp., Lb. helveticus and non-starter lactic acid bacteria (NSLAB) counts, a 10 g test portion was taken under sterile conditions from each cheese model for microbiological analysis. The sample was transferred into a sterile Bag Page with a full-surface filter (Interscience, Argenta Ltd., Poznań, Poland), poured with 90 mL of a sterile 2% sodium citrate solution (Avantor Performance Materials, Gliwice, Poland) having the temperature of 37 °C, and homogenized (Stomacher Lab-Blender 80). Thus prepared first dilutions of cheese samples were used to prepare serial dilutions in sterile Peptobak pepton (BTL, Łódź, Poland) solution. All analyses were carried out in the fourth week of cheese ripening. Determination of TBC was conducted on Plate Count Skim Milk Agar PCSMA culture medium (Merck, Poland), whereas counts of Lactococcus spp., Lb. helveticus and NSLAB were determined on M-17 Agar (Merck, Poland), MRS Agar (Merck, Poland) and Rogosa Agar (Merck, Poland), respectively.
9
Enumerating Streptococcus thermophilus in Probiotic Products
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To determine the presence and cell counts of Streptococcus thermophilus, 0.1 mL of cell suspensions ranging from 10−1 to 10−4 dilutions, were spread on M17 Agar (Merck, Darmstadt, Germany) according to Terzaghi [22 ] and the plates were incubated at 42 °C under aerobic conditions for 24 to 48 h in duplicate. Presence of white colonies with 1–2 mm in diameter were presumed to be representative of Strep. thermophilus and were counted to obtain the CFU/g of probiotic product.
10
Microbial Analysis of Kefir Samples
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To evaluate the microbial content of both kefir samples, 10 mL of kefir was aseptically taken, dispersed with 90 mL of sterile Ringer solution (1:9, w/v; Merck, Darmstadt, Germany), and homogenized for 1 min in a stomacher (Lab-Blender 400; London, UK). Decimal dilutions were prepared and plated in triplicates for bacterial and yeast counts.
Lactobacilli spp. counts were determined on de Man Rogosa and Sharpe agar (MRS; Merck 1.10660) at 30 °C under anaerobic conditions for 3 days. Lactococcus spp. were counted on M17 agar (Merck) at 37 °C under anaerobic conditions for 2 days. Leuconostoc spp. counts were determined on de Man Rogosa and Sharpe agar (MRS; Merck) incorporated with vancomycin hydrochloride (Sigma Aldrich, Poznań, Poland) at 30 °C for 3 days. For enumerating the lactic acid bacteria, samples were plated on de Man Rogosa and Sharpe agar (MRS; Merck) and anaerobically incubated at 37 °C for 3 days. Yeasts were cultured on potato dextrose agar (PDA; Merck, 1.10130; pH 3.5) with 10% added tartaric acid at 25 °C for 3 days [17 (link),37 (link)]. The results were expressed as the logarithm colony forming units per milliliter of kefir (log CFU/mL).
Lactobacilli spp. counts were determined on de Man Rogosa and Sharpe agar (MRS; Merck 1.10660) at 30 °C under anaerobic conditions for 3 days. Lactococcus spp. were counted on M17 agar (Merck) at 37 °C under anaerobic conditions for 2 days. Leuconostoc spp. counts were determined on de Man Rogosa and Sharpe agar (MRS; Merck) incorporated with vancomycin hydrochloride (Sigma Aldrich, Poznań, Poland) at 30 °C for 3 days. For enumerating the lactic acid bacteria, samples were plated on de Man Rogosa and Sharpe agar (MRS; Merck) and anaerobically incubated at 37 °C for 3 days. Yeasts were cultured on potato dextrose agar (PDA; Merck, 1.10130; pH 3.5) with 10% added tartaric acid at 25 °C for 3 days [17 (link),37 (link)]. The results were expressed as the logarithm colony forming units per milliliter of kefir (log CFU/mL).
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