Five grams of each cheese was homogenised in 45 ml of a 2 % tri-sodium citrate buffer (VWR, Dublin, Ireland). Cheese homogenate was then subjected to enzymatic lysis using lysozyme (1 mg/ml), mutanolysin (50 U/ml) (Sigma Aldrich, Dublin, Ireland) and proteinase k (800 μg/ml) and incubated at 55 °C for 30 min as per Quigley et al [39 (link)]. DNA was extracted using the PowerFood Microbial DNA Isolation Kit (MoBio Laboratories Inc, Carlsbad, CA USA). After extraction, DNA was concentrated via ethanol precipitation. DNA was re-suspended in 20 μl TE buffer (Sigma-Aldrich, Dublin, Ireland). Quality and purity of extracted DNA was assessed using the NanoDrop 1000 Spectrophotometer (Thermo-Fisher Scientific, Wilmington, VA, USA), as per manufacturers guidelines.
Tri sodium citrate buffer
Manufactured by Avantor
Sourced in Ireland
Tri-sodium citrate buffer is a commonly used buffer solution for various laboratory applications. It is composed of trisodium citrate and citric acid, which together maintain a specific pH range. The primary function of this buffer is to provide a stable and controlled pH environment for various biochemical and analytical processes.
Automatically generated - may contain errors
Lab products found in correlation
Lysozyme, by Merck Group (1 mentions)
Powerfood microbial dna isolation kit, by Qiagen (1 mentions)
Mutanolysin, by Merck Group (1 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Kanamycin sulfate, by Merck Group (1 mentions)
Anaerogen system, by Thermo Fisher Scientific (1 mentions)
2 protocols using tri sodium citrate buffer
1
Cheese DNA Extraction and Quantification
2
Enumeration of Cheese Microbiota
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Samples were aseptically removed from cheese wheels using a cheese trier at 1, 11, 41, 65, 97, 140, and 180 d of ripening. The cheese samples (10 g) were placed in a sterile stomacher bag (Grade, Leicestershire, UK), diluted (10-fold) with 2% (wt/vol) trisodium citrate buffer (VWR, Dublin, Ireland), and stomached for 10 min using a stomacher (Iul Instruments, Barcelona, Spain) . Serial dilutions of 10-fold diluted cheese samples were made using maximum recovery diluent, containing low levels of peptone (1 g/L) and sodium chloride (8.5 g/L). Total numbers of nonstarter lactic acid bacteria (NSLAB) cells were enumerated on Lactobacillus selection agar (BD, Oxford, UK), with an overlay, after aerobic incubation for 5 d at 30°C. Viable cells of PAB were enumerated on sodium lactate agar, supplemented with kanamycin sulfate (Sigma-Aldrich, Arklow, Ireland) at a level of 4 mg/100 mL of sodium lactate agar, after anaerobic incubation for 7 d at 30°C, and only light brown colonies were counted as PAB (Rehn et al., 2011) (link). Lactobacillus helveticus cells were enumerated on de Man, Rogosa, and Sharpe agar (BD) at pH 5.4 after anaerobic incubation for 3 d at 42°C (Hickey et al., 2017) (link). Anaerobic conditions were maintained through the use of anaerobic gas jars and AnaeroGen system (Oxoid, Basingstoke, UK).
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