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Method GS2/3-41 (2011) The Determination of the Total Mesophilic Bacterial Count in Refined Sugar Products by the Pour Plate Method or the Membrane Filtration Method - Official 1 Scope and Field of Application This method is use¢ for the determination of the total mesophilic bacteria count in refined sugar products (General Subjects 2 and 3) [1,2,3,4,5,6,7]. With higher colony counts and/or for dark-coloured products use the pour plate method. With low levels of microorganisms use the membrane fiter method [12]. This method isa combination of the previous GS2/3-41 (1998) and GS2/3-43 (1998) methods. 2 Definitions 2.1 Colony counts ~ are the enumeration of colony forming units (CFU). The results are reported as CFU per 10 g of sugar. 2.2 Mesophilic bacteria. Mesophilic bacteria have their growth optima between 20°C and 45°C. The Incubation temperature selected is an average for optimum growth. Conditions favouring the growth of a broad spectrum of bacterial species are established by using a general nutrient medium. 2.3 Pour plate. These are agar plates which have been inoculated by mixing known amounts of diluted sample with liquid agar medium (47 21°C). 2.4 Membrane filter. These are sterile filters of knowin pore size which retain microorganisms on their surface for further culture. 3 Principle + Pour plate method. Diluted aliquots of the sample are mixed with ‘small vclumes of cooled molten agar in Petri dishes and incubated when the agar medium has solidified. The microorganisms ara detected as colony forming units. + Membrane filter method. Test solutions of sugars are fitered through sterile membranes of known pore size. The microorganisms are retained on the surface. The membrane is transferred onto a nutrient medium or pad and incubated. The microorganisms are detected as colony forming units. Sterile equipment and an aseptic technique are used throughout the method. 4 Diluents, Culture Media, Reagents and other Products 4,1 Sterile distilled water - autoclaved at 121°C for 15 min. 4.2 Culture media [3,4,8]. Use either nutrient agar or plate count medium, Nutrient Agar (1 L): Beef extract 19 Sodium chloride 5 9 Yeast extract, 20 Agar 150 Peptone Sa pH 7.4 £0.2 (This is commercial available as Oxoid CM0003).. Plate Count medium (1 L): Peptone 3a Agar 109 Yeast extract, 259 pH 7040.2 Glucose 1g (This is commercial available as Merck 5463, Oxoid CM0325, BBL 11638). Alternatively use: Nutrient pad sets in Petri dishes with membrane filters (0.45 um pore-size) commercially available from Sartorius Stedim, Standard SM 140 55 ACN. 4.3 Disinfectant - for the working area. 4.4 Alcohol - for flaming the filter funnel assembly (5.13). 5 Apparatus and Glassware [8,9,11] ition at 180°C - for glassware 5.1 Apparatus for dry ste sterilisation. 5.2 Autoclave, 121 = 1°C - for water and culture media sterilisation. 5.3 Incubator ~ operating at 30 + 1 °C. 5.4 Bottles - for culture medium. 5.5 Erlenmeyer flasks - 200 mL with 2 100 mL mark and plugs for autodaving. 5.6 Test tubes - or preparation of dilutions. 5.7 Graduated Pipettes - 1 mL to 5 ml for sample distribution; 10 ML for dilutions, glass or sterile disposable plastic. 5.8 Petri dishes - diameter 90 to 100 mm, sterile disposable plastic oF glass; alternatively nutrient pad sets. 5.9 Water bath or similar apparatus - operating at 47 + 1 °C. 5.10 pH meter - accurate to + 0.1 pH units at 25°C. 5.11 Colony counting equipment. 5.12 Bunsen burner. The following additional apparatus is required for the membrane fitration technique. 5.13 Filter funnel, base with support and clamp. These can be stainless steel, autoclavable plastic, class or sterile disposable plastic, 50 mm diameter with a capacity of 100 mL. 5.14 Flask for mounting filter apparatus ~ 2 with side arm. 5.15 Vacuum pump or vacuum supply. 5.16 Forceps ~ fer handling membranes. 5.17 Membranes - sterile 0.45 um pore size, 45 mm diameter. 6 Samples Collect representative samples in sterile containers, 200 g minimum. size for sugar or 200 mL for liquid sugar. Retain the samples until the analysis has been completed (see Method GS2/3-42).. 7 Procedure [2,8,9] 7.1 Preparation. Clean and disinfect the working area before starting the analysis. Mark the sterile Petri dishes with type of medium, sample number and date. 7.2 Medium preparation + Agar medium. Following the manufacturer's instructions rehydrate the culture medium with distilled or de-ionised water. Dispense the medium into bottles and sterilise at 121 °C for 15 min. Check the pH value of the medium. Medium for immediate use should be cooled to 47°C + 1 °Cina water bath. Alternatively the medum could be allowed to solidify and be stored in the dark at 0 to 5 °C for up to 1 month. Media i bottles can be remelted, but only once. + Nutrient pads. Wet the nutrient pads with 3 to 3.5 mL of sterile distilled or de-ionised water. Ideally a slight access of liquid should be visible at the edge of the pad [11]. 7.3 Glassware preparation. Sterile the Erlenmeyer flasks with the plugs and the pipettes in the apparatus for dry sterilisation (180°C) for 2h [10]. 7.4 Sample preparation. aseptically place 10 9 of crystaline sugar or, in the case of liquid sugar, the amount corresponding to 109 of dry matter, in the 200 mL Erlenmeyer flask which is marked to indicate 100 mL. Add sterilised water (distilled or de-ionised) up to the 100 mL mark. Shake thoroughly to dissolve and mix the sugar solution. NOTE - 20 g/100 mL must be taken according to the NCA Standard for “Canners Sugars’. 7.5 Inoculation and incubation. For the pour plate method take two sterile Petri dishes. Transfer to each dish 1 mL of th q sample solution (7.4). If necessary prepare serial dilutions from the initial solution (10-*) adding 1 mL to mL of sterle water In a test tube to give a serles 107, 10? etc and plate out similarly. NOTE - 5 plates must be taken according to the NCA Standard for “Canners Sugars” Add approximately 15 mL of the medium, previously melted and maintained at 47 + 1°C in a water bath, into each Petri dish. Mix the Inoculum carefully with the medium by swirling the plate and allow the mixture to solicify. Prepare a control plate just containing culture medium to check its sterility. Invert the Petridishes and inaubate at 30°C for 48-72 h. NOTE ~ 72 h incubation is specified in the NCA Standard for “Canners Sugars’. 7.6 Filtration and incubation. Using the membrane filter method connect the filter apparatus to the vacuum pump or vacuum supply. If not already sterile, flame the fiter funnel and base with alcohol. After cooling place the membrane centrally on the base with sterile forceps. Place the sterile funnel on the top and clamp in place. Pour the sample (7.4) into the funnel, apoly the vacuum and fiter the sample. Turn off the vacuum. Rinse the funnel with sterile water and apply the vacuum once again. Remove the funnel and use flamed forceps to place the membrane on an agar plate or moistened nutrient pad. When posttioning the membrane on the surface ensure that no air bubbles are trapped between them. Invert the dishes an tubate at 30°C for 48-72 h. NOTE - 72 h incubation is specified in the NCA Standard for “Canners Sugars’ 8 Expression of Results Count the colonies in each dish usingthe colony counting equipment. For samples showing more than 100 cfu/plate examine the dilutions. The pour plate method is not suitable when colony counts are below 10 cfu/plate. The membrane filtration method should be used instead. Calculate the number of CFU per 10 g sugar or 10 a dry matter by the following calculations. 8.1 Pour plates. Ye (em 404-1) -d CFU/10 9 of sugar or dry matter where: Ze — cum of the colonies counted on all the plates, ny = number of plates counted in the first Dilution, m2 = number of plates counted in the second Dilution, ¢ = dilution from which the first counts were obtained (10-!). Example: Two Petri dishes from two dilutions were incubated and the observed colony counts were: Dilution 10-2 tstplate = 97 CFU 2né plate = 110CFU Dilution 10-2 tstplete = 35 CFU 2nd plate = 46 CFU 97+ 110435446 288 (1-240.1-2)-107 0.22 1309 CFU /g sugar For 10 g of sugar or dry matter, the result is multiplied by 10 giving the final result: 1.3 x 104 CFU/10 9 of sugar or dry matter. 8.2 Membrane filter. Ne n CFU/10 9 of sugar or dry matter where: Ec = sumof the colonies counted on all the plates, n= number of plates. Example: ‘Two Petri cishes were incubated and the observed colony counts were: istplte = 98 CFU 2nd plate = 110 CFU 98 +110 104 CFU / 10g 2 WARNING AND SAFETY PRECAUTIONS PLACE THE USED DISPOSABLE PLASTIC PETRI DISHES IN AUTOCLAVABLE PLASTIC BAGS AND STERILISE PRIOR TO DISPOSAL ACCORDING TO LOCAL REGULATIONS. GLASS PETRI DISHES REQUIRE IMMEDIATE STERILISATION. 1 2. Proc. 16th Session ICUMSA, 1974, 274-289, 3. Proc. 17th Session ICUMSA, 1978, 329-340 4. Proc, 19th Session ICUMSA, 1986, 356-376 5, 6. 7, Proc. 20th Session ICUMSA, 1990, 291-307 - Proc. 21st Session ICUMSA, 1994, 367-383 - Schneider F, ed. (1979): Sugar Analysis, ICUMSA Methods, 154- 158 8. Alcohol free Soft Drinks Handbook, Vol 1, 1993, 39-83, Sudaucker, Mannheim/Ochsenfurt (in German) 9, 150.4833 (1991): Microbiology ~ General guidance for the enumeration of micrc-organism colony count technique at 30 °C 10. 150 7218 (1996): Microbiology of food and animal feedingstuffs - General rules for micro-biological examinations 11. Application Notes, Microbiological Testing of Foods, Beverages and Pharmaceuticals, Sartorius, Géttingen 12. Proc. 27th Session ICUMSA, 2010, 168

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