Standardized procedures for microbiological examinations. of food products. n acc. EN-ISO Standards n

Transcription

1 Standardized procedures for microbiological examinations of food products n acc. EN-ISO Standards n

2 Scharlau microbiology History Scharlau Chemie S.A. has been manufacturing laboratory chemicals since In 1979 ADSA-MICRO started the manufacture of dehydrated media. This company was acquired by Scharlau Chemie S.A. in 1992 and the brand changed to Scharlau. A new facility for the manufacture of dehydrated media was set up in 2002, which allowed us to increase capacity and to further improve the quality of our media. ISO 9001:2000 quality assurance was put in place since ISO 14001:1996 for environmental care was obtained in In 2002 Scharlau Chemie S.A. started offering also a wide range of prepared media and supplements. Microbiological culture media Scharlau manufactures two separate lines of culture media, dehydrated and prepared media; also stains in solution, reagents, supplements and rapid confirmation tests (see more information at the end of this leaflet). Please ask for the specific catalogues and brochures. NEW Certificates New certificate including picture of typical growth. Additives and supplements in vial 1 2 Prepared culture media New sterilized presentation: fast, easy and less risk of contamination. Click, shake and dispense. Selection of the most common media in different presentations. Dehydrated culture media Scharlau Chemie S.A. manufactures dehydrated culture media for microbiology. Available in 100 g and 500 g bottles Each bottle comes in an individual bag to avoid dust and a COA (certificate of analysis) is included with each bottle Description in 5 languages Tamper-proof cap No inside aluminium foil Perfectly tight-closing cap insures long shelf life NEW Packaging PAC-O-VAC is the novel airtight packing from Scharlau that provides an extraordinary protection to microbiology culture media. Vacuum, followed by replacement of residual air with nitrogen, guarantees that our products are protected from contamination by moisture, dust particles or microorganisms. This assures optimum storage conditions and prevent the products from altering their organoleptic or physical properties. 90 mm Petri plates. Available also with single and double flow pack bag and gamma irradiated for use in clean room. 55 mm filtration plates. Prepared plates of solid medium for the membrane filtration technique. Packed in blister. Rodac plates for surfaces control. Packaging into blisters inside clean room to avoid contamination and dehydration. Agars in flasks. Long shelf life in different volumes for remelting. Broths in flasks. Enrichment media and diluents. Sterility control. Agars in tubes for remelting. For mass inoculation technique. Broths in tubes. Differents volumes depending on working protocol. Index Scharlau microbiology Staphylococcus aureus and other species Escherichia coli Enterobacteriaceae Coliforms Listeria Microorganisms Bacillus cereus Yeasts and moulds Clostridium perfringens Salmonella spp. Food products determinations Technical documentation

3 Presentation In this brochure we have put together the latest ISO directives that have been published for the analysis of food. You will be able to find other media used in microbiological food analysis in our latest Handbook edition no. 8. Microbiological quality control involves the measurement of the general contamination level (total amount of microorganisms) as well as the detection of specific pathogens. Current ISO directives have been published for the analysis of: Staphylococcus aureus E. coli Enterobacteria Listeria monocytogenes Microorganisms Bacillus cereus Yeasts and moulds Clostridium perfingens Salmonella spp. The graphical protocol description will help the microbiologist to apply the official ISO methods without having to read a lot of text. Since new directives are added every year and existing methods changed, please check our website ( for possible updates on this brochure. Apart from the media mentioned in this leaflet, Scharlau offers a full range of standard and chromogenic media. Both in dehydrated and prepared form. Please check our website for the latest editions. CONSIDERATIONS: To choose the dilution fluid, see ISO 6887 (Preparation of tests samples initial suspension and decimal dilutions for microbiological examination) or 8261 and the Specific International Standard dealing with the product under examination. general diluents are saline peptone water/maximum recovery diluent (ref ), buffered peptone water (ref ) and others like peptone water (ref ). Tel Fax

4 Staphylococcus aureus and other species EN-ISO :2000 Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species). Part 1: Technique using Baird-Parker Agar Medium. Isolation medium Spread inoculum over surface of Baird Parker Agar Incubation: 37 ±1 C for h Coagulase sample dilution 0, 0, Discard BHI 37 ±1 C - 24 ± 2 h 0, Coagulase 37 ±1 C up to 24 h S. aureus Others Baird Parker Agar Base BA1033 Egg s Yolk Telurite Sterile Emulsion Baird Parker Medium 064-PA PR0008 Lyophilized Rabbit Plasma (Coagulase Test) 064-PLA-CO Brain Heart Infusion Broth (Coagulase Test) TA0143 EN-ISO :2000 Enumeration of coagulase-positive staphylococci. (Staphylococcus aureus and other species). Part 2: Technique that use Rabbit Plasma Fibrinogen (RPF) Agar Medium. Isolation medium Mass inoculation technique of Rabbit Plasma Fibrinogen Agar Incubation: ±1 C for h Incubate for a further 24 hours if necessary Is not necessary sample dilution S. aureus Discard Baird Parker Agar Base BA1033 Egg s Yolk Telurite Sterile Emulsion RPF Supplement Lyophilized 064-TA-0155 Baird Parker RPF Base 064-BA1028 RPF Supplement Lyophilized 064-TA-0155 Baird Parker RPF Agar 064-BA0RPF (1) 064-PA0145 (1) Set: 4 bottles of Baird Parker RPF base 4 vials of RPF Suppplement

5 Escherichia coli en-iso :2001 Horizontal method for the enumeration of ß-glucuronidase-positive Escherichia coli. Part 2: Colony-count technique at 44 C using 5-bromo-4-chloro-3-indolyl ß-D-glucuronide. Selective medium Tryptone Bile X-Glucuronide Agar (TBX) Mass inoculation Incubation: 44 C for h In case of suspected damaged cells, incubate in advanced at 37 C for 4 h. sample dilution Blue: E. coli 15 ml TBX TBX Other color: Discard TBX TBX Agar BA PA0945 Tel Fax

6 Enterobacteriaceae ISO 8523:1991 General guidance for the detection of Enterobacteriaceae with pre-enrichment. Pre-enrichment medium Enrichment medium Isolation medium Buffered Peptone Water Buffered Glucose-Brilliant Violet Red Bile Oxidase Green Bile Broth (EE Broth) Dextrose Agar (VRBD) Incubation: C for h Incubation: C for h Incubation: C for 24 ± 2 h Glucose fermentation (Glucose Bromocresol Purple) Incubation: 37 C for 24 ± 2 h PRE-ENRICHMENT enrichment Discard Buffered Peptone Water 10 ml Other color > Discard Oxidase Discard Enterobacteriaceae Nutrient Agar Enterobacteriaceae Glucose fermentation Buffered Peptone Water BA TA0100 EE Broth TA0189 Violet Red Bile Dextrose Agar (VRBD) BA TA PA PR0006 Oxidase Test Sterile Swabs Oxidase Reagent in powder Glucose Bromocresol Purple Agar TA0129* Nutrient Agar PA0059 * Please check availability

7 Coliforms en-iso 4832:1991 General guidance for the enumeration of coliforms - Colony-count technique. Isolation medium Mass inoculation with Violet Red Bile Lactose Agar (VRBL) Incubation: 30 C, 35 C or 37 C for 24 ± 2 h Characteristic colonies Violet red with ø 0,5 mm sample dilution 15 ml VRBL Red 0,5mm ø Count Discard Violet Red Bile Lactose Agar (VRBL) BA TA PA PR0018 Tel Fax

8 Listeria en-iso :1997 Horizontal method for the detection and enumeration of Listeria monocytogenes. Part 1: Detection method. Enrichment media Isolation media Primary enrichment Spread inoculum over surface of PALCAM Agar and OXFORD Agar Genus confirmation: in Half-concentrated Fraser Broth Incubation: Aerobic or microaerobic 30 C, 35 C or 37 C for 24 ± 2 h Catalase Incubation: 30 C for 24 ± 2 h and continue for another 24 ± 2 h if there are no colonies or they are small Gram Motility Secondary enrichment: Specie confirmation: 0, of primary culture Haemolysis in 10 ml of Fraser Broth Fermentation of: Incubation: 35 or 37 C for 48 ± 2 h Ramnose Xylose CAMP test Note: This standard has been revised. The new version include as an method a chromogenic medium, like ALOA Agar, or other lecithin based ones, and also another medium of the laboratories choice. enrichment (See below) Fraser halfconcentrated Oxford Discard 225 ml Fraser 0, (See below) TSYEA (35 C or 37 C for 18 or 24 h) Fraser halfconcentrated Discard 225 ml Palcam TSYEB Catalase Add 1 drop of hydrogen peroxid Gram Stain (Gram ) Motility Broth (25 C for 48 h until 5 days if it is necessary) Haemolysis in Blood Agar n. 2 (37 C - 24 ± 2 h) Ramnose (35 C or 37 C until 5 days) Xylose (35 C or 37 C until 5 days) Camp test in Blood Agar n. 2

9 Half-concentrated Fraser Broth (1) 064-BA5002 Fraser Base Broth (1) Listeria Selective Suppl. for Fraser CASE Ferric Ammonium Citrate CASE Fraser Broth 064-TA1125 Oxford Agar 064-PA0071 Oxford Base Agar Oxford Selective Supplement CASE Palcam Agar 064-PA0070 Palcam Base Agar Palcam Selective Supplement CASE Listeria Chromogenic Medium acc. ISO PA0LCM Catalase AC TA9999* Crystal Violet, solution according Gram Lugol s solution Bleaching agent, solution according Gram Safranine, solution according Gram Motility Broth 064-TA9998* Blood Base Agar n Defibrinated Sheep Blood (2) 064-SS0003 Blood Agar n PA9997 Ramnose Test 064-TA9996* Xylose Test 064-TA9995* TSYEB TA9993* TSYEA 064-PA9994* TSYEB (3) TA9993* Agar- Agar (1) Add 1 vial of CASE and 2 vials of CASE to , to obtain 1 l of half-concentrated Fraser Broth; (2) Only available in spanish market; (3) Add to TSYEB 9-18g/l of Agar-Agar to obtain TSYEA formulation * Please check availability Listeria EN-ISO :2000 Horizontal method for the detection and enumeration of Listeria monocytogenes. Part 2: Enumeration method. Dilution and revivification Isolation medium Buffered Peptone Water or Fraser Broth Base Spread inoculum over surface of PALCAM Agar See EN-ISO :1997 obtain 1/10 dilution Incubation: Aerobic or microaerobic 35 C or 37 C for 24 ± 2 h Revivification: 20 C for 1 h and continue for another 24 ± 2 h if there are no colonies or they are small Note: This standard is being revised. The new version it is envisaged, will include as an method a chromogenic medium, like ALOA Agar, or other lecithin based ones, and also another medium of the laboratories choice. Warning!: With the object of safeguarding the health of the laboratory staff, it is recommended that care is taken and the tests for Listeria are performed in properly equipped labs under the supervision of qualified microbiologists and that all contaminated materials are discarded safely. In particular it is recommended that female personnel are advised of the risk to the developing foetus if the mother is infected after exposure to Listeria monocytogenes. National legislation may establish the exact requirements. dilution and revivification (See below) 0, Buffered Peptone Water or Fraser Broth Base Palcam Discard TSYEA (35 C or 37 C for 18 h or 24 h) See on page before, the confirmation references of EN-ISO :1997 Buffered Peptone Water BA BA TA0100 Fraser Base Broth (1) Listeria Selective Suppl. for Fraser Ferric Ammonium Citrate Fraser Broth 064-BA TA1125 Palcam Agar Palcam Base Agar Palcam Selective Supplement CASE CASE CASE 064-PA0070 Tel Fax

10 Microorganisms en-iso 4833:2003 Horizontal method for the enumeration of microorganisms. Colony-count technique at 30 C. Isolation medium Plate Count Agar (PCA) Mass inoculation Incubation: 30 C for 72 ± 3 h sample dilution 15 ml Enumeration Plate Count Agar BA TA PA PR0001 Plate Count Skim milk Agar (1) (1) When milk and dairy products are examinated 15x15 Bacillus cereus en-iso 7932:1998 Horizontal method for the enumeration of presumtive Bacillus cereus. Colony-count technique at 30 C. Isolation medium Spread inoculum over the surface of Mannitol-Egg Yolk-Polymyxin Agar (MYPA) ß-haemolysis test on Sheep Blood Agar Incubation: 30 C for h (Blood Agar n. 2) Incubate for further 24 hours if colonies are not easily visible Incubation: 30 C for 24 ± 2 h SAMPLE DILUTION Isolation 0, ß-haemolysis 0, MYP Sheep Blood Agar MYP Base Agar (Bacillus Cereus Agar) BA2129 Egg s Yolk Sterile Emulsion Polymixin B Sulfate Selective Supplement CASE MYP Agar (Bacillus Cereus Agar) 064-PA0032 Blood Base Agar n Defibrinated Sheep Blood (1) 064-SS0003 Blood Agar n PA9997 (1) Only available in spanish market

11 Yeasts and moulds ISO 7954:1987 General guidance for enumeration of yeasts and moulds. Colony-count technique at 25 C. Isolation medium Chloramphenicol Glucose Agar, CGA (Yeast Extract Glucose Chloramphenicol Agar, YGC) Mass inoculation technique lncubation: 25 C with readings on day 3, 4 and 5 Microscopical observation if necessary sample dilution Yeasts 15 ml CGA Penicilium or others CGA Agar (YGC) BA PA9992 Tel Fax

12 Clostridium perfringens EN-ISO 7937:2005 Horizontal method for the enumeration of Clostridium perfringens. Colonies enumeration technique. Isolation medium : Two options Mass inoculation (poured-plate technique) Option 1: in Sulfite-Cycloserine without Egg Yolk (SC) Lactose Sulfite Broth (LS) (This was originally designated Egg-Yolk-free Tryptose-Sulfite-Cycloserine without Egg Yolk, TSC) Incubation: Incubate anaerobically 46 C for h in bath Incubation: Under anaerobic conditions 37 C for 20 ± 2 h With prior inoculation in Thioglycollate Broth Incubation: Incubate anaerobically 37 C for h Option 2: Motility-Nitrate Medium Stab-inoculate colonies into freshly deaerated medium 37 C for 24 h Incubation: Incubate anaerobically 37 C for 24 h Lactose-Gelatine Medium Incubation: Incubate anaerobically 35 C or 37 C for 24 h plus another 24 h to check Gelatin test, previous cooling 5 C for 1 h sample dilution confirmation Option 1: Lactose Sulfite Broth (LS) 15 ml 5 drops Thioglycollate Broth Lactose Sulfite Broth TSC TSC Option 2: Motility-Nitrate Medium Lactose Gelatin Medium TSC Agar Base BA6016 Cycloserine Selective Supplement CASE TSC Agar 064-BA6020 Thioglicollate TA0134 Lactose Sulfite Base Broth Ammonium Ferric Citrate CASE Sodium Metabisulfite solution CASE Lactose Sulfite Broth 064-TA0846 Motility-Nitrate Medium TA9991* Lactose-Gelatin Medium TA9990* A Reagent for Nitrates Reduction test B Reagent for Nitrates Reduction test * Please check availability

13 Salmonella spp. EN-ISO 6579:2003 Horizontal method for the detection of Salmonella spp. Pre-enrichment medium Enrichment media Isolation media Buffered Peptone Water Rappaport-Vassiliadis Soya Broth (RVS) Isolation in Xylose Lysine Biochemical Incubation: 37 C for 18 ± 2 h Incubation: 41,5 C for 24 ± 3 h Deoxycholate Agar (XLD) Triple Sugar iron Agar ( TSI) and a second agar Urea Agar choosed by the laboratory Lysine Descarboxilation Incubation: 37 C for 24 ± 3 h ß-galactosidase Voges-Proskauer Tetrathionate Muller-Kauffmann Broth Isolation in Xylose Lysine Indole with Novobiocine (MKTTn) Deoxycholate Agar (XLD) Incubation: 37 C for 24 ± 3 h Incubation: 37 C for 24 ± 3 h and a second agar Serological choosed by the laboratory Antigens O, Incubation: 37 C for 24 ± 3 h H and Vi pre-enrichment enrichment 25 g or 25 ml XLD and other second agar chosen by the laboratory 0, 0, Rappaport-Vassiliadis Soya Broth Brilliant Green Agar (BGA) Hektoen Agar Salmonella Shigella Agar (SS) Chromogenic Buffered Peptone Water 225 ml XLD and other second agar chosen by the laboratory MKTTn Brilliant Green Agar (BGA) Hektoen Agar Salmonella Shigella Agar (SS) Chromogenic TSI Agar Urea Agar Lysine descarboxilation ß-galactosidase Voges-Proskauer Indole See the ordering information, on the next page. Tel Fax

14 Buffered Peptone Water BA6012 Rappaport Vassiliadis Caldo TA0198 Tetrathionate Muller-Kauffmann Base Broth Brilliant Green-Novobiocin Supplement CASE Iodine-iodide solution for Muller Kauffman 064-V11108 Tetrathionate Muller-Kauffmann Broth 064-TA1024 Iodine-iodide solution for Muller Kauffman 064-V11108 Xylose Lysine Deoxycholate Agar (XLD) PA0034 Brilliant Green Agar (BGA) PA0045 Hektoen Agar PA0014 Salmonella Shigella Agar (SS) PA0028 Salmonella Plus Chromogenic Agar (Colorex ) 064-PA0083 TSI Agar TA0177 Urea Base Agar Urea Sterile Solution 40% Urea Agar 064-TA0148 Lysine Descarboxilation 064-TA0125* ß-galactosidase (ONPG) 064-TA0203 Methyl Red Voges-Proskauer Broth (MRVP Broth) TA0261 Barrit s Reagent for Voges-Proskauer Test O Meara s Reagent for Voges-Proskauer Test Indole Test 064-TA0132 Kovac s reagent for Indole Test Colorex is the trademark for CHROMagar prepared media. * Please check availability Food products determinations Microorganisms Enterobacteriaceae E. coli S. aureus Salmonella Yeasts and moulds Other parameters/ /Coliforms L. monocytogenes Comments Bottled drinking water (microbiological rules at the bottle) Coliforms Enterococci, P. aeruginosa, Clostridia Bottled drinking water (water rules from water source) Coliforms Fecal enterococci, P. aeruginosa, Clostridia Drinking water for public consumption Coliforms Enterococci, C. perfringens Broth, consommé, soups and creams C. perfringens, B. cereus Sweets and chewing gums Enterobacteriaceae Yeasts and moulds Refrigerated and frozen meat Enterobacteriaceae C. perfringens, Shigella Fresh poultry and rabbit meat Enterobacteriaceae C. perfringens, Shigella Minced meat from the butcher s C. perfringens, Shigella Minced meat from the meat industry Meat derivatives (ham, cold meat, sausages) Butcher s products (cooked and pasteurized) Enterobacteriaceae Shigella, Anaerobic Butcher s products (in cans) Enterobacteriaceae Shigella, Anaerobic Ham and cold ham Enterobacteriaceae Shigella, Cl. sulfite reductors Roasted join and cold bacon Enterobacteriaceae Shigella, Cl. sulfite reductors Cooked lean meat and and cold lean meat Enterobacteriaceae Shigella, Cl. sulfite reductors Cereals Coliforms Yeasts and moulds Breakfast cereals Yeasts and moulds B. cereus Beer Enterobacteriaceae Moulds Group A: Non-heat-treated processed food and heat-treated Coliforms L. monocytogenes 1 processed food with non-heat-treated ingredients Group B: Heat-treated processed food Coliforms L. monocytogenes 1 Group C: Sterilized processed food 1 Group D: Packed food with raw vegetables L. monocytogenes 1 Herbs and spices Sulfite reductors Canned food Enterobacteriaceae Shigella, Cl. sulfite reductors Curd milk and coagulant enzymes Enterobacteriaceae Yeasts and moulds Cl. sulfite reductors Special food supplements for breastfeeding Coliforms Yeasts and moulds Fruits and vegetables Coliforms Yeasts and moulds Frozen fruits and vegetables Coliforms Yeasts and moulds Shigella, Sulfite reductors, aerobic psicrofilic Plain biscuits Enterobacteriaceae B. cereus Non-plain biscuits Enterobacteriaceae B. cereus Edible gelatins C. perfringens Edible fats Yeasts and moulds Arizona Semolina and flour Moulds Ice-creams and frozen products Coliforms L. monocytogenes Syrups Enterobacteriaceae 1 Without pathogenic agents

15 Food products determinations Microorganisms Enterobacteriaceae E. coli S. aureus Salmonella Yeasts and moulds Other parameters/ /Coliforms L. monocytogenes Comments Raw cow smilk heat-treated dairy industry Raw cow s milk for non-heat-treated dairy products Raw cow s milk, non-heat-treated between 36 hours Raw buffalo s milk for heat-treated dairy products Raw buffalo s milk for non-heat-treated dairy products Raw goat and sheep s milk for heat-treated dairy products Raw goat and sheep s milk for non-heat-treated dairy products Raw cow s milk for direct consumption Pasteurized or heat-treated cow s milk Pasteurized milk Coliforms L. monocytogenes Sterilized and UHT milk Coliforms Powdered milk Coliforms L. monocytogenes Cheese from raw or heat-treated milk L. monocytogenes Cream cheese (from heat-treated milk) Coliforms L. monocytogenes Cottage cheese L. monocytogenes Pasteurized cheese Enterobacteriaceae L. monocytogenes Shigella Cream cheese Enterobacteriaceae L. monocytogenes Shigella Cream, grated or powdered cheese or with vegetable ingredients Enterobacteriaceae L. monocytogenes Shigella Frozen dairy products Coliforms L. monocytogenes Liquid dairy products Coliforms L. monocytogenes Sterilized dairy products, UHT liquids or jellies. Liquid dairy products, heat-treated, non-sterilized Coliforms L. monocytogenes and non-fermented (cream, condensed milk ) Butter from milk or pasteurized cream Coliforms L. monocytogenes Shigella Curd cheese Enterobacteriaceae L. monocytogenes Shigella Yogurt Coliforms L. bulgaricus, S. thermophilus Honey Enterobacteriaceae Moulds Shigella Egg products Enterobacteriaceae Cakes and pastries Yeasts and moulds Shigella, Cl. sulfite reductors Crisps and snack food Enterobacteriaceae Streptococcus faecalis, Shigella Fresh, salted, refrigerated and frozen fish products Enterobacteriaceae Shigella Cooked fish products Enterobacteriaceae Shigella Canned seafood with vinegar Enterobacteriaceae Shigella Anchovies in oil Enterobacteriaceae Anaerobes Seafood Coliforms Dried, salted fish products Enterobacteriaceae Shigella Smoked fish Enterobacteriaceae Shigella Smoked salmon, cod and others Coliforms L. monocytogenes Smoked herring and anchovies in brine Coliforms L. monocytogenes Smoked salmon, vacuum-packed Coliforms L. monocytogenes Cooked seafood Coliforms Seafood with shell Seafood without shell Coliforms Crab meat Packed salt 1 Table sauces: Coliforms Fried tomato Shigella, B. cereus Ketchup Enterobacteriaceae Shigella Mayonnaise Enterobacteriaceae Shigella Mustard Enterobacteriaceae Shigella Others sauces Enterobacteriaceae Shigella Tea and derivatives Shigella, B. cereus Nougat and marzipan Enterobacteriaceae Yeasts and moulds Shigella 1 Without pathogenic agents Tel Fax

16 Technical documentation Culture Media Handbook Edition no. 9 The latest edition no. 9 is ready. Over 276 pages with helpful information and photos of our media. Spiral bound for easy and space-saving manipulation on the work bench. International edition written in English language. Cross reference charts available separately in Excel format. New MSDS can be downloaded from our website: Contains over 350 different products. Microbiology catalogues 1 2 NEW 3 NEW 4 5 NEW 6 7 NEW CAT-FOOD07 (7-2007) : Dehydrated culture media handbook 2: NEW Prepared culture media for microbiology leaflet 3: NEW Microbiological water testing leaflet 4: Clinical microbiology leaflet 5: NEW Food analysis leaflet 6: RPF leaflet 7: NEW CHROMagar leaflet 8: Colinstant leaflet 9: Supplements leaflet 10: Dehydrated/prepared culture media and culture media-supplements poster Scharlab S.L. Tel Fax