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Food Microbiology – The basics Scott Colbourne Business Manager NSW – ALS Food & Pharmaceutical R IG H T S O L U T I O N S | R I G H T PA RT N E R Contents • Common Microbiological Tests - what and why • Method Turn Around Times (TAT) • The Confirmation Process • Causes of counts being high initially, then low when retested Food Microbiology - The Basics 2 Common Tests – what and why? • Standard Plate Count (SPC) What? A generic count of micro-organisms present. The results are non-specific Why? Gives a general indication of the level of micro-organisms present in the sample tested Can be referred to as… Total Microbial Count (TMC), Total Aerobic Microbial Count (TAMC), TVC, TVAC Food Microbiology - The Basics 3 Common Tests – what and why? • Yeast and Mould What? Generic test for the levels of these present. Non-specific. Why? • They cause biodegradation of natural materials, which may become food spoilage • Some yeasts are useful in fermentation (e.g. Bread and Beer) • However some (e.g. Candida albicans) are opportunistic pathogens and can cause infections Food Microbiology - The Basics 4 Common Tests – what and why? • Salmonella What? Pathogen found in cold/warm blooded animals and the environment Why? • It causes food poisoning, such as gastrointestinal issues • To ensure there is no presence of pathogens Food Microbiology - The Basics 5 Common Tests – what and why? • Listeria What? A genus of bacteria that contains 10 species, the most important genus, L. monocytogenes is a serious human pathogen. Why? Healthy people and pregnant women may have mild or no symptoms, but Listeria infection may still result in miscarriage, premature birth or stillbirth. In people at risk, Listeria infection can result in serious illnesses including meningitis and septicaemia. Food Microbiology - The Basics 6 Common Tests – what and why? • E. coli and coliforms What? Coliforms can be found in the aquatic environment, in soil and on vegetation; they are universally present in large numbers in the faeces of warm-blooded animals and humans. Why? • Commonly used bacterial indicators of hygiene and sanitary quality of foods and water. • E. coli are almost exclusively of faecal origin and their presence is thus an effective indicator of faecal contamination. • Most strains of E. coli are harmless, but some can cause serious illness in humans. Food Microbiology - The Basics 7 Common Tests – what and why? • Staphylococcus (coagulase positive) What? A common bacterium that lives on the skin or in the nose of humans. Why? Coagulase positive species of Staphylococcus can produce toxins in foods. Food Microbiology - The Basics 8 Common Tests – what and why? • Bacillus cereus What? A common bacterium found in the environment; typically associated in soil as well as a variety of foods. Why? The toxin producing spore forming bacterium can cause vomiting and diarrhoea. Bacillus cereus spores are able to survive harsh environments which include normal cooking temperatures. Food Microbiology - The Basics 9 Method Turn Around Times Turnaround Time (Days) Method If Clean If Confirmation is Required 3 N/a 5–7 N/a Standard Plate Count Yeast and Mould Salmonella ELISA (Automated rapid method) 2 5 Australian Standard (manual method) 4 7 ELISA (Automated rapid method) 2 7 Australian Standard (manual method) 5 7 Listeria Food Microbiology - The Basics 10 Method Turn Around Times Turnaround Time (Days) Method If Clean If Confirmation is Required 3 N/a 5–7 N/a Standard Plate Count Yeast and Mould Salmonella ELISA (Automated rapid method) 2 5 Australian Standard (manual method) 4 7 ELISA (Automated rapid method) 2 7 Australian Standard (manual method) 5 Listeria 7 Food Microbiology - The Basics The ELISA method (e.g. DS2) is quicker, more reliable and cheaper The confirmation process is identical to the Australian Standard method The next slide has more details… 11 Pathogens: ELISA is superior to the Australian Standard Comparing Listeria and Salmonella test methodologies Parameter ELISA Australian Standard Price High level of automation Labour intensive Speed - negative 2 days 4-5 days Speed – confirmed positive 5-7 days 7 days Interpretation of results Automated Labour intensive Human interaction Low High NATA accreditation Yes Yes Internationally recognised e.g. AFNOR Yes Yes Client preference 95% 5% Food Microbiology - The Basics 12 Method Turn Around Times Turnaround Time TAT (Days) Method If Confirmation is Required If Clean Coliforms Petrifilm 2 N/a MPN 2 4 Presence/Absence 2 4 Plate Count 1 N/a E.Coli Petrifilm 2 N/a MPN 2 6 Presence/Absence 2 6 Enterobacteriaceae Plate Count 1 3 Food Microbiology - The Basics 13 Method Turn Around Times Turnaround Time TAT (Days) Method If Confirmation is Required If Clean Coliforms Petrifilm 2 N/a MPN 2 4 Presence/Absence 2 4 Plate Count 1 N/a E.Coli Petrifilm 2 N/a MPN 2 6 Presence/Absence 2 6 Enterobacteriaceae Plate Count 1 Advantages: - Quick - Generally, no confirmation step for coliforms Disadvantages: - Sample matrix sensitive - Cannot use for samples with: - High colour - High micro background - High acidity 3 Food Microbiology - The Basics 14 Method Turn Around Times Turnaround Time TAT (Days) Method If Confirmation is Required If Clean Coliforms Petrifilm 2 N/a MPN 2 4 Presence/Absence 2 4 Plate Count 1 N/a Advantages: - Quantitative - Can report to low levels (0.3) - Australian standard E.Coli Petrifilm 2 N/a MPN 2 6 Presence/Absence 2 6 Disadvantages: - Labour intensive - Statistical value probable number – most Enterobacteriaceae Plate Count 1 3 Food Microbiology - The Basics 15 Method Turn Around Times Turnaround Time TAT (Days) Method If Confirmation is Required If Clean Coliforms Petrifilm 2 N/a MPN 2 4 Presence/Absence 2 4 Plate Count 1 N/a Advantages: - Great if not detected E.Coli Petrifilm 2 N/a MPN 2 6 Presence/Absence 2 6 Disadvantages: - Qualitative only Enterobacteriaceae Plate Count 1 3 Food Microbiology - The Basics 16 Method Turn Around Times Turnaround Time TAT (Days) Method If Confirmation is Required If Clean Coliforms Petrifilm 2 N/a MPN 2 4 Presence/Absence 2 4 Plate Count 1 N/a Advantages: - Quick - Quantitative E.Coli Petrifilm 2 N/a MPN 2 6 Presence/Absence 2 6 Disadvantages: - Not available for E.Coli Enterobacteriaceae Plate Count 1 3 Food Microbiology - The Basics 17 Method Turn Around Times Turnaround Time TAT (Days) Method If Confirmation is Required If Clean Coliforms Petrifilm 2 N/a MPN 2 4 Presence/Absence 2 4 Plate Count 1 N/a E.Coli Petrifilm 2 N/a MPN 2 6 Presence/Absence 2 6 Alternative to Coliforms as a faecal contamination indicator Advantages: - Quick - Quantitative - Salmonella is included Disadvantages: - Generic and positive results may require specific testing Enterobacteriaceae Plate Count 1 3 Food Microbiology - The Basics 18 Method Turn Around Times Turnaround Time (Days) Method If Clean If Confirmation is Required Staphylococcus Plate Count 2 4-6 Presence/Absence 4 6 Food Microbiology - The Basics 19 Confirmation Process • Have you ever received an email like this? Hi John, Please note that the below sample is suspect for E.Coli: Lot: 12345 Food sample A, DOM 15/05/2015 The sample is under confirmation and the final result will be updated once complete Thank you and best regards Jenny Bloggs Microbiologist • What is a suspect or presumptive result? • What is confirmation or follow up? Food Microbiology - The Basics 20 Confirmation Process • Have you ever received an email like this? Hi John, Please note that the below sample is suspect for E.Coli: Lot: 12345 Food sample A, DOM 15/05/2015 What is a suspect result? The sample is under confirmation and the final result will be updated once complete Thank you and best regards Jenny Bloggs Microbiologist • What is • What is For a number of microbiological tests the initial test is generic and any positive or SUSPECT result requires more testing. A suspect may be: - The micro-organism being analysed a suspect or presumptive - Other result? micro-organisms present that interfere confirmation or follow up?interference - Matrix Synonyms: Presumptive Food Microbiology - The Basics 21 Confirmation Process • Have you ever received an email like this? Hi John, Please note that the below sample is suspect for What E.Coli:is confirmation? Lot: 12345 Food sample A, DOM 15/05/2015 Carrying out further steps of a test for The sample is under confirmation and the final result will be a updated oncetype complete identifying particular of bacteria. Therefore CONFIRMING its presence. Thank you and best regards Jenny Bloggs Microbiologist • What is a suspect or When the steps have been completed we say that the test has been “Confirmed”. After confirmation, the result will be presumptive result? finalised. • What is confirmation or followSynonyms: up? Follow up Food Microbiology - The Basics 22 Confirmation Process • What tests can require confirmation? – Coagulase Positive Staphylococcus – E.coli and Coliforms – B. cereus – Salmonella – Listeria – Clostridium perfringens – Enterobacteriaceae Food Microbiology - The Basics 23 High to Low Counts • How can a result be high in one test and then low when retested? • This can be caused by a number of factors. – The sample may not be completely homogenous. – Lab error – The sample has anti-bacterial properties. – Natural sample variation – e.g. due to the make-up and/or components of the sample. Food Microbiology - The Basics 24 High to Low Counts: Sample Not Homogeneous • The laboratory generally uses 10g of the sample in the required media, e.g. 90g • Of that 100g, 0.1g to 1.0g is removed for each test • A reported result of 500 cfu/g, may only be 5 colonies on an agar plate. This is a low number and with repeat testing small variations will affect the result by 100’s of cfu/g • In general, acceptable variation would be within half a log, e.g. Result Possible Variation 5,000 (or 5 x 103) ± 500 3,000,000 (or 3 x 106) ± 500,000 Food Microbiology - The Basics 25 High to Low Counts: Anti-Bacterial Properties • The sample may contain substances that are anti-bacterial • Some samples have the capacity to kill bacteria, e.g. cinnamon, vinegar, salt and preservatives • Samples are stored for 3-5 days from initial testing & before a retest generally occurs. Substances in the sample itself may lower the bacterial count e.g water activity, available nutrients & temperature • Therefore possible high levels upon initial testing have been killed off or had their numbers lowered before the retest. Food Microbiology - The Basics 26 High to Low Counts: Lab Error • This is always a possibility and our quality procedures and controls minimise this risk. However it can never be 100% removed. • If we believe lab error is a possibility we will try to look for a pattern, e.g. • Do all the similar samples have a high count? • Does the sample have a history of high counts? • Do unrelated samples tested concurrently have high counts? • We also look at other factors, e.g. • Experience of the technician • Environmental monitoring results • Media testing, including daily positive/negative controls Food Microbiology - The Basics 27 End. • Thank you very much for your attention • If you would like more information on ALS Food, please visit our website or contact one of our team Food Microbiology - The Basics 28