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Food Microbiology – The basics

Scott Colbourne Business Manager NSW – ALS Food & Pharmaceutical

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

2 Food Microbiology - The Basics

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

3 Food Microbiology - The Basics

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

Food Microbiology - The Basics 9

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.

Method Turn Around Times

Food Microbiology - The Basics 10

Method Turnaround Time (Days) If Clean If Confirmation is Required Standard Plate Count 3 N/a Yeast and Mould 5 – 7 N/a Salmonella ELISA (Automated rapid method) 2 5 Australian Standard (manual method) 4 7 Listeria ELISA (Automated rapid method) 2 7 Australian Standard (manual method) 5 7

Method Turn Around Times

Food Microbiology - The Basics 11

Method Turnaround Time (Days) If Clean If Confirmation is Required Standard Plate Count 3 N/a Yeast and Mould 5 – 7 N/a Salmonella ELISA (Automated rapid method) 2 5 Australian Standard (manual method) 4 7 Listeria ELISA (Automated rapid method) 2 7 Australian Standard (manual method) 5 7 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…

Pathogens: ELISA is superior to the Australian Standard

Comparing Listeria and Salmonella test methodologies

12

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

Method Turn Around Times

Food Microbiology - The Basics 13

Method Turnaround Time TAT (Days) If Clean If Confirmation is Required 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

Method Turn Around Times

Food Microbiology - The Basics 14

Method Turnaround Time TAT (Days) If Clean If Confirmation is Required 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 Advantages:

• Quick
• Generally, no confirmation

step for coliforms Disadvantages:

• Sample matrix sensitive
• Cannot use for samples with:
• High colour
• High micro background
• High acidity

Method Turn Around Times

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Method Turnaround Time TAT (Days) If Clean If Confirmation is Required 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 Advantages:

• Quantitative
• Can report to low levels (0.3)
• Australian standard

Disadvantages:

• Labour intensive
• Statistical

value – most probable number

Method Turn Around Times

Food Microbiology - The Basics 16

Method Turnaround Time TAT (Days) If Clean If Confirmation is Required 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 Advantages:

• Great if not detected

Disadvantages:

• Qualitative only

Method Turn Around Times

Food Microbiology - The Basics 17

Method Turnaround Time TAT (Days) If Clean If Confirmation is Required 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 Advantages:

• Quick
• Quantitative

Disadvantages:

• Not available for E.Coli

Method Turn Around Times

Food Microbiology - The Basics 18

Method Turnaround Time TAT (Days) If Clean If Confirmation is Required 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 Alternative to Coliforms as a faecal contamination indicator Advantages:

• Quick
• Quantitative
• Salmonella is included

Disadvantages:

• Generic and positive results

may require specific testing

Method Turn Around Times

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Method Turnaround Time (Days) If Clean If Confirmation is Required Staphylococcus Plate Count 2 4-6 Presence/Absence 4 6

Confirmation Process

• What is a suspect or presumptive result?
• What is confirmation or follow up?

Food Microbiology - The Basics 20

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

• Have you ever received an email like this?

Confirmation Process

• What is a suspect or presumptive result?
• What is confirmation or follow up?

Food Microbiology - The Basics 21

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

• Have you ever received an email like this?

What is a suspect result? 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
• Other micro-organisms present that

interfere

• Matrix interference

Synonyms: Presumptive

Confirmation Process

• What is a suspect or presumptive result?
• What is confirmation or follow up?

Food Microbiology - The Basics 22

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

• Have you ever received an email like this?

What is confirmation? Carrying out further steps of a test for identifying a particular type of bacteria. Therefore CONFIRMING its presence. When the steps have been completed we say that the test has been “Confirmed”. After confirmation, the result will be finalised. Synonyms: Follow up

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.

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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.

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Result Possible Variation 5,000 (or 5 x 103) ± 500 3,000,000 (or 3 x 106) ± 500,000

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

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