Technological advances in Detecting Microbial Hazards in Food. - - PowerPoint PPT Presentation

Technological advances in Detecting Microbial Hazards in Food.

Rahul Warke

Microbial Hazards

• Survival and growth of microorganisms in food processing

environments may lead to contamination of the finished product that may, in turn result in a reduction of microbiological safety and quality causing a variety of illness and diseases.

• Testing has a very intrinsic role to play in ensuring food safety and in

detecting microbial hazards.

Not all iroial testig is the sae.

Three types of testing:

• Traditional batch testing.
• Environmental testing.
• Process control testing for verification.

Time is money.. …...

Technological Advances : Rapid Methods:

• Methods for detection, identification and enumeration of

microorganisms offer savings in both time and labor.

Tests:

• Sample Preparation
• Enumeration.
• Viable cell counts
• Detection
• Presence or absence
• Identification
• To confirm if hazardous or non hazardous.

Advances in Viable Cell Counts.

• Total Viable count a successful test to gauge
• Sanitary quality
• Organoleptic acceptability
• Adherence to good Manufacturing practices.
• An indication of Safety.

Classical test:

• Pour Plate Techniques.
• Surface or Spread Plate Method.
• Membrane Filtration

Newer Techniques:

• Spiral Plating:
• Reduces human error.
• Consistency in results.
• Automation in reading and platting.
• Petrifilm
• Reduced Labour costs and time.
• Consistent and accurate results.
• User friendly.

• Redigel
• Pectin based gels.
• Saves time.
• Convenient for use
• Simplate
• Rapid Enumeration of Total Aerobic Bacteria
• Reduced test costs.

Identification

• Biochemical and Enzymatic methods:
• Use of Metabolic activity of microorganisms for identification purpose.

Automated systems:

• API sytems.
• BD Crystal ID system.
• Biolog
• Micro ID.
• VITEK
• HiBioID.

• Antibody-Based Methods:
• ELISA :
• VIDAS : 45 mins to 2 hrs.
• Reliable and convenient.
• Read and interpreted by machines
• Lateral Flow Technology:
• Simple to use
• Results can be available within 15 minutes
• Low cost with no requirement for additional equipment
• Stable and robust in storage
• Immuno-magnetic seperation (IMS)

Chromogenic Media:

Conventional Media Chromogenic Media

Sample Sample ↓ ↓ Pre-Enrichment (24 hrs) Pre-Enrichment (24 hrs) ↓ ↓ Selective Enrichment (24 hrs) Direct streaking on chromogenic media (24-48 hrs) ↓ ↓ Isolation (24-48 hrs) Biochemical identification (For further confirmation)

• 1. General Media
• 2. Selective Media

↓ Biochemical Identification (24-48 hrs)

Nucleic acid based Methods:

• Polymerase Chain Reaction (PCR)
• Amplification of target DNA and detection of target PCR products.

Most Popular brands for PCR based products: BAX, TaqMan, Molecular Beacon, Invitrogen, Cephid, Qiagen and Applies Biosystems. Features:

• Validated and accurate.
• Extremely expensive.
• Closed systems.

Collection of Food Sample of Interest Homogenization of Sample

Enrichment Procedure Open Systems.

Template Extraction

Mini-prep or 96 well Format (DNA Extraction or Heat Lysis)

PCR PCR Machine Series

Sae Day ‘esults…

FOOD PATHOGEN DETECTION - Prima-96

Salmonella spp. Detection by Heat Lysis

Milk Powder

Colusio: No Aplifiatio see i No-Spiked saples. Spiked Saples ould e aplified.

Environmental testing or Hygiene monitoring:

• Environmental swabs.
• Traditionally used for checking surface contamination.
• ATP Bioluminescent Assay swabs.
• Instant results.
• Organic matter interference.
• Prohibitive costs.

M1353 HiCrome UTI Agar

For presumptive identification, differentiation and confirmation of microorganisms mainly causing urinary tract infections. It can also be used for testing water, food, environmental & other clinical samples

• The medium contains two chromogens which detects b-glucosidase and b -D-galactosidase.
• Enterococci possesses the enzyme b-glucosidase – blue-green colonies
• Escherichia possesses the enzyme b -D-galactosidase – purple-magenta colonies further confirmation by DMACA
• reagent
• Coliforms possesses both the enzymes – blue coloured colonies.
• Proteus species – brown coloured colony due to tryptophan deaminase activity.
• Staphylococcus and Pseudomonas do not possess either of the enzyme , hence forms golden yellow and
• colourless colonies respectively.
• Prepared medium: Light amber coloured clear to slightly opalescent gel.

2 1 4 3

1) P. mirabilis (ATCC 25933)- Light brown 2) E. coli (ATCC 25922)- pink-purple 3) E. faecalis (ATCC 29212)- blue, small 4) K.pneumoniae (ATCC 13883) - blue, mucoid

Total Time Taken for Detection

PRIMA Series : 15-19 hours Pal PC‘™ 14-18 hours

Cost per test INR 200

Fung’s Ten Predictions for Food microbiology from 1995 to 2015 are:

• 1. Viable cell counts will still be used
• 2. Real-time monitoring of hygiene will be in place
• 3. PCR, Ribotyping and genetics test will become reality in food laboratories
• 4. ELISA and immunological tests will be completely automated and widely used.
• 5. Dipstick technology will provide rapid answers
• 6. Biosensors will be in place for HACCP programs
• 7. Instant Detection of target pathogen will be possible
• 8. Effective separation, concentration of target cells will greatly assist in rapid identification
• 9. Microbiology alert systems will be in food packages
• 10. Consumers will have rapid alert kits for pathogens at home.