Rapid Microbiological Methods:

Why and what!

Dr. Lorenzo Castigliego, PhD

OK SERVIS BioPro, s.r.o. Bořetická 2668/1, 193 00 Praha

XIV. VEDECKÁ KONFERENCIA S MEDZINÁRODNOU ÚČASŤOU

BEZPEČNOSŤ A KONTROLA POTRAVÍN

29. – 31. March 2017

Piešťany, Slovenská Republika

is on the market of effective and advantageous for pathogen detection and count of quality indicators

Why rapid microbiological methods (RMM) are often more convenient for food industry

What

Content of the presentation

1

2

Traditional methods (standard)

“Good”

Long

Labor demanding

Apparently less expensive

Sensitivity

Accuracy

Reproducibility

Direct costs

Costs of labor

Other indirect costs

• Multiple incubation and plating procedures • Use of several different materials

PROS & CONS

• Regulatory approved solutions

What food industry and service labs need ?

Direct costs Indirect costs

• Robust and simple methods

• Short time to result

• Solutions improving lab efficiency and decreasing expenses

RAPIDITY

SIMPLICITITY

Rapid Microbiological

Methods

RMM

What food industry and service labs need ?

• Regulatory approved solutions

• Robust and simple methods

• Short time to result

• Solutions improving lab efficiency and decreasing expenses

Rapid Microbiological Methods: advantages

Simplicity

Rapidity

Simple methods

HR management • Less work for operators • More or different tasks • Better organization of time

Temporary lack of personnel (stuff turnover; sick or maternity leaves)

Activity peaks

No specialized personnel

Analysis directly in small facilities of the industry

No need for big or complex equipment

Costs of personnel

Easier turnover of technicians

Staff training needs

Cost of labor

Simple methods

Short time to result

Increase of sample throughput Earlier in-process notification

Speed of corrective actions (decisions)

Raw material orientation Finished or semi-finished products Reactivity after cleaning

Product loss Delay in production and delivery

Production

Lab analyses

Short time to result

Release time of products

• Lower costs of storage (and energy)

• More rapid cash flow

• More time for operators

Faster placing on the market of the products

Hidden costs

Short time to result

Release time of products

Carry Costs (holding costs of inventory w/o sales (invoicing))

Finance (daily cost of capital: 5%/365=0,0137% per day)

Damage (longer to hold => higher risk of damage)

Obsolescence (shortened shelf life)

Warehousing costs… Electricity, rent, overtime, counting, cooling,…

Lost sales (because you can´t deliver)

Lost sales = lost growth profit

Expedite fees (supplier, contract lab,..)

Extra costs (overtime in lab, extra services)

Additional manufacturing costs

Additional packaging , additional QC time(!) ->$

Freight out (express delivery!)

Cost effectiveness – inventory costs

One real practical example:

Company: Very important brand in the global food market

Stock value (in the period of storage)…………..488.000.000

Cost for 58 days of stock cover.........39.040.000

Cost for 1 day of stock cover…………673.103

Kind of products: various (perishable and long-term storable)

How much money could be saved if the period of storage could be reduced of

about 1\3 or 2\3?

2,7% 5,3%

Spaces Labor

Energy Invoicing time

Cash flow Etc……..

Of the stock value

Real profit Much higher % Much higher %

Actual costs Analysis

Labor

Instrumentation

Storing

RMM Standard methods

** *

**/*** **

* **

* ***

Time to solve crisis events * ***

* *** Finance

Spaces Energy

Management Indirect costs

!

!

Actual costs: a final comparison

Investment

Fast methods

Less money to spend = Simple methods

=

A straightforward equation:

Less money to spend

Which rapid microbiological methods

What possibilities for RMM in pathogen detection?

Rapid chromogenic media

Nowadays very evoluted (specific growth factors + chromogenic

substances)

Automated immunoenzymatic methods

Direct detection of pathogens w/o plating

PCR Detection of pathogens` DNA

w/o plating

Fast

Fast

Very fast

Very fast

Rapid chromogenic media

Nowadays very evolved (specific growth factors + chromogenic

substances)

Fast

Which possibilities for RMM in quality indicator counting?

MPN-based methods

Automatic detection of fluorescence associated to microbial growth Fast

Flow cytometry Detection of single microbial cells ULTRA

fast

Validation

National and International

ISO 16140

AOAC OMA

Assure a high level of performance Simplify workflow of exporting companies

Rapid chromogenic media

Immunoenzymatic

PCR

Which possibilities for RMM?

MPN-based methods

Flow cytometry

They can replace standard reference methods

Peace of mind - VIDAS

• Ready to use reagents (minimum risk of error) in 1 single strip

• Easy to use system and protocols

Pathogen detection

Enzyme Linked Fluorescence Assay

Automatic

As easy as 1, 2, 3...

1- Load sample

2- Insert test

3- Press button

and….

Walk Away

Standard methods VIDAS 48 h VIDAS NEXT day

VIDAS/standard methods workflow

Pre-enrichment

Enrichment

Plating

Results

• LMX (L. mono. )

• LPT (L. spp )

• SPT (Salmonella)

• ECPT(E. coli O:157 incl. H7)

• LDUO (Listeria spp + L. mono)

• LMO2 (Listeria mono.)

• SLM (Salmonella)

• CAM (Campylobacter)

- 24 h

Next day solutions 48 hours solutions

VIDAS applications

(even less than 24h)

Count of quality indicators

Automated solution based on traditional method (MPN) to enumerate Quality Indicators in food and environmental samples

Only 1 dilution

No dilution No inoculation No counting

TEMPO Reader TEMPO Filler

1 ml

10-3

1 ml

10-4

Incubation

3 + 2 + 2 +

Counting

X CFU/ g

Interpretation using statistical

tables

10-1 10-2

1 ml

TEMPO: the principle of MPN

• The core of the system: an ENUMERATION card 48 tubes (or wells) of 3 different sizes for the 3 dilutions…

TEMPO: the principle

• The core of the system: an ENUMERATION card … integrated in a molded plastic card…

TEMPO: the principle

• The core of the system: an ENUMERATION card

… after incubation, fluorescence reading (under UV lamp)

TEMPO: the principle

Selective medium

+

Precursor of a fluorescent molecule

More than 100 tests performed in 1 hour only

6 cards filled in only 3 minutes

with 1 TEMPO Filler

During this period, preparation of 6 additional cards

Time and labor saving

Parameter Incubation time

AC Aerobic count Next day

EB Enterobacteriaceae Next day

EC Escherichia coli Next day

STA Staph. Coag. + Next day

TC Total coliforms Next day

CC Coliforms` count Next day

YM Yeasts and molds 72 – 76 h VS 5 days!

LAB Lactic Acid Bacteria 40 – 48 h

BC Bacillus cereus Next day

TEMPO applications

ULTRA-rapid microbiological tests

LASER DETECTION OF SINGLE VIABLE MICROBIAL CELLS

Presence/absence

Direct count

• Complete automation

• Digital data process with full traceability

• Up to 50 samples per hour

• Ready-to-use reagents

Flow cytometry

Criteria of Viability :

Membrane integrity

Enzyme activity

Enzyme

Viability substrate

free fluorochrome

micro-organism

The technology of D-count

Labelled cells are forced through a micro linear fluid stream

Microorganism cells are hit by a laser beam

Emitted fluorescence is detected by sensitive photo-detectors

Microorganism cells are counted one by one

Detector 1

Detector 2

Waste

Laser

Sample

• Counting every single viable cell

The technology of D-count

The first and only alternative method based on

Flow cytometry being ISO 16140 AFNOR validated

Patented cell labeling technology +++ sensitivity

From 1 log to 6 log more sensitive than the other alternative methods

Shorter pre-incubation time for analysis

Best analytical sensitivity

Validation

D-count

• Yeasts in fermented milk products

• Enterobacteriaceae

• Yeasts & molds in fruit preparations

• Sterility testing UHT milk

• Sterility testing dessert

Direct count in process water

Direct count in milk

Applications in DAIRY PRODUCTS

PRESENCE/ABSENCE

DIRECT COUNT

Commercial sterility

Yeasts & molds in soft beverages and fruit juices

Direct count in process water

Yeasts in fruit concentrates

Bacteria in fruit concentrates

PRESENCE/ABSENCE

Applications in BEVERAGES

DIRECT COUNT

Economical BENEFITS

Global benefits for the company

Rapid methods in microbiology

To make money

TAKE HOME MESSAGE

To save money Labor Financial Warehousing Inventory

What we propose…

Pathogen detection

Count of quality indicators

Presence/absence

Direct count

Ultra-rapid microbiology

What we propose…

You are welcome to speak with us about your needs and projects…