MERCK LIFE SCIENCE

BIOMONITORING

Beirut, Lebanon, September 2016

RNDr.Robert Nadaskay
BioMonitoring MENATP
Table of Contents

1 Bioburden Testing / The Milliflex System

2 Rapid Detection Systems for Bioburden Testing

3 Air Monitoring

4 Hygiene Monitoring

5 Culture Media for Microbiology

6 Rapid Detection of Pathogens

7 Sterility testing: The Steritest System

Bioburden testing
Sources of Contamination
Employees
Packaging
AIR

Pests

Equipment
Buildings and facilities
Product

WATER Raw
Deliveries Materials
 Sources of Contamination: Filling Process

5 - Gas,
Auxiliary
1 – Final Beverage 2 - Piping
Fluids
11 - Atmosphere

9 - Jaws & Closure

Equipment
6 - Filler Bowl

7 - Filler
Heads
3 - Containers

8 - Closures

10 - People

4 - Conveyors

5
Sources of Contamination: Human origin

Humans constantly shed both viable and

non-viable particles
The skin of a healthy adult typically
sheds
2 to 6 million CFUs in 30 min of
vigorous activity !
Clothing fibers and body hair
Moisture
Non-viable particles and fibers can
transport microbes to surfaces and
products

6
Sources of contamination
Sampling and Filtration
 Raw water
 Non-sanitary sampling ports

 Raw materials  Non-sterile collection container

 Non-sterile filtration systems for

 Still/stagnant water analysis

 Non-sterile sanitants
 Dead legs in piping
 Poor sampling technique
 Uncovered tanks  Non-sterile analytical waters

 Poor maintenance of H2O system

 Entrance of non-sterile air to tanks when draining or

venting

 Biofilm build up due to insufficient sanitising or low

dosage sanitant. 7
Biofilm

1- initial attachement
2- irreversible atachement
3- maturation I
4- maturation II
5- dispersion

8
 MOLD & YEAST

BACTERIA
Microbial contamination

VIRUS
PROTOZOA

http://www.sdnhm.org/exhibits/epidemic/naturalhistory.html
Micro organisms commonly tested ?

 Pathogens
 Spoilage micro organisms
Example:

Water fecal contamination:

Bacteria: E Coli, Salmonella, Shigella, Vibrio (cholerae)
 Indicators are easy to detect and corelate to pathogens
 ex: Coliforms (enterobacter, E Coli)

Beverage spoilage:
Yeast & Molds
 source of “bad taste” and pH change
 Ex Candida davenportii in soft drinks
Bioburden (Microbial contamination) testing:

• Food and Beverage

• Process water
• Final products

• Water and waste water testing

• Municipal water, recreational water, swimming pools
• Environmental water samples

• Medical
• Multiple water types

• Pharmaceutical / Cosmetic applications

• Purified water, WFI, final products
QC Bioburden Testing in Micro Labs

Microbiology
Raw material Laboratory
Final Product

In-Process Environmental
Water Monitoring

Support Data
Product Release
Methods of Microbiology

Membrane Filtration Direct Inoculation

Advantages
Advantages
Rinse away inhibitory or
preservatives agents  Fast, Easy
Wide selection of media
Sample concentration :


 high volume sample can be filtered  Non-filterable samples

 Statistically more valid
 More sensitive: 1 CFU/volume
Limitations Limitations
Non filterable samples  Limited sample size: lower
High solids may plug sensitivity
membrane  Small Colonies
 Inhibition issues
USP: Pharmaceutical Water

Suggested Methods
Purified Water
 Pour Plate or MEMBRANE FILTRATION METHOD
 Sample Volume—1.0 mL minimum
 Growth Medium—Plate Count Agar
 Incubation - 48 to 72 hours min., 30-35 °C

Water for Injection

 MEMBRANE FILTRATION METHOD
 Sample Volume—100 mL minimum
 Growth Medium—Plate Count Agar
 Incubation Time—48 to 72 hours min.,30 - 35 °C
EP: Pharmaceutical Water

EP : Purified water in bulk

 Membrane filtration
 Size of the sample ... in relation to the expected results
 Agar R2A
 Incubation: 30 - 35°C, 5 d minimum
 Appropriate Action limit: Total viable count < 100 cfu/ml

EP: WFI, Higly Purified Water

 Membrane filtration
 At least 200 ml
 Agar R2A
 Incubation: 30 - 35°C, 5 d minimum
 Appropriate Action limit: Total viable count < 10 cfu/ 100 ml
The Basics of Membrane Filtration for
Microbiology
The 5 Step Method

2. Filtration 3. Adding of media

1. Sampling

4. Incubation 5. Examination
Membrane Filtration: principle
Filtration Retention Recovery

3193JE
Membrane Filtration: Retention & Recovery

Retention Recovery
Ability to capture all Ability to grow captured
microorganisms on microorganisms
membrane surface filter matrix must allow free flow
of nutrients
proper nutrients, temperature,
and time
cells must be able to replicate

3193JE
Pore Size –
Actual Regulations USP <1231>

Membrane Filtration
 Method sensitivity can be increased via the use of larger
sample sizes. This strategy is used in the membrane
filtration method

 “A membrane filter with a rating of 0.45 µm is generally

considered preferable even though the cellular width of
some of the bacteria in the sample may be narrower than
this. The efficiency of the filtration process still allows the
retention of a very high percentage of these smaller cells
and is adequate for this application. Filters with smaller
ratings may be used if desired, but for a variety of reasons
the ability of the retained cells to develop into visible
colonies may be compromised, so count accuracy must be
verified by a reference approach.”
 Filtration: Membrane Selection

Membrane filter: pore size selection as the best compromise between retention and recovery of microorganisms

 0.45m pore size filter: recommended by Pharmacopeia

 Best Retention of bacteria
 Best Recovery
 0.8 m pore size filter Best General Membrane:
 Complete Retention of yeast & mold
 Improved Throughput (hard to filter samples) White
 0.2 m pore size filter 0.45 m
 Sterilization purposes Grid
Individual
Sterile
Cellulose
 E. coli on membrane surface

E. coli

24
Enterococci retained on a Microporous membrane

Most membranes are made of esters of

cellulose – Millipore Mixed Cellulose Esters
(MCE) membranes
Membrane Filtration
What products are available ?

Filtration membranes and

membrane dispensers

Funnels & manifolds

Filtration monitors

Liquid medias & culture dishes

Accessories, pump
Bioburden membrane filtration products:
EZ Family

The EZ-Family complete solution

AO/SO Membranes
Specification

47mm Non-sterile or Sterile membranes + pad support (pad for liquid media) bulk packed
Mixed Ester of Cellulose
Autoclave pak (AO)
10 resealable envelopes containing 10 filters and 10 pads, requires autoclaving
Sterile pak (SO)
10 resealable envelopes containing 10 filters and 10 pads, presterilized
Materials of Construction
Membranes: Mixed esters of cellulose
Pads: Cellulose
S-PAK Membranes

- Sterile gridded membrane filters made

of Mixed Esters of Cellulose *
- Individually packed
- 4 boxes of 150 membrane
- Certificate of Quality enclosed confirms
Standard Methods compliance
- Offering based on Filter colour, pore size
selection and diameter
(standard 47 mm, also 50mm diameter)

* Except HVWP047S6 made of PVDF

S-PAK Membranes
Why the blue paper protection?

Film laminated with glue in order to be

able to seal the film with medical paper.

Blue paper to avoid any transfer of glue

from film to the membrane.

The blue paper avoids:

•Hydrophobic spots
•Inhibition growth issues

30
S-PAK Membranes

Membrane Selection

Pore size: compromise between flow and retention

0.45 m the “universal” standard for drinking water
0.8 m for yeast and mold testing
1.2 m for yeast and mold testing in hard to filter samples
0.22 m when regulation imposes

Color: choose for contrast between filter and colony color

White for testing water
Black for testing for yeast

Special cases:
Antibiotics and preservatives: hydrophobic edge membranes or Durapore HV
Solvents: Durapore HV – HVWG047S6, 0.45µm, 600 pack)
EZ-Pak Filtration membranes

S-Pak filters in pleated band

packaging
4 cartridges of 150 membranes
Catalogue number, lot number, pore
size on each membrane cover
Used with EZ Pak Curve dispenser
EZ-Pak dispenser Curve
 EZ-Pak dispenser Curve positioning
The EZ-Pak dispenser Curve is the best dispenser
on the market, ideal for beverage, municipal water and
pharmaceutical industries.
Reliability, contamination safety and ease of use strong points,
it is particularly suited to high-throughput laboratories
First automated dispenser with membrane exposure adjustment
First battery powered dispenser enabling portability
Fastest membrane loading on the market
It is part of the EZ family, a complete solution for reliable
routine bioburden analysis.
Reduce False result Rate
 No-touch operation
 One-handed membrane transfer
Industrial IR sensor
No touch detection
Move your hand toward the sensor
and a membrane is unwrapped,
dispensed and automatically
positioned for transfer with forceps.
Software controls prevent the
accidental dispensing of multiple
membranes.
Optimize workflow
 Fast membrane loading
 Membrane auto-adjustment
 Fast membrane dispensing (<1sec)

Easy membrane loading

New design with cover
allows you to load a
cartridge in less than 30
seconds. Membrane is
automatically positioned
for easy transfer with
forceps.
Optimize workflow
 Cable less and portable
 Easy decontamination design
 Lightweight
 Robust
 Long lasting components
EZ-Pak Curve video …
… discovering the key benefits & Features
 Optimized workflow
 Reduced false results rate

Type EZ-Fit Manifold on our website and get access to the video
EZ-Fit Manifold
EZ-Fit Manifold positioning
The EZ-Fit Manifold is the best filtration support
on the market, ideal for municipal water, beverage, and
pharmaceutical industries.
Its unique features allows efficient hardware cleaning &
decontamination reducing false results rate and optimizing
laboratory operations

It is part of the EZ family, a complete solution for reliable

routine bioburden analysis.
Reduce False result Rate: decontamination
 All components can be removed without any tools !
 Easy access to all inner parts to remove biofilms (1)
 Manifold and components can be autoclaved

1 Quick connections
No tools !
Reduce False result Rate
 Check valve to avoid liquid back-flow from vacuum source (2)
 Valve design prevents manifold contamination (3)

2
3
 Optimized workflow
 Quick-fit filtration heads for
all MM consumables or
reusable funnels

Glass & SS funnels

Microfil Monitor 55+ Microfil V/S

Optimized workflow
 Valves are easy to operate (1)
 Low height increases comfort (2) .. And low weight too
 Tubing to vacuum source can be connected to either side

2
Optimized workflow
 1 to 6 places
EZ-Fit Manifold video
… discovering the key benefits & Features
 Optimized workflow
 Reduced false results rate

Type EZ-Fit Manifold on our website and get access to the video
MICROFIL FUNNELS

MICROFIL EQUIPMENT
Membrane Filtration: Filtration apparatus:

1. Traditional re-usable funnels

2. Disposable single use funnels

Traditional Filtration Methods Limitations

Preparation/sterilization of the funnels is painful

Glass funnel
Breakage
Stainless steel funnel

Long to cool
Other autoclavable funnels Not transparent

Sterilization
labour
Microfil
Disposable funnels / membranes

Pre-sterilized funnels
Push-fit funnel
Patented flexible seal
Stackable
Capacity :100 & 250 ml

Available with S-Pak or EZ-Pak membranes ONLY

Optimized workflow
 New Microfil head with rim for easy membrane transfer with forceps

Rim
Microfil consumables

Microfil + S-Pak
Box content:
•150 S-Pak membranes
•150 funnels
(6 x 25 for 100ml and 5 x 30 for 250ml)

Microfil + EZ-Pak
Box content:
•150 EZ-Pak membranes
•150 funnels
(6 x 25 for 100ml and 5 x 30 for 250ml)

Each box contains user’s guide and COQ 52

MICROFIL V
MICROFIL S
Funnels/membranes

MICROFIL V/S

Pre-sterilized 100ml
funnels+membrane assembly with
individual support
 No Manifold decontamination
 No membrane handling before sampling
Easy membrane removal
Microfil V/S - handling

1. 2.
With convenient Place device
carrying tray placed onto standard number
near manifold or 8 stopper, pour
vacuum flask, sample, and apply
remove stopper and vacuum
snap device off tray.

4.
3. Place membrane
onto solid or
After filtration, liquid medium
remove the funnel, and incubate.
You only need one
hand, and discard it
(it’s recyclable) Discard the base
No adapter required (it’s recyclable)
Microfil S

Sterile, Individually
Packed
Convenient Blister Packed
Microfil 100 ml Funnels
24 per box

Also available with PVDF membrane

Filtration Monitor

55-Plus Monitor
 Filter holders with 55mm filtration area
Sterile, includes membrane
 Ready-to-use
 For use with liquid media only

- For testing of beverages and other liquids

faster and easier

- disposable and sterilized units are a

convenient and easy system that enables
you to optimize the analysis time for routine
testing

-after sample filtration, simply add 2 mL of

liquid culture media and convert the base of
the unit into a Petri dish for incubation.
MicropreSure Online Filtration System

The MicropreSure on-line filtration sampler improves

microbiological testing of purified water at the point-of-use
They let you use the pressure in reservoirs or pressurized lines
to sample and filter through a microporous membrane within an
enclosed chamber. This on-line filtration sampler complies with
USP/EP guidelines.

Closed Design for Accurate Sampling

The filtration chamber remains closed during all stages of
sampling, so operator skill and environmental control are less
critical.

Sampling and Filtration in One Step

The MicropreSure samplers and monitors are sterile and ready
to use on-site. With MicropreSure on-line filtration samplers,
you sample and process in one step.

Tests Larger Volumes or During an Extended

Period of Time
The MicropreSure sampler and monitor allows for processing
liters of water through its enclosed housing.

Convenient In-Process Microbiological Analysis

No assembly
No sterilization
No sample containers to transport
No risk of external contamination
MicropreSure handling

Sampler

Monitor
New EZFit Filtration Unit
Features & Benefits
Efficient laboratory workflow
 Clear funnel material (K resin)
 All-around level indicators
 100mL & 250mL funnels
 Stackable devices for space conservation
Features & Benefits
Efficient laboratory workflow
 Base shape facilitates membrane transfer (1)
 Combined with EZ-Fit manifold
 Unit is positioned easily on manifold
 One handed funnel removal (2)

(1) (2)
Features & Benefits
Reduced risk of contamination
 Protective rim (1)
 Forceps are stopped
 Safe membrane transfer
 Vented lid (2)
 Sample protected during filtration

(1) (2)
Features & Benefits
Designed for optimal microbial recovery
 Flat membrane on agar (1)
 No sample residual liquid (2)
 Funnel shape
 Funnel material

(1) (2)
EZFF Unit for liquid media

Funnel converts to a culture media plate!!!

 No membrane transfer
 Stackable in the incubator
Quality assurance, Certificate Of Quality
Left: Manufacturing Middle: qualification Right: Controls per lot
8 devices available, 48 units/box
 White & black membrane
 100ml & 250ml funnels

 individually bagged & Bulk packed

 Individual bag
 No plug

 Protective plastic bag

 Protective plug
 Lid
Vacuum designed for microbiology

EZ Stream Vacuum Pump

New EZ-Stream Vacuum Filtration Pump

The New pump that streamlines your microbial

testing

… No handling of liquid waste !

 Designed for microbiology filtration

 Optimal noise reduction
 Compact design
 Time saving

72
Liquid transfer technology

Oscillating Diaphragm made of PTFE

• Direct Liquid transfer, no waste containers needed

• Highly durable with broad chemical compatibility
• Maintenance-free (no preventative maintenance)
73
New EZ-Stream Vacuum Filtration Pump

Direct connection simplifies

operation
Filtered liquid transferred
to waste
- Time saving
- Save space on lab bench
- Reduced handling

Noise reduction design

ISO 8199 compliant vacuum

level
Direct connection
Easy decontamination

Maintenance-free

74
Performances

High Flow rate

• Filtration time equivalent to • Can handle a six place manifold !

conventional vacuum/pressure
pumps
• 3, 8 L / min to 4.0 L / min

75
 ReadyPlate™55
The „Small but Mighty“ Ready-to-Use (RtU)
Media for Membrane Filtration

76 Presentation title in footer | 00 Month 0000

The Small but Mighty
ReadyPlate™ 55

ReadyPlate™ 55
Name
ReadyPlate™ 55 KIT

Application Membrane Filtration

Municipal Water
Target Customer Beverage companies
Testing Laboratories

Plate size 55mm

Shelf Life 6-9 months

Storage 15-25°C

AO declaration
Stated in the Certificate of Analysis
availability

Released through an ISO 17025 (all kits) in

Compliance
compliance according to ISO 11133:2014

77 Presentation title in footer | 00 Month 0000

 ReadyPlate™ 55
For standard water application

Ordering
Media Name Format
Information
Coliforms and
1467570020 ReadyPlate™ 55 CCA ISO 9308 55mm plates
E.coli
Coliforms and
1467570200 ReadyPlate™ 55 CCA ISO 9308 55mm plates
E.coli
Kit: 150 plates +
Coliforms and
1467580150 ReadyPlate™ 55 Kit CCA ISO 9308 150 EZPAK + ISO
E.coli 17025
ReadyPlate™ 55 Slanetz and Bartley ISO
1467650020 Enterococcus 55mm plates
7899
Kit: 150 plates +
ReadyPlate™ 55 KIT Slanetz and Bartley Enterococcus
1467660150 150 EZPAK + ISO
ISO 7899 17025
1467670020 ReadyPlate™ 55 CN Agar ISO 16266 Pseudomonas 55mm plates
Kit: 150 plates +
Pseudomonas
1467680150 ReadyPlate™ 55 KIT CN Agar ISO 16266 150 EZPAK + ISO
17025

78 Presentation title in footer | 00 Month 0000

ReadyPlate™55
For Beverage application

Ordering
Media Name Format
Information
1467590020 ReadyPlate™ 55 Lactose TTC Coliform and E.coli 55mm plates
Kit: 150 plates + 150
1467600150 ReadyPlate™ 55 KIT Lactose TTC Coliform and E.coli EZPAK + ISO 17025
Coliform and
1467610020 ReadyPlate™ 55 TGE Agar 55mm plates
E.coli
Kit: 150 plates + 150
1467620150 ReadyPlate™ 55 KIT TGE Agar Total Count EZPAK + ISO 17025
ReadyPlate™ 55 Plate Count
1467630020 Total Count 55mm plates
Agar
ReadyPlate™ 55 KIT Plate Count Kit: 150 plates + 150
1467640150 Total Count
Agar EZPAK + ISO 17025

1467690020 ReadyPlate™ 55 m-Green Agar Yeast and Moulds 55mm plates

ReadyPlate™ 55 KIT m-Green Kit: 150 plates + 150

1467700150 Yeast and Moulds
Agar EZPAK + ISO 17025

79 Presentation title in footer | 00 Month 0000

Summary – EZ Family of membrane filtration products for Bioburden testing
Milliflex ® System
THE BEST SOLUTION
Premium level of Bioburden Testing

• Market: Pharmaceutical & Biotech & Beverage

• Applications: Water & bioburden testings

• Milliflex family:

• Pump

• Filtration units

• Media Cassettes
What is Milliflex?
 Milliflex is a Membrane filtration
system with filtration unit based
on a membrane welded under
sterile funnel
 Funnel breakes-off after the
membrane is adapted to a media
cassette.

 Complies with WW
Pharmacopeial requirements
(EP,JP,USP)
Milliflex positioning statement

The Milliflex System is the first choice for

Pharmaceutical users who want a fast,
convenient and reliable/traceable
filtration method to monitor the microbiological
quality of their liquid samples

No membrane handling !!
 Milliflex: how does it work?

Place a Milliflex unit on the Start the pump to Remove the funnel and
pump and pour the sample perform the membrane filtration place onto an agar cassette

or

Break off the funnel Incubate

MILLIFLEX HARDWARE
Multi Module:
Upgrade from 1, 2 or 3 modules for high throughput
EXPENDABLES
Milliflex Expendables

 Funnels : 100 mL
 MX GS WG 124 (0.22µm, white)
 MX HA WG 124 (0.45µm, white)
 MX HA BG 124 (0.45µm, black)
 MSP 010044 (0.65µm, white) Kit (include LMC)
 MX HV WP 124 (0.45µm, white)
 RM HV MFX 24 (0.45µm, white) MFX Rapid funnels

 Funnels : 250 mL
 MX HA WG 224 (0.45µM white)

 Extension Funnels : 500 mL

 MX EF 50024
 Milliflex Evolution

Milliflex Trays

. Trays under packaging

(Tyvek bags)

. Label directly on trays (with

lot number, part number, data
matrix, expiration date
information)
Milliflex Evolution

Catalogue number & lot number laser marking on each

funnel cover for full traceability
MILLIFLEX MEDIA
Milliflex Expendables

 Empty cassette
 for liquid media:MX LM C0 120
 for solid media: MX SM C0 120

 Prefilled media cassettes

Pre-filled Milliflex Agar Cassettes

Advantages to Customer
 Reduced Labor
 QC Tested and Certified
 pH
 Sterility
 Growth Promotion of ATCC strains per USP/EP/JP
 Shelf Life
 Months vs. weeks of in-house prepared media
 Real-time data collected to establish shelf life

 17 varieties available worldwide

 Attaches directly to base of filter
funnel
 No need to manipulate the
membrane
 Liquid media ampoules

All Ampoule
 Liquid ampoules have a 12 month shelf life and are packed in packs of
Media
50.

 2ml of media per ampoule

 Related to the with MXLMC0120 (empty liquid cassettes)

Why Milliflex choice?
Microbial Testing General Critical Needs

 Reduce time to perform a test

 Rapid results to detect a problem before a complete break in
system
 Time & Labor costs saving (fast reaction)

 Reduce false negative

 Repeatable & sensitive method (detect bugs in big volume)
 Quality control & validation

 Reduce false positive

 Sampling, handling and sanitizations

 Optimize Ease of use

 Why Milliflex choice?

Avoid false positive results

Milliflex Solution
Sterility of the filtration setup
Sterile, disposable Technology
Quality certificate
Automated sanitization

No filter handling
Eliminate Membrane handling and
transfer steps

Easy to use
 Why Milliflex choice?

Avoid false negative results

Milliflex Solution
Sealed membrane
 100 % certified Integrity – impossible by-pass
 Perfect membrane/media contact by convex membrane shape
(no risk of bubbles)
 Filtration area almost doubled:
 reduce contact time
 better colonies distribution & Reduce biological competition
Adapted membrane
 PVDF- Durapore : low adsorption
Controlled vacuum
 Conformity certificates
 Preventive maintenance
Filtration area almost doubled (20 cm2)
 reduces total filtration time
Validation protocol
Millipore Validation Service & Customer benefits
Milliflex PLUS Validation Protocols
 Protocols are dedicated to the Milliflex
consumables

QA-QC services = Millipore

expertise
Rapid Detection Systems
Time to result

2. Filtration 3. Adding of media

1. Sampling

Time

5. Examination
4. Incubation
Milliflex Quantum System
Rapid detection of microorganisms for
Bioburden testing based on membrane
filtration with fluorescence staining
Milliflex Quantum Principle

Microroganism before staining

Membrane is incubated on a pad pre-soaked

with 2ml of fluorescence reagent

• Fluorescence principle
• Marker is non fluorescent when outside cells
• Only viable microorganisms are stained (viability marker)
• Fluorescence accumulates inside cells after cleavage Stained micro-organisms are then exposed to a
by the bacterial metabolism (enzyme cleavage) after 30 min incubation specific wavelength (LEDs exposure) in Milliflex
Quantum reader and emit fluoresence
• Natural amplification of the signal by accumulation of reagent
inside the microorganisms
 MFX Quantum / Standard Protocol

Filtration & Incubation

Membrane transfer on a Illumination by LED
1 (xx h at 2 liquid cassette with a pad 3 and visual count (or
32.5°C±2.5°C) soaked of staining solution CCD camera capture)

Incubation 30 min
at 32.5°C±2.5°C

Re-Incubation is
4 possible

http://www.millipore.com/process_
monitoring/cp1/milliflex_quantum
Time to Results

Escherichia coli ATCC 8739 8 hours

Pseudomonas aeruginosa ATCC 9027 TSA 12 hours
Bacillus subtilis ATCC 6633 32.5 ± 2.5°C 8 hours
Staphylococcus aureus ATCC 6538 12 hours

Candida albicans ATCC 10231

SDA
24 hours

Aspergillus brasiliensis ATCC 16404 22.5 ± 2.5°C 30 hours

Caulobacter sp. 30 hours

R2A
Micrococcus sp. Environmental strains 18 hours
32.5 ± 2.5°C
Ralstonia sp. 24 hours
Detection & Enumeration of Microorganisms
 Use simple protocol and workflow (3 steps)
 Get rapid results with full ID option (1/4, 1/3 TTR
reduction)
 Reduce your implementation Risk (Growth based)

Application
 Filterable samples (water and beverages)
 Total count
 Non destructive (compatible with ID)
Growth Based Method, Facilitated Validation

Traditional MFX Quantum

0 Filtration Filtration

Incubation

Incubation Staining & Counting

Re-incubation possible

Counting Counting
5/7 DAYS
Milliflex Quantum Camera Assistance
The camera reading assistance and its associated PC software are
developed to facilitate the spot counting in fluorescence.
Re-incubation for Characterization
Milliflex Quantum benefits

• Non-destructive

• Full Identification capabilities

• Simplicity of the rapid method

• Result in 24 to 48H

• Easy data comparability with traditional method

(method close to compendial)

• Reduce implementation Risk

• Closeness to compendial testing
• Low operator training => fast implementation for routine use

• Affordable prices for a Rapid methodology

EZ-Fluo™ Rapid Detection System
A Fast, non-destructive, fluorescent staining based
system for microbial detection
EZFluo Principle

Microroganism before staining

Membrane is incubated on a pad pre-soaked

with 2ml of fluorescence reagent

• Fluorescence principle
• Marker is non fluorescent when outside cells
• Only viable microorganisms are stained (viability marker)
• Fluorescence accumulates inside cells after cleavage Stained micro-organisms are then exposed to a
by the bacterial metabolism (enzyme cleavage) after 30 min incubation specific wavelength (LEDs exposure) in Milliflex
Quantum reader and emit fluoresence
• Natural amplification of the signal by accumulation of reagent
inside the microorganisms
EZ-Fluo System – membrane filtration and Petri plates
incubation
Protocol Overview
Membrane transfer into Membrane transfer into a
a Petri dish containing Petri-Pad Petri dish
solid medium containing staining solution

Filtration Petri plate Incubation Staining

for 30 minutes at 32.5 ±
2.5°C
Staining solution diffusion
Substrate cleaved by
microorganisms

Counting
Re-incubation of fluorescent micro-colonies with
into a Petri dish containing the reader and camera reading
medium for identification assistance to facilitate counting
(optional)
117
Re-incubation for characterization
Non-destructive method, compatible with ID technique

Result after staining Same membrane after

re-incubation

View of membrane in the reader CFUs are visible to the eye

Staphylococcus aureus
16 hrs 5 days
Re-incubation for characterization
Non-destructive method, compatible with ID technique

Result after staining Same membrane after

re-incubation

View of membrane in the reader CFUs are visible to the eye

Candida albicans
30 hrs 7 days
Faster Results
Get your results in 1/3 of the time required using your
current traditional technique !
EZ-Fluo™
System

Examples of Results
EZ-Fluo System - Microbial Detection

E. Coli Burkholderia cepacia Candida albicans

9 hours 18 hours 28 hours
TSA/30-35°C TSA/30-35°C SDA+CAL/20-25°C
White membrane White membrane Black membrane
EZ-Fluo System - Microbial Detection
Mixed contamination:
Bacillus subtilis ATCC 6633
Pseudomonas aeruginosa ATCC 9027
Staphylococcus aureus ATCC 6538
20hrs on TSA @ 32.5°C
0.45µ white membrane

Mixed contamination:
Candida albicans ATCC 10231
Aspergillus Brasiliensis ATCC 16404
30hrs on TSA @ 32.5°C
0.45µ white membrane

123
EZ-Fluo System - Microbial Detection
Mixed contamination:
Bacillus subtilis ATCC 6633
Pseudomonas aeruginosa ATCC 9027
Staphylococcus aureus ATCC 6538
Candida albicans ATCC 10231
Aspergillus Brasiliensis ATCC 16404
30hrs on TSA @ 32.5°C
0.45µ white membrane

Mixed contamination:
Candida albicans ATCC 10231
Aspergillus Brasiliensis ATCC 16404
40hrs on SDA with Chloramphenicol
@ 32.5°C
0.45µ black membrane

124
Applications

EZ-Fluo can detect and quantify microbial contamination in

any filterable samples. Potential applications are numerous
and include testing of the following sample types:

• Raw materials
• In-process samples
• Final product
• Environmental samples
Easy to integrate in any laboratory

• Simple hardware, simplified validation

• Limited space required in the laboratory for
implementation
• Method using regular filtration and standard
media
• No particular skill required
• Limited system set up & clean up
• Limited maintenance on hardware
Limited hardware investment, cost effective
solution

• Only the EZ-Fluo Reader is necessary

• Optional EZ-Fluo camera
• Limited maintenance on hardware
• Affordable price for rapid microbiology
equipment and Rapid reagent
EZFluo Video
Services & Support

• EZ-Fluo Validation Protocol (IQ/OQ/PQ)

• EZ-Fluo Validation Summary (EU and US
Pharmacopeia)
Milliflex Rapid System
Rapid detection and enumeration of
microorganisms
Milliflex Rapid Technology

MilliFlex Rapid is based on ATP technology

ATP+ D-luciferin + Oxygen (O2)

Luciferase
Magnesium

AMP + PPi + Oxyluciferin + CO2

LIGHT Number of photons proportional to

quantity of ATP in the microorganisms
Detection with Milliflex Rapid
Based on ATP Technology

Milliflex RMHV membranes

Durapore (RMHVMFX24)

AutoSpraystation
(MXRPSPRKT)

Reagents
(MXRPBLRST)
 Milliflex Rapid features

Application of Reagents - Autospray station

Design and efficient dispensing system:
 Fully automatic: Good uniformity of reagents
 One Spraystation for both reagents (ATP releasing reagent /
Enzyme)

“User friendly” interface

Milliflex Rapid features

21 CFR 11
Compatible
Windows
platform
Separate
Administrator
Module to
control users &
access
Backup &
Export
Functions
Milliflex Rapid features

Milliflex Rapid replaces the visual readout of MF results by a highly

sensitive detection method while maintaining all other features of the
compendial MF method

Results are enumerated in around 1/4 of the time of traditional

methods, with equivalent results (CFU)

Limit of detection: 1 CFU / volume filtered

The validated detection limit of the system is 1 CFU

Ease of use and throughput by using the Milliflex sample preparation
system.
MFX Quantum vs MFX Rapid

MFX Rapid when…

• User wants to use Milliflex HV (Durapore, PVDF)

General approach is to sell MFX Quantum
membranes
in aallresult
• User looks for situations except…< 24H
on Y&M detection
• User looks for a result in less than 24/30 hours
• KF Strep/ Mac Conkey/ M-endo agar media are used
• User looks for an automated counting
• User looks for a high laboratory throughput
Environmental Monitoring
Microbiology of Air Sampling
Human source

• There are more microorganisms in and on our body than human cells

Skinhead
Skin (front) Nose-throat (secret)
1.5 x 106/cm2
0.2 x 106/cm2 106 - 107/ml

Mouth (Saliva)
106 - 107/ml Centers of sweet (armpit)
2.4 x 106/cm2
Skin (back)
5 x 102 - 103/cm2 Hand
Stomach 103 - 6 x 103 /cm2
103 - 105/g
Genitals (Urin)
Rektal 0 - < 103/ml
ca. 1011/g
(10-20% of feces) Feet
102 - 103/cm2
 Definitions / Recommendations

Source Personnel
People are the largest contributor of airborne particles
in a Clean room.
- 1 Person shed 1 billion skin flakes per day.
How many 0.3 micron particles does a person shed in 1 Minute?
Activity Particles per Minute
Person sitting or standing 100,000
Moving arm 500,000
Walking slowly 5,000,000
Walking at an average pace 7,500,000
Walking quickly 10,000,000
Microbiological Air Monitoring

Which airborne bacteria are isolated

usually ?
Airborne bacteria Possible source
Staphyloccoci from skin

Micrococci from skin

Pseudomonas from water

Bacilli from soil, environment

Coryneform bacteria from skin

Sources of Microbial Contaminants

 Components
 e.g. water, raw materials, packaging
 Process
 compounding, mixing, filtration,
storage, filling, sampling
 Instruments
 cleaning, decontamination,
sterilisation, drying
 Personal
 training, behaviour
 Environment
 air conditioning, surfaces
Regulations applied to Clean Rooms

 ISO 14644-1/2 (Classification of

Cleanrooms and Controlled Areas)
 ISO 14698 (Biocontamination Control)
 Pharmacopeia (EP, USP, JP)
 <1116> Microbiological Evaluation of
Clean Rooms and Other Controlled
Environments
 <797> Pharmaceutical Compounding-
Sterile Preparations
 FDA Guidance on Aseptic Production
(cGMP)
 EU cGMP Annex 1 on Sterile Medical
Products
 Other international and local
regulations
 Requirements of the EP/ USP

"Microbial Monitoring must ensure that the

controlled environment is consistently and
consequently maintained"

In accordance with the given

clean room class it is required
to monitor
 Surrounding Air
 Compressed Gas
 Surfaces
 Personnel
 Equipment
 Air monitoring in a
nutshell…..

ISO 14 698-2 Evaluation and Interpretation of Bio-contamination Data

Two main methods active and passive Air sampling

Microbial Air / Typical Areas to monitor include: In the critical zones e.g. ISO 5 - near
product exposure areas

In the support / non critical areas e.g.

ISO 7 based on the area usage
Air monitoring Monitoring

ISO 14698-1

Detection and monitoring of

microbial Contamination of the air
in a risk zone is carried out by
collecting viable particles with
appropriate sampling devices
according to a sampling plan,
when the risk zone is in the as-
built and at-rest states, as
appropriate, and routinely under
normal operation
in the risk zone.
Air Monitoring

 Two main methods:

Passive sampling devices,
such as settle plates
(not generally recommended
in USA)

Active sampling devices, such

as Air samplers.
MM active air monitoring technologies

 Merck Millipore air sampling technologies are based on the Impaction

Principle – sampling of microbes directly on solid agar media
 Different system strategies can be offered to our customers
 Sampler using agar plates: MAS
 Sampler using agar cassettes: M Air T
 Samplers using agar strips: RCS

System Technology Strategy Agar

MAS Anderson Open Agar plates, any source
Principle (We recommend Heipha
plates!)
M Air T Anderson Closed Complete system with M
Obsoleted by Principle Air T Cassettes from MM
end of 2014

RCS Centrifugal Closed Complete system with

Sampler Hycon agar strips from MM
 Air Monitoring
Merck-Millipore product portfolio to support your needs

All MAS family has been validated to ISO 14698 in combination with
heipha ICR plates
MAS-100 family
The MAS-100 NT is a
Air inlet
high-performance instrument
that is based on the principle
of the Andersen air sampler
Petri dish
which aspirates air through
a perforated lid.
Anemometer

Impaction principle
Blower • most used and documented method
• open for standard Petri dishes
• best known system on the market
Air outlet • preferred method of FDA
MAS-100 NT

General Description
 The MAS-100 NT is designed for microbiological air monitoring. The sampling is
based on the well known and as standard method established impaction
principle.
 It works with 90 mm standard Petri dishes
 Fully automated calibration process in combination with DA-100 NT
 Validated according to ISO 14698 Standard
 Dedicated for Pharma Industry
MAS-100 NT®
Features:
• Graphic illuminated display
• Easy navigation (hotkeys and up and down keys
• Electronic communication RS232 and USB
• Audible alarm
• up to 100 samples are logged
• up to 100 alarms are logged
• Lithium-Ion batteries with intelligent charging
• Fully automated calibration
• Validated according to ISO-14698
Technical Specification:
Flow rate: 100 Liters / Min100 liters / min. ± 2.5%,
Standard sampling volumes: 50, 100, 250, 500, 1000, volumes, individually
selectable between 0 and 2000 liters.
Battery pack: (Li-Ion)
Charging time: Full recharge time approx. 3.5 hours
Running time: Total running time approx. 7 hours
Total aspiration volume: Total aspiration approx 42000 liters
MAS-100 NT

MAS-100 NT - Carrying case

• Sampling head 300x0.6

• Dust cover
• Main charger
• CD-ROM (Manual and software)
• Allen key
• Calibration certificate (300x0.6 and 400x0.7)
• Feller table (400 x 07. and 300 x 0.6 mm)
• USB connection cable
MAS-100 VF®
Key features‘:
• ISO 14698 evaluated in combination with heipha ICR plates
• New slide controls
• Very easy to use controls
• 5 preset volumes
• New on board software platform, No PC software needed
• Instrument is a Server similar to an internet node
• PDF of Calibration certificate stored in the instrument
MAS-100 Iso NT®
•The MAS-100 Iso NT is based on the same impaction principle as
the MAS 100 NT ®
• Active sampling flow control
• Active disinfection flow control
• Protected mode / user protection
• Alarm log file / Sample log file
•Designed for Clean Rooms and Isolators
• In-/outlet same side
• Easy installation - 360° movable display
• Easy software navigation
• Double valve security system

• Petri dish 85-90mm

• Petri dish 85-90mm • Not adjustable
• adjustable • Only horizontal use possible
• Horizontal and vertical use • Autoclave-able and easy
• Autoclave- able cleaning, no dead spaces
MAS-100 Iso MH®
•The MAS-100 Iso NT is based on the same impaction
principle as the MAS 100 NT ®
• Up to 4 sample heads from one control point and pump
• Same collection efficiency as all other MAS 100 instruments
• H2O2 Decontamination cycle according to
tubing length
Up to 1 meter
• Up to one meter of tube from Control unit
to manifold
• Up to 9 meters of tube from valve
mounted on manifold to sample head
• Ethernet or ProfiBus Interface / potential
free in/out contacts
• Each head can be fitted with a filter
(opticap 4 inch)

Up to 9 meter
 MAS-100 CG Ex
 Common Compressed Gases used:  Why Test Compressed
 Air
Gases?
 Nitrogen
 Carbon Dioxide • Gas that contact product can
 Argon effect product quality.
 Oxygen

• These gases should be

qualified and monitored with
appropriate actions taken if
limits are exceeded.
Applications in Pharma/Biotech

•Blanketing
To maintain constantly a protective layer of gas
on top of a substance
In process (tank)
Final packaging
• Purging
To fill and rinse a reactor
• Sparging
To remove oxygen when injected into liquids in
the form of tiny bubbles
• Stirring
To mix and store liquids
MAS-100 CG Ex – Principle of measurement
 Physical characteristics comparable to
MAS-100 NT
 Use of standard 90 mm petri dishes
 Samples are taken under pressure
 Completely compatible with all
common compressed gases, i.e.
Compressed air, nitrogen, carbon
dioxide, argon, oxygen  preset
programs per gas type open sampling head
 Up to 10 gas types are programmable with Petri dish
Technical specification

 Dimensions (with sampling head): 49 x 32 x 11cm; 12,5 kg

 Flow rate: 50 L or 100 L/min (auto-regulated) Instrument precision + 10%
 Impaction speed: 11 or 20 m/s (flow rate dependent)
 Pressure: 1,5 to 10 bar
 Sensors: Flow rate sensor, pressure sensor
 Battery capacity: 3,5 hours / Recharging time: approx. 2 hours
 Programs: Pre-set programs per gas type
 Material Housing: coated aluminum, Sieve: anodized aluminum, autoclave able
MAS-100 CG Ex - Sampling head
• Properties identical to the one of MAS-100 - but adapted to the high pressure
conditions
• Unique features:
• Tested and certified for up to 20 bar
• Autoclave able for 20 min at 121°C.
• This head is classed as a pressure vessel and has its own test certificate

•The MAS-100 CG Ex is
based on the principle of the
Andersen air sampler.
•Use of standard 90mm Petri
dishes
•Samples are taken under
pressure
MAS-100 CG Ex (Merck Cat.-No. 1.09327.0001)

Sampling head: MAS-100 EC Ex

The sampling head is identical to the one of the MAS-100 but adapted to the
high pressure conditions.
It is tested up to 20 bar and certified.
Sterilize for 20 min at 121°C.
This head is classed as a pressure vessel and has its own test certificate
No other manufacture has this.
Air Monitoring
Common Compressed Gases used:
Air
Nitrogen
Carbon Dioxide MAS-100 CG Ex
Argon (Merck Cat.-No. 1.09327.0001)

Oxygen
MAS-100 family:

All MAS family has been validated to ISO 14698 in combination with
heipha ICR plates

Physical efficiency:
Is the ability of the microbial air sampler to collect various sizes of particles, and is the
same regardless of particle composition (i.e. whether the particle is a microorganism,
carries a microorganism, or just a particle
Biological efficiency:
Is the efficiency of the air sampler in collecting microbe-carrying still viable particles.
MAS-100 Eco

Microbial Air Sampler

for Food and Beverage Industry
Microbiological Air Monitoring

MAS-100 Eco®

• designed for microbiological air monitoring

• wellknown MAS-100 impaction principle
• smart design
• volumes between 1 and 1000 liters
• for standard Petri dishes
HYCON® RCS Instruments
Microbial Air Sampling Portfolio
 RCS® High Flow Touch
100 L/ min; renowned RCS High Flow Technology
+ RCS® CG Adapter
for monitoring compressed gas lines
 RCS® Isolator
HF Technology, Separation of sampling &
operation unit for restricted access (barrier) areas RCS Plus Ex
 RCS® Plus Ex
50 L/min, Explosion-proof RCS Plus Technology
 (RCS ® Standard – 1st model 1979)
non-regulated areas!!! RCS High Flow Touch
40 L/min (320 in total)

 Hycon® agar strips

 Hycon® anemometer & CalibSo Software
 Hycon® services RCS High Flow Touch +
RCS Isolator
CG Adapter
RCS High Flow Touch -
Microbial safety at your fingertips

1. RCS High Flow Touch

overview
 Proven RCS Technology
 RCS High Flow Touch: Key
features and benefits
 RCS® systems for microbial air sampling
 Air sampler, agar media, accessories
 Production and service according to high
quality standards
 Closed system, completely evaluated
according to ISO 14698
 Gentle Sampling (Centrifugal Sampling)

 Airborne Microorganisms
contained in the air are
accelerated through centrifugal
forces and are equally
distributed onto an agar strip
contained in the rotor
 Air outlet is directed in parallel
to the device
RCS High Flow Touch – Technical Specifications
 Principle: Centrifugal Sampler
 Dimensions: 30x 13x11 cm; 1.5 kg
 Flow rate: 100 L/min; 10 minutes per 1m3
 Impaction speed 0.07-7 m/s
 D50: 1.2 µm
 Battery capacity: 5-6 hours
 Recharging time: approx. 2-3 hours
 Volumes: 7 preset, 3 individual (1-2000 Liters)
 Timer: 1 to 120 minutes
 Interval Sampling: up to 4 hours (agar validation!)
 Housing material: polycarbonate (Lexan)
Autoclaveable sampling head (Air direction ring:
APEC, Rotor: aniodied aluminium, Prot. Cap stainless
steel)
 Operation: Touch screen
 Instrument precision +/- 5%
 Calibration: Hycon Anemometer and CalibSo
Software
Hardware Cleaning & disinfection

 Housing
 70% ethanol or isopropanol (p.A. – no technical)
 Any disinfectant compatible with polycarbonates

 Sampling head
 70% ethanol or isopropanol (p.A. – no technical)
 autoclaving
 UV bath

 Sterilization: Instrument can be subjected to VHP gassing (H202 ,only, peracetic

acid not recommended)
Validation & Qualification Handbook

 Experimental Determination of the Collection Efficiency of RCS High Flow F, RCS

Plus, RCS Isolator
 Basics of aerobiology
 Introduction
"Complete system fully
 Physical collection efficiency
 Biological collection efficiency
validated to ISO 14698
 Validation of agar strips
(v. Jan 2007)"
 Operative efficiency and performance qualification
 Qualification Plan for RCS HF (guideline)
 Installation Qualification
 Operation Qualification
 Performance Qualification (recommendations)
 Qualification Report
Monitoring of Compressed Gas
 RCS High Flow Touch is used for monitoring of ambient air and
compressed gas
 For monitoring of compressed gas the compressed gas adapter is used
 Recommended sample volume: 1m3
 6 nozzles to cover a pressure range of 0.1 to 7.0 bar
 no external pressure reducer required (potential source of contamination)
 Adapter is autoclave-able, can be disinfected and sterilized with H2O2
 To be applied for all non-flammable and non-toxic gases.
RCS Isolator
 Same air stream characteristics as RCS High Flow
(Touch)
 Instrument separated in 2 parts
 Sample unit (inside the isolator, RABS or sensitive
production area)
 Control unit (outside the sensitive area)
 Air flow directed through the instrument, exhaust can
be “vented” away
 Gas-sterilized after production
Other Accessories

940 376 Docking Station

940 335 Table Top Tripod
940 330 Tripod
940 215 Sterile Sleeves

940 244 Air Direction Ring

940 230 Rotor
940 415 Protection Cap
Validated, Standardized, Ready-to-use

Total count
TC Total count (TC) Tryptic Soy Agar
TSM Total count TC Agar modified for increased sensitivity
TC- Gamma-irradiated TC Agar for sterile environments
TCI- Gamma-irradiated TC Agar with H2O2 neutralizers
(antibiotics containing air)
PEN-  Gamma-irradiated TC Agar with Penase
LAC-  Gamma-irradiated TC Agar with Lactamase (Cephase) and Penase

Specific
YM Rose Bengal Agar for yeasts and molds - restricts (not inhibits) spreading
SDX Sabouraud Dextrose Agar (for yeasts and molds) - spreading not restricted
SDX- Gamma-irradiated modified SDX
DG18 Dichloran Glycerol-18 Agar (for xerophile yeasts and molds) - spreading restricted
C MacConkey Agar coliform bacteria (gram negative)
S Manitol Salt Agar for Staphylococci (gram positive)
Extensive
Quality Control
Certificate of Analysis
 Batch Control
 Shelf Life Information
 Storage Conditions
 Regulations
 Performance
 Sterility Test
 pH

Production
 100% optical control
 Gamma-irradiation
 Performance testing at different points
 In process control
 Environmental Monitoring Culture Media

The perfect solution for

Air monitoring MAS-100®

Envirocheck Settle Plates Envirocheck Contact Plates

1.18410.0020 TSA w. neutr. 1.18408.0020 TSA w. neutr.

1.18411.0020 SDA w. neutr. 1.18409.0020 SDA w. neutr.
Ready-to-Use Culture Media Envirocheck® Settle plates

Envirocheck® Settle plates for Isolators and Clean Rooms

For Active and Passive Air Sampling

There is increasing need for effective air monitoring in all areas where
airborne micro organisms may contaminate or affect industrial products and
processes. Air monitoring is therefore highly important in all industries, which
do have clean room areas with filtered air.

The Envirocheck® Settle plates, 90 mm diameter plates, can easily be

used for Active Air Sampling in combination with the different MAS – 100®
Microbial Air Monitoring Systems (for further information concerning our
Air Monitoring program, please refer to our brochure “MAS-100® Microbial
Air Monitoring Systems, W286138), Of course, the settle plates can also be
used for Passive Air Sampling
Ready-to-Use Culture Media Envirocheck® Settle plates

Envirocheck® Settle plates for Isolators and Clean Rooms

For Active and Passive Air Sampling

To fulfil the requirements for protected areas the Envirocheck® Settle plates
in there finale packaging are, triple wrapped and gamma-irradiated,
The 90 mm Settle plates are filled with 25 ml of media to avoid desiccation
after active air sampling of 1000 litres and long incubation of up to 7 days
for the detection of e.g. Yeast and Moulds.
The Settle plates are packed in boxes of 2x 10 plates, the inner bag is
impermeable to H2O2 and includes a pouch containing a drying agent to
minimize the formation of condensation.
Ready-to-Use Culture Media Envirocheck® Settle plates

Envirocheck® Settle plates for Isolators and Clean Rooms

For Active and Passive Air Sampling

All plates are stored at room temperature (+15°C to +25°C), hence

cooling capacities won’t be blocked. Storage at site of application
is possible.

Product name, expiry date, lot number and serial production

number are imprinted on each Envirocheck® Settle plate for safe
and easy product identification and shelf life monitoring.
Ready-to-Use Culture Media

Envirocheck® Settle plates

Envirocheck® Settle plates for Isolators and Clean Rooms

For Active and Passive Air Sampling
Ready-to-Use Culture Media

Envirocheck® Settle plates

Envirocheck® Settle plates for Isolators and Clean Rooms

 Storage at +15°C to +25°C

 90 mm diameter plates
 Gamma irradiated
 Triple wrapped – (2 sets of 10 plates each)
 H2O2 –impermeable inner bag
 4 neutralizers
 Reduced formation of condensation water
 Long incubation time possible
 Filling Volume 25 ml
 Long shelf life - up to 9 month
Ready-to-Use Culture Media

Envirocheck® Contact plates

Envirocheck® Contact plates

for Isolators and Clean Rooms

As a simple and efficient way to detect microbiological contaminations,

Envirocheck® Contact plates are used to screen for bacteria, yeasts and
moulds related to contaminations in the production process. The contact
plates serve to examine surfaces in Isolators and / or Clean Rooms
In order to fulfil the requirements for protected areas, all Envirocheck®
Contact plates for Isolators and Clean Rooms are, triple wrapped and
gamma-irradiated, when looking at the final packaging - The Contact plates
are packed in boxes of 2x 10 plates, the inner bag is impermeable to
H2O2 and includes a pouch containing a drying agent to minimize the
formation of condensation water.
Ready-to-Use Culture Media

Envirocheck® Contact plates

Envirocheck® Contact plates

for Isolators and Clean Rooms

To inactivate any residual disinfectants on the surface to be tested, the

Envirocheck® Contact plates for Isolators and Clean Rooms additionally
contain 4 neutralizing agents.
All plates are stored at room temperature (+15°C to +25°C), hence cooling
capacities won’t be blocked. Storage at site of application is possible.
Product name, expiry date, lot number and serial production number are
imprinted on each Envirocheck® Contact plate for safe and easy product
identification and shelf life monitoring
Ready-to-Use Culture Media

Envirocheck® Contact plates

Envirocheck® Contact plates

for Isolators and Clean Rooms
Ready-to-Use Culture Media

Envirocheck® Contact plates

Envirocheck® Contact plates for Isolators and Clean Rooms

 Storage at +15°C to +25°C

 Gamma irradiated
 Triple wrapped – (2 sets of 10 plates each)
 H2O2 –impermeable inner bag
 4 neutralizers
 Reduced formation of condensation water
 Long incubation time possible
 Long shelf life - up to 9 month
Ready-to-Use Culture Media

Envirocheck® Contact plates

Envirocheck® Contact plates for Isolators and Clean Rooms

 Storage at +15°C to +25°C - no cooling capacity blocked

- store at site of application
 Triple wrapped - easy to open
- maximum protection
 4 neutralizers - inactivation of additional
disinfectants
 Long shelf life - less documentation
- less quality control
Surface Monitoring
4
Surface Testing with RtU agar media

Heipha Contact Plates Hycon Contact Slides (OKI)

Merck Envirocheck Contact Plates
Requirements for contact plates and contact slides

EG-GMP-Guide: 55 mm surface area

USP 24 to 30 cm2 surface area

 contact plates used for flat surfaces

 contact slides are used for flat, uneven or curved surfaces
 used for contact areas or adjacent to contact areas (of product)
Surface Testing – Contact Slides
HYCON® Contact Slides: the flexible way of monitoring surfaces
 Designed specifically for use in aseptic cleanrooms and isolation areas.
 Standardized contact area of 25 cm2 meets international guidelines
Individually wrapped
 For prevention of secondary contamination. Resealing with included cover slides for safe
transport after use
Flexible media carrier
 Easy and flexible way to determine bacteria, yeasts and molds on curved surfaces.
100% visible quality control
 For highest level of safety

Incubation Rack
for Contact Slides with time incubator
Handling of agar media for surface
monitoring
 Contact Plates and Contact Slides: Direct contact of a
defined surface
 Incubation of the agar media
Determination of Days Temperature

total count 1-5

30-35°C
yeast and 3-7
20-25°C
molds
 Counting of the colonies
 Result calculation per surface unit
Comparison Contact Slide vs. RODAC Plate

HYCON Contact Slide Contact Plate

• is flexible and • is easy to use by simply opening
thus applicable on curved or and closing the lid
irregular surfaces
• is quite cheap by low cost of the
• Individual packaging material
- reduces drying process
- allows safe transporation • common used - 80% of surface
- provides sterile incubation monitoring is done by RODAC

• recommended for • recommended for

 on irregular surfaces  on even surfaces
 for areas with frequent sterilization  surface and personnel monitoring
processes
Basic Product Info heipha
Environmental Monitoring Media
Environmental Monitoring
Application – Product Selection

surface contact plates,

monitoring swabs

active air monit. settle > contact >

Hycon strips
air monitoring
passive air monit. settle

contact > settle >

personnel
140/150 mm >
monitoring
specialities
Product Technology & Features – Media packaging
Product Technology & Features
The ICRplus Contact Plate

Plus
Two positions of the lid
„Closed“
„Vent“
→ Aerobic/Anaerobic
Incubation
→ Safe Transport
IsoBagTM

Product Objective and Scope

Product Objective
 Ready-to-use solution for a rapid introduction of culture
media into isolators by combining existing Getinge La
Calhène technology (bidirectional rapid transfer system;
190 mm alpha port) with our ICR products

Product Scope
 TSA + LTHTh ICR/ICR+ in contact and settle plates
(articles 146527, 146231, 146683 and 146069)
 These products will be offered in isolator transfer bags
from Getinge La Calhène with 190 mm DPTE® beta
port

IsoBags™ help to prolong production times by

reducing numbers of decontamination cycles
and saving storage place in production
isolators

199 New IsoBag - A.-G. Klees January 2016

IsoBagTM
Features of new IsoBagTM

• Ready to use single bagged ICR plates for Environmental

Monitoring in in Getinge La Calhène DPTE Beta Bag® compatible
with 190 mm DPTE® Alpha Port
• No need for decontamination before use in production isolators 
provides probability for increased production time
• Multiple connections possible  introduction of minimum required
amount of plates possible.
• Single bagged ICR plates inside  reduced workload and waste
compared to removal of triple bags
• Sterilized zip lock transport bags inside  prevention of handling
contamination outside of the isolator at incubation/ examination
• IsoBag™ itself is double bagged and finally irradiated at 10-20
kGy  provides safe passaging of material locks at minimized
contamination risk
• Use of Standard ICR plates  features of validated culture media
do not change (e.g. growth promotion, shelf life, inner packaging,
sterility)

200
Use of IsoBag™

Outside the isolator before connection Inside the isolator after connection:
Closed Alpha Port Open Alpha Port
Closed IsoBag™ Open IsoBag™

Environmental Monitoring in Isolators and RABS - September 2016

IsoBagTM

Ordering Information

Order Number Product Description Long product Description

1467530100 IsoBag TSA+LTHTh Contact+ IsoBag™ TSA+LTHTh Contact+ for 190 mm

alpha-port lockable 55 mm Contact Plates
irradiated

1467540100 IsoBag TSA+LTHTh Contact IsoBag™ TSA+LTHTh Contact for 190 mm alpha-
port 55 mm Contact Plates irradiated

1467550080 IsoBag TSA+LTHTh Settle+ IsoBag™ TSA+LTHTh Settle+ for 190 mm alpha-
port 90 mm lockable Settle Plates irradiated

1467560080 IsoBag TSA+LTHTh Settle IsoBag™ TSA+LTHTh Settle for 190 mm alpha-
port 90 mm Settle Plates irradiated

(lead time approx. 8 weeks, if no stock available)

202 New IsoBag - A.-G. Klees January 2016

 The ICR Swab

Polyester swab moisturized with NaCl

Reservoir: TSB + LT
Triple bagged, γ-sterilized at > 25 kGy
Presence/Absence Test
Surface and Liquid Monitoring
Product Description

• Swab
 Swab fixed on an ergonomic cap + transparent chamber filled with sterile
buffer for surface sampling
 Used with standard method (filtration) or in combination with a dip tester
• Dip testers
 Paddle with media impregnated pad and gridded membrane +
transparent chamber for the enumeration of
Total Coliforms (Blue)
Heterotrophic Count (Red)
Yeast and Mold (Yellow)
Total Count (White)
Merck Millipore Particle counter’s

Coming soon

APC SmartTouch APC ErgoTouch Pro 2

206
Particle Counting

207
Standards for particle counting

ISO 14644 General standards for all clean rooms

 Particle limits for sizes/classes
 EU-GMP
 C-GMP
 Industry specific (pharmaceutical)
 More stringent levels
 At rest v. operational
 ISO 21501

describes a calibration and verification method for a light scattering airborne particle counter (LSAPC),
which is used to measure the size and particle number concentration of particles suspended in air. The
typical size range of particles measured by this method is between 0,1 µm and 10 µm in particle size.
Instruments that conform to ISO 21501-4:2007 are used for the classification of air cleanliness in
clean-rooms and associated controlled environments in accordance with ISO 14644-1, as well as the
measurement of number and size distribution of particles in various environments.

20
8
Main Features
Coming soon
Flow rates
•New idea coming from the market: combination 100 LPM & 1 CFM
•Different models based on flow rate:
-1 CFM, 50 LPM, 100 LPM, 100 LPM & 1 CFM

Reports
• Raw data
• ISO 14644, FS-209E & GMP reports
• Minimum, maximum and average report
• Trending of measurements through the download software

A report will be generated for

the selected particle counting
regulation like the ISO 14644
Features
• Visual presentation of the sample area on the screen
• Filtered Exhaust outlet for sampled air
• Active flow control
209
ErgoTouch Pro 2 Specifications
• 3.7 inch color touch screen, icon driven menus, easy to navigate
• Size channels can be configured by user
• User password
• Built-in flow meter
• Ergonomic design
• Memory for 10.000 sample records
• Reports: ISO 14644 & EU GMP
• Integrated alarms
• Offering features of a portable counter

 Size Range: 0.3 - 10 m

 Channel Sizes: Standard: 0.3, 0.5, 1.0, 3, 5, 10 m
 Counting Efficiency: 50% @ 0.3μm; 100% for particles > 0.45μm (per JIS)
 Concentration Limits: 2,000,000 particles/ft3 @ <5% coincidence loss
 Light Source: Laser diode
 Zero Count Level: <1 count/5 minutes (<2 particles/ft3) Meets JIS B9921
 Flow Rate: 0.1 CFM (2.83 LPM) with 5% accuracy
 Flow Control: Automatic
 Calibration: NIST traceable National Institute of Standards and
echnology
210
Hygiene Monitoring in Food and
Beverage Industry
Food Safety

Food safety is a scientific discipline describing

handling, preparation, and storage of food in ways
that prevent food borne illness.

Food borne illness (also food borne disease) is any

illness resulting from the consumption of
contaminated food.
Reasons for increasing problems with foodborne
diseases

Population growth
Urbanization
Increase in population at risk
Increase in consumption of animal
products
Increase in international trade in food &
feed
Increase in international tourism
Food safety

Tip of the iceberg – only

a small number of cases
of foodborne illness are
reported
An even smaller number
are laboratory
investigated
HACCP

Hazard
Analysis (of)
Critical
Control
Points
HACCP

History:

HACCP was 1st developed to ensure a

safe food supply to NASA astronauts
during space flights.
It is still used by NASA until today.

1958 Concept discussions at NASA

1959 Development of the HACCP concept to assure
100% Food safety
History:
HACCP
1971 The HACCP system was published and documented in the USA
1985 NAS (National Academy of Science) recommend the use of HACCP

Nowadays HACCP expanded in all areas of the food industry, going

into meat, poultry, seafood, dairy, and has spread now from the farm
to the fork.

The HACCP concept is also part of the WHO (World

Health Organization) Codex Alimentarius (Latin for "food
code" or "food book") is a collection of
internationally recognized standards, codes of
practice, guidelines and other recommendations
relating to foods, food production and
food safety.
HACCP
HACCP is a safety system
Preventive, not reactive
A management tool used to protect the food supply against biological, chemical
and physical hazards
Recognized internationally as an effective system of food production controls.

But HACCP is not a Stand-Alone-Program and prior to its full

implementation, the necessary prerequisite programs must be
in place!!!
Typical Prerequisite programs:
GMP = Good Manufacturing Practice
GHP = Good Hygiene Practice
SSOP = Sanitary Standard Operating Procedures
Vendor certification
Recall and trace back procedures
HACCP

How does HACCP work?

HACCP is based on 7 principles!!

1. Assess the hazards

2. Identify critical control points
3. Establish critical limits for each CCP
4. Establish Monitoring procedures
for each critical control point
5. Establish corrective action
6. Verify the system is working
7. Develop Effective Record Keeping
HACCP

Principle 1:
Perform a hazards analysis
- Assemble HACCP team (multidisciplinary team)
- Describe your product (pH, Composition,
storage,…)
- Construct a flow of all steps
- List all potential hazards for each step

Hazards can be:

- Biological
- Chemical
- Physical
Hazards
HACCP
can be:

- Biological
Biological hazards are yeast, mold, bacteria, viruses, protozoa and parasitic worms
Bacteria: causes most cases of food borne illness
Viruses: focus on personal hygiene
Parasites: need a host to survive
Viruses 6%
Fungi: spoilage microorganism Chemical 2%
Parasites 1%

Bacteria
90%
HACCP

Hazards can be:

Chemical
•Intentionally added chemicals
 Food Additives, Preservatives
•Unintentionally or incidentally added chemicals
 Agricultural chemicals
 Toxic elements and compounds
 Secondary direct and indirect

Physical
Any potentially harmful material not normally found in food such as
Glass, Wood, Stones, Metal or Plastic
Principle 2:
HACCP
Identify critical control points (CCP) in the process

A control point is any point, step, or procedure where biological, physical, or chemical factors can be controlled.
A critical control point (CCP) is a point, step, or procedure where an identified hazard can be prevented, eliminated, or
reduced to acceptable levels.
Critical control points are monitored much more frequently than are control points.
HACCP

Principle 3:
Establish critical limits for each
CCP

This step involves establishing criteria that must

be met to prevent, eliminate, or the reduce
the identified hazard at the CCP so that the food
is safe to eat.
Examples of critical limits are:
•temperature, time, physical dimensions, water activity, pH, and
available chlorine

Critical limits can come from regulatory

standards and guidelines, scientific literature,
experimental studies, and consultation with
experts.
HACCP

Principle 4:
Establish Monitoring procedures for each critical control
points
Monitoring is a planned observation or measurement:

 to determine if a CCP is under control

Examples of monitoring include:
 Visual observations
 Temperature measurements
 Time assessment
 pH measurements
 Water activity measurements
Deviation - Corrective Actions
Principle 5: Establish corrective action
Deviation Corrective Action

Temperature of milk at pasteurizer drops below critical - Milk flow is diverted until
limit temperature recovers.

- Diverted product is re-pasteurized

- Check the operation of the

heating/cooling units to determine
the reason for the temperature
deviation that caused the flow
diversion.

- Repair if necessary, re-establish

control and resume production

Product (e.g., hot-smoked fish) does not reach  Re-cook or

required internal temperature for the required time  destroy product
HACCP

Principle 6:
Verify the system is working

 Establish Procedures That Show System Works Correctly

 Both the Facility and outside third-party have a role to
verify that a HACCP Plan is Working Effectively

Verification methods

 Plant internal audits

 Government agency audits
 Private sector third party audits
HACCP

Principle 7:
Develop Effective Record Keeping:

Records demonstrate control over CCP's

Records can be used to:
 Provide early detection of areas of concern
 Isolate potential problems
 Identify trends
ISO 22000
The ISO 22000 international standard specifies the requirements for a food safety management system that involves the
following elements:

 HACCP Principles
 System Management
 Interactive communication
 Support Programs

Food safety is linked to the presence of food-borne hazards in food at the point of consumption.
Since food safety hazards can occur at any stage in the food chain it is essential that adequate control be in place.
Therefore, a combined effort of all parties through the food chain is required.

229
ISO Documents
ISO 22000 - Food safety management systems - Requirements for any
organization in the food chain.
ISO 22001 - Guidelines on the application of ISO 9001:2000 for the food
and drink industry (replaces: ISO 15161:2001).
ISO/TS 22002- Prerequisite programmes on food safety—Part 1: Food
manufacturing
ISO TS 22003 - Food safety management systems for bodies providing
audit and certification of food safety management systems.
ISO TS 22004 - Food safety management systems - Guidance on the
application of ISO 22000:2005.
ISO 22005 - Traceability in the feed and food chain - General principles
and basic requirements for system design and implementation.
ISO 22006 - Quality management systems - Guidance on the application
of ISO 9002:2000 for crop production.
ISO 22000 is also used in the Food Safety Systems Certification (FSSC)
Scheme FS22000. FS22000 is a Global Food Safety Initiative (GFSI) approved
scheme.
EU Regulations
Commission Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs
 Commission Regulation (EC) No 2074/2005 of 5 December 2005 laying down implementing measures for certain
products under Regulation (EC) No 853/2004 and for the organisation of official controls under Regulations (EC) No
854/2004 and No 882/2004, derogating from Regulation (EC) No 852/2004 and amending Regulations (EC) No
853/2004 and No 854/2004
 The link below is very helpful with these regulations.
http://europa.eu.int/comm/food/food/foodlaw/guidance/index_en.htm)
HACCP = Hazard Analysis Critical Control Point
HACCP Summary

Prevention of foodborne illnesses

Reduction of costs of food analysis
Protection of brand and reputation
Reduction of losses due to product recall or withdrawal
Hygiene Monitoring

Products
Hygiene monitoring

Cleaning monitoring
Limitations of microbiological hygiene control with Contact
Plates/Slides:

 Does not detect food residues

 Does not detect (recover) all microorganisms
 Detects microorganisms after 1- 7 days
Hygiene monitoring

Cleaning monitoring
Rapid Testing

HY-LiTE®

HY-RiSE®

To be of practical value to the manufacturer, a sanitation monitoring system

must give results fast enough to allow necessary corrective action before
restart of production.
Hygiene Monitoring

 ATP Testing
 HY-LiTE® 2 System
Hygiene monitoring

How can ATP-Bioluminescence be

used for measuring cleanliness?
RLU
 Levels of ATP can be used to indicate the
cleanliness of surfaces that could come into
contact with food.

 Relative Light Units are directly

RLU proportional to ATP

 Measuring the light released by this reaction,

allows an accurate determination of the quantity
of DIRT+ MICROORGANISMS =ATP present.

RLU  The HY-LiTE® 2 system is a luminometer,

counting photons out of the bioluminescence
light reaction.
Cleaning monitoring
AMP
AT Mg2+  The HY-LiTE® 2 system is a
P luminometer, counting photons out
Firefly of the bioluminescence light
O2 Luciferase CO2
Enzyme +
reaction.
PP

Luciferin oxy - luciferin  Display of light intensity (from

Substrate bioluminescence) in Relative Light
Units (RLU).

RLU  Relative Light Units are directly

proportional to ATP
Hygiene monitoring

Experimental results
Bacterial growth can only occur on Food residues
Therefore Food residues must be completely
removed by cleaning

 Microbiology can not detect Food residues

Experimental
Hygiene monitoring
results

Sterile = clean ??

Even on STERILE surfaces

HY-LiTE detects up to 1000 RLU

„Sterile“ therefore does NOT mean clean

„Not clean“ means RISK for contamination

Experimental results

Time factor

HY-LiTE can detect ATP from Food residues

and other contaminations – Rapidly

Microbiology can detect living Microorganisms

which are growing on the used medium
- But only after 1-2 days
Hygiene monitoring
Experimental results
Dirty surfaces detected before
production

100

90

80

70

60 Dirty
50

40

30

20

10
Dirty
0

HY-LiTE Visual Microbiology

= 100 %  20 % 0%
PASS / FAIL Limit setting
Hygiene monitoring

Values are only a rough guideline

Production environment Pass Fail

Raw Milk 100 300
Raw Meat / Fish / Egg 300 1000
Raw Vegetable / Fruit 500 1500
Processed Milk / Milk products 70 200
Processed Meat / Fish / Egg 70 200
Processed Vegetable / Fruit 200 600
Mass Catering / Flight Catering Facilities 500 1500
Beverages 50 100
Limits setting

It is best to set limits based on real (own) data from routine cleaning but:
 The HY-LiTE 2 manual contains a few suggestions
Once you have accumulated some measurement data
 You decide what an appropriate Pass and Fail % is
 Trend 2 software will calculate suggested limits based on any desired dataset
Hy-LiTE 2 System - Industries

Meat & Fish

Frozen Foods, Pizza, Ice-Cream
Vegetables & Potato Processing
High Care areas eg Baby Food
Soft drink industry (also fruit juice)
Beer & Wine industry
Catering and large kitchens
Industrial applications eg Automotive industry,
Fuel applicatiomn

250
Hygiene monitoring

HY-LiTE® 2 System
For the rapid detection of total ATP, this is a completely
new method to measure cleanliness.

HY-LiTE® 2 Luminometer with built-in printer

HY-LiTE® Pens

TREND 2 Software
HY-LiTE 2 Instrument

Built-in light-source
Automatic check of instrument calibration on every start-up
Temperature compensation
accurate readings from 5-35°C
outside this range instrument displays a warning message
Stored data secure
Data is stored in non-volatile memory and will not be lost even
if instrument looses power
In HACCP mode, data can only be deleted via PC
The HY-LiTE® pen
2 formats
- Surface test pen with rinse tube and swab
- Liquid test pen for CIP rinse water samples
• Very resistant to chemical interference
- Dilution of sample in rinse tube
- High buffer to sample volume in pen
- Efficient neutraliser against QUATs and similar detergents
- High buffer capacity against extreme pH
Special Application:
HY-LiTE Jet A1 Fuel Test: to detect biomass in Civil Aviation fuel (Jet A / Jet A1)
HY-LiTE 2 System

254
Trend 2 Software package
Windows XP compatible
3 access levels for data security
Set up sampling plans and transfer to HY-LiTE
instrument
Retrieve data from Instrument for
 Data analysis
 Pass/Fail Limit setting
 Trend Monitoring
Comprehensive Help function
Easy learning through PC tutorials (PowerPoint tours)
Trend 2 Software package – scatter graph
Hygiene monitoring
Cleaning monitoring
HY-RiSE® NAD (nicotinamide adenine dinucleotide) colour test
strip

Visual test (no reader needed)

Qualitative test
Simple to perform
Results in 5 minutes
Reagents stable at room temperature
More sensitive to micro-organisms than similar
colorimetric tests for sugar or protein
Suitable for confirmation of cleaning operations in Hotels
/ Restaurants / Catering / Retail
Hygiene monitoring
Cleaning monitoring

Is it clean or dirty? A simple answer...

Before After
cleaning cleaning

5 Min.
to
results

DIRTY CLEAN

movie
Hygiene monitoring

Cleaning monitoring
HY-RiSE® protocol
Standard Procedure:
Thank you for your
attention !
Culture Media
Culture Media Requirements
Demands to a reliable manufacturer of culture media:
 Media manufacturer must be certified according to at least ISO 9001 standards
 Media manufacturer should produce culture media in full compliance with
ISO 11133 Quality control methods

Merck as manufacturer of culture media:

 Merck is certified as culture media manufacturer acc. various ISO norms.
 Merck has available a clear documentation about the performance of their culture
media with values that exceeds ISO 11133

Customers:
 Customers labs which are certified acc. ISO standards need:
- To use validated culture media
- To follow validated enrichment procedures
 Why was ISO 11133 developed ?
1. No International Standard exist in
which the Quality Control Specifications of
commercial Culture Media are described

2. Each Culture Media Manufacturer has established

it´s own QC Specification

3. User of Culture Media have no information which

Growth limits are used by the Manufacturer

4. The new ISO 11133 describes now for the first time
which limits for Productivity and Selectivity each
Culture Medium MUST fulfil,
ISO 11133
part 1 and 2
ISO 11133-1 (2000-06-01) ISO 11133-2 (2003-12-15)
Microbiology of food and Microbiology of food and
animal feeding stuffs — animal feeding stuffs –
Guidelines on preparation and Practical guidelines on
production of culture media performance testing of culture
media

This new standard describe for the first time in history of

Microbiology the definition of quality control for commercial
culture media.

Quantitative testing and reporting of results ( CoA )

with recovery rates is the new preferred testing method
General Quality Criteria

Quality of Basic Media Components

Culture media described in the International Standards were judged
satisfactory; however, due to the variability of their quality, it may be
acceptable for media manufacturers to modify the concentration of
some basic biological ingredients, as listed below:

peptones and meat or yeast extracts variable in their nutritive properties

agar variable in its gelling properties
buffering substances
bile salts, bile extract and deoxycholate, antibacterial dyes, depending on
their selective properties
antibiotics depending on their activity
 Dehydrated Culture Media Merck –
granulated: GranuCult brand name

-Merck Microbiology Manual 12th Edition: available on MM website

Compliance of ISO - relevant Culture media
from Merck Millipore
New Brand names for ISO-relevant culture media from Merck Millipore

Merck Millipore offers culture media for

quality control applications that are
regulated by reference standards.
ReadyPlate*,
ReadyTube*,
The portfolio serves different Compliant RtU media
convenience needs for customers in
terms of handling, storage, QC
parameters ReadyCult, ReadyBag
Conveniently packaged
compliant DCM
Typical composition of branded
products are fully compliant to ISO
and other standards and directly
applicable in ISO workflows GranuCult*, ChromoCult
Packaged compliant DCM media

Branded products are fully compliant

to newest ISO11133 QC standards
*new brand
names with own
trademarks
Differentiating the Merck Millipore culture media brands for reference
standards

GranuCult (DCM Darmstadt) ReadyTube & ReadyPlate (heipha)

 Granulated culture media  Ready-to-use culture media in bottles,

 Easier and faster media preparation: tubes and plates
superior flow properties and non-sticking;
prevents component separation and  EN ISO 11133:2014 compliant No time-
clumping consuming preparation
 Minimize airborne toxic and allergenic
dust, preventing allergic responses and  High number of test strains exceeds all
contamination of workplace regulatory demands

 EN ISO 11133:2014 compliance  Safe testing at maximum convenience

 Highest batch to batch reproducibility  High reproducibility with low error-rate

 Additional number of test strains to

exceed regulatory demands
GranuCult™

- New brand name for our existing granulated DCM for testing of
food & water samples

- All DCM in accordance with newest EN ISO 11133:2014 and

various other ISO standards and FDA-BAM / USDA-FSIS methods
for testing of food and water samples.
- All DCM are currently being updated to be fully compliant to the
latest standards and regulations: typical composition & Quality
Control (finished by Oct 2015).

- Visible changes:
- New labels with updated article name to demonstrate compliance of the
product & updated typical composition / pH (if needed)
- New Certificate of Analysis to be fully ISO compliant

- With each new batch production, updated product & CoA is

available
27
9
279
WHY GRANULATED
CULTURE MEDIA?
CULTURE MEDIA: SAFETY.....??

IF IT IS GOOD FOR MICROORGANISMS, IT IS GOOD FOR HUMANS TOO ?

CLASS

SODIUM AZIDE T+
SELENITE T
LITHIUM CHLORIDE Xn
DEOXYCHOLATE Xn
MALACHITE GREEN Xn
SODIUM CARBONATE Xn
NALIDIXIC ACID Xn
CYCLOHEXIMIDE T+
AMPHOTERICIN B Xn
TELLURITE T+

Page 281
POTENTIAL HAZARDS related to the CULTURE MEDIA

Specific
Inhalation toxic Skin exposure
substances

Skin
Uptake via
Eye mouth
contamination

Environment

Page 282
 HEALTH AND SAFETY

LABEL INFORMATIONS

Page 283
 HEALTH AND SAFETY

Slanetz &
Bartley Agar
Sodium - Entero-
azide Azide coccus
Dexrose
Broth

Page 284
 HEALTH AND SAFETY

Selenite
Selenite Cystine Salmonella
Broth

Page 285
 HEALTH AND SAFETY

SELENITE

R SPECIFIC HAZARDS
TOXIC DUE TO INHALATION; INTAKE CAUSES
HAZARD DUE TO CUMULATIVE EFFECTS
Page 286
 HEALTH AND SAFETY

Selenite can / will damage

LIVER

KIDNEYS

CENTRAL NERVE SYSTEM

ALLERGIC REACTIONS
Page 287
 HEALTH AND SAFETY

XLD Agar • Salmonella

Sodium -
• Coliforms
Deoxy VRB Agar
- • Entero -
cholate
VRBD Agar bacteriaceae

Allergic reactions
Page 288
 HEALTH AND SAFETY

Baird-Parker Staph.
Agar aureus
Lithium
- -
chloride
OXFORD Listeria
Agar

Page 289
 HEALTH AND SAFETY

LITHIUM CHLORIDE

INHALATION MUCOUS IRRITATION

COUGHING

SKIN IRRITATION
EYE CONTACT IRRITATION
CONSUMPTION VOMITING, DIARRHOAE

INTAKE OF DROUSINESS
TOXIC DOSE IMPIRED SIGHT
HEART FAILURE
Page 290
 HEALTH AND SAFETY

Malachite Malachite • Pseudom.

Green Green broth aeruginosa

Page 291
 HEALTH AND SAFETY

Listeria Broth • Listeria

Nalidixic - • Pseudom.
acid Cetrimide Agar aeruginosa

Page 292
 HEALTH AND SAFETY

Listeria Broth
-

Listeria Agar • Listeria

-

Cyclo - Fungi Agar • Campy

• Fungi
-
heximide
Bolton broth
- • Legionella
Legionella
Agar

Page 293
 HEALTH AND SAFETY

BPA • Staph.aureus
Tellurite -
CT SMAC • E.coli 0157

Page 294
 HEALTH AND SAFETY

• Coliforms
Fuchsin ENDO Media
• E.coli

Page 295
 HEALTH AND SAFETY

CONTROL OF HAZARDS
 USE OF GRANULATED CULTURE MEDIA

 USE A SAFETY CABINET

 HYGIENE

 GLP

 INFORM STAFF

 DISPOSSAL ACCORDING RULES

Page 296
World’s best known Granules

NESCAFE Merck DCM

- 12
DCM – Health and Safety
- Granulated Media MERCK since 1970

During weighing process high

level of dust is formed.
Less dust = less risk of inhaling
dangerous / toxic substances

 Granulated media reducing

formation of dust by a factor of
3-4 compared to powder media

Page 298
DCM – Health and Safety - Dust Formation

Spread of Dust around Weighting Booth

MANUFACTURER

Powder M4

Powder M3

Powder M2
VRBG
Powder M1
BPA
MCB
Granulated

0 20 40 60 80
Page 299 SETTLED DUST IN mg
DCM – Health and Safety - Powder Media Problems

Powder
Media
Problems

Separation
Contamination Clumping Poor of components
from solubility during storage
dust particles
= DECOMPOSITION

Page 300
DCM – TAT Broth – Clumping - Powder vs. Granules

Tween, Lecithin, Histidine are very hygroscopic substances

cause rapid clumping of the culture medium

POWDER MERCK
Page 301
DCM – TSB - Granules vs. Powder

Page 302
DCM solubility - Granules vs. Powder

Page 303
 HEALTH AND SAFETY

Advantages of granulated media

 Significantly reduces inhalation

of toxic substances

 Reducing allergic responses

 Degree of contamination of
laboratory equipment and
clothing

Page 304
 HEALTH AND SAFETY

Advantages of granulated media

 Better flow properties

 Easily weighed out w/o sticking

to the side of vessels or
equipment

Page 305
 HEALTH AND SAFETY

Advantages of granulated media

 Dissolves rapidly and uniformly

 No lumps formed

Page 306
 HEALTH AND SAFETY

Advantages of granulated media

 Homogenous distribution of
the ingredients

 Guarantees reproducible
composition even when
small amounts are produced

Page 307
 HEALTH AND SAFETY

 Extended shelf life ( 5 years)

 Storage at room temperature

 No settling of the heavier

components

 Antibiotics included

Page 308
Antibiotics / Inhibitors included

Listeria Enrichment Broth

mECn+N and mTSB+N
MKTTn Broth
Selenite Cystine Broth
YGC Agar
DRBC Agar
RBC Agar
DG18 Agar
Pathogenic fungi Agar
Pseudomonas Agar (Cetrimide)
Yersinia Broth
Page 309
SSDC Agar (Yersinia)
Antibiotics / Inhibitors included

Listeria Enrichment Broth

mECn+N and mTSB+N
MKTTn Broth
Selenite Cystine Broth
YGC Agar
DRBC Agar
RBC Agar
DG18 Agar
Pathogenic fungi Agar
Pseudomonas Agar (Cetrimide)
Yersinia Broth
Page 311
SSDC Agar (Yersinia)
Examples of Culture Media
 Chromocult® Coliform Agar
Chromogenic Agar for the simultaneous detection of total coliforms and
E. coli in drinking water and processed food samples within 24 hrs

Coliforms

non Coliforms
Gram (-)

E.coli

Chromocult® Coliform Agar # 1.10426.0500

The new ISO 9308 part 1 (2014) describes the usage of membrane filtration
and a chromogenic coliform agar for enumeration of Escherichia coli and
coliform bacteria in drinking water, disinfected pool water, finished water from
treatment plants.

Merck Millipore Chromocult Coliform Agar (CCA) is the original: the only
chromogenic culture medium in combination with Merck Millipore mixed ester
filter for membrane filtration was used for all validation studies that were
needed to prepare this new ISO 9308 - 1 standard.

The procedure takes much less time for positive results than the 48 hours
needed for the previous ISO 9308 part 1 (2000) method using Lactose TTC
agar, and the new method requires no additional culture media for result
confirmation.
 Chromocult ® Coliform Agar
Simultaneous detection of coliform bacteria
and E.coli in water

Innovative features
 Save time: Up to 24 hours faster than the old
Lactose TTC agar method
 Clear results: Easy counting of E.coli and
coliform bacteria
 Save costs: Nod additional culture media
needed for confirmation
 Easier-to-do: Much easier and shorter
workflow with CCA compared to the
Lactose TTC Agar Method
 Certification : Validation of Alternative
Methods

Go ahead and replace it

with your own text. This is
an example text.
Pathogen Testing

“Convenient solutions for Salmonella & Listeria testing"

318 Presentation title in footer | 00 Month 0000

New Readybags

Readybag Buffered Peptone Water (BPW)

Readybag Half Fraser Broth (HF)

“Maximum convenience - minimum effort”

Pain point for customers with large test volumes of Salmonella and Listeria
tests is the preparation (weighing and autoclaving) of enrichment media.
Readybag is irradiated sterile DCM in pre-filled sachets filled with DCM for
enrichment of 25 gr food samples.

Benefits: - No weighing of media

- No autoclaving
- No separate addition of supplement (already included in Readybag
HF)

With Lab Water‘s Elix Advantage

- for prewarmed & sterile water supply -
 Readybag® Product Overview

1.02448.0060 Readybag® Buffered Peptone Water (Salmonella testing: for 25 g food sample +
225 ml water)
1.02449.0060 Readybag® Half Fraser Broth (Listeria testing: for 25 g food
sample + 225 ml water)

Food sample
+
Readybag® content
+
Sterile water
=
Ready to incubate!
Elix Advantage
No media preparation: - no weighing with sterile filter
- no autoclaving
- no supplement handling
Readybag concept

Speed up and simplify your workflow!

 Pre-weighed

 Gamma-irradiated
 No media preparation needed!
 Eliminates autoclavation
 Sterile lab water provided with ELIX advantage

321
Convenience Readybag DCM’s
Handling of Readybag

1. Weigh 25 g 2. Open Readybag and add

of pre-weighed medium into
food sample stomacher bag.
in
stomacher
bag

3. Add 225 ml of 4. Mix by hand and

sterile water. place into stomacher
to homogenize sample
in enrichment broth

5. Ready to incubate!
Pathogen enrichment - Traditional workflow

1. Media preparation

1 Weighing +
Dissolving media 5 min

•Add water 01 min

•Stir and dissolve 05 min
•Load autoclave 02 min
2 •Autoclaving 25 min
•Cool down autoclave 30 min 98 min
•Unload autoclave 05 min
•Cool down media 30 min

3 Supplementation
Selective Media 6 min
Pathogen enrichment - Traditional workflow

2. Food sample handling

4
Weighing food sample 3 min

5 Adding media 3 min

6 Homogenization 2 min
-------
117 min
Pathogen enrichment – Readybag Workflow

1
Weighing food sample 3 min

Add Readybag content 1 min

2
Add sterile water 2 min

3 Homogenization 2 min
-------
8 min
Discover the new Readybag pouches

with dehydrated culture media for

fast , flexible and easy pathogen testing

Movie readybag
Pathogens Testing
Rapid Methods
Merck Millipore Lateral Flow
Winning combination for rapid detection

Superior Granulated Dehydrated Innovative Immunoassays – Lateral FlowTests

Culture Media - Singlepath® and Duopath®
Merck Lateral Flow Tests

Singlepath
® and Duopath
® products
 General Application of Lateral Flow Tests

1 ADD MEDIUM 2 ADD SAMPLE 3 BLENDING 4 INCUBATE 5 PIPETTE

SAMPLE

6 PLACE NEGATIVE POSITIVE

SAMPLE

Read
after
20 -25
min
 Advantages of Lat. Flow tests vs. ELISA

1. Control reaction in-built in LFT.

No need for separate reaction to demonstrate proper function,
as necessary for ELISA.
Can save costs esp. when low numbers of samples to be tested

2. LFT’s are 1-step tests.

After application of test sample to sample port, NO further pipetting steps
necessary.
No separate color reaction for LFT’s necessary to make reactions visible.

3. LFT are provide results faster than ELISA

Only incubate test on lab bench for 20 – 30 min and read result.
Example: Singlepath® Salmonella:
Validated alternative method

-For detection of Salmonella in foods directly after 2-step enrichment

-No Post-enrichment step needed:

sample directly taken from RVS
+ 15 min heating,
transfer to sample port
read results within 20 min

-Lower detection limit: 105 cfu / ml

-Is an AOAC-validated alternative method

FDA-BAM Standard for Detection of Salmonella: Traditional

4 days
25 g/ml test sample
Day in 225 ml Lactose Broth
16 - 20 h / 35 - 37 °C
1
1 ml BPW to 1 ml BPW to
3 plates from each
Day 10 ml SC Broth selective broth
10 ml TT Broth
24 h at 35°C h 24 h at 35°C
2
Hektoen Agar XLD Agar Bismuth Sulfite Agar
Day 24± 2 h at 35 °C
24± 2 h at 35 °C 24± 2 h at 35 °C
3

Day
4 Interpretating of Growth on Plates 9 media
Day For confirmation take 5 suspected colonies from each plate
5 and streak onto Nutrient agar 18 - 24 h / 35 - 37 °C

Day Biochemical / Serological Confirmation Tests

6 TSI / Urea / Lysin / -Gal / VP / Indol
Immunological Rapid Test Method
Singlepath® Salmonella Screening of foods

25 g/ml TEST SAMPLE

Day
In 225 ml Buff. Peptone Water
1
35-37 °C for 16-20 h

only only
Day
2 2 days 0.1 ml BPW to 9.9 ml RVS Broth
42°C for 24 h 2 media

NO YES
Salmonella Salmonella
Day
3 present present

Day STREAK OUT

If positive, confirmation by culture and ON RAMBACH
4 biochemical tests required 37 °C FOR 24 h
Singlepath® Product Range

Singlepath® Campylobacter Rapid test for the

detection of Campylobacter in foods
104143

Singlepath® E.coli O157 Rapid test for the

104141
detection of E.coli O157 in foods

Singlepath® L'mono Rapid test for the detection

104148
of Listeria monocytogenes in foods

Singlepath® Salmonella Rapid test for the

104140
detection of Salmonella in foods
Duopath® Product Range

Duopath® Cereus Enterotoxins Rapid test

104146
for the determination of the NHE and HBL toxins
in foods

Duopath® Legionella Rapid test for the

104147
determination of Legionella spec. and Legionella
pneumophila in water and environmental
samples

104144 Duopath® Verotoxin Rapid test for the

detection of verotoxins in pathogenic E.coli
 MM‘s unique & broad Lateral Flow Test portfolio
Listeria: Salmonella: E. coli O157&VTEC: Campylobacter: Bacillus cereus: Legionella:
LEB Tetrathionate mEC + n Bolton Broth M.Y.P. Agar CYE Agar Base
Fraser Rappaport.-Vas. mTSB + n CCD Agar CGY Broth BCYE Suppl.
UVM Selenit Cystin CT-SMAC BHI GVPC Suppl.
PALCAM Salmosyst SMAC CYE-Plates
OXFORD M Broth BHI GVPC-Plates
Chromocult® XLT4 , XLD, CAYE Anaerocult® C
Listeria Agar Rambach®

Movie Lateral flow test

Simplified Microbial WaterTesting

Readycult
COLIFORMS
Readycult® Coliforms Procedure

Add one Readycult® snap

pack to 100 ml of water
in a sterile 120 ml to
150 ml bottle.
Seal vessel and shake
completely to dissolve
granules.
Incubate 24 hr +/- 1 hr
at 35-37°C
Interpretation of results
 Interpretation of Results
POSITIVE TOTAL COLIFORMS

Coliforms Present
β–D-Galactosidase Positive

Distinct Color Change

to Blue-Green
 Interpretation of Results
E. Coli POSITIVE
If the Broth is Total Coliform Positive,
(Blue-Green in Color) Check with UV
Light for FLUORESCENCE.

If the Broth Fluoresces then:

E. coli is Present

β-D-Glucuronidase Positive
 Interpretation of Results

E. coli
BIOCHEMICAL CONFIRMATION WITH THE
99 %
INDOLE REACTION

 Indole Reaction 99%

Accurate.
Tryptophanase Positive.

Overlay the Total Coliform or INDOLE POSITIVE

E..coli Positive Sample with
Kovac’s Indole Reagent. The
Formation of a Red Ring Indicates E. Coli Confirmed 99%
a Positive Indole Test

Movie Readycult
Tools for Microbiology
Anaerocult System for the cultivation
of Anaerobic & Microaerophilic bacteria

 What is expected from an Anaerobiosis syste

1. Rapid establishment of virtually

oxygen-free conditions
2. Eh values of below 100mV-redox potential
3. Sufficient high levels of carbon dioxide
4. Ease of use
5. High degree of safety
6. Reliable performance
7. Long term-stability and problem-free storage
8. Economy
 The Anaerocult portfolio

1. Anaerocult A : For the incubation of up to 12 petri dishes in an Anaerobic jar

2. Anaerocult A mini : For up to 4 petri dishes in an incubation bag

3. Anaerocult P : For the incubation of 1 petri dish in an incubation bag

4. Anaerocult IS : For the incubation of Identification systems and microtiter plates

5. Anaerocult C : For the incubation of microaerophilic and capneic

microorganis in an Anaerobic jar

6. Anaerocult C mini : For the incubation of microaerophilic and capneic

microorganisms of up to 2 petri dishes in an incubation bag
Anaerobic jar for up to 12 petri dishes
Anaerocult A for up to 12 petri dishes in a jar
Anaerocult A mini for up to 4 petri dishes
Anaerocult P – for 1 petri dish
Sterikon Bioindicator for Autoclaving control

according to Pharmacopoeia requirements

Biological Indicators for sterilization
USP 1035 and 5.1.2 EP

Sterikon® plus
Bioindicator for
checking steam
sterilization
efficiency
acc. to USP
and EP
Sterikon® Plus Bioindicator

Growth of Geobacillus. No growth of Geobacillus.

Color of indicator
Color of indicator did not
changed from Purple to
changed
Yellow
Application Using the MERCK Sterikon® plus
Bioindicator System it is possible to check
the efficiency of autoclavation for 15 minutes
at 121 °C.

Furthermore it is possible to control the

sterilization success of any kind of autclave-
loading after auto-clavation.

As for example:
pharmaceuticals, especially drugs in
ampoule form, canned food, culture media
etc.
Principle: The Sterikon® plus Bioindicator consists of an
ampoule that contains:

• Nutrient broth
• Carbohydrates
• ph indicator (Bromoxylenalblue)
• Spores of an apathogenic organism,
Geobacillus stearothermophilus

The thermal resistance is such that the spores

are totally killed after 15 minutes, when treated
in com-pressed steam at a temperature of
121°C ± 0.5°C (1 bar).
 An appropriate number of ampoules
Procedure: are included in the batch to be
autoclaved.

Autoclaves with a capacity of up to

250 litres should be checked with at
least 2 ampoules, whilst those with a
capacity of more than 250 litres
should have at least 6 ampoules.

The ampoules are placed in the

autoclave at sites where the most
unfavourable conditions for
sterilization are thought to exist, i.e.
at the bottom and in the middle of
the autoclave.
Procedure: Sterikon® can also be used to
check the functional efficiency, i.e.
to test whether the prescribed tem-
perature of 121°C is reached
within the entire autoclave and
whether the temperature remains
constant over the whole prescribed
period of 15 minutes.

After sterilization, the ampoules

are removed and incubated 48
hours at 60°C ± 2°C.
 If sterilization is adequate, the
Bacillus stearothermophilus spores
Evaluation: are killed off.

The contents of the ampoule remains

a red-violet colour.

If sterilization is inadequate, low

temperature, short heating period, the
spores of Bacillus stearothermophilus
survive.

The contents of the ampoules then

Non-sterile = yellow, turbid Sterile = red-violet usually turn yellow within 24 hours
(growth) (no growth)
due to the formation of acid as a
result of sugar fermentation and also
become turbid due to microbial
growth.
Sterility Testing
Programs in Pharmaceutical Microbiology

Sterility
Testing
Microbiology
Laboratory
Final
In-Process
Product

Raw Environmental
Materials Water Monitoring

Support Data

Product Release
What is a sterility test ?

 Presence / Absence test

 Reference test for Pharmaceutical industry
 Quality assurance requirement
 Represents one set of data which contributes to
the decision of whether or not the product lot
meets the stated claims.
 Not intended as a sole product release test
 Not for viruses and mycoplasma detection
Sterility Testing
Why to conduct a sterility test ?

cGMP requirements
“For each batch of drug product purporting to be sterile and/or pyrogen free, there shall be
appropriate lab testing to determine conformance to such requirements” 21CFR211.167 (a)

EP7/USP33
„The test is applied to substances, preparations or articles which, according to the
Pharmacopoeia, are required to be sterile.“
Where to conduct the test

Aseptic conditions for performance of the test can be achieved using for example a class A
laminar-air-flow cabinet located within a class B-room, or an isolator (EP, 5.1.9)

The sterility test should be conducted within a class A laminar airflow cabinet located within a class
B clean room, or in an isolator that need not be located within a controlled environment. The test
may also be performed within a class A clean room, if available. (PIC/S, 2007)
Sterility Testing Methods: Membrane Filtration

The US and European Parmacopoeias state: « Membrane filtration is the

method of choice for sterility testing».

Required conditions and specifications for a membrane filter dedicated for

Sterility testing:

o Pore size diameter (nominal) 0.45 µm maximum

o Membrane diameter approximately: 50 mm
o Flow rate of water per minute / per cm², at a pressure of 13,5 PSI (70 cm de HG):
55 – 75 ml
Sterility Testing History

1957: membrane filtration (MF) for products with

antimicrobial activity (FDA)

1966: membrane filtration method improvement

with filtration manifold (FDA). Usage of rinsing fluids

1977: Closed concept system Steritest (Millipore)

Since the early 80s: first isolators for sterility testing

1983: specific membrane (Durapore, Millipore) for

Antibiotics

 2011: Milliflex Rapid for Sterility Testing: 5 days

result!

 2014 New Steritest Symbio pump launched

Sterility Testing Methods: Membrane Filtration

(Open-) Funnel method

RISK of FALSE POSITIVE
RESULTS !

OR
Closed concept
system:
Steritest
Membrane Filtration:
Steritest Closed concept system

Steritest
Leader in this field (44 years of experience in sterility
testing)
Method accepted from all Pharmacopoeias
Fast filtration through pressure, not vacuum
Closed system (no risk of false positive)
Pre-sterilised, ready-to-use units
Steritest System
Components for the test

The Steritest System is a closed system for sterility testing by

membrane filtration. Three Components:
1. Hardware & Accessories

o NEW Steritest Pumps:

o Symbio LFH, ISL, FLEX
o Foot Pedal
o Communication Hub
o Symbio replaces the Equinox pump

1. Sterility Testing Devices

o Different Steritest Units (canisters) with

different adapters and membranes

2. Media & Rinse Fluids

o Recommended from regulatory agencies

 Steritest Symbio LFH

• Designed to ease
manipulation, improve
screen visibility and
free space under a
laminar flow hood or in
a clean room
Steritest Symbio ISL

• Small footprint and low profile

to preserve loading and
working space in isolators.

• The motors and electronics

are easily accessible for
maintenance in the housing
located under the table
(outside the isolator)
Designed to fit within all testing environments

Steritest Symbio pump vs Equinox pump:

Reduced height (pump head is 10 centimeters lower)

250

150

-> easy tubing loading under Laminar Flow Hoods windows

-> improved ergonomics (better view of control panel and easy access to knobs).
User friendly pump: Drain tray

Easy sliding of the 2 parts

Minimized splashing

Transportation
position (LFH pump
only)

Locked position
User friendly pump: Communication hub

More than a simple power supply box

Switch in the front !

• Network port

• USB port

• Footswich port

• Syringe support port

• Waste overfill sensor

port
User friendly pump:
Ergonomic buttons and knob

Bigger buttons designed to be easily operated with isolator gloves

User friendly pump: screen

4,3 inch (11 cm) colored LCD screen, ergonomically positioned

Profiled for both vertical and horizontal flows
Adjustable viewing angle and depth without screw
User friendly pump: languages

Chose your operating language (French, English, Simplified Chinese,

Japanese, Italian, Portuguese, Spanish, German)

-> Lab technicians not fluent in English can configure and use all pump functionnalities, including Test Methods
Library
Safety: Pressure detection

Improved pressure mode detection:

• Selected mode always visible (Pressure Monitoring or P.
Regulation)
• Easy switch from one to another
• Visual alert and increased buzzer power
User friendly pump: Test Method Mode

follow them step by step on the pump screen

Win time with preset speed and timer values for each filtration steps
Never forget or mix test protocols
 User friendly system: Test Method software

Test methods library: create up to 250 filtration protocols of 30 steps each

transfer them with a simple USB flashdrive (or a network cable)

Available directly from the new Merck Millipore website

Free of charge (registration needed)
Import of Equinox test methods is possible
Check the related movie on the Web site !
Reliability: Small volumes sampling precision
Market’s most precise Timer
function: small volumes are
sampled with even higher
precision vs Equinox.
An exclusive rotor design has been
added to the compensation of
the rotor position (launched with
Equinox)1,04 mL
Volume: Steritest Equinox Steritest Symbio
CVr 2,45% 1,96%
Speed 2 & Timer 1 Speed 2 & Timer 1

-> More reproducible sampling

-> Higher test safety (no additional air aspirated)
Safety: Particles generation

Clean room friendly hardware: air-tight housing and passive cooling are
preventing particles emission

Gasket

No fan
Steritest Consumables
Steritest Product Range

Select the best appropriate Steritest Unit to make the handling easier according
the sample type and sample packaging
 Product characteristics
o Filtrability / Viscosity
o Compatibility
o Growth inhibition

 Product packaging
o Small vials
o Large vials
o Ampoules
o Bags
o Syringes
o Medical devices
Steritest BLUE Base
 Canisters: SAN (Styrene Acrylo Nitrile)
 Tubing: PVC

 MCE membrane
 Heat sealing of the membrane to the base
followed by ultrasonic welding of the Top

 Small Drainage Channels - Prevents “cracking”

of membrane while under pressure.

APPLICATIONS Products WITHOUT microbiological growth inhibition activity

Products “easy to filter” (saline solutions, low viscosity
products diluted/dissolved products).
Steritest BLUE Base TZHALA210
 For IV Bags and Ampoules
 Single unvented needle
oOpen ampoules do not need to be
vented
oCollapsible bags normally require no
venting
oSome IV bags may also interface with
the medical device Steritest
configuration
 Separate vent filter provided
ofor rinse buffer and media

 Exist also in double pack

(TZHALA205)
Steritest BLUE Base

TZHALV210 (TZHALV205 for DP)

Large Volume Parenterals
Liquid products in bottles larger than 100 ml
Products are typically saline, glucose solutions
Single Vented Needle draws air into container
as liquid is withdrawn.

TZHASV210 (TZHASV205 for DP)

Small Volume Parenterals
Liquid products in 100 ml bottles or smaller
Two small diameter needles with internal vent
draw air into container as liquid is withdrawn.
Steritest BLUE Base TZHAPC210

 For Plastic bottles

 Non-coring needle adapter with
aperture
on side of needle minimizes
blockage
when piercing plastic containers
Separate vent needle included for
transfer
of culture media or rinse buffer
Steritest BLUE Base TZHASY210
 For control of pre-filled syringes with needle
 Specialized tube & adapter with vent to test
BOTH the syringe needles and its contents
simultaneously.
 This unit may be used with a syringe actuator system
Steritest BLUE Base TZHAMD210

 For Medical Device (perfusion lines,

catheters,
blood pumps, transfusion lines, urine
pouches
and artificial kidneys)
 Use with Novaseptum sampling devices
 Includes 3 adapters (tubing sets)
omale luer
ofemale luer
osampling needle
 Flush out the medical device by pumping
the
appropriate rinse buffer and then filter
the buffer
through Steritest Canisters.
Steritest BLUE Base TZHADV210
 Sterisolutest: designed for Lyophilized Products
 Lyophilized and soluble powders in vials

 This unit contains two small diameter needles to

simultaneously dissolve the powder in sterile
diluent and then transfer and filter the resulting
solution
Steritest BLUE Base TZHADA210
 Sterisolutest: designed for Lyophilized Products
 For Lyophilized and soluble powders in vials and
medical devices in various packaging configurations

 Solubilize and filter in one step.

 Sterile diluent may be pressurized (via the vent filter)
so that it flows into the ampoule.
 Insert needle aseptically into vial or ampoule (while
holding the ampoule at a 45° angle.)
 To preserve the sterility of the closed system: Do not
remove needle from rinse bottle (this could allow
contaminated air to enter the canisters).
 Steritest RED Base

 Canisters: SAN (Styrene Acrylo Nitrile)

 Tubing: PVC

 Durapore membrane
 Heat sealing of the membrane to the base followed by
ultrasonic welding of the Top

 Flat Base Design & Small Drainage Channels with

Additional “drainage ring”
o Improves antibiotic drainage away from the membrane
o Improves rinsing by eluting the antibiotic from the membrane

APPLICATIONS Products WITH microbiological growth inhibition activity =

Antibiotics or products containing preservatives
Steritest RED Base

How antibiotics/products containing preservatives should be

tested ?

As risk of inhibitory effects on the growth of microorganisms in the

product
to be tested exists (false negative results), we have to:

 Choose the right Steritest device with right testing procedure

 Prove that the Sterility Testing procedure will allow a perfect
elimination
of any growth inhibition before the incubation
The right testing procedure for Antibiotics
3 GOLDEN rules to follow

Maximize Solubility
oPre-wet membrane; In some cases, retain fluid on the membrane
oUse Steridilutor OR Sterisolutest to completely dissolve and dilute the
product

Minimize contact time with the membrane

oUse Durapore membrane
oMaximize Pump Speed for product filtration

Optimize Rinsing
oIncrease the number of rinses using right Fluid types (D, K)
oOptimize Drainage with additional drainage ring base design
oReduce Pump Speed for rinsing steps
Steritest RED Base – TZVC00010 & TZHVAB210

 Used to test antibiotic powders or strong binding

antibiotics
Steridilutor TZVC00010 + Steritest EZ TZHVAB210

 Steridilutor facilitates dissolution and pooling

of all the drug samples in 1 bottle
 The pooled samples are then filtered using
the Steritest EZ for Antibiotics
 Separate Steridilutor vent should be kept until
the last rinsing step
 For media transfer use the separate vent
delivered with TZHVAB210
Steritest RED Base – Sterisolutest TZHVDV210

 Used to test liquid antibiotics or low binding

antibiotics
 MAIN ADVANTAGE: Time saving!
oIn 1 step : dilution of the liquid antibiotic AND filtration through the
membrane

 Drawbacks vs. Steridilutor + TZHVAB210

oNO liquid recirculation = will not dissolve powders correctly.
oYou do not pool all liquid in 1 container and then filter quickly in 1step
=> longer contact time than with Steridilutor + TZHVAB210
Steritest RED Base TZHVMD210

 For Medical Device (perfusion lines,

catheters,
blood pumps, transfusion lines, urine
pouches
and artificial kidneys)
 Use with Novaseptum sampling devices
 Includes 3 adapters (tubing sets)
omale luer
ofemale luer
osampling needle
 Flush out the medical device by pumping
the
appropriate rinse buffer and then filter
the buffer
through Steritest Canisters.
Steritest RED Base

 Testing other products with antimicrobial activity !

Steritest TZHVLV210
For Liquid products in Large vials

Steritest TZHVSV210
For Liquid products in Small vials

Main advantage of these Steritest versions :

Easy to use and avoid additional handling of the separate vent
needle
Steritest GREEN Base

 Canisters: Grilamid (Nylon)

 Tubing: PVC

 Durapore membrane
 Heat sealing of the membrane to the base followed
by ultrasonic welding of the Top

 Flat Base Design & Small Drainage Channels with

additional “Draining Ring”
 Internal chimney

APPLICATIONS
• SOLVENTS : GRILAMID compatibility vs. wide range of solvents
• CREAMS, OINTMENTS, PETROLATUM PRODUCTS: GRILAMID Compatibility vs.
Isopropyl Myristate.
• Internal chimney on canister for highest resistance to the Pressure
Steritest EZ – Application Summary
MEMBRANE MAIN FEATURES APPLICATIONS

Products without microbial

HA (MCE) HIGH FLOW RATE (1000 ml/mn growth inhibition activity =
membrane at 10 psi with water) Products “easy to filter” (saline
solutions, low viscosity product,
diluted/ dissolved products)

Heat sealing : membrane to the

HV Durapore base followed by ultrasonic
membrane welding of the Top
= NO risk of antibiotic binding Antibiotics and Products with
Low binding around sealing. anti-microbial activity
vs. Antibiotics Additional “Drainage ring”
Improves Rinsing by eluting the
antibiotic from the membrane.
Same as RED base +
HV Durapore - Grilamid instead of SAN.  Solvents (Veterinary
membrane Compatibility vs. solvents and products).
IPM.  Creams
Low binding - Underside “Faucet Design”  Ointments
vs. Antibiotics Resistance to the pressure  Petrolatum Products
- Inside chimney on top. soluble
Resistance to the pressure
Steritest Quality

Certificate of Quality : TZHALV210 / TZHALA210

21.9 and 40 kGy. 40 kGy is not inhibiting

thegrowth
 GMP and Quality Standard compliance
Manufacturing and controls are done according to international regulations and standards

Certificate of Quality

Production steps and critical integrity checks are automated

Steritest Services
Validation Services

Application Support
• Support to customers on test method
development and issues
• Operator training

Validation Services

• Validation Protocol available for IQ/OQ and PQ

support
• On-Site IQ/OQ Service Execution
• PQ Consultancy Service

Instrument Service

• Service plans
• Maintenance & repair
 Sterility Testing Validation Protocol
Complete & Ready to use IQ, OQ, PQ Protocol
Complete descriptions of all the activities necessary for both proving the suitability
of the test methodology and writing SOPs for the test method

Shortens Validation Times

Ideal for busy QC/QA departments

Complies with International Compendia

The Validation Protocol complies with the United States, European, and Japanese
Pharmacopeias

Accurate, Knowledgeable Advice

The Validation Protocol has been prepared by Millipore, a company with 44 years
experience in sterility testing and helping customers successfully validate
microbial test methodologies.

Detailed Protocol
The Validation Protocol includes all the information needed to organize and
execute step by step the validation.
 Sterility Testing Method Development
What for?
To develop/optimize a sterility test method for your new/existing products compliant with current regulations (EP/USP)

 What is included?
 Definition of the required sample volume.
 Product filterability assessment.
 Selection of the appropriate Steritest device(s).
 Selection of the pre-wetting and rinsing fluid(s).
 Determination of filtration and rinsing procedures.
 Recovery evaluation on the most sensitive micro organism.
 Checking the recovery with all other Pharmacopoeia micro organisms.
 Complete report including detailed Sterility test method proposal
Sterility Testing Media & Fluids
Merck Millipore Media

Fluid thioglycollate medium (FTM/CTM):

Primarily intended for the culture of anaerobic bacteria but will also detect
aerobic bacteria (EP/USP).
Incubation: FTM is to be incubated at 30-35°C (EP) / 32,5 + 2,5°C (USP)

Soya-bean casein digest medium (TSB):

Is suitable for the culture of both fungi and aerobic bacteria (EP/USP).
Incubation: TSB is to be incubated at 20-25°C (EP) / 22,5 + 2,5°C (USP)
Merck Millipore Rinse Fluids
Role of sterile buffers (rinse fluids): dissolve, dilute, pre-wet,
rinse
TYPE APPLICATIONS
o Suitable as a general rinse buffer
Fluid A o Works well with most samples
(USP) / neutral solution of meat or o Excellent to dissolve or dilute samples
casein peptone (EP) o Excellent to reconstitute commercial microorganisms
o Excellent transport medium for microorganisms
o Suitable for testing specimens that contain lecithin or oil
o Excellent for rinsing sterile pathways of devices
Fluid D
o Works well with most antibiotics
(USP)
o Needed for rinse method testing of Medical Devices

o Suitable for testing specimens that contain petrolatum

Fluid K o Suitable for oils and oily solutions
(USP) / neutral solution with emulsifying o Excellent for rinsing pathways of Medical Devices
agent (EP) o Good for “difficult” to filter or dissolve samples
 Merck Millipore
Double Packed Media and Fluid A

Ethylene oxide sterilized media and Fluid A bottles

• Sterilized bottle surface, including septum and protective cap
• 2 step unpacking fitting with clean room procedures:
 First Tyvek® bag removed to enter the lab/cleanroom
 Second bag removed before loading the testing environment (Laminar flow hood, Isolator, etc)
• Same features as standard 100 mL bottles:
• Easy and safe septum decontamination (Rimless design)
• Easy piercing of the septum (large surface)
• Easy scanning (2D bar code)
• 12 months shelf life
• Meck/EMD Millipore quality
Product Portfolio

Description Volume Closure Packaging Size Catalogue Nber

(mL)

Trypcase Soy Broth (Soybean- Screw cap with

100 12 STBM TSB 12DP
Casein Digest Medium septum

Screw cap with

Fluid Thioglycollate Medium 100 12 STBM FTM 12DP
septum

Screw cap with

Clear Fluid Thioglycollate Medium 100 12 STBM CTM 12DP
septum

Screw cap with

USP Rinse Fluid A 100 12 STBM RFA 12DP
septum
Double Packed Steritest units

Double Packed media and Double

packed Steritest units :
A perfect team to reduce
the false positive risks

Description Packaging Catalogue Nber

Steritest EZ Double Packed

2 bags of 5
for Liquids in Ampoules TZHA LA 205
and Collapsible Bags devices

Steritest EZ Double Packed 2 bags of 5

TZHA LV 205
for Liquids in Large Vials devices

Steritest EZ Double Packed 2 bags of 5

TZHA SV 205
for Liquids in Small Vials devices
Steritest: what configuration to choose?

 What is the nature of the product?

oi.e. normal easy to filter liquid, viscous, suspension,
antibiotic or preservative added?

How is the product packaged?

oi.e. glass bottle, collapsible bag, ampoule, syringe,
aerosol or other?

Special considerations
oDoes the product need to be diluted or dissolved?
oDo we need to use vent needles or other accessories?
Remember!!
Merck Millipore propose a complete portfolio for Sterility
testing!

 Steritest Symbio Pumps – LFH and Isolator Versions

 Steritest consumables – MCE & PVDF Membranes
 Steritest Media & Rinse Buffers
 Sterility Testing Method Development
 Validation Protocol – VMP, IQ, OQ, PQ, Final Report
 On-Site Validation Services – IQ/OQ Execution and PQ Consulting
Thank you for your attention !
Enjoy the Art of Microbiology
with Merck Life Science. Thank you!