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The expertise in culture media

preparation and distribution


PREPARATION
High Quality Medium's Requirement
The Steps of Bacteriological
Diagnosis
1- Specimen collection/transportation

2- Microscopic Examination

Direct examination Influence on what type of media I have touse


The Steps of Bacteriological
Diagnosis
3- Culture 5- Antibiotic Susceptibility Testing
The choice & quality of the
media will define the quality
of the analysis

4- Identification
Which Atb we must
choose? Possibility
to improve the result
with the MIC*

For the identification, we


must obtain the growth
of microorganisms
Choose the right culture
media

➢ depends on the specimen


➢ depends on the microscopic examination
➢ depends on the bacteria to be detected

Basic medium ? Atb Resistance


detection medium ?

Rich medium ? Selective medium ?


Common Mistakes/Problems of
Preparation Culture Medium

Media Concentration
 Weighing, Water Quality, Pre-Warmed – mixing.
Media Formulation
 Adding supplement, pH, Indicator dye.
Nutrient levels
 Sterilization, Growth Promotion.
Filling & Storage
 Precision of Volume, Temperature, Moisture,
Shelf-life.
What are the effect of incorrect media
preparation

Inhibition of target organisms

Atypical colony morphology

Atypical physical characteristic

Reduced growth / Recovery


rates

Faillure to inhibit competing


flora

Reduced shelf – life of prepared


medium
Preparation of Media

1. Weighing

2. Sterilization

3. Filling

4. Storage
Preparation of Media - Weighing

• Safety
protect user from powder – extraction hood, personal protective
equipment (PPE)

• Weighing
must be accurate

• Water Quality
conductivity should be less than
15 micro-siemens(μs) microbial contamination
should be low
Preparation of Media - Sterilization

Sterilization
• Vessel -Glass / Stainless Steel
ensure grade of glass/stainless steel is suitable for culture media
preparation

• Quantity and Volume


ensure that validation is carried out for quantity/volume prepared

• Holding Time
ensure that holding period (i.e. as molten medium) does not effect
performance

• Follow Manufacturers Instructions


Preparation of Media - Sterilization

Sterilisation is the area where most media damage occurs

Ensure the medium is not over-heated. Validate process using relevant volumes

• Chemo-oxidation - occurs on exposure to heat.


peptones also damaged - not only the selective agents this may lead to the
formation of peroxides and superoxides medium may become toxic when over-
heated

• Reduced growth
• Reduced selectivity
• Low pH
• Low gel strength
• Precipitation
• Atypical colour
Example :BLOOD AGAR BASE,
CM0055
A non-selective general purpose medium which may be enriched with blood or serum.

Directions
Suspend 40g in 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilise by
autoclaving at 1210C for 15 minutes.

For blood agar, cool the Base to 500C and add 7% of Defibrinated Horse Blood SR0050. Mix
with gentle rotation and pour into petri dishes or other containers.
Example : CLED MEDIUM , CM0301
Preparation of Media -
Sterilization
CM0333
TCBS Cholera Medium

Over-heated medium, reduced growth of Medium prepared correctly good


Vibrio cholera Medium growth of V.cholera
Preparation of Media -Sterilization

Brilliant Green Agar CLED Medium CM301


CM329
Preparation of Media -Sterilization

Brilliant Green Agar CLED Medium CM301


CM329

Over-heated medium
reduced growth of
Over-heated medium Staphylococcus aureus
-no inhibition of Proteus
sp.

Medium prepared
correctly
Medium prepared correctly -good growth of S.
-inhibition of Proteus sp. aureus
Preparation of Media -Sterilization

VS

Over-heated medium: Prepared


-dark colour correctly
-low pH
-reduced growth of
fastidious organisms • TryptoneSoya Broth CM129 Medium
Preparation of Media -
Sterilization

Selenite Broth CM0395

Over-heated medium,
Medium prepared
pink with precipitate.
correctly.
Growth of Salmonella
sp. could be reduced.
Preparation of Media – Plate Filling

• Temperature - pour plates below 50C

• Volume – uniform and sufficient to allow storage and incubation without plate drying

• Holding Time - time held in molten state should be validated

• Aseptic Technique – essential

• Contamination - ensure medium does not become contaminated from packaging material

• Moisture Loss/Drying-keep moisture loss to a minimum


increased concentration of selective agents in medium
surface of medium will become hard -reduce colony size
reduced water availability (aw)

• Physical Appearance -ensure the plates are poured


on a level surface and medium is free from bubbles, cracks etc.
Preparation of Media - Storage

• Temperature - should not fall below 2 C; extreme low temperature will


cause loss of moisture (condensation) and lysis of blood plates.

• Photo-Oxidation-exposure to sunlight or artificial light may cause the


production of toxic peroxides. Store prepared media in the dark

• Packaging- packaging should protect media from contamination and


moisture loss.

• Site-stable temperature, in dark


(artificial light may be used if at suitable wavelength)

• Shelf-Life -must be validated.


Effect of incorrect preparation media for
fastidious bacteria testing

Fastidious bacteria is any organisms that has a complex


nutritional requirement or will only grow when specific nutrient
are included/present in culture medium

Neisseria gonorrhoeae  requires blood or hemoglobin


and several amino acid and vitamins
Campylobacter spp  requires elevated CO2
Helicobacter spp  requires elevated CO2
Preparation of Media -
Tracebility

TRACEABILITY
❑Operator’s name
❑Batch number
❑Program used
❑Date/time
❑Cycle curve
Preparation of Media – ISO
17025
Quality Control of Homemade Media
Culture Media Quality Control
 Validate the quality of all media produced for each
batch

 Validate the shelf life of each media

Quality Control protocol :


For Conventional non selective media (e.g. TSA, Blood agar …), we have to
control the fertility for the main microorganisms detected on the media. Two or
three strains must be tested (try to test different gram and respiratory strains)
For a selective media (e.g. Sabouraud, Mc Conkey ..) we have to control the
fertility of the microorganisms targeted and also the selectivity of strains which
have not grow on the media.

We need to still control one parameter, the sterility. For this point, we must
incubate plates, from the begin and the end of the batch, at the optimum time
& temperature. In case of using a Media Preparator, the ticket with the
traceability of the cycle avoids to realize this control.
Shelf Life Validation
Shelf Life validation:
To preserve the better quality of a media till the end of the analysis, we
have to validate the shelf life.

Storage conditions :
Media includes thermo-sensitive ingredients ?

YES => Fridge storage (2 to 8 deg.C)


NO => Extended storage (2 to 25 deg.C)

Media includes light-sensitive ingredients ?


Shelf Life Validation
Stability Study :
To preserve at the end of the shelf life :
- Physicochemical attribute (pH, colour, texture…)
- Microbiological state
- Cultural attribute (fertility, selectivity, neutralization...)

On 1 batch storage at the predicted conditions – control at regular interval


in accordance with the QC protocol and compare with
Finally, for a validated stability of 10 weeks for example, we will take on 8
weeks of shelf life.
And for 7 months, 6 months will be took on.
Media Preparation
Pembuatan Media (Culture Media) - Konvensional

1 2 3

1. Menimbang dry media 2. Stirring: dry media+air pada 100 °C 3. Autoclaving

4 5

4. Keeping in water bath 5. Manual dispensing


MANUAL MEDIA PREPARATION

1. Weighing dry media 2. Stirring: dry media


Drawbacks: + water at 100°C

Risk of burn with agar at 100°C

Glass handling:
 Risk of cut

 Time consuming ~ 4min/ bottles (1 bottle = 1 sample)

No traceability
CLASSIC MEDIA PREPARATION

3. Autoclaving bottles
Drawbacks:

No accurate temperature control (+/- 3°C)

Temperature different for top/base

No homogeneity of the temperature (no stirring in the bottle)


CLASSIC MEDIA PREPARATION

3. Autoclaving bottles
Drawbacks:

No accurate temperature control (+/- 3°C)

Temperature different for top/base

No homogeneity of the temperature (no stirring in the bottle)


Automatic Preparation Culture Media

Manufacturing ------ Masterclave


Benefit :
- Produktif
- Standarisasi
- Fleksibel
- Easy to Use
- Traceability
- Quality
- Safety

Automated system to
prepare high quality broth
and Agar Media
Masterclave

Core temperature by a temperature probe


controller inside the media

Rapid cooling by a
water cooling
device

Homogeneous
temperature by
large paddle stirrer

Masterclave 60

 Homogeneity and temperature accuracy


Masterclave

Produktif : 1 2

Time & Money Saving


 Less steps
 Less accessories
 Less handling
 Less washing
1. Menimbang dry media 2. Stirring: dry media+air pada 100 °C

3 4 5

3. Autoclaving 4. Keeping in water bath 5. Manual dispensing


Masterclave

Standarisasi :
- Akurasi Terjamin (heating, sterilization, cooling, dispensing)
- Automatis
- Terkontrol (software)
Masterclave

Fleksibel :
- Setting metode preparasi (Single step, 2 step, Special Step)
- 3 Type Masterclave (Type 09, Type 528, Type 60)
Masterclave
Masterclave

Step 2

Step 3
Step 1 Dispensing

Temperature

Time
Addition
Addition Addition
Masterclave

Easy to Use :
- Timbang Media, Tambahkan Air, Star Cycle
(Sterilisasi)
Masterclave

Traceability :
 Data Oprational Ter Record
(Operator, No.Batch, program, time, cycle curve)
Masterclave

Quality :
 Kualitas Media Terjamin (Panas, homogen, hygienis
terkontrol)
Safety :

 Safety devices:

 Several alarms:
Masterclave

Capacity: 1to9L

Preparation time:
 45min for 1 liter (sterilization 121°C, 15min)

Dimensions
 w x h x d= 500 x 435 x 590mm

Installation
 Electrical connection: 230V, 16A

 Water supply:
- Maximum hardness: 7°TH
- Temperatures: 6 to 20°C
- Pressure: 1 to 3 bar
- Minimum flow: 6liters per minute
Masterclave

Capacity: 5 to 28L
MAIN APPLICATIONS
– Combine system for contract labs:
Preparation time: • 28 liters of diluent in the morning
 90 min for 28liters of media (121°C/15min) • 5-10 liters of agar in the afternoon

– Medium autoprep for manufacturer


Dimensions
ECO MODE
 w x h x d= 760 x 1100 x 580mm
• Fast or Eco mode for
saving cooling water or
Installation time
• Eco mode for stop
 Electrical connection: printing once in
- 400V triphase with neutral, 16A per phase dispensing phase.
OR
- 230V triphase without neutral 20A per phase
 Water supply:
- Maximum hardness: 7°TH
- Temperature: 6 to 20°C
Pressure: 1 to 3 bar
• Minimum flow: 6liters per minute
Masterclave

Capacity: 10 TO 60L
MAIN APPLICATION
Preparation time: - Agar for agar manufacturer
 120 min for 60 liters of media (121°C/15min) - Diluent for labs

Dimensions
 w x h x d= 1,03 x 0,65x 1,10m

Installation
 Electrical connection:
- 400V triphase with neutral, 20A per phase
OR
- 230V triphase without neutral 32A per phase
 Water supply
- Maximum hardness: 7°TH
- Temperatures: 6 to 20°C
Pressure: 1 to 3 bar
• Minimum flow: 6liters per minute
Dispensing

Dispensing ------ APS One, PMi, XY500

PMi

APS One XY500


APS One

 PRODUCTIVITY

 Petri dish shuttle: petri dish filling revolution


 Dish opened time: 4 seconds
 High filling rate:
- 650 plates/hour (18 ml) as standard
- 850 plates/hour (18ml) w/turbo option
 Large Capacity :
- 10liters of agar (560plates) in 50 min
APS One

COMPACITY

 Compact device: Small foot print


 Carrousel hight adapted to technicians size: easy loading/unloading

Optional AES Standard


540 plates carrousel 560 plates carrousel
H=1405mm H=845mm
APS One

Fleksible
- Kompatible 80% Petri (55mm & 90mm Plates)
- Biplates
- Line & Tubing Sterile

Petri Dish

Sterile line tubing


Biplate kit
APS One

Simple

 User friendly interface:


• Big colour screen
• Intuitive display (icon)

 Easy to load: reduced plate stacking hight

 easy cleaning
APS One

Safety
 Sterility warranty:
• Dish shuttle
• UV lamp
• Motorised nozzle

 Easy to clean:
• Nozzle holder removable
• Shuttle removable
• No corner
Dispensing

PMi XY500
Semi-automatic Complete walk away system

 Automation and volume accuracy


PMi

Benefit
 Sterility warranty
 Easy to Use (Large Display & Touch Screen)
 Wide Range (2 – 20 ml)
 Speed Dispensing (up to 600rpm)
 Dispensing Accuracy (1% ≤ 225ml & 2% >20L)
 ISO (7218, 6887-1, 16189)
PMi
 4 different Tubing (1.6mm, 3.2mm, 4.8mm, 6.4mm)
XY 500

Benefit
 Sterility warranty
 Easy to Use (Tube Loading with optional stand)
 Easy to Program
 Walk away Program
 All Kind of containers (Vials, Tube & Bottle)
 High Productivity
- 1500 tubes/hour (9ml)
- 250 bottle/hours (225ml)
- Large capacity: up to 500 tubes in one cycle

XY500
Media Preparation Full Automatic

Preparation Mixing &


Dispensing Ready Media
Water + Powder Sterilization
MEDIA PREPARATION

Masterclave 528
(5-28 litres)

Masterclave 60
(10-60 litres)

Masterclave 09
(1-9 litres)
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