Microbiological Control Procedure

Microbiological control of product

Document code: CPM111.1 Version: 01 Page: 1/3

Aseptic R&D
Author: B. Botti Validation: A. Silvestri Date: 20/05/2008

1. Principle & scope

Control of product own contamination level inside finished bottles

2. Equipment, instruments and reagents used

• Laminar flow box
• Sterile Petri dishes (or equivalent sterile plates), 90 mm diameter
• Sterile disposable plastic pipette, individually packed
• Sterile syringe with sterile needle
• Culture media (acidified at product pH): Plate Count Agar (PCA) for Aerobic Colony Count1 and
Malt Extract Agar (MEA) for yeasts and moulds
• Incubator at 30°C for Aerobic Colony Count
• Incubator at 25°C for yeasts and moulds research
• Disposable gloves
• Isopropyl/ethylic alcohol
• Bunsen
• Fridge at 4°C
• Optical microscope

3. Test protocol

3.1 Preliminary operations

Internal well equipped microbiological laboratory to perform the analysis is needed.
Perform the analysis after enrichment of product obtained by incubation of bottles for 7 days at 30°C (in
case of products containing preservatives do not incubate bottles, but perform analysis right after
sampling). Depending on the product sampling approach, adopt one of the following step before
incubation:
1. Finished bottles: put samples directly inside incubator in vertical position. At the 6th day (one
day before analysis) take from the incubator the bottles, put them under laminar flow. Spray the
sampling area on the bottles with alcohol, put one drop of silicone rubber on the disinfected
area and let it dry for 24 hours carrying on the incubation at 30°C till the 7th day
2. Glass flask sampler: under the laminar flow, replace specific cap (used to do sampling from
the line) with a standard cap previously sterilized in autoclave for 15 minutes at 121°C wearing
sterile gloves. Put samples inside incubator in vertical position
3. One-way sterile plastic bag: put samples directly inside incubator

3.2 Execution method

3.2.1 Filterable/Non filterable products
• Finished bottles
a. Wear gloves and disinfect them with alcohol
b. Put samples inside laminar flow box (or in a sterile area from a gas flame)
c. Spray the external surface of the bottle with alcohol
d. Remove syringe and needle from their packages inside sterile area and connect them
e. Hole the silicone deposit on the bottle and take 1 ml of sample with syringe
f. Put this volume inside Petri dish
g. Put liquid medium into Petri dish with 3-4 mm layer (temperature of medium around 50°C)
h. Rotate Petri dish in order to include product inside medium
i. Wait the medium becomes solid under laminar flow
j. Close Petri dish with cover

1
Ref.: ISO 4833:2003 (E) – 5.2
Sidel S.p.A.
Via La Spezia, 241/A
43100 Parma, ITALY
Tel.: +39 0521 9991, fax: +39 0521 959009
 Microbiological Control Procedure

Microbiological control of product

Document code: CPM111.1 Version: 01 Page: 2/3

Aseptic R&D
Author: B. Botti Validation: A. Silvestri Date: 20/05/2008

k. Invert Petri dishes and place them in the incubator at a suitable temperature for the
appropriate number of days (72 hours)
l. Repeat 10 samplings for each bottle
m. Store samples at 4°C for one week

• Glass flask sampler

a. Wear gloves and disinfect them with alcohol
b. Put samples inside laminar flow box (or in a sterile area from a gas flame)
c. Working in sterile conditions, open the bottle
d. Take 1 ml of sample using sterile disposable plastic pipette
e. Put this volume inside Petri dish
f. Put liquid medium into Petri dish with 3-4 mm layer (temperature of medium around 50°C)
g. Rotate Petri dish in order to include product inside medium
h. Wait the medium becomes solid under laminar flow
i. Close Petri dish with cover
j. Invert Petri dishes and place them in the incubator at a suitable temperature for the
appropriate number of days (72 hours)
k. Repeat 10 samplings for each sample
l. Flame bottle mouth, plug it up and store sample at 4°C for one week
• One-way sterile plastic bag
a. Wear gloves and disinfect them with alcohol
b. Put samples inside laminar flow box (or in a sterile area from a gas flame)
c. Spray the external surface of the sampling system with alcohol
d. Take 1 ml of sample using sterile syringe (make the hole in the silicone tube)
e. Put this volume inside Petri dish
f. Put liquid medium into Petri dish with 3-4 mm layer (temperature of medium around 50°C)
g. Rotate Petri dish in order to include product inside medium
h. Wait the medium becomes solid under laminar flow
i. Close Petri dish with cover
j. Invert Petri dishes and place them in the incubator at a suitable temperature for the
appropriate number of days
k. Repeat 10 samplings for each sample
l. Throw away sample

3.3 Special precaution

Sampling can be done at the end of the line, taking a number of bottles sufficient to perform the analysis
(as defined in the sampling plan), or on product pipe, using glass bottle sampler or one-way sterile
plastic bag, during production.

4. Result evaluation
Colonies count and visual inspection using optical microscope

5. Acceptance criteria
According to product nature: no cloudiness, no flakes, no filaments, no swelling, no off-flavours.

Sidel S.p.A.
Via La Spezia, 241/A
43100 Parma, ITALY
Tel.: +39 0521 9991, fax: +39 0521 959009
 Microbiological Control Procedure

Microbiological control of product

Document code: CPM111.1 Version: 01 Page: 3/3

Aseptic R&D
Author: B. Botti Validation: A. Silvestri Date: 20/05/2008

6. Link with other methods:

Level 1
Code Title
CPM120.1 Culture media
DI100.1 Novaseptum bag
DI101.1 Glass flask sampler

7. Document updates and versions:

Version Explanation of the update(s) Writer Date
01 Logos change B. Botti 20/05/2008

Sidel S.p.A.
Via La Spezia, 241/A
43100 Parma, ITALY
Tel.: +39 0521 9991, fax: +39 0521 959009