1.0
Purpose
1.1 To establish a formal procedure for the
drug products to ascertain shelf-life & storage condition for all the
future batches to come which will be manufactured & packaged under similar
condition.
2.0
Scope
2.1 This procedure
is applicable for all the dehydrated culture media to be used for microbiological
testing.
3.0
Responsibilities
3.1
Microbiology
personnel are responsible for preparing, updating and issuing of this SOP.
3.2
Microbiologist
is responsible to follow the SOP.
3.3
Head
of Quality Control/In-Charge is responsible for compliance of the SOP.
3.4
Head
of QA is responsible for SOP compliance.
4.0
Abbreviations and Definitions
4.1
LAF
- Laminar Air Flow
4.2
LAFU
- Laminar Air Flow Unit
4.3
DHS
- Dry Heat Sterilizer / Sterilization
5.0
Materials
and Equipment
5.1
Equipment’s required
5.1.1
LAFU
5.1.2
Incubators
5.1.3
DHS
5.1.4
Autoclave
5.2 Material
Required
5.2.1 Dehydrated
culture media
5.2.2
Sterile Saline solution
5.2.3
Sterile Petri Dish
5.2.4
Measuring Cylinder
5.2.5 Test
organism – E. coli, Staph. aureus, Ps.
aeruginosa, C. albicans, A. Niger, Salmonella. B. subtilis, Cl. Sporogenes, S. epidermidis.
6.0
Precaution
/ Health and Safety Considerations
6.1 The dehydrated culture media as well as their ingredients are highly hygroscopic and
must be stored in a cool dry place away from bright light. These media are meant for
laboratory use only and shall never be used for human or animal consumption.
6.2
Use fresh sterile pipette for each
transfer.
6.3
The medium to be poured in Petri plates
should have a temperature of 40 - 45° C.
6.4 The plates should be incubated in an inverted position to prevent collection of
condensation on the plate surface.
6.5
If any spillage of cultures, immediately
wash with 70% IPA solution.
6.6 Entire operation inside the microbiology room should be carried out under the laminar air
flow unit using gas burner.
6.7
Examine the physical nature of the
dehydrated medium. If any unusual colour, odour or
physical appearance is noticed, discard the medium.
6.8
Always use a dry spoon or spatula for
weighing the dehydrated media. The weighing
operation shall be completed as quickly as possible to prevent
absorption of moisture by the hygroscopic contents. Wear a face mask while
weighing the dehydrated media to avoid inhalation of fine particles of media.
6.9
All dehydrated media must be retest after
the release of three months interval and finally
media must be discarded after release of one year.
7.0 Procedure
7.1 After
receipt of dehydrated culture media, make a necessary entry in receipt register
7.2 Collect
minimum 5.0 g sample from each received pack (i.e., Batch wise) and mix
properly.
7.3 Prepare
culture media & sterilize as per SOP for preparation of culture media SOP.
7.4 After
sterilization, transfer the media to microbiology analysis room and allow it to
cool at 40 to 45° C.
7.5 Start
the LAFU as per SOP and further proceed works under LAFU.
7.6 Test
For Growth Promoting Properties, Solid Media
7.6.1
Label the plates with
culture name, Media B. No, Date of incubation on the base of petri plates.
7.6.2
Add 1.0 ml suspension
of specific culture containing 10 to 100 cfu/ml (as specified in Table– I)
into two sterile petri plates.
7.6.3
Aseptically pour the
cooled media at 40 to 45° C into both the labelled plates, mix the
plates by gently rotating clock wise and anti-clock wise direction.
7.6.4
Allow the plates to
solidify at room temperature under Laminar Air Flow.
7.6.5
Simultaneously run
negative control to verify testing conditions, using the same procedure in
place of the test organism use diluents i.e., 1.0 ml of saline solution.
7.6.6
Incubate the plates at
specified temperature and period as listed in Table – 1.
7.6.7 Observe the plates for number of colonies, and count the cfu observed on both plates and express the result in cfu by following formula.
P1
+ P2 / 2
Where P1 and P2 are plate 1 and plate 2
7.6.7.1 Calculate the Microbial recovery in percentage by following equation -
Mean cfu observed
% Recovery =------------------------------------- X 100
Inoculated cfu ml
7.7 Test
For Growth Inhibitory Properties, Solid Media
7.7.1
Label the plates with
culture name, Media B. No, Date of incubation on the base of petri plates.
7.7.2
Add 1.0 ml suspension of
specific culture containing 100 cfu/ml (as specified in Table – I of Growth
inhibitory property) into two sterile petri plates.
7.7.3
Aseptically pour the
cooled media at 40 to 45° C into both the labelled plates, mix the plates by
gently rotating clock wise and anti-clock wise direction.
7.7.4
Allow the plates to
solidify at room temperature under Laminar Air Flow.
7.7.5
Incubate at specified
temperature and period as listed in Table – 1.
7.7.6
Observe the plates for
number of colonies. No growth of the test organism occurs.
7.8 Test
For Growth Promoting and Inhibitory Properties, liquid Media
7.8.1
Prepare required
quantity of liquid culture media, dispense 100 ml in test tubes and sterilize
as per manufacturers instruction.
7.8.2
After sterilization,
transfer the media to microbiology analysis room and allow it to cool at room
temperature.
7.8.3
Start the LAF as per
SOP and proceed further work under LAF
7.8.4
Add 1.0 ml of positive
culture of Growth promoting properties, containing 100 cells (as per table – 1)
into broth tube and label with Media B. No, name of positive culture & date
of inoculation.
7.8.5
For Growth Inhibitory
Test, add 1.0 ml of positive culture of Growth inhibitory properties,
containing 100 cells (as per table – 1) into broth tube and label with Media B.
No, name of positive culture & date of
inoculation.
7.8.6
Simultaneously run
negative control to verify testing conditions, using the same procedure in
place of the test organism use diluents i.e. 1.0 ml of saline solution.
7.8.7
Incubate all the tubes
at specific temperature as specified in table –1.
7.8.8
Daily observe the tubes
for growth for turbidity.
7.8.9
Satisfactory growth
should be observed within 3 days of incubation in the test. There should be no
growth in growth inhibitory test & negative control.
7.8.10
In case the media
passes the growth promotion test, a Approved label shall be affixed on the
media container, then the same should be used for analysis.
7.8.11
In case the media fails
for the growth promotion test then a rejected label shall be affixed on the
container then the same shall be rejected and accordingly the rejection entry
should be made in the stock register.
7.8.12
The rejected media
should be discarded or returned back to the supplier.
Table-1
|
Name of Media |
Positive culture to be used for Growth Promotion Test |
Positive culture to be used for Growth Inhibitory Test |
Expected growth characteristics of the test organism for
the respective media |
Incubation
temperature |
Incubation
period |
|
|
Fluid Thioglycollate
Medium |
B.subtilis Ps.aeruginosa S.aureus
|
NA |
-Growth (Turbidity) Observed in the
respective media tubes |
30 to 35°C |
72 hrs |
|
|
Soybean Casein digest
Medium |
B. subtilis
C.albicans A. Niger |
NA |
-Growth (Turbidity) Observed in
the respective media tubes |
20 to 25°C |
72 hrs |
|
|
Soybean Casein digest
Agar |
E.coli Ps.aeruginosa S.aureus |
NA |
-Opaque white colonies
on SCDA Plates -Large grayish colonies on SCDA Plates -Tiny white colonies on
SCDA Plates |
30 to 35°C |
48 hrs |
|
|
MacConkey Agar |
E.coli
|
S.aureus |
- Brick red
colonies on MacConkey Agar Plates |
30 to 35°C |
48 hrs |
|
|
MacConkey broth |
E.coli |
S.aureus |
- Yellow
colour Change |
30 to 35°C |
48 hrs |
|
|
Mannitol salt Agar |
S.aureus
|
E.coli
|
- Yellow colonies on
Mannitol SaltAgar Plates |
30 to 35°C |
48 hrs |
|
Table-1 (continue…)
|
Name of
Media |
Positive
culture to be used for Growth Promotion Test |
Positive
culture to be used for Growth Inhibitory Test |
Expected
growth characteristics of the test organism for the respective media |
Incubation temperature |
Incubation period |
|
Cetrimide agar |
Ps.aeruginosa |
E.coli
|
- Greenish Colonies on
Cetrimide Agar Plates |
30 to 35°C |
48 hrs |
|
Antibiotic assay
Medium No. 11 |
S. epidermidis |
NA |
- Good Luxuriant
colonies on therespectivemedia Plates |
30 to 35°C |
24 hrs |
|
Peptone water |
E.coli
|
NA |
Luxuriant
(Turbid)growth in the respective media tubes |
30 to 35°C |
48 hrs |
|
Sabouraud Dextrose
Agar |
C.albicans |
NA |
-white colonies
on Sabouraud Dextrose Agar Plates |
22 to 25°C |
120 hrs |
|
Selenite F broth |
Salmonella
abony |
S.aureus NCIM 2079 |
-Red ppt.
observed in the Selenite F broth |
30 to 35°C |
24 hrs |
|
EMB agar |
E.coli |
S.aureus |
- Blue – Black
colonies with metallic sheen on EMB Agar Plates |
30 to 35°C |
24 hrs |
|
Brilliant green agar |
Salmonella
abony |
S.aureus |
- Opaque pinkish to
white colonies on Brilliant green agar Plates |
30 to 35°C |
48 hrs |
|
Nutrient Broth |
E.coli
|
NA |
-Good Luxuriant
(Turbid) growth Observed. |
30 to 35°C |
24 hrs
|
Table-1
(continue…)
|
Name of Media |
Positive culture to be used for Growth Promotion Test |
Positive culture to be used for Growth Inhibitory Test |
Expected growth characteristics of the test organism for
the respective media |
Incubation temperature |
Incubation period |
|
|
Triple sugar Iron agar |
Salmonella
abony |
S.aureus
|
-Red and Yellow butt
(With or without blackening) |
30 to 35°C |
48 hrs |
|
|
Vogel Johnson Agar |
S.aureus |
E.coli
|
- Black Colonies on
Vogel Johnson Agar Plates |
30 to 35°C |
48 hrs |
|
|
Baired Parker Agar |
S.aureus |
E.coli |
- Black Colonies
surrounded by a clear zone |
30 to 35°C |
48 hrs |
|
|
Pseudomonas agar(For
Fluorescein) |
Ps.aeruginosa |
S.aureus |
- Yellowish
Colonies on Pseudomonas Agar Plates |
30 to 35°C |
72 hrs |
|
|
Pseudomonas agar(For
Pyocyanin) |
Ps.aeruginosa |
S.aureus |
- Greenish Colonies on
Pseudomonas Agar Plates |
30 to 35°C |
72 hrs |
|
|
Urea broth |
Salmonella
abony |
S.aureus |
-Luxuriant growth with
no colour change Observed in the respective media tubes |
30 to 35°C |
24 hrs |
|
|
Tetrathionate
Brilliant Green Bile Broth |
Salmonella abony |
S.aureus |
-White ppt. observed
in the TBGB broth tubes |
30 to 35°C |
24 hrs |
|
|
Bismuth sulphite agar |
Salmonella
abony |
S.aureus |
- Black or green
Colonies on Bismuth Sulphite Agar Plates |
30 to 35°C |
48 hrs |
|
|
Xylose lysine
deoxycholate agar |
Salmonella
abony |
S.aureus |
- Red Colonies on
Xylose lysine deoxycholate Agar |
30 to 35°C |
48 hrs |
|
8.0 Reference Document
8.1 WHO
Guidelines, volume 2, second edition.
9.0 Annexure
9.1 No
Appreciable
10.0 Revision History
|
Revision
No. |
Brief reason for the revision |
Effective Date |
Remarks |
|
01 |
New |
|
|
11.0 Training
11.1 Head of Quality Control or his/her nominee shall give the SOP training before effective date.
