1.0
Purpose:
1.1 To
establish a standard test procedure for the quantitative enumeration and
identification of the microbial contamination (Bacteria and Fungi) present in
raw material, articles and finished products of Liquid dosage forms. The tests
are primarily designed to determine whether a substance or preparation complies
with an established specification for microbiological quality.
2.0
Scope:
2.1 This procedure is applicable for the Microbiology
Laboratory.
3.0
Responsibilities:
3.1
Executive Microbiology is responsible for performing
Microbial Limit Test.
3.2
Head of Quality Control/In-Charge is responsible for
ensuring that Microbial Limit Test is performed properly.
3.3
Head of Quality Assurance is responsible for SOP
compliance.
4.0
Abbreviations
and Definitions
4.1
LAFU: Laminar Air Flow Unit.
4.2
CFU: Colony Forming Unit.
4.3
TAMC:
Total Aerobic Microbial Count.
4.4
TYMC:
Total Combined Yeast and Mold Count.
5.0
Materials
and Equipment
5.1 Apparatus: Standard laboratory equipment,
Glassware (flasks, test tubes, pipettes, petri dishes), Balance, Spreader,
Inoculation Loop, Burner etc.
5.2
Purified Water
5.3 Media: (Casein digest broth(Tryptone
Soya Broth-TSB), Soyabean Casein Digest agar(Tryptone Soya Agar-TSA), Rappaport
Vassiliadis (RV), MacConkey Broth(MB),etc.)
6.0 Precaution / Health and Safety Considerations
6.1
Perform
the test under LAFU.
6.2
Use
lab coat and sterile gloves during performing the test.
6.3
Use sterile glassware and media.
6.4
Do not pour agar media into the plate above 45° C.
7.0
Procedure:
7.1 Apparatus: Standard laboratory equipment,
Glassware (flasks, test tubes, pipettes, petri dishes), Balance, Spreader,
Inoculation Loop, Burner etc.
7.2
Reagents / Media: Purified
Water, Buffered Sodium Chloride-Peptone Solution pH 7.2, Soyabean Casein digest broth(Tryptone
Soya Broth-TSB), Soyabean Casein Digest agar(Tryptone Soya Agar-TSA), Rappaport
Vassiliadis (RV), MacConkey Broth(MB),
Sabouraud-Dextrose Agar(SDA), MacConkey Agar(MA), Mannitol-Salt Agar(MSA),
Cetrimide Agar(CA), Xylose-Lysine Deoxycholate Agar(XLD), Eosin Methylene Blue
Agar(EMB), Triple Sugar Iron Agar(TSI),Motility Indole Lysine Medium(MIL), Crystal Violet, Iodine, Alcohol and Safranin
solution, N,N-dimethyl-p-phenylenediamine
dihydrochloride, Polysorbate 20, Lecithin etc.
7.3
Sample Preparation: The Methods for sample preparation
depends upon the physical characteristics of the material/product to be tested.
Sample preparation can be carried out as follows:
7.3.1
Water-soluble product: Dissolve or dilute (usually 1 in 10
dilution prepared) the product to be examined in buffered sodium chloride
peptone solution pH 7.0 or phosphate buffer solution pH 7.2 or Soyabean casein
digest broth. If necessary, adjust to pH 6-8. Further dilutions where necessary
are prepared with the same diluents.
7.3.2 Non-fatty
products insoluble in water: Dissolve
or dilute (usually 1 in 10 dilution is prepared) the product to be examined in
buffered sodium chloride peptone solution pH 7.0, or phosphate buffer solution
pH 7.2 or soyabean casein digest broth. A surface-active agent such as 1 g/l of
Polysorbate 80 may be added to assist the suspension of poorly wettable
substances. If necessary, adjust to pH 6-8. Further dilutions where necessary
are prepared with the same diluents.
7.3.3 Fatty
Products: Dissolve in
Isopropyl Myristate sterilized by filtration, or mixed the product to be
examined with the minimum necessary quantity of sterile Polysorbate 80 or
another noninhibitory sterile surface-active reagent heated, if necessary, to
not more than 40°C or in exceptional cases not more than 45°C. Mix carefully
and if necessary, maintain the temperature in a water bath. Add a sufficient
amount of prewarmed chosen diluents to make a 1 in 10 dilution of the original
product. Mix carefully while maintaining the temperature for the shortest time
necessary for the formation of an emulsion. Further serial 10 fold dilution may
be prepared using the chosen diluents containing a suitable concentration of
sterile Polysorbate 80 or another noninhibitory sterile surface-active reagent.
7.3.4
Neutralization/removal of antimicrobial
activity of the material/product to be tested: If growth is inhibited (reduction by a
factor greater than 2), then modify the procedure for the particular
enumeration test to ensure the validity of the results. Modification of the
procedure may include, for example,
a)
An increase in the volume of the
diluents or culture medium.
b)
Incorporation of a specific or general
neutralizing agent in to the diluents.
c)
Membrane filtration.
d)
A combination of the above measure or
e)
Readymade dehydrated media containing
neutralizing agents.
7.3.5 Neutralizing agents can be added to the
chosen diluents or the medium preferably before sterilization. Efficacy and absence
of toxicity of the neutralizer for microorganisms must be demonstrated by
carrying out a blank with neutralizer and without product. Common neutralizing
agents for interfering substances are shown in Table 1.
Table 1: Common Neutralizing agents
|
Interfering
Substances |
Potential
Neutralizing agents |
|
Glutaraldehyde, Mercurials |
Sodium hydrogensulphite (Sodium
bisulphate) |
|
Phenolics, alcohol, Aldehydes, Sorbate |
Dilution |
|
Aldehydes |
Glycine |
|
Quarternary Ammonium Compounds (QACs),
Para hydroxybenzoates (Parabens), Bis-biguanides |
Lecithin |
|
QACs, Iodine, Parabens |
Polysorbate |
|
Mercurials |
Thioglycolate |
|
Mercurials, halogens, Aldehydes |
Thiosulphate |
|
EDTA |
Mg2+ or Ca2+
ions |
7.3.6 Amounts
of sample to be used for the test:
Unless otherwise prescribed use 10 g or 10 ml of the material or product to be
examined. Select the samples at random from the bulk material or from the
available finished product containers of the preparation. If the presence of
active substance in the formulation is less or equal to 1 mg per dosage unit or
less or equal to 1 mg per g/ml, in these cases amount of sample to be tested
not less than amount present in 10 dosage units or 10 g or 10 ml of the
product. The materials which sample quantity or batch size is extremely small
(i.e., less than 1000 ml or 1000 g), the amount to be tested is 1%. For the
product where the batch size is less than 200 and 100, the sampling plane also
reduced to 2 units and 1 unit respectively.
7.4
Pour-plate method:
7.4.1 Prepare the sample to be tested using a
method that has been shown to be suitable as described in section 7.3.
7.4.2
With
a sterile pipette transfer each 1 ml of the sample into two 90 mm sterile
petridishes for TAMC.
7.4.3
Add
15-20 ml of sterile Soyabean casein digest agar media being at not more than 45
°C.
7.4.4
Again,
with a sterile pipette transfer each 1 ml of the sample into two 90 mm sterile
petridishes for TYMC.
7.4.5
Add
15-20 ml of sterile Sabouraud dextrose agar media being at not more than 45 °C.
7.4.6
Mix
the sample with the agar by rotating clock wise and anti-clock wise and allow
solidifying.
7.4.7
Invert
all the plates and incubate the plates of Soyabean casein digest agar at 30-35
°C for 3-5 days and the plates of Sabouraud dextrose agar at 20-25 °C for 5-7
days.
7.4.8
Take
1 ml diluents, used for dilution purpose into each of the two sterile
petridishes for negative control. Aseptically add 15-20 ml of sterile Sabouraud
dextrose agar media being at not more than 45 °C into one plate and Add 15-20
ml of sterile soya bean casein digest agar media being at not more than 45 °C
into the other plate. Finally follow the steps 7.4.6 and 7.4.7.
7.5
Spread-plate method:
7.5.1
Add
15-20 ml of sterile Soyabean casein
digest agar and Sabouraud dextrose agar
to each 90 mm petri dishes at about 45 °C and allow solidify.
7.5.2
Dry
the plate surface in a Laminar Air Flow Unit.
7.5.3
Transfer
0.1 ml of the sample prepared as per section 7.3 over the surface of the media
plates with a sterile pipette.
7.5.4
Spread
the sample evenly over the surface of the media plate with the help of sterile
L-shaped spreader.
7.5.5
Always
prepare at least 02 Petri dishes for each of the TAMC and TYMC.
7.5.6
Transfer
0.1 ml of diluents, used for dilution purpose, over the surface of both Petri
dishes containing Soyabean casein digest
agar and Sabouraud dextrose agar for negative control with a sterile
pipette. Finally follow the step 7.5.4.
7.6
Evaluation of Results:
7.6.1 Examine all plates after incubation
period for growth and count only those plates contain between 30-300 colonies.
Report the average number of
microorganisms per g or ml of sample by the following Formula:
AV X
DF=Microorganisms/g or ml.
Here, AV= Average Number of colonies on
two plates of an appropriate dilution.
DF= Dilution Factor.
7.7
Test for Specified microorganisms:
7.7.1 Escherichia coli
7.7.1.1 Sample
preparation and pre-incubation: Prepare
sample using a 1 in 10 dilution of not less than 10 g/10 ml of the Material /Finished
product to 90 ml of Soyabean casein digest broth. Mix well and incubate at
30-35 °C for 18-24 hours.
7.7.1.2 Selection
and Subculture: Shake
the container, transfer 1 ml of Soyabean casein digest broth to 100 ml of
MacConkey broth and incubate at 42-44 °C for 24-48 hours. Subculture on plate
of MacConkey Agar and incubates at 30-35 °C for 18-72 hours.
7.7.1.3 Interpretation
of result: Growth of
colonies similar to the description of the table 2 indicates the possible
presence of E.coli. This is confirmed by identification test. The product complies with the test if
no colonies are present or if the identification tests are negative.
Table
2: Morphological Characteristics of Escherichia coli.
|
Medium |
Description of colony |
Interpretation |
|
MacConkey
Agar |
Pink to
red generally non-mucoid colonies sometimes surrounded by a reddish
precipitation zone. |
Positive result |
7.7.1.4
Confirmatory Test: Confirmatory test is to be carried out
by using two different media.
Motility Indole lysine
(MIL) Medium: Pick two
or more suspected colony from selective agar media with a sterile inoculating
needle and inoculate tube of MIL by stabbing into the media. Incubate the tubes
35 ± 2 °C for 24 hours.
Evaluation of Results:
a) Motility:
Motility positive is
indicated by diffused growth away from the line of inoculation. Non motile
cultures grow along the line of inoculation.
b) Lysine deaminase:
Is indicated by a red or red-brown color in the top centimeter of the medium.
c) Lysine Decarboxylation: It produces purple color throughout the
medium.
d) Indole
Production: Add 3-4
drops of Kovac`s reagents to each tube. Do not shake.
The appearance of a pink to red color in the reagent is interpreted as a
positive indole test.
Culture showing the following reaction
is indicative of the presence of
Escherichia coli.
Table 3: Confirmatory
Characteristics of Escherichia coli.
|
Name of the test |
Results for Escherichia coli. |
|
Motility |
Positive |
|
Lysine
decarboxylase |
Positive |
|
Lysine
deaminase |
Negative |
|
Indole |
Positive |
Triple Sugar Iron (TSI) Agar: Pick one or more suspected colony from selected agar medium with
a sterile inoculating needle and inoculate by stabbing into the butt and by
streaking on the surface of slant. Incubate the tube at 32 ± 2.5 °C
for 24 hours. Simultaneously carry out a positive control by inoculating known Escherichia coli by stabbing into the
butt and streaking on the slant of another TSI tube.
Evaluation of Results: After incubation if the Tube of TSI
medium both slant and butt shows acidic reaction (yellow), without production
of hydrogen sulfide (H2S) (blackening of the agar) and occasional
production of gas then the test confirms the presence of Escherichia coli. Then perform Gram Staining. Pink color indicates the Gram- negative
organisms. If the smear represents gram negative rod, Escherichia coli is present.
Table 4: Confirmatory
Characteristics in TSI Media
|
Name of the
Microorganisms |
Reaction type/Changes in
TSI media (Interpretation) |
||
|
Slant |
Butt |
H2S |
|
|
Escherichia coli |
Yellow |
Yellow and Gas |
No |
7.7.2
Salmonella
7.7.2.1 Sample
preparation and pre-incubation: Prepare
the sample to be examined as described in section 7.3.1 and use the quantity
corresponding to not less than 10 g or 10 ml to inoculate 90 ml of Soya bean
casein digest broth. Mix well and incubate at 30-35 °C for 18-24 hours.
7.7.2.2 Selection
and Subculture:
Transfer 0.1 ml of casein soya bean digest broth to 10 ml of Rappaport
Vassiliadis Salmonella enrichment
broth and incubate at 30-35 °C for 18-24 hours. Subculture on plates of Xylose
Lysine Deoxycholate Agar and incubate at 30-35 °C for 18-48 hours.
7.7.2.3 Interpretation
of result: The possible
presence of Salmonella is indicated by the growth of well-developed, red
colonies, with or without black centers. This is confirmed by identification
test. The product complies with the test if colonies of the types described are
not present or if the confirmatory identification tests are negative.
Confirmatory Test: Confirmatory test is to be carried out
by using two different media.
Triple Sugar Iron (TSI) Agar: Pick one or more suspected colony from selected agar medium with
a sterile inoculating needle and inoculate by stabbing into the butt and by
streaking on the surface of slant. Incubate the tube at 32 ± 2.5 °C for 24
hours. Simultaneously carry out a positive control by inoculating known Salmonella Spp by stabbing into the butt
and streaking on the slant of another TSI tube. Characteristics of Salmonella
given in table 5.
Evaluation of Results:
Following incubation if
the slant of TSI medium shows alkaline reaction (red) acidic butt, with
production of hydrogen sulfide (blackening of the agar) and occasional
production of gas then the test confirms the presence of Salmonella Spp. Then perform Gram Staining. In gram staining Pink
color indicates that the sample is Gram- negative organism. If the smear
represents gram negative rods, Salmonella spp is present.
|
Name of the Microorganisms |
Reaction type/Changes in TSI media
(Interpretation) |
Gram Staining reaction |
||
|
Slant |
Butt |
H2S |
||
|
Salmonella Spp. |
Red |
Yellow and Gas |
Yes |
Gram
negative |
7.7.3 Staphylococcus aureus
7.7.3.1
Sample preparation and pre-incubation: Prepare the sample to be examined as
described in section 7.3.1 and use 10 ml or the quantity corresponding to 1 g
or 1 ml to inoculate 90 ml of Soya bean casein digest broth. Mix well and
incubate at 30-35 °C for 18-24 hours.
7.7.3.2
Selection and Subculture: Subculture on a plate of Mannitol
Salt Agar and incubate at
30-35 °C for 18-72 hours.
7.7.3.3
Interpretation of result: The possible presence of S. aureus is indicated by the growth of
yellow or white colonies surrounded by a yellow zone. This is confirmed by
identification test. The product complies with the test if
colonies are not present or if the confirmatory identification test is negative.
Table
6: Morphologic characteristics of Staphylococcus
aureus on Selective Agar Medium.
|
Selective Medium |
Characteristic Colonial Morphology |
Gram Stain |
|
Mannitol -
Salt Agar Medium |
Yellow
colonies with yellow zone |
Positive
cocci (in clusters) |
7.7.4 Pseudomonas aeruginosa
7.7.4.1
Sample preparation and pre-incubation: Prepare the sample to be examined as
described in section 7.3.1 and use 10 ml or the quantity corresponding to 1 g
or 1 ml to inoculate 90 ml of Soya bean casein digest broth. Mix well and incubate
at 30-35 °C for 18-24 hours.
7.7.4.2
Selection and Subculture: Subculture on a plate of Cetrimide agar
and incubate at 30-35 °C for 18-72 hours.
7.7.4.3
Interpretation of result: Growth of colonies indicates the
presence of P. aeruginosa. This is
confirmed by identification tests. The product complies with the test if
colonies are not present or if the confirmatory identification test is
negative.
Table
7: Morphologic characteristics of Pseudomonas on Selective and Diagnostic Agar
Media
|
Selective Medium |
Characteristic Colonial Morphology |
Fluorescence in UV light |
Gram Stain |
|
Cetrimide
Agar Medium |
Generally
greenish |
Greenish |
Negative
rods |
7.7.5
Acceptance
Criteria for microbiological quality of non-sterile doses forms
Acceptance
criteria for non-sterile pharmaceutical products based upon the total aerobic
microbial count (TAMC) and the total combined yeasts/moulds count (TYMC) are
given in table 8 and table 9.
When an
acceptance criteria for microbiological quality is prescribed it is interpreted
as follows:
-
101 CFU;
maximum acceptable count = 20
-
102 CFU;
maximum acceptable count = 200
-
103 CFU;
maximum acceptable count = 2000, and so forth.
Table 8: Acceptance Criteria for microbiological quality of non-sterile dosage forms.
|
Route of
administration |
TAMC (CFU/g or CFU/ml) |
TYMC (CFU/g or CFU/ml) |
Specified
Microorganisms |
|
Non-aqueous
preparation for oral use |
103 |
102 |
Absence
of E.coli(1 g/ 1ml) |
|
Aqueous
preparation for oral use |
102 |
101 |
Absence
of E.coli(1 g/ 1ml) |
|
Rectal
Use |
103 |
102 |
- |
|
Oromucosal
use Gingival
use Cutaneous
use Nasal
use Auricular
Use |
102 |
101 |
Absence
of Staphylococcus aureus (1 g/ 1ml) Absence of Pseudomonas aeruginosa (1 g/ 1ml) |
|
Vaginal
use |
102 |
101 |
Absence
of Staphylococcus aureus (1 g/ 1ml) Absence of Pseudomonas aeruginosa (1 g/ 1ml) Absence
of Candida albicans (1 g/ 1ml) |
|
Transdermal
Patches |
102 |
101 |
Absence
of Staphylococcus aureus (1 g/ 1ml) Absence of Pseudomonas aeruginosa (1 g/ 1ml) |
|
Inhalation
use(Special requirements apply to liquid preparations for nebulization) |
102 |
101 |
Absence
of Staphylococcus aureus (1 g/ 1ml) Absence of Pseudomonas aeruginosa (1 g/ 1ml) Absence
of bile-tolerant Gram negative bacteria (1 g/ 1 ml) |
|
Special
ph.Eur. provision for oral dosage forms containing raw materials of natural
(animal, vegetal or mineral)origin for which antimicrobial pretreatment is
not feasible and for which the competent authority accepts TAMC of the raw
materials exceeding 103 CFU/g or CFU/ml |
104 |
102 |
Absence
of Salmonella (10 g/ 10ml) Absence
of E.coli(1 g/ 1ml) Absence
of Staphylococcus aureus (1 g/ 1ml) |
Table
9: Acceptance
Criteria for microbiological quality of non-sterile Substances for
Pharmaceutical Use.
|
|
TAMC (CFU/g or CFU/ml) |
TYMC (CFU/g or CFU/ml) |
|
Substances
for pharmaceutical Use |
103 |
102 |
8.0
Reference Document
8.1
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.
