MICROBIAL
LIMIT TEST
v 10gm sample weighing on
calibrated weighing balance. Exact volume of sample transfer to 90 ml sterile Tryptone
Soya Broth with 0.5% Soya lecithin & 4% Tween 20 in screw caped Glass
Bottle.
v Mix well to obtain a
uniform 1:10 (v/v or w/v) dilution.
Determination of Total Aerobic Microbial Count by (Pour Plate
Method)
1.
Pour 1 ml or required quantity from prepared dilution of sample
using sterile calibrated Micropipette to sterile duplicate Petri plates.
a.
Pour 20-25ml of sterile Soybean Casein Digest Agar/Tryptone Soya Agar
in both plates.
2. Mix the content of petri
plates by rotating clock and anticlock wise the plate, and allow medium to
solidify.
3.
Incubate the plates in an inverted position at 30 to 35o
C for 3-5 days.
4.
Determination of Total Yeast & Mold Count by (Pour Plate
Method)
5.
Aseptically transfer 1ml or required quantity from the prepared
dilution into two sterile duplicate Petri plates.
6.
Add 20-25 ml of sterile Sabouraud Dextrose Agar in both petri
dishes.
7.
Mix the contents of petri plates by rotating clock and anticlock
wise the plate and allow medium to solidify.
8.
Incubate all the plates at 20 to 25oC for 5-7 days in
an inverted position.
9.
After incubation period count the number of colonies from each
plate and find out the average.
10. Express the result as
Colony Forming Unit (cfu) per gm/ml divided by volume taken by multiplying
average number of cfu/plate with dilution factor. If no colonies are observed
express the result as number of colonies less than dilution factor.
11. If counting of colonies
is not possible due to high count or merged colonies total
count test shall be repeated after further diluting
solution/suspension.
12. If total count observed
in retest are not conforming to specified limits the material will be
considered failing in the tests.
Carry out a control test by adding 1 ml of
Microbial Culture Suspension of all objectionable organisms containing 10-100
cfu/ml and pour SCDA media & Incubate 30-35° C for 3-5 days and C. albicans containing 10-100 cfu/ml and
pour 20-25 ml SDA Media & after solidify Incubate at 20-25°C for 5-7 days.
Negative Control
Perform negative control using 1 ml diluent and
pour SCDA Media & after solidify Incubate at 30-35°C for 3-5 days & SDA
media at 20-25°C for 5-7 days.
Determination of Total Aerobic Microbial Count
by (Filtration Method)
Procedure: Microbial Limit Test for Pharmaceutical Products
1. Arrange
sterile membrane filtration assembly. Add 10 ml sample to the 100 ml sterile
purified water with the help of Micropipette and mix thoroughly. Filter the
solution through NMT 0.45μm sterile filter and Rinse the filter with 5x 100
ml sterile purified water or saline.
a.
Transfer the filter paper with sterile forceps on SCDA plate and
Incubate in inverted condition at 30-35oC for 3- 5 days. After
incubation period counts the number of colonies.
2.
Express the result as Colony Forming Unit (cfu) per gm/ml, if no
colonies are observed express the result as number of colonies less than
dilution factor.
3.
If counting of colonies is not possible due to high count or
merged colonies total count test shall be repeated after further diluting
(1:10) solution/suspension.
Positive Control
1.
Take 1 ml of Culture Suspension containing 10-100 cfu/ml of objectionable
organisms and add to 100 ml sterile purified water and mix thoroughly.
2.
Filter the solution through 0.45μm sterile filter and Rinse the
filter with 200 ml sterile purified water.
3.
Transfer the filter paper by sterile forceps on SCDA plate and
Incubate in inverted condition in incubator at 30-35oC for 3-5 days. After
incubation period counts the number of colonies. Repeat the steps for all
organisms.
Negative Control
Take 10 ml of Diluent in
100 ml Sterile purified water and filter through 0.45μm sterile
Filter and Rinse the filter with 200 ml Sterile
purified.
Transfer the filter paper by sterile forceps on
SCDA Plate and Incubate in inverted condition in incubator at 30-35oC for
3-5 days. After incubation period counts the number of colonies.
Determination of Total
Yeast & Mold Count by (Filtration
Method)
Procedure: microbial limit test
for pharmaceutical products
a.
Arrange sterile membrane filtration assembly. Add 10 ml sample to
the 100 ml sterile purified water with the help of Micropipette and mix
thoroughly. Filter the solution through NMT 0.45μm sterile filter and
Rinse the filter with 200 ml sterile purified water.
b.
Transfer the filter paper with sterile forceps on SDA Plate and
Incubate in inverted condition at 20-25oC for 5-7 days. After
incubation period counts the number of colonies.
c.
Express the result as Colony Forming Unit (cfu) per gm/ml, If no
colonies are observed express the result as number of colonies less than
dilution factor.
d.
If counting of colonies is not possible due to high count or
merged colonies total count test shall be repeated after further diluting
(1:10) solution/suspension.
Positive
Control
1.
Take 1 ml of Culture Suspension containing 10-100 cfu/ml of C. albicans and add to
100 ml sterile purified water and mix thoroughly.
2.
Filter the solution through 0.45μm sterile filter and Rinse the
filter with 200 ml Sterile purified water.
3.
Transfer the filter paper by sterile forceps on SDA Plate and
Incubate in inverted condition in incubator at 20-25oC for 5-7 days.
After incubation period counts the number of colonies.
Negative Control
i.
Take 10 ml of Diluent in 100 ml sterile purified water and filter
through 0.45μm sterile.
ii.
Filter and Rinse the filter with 200 ml Sterile purified.
iii.
Transfer the filter paper by sterile forceps on SDA Plate and
Incubate in inverted condition in incubator at 20-25oC for 5-7 days.
After incubation period counts the number of colonies.
iv. The maximum
countable number on a plate is 250 CFU for TAMC and 50 for TYMC
Procedure: Escherichia coli
v After incubation shake
the broth (under the section enrichment of sample) and transfer 1 ml of
enriched sample to 100 ml of MacConkey broth and mix thoroughly.
v Incubate at 42 to
44oC for 24 to 48 hrs.
v If growth observed in
MacConkey broth medium, streak on a plate of MacConkey agar and incubate
in inverted condition in incubator at 30 to 35oC for 18 to 72
hrs. If pink color, non-mucoid colonies observed on MacConkey agar
medium, it indicates the possible presence of Escherichia coli and carry out biochemical tests.
Biochemical Test
Indole Test
Transfer suspected
colonies from MacConkey agar to 10ml of peptone water. Incubate at 30 to 35°C
for 24 to 48 hrs. After incubation, add 3 to 4 drops of Kovac’s reagent
in peptone water tube. If red color ring produced in
tube. it indicates the presence of Escherichia coli.
Positive Control
Carry out a control test
using 0.1ml of the enrichment culture containing 10 to 50 Escherichia coli. if pink color,
non-mucoid colonies not observed test is invalid.
Perform negative control
using Autoclaved media as blank.
Salmonella sp.
Pre-treatment of sample
Clean the outer Surface
of the sample with 70% IPA and Actuate 10 ml sample under sterile condition in
sterile class A volumetric measuring Cylinder or Approx 10gm sample
weighing on calibrated weighing balance and transfer to (required
dilution) sterile Tryptone Soya Broth with 0.5% Soya lecithin & 4% Tween 20
in screw caped Glass Bottle.
If required add exact 10
ml or 10 gm sample to 10 ml tween 80 for neutralization, Add this solution 180
ml sterile Tryptone Soya Broth with 0.5% Soya lecithin & 4% Tween 20 in
screw caped Glass Bottle.
Note: If
required, make further dilution with concern to specification limit.
Procedure: Salmonella sp.
After incubation shake
the broth and transfer 0.1 ml of enriched sample to 10 ml of Rappaport
Vassiliadis Salmonella enrichment broth and mix thoroughly. Incubate at 30-35oC
for 18 to 24hours. If growth observed in Rappaport Vassiliadis Salmonella enrichment
broth, streak on a plate of Xylose lysine Deoxycholate Agar and incubate in inverted
condition in incubator at 30o to 35o for 24 to 48 hrs. If
red colonies with or without tests.
H2S Production
Inoculate suspect
colonies of Salmonella sp. in triple
sugar agar from Xylose lysine Deoxycholate agar, incubate the tubes at 30
to 35°C for 18-24 hrs. If black coloration along the line of stab inoculation
observed (due to H2S production), it indicates the presence of
Salmonella sp.
Interpretation of the results
If there is no growth of
colonies with black center and in 48 hrs. The colonies become uniformly black
surrounded by a dark zone and metallic sheen and identification tests are
negative it indicates absence of Salmonella
sp. and the sample passes the test.
Positive Control
Carryout a control test
using 0.1ml of the enrichment culture containing 10 to 5 Salmonella spp.,
prepared from 24 hrs. old culture. The test is invalid if the result does
not indicate that the control contains Salmonella spp.
Negative Control
Incubate Sterilized
media as blank.
Procedure: Pseudomonas
aeruginosa
Streak loop full
pretreated sample on the surface of Cetrimide Agar and
incubate in inverted condition in incubator at 30-35oC for 18-72 hrs. If
greenish colored colonies are observed, then carry out the identification
test (oxidase) and pigment test.
Pigment Test
Streak respective
suspect colonies from the surface of Cetrimide Agar on the surface of Pseudomonas
Agar Medium for detection of fluorescein and Pseudomonas Agar medium for
detection of Pyrocyanin contained in petri dishes. Cover the plate and incubate
in an inverted position at 30-35oC for 48hrs. Examine the colonies (Table 1).
Oxidase Test
Oxidase disc touch in
colonies rapidly colour change in blueish within 20 sec. If there is no
changing to blue, the sample meets the requirements of the test for the absence
of Pseudomonas
aeruginosa.
|
Medium |
Colony Characteristics |
Fluorescence in UV light |
Oxidase Test |
Gram Strain |
|
Cetrimide Agar Base |
Generally greenish |
Greenish |
Positive |
Negative Rods |
|
Pseudomonas Agar Medium for detection of Fluorescein |
Generally colorless to yellow |
Yellowish |
Positive |
Negative Rods |
|
Pseudomonas Agar Medium for detection of Pyrocyanin |
Generally greenish |
Blue |
Positive |
Negative Rods |
Interpretation of the
results
If there is no growth of
such type of colonies (table 1) and the identification tests are negative,
it indicates the absence of Pseudomonas
aeruginosa and the sample passes the test.
Positive Control
Carryout a control test
by using 0.1ml of the enrichment culture containing 10 to 50 Pseudomonas aeruginosa. The test is
invalid if the result does not indicate that the control contains Pseudomonas aeruginosa.
Negative Control
Incubate pre incubated
Cetrimide Agar media as blank.
Staphylococcus aureus
Procedure Staphylococcus aureus.
Streak loop full
pretreated sample (under the section enrichment of sample 4.9.2) on
the surface of Mannitol-Salt Agar Medium. Incubate the plates
in an inverted position at 30-35°C for 18 to 72 hrs. If upon observation
yellow or white colonies with yellow zones are observed carry out the
coagulase test.
Coagulase Test
Transfer representative
suspect colonies from the agar surface of Mannitol Salt agar to tube
containing 0.5 ml of mammalian, preferably rabbit or horse, plasma with or
without additives. Incubate at 30-35°C and examining the tubes at
three hours and subsequently at suitable intervals up to 24 hours. If
coagulation in any degree not observed, the sample meets the requirements
of the test for the absence of Staphylococcus
aureus.
Interpretation of the
results
If there is no growth of
yellow or white colonies with yellow zones and the identification tests
are negative, it indicates the absence of Staphylococcus
aureus and the sample passes the
test.
Positive Control
Carry out a control test
using 0.1ml of the enrichment culture containing 10 to 50 Staphylococcus aureus. The test is invalid if the result does
not indicate that the control contains Staphylococcus
aureus.
Negative Control
Incubate pre incubated
Mannitol Salt Agar media as blank.
Transfer 10 ml of
pretreatment sample from (4.9.1) in 90 ml Tryptone Soya Broth or Soyabean
Casein Digest Broth with 0.5% Soya lecithin & 4% Tween 20 in screw
cap Glass bottle and mix thoroughly and Incubate at 30-35oC for 48
hrs.
Procedure: Fungi identification:
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