Biological methods in quality control

p a r ed b y : Fitsum .D
Pre 1
 Biological methods

• Out line:
– Introduction
– Micro-biological assay
– Pyrogen and pyrogen test(in vivo & in vitro)
– Microbial limit test
– Evaluation of preservatives
– Principles of sterility testing

2
 Biological assay

• Biological assay
• It is a practical procedure where by the potency of a
material of unknown potency is estimated by comparison
of its effect in a biological system with that of the
reference standard of known or defined potency.

• Microbiological assay
• The biological system is a culture of MOs

3
 Introduction
• Microbiological tests for pharmaceuticals fall into several

categories.
 Microbiological Assay of Antibiotic

 Microbial Limits Test

 Microbiological Identification Tests

 Sterility Test

 Bacterial Endotoxins Test (BET) for Pyrogen,

 Test for Effectiveness of Antimicrobial Preservatives,

Most of these tests involve growth of microorganisms

4
 Introduction …

• According to their uses the MBA test methods can be grouped into :
– Qualitative tests : provide a Yes/No answer to the question of microbial
contamination.
• Sterility test is probably the most common qualitative test.

– Quantitative test : methods provide a numerical value for the microbial

contents of a sample

• conventional microbial limits tests

– Identification tests : provide a name (or at least a description) of a
microorganism.
• Specific designated test
5
 1.Microbiological Assay for Pharmaceuticals
 In this section, emphasis will be given to

1. Antimicrobial compounds (antibiotics),

2. Growth promoting compounds

Amino acids, vitamins of B group

 Microbial Assay for Antibiotics

 It requires the fulfilment of 3 factors:

i. The test compound → Antibiotic

ii. Reference standard

iii. Sensitive reference strain
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 Antibiotic -Microbiological Assay

• Why antibiotic should be analysed ?

– The use of antimicrobial agent are increasing rapidly

• resistance of many pathogenic microbes

– To confirm the effectivity of available antimicrobial agent against
new strains
• Concentration: amount of active ingrident per unit volume

• Potency: measurement of power to kill or inhibit the growth of

certain microorganism

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 Antibiotic -Microbiological Assay ...

• Expected effect:

• Inhibition of growth of the sensitive test strain.

 the basis of microbial assay of antibiotic is the quantitative

comparison of the effect of the test cpd and the reference
standard on the growth of a suitable or susceptible
organism in a nutrient medium.
• The test micro-organisms should:
be sensitive to the antibiotic
Show graded response
8
Reference standard to be used for Microbiological
Assay:

 Is required to be PURE.
– Since the basis of any Microbiological Assay (MBA)
is comparison of the test substance with
reference standard,
=> the success of MBA is ensured only when the
reference standard itself is pure.

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 Media and Diluents → for an MBA

 The USP protocol is the standard protocol for the selection of both
media and diluents.
 The media, chosen according to USP, will be used for both preparation of
test org. and for the actual conduct of assay
 Media must provide adequate nutrient/physical conditions and also
facilitate antibiotic diffusion
 While being sterilized, the media should not be heated excessively,

 Media should always be stored under defined condition: RT, Refrigerated

 Before use, all media should be checked for sterility & growth promotion

10
 Solution (Sample &RS) Preparations:

 It requires taking extreme care specially during the preparation of stock

solutions and dilutions.
• The essential requirement is that:

– The antibiotic and reference material be completely soluble in the

solvent/diluent,
 The most common diluents is phosphate buffer (pH 4-8)

– If the antibiotic is to be extracted from an ointment/cream base, then

the extraction process should be validated.

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 Antibiotic -Microbiological Assay ...
• Objective of the test
– As a standard to confirm the potency of antibiotic to kill or
inhibit the growth of certain microorganism

• General Method
– Diffusion Assay /Plate/ Cylinder method

– Turbidimetric/Tube assay method

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 A. Plate Assay

Steps performed:
1. Keep ready sterile glass plates /petridish
2. Pour the seeded agar medium into plates
3. Keep the plates in refrigerator before cutting
cups or placing cylinders → solidification
makes the cutting easier
4. Use stainless steel /porcelain cylinders of:

 8mm outside D & 6mm inside D

 Drop them from a height of 12mm
and use 6 cylinders per plate
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 Plate Assay …

5. Add a volume of 100µl of test/reference solution with an accuracy of ± 2% from

a caliberator

 Keep standard/test in alternative cylinder

6. After addition of solutions, the plates are kept in refrigerator for 1-2 hrs. → this
will ensure complete diffussion of antibiotics

7. Incubate at 32-35 oC (37 oC) for 12-18 hrs

8. Observe the inhibited zone diameter.

• Consider only those inhibition zones of not < 14mm.

9. Inhibition zone should be perfect circle.

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 Cut-off point for the determination of Antibiotics
susceptibility/resistance patterns

Antibiotic Disc Inhibition Zone Diameter (mm)

conc. Susceptible(s) Intermediate(I) Resistant
(R)
amoxacillin (Aml), (25µg) 14 or more 12-13 11 or less
ampicillin (Amp) (10µg) 14 or more 12-13 11 or less
cephalothin (Cep) (30µg) 18 or more 15-17 14 or less
erythromycin (Ery) (15µg) 18 or more 14-17 13 or less
gentamycin (Gen) (10µg) 13 or more - -
kanamycin (Kan) (30µg) 18 or more 14-17 13 or less
methicillin (Met) (5µg) 14 or more 10-13 9 or less
penicillinG (Pen) (10µg) 22 or more 12-21 11 or less
streptomycin (Str) (10µg) 15 or less 12-14 11 or less
tetracycline (Tet) (30µg) 19 or more 15-18 14 or less
vancomycin (Van) (30µg) 12 or more 10-11 9 or less

Data adapted from Atlas 15

(1997)
 Turbidimetric Method

• Turbidimetric Method

Basics:
– The antibiotic will inhibit the growth of microbes in liquid media
in test tubes.
– Turbidity intensity of the culture will express the population of
microbes
– Turbidimetric method is suitable for antibiotic which is not
soluble in water

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 Tube Assay/Turbidimeteric Assay/ of Antibiotics
• Activities:
1. A series of concentrations of Ref/samples/ are prepared (3x)
2. 1ml of each solution is added to a separate tube,
3. About 9ml of Nutrient broth is added to the above tubes, followed by
inoculation of the test microbe,
4. The tubes are incubated for 4 hrs,
5. Growth is stopped by heating at 80 0C.
6. The inhibition of growth is estimated by measuring Turbidity using
Spectrophotometer .
7.  the lesser the Turbidity, the higher the efficacy of the Test sample
(antibiotic)/

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 2. Pyrogens/ endotoxin testing

– Pyrogens/Endotoxins /are defined as substances that when

injected in sufficient amounts gives rise to a variety of
responses and symptoms.
• The most prominent response/symptom is raise in body
temperature.
– All microbes appear to be capable of producing pyrogens but
the most potent are associated with Gram-negative bacteria.
Gram-negative bacteria vs Pyrogen????

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 Nature/ source of pyrogens

 Gram-negative bacteria are characterized by their production of

endotoxins, which consist of a lipopolysaccharide (LPS) layer (outer
membrane) of the cell envelope.

=> Endotoxin/pyrogen is a lipopolysaccharide present in the cell wall of

gram negative bacteria which can cause fever if introduced into
the body

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 Properties of pyrogens
Chemical property:
 Pyrogens are polysaccharides attached to a lipid, thus called
Lipopolysaccharides.
Physical property:
– Highly stable at higher temperature

– Long drastic heating of more than 180 oC is necessary.

Biological Properties:
 Induce Fever,
 Intravascular coagulation,
 Bone marrow necrosis, etc

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 Assay of endotoxins /pyrogen:
both in-vitro & in-vivo
• Endotoxin Test/Pyrogens Test
– Endotoxin Testing

• Parenteral products should be free from endotoxin

• Raw materials and some finished product must be tested
for endotoxin
– Pyrogens tests:

• In vivo – Rabbit Pyrogen Test ( biological)

• In vitro – Limulus Amebocyte Lysate (LAL) Test (chemical)
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 Pyrogen Testing
1. In vivo Rabbit Pyrogen Testing
WHY rabbit?
i. Inexpensive, Easy to handle,
ii. Rabbits show some response to pyrogens as humans on per kg body
weight basis,
iii. Rabbits have labile thermoregulatory mechanisms
BUT, rabbits frequently produce false positive reactions.
Basic principles: after administration of the product;

– If ↑ in body To of rabbit above the limit → the product is pyrogenic;

– If the body To of the rabbit is below the limit →the product is free from
pyrogen

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 Pyrogen Testing …
Test Procedure:
1. Three healthy rabbits are selected ,
2. Accurate temperature is initially taken by a thermometer. (> 30
min) (Control To)
3. Test solution is warmed to 37 oC ±2 oC and administered, (≈
10ml/kg)
4. Rectal temperatures are detected every 30 min for 1-3 hrs,
5. Conclusion: The product passes if no rabbit shows a temperature
raise of 0.5 oC or more,
6. If any rabbit shows > 0.5 oC raise, the test is repeated on 5 more
rabbits
7. Now, if not more than 3 of the eight rabbits show an individual To
raise o.5 oC and if the sum of the eight individual rabbits To
increase is not greater than 3.3, then the product passes.
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 Pyrogen Testing …
Advantages
 Not specific to any one class of pyrogens

 Best replicate & domenstrate the production of fever in human

• Disadvantage

 Time taking, laborious

 Chance of biological variation ( extremly sensitive to Env’t)

 Development of tolerance by the rabbits

 Less sensitive

 Interferance from pharmaceutical ingrident; eg. Anticancer drugs

 A pass/fail test, rather than an assay

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 Pyrogen Testing …

2. In-vitro pyrogen Testing

• Limulus Amoebocyte Lysate (LAL)Test
 A test for the determination of G-ve bacterial endotoxin
 It employs a lysate protein obtained by lysing the blood cells
(Amoebocytes) of the horseshoe crab (Limulus polyphemous).
 The lysate protein is the most sensitive substance known for
endotoxins.
• Principle: If the lysate protein (diagnostic reagent) is added to the test
material with endotoxin, there will be formation of a ‘firm gel’ in < 60
min at 37 0C.
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 Pyrogen Testing …

• Three ways of conducting LAL Test

• Gel-clot End Point Test
• Turbidimeteric Test
• Chromogenic Substrate Test

1. Gel-clot Test
• The specimen is incubated with LAL of a known
senstivity.
• Formation of a gel clot is positive for endotoxin.

26
 Gel-clot Test …
Steps:
– Equal volumes of test solution and LAL reagent (usually 0.1ml each) are mixed in
depyrogenated test tubes of 10x75 mm size
– Spin gently and keep for incubation at 370C ± 1 0C for 60 min ± 2 min and then carefully
remove to observe
• A positive reaction is characterized by the formation of firm gel that will not
collapse on inverting the tube.
• A negative rxn is characterized by absence of a gel OR formation of a gel that does
not maintain its integrity

– Compare with +ve control (Reference Standard Endotoxin, RSE) and –ve control ///

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 2. Turbidimetric Method
• During the LAL– endotoxin reaction, the solution mixture becomes
increasingly turbid.
=> The turbidity is proportional to the amount of endotoxin present.
• The endotoxins concentration of a sample can be estimated by comparing
its turbidity to that of an endotoxin standard.
• Turbidity is read by a spectrophotometer.
• Usually more sensitive than gel-clot end point tests because turbidity
occurs prior to gelation,
=> it is thus possible to detect endotoxin concentrations that are lower than needed
to form a solid gel clot.
• Quantitative

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• Steps:
Chromogenic Method
Endotoxin + LAL reagent

Activation of proenzyme
- substrate
Chromophore is released

Color change
• Intensity of the color produced is proportional to the amount of
endotoxin present in the test sample.
• Measured by spectrophotometer

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 Pyrogen Testing ....
• Advantage of LAL Test
 More sensitive
 Less variable
 Quantification possible
 Easier, Not time consuming
• Disadvantage
 Done mainly with reference to Gram-ve Endotoxins,
 Interference problems from chemicals
 It can not measure the fever producing potential of pyrogen in human

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 3. Microbial Limit Tests & Standards
• Non-sterile medicaments (products)
– May not be totally free of microbes
– Limited permissible level of non-pathogenic microbes.
– The Microbial Limit Tests are designed to perform the qualitative and quantitative estimations of specific
viable microorganisms present in samples.
– It includes tests for total viable count (bacteria and fungi)
– Care must be taken in performing the tests, so that microbial contamination from the outside can be
avoided.

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 Microbial Limit Tests …
 Tests for Microbial Limits are designed for the
determination of level of bioburden and expressed as:
1. The extent of contamination  the microbial load (Total
Aerobic Microbal Count)
2. Nature of contamination  species composition
 Species of concern:
According to USP, the four major species of concerns are:
i. Escherichia coli
ii. Staphylococcus aureus
iii. Pseudomonas aeruginosa
iv. Salmonella species

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 Microbial Limit Tests …

• Microbial contamination could be a critical phenomena in:

1. Health significance - infection/disease

2. Economic significance - deterioration of products

(ex. Syrups, emulsions, suspensions)

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 Preparatory Testing

• When test samples have antimicrobial activity or when they

include antimicrobial substances → these antimicrobial
properties must be eliminated by:
– dilution, filtration, neutralization, inactivation, or

– other appropriate means.

After dilution, the actual Microbial Limit Test could be conducted
quickly. Why?

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 A. Total Viable Aerobic Count (TVAC)
This test is conducted to determine bacteria and fungi which grow
under aerobic conditions.
 There are four methods for this test (TAC):
1. Membrane filtration method,
2. Pour plate method,
3. Spread plate method, and
4. Serial dilution method (Most Probable Number method).
→The serial dilution method is applicable only to bacteria.
 An appropriate method should be taken from among these four,
depending on purposes.

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 TVAC …

• Culture media preparation→ should be as per the USP specification

Fresh
Specificity, selectivity, and sensitivity
• Different media and incubation temperature are required for the growth
of bacteria and fungi (molds and yeasts).
– Mac Conkey Agar Bacteria ; 30-35 0C, 24-
48h
– Soyabean-Casein Digest Agar,

– PDA (Potato Dextrose Agar) Fungi ; 25-28 0C, 2-5 days ;

– SA (Sabouraud glucose) Antibacterial should be included.
– SGA (Sabouraud Glucose Agar)
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 How to conduct this Test (TVAC)?

Preparation of test fluids:

 To dissolve or dilute the sample, use:
 phosphate buffer (pH 7.2),
 sodium chloride, or
 peptone buffer solution.
 Unless otherwise specified,

 use 10 g or 10 ml of the sample.

 Adjust the test fluid to pH 6-8.
 Use the test fluid within one hour after preparation.

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 Procedure (For Total Viable Aerobic Count)

1. Membrane Filtration Methods

 Applicable to sample which contains antimicrobial substances.

• Use membrane with a pore size of < 0.45 μm,

• Filters of about 50 mm diameter are recommended

• Sterilize the filters, filtration apparatus, media, and other

apparatus to be used

38
 Steps:
 Usually, measure two test fluids of 10 ml each, → pass each sample through
a separate filter.
 Dilute the pre-treated test fluid if the bacteria conc. is high, So that 10-
100 colonies can develop per filtration.
 After filtration, wash each filter three times or more with an appropriate
liquid such as phosphate buffer, sodium chloride-peptone buffer, or fluid
medium.

→The volume of the washings should be about 100 ml each.

 If the sample includes lipid, polysorbate 80 or an appropriate emulsifier
(surface-active agents) may be added to the washings.
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 Steps:
 After filtration:
 for bacteria detection, place the two filters on a plate of soybean-casein
digest agar medium, and
 for fungi detection, add an antibiotic to the medium and place them on a
plate of one of Sabouraud glucose agar, potato-dextrose agar, or GP agar
media.
 Incubate the plates:
 at least for 5 days at 30-35 oC for bacteria detection and at 20-25 oC for fungi

detection, and
 Count the number of colonies.

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 2. Pour Plate Method
 Dissolve/suspend 10g /10ml of the product in:

 Phosphate buffer, or
 Fluid Soybean casein Digest broth and make up to 100ml
(1: 10 dilution)
 Pipette 1 ml of the test fluid or its dilution into each petri dish aseptically,
→ Use petri dishes 9-10 cm in diameter. Use at least 2 agar media for each dilution
(Duplicate experiment///)
 Add 15-20ml of the Soybean casein Digest Agar medium

 for detection of fungi, use either Sabouraud Glucose Agar, Potato-

dextrose agar, or GP agar media, to which antibiotic has previously
been added.

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 Pour Plate Method …

• Cover the plates, mix, & incubate for 48-72hrs at 30-35oC for bacteria
detection and at 20-25 oC for fungi detection.
• Calculate viable counts based on counts obtained:
 From plates with not more than 300 colonies per plate for bacteria
detection
 from plates with not more than 100 colonies per plate for fungi detection.

• If no colonies are observed in 1:10 dilution,  express the result as < 10

CFU/g or < 10CFU/ml of the specimen.
– Normally, if the microbial count is less than 100 cfu/ml, it has been
expressed as BELOW DETECTABLE LEVEL.
42
 3. Spread Plate Method
(Surface-spread method)

• Place 0.1 ml of the test fluid on the solidified and dried

surface of the agar medium, and
• Spread it uniformly using a Spreader.
→Proceed under the same conditions as for the Pour Plate Method:
- The size of petri dishes,
- agar media,
- incubation temperature and time, and
- calculation method.

43
Microbial Limit Tests...

44
 4. Serial Dilution Method
[Most Probable Number (MPN) Method]
 Use 12 test tubes:
9 containing 9 ml of soybean-casein digest medium each &
3 containing 10 ml of the same medium each for control.
 Prepare dilutions using the 9 tubes.
First, add 1 ml of the test fluid to each of three test tubes and mix to
make 10- times dilutions.
Second, add 1 ml of each of the 10-times dilutions to each of another
three test tubes and mix to make 100-times dilutions.
Third, add 1 ml of each of the 100-times dilutions to each of the
remaining three test tubes and mix to make 1,000-times dilutions.
 Incubate all 12 test tubes for at least 5 days at 30 – 35 oC.
 No microbial growth should be observed for the control test
tubes.

45
 Serial Dilution Method …

 If the determination of the result is difficult or if the result is not

reliable;
 take a 0.1ml fluid from each of the 9 test tubes and place it to an agar
medium or fluid medium,
 incubate all media for 24-72 hours at 30-35 oC, and check them for
the absence or presence of microbial growth.

46
 B. Specific Tests for the designated microbes

If the product microbial colony is above the limit  initially the

product failed
If the product microbial colony is below the limit  use specific
designated test to check whether the product contain
pathogenic microbe or note
 Escherichia coli- IMViC tests
 Salmonella species-Brilliant Green Agar
 Staphylococcus aureus-Coagulase Test
 Pseudomonas aeruginosa-Oxidase Test

47
 4. Evaluation of preservatives
• Essence of a preservative
• to protect the user against possible infections
• to protect the product against deterioration during storage and use.
• Preservatives:
 should not be irritant or toxic to tissues to which they will be applied,
 must be effective in preventing growth of microorganisms likely to
contaminate them, and
 must have sufficient solubility and stability to remain active.
 Substances added to products to preserve quality and safety should
themselves be safe.

48
 Microbial methods
 Why Microbial methods?
• The intrinsic antimicrobial activity of the preservative
may be modified. (by interaction with excipients)
• Change in product pH or water activity during storage.
• NB: Preservative test is qualitative i.e to check the efficacy of the
preservative.
– Used to differentiate a good preservative from a poor preservative.

49
 Evaluation of preservative…
• Test organism:
– Standard species/ strains are employed
– Could be obtained from culture collection centres:
• Bacteria: S. aureus; P. aeruginosa; E.coli
• Yeast: Candida albicans
• Mold: Aspergillus niger
• Why the above organism?
• representatives of typical contaminants during manufacture
• arise as contaminants during use/treatment
• they have undemanding nutritional requirements (thus
easy to cultivate, also grow on medicaments in presence of
ineffective preservative)  preservative resistance
• they have pathogenic potentials

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 Preparation of inoculum

• Inoculum- actual suspension of MO that are added to the product.

• Volume of inoculum = 0.1 ml for 20ml i.e 0.5%

• Inoculum concentration :
– 105-106 cells/ml or gm of product  affords margin of safety

• to challenge the preservative efficacy

• for safety reasons ( i.e if the preservative is effective for

such large no, it is highly efficient in the actual test where
there are very small no of bacteria ( contaminant) present.
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 Preparation of inoculum …

 Is the most critical step in the evaluation of efficacy of

preservatives. Why?
 The following factors, related to inoculum preparation, affect
the reliability of test result:
a. The way to grow the cells from the stock culture,
b. The incubation period for the sub-culturing,
c. The inoculum size  the need for standardization
d. The growth phase of test organism,
e. The composition of the sub-culturing medium.

52
 Basic test protocol:

i. Where product container can be entered aseptically- conduct

the test in five original containers.

ii. If container can not be entered – then transfer 20 ml samples or

enter ( if < 20 ml) to 5 sterile capped tube.

iii. Inoculate each tube or container with standard inoculum at a

ratio of 0.1 ml inoculum to 20 ml of product and mix.
{Ensure that the initial inoculum size or no of cells is between 105- 106 cfu/ml}

iv. Incubate the containers at 20-25 oC; examine at 7, 14, 21, 28,
days; record any change, find total count (TC) and express as %
age change in the number of microbes. 53
 Interpretation of results;

• The preservative is considered efficient if:

– the number of viable bacteria are reduced by at least 0.1% of the
initial count by the 14th day
– The concentration of yeast/mould remain at or below that of the
original concentration during the first 14th day
– The concentration of each test organism remains at or below the
designated levels during the remainder of 28th days of test period.

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 5. Sterility Test

• Introduction
 ☻Required for all articles or substances to be introduced into raw
tissue (injections and ophthalmics).

 Sterility testing attempts to reveal the presence or absence of viable

micro-organisms in a sample number of containers taken from batch of
product.

 Based on results obtained from testing the sample a decision is made as

to the sterility of the batch.

 The test shows that samples tested were free from living bacteria and
fungi but not viruse.
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 Sterility test con…
☻It is not possible to claim thabatch is sterile unlessa;
i. The entire contents of every container is tested &

Ii. The test provides optimum conditions for growth of all possible
microbes. BUT, Neither of the above two conditions can be fulfilled.

►Thus, sterility test can only show that “ microbes capable of growing in
the tested media employed under selected conditions are absent from
a fraction of a batch tested.”

56
 Sterility Test …

Three generalization could be made:

1. To obtain reliable results, it is necessary to take sufficient
samples and use sensitive enough media.

2. Sterility testing should not be used as a sole means of

controlling sterile processing.

3. Sterility testing should be carried out under the same

conditions as aseptic manufacture

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 General Methodology

• Sterility test methods defined in Pharmacopoeia are carried out by:

• Membrane filtration is the preferred method if product is filterable
• Direct inoculation is alternative
• Media: media generally used for tests for sterility are:
• Soybean Casein Digest medium (SCD) - used to detect aerobic MOs
• Fluid Thioglycollate medium (FTM) is usually used to detect anaerobic
organisms

=> Should be tested for growth promoting qualities prior to use (low

number of organisms)
• Incubation Period - At least 14 days incubation
58
 Sampling

• No of containers and volume to be tested defined in

Pharmacopoeia
• Sampling should be sufficient to allow for re-tests if needed
Stages of sampling:
1. For terminally sterilized products – the final end product stage is
where samples are collected,

2. For aseptically processed products – two sampling stages are

required:
i. The bulk from which final container are filled
Ii. The final container after they are sealed.
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 Selection of samples
• The selected sample must be representative of the entire lot of final
container,
• For bulk product, the entire liquid/material be well mixed.
• For terminally sterilized lot, select at random, BUT:
1. When a lot from heat sterilized process is being tested, samples been
taken from every shelf and from every lot,
2. For aseptically processed preparations, sampled be taken through out
the filling operation,
3. For radiation/Gas sterilized products, samples be taken from a batch of
similar items, subjected to uniform sterilization during a given period.

60
 Control tests
• Negative Contols
– media should be incubated for 14 days prior to use
– growth is not expected,
– but growth may occur if

i. improperly sterilized medium

ii. Contamination during testing
– So what???
• each of the media is incubated at the same time as test media
• Purpose of the –ve control ; if no growth is seen:
 it confirms that medium is sterile
 serves as a standard with which test can be compared

61
 Control tests …
• Positive Test Controls
– should demonstrate that media are capable of supporting growth
of a range of low numbers of organisms in the presence of
product.
• growth should be evident after 3 days (bacteria), 5 days (moulds)

– growth is expected
– growth may not occur if
i. Inadequate medium
ii. Overheating of medium during sterilization
iii. Inhibition by active ingredient or excipient
• So what is done?

62
 Test procedure

• Opening the container:

– Cleanse external surface with a suitable disinfecting agent and gain
access inside aseptically.

• How to inoculate:
– If it is a liquid, remove sample with a sterile pipette or syringe; add
to medium & mix gently.
– Incubate as directed for not less than 14 days
• Bacteria- 30-35 oC, Fungi-20-25 oC

63
 Test procedure cont…

• Immersion method or direct inoculation method

– Prepare the appropriate media
– Directly inoculate the product or test solution
– Incubate the inoculums for14 days (check the + / - of MOs in
certain interval of time)
Advantage
• It require small number of samples
• used for non liquid article – cream, ointment, etc
Limitation
• Not used for article having antimicrobial activity
– It require pretreatment step

64
 Test procedure

• Membrane filtration method

– Test article is made to pass through a filter
• Organisms of interest are retained in the filtering material
• Test article pass through the filter
– Incubate the filtered microbe for 14 days
• Check if growth is present in certain interval of time
Advantage; it used for
• Large volume of sterile product
• Articles having antimicrobial activity
Limitation:
– Used only for liquid samples
– Require large volume of sample
65
 Recording and interpretation of results:

• Examine the media for growth at least as often as on

3/4/5/7/8/14th day
• First stage
– At various times - no evidence of growth  product passes

– If growth is seen, but a review of procedure, materials suggests a

possible contamination and also the –ve control shows growth,
the entire test is declared invalid and repeated.

66
 Recording and interpretation of results: …

• Second stage
• In this repeat testing- the minimum number of samples to be tested is
doubled.
• However; the volumes of each test substance & medium remain same as in
1st test.

• If no growth is observed (–ve control & test cpd)– product passes

• But if growth occurs again – it conclusively proves that product is non-

sterile [to be repeated if –ve control also shows growth]

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 Sterility test …

• By definition a product that is sterile contains zero viable

microorganisms.

• The sterilization process must be validated and audited to

insure that each product labeled as sterile is truly free of all
contaminating microorganisms.

=> A manufacturer that chooses to make and sell sterile products must
do sterility testing.

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THE END !!!

Thank you

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