Microbiological
Quality Control in
Pharmaceutical
industry
Prepared by:
Roaa Khaled Elsayed
Assistant lecturer, Microbiology and
Immunology Department
Microbiological Quality Control
• Microbiological QC unit must ensure the
quality and safety of the manufactured
products.
• This is done by thoroughly testing the
following items for microbial contaminants
that may have been introduced accidentally
during or subsequent to the
manufacturing process:
• Raw materials
• Environmental surfaces
• Finished product
General considerations
• Carry out the microbiological QC tests under conditions
designed to avoid extrinsic microbial contamination.
• The precautions taken to avoid contamination must be
that they do not affect any microorganisms to be revealed
in the test.
• Since not every container can be tested, a sufficient
amount of the containers –indicated by the
pharmacopeia- should be examined to give a suitable
degree of confidence in the tested result.
• The result of each test assumes that every container of
this batch will give the same result.
Microbiological QC in
pharmaceutical industry
Pharmaceutical
Environmental QC
preparations QC
Non-sterile
products
Sterile products
QC of Pharmaceutical Dosage forms
Sterile Non-sterile
Microbial enumeration tests
• Quantitative enumeration of mesophilic
bacteria and fungi that may grow under
aerobic conditions.
• Microbial enumeration tests are not
applicable to products containing viable
microorganisms as active ingredients.
Microbiological
examination of non-sterile
products
Microbial enumeration test Test of specified organism
(Microbial Limit Test (MLT))
Membrane Plate count Most probable
filtration method number (MPN)
(least accurate)
Pour plate Determine the presence
or absence of
Spread objectionable organism
plate
❖Sample Preparation:
✓Products to be tested are prepared as stated in the
Microbial pharmacopeias.
✓Usually, 10 gm or 10 ml of the product are sampled.
✓Sample preparation of common dosage forms:
Enumeration o Water-Soluble Products:
• Dissolve or dilute (usually 1:10 dilution) the
test product to be examined in Phosphate Buffer
Solution (PBS) pH 7.2.
(Sample preparation) o Fatty Products:
• Mix with the minimum necessary quantity of
sterile tween 80 heated, if necessary.
• Mix carefully with pre-warmed diluent (1:10)
to form an emulsion.
Microbial Enumeration test
(Test Methods)
1. Plate-count methods:
A. Pour-Plate Method
✓Add to the dish 1 mL of the prepared
sample
✓Add 15 to 20 mL of Soybean–
Casein Digest Agar or Sabouraud
Dextrose Agar (maintained at not
more than 45°C).
✓Incubate the plates as mentioned
previously.
✓Perform the counting.
Microbial Enumeration test
(Test Methods)
1. Plate-count methods:
B. Surface-Spread Method:
✓Add 15 to 20 mL of Soybean–Casein
Digest Agar or Sabouraud Dextrose
Agar at about 45°C to each Petri dish
and allow to solidify and dry.
✓Spread a measured volume of not less
than 0.1 mL of the sample.
✓Incubate the plates as mentioned
previously.
✓Perform the counting.
Microbial Enumeration test
(Test Methods)
2. Membrane Filtration:
1) Use membrane filters with pore size not
greater than 0.45μm.
2) Transfer a suitable quantity of the
prepared sample to the membrane filter,
and filter immediately.
3) For the determination of:
❖Total aerobic microbial count (TAMC),
transfer the membrane filter to the surface of
the Soybean–Casein Digest Agar. (Incubate at
30°–35° for ≤3 days).
❖Total combined yeasts and mold count
(TYMC), transfer the membrane to the surface
of the Sabouraud Dextrose Agar (Incubate at
20°–25°for ≤5 days).
4) Perform the counting.
Check appendix in lab book
Validation studies before testing
Test Media suitability; Test Method suitability;
• Media sterility test • Eliminating effect of
• Media supporting the growth antimicrobials so that if
contamination is present, it will be
detected
• Once method suitability is
established, it does not need to be
repeated unless there is a change
in formulation, production
method, or test procedures
Before testing (Media suitability)
Negative control
• Used to ensure media sterility, therefore, exclude a “False positive result”.
• Sterilize media and incubate at the specified conditions.
• Medium is accepted sterile if no growth is found after the specified incubation
time.
Growth promotion test (Positive control):
• Test the ability of the medium to support the growth of the microorganism,
therefore, exclude “False negative result”.
• The medium should be inoculated with less than 100 CFU of each of the 5
challenge organisms (See Table in the appendix).
• For solid media, growth obtained on test media must not differ by a factor
greater than 2 compared to growth on previously tested and approved media.
• For liquid media, clearly visible growth should be obtained on test media
comparable to growth on previously tested and approved media
Before
Perform the standard testing
procedure of the selected test method
(Suitability of counting method) (filtration/pour-plate) using each of the standard challenge
(Suitabilityorganisms
of counting method)
(≤ 100 CFU)
Media + Challenge organism
Media+ Pdt. + Challenge organism
Before testing
(Positive control)
If count within a If count is
Determine count
factor of 2 from reduced by a
(X)
(X) factor > 2
No antimicrobial activity under
the conditions of the test OR the product possesses antimicrobial activity that
has not been satisfactorily eliminated under the
such activity has been conditions of the test
satisfactorily eliminated.
Modify the method to eliminate the
Method is accepted >> Do the test antimicrobial activity and repeat the
suitability test.
Neutralization/removal of
antimicrobial activity
If growth is inhibited, then modify the procedure to ensure the validity
of the results.
Modification of the procedure may include:
1. Increasing the volume of the diluent or culture medium.
2. Incorporation of specific or general neutralizing agents into the
diluent.
3. Membrane filtration (only in microbial enumeration).
4. A combination of the above measures.
Neutralizing Agents
• Neutralizing Agents: are agents used to
neutralize the activity of antimicrobial
agents.
• They may be added to the chosen
diluent or the medium preferably
before sterilization.
• If used, their efficacy and their absence
of toxicity for microorganisms must be
demonstrated by carrying out a blank
with neutralizer and without product.
Check the appendix in lab book
USP-Chapter 〈71〉
Test for absence of specified
microorganisms
Prepare a sample using a 1:10 dilution
Use 10 mL or the quantity corresponding to 1 g or 1 mL, to inoculate Soybean–
Casein Digest Broth (TSB), mix, and incubate at 30° to 35° for 18 to 24 hours.
E. coli S. aureus P. aeruginosa
MacConkey broth Mannitol
Cetrimide agar
salt agar
MacConkey agar
Tests for specified
microorganisms
A) Test for Escherichia coli
10 ml or the
quantity
corresponding to
1 g or 1 ml Shake &
Mix Transfer
1 mL
100 mL
Sample preparation Suitable amount of
(1:10) dilution
Incubate at 30 to MacConkey broth
soybean casein digest
*NOT Less than 1 g* broth 35˚C
Mix
for 18-24 hours
Subculture
Incubate at 42 to 44˚C
Incubate at 30 to 35˚C MacConkey agar for 24-48 hours
for 18-72 hours
Tests for specified
microorganisms
B) Test for Cutaneous microorganisms
(Pseudomonas aeruginosa & Staphylococcus
aureus)
10 ml or the
quantity
corresponding to
1 g or 1 ml
Mix
Sample preparation Suitable amount of Incubate at 30 to 35˚C
(1:10) dilution soybean casein digest for 18-24 hours Mix
*NOT Less than 1 g* broth &
Subculture
on
Mannitol salt
agar
Cetrimide
Incubate at 30 to agar
35˚C
for 18-72 hours
Check appendix in lab book
Test for specified microorganisms
E. coli S. aureus and P. aeruginosa
Oromucosal use
Gingival use
Aqueous and Non-aqueous
preparations for oral use Cutaneous use
Nasal use
Auricular use
Vaginal use
Inhalation use
Transdermal
patches
Before testing (Media suitability)
Negative control
• Same as in Microbial enumeration tests
Growth promotion test (Positive control):
• The medium should be inoculated with less than 100 CFU of appropriate
organism and incubated at the specified temperature for a period of time within
the range specified in the test.
• For solid media, perform surface-spread method. Growth comparable to that
previously obtained with a previously tested and approved batch of medium
occurs.
• For liquid media, clearly visible growth should be obtained on test media
comparable to growth on previously tested and approved media
• Test for inhibitory properties of liquid or solid media: No growth of the
test micro-organism occurs.
• Test for indicative properties: perform surface-spread method. Colonies are
comparable in appearance and indication reactions to those previously obtained
with a previously tested and approved batch of medium.
Suitability of absence of
specified organism test method
Perform the standard procedure of the test using the specified
organism (≤ 100 CFU)
Media + Pdt. + specified organism Media + specified organism
(Positive control)
Specified organism is detected with appropriate indication reactions
Reference pharmacopeia chapters
• USP chapter 〈61〉 Microbiological Examination of Nonsterile Products: Microbial
Enumeration Tests.
• USP chapter 〈62〉 Microbiological Examination of Nonsterile Products: Tests for
Specified Microorganisms
• USP chapter 〈 1111 〉 Microbiological examination of nonsterile products:
acceptance criteria for pharmaceutical preparations and substances for
pharmaceutical use
Practical
Microbial limit test practical procedure
Sample preparation Pour plate technique
Product 1 Tablet OR
1 spatula of
1. Oral non- granules 1 TSA
aqueous
Aseptically 9ml PBS (2) Pour into plate
Molten agar
1ml 2 Vortex
2. Oral (till no big (1) Add 1ml to
Aseptically clumps) empty plate (3) Swirl
aqueous 9ml PBS Empty
Incubation
1 Spatula/ plate
3. Topical
1ml 3 1ml MacConkey’s
Aseptically 2-Test for M.B broth
9ml PBS specified M.O TSB Oral
pdt.?
1 Wipe 4
4. Topical Swirl
(wipes) Turbid Topical Cetrimide
Mannitol
Aseptically Salt Agar
90ml PBS Provided pdt.? Agar
Streaking on Agar plate
Name
1 & seat number 2 A
A
B C B C
Follow the
aseptic
4 technique 3
A A
B C B C
