PHAR457

Asst. Prof. Dr. Mehmet İLKTAÇ

Lecture 2
 NON-STERILE PHARMACEUTICALS
 Contain particular number of microorganisms as a
result of their nature.

 Quantities higher than acceptable threshold

and/or presence of objectionable microorganisms

Spoilage of the Health threat to

pharmaceutical consumers

1. Product recalls CONSEQUENCES

2. Production shutdown OF
3. Losses in labor and manufacturing CONTAMINATION
4. Financial loss
 NON-STERILE PHARMACEUTICALS
Although non-sterile,
 Quantity determines Safety of the product and
+
efficacy of manufacturing
 Types
process
of m.o

‘’OBJECTIONABLE MICROORGANISMS’’
‘’Microorganisms which might cause serious health
threat to consumers, might cause drug spoilage or
indicate the presence of other pathogenic bacteria’’
Microbiological testing of non-sterile pharmaceuticals
=
Microbial limit tests
QUALITY ACCEPTANCE CRITERIA OF NON-
STERILE PHARMACEUTICALS

1. Quantity of microorganisms
(Quantitative tests)
a. Total Aerobic Microbial Count

b.Total Combined Yeast and Mould Count

2. Absence of specific indicator microorganisms
(Qualitative tests)
 INDICATOR OBJECTIONABLE MICROORGANISMS
(SPECIFIED MICROORGANISMS)
 Salmonella Gastrointestinal system disorder (AGE)

 Escherichia coli Fecal coliform and some strains

cause GIS diseases

 Pseudomonas aeruginosa Opportunistic

infections

 Staphylococcus aureus Skin and GIS infections

and toxic shock
syndrome
 Candida albicans Vaginitis
 Bile tolerant Gram negative Coliform and
Opportunistic pathogen
bacteria
 Clostridia Wound, intestinal and neurological infection
 INDICATOR OF FECAL CONTAMINATION
Coliform bacteria: E. coli
 Rod shaped Klebsiella
 Gram negative Enterobacter
 Non-spore forming Citrobacter
 Ferment lactose with the production of acid and gas
when incubated at 35–37°C for 24 hours.

Fecal Coliform bacteria:

BETTER INDICATOR  Rod shaped E. coli
FOR FECAL  Gram negative
CONTAMINATION  Non-spore forming
 Ferment lactose with the production of acid and gas
when incubated at 44°C for 24 hours.
COLIFORMS: INDICATOR OF FECAL
CONTAMINATION OF PRODUCTS
Is the indicative
of failed GMP in
industry.
 BACTERIAL INDICATORS DO NOT INCLUDE ALL THE
OBJECTIONABLE BACTERIA PRESENT IN THE
ENVİRONMENT
THE USE OF SEVEN INDICATOR BACTERIA DOES
NOT MEAN THAT THE PRESENCE OF OTHER
BACTERIA MIGHT NOT BE A PROBLEM DURING
QUALITY EVALUATION.
 ROUTE OF APPLICATION AND WILL DETERMINE IF
DELIVERY SYSTEM OF DRUG
 INDICATION FOR USE (INTENDED THERE IS A RISK
CONSUMER) INVOLVED WHEN
 CHEMICAL COMPOSITION OF OTHER
PRODUCT MICROORGANISMS
 PRODUCTION PROCESS (FILTRATION, PRESENT
HEATING, DRYING...)
 NON-STERILE PHARMACEUTICALS
 Manufactured under aseptic conditions
 GMP, Necessary to
 aseptic thecniques control the
 Environmental control presence and
 Personnel and equipment the number of
sanitation microorganisms
BUT
 Processes are NOT controlled As regularly as
Water, air and environmental sterile
monitoring are NOT performed manufacturing

Limit testing is important for quality control analysis

of non sterile products.
 RAW MATERIALS THAT MAY NEED
MICROBIAL LIMIT TESTING
Microcrystalline Cellulose Copovidone
Capsule Shells Hydroxyethyl Cellulose
Magnesium Stearate Hydroxypropylcellulose
Xanthan Gum Sugar
Sodium Starch Glycolate Mannitol
Lactose Monohydrate NF Corn Starch
Lactose Anhydrous Glucose
Talc Croscarmellose Sodium NF
Powdered Cellulose Cotton Coil
Cellulose Acetate Sugar Spheres
Povidone Opadry
CATEGORIES OF NONSTERILE PHARMACEUTICAL
PRODUCTS
Metered-dose and dry powder inhalants
 Nasal sprays Antimicrobial
activity
Antimicrobial
preservative
Otics
Vaginal suppositories Decreasing risk
of contamination
Topicals Water activity

Rectal suppositories Exipient microbial

content

Oral liquids (aqueous)

Manufacturing
Liquid-filled capsules processes

Oral tablets and powder-filled capsules

 WHAT ARE THE
SOURCES OF
MICROBIAL
CONTAMINATION
OF NON-STERILE
PRODUCTS
 SOURCES OF CONTAMINATION
1.) Pharmaceutical ingredients (API, exipient, raw mat)
Animal and plant origined raw materials naturally
contain microorganisms.
Raw materials should be tested prior to use in
manufacturing.
2.) Water
Water system must be validated.
Water system and water should be monitored
regularly to reduce the microbial load.
Water system must be sanitized regularly to prevent
microorganism colonisation and biofilm formation.
Heat treatment UV treatment
Chemical treatment Filtration
 SOURCES OF CONTAMINATION
3.) Equipment and building areas

Building areas and equipment

should be sanitized properly.

Environmental monitoring is
important in order to reduce
microbial contamination.
 SOURCES OF CONTAMINATION
4.) Manufacturing Personnel and noncompliance with GMP
Normal flora in oral cavity, skin, nasopharynx etc.

Face masks, gloves, laboratory

coat, hair coverers and safety
glasses should be worn

Training of manufacturing and lab.

personnel
 SOURCES OF CONTAMINATION
5.) Air quality

Air is generally contaminated with moulds and

bacterial spores.

Air quality control of both living and non living

particulates should be carried out.
 1
4 pt
3
4 pt

4 pt

4 pt

4 4 pt

4 pt
2
4 pt 4 pt
 NON-STERILE PHARMACEUTICAL
CONTAMINATION
 Less stringent GMP compliance INCREASES THE
RISK OF
 Lack of functional microbial limit CONTAMINATION,
ALLOWS
testing (raw materials and finished OBJECTIONABLE
products) program MICROORGANISMS
TO CONTAMINATE
PRODUCTS AND
 Lack of equipment sanitization and
INCREASES THE
environmental sampling LOAD OF
MICROBIAL
 Lack of properly trained personnel CONTAMINATION
 MOST COMMON CONTAMINANTS
Gram (-) rods are the most common contaminants.
Lack of
process
Pseudomonas control of
environment
Burkholderia cepacia Water and raw
Ralstonia pickettii material
origined
Molds and yeasts are also among common
contaminants. Lack of personnel
hygiene
Staphylococcus
Gram (+) bacteria Streptococcus
Bacillus Personnel
origined
Clostridium
I.) Health threat to consumers

II.) Spoilage of pharmaceuticals

IMPORTANCE OF USP BACTERIAL INDICATOR
I.) Health threat to consumers
Salmonella GIS disease in healthy
patients

Some strains of E. coli GIS disease in healthy

patients

S. aureus Skin and GIS disease in healthy patients

Pseudomonas,
Opportunistic
Enterobacteriaceae
infection
and others
 IMPORTANCE OF USP BACTERIAL INDICATOR
Opportunistic: Infections not seen in healthy patients but in :
FOR THESE
PATIENTS,
 Immuncompromised patients LIMITS OF THE
PRODUCTS
 Patients with underlying disease MUST BE
LOWER THAN
 Newborn infants FOR PEOPLE
WITH
 Elderly people FUNCTIONAL
IMMUNE
SYSTEM
 MICROBIAL LIMIT TESTING

Microbial limit tests of raw material and

finished products determine the
MICROBIOLOGICAL QUALITY of non-
sterile pharmaceutical products.
 MICROBIAL LIMIT TEST METHODS
According to USP, EP and JP, microbial limit testing
is divided into 2 different tests:
 Quantitative tests
 DETERMINES TOTAL THE NUMBERS OF BACTERIA,
YEAST AND MOULDS PRESENT IN THE SAMPLE

Microbial Bioburden: Total number and type of

microorganisms present in a pharmaceutical.

 Qualitative tests
 DETERMINES THE PRESENCE OF SPECIFIC
INDICATOR (OBJECTIONABLE) MICROORGANISMS
 MICROBIAL LIMIT TESTS (MLT)

Qualitative
tests

Quantitative
tests
MICROBIAL LIMIT TESTS (MLT)
 MICROBIAL LIMIT TEST METHODS
Three pharmacopoeia

USP EP JP
Prior to 2006, used test methods that were similar
but widely variable in practice and acceptance
criteria.
They harmonized their testing methods,
specifications and acceptance criteria for non-sterile
pharmaceutical products.
MICROBIAL LIMIT TEST METHODS

I. QUANTITATIVE TESTS
(ENUMERATION TESTS)
 QUANTITATIVE TESTS
The ability of the test to detect and
queantify bacteria, yeasts and moulds in
the presence of the product to be
tested must be established.

SUITABILITY OF QUANTITATIVE TEST

 SUITABILITY OF QUANTITATIVE TEST
Preparation of test strains
 Staphylococcus aureus
 Pseudomonas aeruginosa Test strains
 Bacillus subtilis for
 Candida albicans quantitative
validation
 Aspergillus brasiliensis
S. aureus, P. aeruginosa and Bacillus subtilis are
grown on Soybean Casein Digest broth (SCDB) at 37
°C for 18-24 hours.
C. albicans is grown on Sabouraud Dextrose broth at
20-25 °C for 2-3 days and A. brasiliensis is grown on
Sabouraud Dextrose agar at 20-25 °C for 5-7 day.
 SUITABILITY OF QUANTITATIVE TEST
Preparation of test strains
 Buffered sodium chloride peptone (BSCP) solution,
phosphate buffer solution (PBS) or SCDB is used as
diluent to make test suspension.

Need to do 10 fold serial dilutions because maximum

100 cfu microorganism inoculum is needed to be
obtained in the further steps.

 Use suspensions within 2 hours (stored at room

temperature) or 24 hours if stored at 2-8 °C.
 Preparation of test strains

Negative control
 Negative control is performed with only diluent
solution having no microorganism. Should be no growth
in the final analysis.
Growth promotion of the media
 All media should be tested for growth promotion.
 Growth obtained must not differ by a factor greater
than 2 from the calculated value for standardized
inoculum.
Preparation of Microorganisms and Growth Promotion Tests
 SUITABILITY OF QUANTITATIVE TEST
Preparation of Drug Samples To be Tested
Water soluble products: Dilute the 10 ml or gr of
the product 1:10 in BSCP, PBS or SCDB.

Non fatty products Insoluble In Water: Add

polysorbate 80 (1g/lt) after diluting 10 ml or gr of the
product 1/10 in BSC, PBS or SCDB.
Fatty Products: Dissolve in isopropyl myristate or
mix with minimum quantity of sterile preheated (40
°C) polysorbate 80. Dilute 1/10 with diluent.
Transdermal patches: Remove protective cover
sheets of 10 patches, cover adhesive surface with
sterile gauze and transfer to 500 ml diluent. Shake 30
minutes.
 SUITABILITY OF QUANTITATIVE TEST
Inoculation and dilution
Sufficient volume of diluted microbial suspension is
added to the diluted product and to the POSITIVE
CONTROL with no product (positive control will be
compared with the result of product to asses the
suitability of method ).

The final inoculum of microorganism should not be

more than 100 cfu/ml.

The volume of suspension of microorganism should

not be more than 1% of that of diluted product.
 INOCULATION AND DILUTION

Diluted Diluted
microorganism product
 SUITABILITY OF QUANTITATIVE TEST
RECOVERY AND ENUMERATION OF MICROORGANISMS
IN THE PRESENCE OF PRODUCT
1. PLATE COUNT METHODS

2. MEMBRANE FILTRATION METHOD

3. MOST PROBABLE NUMBER (MPN)METHOD

 The least accurate method for microbial counts

Which one
to choose ?  Required limit of microorganisms
 Nature of the product
depends
RECOVERY AND ENUMERATION OF MICROORGANISMS
1. PLATE COUNT METHODS
Perform plate count methods at least in duplicate
for each strain and use the mean (avarage) count.
A. POUR PLATE METHOD
1 ml of sample (diluted product + diluted m.o +
neutralization agent)
+
15-20 ml of Soybean-Casein Digest agar or Saboraud
Dextrose agar at 45 °C

Add to petri dish, stir and incubate as

stated in prep. of test strains
 POUR PLATE METHOD

1 ml

Inoculate empty plate

Diluted
product
and
bacteria
Add melted SCDA
(15-20 ml)

Swirl to mix

Colonies grown on
solidified media
STREAK OF DIFFERENT DILUTED SERIES OF MICROORGANISMS

CHOOSE THE LOWEST DILUTION WHICH HAS

LESS THAN 100 CFU
RECOVERY AND ENUMERATION OF MICROORGANISMS
1. PLATE COUNT METHODS
B. SPREAD PLATE METHOD
Add 15-20 ml Soybean-Casein Digest agar or
Sabouraud Dextrose Agar into a petri dish, allow it to
solididy.

Spread at least 0.1 ml of the sample (diluted product

+ m.o + neutralization agent) over the surface.

 Incubate as stated in prep. of test strains.

Count cfu of the duplicates, take the average and

calculate the number of cfu in original inoculum
 SPREAD PLATE METHOD
0.1 ml

Inoculate petri dish

containing solid medium
Diluted product
and bacteria

Spread inoculum over

surface evenly

Colonies grow on
surface of medium
RECOVERY AND ENUMERATION OF MICROORGANISMS
2. MEMBRANE FILTRATION METHOD
Membrane filters with 0.45µm pore size

Sample (representing 1 gr of the sample; generally 10

ml) is transferred into membrane filter and filtrated.

For total aerobic microbial count (TAMC), place the

filter onto Soybean-Casein Digest agar.

For total combined yeasts and moulds count

(TYMC), put the filter on to Sabouraud Dextrose agar.
RECOVERY AND ENUMERATION OF MICROORGANISMS
3. MOST PROBABLE NUMBER (MPN) METHOD
The accuracy of MPN method is less than that of
plate count methods and membrane filtration.
Not suitable especially for yeasts and moulds.

The MPN is particularly useful for low

concentrations of organisms
METHOD
Prepare 10-1, 10-2 and 10-3 dilutions of sample
From each dilution, add 1 ml to to each of three
tubes containing 9 ml Soybean-Casein Digest Broth.
Incubate and analyse according to the table.
MPN TABLE
 SUITABILITY OF QUANTITATIVE TEST
RESULTS AND ANALYSIS
To verify suitability of,
Mean count of test
organism with product must
1. Plate Count Methods
not differ by a factor greater
than 2 from the value of
2. Membrane Filtration
POSITIVE CONTROL
(contains no product)
> 50% and < 200%
The calculated value from
3. MPN
inoculum must be within
95% confidence limits of
results obtained with
CONTROL.
 SUITABILITY OF QUANTITATIVE TEST
Neutralization and Removal of Antimicrobial Activity
If the number of microorganisms recovered from the
diluted product (after the end of the test) is less than
a factor greater than 2 compared to the number of
microorganism recovered from positive control
preparation

THE PRODUCT HAS ANTIMICROBIAL ACTIVITY

HAVE TO NEUTRALIZE/REMOVE IT
 SUITABILITY OF QUANTITATIVE TEST
Neutralization and Removal of Antimicrobial Activity
Increase the volume of diluent (eg. dilute 1/100)
Membrane Filtration
Add neutralizing agent into diluent
 Glycine
Thiosulphate
Lecitin
Polysorbate

Agents used for neutralization and

as surface active substance
SHOULD HAVE NO TOXICITY ON
MICROORGANISMS
Thioglycollate
Ca+2 Sodium
Mg+2 bisulphite
Neutralization and Removal of Antimicrobial Activity
If all neuralization methods fail to neutralize the
product, failure to isolate the inoculated organism is
due to microbicidal activity of the product.

The product cannot be contaminated with that

microorganism species.

However, other microorganisms not included

among the test strains can contaminate this product.
Therefore, microbial limit testing must be performed
in case of the contamination of other microorganisms.
QUANTITATIVE TESTING
OF PRODUCTS
QUANTITATIVE TESTING OF PRODUCTS
After methods are validated (suitability testing), non-
sterile products can be tested by quantitave tests
routinely.
AMOUNT OF PRODUCT USED FOR TEST
Generally 10 g or ml

 Aerosol dosage forms: 10 containers

 Transdermal patches: 10 patches
 Tablet, capsule and injection: 10 units

 Drug substances that is less than 1000 ml or

1000 gr: 1% of the batch
 QUANTITATIVE TESTING OF PRODUCTS
AMOUNT OF PRODUCT USED FOR TEST
Distribution of microorganisms
in a batch is not homogenous.

They can clump together only in

some containers in the same
batch.

The sample of volume of 10 ml or gr

MUST BE A COMPOSITE OF 10 different
containers.
 QUANTITATIVE TESTING OF PRODUCTS
EXAMINATION OF THE PRODUCT
MEMBRANE FILTRATION
 Transfer the appropriate amount (representing one
gram of sample) of prepared product to each of two
0.45 µm membrane filters. 50 ml of prepared product
for transdermal patches are filtered.
After filtration
Total aerobic Transfer filter Incubate 3-5
microbial count onto SCDA days at 37 °C

Total yeast Transfer filter Incubate 5-7

mold count onto SDA days at 25 °C
MOST APPROPRIATE METHOD FOR PRODUCTS CONTAINING
ANTIMICROBIAL SUBSTANCES
 QUANTITATIVE TESTING OF PRODUCTS
EXAMINATION OF THE PRODUCT
POUR PLATE METHOD
 Ten fold serial dilution of sample is prepared. 10-1,
10-2, 10-3
 For each dilution 2 SCDA (for TAMC) and 2 SDA
(TYMC) are used.
 Incubate media for 3-5 days at 35-37 C for bacteria
and 5-7 days at 20-25 C for fungi.
 After incubation, select the dilution showing the
highest number of colonies less than 250 for TAMC
and 50 for TYMC.
 Take the arithmetic mean of counts of 2 media.
ANALYSIS OF PRODUCT WITH POUR PLATE METHOD
 QUANTITATIVE TESTING OF PRODUCTS
EXAMINATION OF THE PRODUCT
SPREAD PLATE METHOD

Follow the the same procedures as in Pour plate

method.

The only difference is that the sample and the media

are not poured together. First media is poured into
petri dish and after it has solidified 0,1 ml of the
product is spread over the media.
 QUANTITATIVE TESTING OF PRODUCTS
EXAMINATION OF THE PRODUCT
MOST PROBABLE NUMBER
Prepare dilutions of 10-1, 10-2 and 10-3.

Transfer 1 ml each of dilution to 3 tubes containing 9

ml SCDB.

Incubate at 37 °C for 5 days.

Record the number of tubes that are positive for

growth for each dilution.
Determine the most probable number of organism
according to table.
 QUANTITATIVE TESTING OF PRODUCTS
INTERPRETATION OF RESULTS
Total Aerobic Microbial Count

TAMC Number of cfu found on SCDA.

Total Combined Yeast and Mould Count

TYMC Number of cfu found on SDA.

Acceptance criteria for nonsterile pharmaceutical

products based upon the total aerobic microbial count
(TAMC), the total combined yeasts and molds count
(TYMC) and the absence of specified microorganisms.
QUANTITATIVE TESTING OF PRODUCTS
INTERPRETATION OF RESULTS
CALCULATION OF CFU/ml

Dil Fac=10/ml per plate

QUANTITATIVE TESTING OF PRODUCTS
INTERPRETATION OF RESULTS
When an acceptance criterion for microbiological
quality is analysed, it is interpreted as follows:

If the acceptance criterion is;

101 cfu : maximum acceptable count = 20;

102 cfu: maximum acceptable count = 200;
103 cfu: maximum acceptable count = 2000
....
RECOMMENDED ACCEPTANCE CRITERIA FOR MICROBIOLOGICAL
QUALITY OF PHARMACEUTİCAL PREPARATIONS (TOTAL COUNTS)

103 102
102 101
103 102
102 101
102 101
102 101
102 101
102 101
102 101
102 101
102 101
 RECOMMENDED ACCEPTANCE CRITERIA FOR
MICROBIOLOGICAL QUALITY OF RAW MATERIALS FOR
PHARMACEUTICAL USE (TOTAL COUNTS)

103 102
 QUANTITATIVE TESTS

Quantitative testing determines only mesophilic

bacteria and fungi which grow under aerobic
conditions.

Psychrophilic, thermophilic, basophilic and anaerobic

bacteria and microorganisms which require specific
ingredients for growth may give a NEGATIVE RESULT,
even if they exist in a significant number.
 MICROBIAL LIMIT TEST METHODS

II. QUALITATIVE
TESTS
QUALITATIVE: PRESENCE OR ABSENCE
The presence of certain microorganisms in nonsterile
preparations may have the potential:
 To reduce or inactivate the therapeutic activity
of the product

 To adversely affect the health of the patient.

 To be indicative of pathogen bacteria that may

present
These certain risky microorganisms are refered to as
‘’OBJECTIONABLE MICROORGANISMS’’.
In qualitative quality control tests of nonsterile
products, the presence/absence of some indicator
‘’objectionable microorganisms’’ are investigated.
INDICATOR (SPECIFIED) MICROORGANISMS
 Salmonella
INDICATOR
 Escherichia coli ORGANISMS WHICH
USP/EP/JP PROVIDE
 Pseudomonas aeruginosa PROCEDURES AND
TEST CONDITIONS
 Staphylococcus aureus FOR DETERMINING
WHETHER THE
 Bile tolerant Gram negative
bacteria PRODUCT MEETS THE
 Clostridium ACCEPTANCE
CRITERIA OF QUALITY
 Candida albicans
 INDICATOR (SPECIFIED) MICROORGANISMS
 Salmonella Gastrointestinal system disorder (AGE)

 Escherichia coli Fecal coliform and some strains

cause GIS diseases

 Pseudomonas aeruginosa Opportunistic

infections

Skin and GIS infections

 Staphylococcus aureus
and toxic shock
Vaginal syndrome
 Candida albicans infections
 Bile tolerant Gram negative Coliform and
bacteria Opportunistic pathogen
 Clostridium Wound, intestinal and neurological infection
 INDICATOR (SPECIFIED) MICROORGANISMS
Indicators donot include all the objectionable
microorganisms found in the environment.

Other types of bacteria can also contaminate and be

responsible of product recalls.
 Burkholderia cepacia
 Acinetobacter
 Pseudomonas putida
 Bacillus cereus ......
 INDICATOR (SPECIFIED) MICROORGANISMS

Objectionable screening testing MUST NOT

BE LIMITED TO THESE INDICATORS. MUST
INCLUDE OTHER PATHOGENS OR
OPPORTUNISTIC MICROORGANİSMS THAT
MAY GENERATE HEALTH THREAT TO
CONSUMERS OR MAY SPOIL THE PRODUCT
FORMULA.

Some of the frequently isolated

microorganisms from a production facility can
be added as an indicator.
Pharmacopoeias donot include all objectionable microorganisms.
The significance of other microorganisms recovered should be
evaluated in terms of the following:
Route of administration: Hazard varies according to
the route of administration (oral,ocular, aural, topical,
injection, inhalation, rectal, vaginal ....).
The chemical composition of the product: Does the
product support growth? Does it have adequate
antimicrobial preservation?
The intended consumer: Risk may differ for
neonates, infants, the debilitated. Use of
immunosuppressive agents, corticosteroids. The
presence of disease, wounds, organ damage.
Production process: Heating, blending, filtration,
drying, shearing ....
Number of microorganism on plate: If high, then identify
 II. QUALITATIVE TESTS
Qualitative tests use selective
media for the recovery indicator
microorganisms.
Selective media cannot detect
subletally injured
microorganisms.
As subletally injured
microorganisms are relevant for
the quality of the product, A
RESUSCİTATİON
(PREINCUBATION) STEP MUST BE
INCLUDED.
 II. QUALITATIVE TESTS
TESTING OF PRODUCTS
Preparation of sample (product) is carried out as
described in quantitative methods.

If the product has antimicrobial activity it has to be

removed or neutralized by a neutralization agent
which has no toxicity on microorganisms.

Surface active substances must also have no

antimicrobial activity against microorganisms.
 II. QUALITATIVE TESTS
TESTING OF PRODUCTS
SALMONELLA
1.) Sample preparation and Preincubation:
The product is diluted 1:10 using not less than 10 gr
or 10 ml of product (as in enumeration method).

Inoculate Soybean Casein Digest Broth with 10 ml

of the sample solution.

Incubate for 18-24 hours at 35-37 C.

 II. QUALITATIVE TESTS
TESTING OF PRODUCTS
SALMONELLA
2.) Selection and subculture:
Transfer 0.1 ml of SCDB to 10 ml RAPAPORT-
VASILIADIS SALMONELLA enrichment broth and
incubate 35 C for 18-24 hours.

RAPAPORT-VASILIADIS IS A SELECTIVE ENRICHMENT

BROTH FOR SALMONELLA

From Rappaport, subculture on Xylose Lysine

Deoxycholate agar. Incubate at 35-37 °C for 18 to 48
hours.
 II. QUALITATIVE TESTS
TESTING OF PRODUCTS
SALMONELLA

There is an enrichment step

for Salmonella spp. using
Rappaport-Vasiliadis broth.
 II. QUALITATIVE TESTS
TESTING OF PRODUCTS
SALMONELLA
3.) Interpretation:
Red colonies with black centers are indicative of
Salmonella.
Confirm with biochemical tests even if the colony
morphology is different than expected.
II. QUALITATIVE TESTS
TESTING OF PRODUCTS
SALMONELLA
Rappaport Vasiliadis

Xylose Lysine
Deoxycholate
II. QUALITATIVE TESTS - SALMONELLA

SCDB RVSEM

XLDA
 II. QUALITATIVE TESTS
TESTING OF PRODUCTS
OTHER STRAINS APART FROM SALMONELLA
1.) Sample preparation and Preincubation:
For the other strains apart from Salmonella, at least
1 gr or 1 ml of the product is diluted 1:10 except for
Clostridium in which 2 gr or 2 ml of the product is
diluted.
For C. albicans Sabouraud Dextrose broth is used
instead of SCDB in preincubation for 24-48 hours.

For Clostridium, diluted (1:10) product is divided

into 2 portions of at least 10 ml. One portion is heated
at 80 C; the other is not.
 II. QUALITATIVE TESTS
TESTING OF PRODUCTS
OTHER STRAINS APART FROM SALMONELLA
2.) Selection and subculture:

Do not forget to incubate Clostridium under

ANAEROBIC conditions.
II. QUALITATIVE TESTS
TESTING OF PRODUCTS
E. coli
MacConkey Broth

MacConkey Agar
II. QUALITATIVE TESTS
TESTING OF PRODUCTS
Pseudomonas aeruginosa

Cetrimide agar
II. QUALITATIVE TESTS
TESTING OF PRODUCTS
Staphylococcus aureus

Mannitol salt agar

II. QUALITATIVE TESTS
TESTING OF PRODUCTS
Candida albicans
Sabouraud Dextrose broth
II. QUALITATIVE TESTS
TESTING OF PRODUCTS
Clostridium
MEDIA USED IN SELECTION AND SUBCULTURE FOR MICROORGANISMS
 Acceptance Criteria for NONSTERILE
Pharmaceutical Preparations and Substances for
Pharmaceutical Use
1.) Total aerobic microbial count (TAMC)

2.) The total combined yeasts and

molds count (TYMC)

3.) Absence of specified microorganism

 TESTING FREQUENCY
There is no requirement for every batch of non-
sterile medicine to be tested for microbial quality prior
to release.
Periodic testing or 'skip-lot' testing can be
performed.
The frequency of testing should be determined
based on:

 The bioburden history of the medicine

 The nature of product
 The manufacturing process for the medicine
 The controls that are inherent in GMP
 TESTING FREQUENCY
A BIOBURDEN HISTORY FOR THE MEDICINE
The first 5 to 10 batches of a new medicine should
be tested for microbial quality prior to release.
If test results for these batches are satisfactory,
then testing could be performed periodically, rather
than on every batch.
NATURE OF PRODUCTS
Some products have antimicrobial activity
Require little monitoring
Products which have higher risk of contamination
require more frequent testing than products with low
risks.
CATEGORIES OF NONSTERILE PHARMACEUTICAL
PRODUCTS
Metered-dose and dry powder inhalants
 Nasal sprays Antimicrobial
activity
Antimicrobial
preservative
Otics
Vaginal suppositories Decreasing risk
of contamination
Topicals Water activity

Rectal suppositories Exipient microbial

content

Oral liquids (aqueous)

Manufacturing
Liquid-filled capsules processes

Oral tablets and powder-filled capsules

 TESTING FREQUENCY
MANUFACTURING PROCESS FOR THE MEDICINE
Some processes include filtration, drying,
heating processes which have detrimental effect
on microorgansims.
 Such products are tested less frequently than
those whose manufacturing donot include such
processes.
CONTROLS IN GMP
 The more strictly adherence to GMP, the less
the risk of contamination and the less frequent
the testing.