Advanced Microbiology

PHR558
Lecture 2
Microbiological quality control

Industrial microbiology (pharmaceutical industrial microbiology ) deals
with the microbiological quality control of pharmaceutical products either
sterile or non sterile and the monitoring of the industrial facilities used for
their manufacturing .

Such procedures and tests are governed by laws and regulations so as to
ensure safety of the product

Microbial contamination of a product may lead not only to spoilage of the
product, with the associated physical and chemical changes, but also to risk
of infection for the user.
Therefore, oral and topical pharmaceutical products (capsules, tablets,
suspensions, creams, patches, etc.), which are not required to be sterile,
should be subject to controls for microbial contamination.

Microbiological testing of non-sterile products
Quality assurance and manufacturing controls should be such that
organisms capable of proliferation and contamination of the product are
within acceptable limits.



Microbial limit test

The principle of the Microbial Limit Test is that each viable microbial cell
present in a sample, will, when mixed with an agar medium, and after
incubation, form a visible separate colony.
The enumeration of these colony-forming units (cfu), when taking
into account sample preparation will give an estimate of the
microbial population of the product under the specified incubation
conditions.
Quality control test for non sterile pharmaceutical product includes
the microbiological testing of raw material , final products .
Non sterile pharmaceutical products are allowed certain microbial
bioburden based upon the product specifications .
With the absence of pathogenic microorganisms that change chemical
composition by spoilage and might affect stability and integrity of the
product and package
There are 2 methods used in enumerating the microbial content of a
product. The choice of a method should be based on the nature of the
product and the required limit of microorganisms.
Membrane Filtration uses filtration apparatus.
Total Plate-Count Method (TPC) involves direct plating.
USP divided MLT into 2 types
Quantitative test
Determines number of bacteria, yeast and mold present in a
given pharmaceutical samples.
Qualitative test
Determines the presence of specific pathogen as salmonella
spp, Staphylococcus aureus , Ps. aeruginosa and
Enterobacteriaceae.

o Salmonella and E coli (toxin producers) are gram negative rods
lactose fermentors , indicates fecal contamination.
o Ps aeruginosa gram negative non fermenting rods associated with
opportunistic infection
o Staphylococcus aureus is gram positive cocci usually associated with
skin and gastrointestinal infections.


USP recommendations and limits
1. Plant animal , mineral based formula ..absence of salmonella
2. Orally administered product .. absence of E coli
3. Topical pharmaceutical preparations absence of S.aureus and
Ps. aeruginosa
4.Vaginal,rectal and urethral ..absence of molds and
yeasts

Preparatory testing
It is done to assure that the sample itself do not inhibit multiplication of
microorganisms under test conditions.
It includes the inoculation of diluted samples of the product with separate
viable cultures of Staphylococcus aureus , Ps.aeruginosa ,E.coli and
Salmonella .

Result of the preparatory test
Presence of growth ..substance can be assayed by
this methodology under test conditions
Absence of growth ..invalidate the MLT and
Necessitates modification of the procedure by
1. An increase in the volume of the diluent, with the quantity of test
material remaining the same ex: 1gm /100ml
2. Addition of suitable inactivating agent in the diluents (0.5% soy lethicin
and 4% polysorbate 20 are used to neutralize inhibitory substances present
in the sample
3. Combination of the 1 and 2


Antimicrobial Effectiveness Test (AET) - USP
Antimicrobial preservatives are often added to compendial products for
the following reasons:
1. To prevent proliferation or limit microbial contamination that may
occur subsequent to the manufacturing process.
2. To inhibit growth of microorganisms during normal conditions of
storage and use when microorganisms might be introduced
inadvertently from repeated withdrawing of individual doses from
containment.
3. To prolong the life span of the product.
The Antimicrobial Effectiveness Test is performed to prove that the
added preservative provides adequate protection from adverse effects that
may arise from microbial contamination or proliferation during storage
and use.
This test is not intended to be used for routine control purposes but it
instead designed to put a challenge on a compendial preparation in its final
container with a prescribed inoculums of suitable microorganisms.

Testing sterile pharmaceutical preparations
Sterile pharmaceutical preparations are those intended for injections and
those intended for ophthalmic ,sterile otic and nasal preparations

Sterility tests
These tests are exclusively based upon the principle that in case the
bacteria are strategically placed in a specific medium that contains requisite
nutritive material and water, and maintained at a favourable temperature
(37 2C), the microbes have a tendency to grow, and their legitimate
presence may be clearly indicated by the appearance of a turbidity in the
originally clear medium.
Please notice that Compliance with the tests for sterility individually
cannot certify absolute assurance of freedom from microbial
contamination.
Greater assurance of sterility should invariably originate from reliable
stringent manufacturing procedures vis-a-vis strict compliance with Good
Manufacturing Practices (GMPs).


Tests for sterility are adequately designed to reveal the presence of
microorganisms in the samples used in the tests.
However, the interpretation of results is based upon the assumption that
the contents of each and every container in the batch, had they been tested
actually, would have complied with the tests.
As it is not practically possible to test every container, a sufficient number
of containers must be examined to give a suitable degree of confidence in
the ultimate results obtained of the tests.

Batch : a homogeneous collection of sealed containers prepared in such a
manner, that the risk of contamination is the same for each of the units
present in it.

Sampling
Injectable Preparations
(a) Not more than 100 containers Either 10% or 4 containers whichever
is greater.
(b) More than 100, but not more than 500 containers 10 containers.
(c) More than 500 containers. Either 2% or 20 containers whichever
is less.

Sterility test could be performed either by
(a) Membrane Filtration
(b) Direct Inoculation.

Membrane Filtration
The membrane filtration method is used when you want to overcome the
activity of antibiotics for which there exist practically little inactivating
agents

The solution of the product under investigation is carefully filtered via a
hydrophobic-edged membrane filter that would precisely retain any
possible contaminating microorganisms.
(2) The resulting membrane is duly washed in situ to get rid of any possible
traces of antibiotic that would have been sticking to the surface of the
membrane intimately.
(3) Finally, the segregated microorganisms are transferred to the suitable
culture media under perfect aseptic environment.

Microorganisms for Positive Control Tests :
(a) Bacillus cerreus
(b) Staphylococcus aureus
(c) Klebsiella aerogenes
(d) Enterobacter].
, the membrane filtration is to be preferred exclusively in the case of
the following
(i) an oil or oil-based product,
(ii) an ointment that may be put into solution,
(iii) a non-bacteriostatic solid that does not become soluble in the culture
medium rapidly, and
(iv) a soluble powder or a liquid that essentially possesses either inherent
bacteriostatic or inherent fungistatic characteristic features.

The test should be carried using
(a) a sophisticated laminar sterile airflow cabinet (provided with effective
hepa-filters),
(b) necessary precautionary measures taken to be such so as to avoid
contamination that they do not affect any microbes which must be revealed
duly in the test.
(c) ensuing environment (i.e., working conditions) of the laboratory where
the tests for sterility is performed must always be monitored at a definite
periodical interval by :
sampling the air of the working area,
sampling the surface of the working area, and
perforing the stipulated control tests.
Once the filtration is completed, wash the membrane filter with peptone
water and then clise the device to introduce the liquid culture medium
Fluid Soyabean-Casein Digest Medium*, and incubate at 2025C for a
duration of seven days. And Fluid Thioglycollate Medium,** and
incubate at 3035 C for seven days.






2. Direct Inoculation [or Direct Inoculation of Culture Media]

The three usual methods being used for performing the tests for sterility
are as enumerated under :
(a) Nutrient Broth, for the aerobic microorganisms.
(b) Cooked Meat Medium for clostridium and Thioglycollate Medium, for
anaerobic
(c) Sabouraud Medium for moulds and fungi
This test is suitable for non filterable solutions like oily or viscous solution
sterile ointments Sterile containers , gloves and gauzes
1. Liquid from the test containers must be removed carefully with a with
a sterile syringe.
2. Transfer aseptically the requistite prescribed volume of the substance
from each container to a vessel of the culture medium.
3. Mix the liquid with the medium carefully taking care not to aerate
excessively.
4. Incubate the inoculated media for not less than 14 days (unless
otherwise specifically mentioned in the monograph*)



LAL test (pyrogen test )
It is done to detect the presence of pyrogen in any parentral
pharmaceutical preparation
A pyrogen is frequently described as a fever producing substance. The
most potent pyrogens originate from gram negative bacteria, which are
common water-borne organisms.
Limulus amebocyte lysate (LAL) is an aqueous extract of blood cells
(amoebocytes) from the horseshoe crab, Limulus polyphemus.


LAL reacts with bacterial endotoxin or lipopolysaccharide (LPS), which is
a membrane component of Gram negative bacteria. This reaction is the
basis of the LAL test, which is used for the detection and quantification of
bacterial endotoxins.
The primary application for LAL is the testing of parenteral
pharmaceuticals and medical devices that contact blood or cerebrospinal
fluid

A pyrogen is frequently described as a fever producing substance. The
most potent pyrogens originate from gram negative bacteria, which are
common water-borne organisms
For pharmaceutical preparations
- 1 ml of diluted solution using LAL water is injected into LAL test tube
and incubated at 60 C for 1 hr
Formation of gel indicates presence of pyrogen
Formerly rabbit test was used where dilution was injected into a rabbit and
rabbit watched for pyrexia

Antibiotic assay

The activity (potency) of antibiotics may be demonstrated under suitable
conditions by their inhibitory effect on microorganisms.
Two general methods are employed,
1. the cylinder-plate or plate assay diffusion of the antibiotic from a
vertical cylinder through a solidified agar layer in a petri dish such that
growth of the added microorganism is prevented entirely in a circular area
or zone around hole containing a solution of the antibiotic.

2. the turbidimetric or tube assay. the inhibition of growth of a microbial
culture in a uniform solution of the antibiotic in a fluid medium that is
favorable to its rapid growth in the absence of the antibiotic.

The potency of antibiotics is designated in either Units or
g of activity
The corresponding USP Reference Standard is Each antibiotic is
tested against a specific microorganisms listed used in the
pharmacopeia eg
Microbe under test is inoculated in a concentration of 1 % in muller
hinton agar which do not interfere in activity of all known antibiotics
the seeded agar is left to solidify
Holes are formed using 8 mm cork borer
Then certain volume of serial dilutions of the test and standard is
instilled into the holes left for one hour to diffuse then incubated for
24 hrs the potency is calculated through known formula by using
diameter of inhibition zones



Environmental monitoring

Environmental Monitoring (E/M) is a program designed to demonstrate
the control of viable (living microorganisms) and non-viable particles in
critical areas.
These areas include clean-rooms for drug fill/finish, formulation tank
rooms, laminar flow hoods, molding machines, kit assembly lines,
Intravenous (IV) compounding areas and sterile packaging.

Viable monitoring is :
testing for the detection and enumeration of bacteria, yeast and mold.

It includes the monitoring of personnel, air and area surfaces for
microbial contamination.
Non-viable environmental monitoring is particle counts measured
by a laser counter.


The items that are sampled in a manufacturers clean room include
1. Personnel Personnel are the biggest source of contamination in clean
areas. Personnel harbor bacteria, carrying them with them everywhere they
go. Gowning is the most effective way to protect the cleanroom
environment from ourselves.. Personnel monitoring employs contact
plates to assess microbial contamination of clean room personnel.

How Personnel are monitored in a Clean Room
Contact Plates - Personnel in critical areas may be monitored for microbial
contamination utilizing contact plates. The contact plates monitor areas of
the body that may interact with the sterile field or product exposure areas.
These may include gloved hands, forearms, or other areas. Personnel
monitoring is a good indication of how well personnel are gowning when
they enter the clean room. .
2. Air
the air in a clean room is controlled and Monitored on a regular basis (e.g.,
daily, weekly, quarterly) for particle counts, viable counts, temperature and
humidity.
HEPA filters are used to control the viable and non-viable particulate
counts within the air. HEPA filters have the capability to filter out
particulates down to 0.2 2m in size.
These filters run continuously at a calibrated flow rate in order to maintain
the required air quality within the room. Humidity is usually kept at a low
level in order to help prevent the proliferation of microbes within the room
such as bacteria and mold, which tend to prefer damp conditions in order
to replicate.
Two methods of Air sampling in a Clean Room
1. Air Samplers (active air sampling) Air samplers draw in
predetermined volumes of air. The air is drawn over a sterile media plate,
which is later incubated to reveal the number of viable organisms per cubic
feet or liter. Currently agar impaction is the method of choice throughout
the industries. Using a specially designed, and calibrated piece of
equipment which holds the media plate under a perforated lid and draws
in a known amount of air one can accurately measure the amount of viable
bacteria within the air.

2. Settling plates (passive air sampling) - Petri dishes containing sterile
growth media are exposed to the environment for a specific period of time,
usually between 30-60 minutes but can be exposed up to four hours before
compromising the integrity of the media itself. Viable microorganisms
which settle onto the media surface will grow after the plates are
incubated. However, passive air sampling is tending to be phased out
because it does not reflect microbial contamination with an accurately
measured volume of air.
3. Surfaces (including floors, walls, equipment, etc.) are cleaned and
monitored on a regular basis for viable counts by using specially designed
contact plate
Two methods for surface monitoring in a Clean Room
1. Contact Plates are special Petri dishes which contain sterile growth
medium prepared in a manner so the surface of the media protrudes above
the rim of the plate. These plates that contain a growth medium called
Trypticase Soy Agar (TSA) and Sabouraud Dextros Agar (SDA, TSA at 30-
35 C which is mainly the optimal growing temperature for most
environmental bacteria, and 20-25 C which is the optimal growing
temperature for most mold and yeast species.
The contact plate is pressed against any flat surface the needs to be
sampled. Any viable microorganisms on the surface will stick to the agar
surface and will grow upon proper incubation. This technique reveals the
number of viable microorganisms on a surface.
2. Swabs - are sterile and stored in a suitable sterile liquid. The swabs are
rubbed over the test surface. The microbiologist can determine the type of
microorganisms on the swab by subculturing it to media. Swabs are used
for surfaces that are not flat, and can be used to sample hard to reach areas
of machinery that could not be sampled with a contact plate. Swabbing is
more qualitative than quantitative.

Alert and Action levels should be implemented based on your products,
the intended use of the clean room and the classification of the clean
room.grades A (class 100), B (class 1000), C (class 10,000), and D (class
100,000). Sterile area is 10 under laminar flow.