This document discusses the importance of cleanroom microbiology in various industries to control contamination and maintain product sterility, particularly in pharmaceutical and medical fields. It outlines practices such as aseptic techniques, personnel control, and environmental management to minimize microbial contamination sources like air, surfaces, and personnel. It also addresses quality assurance procedures, including testing for sterility and pathogens in pharmaceutical products to ensure safety and compliance with good manufacturing practices.

1. • Sources of contamination in clean room area:
• many industries from pharmaceutical, biotechnology, medical device,
hospital pharmacies and medical disposables have clean room
operations of differing sizes and complexities.
• Understanding the concepts of cleanroom microbiology is important to
help control the cleanroom environment and ultimately ensure the
consumer is safe.Tainted environments or materials can potentially
contaminate the product harm the product efficacy, or even kill the
patient
The objective of aseptic processing is to maintain the sterility of product
during the handling or preparation process. Therefore, aseptic
processing doesn’t generate sterile outcomes from non-sterile
products, it only maintains sterility throughout the manufacturing,
&packaging,
• Cleanrooms are classified by how clean the air
•

2. Particular practices and procedures must be followed in order to
minimize and control microbial contamination in the cleanroom
environment. One of these practices is widely known as aseptic
technique, a practice used to maintain sterility and prevent the
spread of contamination.
• Some aspects of aseptic technique include:
1-Never breaking first air or reaching over exposed product,
components, or fill lines.
2-Not touching sterile items with non sterile items. Never touch a
critical surface directly i.e. Vials, stoppers and surfaces that are in
direct contact with the product. Only use sterile forceps or an
implement that has been sterilized to touch a critical surface.
3-Not exposing sterile items to nonsterile environments.
4-Using sterile components and gloves.

3. • Sources of contamination : such as, Personnel, Poor facility
design, Incoming ventilation air, Machinery and other
equipment for production, Raw material and semi-finished
material, Packaging material.,
• Clean Room is Classified into Different Grades

4. COMMON sources of contamination & control of them
1.Personnel – Personnel are the biggest source of contamination in clean areas.
Personnel harbor millions of bacteria, carrying them with them everywhere
they go. Gowning is the most effective way to protect the cleanroom
environment from ourselves. To asses the effectiveness of the gowning program
personnel may be monitored on a regular basis for viable counts. Personnel
monitoring employs contact plates to assess microbial contamination of clean
room personnel. N:B Cleanroom clothing is used to prevent substances from
being released off the wearer’s body and contaminating the environment. In
addition to gowning &personnel protective equipment ,the other main reasons
for contamination from the personnel include:
Lack of training, Inadequate personnel cleanliness, Access of unauthorized personnel into production,
storage, and product control areas, Malpractices like eating food, drinking beverages, or using tobacco in
the storage and processing areas.
Gowning should be done as Gloves (disinfected with alcohol). ,Foot wears.
Hood/mask. ,Gown (jump suit). ,Shoe covers. ,Replace the gloves with new ones.

6. • 2. Air - the air in a clean room is controlled and Monitored on a
regular basis (e.g., daily, weekly,) 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 0.3m u in size. These
filters usually run continuously at a calibrated flow rate in order to
maintain the required air quality within the room. Cleanrooms
maintain particulate-free air through the use of either HEPA
employing laminar or turbulent air flow principles..
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.

7. 3. Surfaces (including floors, walls, equipment, etc.) are cleaned and monitored on a
regular basis for viable counts by using specially designed contact plates that
contain a growth medium called Trypticase Soy Agar (TSA) and Sabouraud Dextros
Agar (SDA). The TSA is a growth medium designed for bacteria and the SDA and a
growth medium designed for mold and yeast. TSA and SDA are typically incubated at
different temperatures, 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.
There are 4 grades of cleanroom differ in the recommended limits for microbial
contamination A,B,C,D … A is aseptic area
How to Control Contamination? Three main types are there to Control Contamination
1- Environmental Control. HEPA Filters , HVAC System (Short for heating, ventilation,
and air conditioning.)& Fumigation of Area
2-Equipment &all surfaces Control…..Sterilization & Sanitization
3-Individual Control. Personnel Hygiene & Aseptic Gowning

8. 1. Air Samplers (active air sampling) Slit-to-Agar –
Airsamplers 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. It 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 .the petri dish is
incubated .then count the No. of bacterial &fungi viable count. can
accurately measure the amount of viable bacteria within the air.
Determine bioburden in 1 cubic feet area of air
Advantages:1-it allow both qualitative &quantitative analysis of air.
2-allow shorter sampling of air …10 minutes.3-ideal for controlled area
which tend to have low No. of contaminant.

9. 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. it does not reflect microbial contamination with
an accurately measured volume of air.it determine how much microbes
may settle on 90 mm surface.
Advantages:1- inexpensive .2-easy to sampling.3-offer average
contaminaion level over long sampling time reach hours .
Accordingly both methods are required to give different information
about air in the classified air.

10. surface monitoring in a Clean Room
Contact plates besides using swabs for area not easy to be reached
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. Swabbing is more qualitative than quantitative.
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. Sampling is achieved by gently rolling the domed surface of the agar onto the test
area.Periodic sampling of clothing (gowns and gloves) is used to measure the effectiveness of
aseptic precautions. Gloves can be sampled (prior to removing or replacement) by touching all
fingers and thumbs onto the surface of an agar plate .Personnel monitoring is a good indication
of how well personnel are gowning when they enter the clean room.

12. THE MICROBIOLOGICAL QUALITY OF
PHARMACEUTICALS
• Good manufacturing practice (GMP) refers to systems for ensuring that
products are consistently produced and controlled according to quality
standards. It is designed to minimize the risks involved in any pharmaceutical
production that cannot be eliminated through testing the final product.
•
Microbiological Quality: The incidence & level of contamination in
pharmaceutical products.
• Contamination might originated from raw materials or during
manufacture.Also, Products may be contaminated during storage and use.
Microbial contamination in pharmaceutical products represents potential
hazards due to:
(i) it may cause spoilage of the products.
(ii) products contamination represents a health hazards to the patient
Some of the components in pharm. Products&cosmetics encourage microbial growth.e.g thickening
&suspending agents as gums,starches .Sugers inspite high osmotic pressure allow growth of molds
&fungi.Presence of water increase M.O. differ from oils &ointements.

13. • Classes of pharmaceutical products :
• 1- Sterile Certain pharmaceutical products must be
sterile(injections, ophthalmic preparations,
irrigations solutions, haemodialysis solutions )they
are tested qualitatively for absence of microorganisms.
Since they would contaminate the tissues and cause infections. Such
products require testing for sterility as a QC procedure
• 2 categories of sterile products:
• A- those that can be sterilized in final container(terminally sterilized)
• B-those that cannot be terminally sterilized and must be aseptically
prepared
2- Non-sterile (topical and oral preparation) they are tested
quantitatively for the presence of maximum number of
microorganisms and qualitatively for absence of certain
pathogens .
•

14. In order to control microbial contaminationMicrobial limit tests are
applied to all raw materials and preparations not required to be sterile.
They include:
1)Total microbial viable count (TC) MICROBIALENUMERATION
TESTS(quantitative) The TC must not exceed a certain value.
• ENUMERATION METHODS using either Pour Plate Method or
Membrane Filtration
• TC IS the sum of total aerobic microbial count (TAMC)& total mold and
yeast count (TYMC) & other limit should absence of Specified micro-
organisms Acceptance criteria for microbiological quality of non-sterile
dosage form in EP according to Route of administration:
• Oralprep.non- aqueous TAMC= 103
,TYMC= 102
CFU/g ,absence of E.coli
Aqueous preparations for oral use 102
101
Absence of E.coli
Cutaneous use TAMC102 , TYMC
101
Absence of Staphylococcus
aureus & Pseud.aeruginosa
Vaginal use102 ,
101
Absence of Staphylococcusaureus & Pseudomonas
aeruginosa & Candida albicans

15. in conclusion the non-sterile products must not contain total microbial
count exceed 10divided to 10TABC and 10 2 mold &yeast
• The TC limit ranges between 100 and 10,0000 cfu / ml or g according
to route of administration of product
However higher levels are accepted for Herbal medicinal products .
If the product is determined to have low count of bacteria, yeast and mold, it can still be
harmful if pathogens are present even though they are very low in number. Therefore, it
should also be determined that the product does not have harmful bacteria present.
Harmful organisms and pathogens are supposed to be absent in a product .
2) Test for absence of pathogens(qualitative)
Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, bile-tolerant Gram-
negative bacteria, Clostridia species,Salmonella species and/or Candida albicans. Example:.
For topical products Ps. aeruginosa and S. aureus should be absent
For oral E. coli and Salmonella spp. should be absent.
Oral and topical preparations: (as well as cosmetics) are non sterile preparations,
although prepared by highly trained staff and traditionally contain a
preservative, to prevent infection, however,cases of infection were
associatedalso preservative prevent spoilage of product.

16. Sterile pharm.products
Ophthalmic preparations (eye drops, ointments and lotions) are
applied to infected eye.
Compendial requirements for ophthalmics were introduced including:
a) be sterile upon dispensing
b) be filled in containers which reduce the chances of microbial contamination
during storage and/or use by the patient (the use of single-dose containers
proved impractical) in small volumes (mostly not exceeding 5 ml)
c) contain a preservative which is especially effective against Ps. aeruginosa
(opportunistic pathogen that may destroy the eye), not absorbed by the
container & sufficiently powerful to more or less re-sterilize the preparation
within a few hours of re-contamination.

17. • Microbial quality of *Parenterals
• Parenterals must be sterile otherwise they would
contaminate the tissues and cause infections. Such
products require:
1-testing for sterility as a QC procedure
2-search for any reason cause non-sterile product.
3-test for pyrogen.

18. Test for pyrogen and bacterial endotoxins in pharmaceuticals and
Medical Devices :
pyrogens is 'heat or fever inducing substanceby acting on
the hypothalamic thermoregulatory center .Pyrogens
constitute a heterogeneous group of fever causing
substances which comprise both microbial and non-microbial
substances. The most potent and most widely known are the
endotoxins or lipopolysaccharides (LPS), which are cell wall
components of gram-negative bacteria. In bloodstream,when
bacteria died they are released into the surrounding
environment cause severe patient reactions (cause non-
specific inflammatory response &fever in human). Gram-
positive bacteria are also sources of pyrogens, in particular
lipoteichoic acid (LTA), also are particles from yeasts and
viruses. Non-microbial pyrogens often arise from production
environments ,a typical example is
Small particles of packaging materials .

19. • LPS is a very stable material and is not destroyed easily byheating or
other means. It can persist for very long periods in liquids and on dry
surfaces in the absence of viable bacteria. It can exist in a cell-
associated state or in a free state. Cell associated can be removed
from a solution by micro porous filters. But a free state easily passes
through sterilizing filters, heat stable.
• Sources of Pyrogen Solvents, drugs, additives apparatus used in
manufacture, containers may be sources of pyrogens. The method of
storage in between preparation and sterilization also may cause the
development of pyrogens. Hence every item must be apyrogenic and
method of storage must not allow any bacterial growth
• Clearly the presence of pyrogens in any injectable pharmaceutical
preparation or on the surfaces of medical devices that will have
contact with circulating blood or cerebrospinal fluid is a potential
hazard This means that tests specifically designed to detect
endotoxin/LPS are essential to demonstrate the safety of
pharmaceutical and medical device products

20. Tests for pyrogen and bacterial endotoxins inpharmaceuticals:
1-Rabbit test
2- LAL test(gel clot method ) is most popular and widely used method.
3-MAT
• The oldest test for pyrogen:1-The Rabbit test Rabbit Pyrogen test
Qualitative fever response test measuring the rise in body
temperature in healthy rabbits after the intravenous injection of a
sterile test solution. . Inject the solution being examined slowly into
the marginal vein of the ear of each rabbit.
Rabbits have a pyrogen tolerance similar to humans, so by observing a change
in body temperature in rabbits it is possible to make a determination of
the presence of pyrogens. Itcan detect non-bacterial pyrogens as wellas
bacterialendotoxins. itMeasures the rise in temp. of standardize rabbits(3)
upon I.V injection of a sterile solution of the subs.(or the inside of a container)
to be tested,under standardized conditions. and then monitored it to see if a
fever was induced body temperature is recorded for 180 minutes.using
rectal thermometer Interpretation of pyrogen test <0.5◦C Preparation
being examined passes the test if >0.5◦C fail

21. disadvantages
This test however does not give a quantitative result The rabbit test gives
only a pass/fail result, is ,expensive and is not suitable for products that
may themselves adversely effect the animal. :sensitivity variation .For
many drugs the volume tested is significantly smaller. A gap
between the observed pyrogenicity in rabbits and the
expected pyrogenicity in humans..
Why the Rabbit? Other species not predictable , Rabbit
chosen for economic purposes , Similar threshold pyrogenic
response to humans so reproducible pyrogenic response
• The test is still approved as a method of detecting pyrogens but is
now rarely used,

23. • 2-the LAL endotoxin test (Limulus Amebocyte Lysate):
The LAL test has its origins in the discovery in the 1950s that the blood
of the Atlantic Blue Horseshoe Crab, Limulus polyphemus forms a gel-
like clot when incubated in the presence of endotoxins this reaction is
caused by a clotting factor contained in granules in motile blood cells
called amoebocytes. This method is used to determine if products or
materials are endotoxins free. The presence of even very low levels of
bacterial endotoxin triggers a 'coagulation cascade' of reactions and
resulting in a coagulin gel clot. The product incubated along with a
standardendotoxins(obtained from E.coli) as the positive control and the
unexposed extract fluid as a negative controlAfter the incubation period for 60
minutes, the tubes containing the controls and the extract are
observed for the presence of the gel clot

24. If it clots &remain firm when inverted 180. endotoxin is present . If no clot is
observed, product is freeofendotoxin.the test can be carried on slide with incubation
20 m.
The method can be qualitative or quantitative when comparing a
sample against a dilution series of an endotoxin standard. Endotoxin is
measured in Endotoxin Units (EU). detection limit of the endotoxin
test is 0.06 EU/ml .
LAL test used for : injectable and intravenous drugs, pharmaceuticals,
biological products,vaccines as well as for screening prosthetic devices
such as heart valves or hip replacements ,used in renal dialysis centers
and a wide range of other applications .t he LAL test is now a standard
method for endotoxin testing of parenteral drugs and medical devices .
• In the past, the rabbit pyrogen test (RPT) and the Limulus Amoebocyte Lysate
(LAL) test were the most frequently applied methods to detect pyrogens. Both
methods are limited in the products and range of pyrogens which can be
detected. For example, the LAL test can only detect endotoxins. Both also have a
high level of animal consumption.

25. • Another alternative method is monocyte-activation testing (MAT),
which exploits an immune reaction found in human blood. The
method is not specific to bacterial endotoxin, but will also detect
other pyrogens. It is said to be a better indicator of the human fever
response to pyrogens than the LAL test because it is based on a
human immune response
• 3-the Monocyte-Activation Test (MAT) :In-vitro pyrogen testintroduced
in the European Pharmacopoeia) in 2010
• In short, it monitors the ex vivo reaction of monocyte to pyrogens (Using an innate
immune defense reaction, simulates the human fever reaction) in an in vitro assay
and is therefore the ideal test system for predicting the human reaction to the
pharmaceutical product or device.

26. • . Monocytes ofhuman blood are activated by any pyrogens
the sample may contain and subsequently produce
cytokines, which are detected in an immunological assay
(ELISA) involving specific antibodies and an enzymatic color
change reaction. The easily performed assay allow a broad
range of pyrogens to be detected. better than any animal-
based pyrogen test.

27. .The test sample is mixed with the leucocytes of blood and subsequently
kept in an incubator overnight at 37 °C. If any pyrogens are present in
the sample, the monocytes of the blood will produce IL-1β during the
incubation period
For the detection of IL-1β the incubated mixture is transferred into an
ELISA microplate coated with an antibody which is specific for IL-1β. Any
such interleukin molecules present are bound by the immobilized
antibody. Then an enzyme-linked polyclonal antibody and a substrate
are added. The developed color reaction allows the detection in an
ELISA reader of any bound IL-1β.
The pyrogen concentration in the sample is then deduced from the IL-1β
concentration using an endotoxin standard curve
Itcombines the advantages of the RPT (assessment of pyrogenicity
beyond gram-negative endotoxin) with the benefits of an in-vitro
method (non-animal, high-validity,easy to modify

29. • Comparison of Test principle
• Fever reaction in Rabbit
• Defence mechanism of Limulus (LAL)
• Fever reaction in human blood (MAT)
• it can be expected that the MAT will gradually
replace the rabbit pyrogen test for all those
products where contamination with nonendotoxin
pyrogens seems probable.

30. T F - virucidal agents could be evaluated by index ratio No.
Change in the slope of the phase tolerance curve indicate change in the
incubation temp. of the reaction medium.
Linearity in phase tolerance curve reflects change in the mode of action of an antimicrobial
agent.
Index ratio number depends on the measurement of
a) extinction time b) generation time
c) logarithmic time d) thermal death time