General Principal of Instrumentation and Working in

Pharmaceutical Microbiology Department

In Partial Fulfillment of the Requirements for the Award

Of
Master of Business Administration (MBA)

Submitted by

Ms. GEETA SINGH

1
 CERTIFICATE

This is to certify the dissertation entitled “General Principal of instrumentation and

working in pharmaceutical microbiology department” Submitted by ‘Geeta Singh’ in
partial fulfillment of the requirement for the degree of Master of Science in
Biotechnology, KIIT School of Biotechnology, KIIT Deemed to be University,
Bhubaneswar bearing Roll No. ‘1861010’ & ‘18340562182’ is a bona fide research work
carried out by his/her under my guidance and supervision from ‘16/01/2020’ to
‘18/03/2020’.

(Research Supervisor full Signature)

Full name and Designation

2
 CERTIFICATE

This is to certify that the dissertation entitled “General Principal of instrumentation

and working in pharmaceutical microbiology department” submitted by ‘ Geeta
Singh’, Roll No. 1861010,Registration No. ‘18340562182’ to the School of
Biotechnology, KIIT Deemed to be University, Bhubaneswar-751024, for the degree of
Master of Science in Biotechnology/ Applied Microbiology is his/her original work,
based on the results of the experiments and investigations carried out independently by
him/her during the period from ‘16/01/2020’to ‘18/03/2020’ of study under my guidance.

Further, it is also to certify that the above said work has not been previously submitted
for the award of any degree, diploma, or fellowship in any Indian or foreign University.

Date: (Supervisor Signature)

Place: (Supervisor Name)

3
 AKNOWLEGDGEMENT

I express my deepest sense of gratitude to the allowing me to carry out my dissertation

work at Renova life science pvt. Limit. Rajkot (Gujarat).

I avail this opportunity to thanks my project guide Mr. Ankit Sheth. I am very much
thankful to Mr. Dixit Sitapara, Microbiology Laboratory who have played a deciding role
in the design of my study and constantly guided me throughout the course of the entire
project. I am grateful to them for giving me an opportunity to work in his lab. His vast
knowledge and whole hearted commitment have always inspired and motivated me.

I am extremely grateful to the Director, Dr. Mrutyunjay Suar, Dr. Biswadeep Das, Course
Coordinator of M.Sc. Biotechnology, KIIT Deemed to be University and all the faculties
for their helpful suggestion and timely advice during my dissertation project.

A special thanks to all my friends who always encourage me and walk by my side
throughout my work and helped me to tide over the difficult situations.

At last I extend my deep sense of gratefulness to my loving parents, and family for their
understanding, motivation, constant support that they provided me during my dissertation
work and for their blessings in the completion of my thesis.

4
 Abstract

The aim of our thesis is testing of eyes drop because no any unwanted organism growth
within their particular time. We are not need to any organism and contamination so that
reason the company in our microbiology department focus on eye testing and in this
department provided test i.e. MLT, Sterility, Environmental monitoring, Personnel
monitoring.

In MLT test, in these test for the E.Coli detection from MC broth after sub culturing in
MC agar plate, for the Staphylococcus aureus detection from Mannitol Salt Agar media
with the help of after testing the eye drop follow some standard before production and
packing. In our ophthalmic unit microbiology lab also performed Sterility test for last
procedure testing of eye drop product.

In this test mainly used SCDM and FTM media used these test help for ensuring the
presence or absence of viable micro organism in our eye drop taken from batch of
product and all over ophthalmic unit HEPA filter are placed because of it protect from
contamination and personnel from exposure to contaminated substance. The companies
have several departments for testing of our product. For example (micro department, QC
department, QA department). If the product will be pass by micro department it means no
any growth in our eye drop product then sample product further proceed to other
department.

When SCDM plate exposure in production B and production C area for 4 hrs, if the
microorganism present in their out of the range accordingly production then production
area will be suddenly shut down. For the controlling of micro- organism we have to done
personnel monitoring and also fogging and defogging procedure follow through the
production department.

All production area classified into four class, these are as follows:

Class D and range of classes D - 100000 cubic meter/particles

Class C range of classes C - 10,000 cubic meter/particles
Class B range of classes B - 1000 cubic meter/particles
Class A range of classes A - 100 cubic meter/ particles

5
 Contents
Abstract..........................................................................................................................................5
List of Abbreviations.......................................................................................................................8
List of Figures..............................................................................................................................10
1 Chapter-1: Company Profile.................................................................................................12
2 Chapter-2: Microbiology Department Instrument................................................................14
2.1 PH Meter.......................................................................................................................14
2.2 Electronics Balance.......................................................................................................15
2.3 Autoclave......................................................................................................................17
2.4 Hot Air Oven................................................................................................................18
2.5 BOD Incubator..............................................................................................................19
2.6 Bacteriological incubator..............................................................................................21
2.7 Laminar Air Flow.........................................................................................................22
2.8 Colony Counter.............................................................................................................24
2.9 Pass box........................................................................................................................25
3 Chapter-3: Preparation of Media..........................................................................................27
3.1 Nutrient medium:..........................................................................................................27
3.2 MacConkey Agar:.........................................................................................................28
3.3 Sabouraud Dextrose Agars (SDA):...............................................................................29
3.4 XLD Agar (Xylose Lysine Deoxycholate Agar):..........................................................30
3.5 Mannitol Salt Agar (MSA):..........................................................................................32
3.6 Cetrimide Agar:............................................................................................................33
4 Chapter-4: Growth Promoting Test Media...........................................................................35
4.1 Growth promoting test:.................................................................................................35
5 Chapter-5: Water testing......................................................................................................38
5.1 Tests for Total Microbial Count:...................................................................................38
5.2 Test for Microbial limit test:.........................................................................................40
5.3 5.3 Sampling of water:..................................................................................................42
6 Chapter-6: Environmental Monitoring.................................................................................44

6
 6.1 Settling plate method:...................................................................................................44
6.2 Personnel Monitoring:..................................................................................................46
6.3 Glove Prints (Finger dabs):...........................................................................................46
6.4 6.4 Swab Sampling.......................................................................................................47
6.5 6.5 Contact Plates:........................................................................................................48
7 Chapter-7: Fogging and Defogging.......................................................................................50
8 Chapter-8: Discussion..........................................................................................................51
9 Chapter-9: Result................................................................................................................520
10 Chapter-10: Conclusion………………………………………………………………………………………………………53

References....................................................................................................................................54

7
 List of Abbreviations

SOP= Standard operating procedure

MLT=Microbial limit Test

QA=Quality Assurance

QC=Quality Control

IPA=Isopropyl alcohol

PLC=Programmable Logic Controller

Kg/cm2=Kilogram per square centimeter

°C= Degree Centigrade

Mm= millimeter

PI=Process and Instrumentation

SYS=Systronics

CAL=Calibration

Y= Yes

N=No

UV= Ultraviolet

OP=Operation

8
WFI = Water for injection

% = Percentage

M/ V = Weight by volume

SS = Stainless Steel

Mm = millimeter

Gm = Gram

N = Normal

M = Molar

HEPA= High efficiency particulate air

SCDA= Soybean casein digest agar

SCDM= Sobouraud digest agar

SDA= Soybean casein digest medium

XLD= Xylose-lysine

MA= Maccokency agar

MC=Maccokency agar

NA= Nutrient agar

FTM= Fluid Thioglycollate medium

CA= Cetrimide agar

RV= Reinforced medium clostridia

MSA= Mannitol salt agar

Peptone water

9
Peptone Bacteriologicals

List of Figures

Chapter2:

Figure-2.1 PH meter

Figure-2.2 Electronics Balance

Figure-2.3 Autoclave

Figure-2.4 Hot Air Oven

Figure-2.5 BOD Incubator

Figure-2.6 Bacteriological incubators

Figure-2.7 Laminar Air Flow

Figure-2.8 colony counter

Figure-2.9 pass box

Figure-2.8 colony counter

Figure-2.9 pass box

10
Chapter3:

Figure-3.1 Nutrient Agar

Figure-3.2 MacConkey Agar

Figure-3.3 Sabouraud Dextrose Agar

Figure-3.4 Xylose Lysine Dexoxycholateagan

Figure-3.5 Mannitrol Salt Agar

Figure-3.6 Cetrimide Agar

Chapter4:

Figure-4.1 Growth Promoting Test

Chapter5:

Figure-5.1 Membrane Filtration

Figure-5.2 (A) MC Broth

Figure-5.3 (B) RV Broth

Chapter 6

Figure-6.1 Environmental Monitoring

Chapter 9

11
Figure 9 (A): Laboratory plates Isolated on SCDA plates and observed after 48
hrs.

Figure9 (A): Production C Isolated on SCDA plates and observed after 48 hrs.

Chapter-1: Company Profile

Renova Life sciences Private Limited started its operations in the year 2005 as a domestic
marketing company based in Rajkot, Gujarat. It started with marketing of 8 ophthalmic
products in Gujarat. Over a period time the product range grew to 27 products portfolio.
In 2009 it launched Dermatology marketing division and started marketing its products in
states of Gujarat, Karnataka, Kerala and Madhya Pradesh. Backed by excellent CMO
facilities the business grew year on year. In 2011 Renova filed 5 dossiers of products in
International markets. The first breakthrough in Exports came with the orders in 2013.
Company nearly doubled its revenue in 2 years by 2015. For better compliance company
decided to have its own manufacturing facility and hence started construction of a
Modern formulation plant in Rajkot. The new plant is spread across 27,000 sq. Feet
carpet area of manufacturing, warehouse, QC Laboratory and Microbiology Laboratory.

12
With ultra modern equipments the company boasts of a robust facility available for
contract manufacturing. Now the company has a dedicated QC, Microbiology,
QA and Regulatory team to back its robust product ranges.

With business spread across 17 countries in export markets and 10 states in domestic
markets the company has a vision to reach to newer and newer heights year on year.
Renova now manufactures products for some of the Top companies of India and abroad.

Dedicated workforce, F & D, Superb knowledge of its formulations, Ultra modern

production techniques, highest levels of quality control machines, great regulatory
compliances ensures Renova leads the path it has chosen for itself.

Being an ISO 9001:2015 certified organization and recently audited for WHO GMP
Certification Renova sees a great future ahead.

Vision:
Renova strives to see a world in which each of the disease and ailments are tackled by
innovative, quality and affordable solutions, specially developed to ensure the quality
of life that mankind deserves. In this way Renova strives to become a leading
Pharmaceutical Company offering a first truly specialized commercial platform for all
the therapeutically segments.

Mission:
Our mission is to provide access to safe, effective and affordable medicines and related
health care services to the whole of Mankind. At Renova, our mission of curing and
caring highlights our work, our commitment and our confident steps across the globe.

Values:
In order to align our mission and vision with Renova strategy we have carved our
strong values which define our culture.

13
Strategy:
Our strategy at Renova is to be dedicated and committed to improve good healthy life
with the help of all our knowledge of science. We aim to be a front runner in
healthcare.

Chapter-2: Microbiology Department Instrument

2.1: PH Meter

A pH meter is a simple and speedy device to measure the acidity and alkalinity of a fluid.
When pH meter are used for the volt meter that measure the electronic potential
difference between a pH electrode and reference electrode and a reference and display the
result in it term of the pH value of the solution in which they are immersed. The PH value
of solutions ranges from 3.
The solution having ph value 1 will be acidic, 1 will be Neutral and 1 will be basic. The
acidity and alkalinity of any solution depend upon the concentration of hydrogen ions and
hydroxyl ions respectively. A natural solution as pure water has PH 7.
PH meter used to determine the ph of different solutions in pharmaceuticals. It is a more
accurate method then the ph strip.
In the microbiology department of quality control here they do the daily calibration to
check the proper working of the ph meter. The buffer used for the daily calibration is 4.0,
7.0 and 9.2. Always keep the electrode dipped in 4.0 Ph buffer solutions when not in use.
That time they use three probes for the daily calibration.
Calibration with Standard Buffer Solution:

Auto Made Calibration:

Switch on the instruments. Press the 'CAL' key, the unit will show CAL 2 pt. Press CAL
key for selection of 3 point calibration.
ENTER the key, the display will show put 7.0 PH Dip electrode and temperature probe
in a 7.0 pH buffer solution. Press the ENTER key, display BUSY for a few seconds.

2nd Point Calibration for pH-"4.0"

14
ENTER key, the display will show put 4.0 PH Dip electrode and temperature probe in
4.0 buffer solution. Press ENTER key, display a few seconds.

3rd Point Calibration for pH-"9.20"

ENTER key, the display will show PUT 9.2 PH. Dip electrode and temperature probe in
9.2 pH buffer solution. Press ENTER key, display BUSY for a few seconds and screen
will display "calibration done".

PROCEDURE:
1. Instrument in on and the calibration is done. The display will Display SYS 361
message.
2. ENTER the key and the press "pH" mode.
3. It will show "PUT THE SOLUTION".
4. Wash the electrode with distilled water and Dip the electrode in the solution.
5. ENTER key.
6. The display flashes the BUSY message after a few second displays will show the
temperature and pH of the solution simultaneously.

Fig: 2.1 PH Meter

15
2.2: Electronics Balance

Electronic balances are used for the measure the weight .When balance measure the mass
of an object and are used in science. In many industrial and commercial application
scales and balance to determine the weight and mass of thing raining from feather to
loaded tractor trailer.
Weight balances are instruments that are used to weight a given sample with high
accuracy and precision. Weight of an object various with respect to gravity where as
mass of an object remain constant. The process of determine both mass and weight is
called weighing. A zero indicates recognize any minimal deviation and immediately so
much current that the balance beam hardly moves and remain in its neutral position.
In the microbiology department of the quality control department, they do daily
calibration and balance check of the weighing balance and record it in the weighing
balance format.

Procedure:
1. Clean the balance.
2. The spirit level is in the center of the circle.
3. Main switch 'ON'.
4. Press 'ON' key, all the display will glow.
5. Presses TARE KEY, 0.0 marks appear on the display.
6. The stability is attained, the balance is ready.
7. Keep 10 gram weight in the center.
8. Keep 200-gram weight in the center.
10. Keep 500-gram weight in the center
11. The reading obtained should be in range within the standard value.

16
 Fig: 2.2 Electronics Balance

2.3: Autoclave
The autoclave is a used to remove microorganism (virus, bacteria, fungus etc) and spore
using high pressure and high temperature stem sterilization. The autoclave must reach
and maintain a temperature of 121dgree centigrade for at least 30 minutes by using
standard steam under at least 15 psi of pressure. Increases cycle time may be necessary
depending upon the make-up and volume of the load.
Autoclave sterilization is used to certain biological waste sterilize media, instruments,
and lab ware. Regulated media waste that might contain bacteria, viruses, and other
biological material is recommended to be activated by autoclaving before disposal.
Procedure:
1. The power supply to the autoclave is switched OFF.
2. Drained used water through the drain valve after the chamber is empty.
3. Rinse the chamber with purified water so that the residual matters/particles are
flushed out.
4. After the chamber is empty, clean the chamber with 70%IPA.
5. The drain valve and fill purified water up to the optimum level where heater cover
dipped into the water.

17
6. The growth containing media and culture into disposal biohazard bag as per SOP for
media disposal of growth containing media and culture .keep it into the vertical
autoclave.

Fig: 2.3Autoclave

2.4: Hot Air Oven

An oven provides a temperature higher than that of the atmosphere. The temperature
range covered by ovens is between 70 -85°C. These are used for rapid evaporation of
material, rapid drying and for sterilization of articles that can be sterilization by dry heat.
High temperature than moist heat temperature. Sterilization by dry heat is accomplished
by conduction. The heat is absorbed by the outside surface of the item and then passes
toward the center of the item, layer by layer. The inter-item will eventually reach the
temperature required for sterilization to take place. Dry heat process is claimed to
produce both serial and progeny free commodities, validation studies must be done using
both micro organism and microbial condition. The goal is to validation a heating cycle
that can produce a 12 log reduction in the biological indicator population.
Procedure:
1. Cleanliness of the instruments.
2. Open the validation knob provided on top of the oven.
3. Switch "ON" the power supply.
4. The electronic temperature controller displays the chamber temperature.

18
 5. Set the required temperature by pushing the "PUSH" switch and the first
potentiometer know clockwise or anticlockwise until the temperature comes
to set on.
6. The temperature with the help of the second potentiometer knows.
Release the "PUSH" switch.
7. Indicator bulb glows indicates that the power to the heater is "ON".
8. Switch "ON" the fan switch for air circulation.
9. Use a rotary switch for price control of temperature.

Fig: 2.4 Hot Air Oven

2.5: BOD Incubator

BOD incubators are often called low-temperature incubators used for the growth of yeast
and mold as they require low temperature to grow. This incubator is called
BOD(Biological oxygen demand) because in biological oxygen demand testing there is a
need for a low temperature around 20-25˚C so don't confuse with the term. After all, the
purpose of the BOD incubator is also the same as a bacteriological incubator. The BOD
incubators are both cooling and heating systems that are different from a biological
incubator. BOD incubators are not affected by the surrounding environment.

19
Procedure:
1. Clean the external surface using a lint-free cloth with 70% IPA; clean the entire
internal surface using a lint-free cloth with 70% IPA.
2. The power cord is connected to the incubator. Switch on the mains.
3. Select the set button.
4. The desired temperature with the help of up and down arrow key.
5. Press ENT.
6. The temperature twice per day, temperature shell be recoded which is displayed
on LCD of the controller of the incubator.

Fig: 2.5 BOD Incubator

20
2.6: Bacteriological incubator
The bacteriological incubator is the laboratory equipment that is used for the incubation
of biological products under controlled conditions. This medical laboratory equipment is
available with a digital temperature controller with a thermocouple sensor for better
temperature accuracy.
They are insulated enclosures that are thermostatically regulated to maintain a constant
temperature. Hot air are circulated the racks or shelves of the incubator. The containing
samples are Petri dish flasks and other culture media.
It is generally available with double-walled construction and is made of different
materials. It is a measure device is the perfect product for reliable day to day operation,
draying staining and incubation of antibiotic tests for microbial determination.
In this type of incubator, environmental conditions, such as temperature and humidity,
can be controlled for growing bacterial cultures (32°C), hatching eggs
artificially/providing suitable conditions for a chemical or biological reaction.

Procedure:
1. Clean the external surface using a lint-free cloth with 70% IPA, clean the entire
internal surface using a lint-free cloth with 70% IPA.
2. The power cord is connected to the incubator. Switch on the mains.
3. Press the SET button.
4. Set the desired temperature with the help of an up and down arrow key.
5. Press ENT.

Fig: 2.6 Bacteriological incubators

21
2.7: Laminar Air Flow
Laminar air flow closet or tissue hood is a carefully enclosed bench. Designed to prevent
contamination of semiconductor wafers, biological samples or any particles sensitive
materials. When air to pass through a HEPA (High Efficiency Particles Air) filter which
removes all airborne contamination to maintain sterile condition.
Laminar flow hood consist of a filter pad fan and a HEPA filter. After that profiteered air
has to pass the HEPA filter where contaminating fungi, bacteria, dust etc are removed.
Sterile air flow into the working area where you can do all your flaking work without risk
of contamination. Laminar flow cabinets may have a UV-C germicidal lamp to sterilize
the interior and contents before usage to prevent contamination of the experiment.
Germicidal lamps are usually kept on for 15 minutes to sterilize the interior and no
contacts are to be made with a laminar flow hood during this time. In this time scientists
are normally prepare other material to maximize efficiency
Procedure:
1. Clean the outer surface and workbench with a lint-free duster by mopping with
70% IPA before start-up work.
2. Switch "ON" the LAF blower and UV light at least half an hour before starting
works.
3. After the start, ensure that the pressure on the display shall be as per limit.
4. The display initially will show 7-15 mm WC which is in the limit.
5. If the pressure is not in a range, the display indicates red light and beep sound
continuously.
6. As pressure drop against HEPA filter reaches 25 mm of WC stop using until the
HEPA filter is replaced with a new one.
7. The lower range of pressure indicates the puncturing of the HEPA filter. Replace /
Repair the punctured filter.
8. Switch OFF the UV light after minimum half an hour, Switch ON visible light
and start the gas burner with the help of gas lighter and carry out the work.
Remember that LAF blower will ON during work.

22
 9. After completion of work clean the workbench with 70% IPA with a lint-free
duster, "OFF" the visible light.
10. Switch "ON" the UV light minimum half an hour after completion of work
11. Switch "OFF" the LAF blower and UV light at least half an hour completion of
work

Fig: 2.7 Laminar Air Flow

2.8: Colony Counter

Digital Colony Counter is designed for quick and accurate counting of bacterial and mold
colonies in Petri dishes. Feature-packed and easy to use, this is an indispensable bench
top tool for the busy microbiologist. It is designed for rapid and accurate counting of
bacterial and mold colonies. Simply place the Petri dish on the illuminated pad and touch
the dish with the pen provided to mark each colony in turn.
This causes the account to be registered on the digital display and audible tone confirms
each count made. Marking the dish with the pen avoids missing colonies or doubles
counting. The digital count on the display can be reset manually at any time by pressing
the RESET key provided. Optimum viewing of colonies is aided by peripheral glare-free
illumination. An integral magnifying glass provides for easier counting of small colonies.

23
Procedure:
1. Clean magnifier glass, plate holder and other outer surfaces with 70% IPA with a lint-
free cloth.
2. Connect the plug to the main supply, power ON the supply.
3. Switch ON the main switch.
4. Show 0000 on the digital display, if not displayed press reset switch.
5. Place an invert Petridis on the illuminated dish holder.
6. Check whether the auto mark is connected to the colony counter.
7. Arrange magnifier with suitable height and adjust brightness from which small colony
will clear visual.
8. Remove the cap of the auto mark and touch the marking point of a probe on the invert
Petridis where a colony is located.
9. Press the probe gently. The counter will register a count with a beep sound and ink dot
will appear on the Petridis. Continue till all the colonies are counted.
10. When the counting is over, note down the reading from the digital display.

Fig: 2.8 Colony Counter

24
2.9: Pass box
A pass box reduces the need for personal to transfer item through the door. The pass box
is usually installed on the walls of the clean room act as a temporary storage area to
transfer any material into or out of the clean room.

There are two type of pass box:

1. Dynamic pass box
2. Static pass box
The dynamic pass box is a cubic box that has got interlocked door located on both sides.
A dynamic pass box has got a system know as an interlock guard which helps to control
both the outlet and the inlet so that there is no time that the two are opened. Due to this,
dirt and other loose particles are removed from the material being transferred to the
manufacturing area. A dynamic pass box is a filter with a suction filter of around 0.3
microns that is a stainless steel product and a supply filter of about 10 microns that is
made of aluminum. They have a pressure gauge ranging from 0 to 25 mm.
The static pass boxes are known to transfer material between two clean environments
which are equally clean and designed to work with minimal personal movement. A static
pass box should never be used to transfer material between a clean room and now a clean
room. The static pass box doesn't have the filter. It has designed as an airlock to prevent
ambient air from entering or clean air from disappearing from the clean room.

Procedure:
1. Firstly we can clean the pass box with 70% IPA Solution
2. Make surface dry by wiping the surface with an only clean lint-free cloth.
3. Clean the pass –boxes before starting usages for the first time a day.
4. Close the door o0f pass box.
5. Switch on the UV light.
6. Switch on the UV light for at least 30 min daily before using the pass box.
7. Open the door from one side and place the material to be transferred into the pass box.
8. Close the door carefully, ensuring that the interlocking system has been interlocked
properly.

25
9. Remove the material from the pass box by opening the door of the opposite side.
10. Close the door carefully after the transfer is completed again ensuring that the
interlocking system has been engaged.
11. Both the door should not be opened simultaneously at the same time.

Fig: 2.9 Pass box

26
 Chapter-3: Preparation of Media

Medium:
Any preparation that contains nutrients essential for bacteria growth is called medium.
Any medium that has been successfully inoculated with bacteria is called medium.
Bacteria are capable to growing of media.

3.1: Nutrient medium:

Nutrient medium are used for the cultivation of microbes supporting growth of a wide
range of non-fastidious organisms. Nutrient medium are grow various type of bacteria
and fungi.

Table-3.1: Composition of Nutrient medium

Ingredients Amount
Peptone 0.5 gm
Proteose peptone 3 gm
Lactose 10 gm
Bile Salts 1.5 gm
Sodium Chloride 5 gm
Neutral Red 0.03 gm
Crystal Violet 0.001 gm
Agar 13.5 gm
Distilled Water 1 liter

When pH is maintain in 7.4.

27
 Fig3.1: Nutrient Agar

Uses of Nutrients Agar:

1. It is used for isolation and purification of cultures.

2. It can also be used as a means for producing the bacterial needed for antibiotic
sensitivity tests.

3.2: MacConkey Agar:

MacConkey agar is a selective and differential media. It is used for the isolation of gram
negative bacteria and the differentiation of lactose non fermenting gram negative
bacteria.

Table-3.2: Composition of MacConkey Agar

Ingredients Amount
Peptone 17 gm
Proteose peptone 3 gm
Lactose 10 gm
Bile Salts 1.5 gm
Sodium Chloride 5 gm
Neutral Red 0.03 gm
Crystal Violet 0.001 gm
Agar 13.5 gm
Distilled Water 1 liter

28
When pH is maintain in 7.3.

Fig3.2 MacConkey Agar

Uses of MacConkey Agar:

1. MacConkey ager is used for the gram negative bacteria.

2. It is used for the lactose fermentation for non forming gram negative bacteria.

3.3: Sabouraud Dextrose Agars (SDA):

Sabouraud Dextrose agar is used for the isolation and cultivation of pathogenic and non
pathogenic species of fugues and Yeasts.
Table-3.3: Composition of Sabouraud Dextrose Agars

Ingredients Amount
Peptone 10 gm
Dextrose 40 gm
Agar 15 gm
Distilled Water 1000 ml

When pH is maintain in 5.6.

29
 Fig3.3: Sabouraud Dextrose Agars

Uses of SDA:
1. It is used for the selective cultivation of yeasts, molds and acid uric bacteria.
2. This is used for antibiotics isolation of pathogenic fungi from material containing
large numbers of other fungi or bacteria.

3.4: XLD Agar (Xylose Lysine Deoxycholate Agar):

XLD agar is selective and differential medium. It contains yeast extract as a source of
nutrients and vitamins. It is used to sodium deoxycholate as the selective agent therefore
it is a gram positive microorganism.

Fig3.4: Xylose Lysine Deoxycholate Agar

Table-3.4: Composition of XLD Agar (Xylose Lysine Deoxycholate Agar)

30
 Ingredients Amount

Lactose 7.5 gm

Sucrose 7.5 gm

Sodium Thiosulfate 6.8 gm

L-Lysine 5.0 gm

Sodium Chloride 5.0 gm

Xylose 3.75 gm

Yeast Extract 3.0 gm

Sodium Deoxycholate 2.5 gm

Ferric Ammonium
0.8 gm
Citrate
Agar 15.0 gm

Phenol Red 0.8 gm

When pH is maintain in 7.40.

Uses of XLD Agar:

1. It is a selective differential medium for the isolation of Gram-negative enteric
pathogens from fecal specimens and other clinical material.
2. It is especially suitable for the isolation of Shigella and Salmonella species.

3.5: Mannitol Salt Agar (MSA):

Mannitol Salt Agar (MSA) are reselective and differential medium. The isolation and
identification of staphylococcus. It encourages the growth of a group of certain bacteria
while inhibiting the growth of others.

31
 Table-3.5: Composition of XLD Agar

Ingredients Amount
Pancreatic Digest of
5.0 gm
Casein
Peptic Digest of Animal
5.0 gm
Tissue
Beef Extract 1.0 gm

Sodium Chloride 75.0 gm

D-Mannitol 10.0 gm

Phenol Red 0.025gm

Agar 15.0 gm

When pH is maintain in 7.4.

Fig3.5 Mannitol Salt Agar

Uses of Mannitol Salt Agar:

1. It is used for the selective isolation and differentiation of Staphylococcus aureus from
clinical samples.
2. This medium is also included in the Bacteriological Analytical Manual for cosmetics
testing.

32
3. It is also used in the bacteriological examination of swimming pool water, spas and
drinking water using membrane filtration

3.6: Cetrimide Agar:

Cetrimide Agar is a selective and differential medium used for the isolation and
identification of Pseudomonas aeruginosa from clinical and non-clinical specimens.

Table-3.6: Composition of Cetrimide Agar

Ingredients Amount
Pancreatic Digest of
20.0 gm
Gelatin
Potassium Sulfate 10.0 gm

Magnesium Chloride 1.4 gm

Cetyltrimethylammonium
0.3 gm
Bromide
Glycerine 10.0 gm

Agar 13.6 gm

Distilled Water 1000 ml

When pH is maintain in 7.4.

33
 Fig3.6: Cetrimide Agar:

Uses of Cetrimide Agar:

1. It is primarily used for the selective isolation and presumptive identification
of Pseudomonas aeruginosa from clinical and nonclinical specimens.
2. It is also used for determining the ability of an organism to produce fluorescein
and (Antibiotic)

Procedure:
1. Weight and transfer to the media in 1000ml containing flask purified / distilled water.
2. Heat to boil to dissolve the media completely.
3. Pour about 100ml media into flask, al the flask should be sterilize by autoclave at
121˚C for 20 minutes in Gravity cycle.
4. After the completion of autoclave cycle cool the media at room temperature.
5. Check the pH.

34
 Chapter-4: Growth Promoting Test Media

4.1: Growth promoting test:

Growth promoting testing of the microbial culture media (solid and liquid) used in the
microbial analysis for nutrient quality used different microbial cultures as per USP and
precautions taken during the growth-promoting test.
1. The growth-promoting test of newly incoming dehydrate media or whenever require
specific requirements.
2. Growth promoting tests also perform when used media above one month. Prepare the
suspension of the organism by taking loopful growth from requiring slant of the
respective organism in 1ml of normal saline.
3. Uses test suspension of the organism within 2 to 4 hours if not use then store 2˚to 8˚ in
the refrigerator.
4. Prepare a serial dilution of suspension by adding 1ml of the suspensions in 9ml
consist of normal saline test this called 10-1 dilution, then add 1ml from 10-1to
another 9ml normal saline test tube this called 10-2 dilute after prepare 10-3,10-4 to
10-4 dilution same as discussed above procedure.
5. After serial dilution takes 10-5 to 10-9 dilution tube and pours 1ml on sterile Petri
dish separately for responsible dilution tube, pour 15-20ml approx soybean casein
digest agar for bacteria and sabouraud dextrose ager for fungal into sterile Petri dish.
6. Incubate soybean casein digest agar plate at 30˚to 35˚ for 18 to 24 hours and
sabouraud dextrose agar plate for Candida albicans at 20 to 48 hours and for
Aspergillus Brasiliense at 20˚ to 25˚ for 5 to 7 days.
7. After completion of the incubator, period counts the colony.
8. Select the dilution tube of the organism which is consists of 10-100 CFU.
9. Selection criteria for 10-100 CFU dilution tubes.
10. Priority is given to dilutions that consist of nearby 50 CFU count and give second
priority to dilution tube that consists of more than 50 CFU.

35
Growth promotion test of liquid media:
1.Inoculate 10 ml of the appropriate media with not more than 100CFU of the appropriate
test microorganism, for sterility test media use 100ml of the media for the growth-
promoting test, this concludes as a positive control.
2. Inoculate with the normal saline instead of organism this concludes as a negative
control.
3. Incubate appropriate medium at specified temperature and time which is specified
below table.
4. Visible growth of the microorganism or positive result in positive control and no
growth should be observed in negative control is indicate that the growth-promoting test
of the medium should be a pass.
Growth promotion test of solid media
1. Growth promotion test of solid media
2. Inoculate appropriate plates of media with not more than 100CFU of the appropriate
microorganism this concludes as a positive control.
3. Inoculate with normal saline instead of organism this concludes as a negative control.
4. Incubate appropriate media at specified temperature and time which is specified below
table.
5. Visible growth of a colony of the microorganism or positive result in positive control
plates and no growth should be observed in negative control is indicate that the growth-
promoting test of the media should be a pass.

Fig4.1: Growth promoting Test

36
Table-4.1: Name of the media and appropriate microorganism and their
incubation temperature:

Incubation
Name of media Test microorganism
Temperature Duration
Staphylococcus aureus 30˚to 35˚C 18 to24hours
Soyabean casein Pseudomonas
30˚ to 35˚C 18 to24hours
digest agar aeruginosa
Bacillus subtillies 30 ˚to 35˚C 18 to24 hours

Saboutaud dextrose Candida albicans 20 ˚to 25 ˚C 5 days

agar Aspergillus brasiliensis 20 ˚to 25˚C 5days
Staphylococcus aureus 30˚to 35˚C 18 to24hours
Soyabean casein Pseudomonas
30˚to 35˚C 18 to24hours
digest ager aeruginosa
Bascillus subtillis 30˚to 35˚C 18 to24hours
MacConkeyBroth Escherichia coli 42˚to 44˚C 24 to48 hours
MacConkey agar Escherichia coli 30˚to 35˚C 18to72hours
Rappaport vassiliadis Salmonella typhimuriun
30˚to 35˚C 24 to48 hours
broth or salmonella abony
Salmonella
Xylose lysine
tyohimurium or 30˚to 35˚C 24 to48 hours
deoxycholate agar
salmonella abony
Pseudomonas
Cetrimide agar 30˚to 35˚C 18 to 72 hours
aeruginosa
Staphylococcud aureus
Mannitol salt agar 30˚to 35˚C 18 to 72 hours

Clostridium sporogenes 30˚to 35˚C 3 days

Fluid thioglycollate
Staphylococcus aureus 30˚to 35˚C 3 days
medium
Pseudomonas aerginosa 30˚to 35˚C 3 days
Aspergillus brasiliensis 20˚to 25˚C 5 days
Soyabean casein
Candida albicans 20˚to 25˚C 5 days
digest medium
Bacillus subtillis 20˚to 25˚C 5 days

37
 Chapter-5: Water testing

1. Introduction:
The tests are intended for detecting the Total Microbial Count and microbial limit test
(i.e. Escherichia coli, Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus) in a
water sample.

Table-5.1: Limit of different water samples

Water for
Organisms Raw water RO water Purified water
injection(wfi)

NMT 200 NMT 100 NMT 100 NMT 10 CFU/

Bacterial count
CFU/ml CFU/ml CFU/ml 100ml

<10 CFU/ml 0 CFU/ml 0CFU/ml

Fungal count 1 CFU/ml

5.1: Tests for Total Microbial Count:

a) Membrane filtration method

1. The test by Membrane Filtration Method for all Water Samples.
2. Arrange the Mill flex filtration assembly, close all knobs and connect with vacuum
Supply by a sterile rubber tube. Place a sterile 0.45µ filter aseptically on the mesh disc of
the Filtration Unit.
3. Filter 1 ml of water for injection sample (take 1 ml of water for injection sample and
make it up to 100ml by using sterile water then filter it, if necessary ) and transfer the
membrane to labeled Soybean casein digests Agar and Incubate at 30-35 ℃ for 72 hours
in duplicate. Following the incubation, examine the plates for growth, count the number
of colonies and express the results as a number of Colony Forming Unit (CFU) per ml of

38
sample. If no microbial colonies are recovered from the dish, express the result, as ND
(not detected).
4. Simultaneously filter 1 ml of water for injection sample (take 1 ml of purified water
Sample and make it up to 100ml by using sterile water then filter it, if necessary) and
transfer the membrane to labeled sobouraud dextrose Agar and Incubate at 20-25 ℃for
for 72 hours in duplicate. Following the Incubation, examines the plates for growth,
counts the number of colonies, express the results as several Colony Forming Unit (CFU)
per ml of sample. If no microbial colonies are recovered from the dish, express the result,
as ND (not detected).

Fig5.1 Membrane Filtration

B) Pour plate method

After preparation of the sample, dilution adds 1ml of this to sterile Petri dish and pour
approx 20ml cooled 40-45℃ of sterile Soyabean caseain Digest agar media for the total
bacterial count and same add 1ml of test sample into another sterile Petri dish and pour
approx 20ml. Cooled 40-45℃ of sterile sauboured dextrose agar media for total fungal
count mix the content slowly.
Use at least two Petri dish of soybean casein digest agar for Each dilution and incubate
the plats at 30 to 35℃ for 3 to 5 days similarly, for total fungal use at least two Petri dish
of sauboured dextrose agar for each dilution and indicate at 20 to 25℃ for 5 to 7 days.

39
5.2: Test for Microbial limit test:

Enrichment:
Filter not less than 100ml of the sample through 0.45 membrane filter (diameter 47mm)
and transfer the membrane into 100 ml Soybean Casein Digest Medium and mixed the 10
ml sample( water for injection), Another 90 ml Soybean Casein Digest Medium and
mixed the 10 ml sample (water for injection)and take the 10 ml in 90 ml Soybean Casein
Digest Medium mixed another 100ml Soybean Casein Digest medium and incubate the
100ml two flasks and 90 ml flask are discard incubate at 30-350C for 18 to 24 hours.
Examine the medium for growth (turbidity in medium), and carry out the primary test.

1. Test for Escherichia coli:-

Selection and subculture:- After 24 hours of incubation of Soybean-Casein Digest

Medium, shake the container and aseptically transfer1 ml of Soybean-Casein Digest
Medium to 100 ml of MacConkey broth and incubate at 42-44 °C for 24-48 hours. After
48 hours of incubation take 0.1 ml sample and subculture on a plate of MacConkey agar
at 30-35 °C for 18-72 h.

Interpretation: - Growth of pink, nonmucoid colonies indicates the possible presence of

E. coli. This is confirmed by identification tests. If there is no growth of such type of
colonies it indicates the absence of Escherichia coli and the test sample passes the test.

2. Test for Salmonella:-

Selection and subculture: After 24 hours of incubation of Soybean-Casein Digest

Medium shake and transfer 0.1 ml aseptically 10ml of Rappaport Vassiliadis Salmonella
Enrichment Broth and incubate at 30-35 °C for 24to48 hours. After the 24to 48 hours
take 0.1 mal sample and Subculture on plates of Xylose Lysine Deoxycholate Agar and
incubate at 30-35°C for 24to48 hours.

40
Interpretation: The possible presence of salmonellae is indicated by the growth of well-
developed red colonies with or without black centers. This is confirmed by identification
tests. The product complies with the test if colonies are not present, it indicates the
absence of salmonella and the test sample passes the test.

3. Test for Pseudomonas aeruginosa:-

Selection and subculture: After 24 hours of incubation of Soybean-Casein Digest

Medium take 0.1 mal sample and subculture on a plate of Cetrimide agar. Incubate the
plates at 30-35°C for 18 to 72 hours.

Interpretation: Greenish color of colonies indicates the possible presence of

P.aeruginosa. This is confirmed by identification tests. The product complies with the test
if the colonies are not present it indicate the absence of Pseudomonas aeruginosa and the
test sample are passes the test.

4. Test for Staphylococcus aureus:-

Selection and subculture: After 24 hours of incubation of Soybean-Casein Digest

Medium take 0.1 ml sample and subculture on a plate of Mannitol salt agar Incubate the
plates at 30-35°C for 18 to 72 hours.

Interpretation: The possible presence of S.aureus is indicated by the growth of yellow

or white Colonies surrounded by a yellow zone. This is confirmed by identification tests.
If there is no growth of such type of colonies it indicates the absence of Staphylococcus
and the product passes the test.

41
Fig5.2 (A) MC Broth Fig5.3 (B) RV Broth

5.3: Sampling of water:

Purified Water
1. Clean the sampling bottles.
2. Autoclave it at 121°C for 15min 15lbs (lose the cap)
3. Wear sterile gloves.
4. open sampling valve allows water drain for 1 min.
5. Collect water then close the cap don't touch by hand.
6. Sampled water should be analyzed in 2hr after sampling. Store it in 2-8°C, not more
than 6-8 hrs.
7-Due to sop of the company production has 30 points in purified water they do water
testing daily and cover it in 15 days.
8- There are 5 points
Purify water (Rw1)
Purify water (Rw2)
Loading area (Rw3)
Physiochemical room (Rw4)

42
Filter wash area (Rw5)

Row water:
There is 3 point.
1. Utility (RO plant) (Rw1)
2. Physiochemical lab (Rw2)
3. Media preparation room (Rw3)

Ro water:
There is only one point.
1. Utility (Ro plant) (Ro1)

Water for injection:

There are 6 points.
1. Wash area (Dw1)
2. Loading area (Dw2)
3. Loading area (Dw3)
4. Housekeeping solution preparation (Dw4)
5. Eye drop manufacturing room (Dw5)
6. SIP room (Dw6)

43
 Chapter-6: Environmental Monitoring
Environmental Monitoring Program is a proceduralized recurring set of activities for
evaluating air and surfaces (including personnel, where applicable) for viable or non
viable particles as well as other environmental variables. For completeness for coverage,
EMP compasses the following:
1) There are found that temperature, relatively humidity, pressure differential, HEPA
filtration air velocity, non viable particles count, viable active air count and viable
passive air count.
2) Surface and personnel viable particle counts.

6.1Settling plate method:

Procedure:

1. Prepare the soyabean casein digest agar media for preparation and sterilization.

2. Pre-incubate the plates at 30-35°C for 18 to 24 hours before use.

3. Stack the pre incubated Petri dishes in a canister and takes the canister to the area.

4. Before exposing the plate, ensure that the air flow of the area has been switched ON.

5. Remove the plate for the canister and take it to the point of exposure.

6. The lid of the plate and place it beside the plate in an inverted position.

7. Exposé the plate for a period of 4 hours.

8. After 4 hours, place the lid back on the plate mark the plate, mentioning the date and
point of expose.

9. Put the plate back into the canister.

10. Incubate the plate at 30-35°C for 48 hours the number of colony forming units
clouted and recoded, after that plate transfer 20-25°C for 72 hours. The colony forming
units counted and recoded.

11. If the count exceeds limit, it indicate that fogging was ineffective.

44
12. In the case, defogging the area is required.

Alert and Action levels for environmental monitoring:

An Alert level in EM is that level of microorganisms that shows a potential drift from
normal Operation conditions. Exceeding the alert level is not necessarily grounds for
definitive corrective Action, but it should be at least prompt a documented follow-up
investigation that could include Sampling plan modifications.
An Action levelin EM is that level of microorganisms that when exceeded require
immediate Follow up and if necessary, corrective action.

Table 6.1: Target, warning and action by class

Target Warning Action

Class
Bacterial Fungal Bacterial Fungal Bacterial Fungal
Class A <1 <1 1 1 >2 >1
Class B <5 <1 3-5 1 >5 >1
Class C <50 <1 20-50 1 >50 >1
Class D <100 <1 60-100 1 >100 >1

Fig6.1: Environmental Monitoring

45
6.2 Personnel Monitoring:

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 clean room environment from ourselves. To assess the
effectiveness of the gowning Program personnel may be monitored on a regular basis for
viable counts. Personnel monitoring Employ contact plates to assess microbial
contamination of clean room personnel.
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. Many companies utilize this testing for proficiency based training
programs for clean room personnel.

6.3 Glove Prints (Finger dabs):

Fingertips are the most likely area to come into contact with microbial contamination on
work Surfaces, on materials, or arising from the operator and then be transferred to
products. Glove prints including all five fingers should be taken to monitor this
possibility.
Sampling should be conducted before routine sanitization of gloves with alcohol, or
before Changing of outer gloves in cases where double-gloving is used.
Procedure:
1. Prepare soybean Casein Digest Ager plate and sterilization of media.
2.Pre incubate the plate in invert position at 30-35 °C for 24 hours, after incubation check
plates for any contamination, if there is contamination discard the plates.
3. Clean the container with 70% IPA and level the plates with date, Personal name and
keep the container.
4. Transfer the container in sterile area and call personal to be monitored, open the plate
and tell personal to place her right hand finger with gloves gently on the surface of SCDA
plates after close the plate immediately and take the another plate for left hand finger

46
follow the same procedure, disinfected the finger with 70% IPA and ask personal to
remove gloves and wear another sterile gloves.
5. Positive control by streaking any pure culture and for negative control incubates the
plates.
6. Incubate the plate for 30-35°C for 48 hours and 20-25°C for 72 hours.
7. After incubation count the colonies as CFU.

Table-6.2: Limit of personal finger plate count (CFU/ plates)

Target Warning Action
Bacterial Fungal Bacterial Fungal Bacterial Fungal
<3 <1 3-5 1 >5 >1

6.4 Swab Sampling

Swab sampling detects microorganism contamination in the immediate vicinity of the

work area.

Swabs are sterile and stored in a suitable sterile liquid or other diluents. The swabs are
rubbed over the test surface. It can be used to sample irregular or constrained surfaces
such as equipment, filling nozzles, tubing, or corners. They are useful for sampling large
areas, such as after cleaning or sanitization procedures. They are also 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.
The microbiologist can determine the type of microorganisms on the swab by sub
culturing it to media. Swabbing is more qualitative than quantitative. The recovery of
microorganisms from swabs should be validated, including the chosen sampling method,
the suitability of the swab moisturizing liquid, and the transfer of microorganisms to
growth media. Normally > 50% of microorganisms should be recoverable during
validation studies.

47
If the area to be sampled is large but not standardized, no regulatory limits are applicable
to swabs. However, the detection of microorganisms using this method should be
investigated as part of batch release.

Procedure:

1. Prepare normal saline (0.9%NACl), transfer sterile normal saline and required number
of cotton swab into LAF of testing area through pass box.
2. Sterilize the tubes and cotton swabs in autoclave at 121°C for 15 min at 2.1 bars
pressure.
3. Carry the swabs sterilize saline or peptone water tubes to be area where surface
monitoring to be done.
4. Wear sterile hand gloves.
5. Disinfect the 5×5cm stainless steel template with 70% IPA.
6. Swab the 5×5cm area with the help of stainless steel template and sterile cotton swabs
by horizontal and vertical strokes.
7. Clean the area after swabbing with 70% IPA.
8. Dip the same swab in 10ml saline or peptone water tubes immediately plug the tube
with sterile cotton and label it as name of area, date, and sign.
9. Filter the sample using sterile cellulose nitrate membrane filter.
10. Mark the plate with respective sampling area, date and sign.
11. Incubate at 30 -35°C for 48 hr.
12. Then incubate at 20-25 °C for 72hr.
13. After incubation count the colony.

6.5 Contact Plates:

Contact plates should be used to detect microorganisms on surfaces that could lead to
product Contamination. These surfaces may include working surfaces, equipment
surfaces, and walls and Ceilings of unidirectional air flow systems. When spills dropped
materials are likely to contaminate floors, these should be sampled. When operators work

48
in close proximity to exposed product, such as in an open flow hood, gown fronts,
sleeves, masks, or other representative areas should be sampled.

Contact samples should be taken after completion of production activities or in such a

way that contamination of sterile areas by monitoring does not occur. Samples should be
taken before sanitization of the area. Where frequent sanitization (e.g., through spraying
with alcohol solutions) occur, samples should be taken prior to the sanitation procedure to
maximize the likelihood that microorganisms are detected. Where surfaces are still wet
with sanitization solutions, contact plate measurements are invalid. These are monitored
on a regular basis for viable counts by using specially designed contact plates that contain
a growth medium called Soybean casein digest agar medium(SCDA)

Insert timer in RODAC weight.

Program me the timer for 2min using min button on the time.
Insert the contact plate at the back side of RODAC weight.
Remove plates then press stop button.
Then incubate at 30-35 °C Celsius which is mainly the optimal growing temperature for
most environmental bacteria, and 20-25 °C degree Celsius which is the optimal growing
temperature for most mold and yeast species.
Monitoring shell be done by:
Forehead
Shoulder left and right
Elbows left and right
Waist
Knee left and right
Booties left and right

49
 Chapter-7: Fogging and Defogging

Fogging and defogging process are used for the controlling of microorganism. These
processes are used for killing of microorganism. This is directed by a blower and
Vaporized spray. These processes are required fogging machine and fogging solution. By
these fogger machines solution is sprayed in the area. The small particles of disinfectant
solution suspend in the air from long time and kill the air borne bacteria, fungi and their
spores.

This is very effective way to control the contamination the recommended ultra low
volume fogger used for fogging. These process are used for the regulatory guidelines
because negative effect causing or irritation to the eyes, nose and skin.

Pre start up procedure:

1. 2 hour before the fogging AHU switched off.
2. The area should be fogging shell be completely off and other area and plant should be
cleaned.
3. 20% Silvox and 1.0% bacillocid are used for the measuring cylinder.
Precaution of fogging:
1. Silvox 20 % and BacSillocid 1.0% should wear nose mask, gloves and goggles.
2. Fogging area should not allow.
3. All people coming in contact should wash their hand and face with soap.
4. All material and produced are contaminated by silvox and Bacillocid should be
removing and protected from it.
5. The room and the remaining equipments shell be cleared and disinfected with
approved disinfectants.
Precaution of Defogging:
1. Open the 100% fresh water supply dampers and exhaust and close the return air
damper.
2. The fog has been complete removed, adjust the fresh air damper.
3. Remove the adhesive tape used to seal the area before the start of defogging.

50
 Chapter-8: Discussion

Based on the results we obtained, we were able to deduce that the source of these
microorganisms isolated from the core area of the facility were mainly human
interventions. The microorganisms were mainly isolated from core area. We were ablsse
to isolate them and characterize them based on their species. It was also deduced that
these microorganisms didn’t seem to affect the quality of the product in whatsoever way.

This study is of utmost importance as this study shows what kind of microorganisms
might be present in the facility and what impact they might have on the product.
Identifying these microorganisms is the first step in identifying any potential hazards
present in the core area and only by identifying them we can take various actions which
might help us get rid of these hazards. Working in close quarter with pharmaceutical
industry, I feel it is really important to eliminate any possible source of contamination
which might be potential source of infection for the consumer consuming the product.

All of the air delivered to a clean room passes through HEPA filters, and in some cases
where stringent cleanliness performance is necessary, ULPA filters are used.

Temperature should be maintained: 16 to 19 degree Celsius.

50% humidity is maintained in clean room.

Pressure is maintained in according to classification of clean rooms. Pressure is high in

Grade A than B. Pressure is low in Grade D.

Personnel selected to work in clean rooms undergo extensive training in contamination

control theory. They enter and exit the clean room through airlocks, air showers and/or
gowning rooms, and they must wear special clothing designed to trap contaminants that
are naturally generated by skin and the body.

51
 Chapter-9: Result
Samples were mainly obtained from the core area of the pharmaceutical company
using three techniques: Settle plate, Air sampling and personal monitoring. After
sampling of core area, the plates were incubated in the incubators 22.5±25°C and
32.5±25°C for 72 hrs and 48 hrs respectively.

Fig9 (A): Laboratory plates Isolated on SCDA plates and observed after 48 hrs.

Colonies were further streaked on the SCDA plates for better isolation. The fresh
streaked plates were incubated in 32.5±35°C for 48 hrs.

Fig9 (B): Production C Isolated on SCDA plates and observed after 48 hrs

52
 Chapter-10: Conclusion
These results are in agreement with the findings of other workers. It was found that
bacteria and fugues growth.

To ensure a clean room conforming to the designated classification, constant

monitoring of contaminant sources and identification of the predominant
contaminant bacteria is usually necessary.

This study found that bacteria and fungus growth and manage the limitation
of bacterial or fungus. If the bacterial and fungus growth are out of the limit
mange the growth of the bacteria for Silvox 20 % and BacSillocid 1.0% are
used.

Maintaining the integrity of a clean room is a constant battle.

There are 3 prime sources of contamination. The first is from human errors. To
control this source of contamination, human hands must be washed with disinfectant.
70% IPA is the widely used skin disinfectant because of its mild nature.
Contamination may also result from the room surface areas. To avoid such
contamination, floors, walls and ceilings must be swept with disinfectants. The third
contamination source is from the room air. UV irradiation is the most convenient
way to sterilize room air although it is advised the fumigation with suitable
disinfectant periodically reduce and limit the microbial load in the production area at
pharmaceutical industry

53
References

1. http://renovalifescience.com/aboutus.php

2. https://microbenotes.com/agar-principle-composition-preparation-and-uses/

3. http://pharmaceuticalmicrobiologi.blogspot.com/2016/12/fogging-and-fumigation.html?m=1

4. http://www.ivtnetwork.com/sites/default/files/Environmental_01.pdf

5. https://www.pharmaguideline.com/

6. Microbial identification in pharmaceutical industry

Scott v.w sutton,phd and anthony m.cundeli,phd usp expert committee on analytical
microbiology

7. Bacterial isolates from pharmaceutical industry enviornment and water systems A.M
Ramachandran asst.professor department of microbiology, Dr N.G.P arts and science
college,coimbatore.

54