MICROBIOLOGICAL ANALYSIS OF

PHARMACEUTICAL PRODUCTS
A
Project Report
Submitted In Partial fulfillment of the
Requirement for the award of
The Degree
of

MASTER OF SCIENCE
in
MICROBIOLOGY

Submitted by
Yogesh Krishan
Department of Botany & Microbiology
Gurukul Kangri Vishwavidyalaya
Haridwar- 249 404 (U.K.)
2013

CERTIFICATE
I have the pleasure in forwarding the M.Sc. 4th Semester of
Mr. Yogesh krishan who carried out a piece of project work on
Microbiological analysis of Water and Pharmaceutical
Products at Akums Drugs and Pharmaceuticals Ltd.,
SIDCUL, Haridwar for partial fulfillment of M.Sc. Degree in
Microbiology from this vishwavidyalaya.

Date:

(G.P. Gupta)
(H.O.D.) Department of
Botany and Microbiological

ACKNOWLEDGEMENT
Words are very poor comforters to express the deep debt of gratitude which
one feels in ones corner of the Thank when one is helped to achieve the
ultimate goal, in this boundless & endless field of project work..
I would like to express my sincere gratitude and indeptness to
Dr. G.P. Gupta. Head of Department of Botany & Microbiology Gurukul
Kangri University,Haridwar (Uttarakhand) whose constant guidance and
suggestion helped me throughout my M.Sc. Program and also my
dissertation work.
I am thankful to AKUMS DRUGS & PHARMACEUTICAL Ltd. For
permitted me for project work at HARIDWAR Plant-VI as a part of my
M.sc.Microbiology (IV Sem)
I wish to take this opportunity to thank the following who have assisted with
the completion for selecting me as a candidate to complete my project work
I offer my cordial thanks to Mr. Kuldeep Tyagi ( H.R.Manager ), Mr.
Parvindra Tyagi ( Q.C.Manager) for suggesting me to undergo training in
Q.C. Department.
I deeply convey my homage with regards to all Q.C. Staff members for their
constant encouragement ,and valuable criticism throughout the course of my
work and beyond that.

DECLARATION

This is to be certifying that this project entitled Microbial testing

of pharmaceutical products has been carried out by me at
department of Q.C. (Microbiology) AKUMS
DRUGS &
PHARMACEUTICALS LTD. SIDCUL , HARIDWAR
at
Gurukul kangri vishwvidyalaya No parts of it have been copied
as a whole from any other journals /books/thesis ,it is futher
certified that this dessertation or any part of this has not been
submitted/for diploma degree in any other university.

DATE:
PLACE: Haridwar

YOGESH KRISHAN
M.Sc IV sem Microbiology
G.K.V. Haridwar

CONTENTS
An Overview Of The Company
Environment Monitoring
Water Used For Pharmaceutical Purpose

1. Raw water
2. Purified water
3. Water for injection
Microbial Limit Test
Antibiotic assay
Bioburdon sterility test
Observations
Summary
Referances

AN OVERVIEW OF THE COMPANY

Akums Drugs and Pharmaceutical Ltd. Haridwar was
established 2004 jointly by Mr.D.C. Jain, Mr. Sanjeev jain and Mr.
Sandeep jain , the promoter directors, who had over 30 years of
experience in Pharmaceuticals trading and manufacturing. Akums
is endeavouring to make a name in the global pharmaceutical
industry.

The Present capacity

Standards ,avers Mr. Sanjeev jain . No wonder the motors of the
company are Quality gyrates Around Customer Satisfaction and
Essence of business is to create Customer and Profit will follow
Akums has huge capacity to manufacture 40 million tablet The
company is into contract manufacturing for more than 35 leading

pharmaceutical companies in the country on a third party/lone

licensce and P to P basis. At present, Akums Manufactures over
500 formulations in the form of Tablets, Capsules, Dry Syrups,
Liquid Orals, soft Gelatin preparations, liquid injections, dry
powder injection s, eye and ear drops, etc. Sprawled on an area of
about 1,70,000 sq.ft. Akums is an independent state-of-the-art
manufacturing unit having modernand sophisticated manufacturing
facilities of international standards, which include an ultra modern
plant, machinery and equipment. Besides, the company boasts of
an in-house testing laboratory along with an R & D center
supported by highly qualified, technical and skilled personnel.

ENVIRONMENTAL MONITORING
(E. M.)
Environmental monitoring is done to check the bioburden of
the aseptic area of the controlled environment. The purpose
of this is to understand the various issues that is related to
aseptic processing of bulk drug substance or finished products
(sterile), dose forms, and in certain cases and to establishments
maintenance and control of the microbiological quantity in the
controlled environment.

Methods of E. M.:1)
2)
3)
4)
5)

By
By
By
By
By

Settle plate
Active Air sampler
Swab
Finger Dab
Contact plate

Personal Monitoring:1)
2)

This is done by two methods as follows:By Contact Plate

By Finger Dab

1)E. M. By Settle Plate Method:Plate Preparation:-

Prepare SCDM
Medium.
Aseptically
transfer the
preincubated
plate
into
the
manufacturing room and mention plate code and exposure
time on the plate, expose as per layout. For class 100 expose
the plate for 30 min and in other for area for 2 hrs. After
completion of exposure time close the plates in the same
manner.
Incubate the plate at 30 to 35C for 48 hrs bacterial count
and after 48 hrs transfer the plate in BOD incubator at 20 to
25C for 72 hrs. After completion of each incubation period
observe the result for bacterial and fungal count respectively.
LIMIT:- Bacterial count
CLASS
ALERT
1,00,000
60 CFU
10,000
30 CFU
100 B
03 CFU
100 A
<1 CFU
Fungus should be absent.

(2)

ACTION
80 CFU
40 CFU
04 CFU
<1 CFU

LIMIT
100 CFU
50 CFU
05 CFU
<1 CFU

By Active
Sample:-

Air

Microbial Air Sampler

Plate preparation:Prepare SCDA medium. Aseptically transfer the preincubated
plates into the concern area and mention plate code
sampling time on the plates, take the sample 1000 litre of
air (1m) as per layout. After sampling close the lid of the
plates aseptically. Incubate the plates at 30 to 48 hrs for
bacterial count and after 48 hrs transfer the plates into
BOD incubator at 20 to 25 C for 72 hrs.
LIMITS
Class
100A
100B
10000
1000000

Alert(CFU/m)
<1
6
60
300

Action(CFU/m)
<1
8
80
400

(3)By Contact Plate Method:(a)Surface Monitoring by contact plate:-

Plate preparation:-

Limits(CFU/m)
<1
10
100
500

Prepare De Engle neutralizing agar medium.Aseptically

transfer the pre incubated plate into the concern area and
mention plate code and sampling time on the plates, open the
lid of the plate and press smoothly on the surface (which has
to be monitored) after sampling close the lid of the plate.
Incubate the plate at 30C to 35C for 48 hours for bacterial
count and after 48 hours transfer the plate in the BOD
incubator at 20C to 25C for 72 hours. After completion of
each incubation period observe the plate for bacterial and
fungal count respectively.

LIMITS:Class
100 A
100 B
1000
1000000

Alert(CFU)
<1
3
15
30

Action(CFU)
<1
4
20
40

Limits(CFU)
<1
5
25
50

(b)Personnel Monitoring by contact plate method:-

Plate Preparation:-

Prepare De Engle neutralizing agar medium.

Aseptically transfer the preincubated plate into the concern
area and mention plate code and sampling time on the plates
open the lid of the plate and press smoothly on the surface
(forehead, chest, armpits) after sampling close the lid of the
plate. Incubate the plate at 30C to 35C for 48 hours for
bacterial count and after 48 hours transfer the plate in BOD
incubator at 20C to 25C for 72 hrs. After completion of each
incubation period observe the result for bacterial and fungal
count respectively.
LIMITS:Class

Alert(CFU)

Action(CFU)

Limits(CFU)

100A
100B
10000

<1
3
15

<1
4
20

<1
5
25

(4)Personal Monitoring by finger Dab Method:Plate

Preparation:Prepare SCDA medium.
Aseptically transfer the preincubated plate into the concern
area and mention name of the person and the location
(standing position), aseptically open the lid of the plate and
take the finger impression smoothly of both hand in separate
plate. After completion of the sampling close the lid of the
plates. Incubate the plate at 30C to 35C for 48 hrs for
bacterial count and after 48 hrs for the bacterial count and
after 48 hrs transfer the plates in BOD incubater at 20C to
25C for 72 hrs. After incubation period observed the result for
total microbial count.
LIMITS:Micro
Class
organism (CFU/fing.)
rt
Bacteria

100A Class 100B(CFU/fing.)

Ale Actio Limit Alert

n
<1
<1
<1
3

Action

Limit

WATER USED FOR

PHARMACEUTICAL PURPOSE
Water is used as a raw material, ingredients and solvent in
processing, formulations and manufacture of pharmaceutical
ingredients (APIs ) and intermediates, compendial articals and
analytical reagents.
Control of the chemical purity of these waters is important
and is the main purpose of the monographs. Because
pharmaceutical waters are generally produced by continous
processes soon after generation, the water is likely to have
been tested for several criteria simultaneously.
Water in pharmaceutical industry is of following types:
{1}- Water for Injection
{2}- Purified Water
{3}- Pure Steam
{4}- Sterile WFI

PURIFIED WATER :
Purified water is water obtained by distillation, ion-exchange
treatment, reverse osmosis, or other suitable process. It is prepared from
water complying with the regulations of the U.S. Environmental
Protection Agency (EPA) with respect to drinking water. It contains no
added substances.

The plant of Akums Drugs & Pharamaceuticals limited receives the

source water from bore well. The raw water from the bore well is
collected in the underground tank of capacity of 100kilolitres.
During the purification process, this water is pumped to the
overhead tank of 200,000 liters capacity. During this transfer the
water is dosed with sodium hypochloride in the concentration of 1
PPM. The chlorinated water then passes through Multi grade sand
filter (MGF) for removal of the colloidal matter. The treated water
from MGF is dosed with sodium Meta bi sulphate (SMBS) to
remove the traces of chlorine and passed through softener unit
comprising of cation resins to reduce the hardness. After softening
the water is dosed with Sodium Hydroxide for the pH adjustment
and collected in a soft water tank of 7.5 kilolitres capacity. The
water from soft water tank is pumped to Reverse Osmosis unit.
Antiscalent is dosed into water during this transfer. After RO unit
the water is treated through Electro Deionization (EDI) units and
Ultra Filtration system. Purified water is collected in jacketed tank
of 3.0 kilolitres capacity and distributed after Ultra Violet
treatment.

Two types of aerobic microorganisms count are tested

in purified water.
Bacterial count.
Fungal count.
Total bacterial count:
Media : soyabean Casein digest Agar
Examination of the sample:
Determine the total aerobic microbial count in the water sample to
be tested or examined by the following method.

Pour plate method.

Procedure:
Take sterile empty Petri plates and label the Petri plates by
the name of the sampling point and date of sampling all this
and upcoming work should be carried out under the Laminar Air
Flow. Take Petri plates in duplicate.
Aseptically transfer 1ml of respective samples in the above
labeled Petri plates.
Aseptically pour 30 ml of the sterile soyabean casein digest
agar media to each of the Petri plates.

 As soon as the pouring is over, rotate the plates gently for

uniform distribution of the media.
Carry out the positive control by streaking the culture of E.
coli on the surface of soyabean casein digest agar and
negative control by keeping a blank plate soyabean casein
digest agar.
Dont disturb the plates till the media solidifies completely.

Incubation:
After the media was solidified incubate both the Petri plates
in bacteriological incubator at 32c for two days.

Observation:
Observed that the growth of bacterial colonies and count the
colonies in both plates.

Calculations:
After counting colonies in both the plates, calculate the mean
of the colonies present in both the plates and this give the number
of bacteria present in 1 ml of water sample.
Number of colonies =
colonies inplate 2

No. of colonies in plate 1+ No. of

Total fungal count:

Media: Sabouraud Dextrose agar media.
Examination of the sample:
Determine the total aerobic fungal count in the water sample to be
tested or examined by the following method

Fungus count
Pour plate method

Procedure:
Take sterile empty Petri plates and label them with the
name of the sampling point and date of sampling, all this
and upcoming work should be done under the Laminar Air
Flow. Take Petri plates in duplicate.
Aseptically transfer 1ml of the respective samples in the
above labeled Petri plates.
Aseptically pour 30ml of the sterile Sabourd dextrose agar
media to each of the Petri plates.
As soon as pouring is over, rotate the plates gently for
uniform distribution of the media and water sample.
Carry out the positive control by streaking the culture of E.
coli on the surface of Sabouraud dextrose agar and

negative control by keeping a blank plate Sabouraud

dextrose agar.
Dont disturb the plates till the media gets solidified.

Incubation:
After the media was solidified incubate all the test plates in
BOD incubator at 22c for 3 days.

Observation:
After the incubation period of 3 days is over, observe the
Petri plates for the presence of fungal colonies in both the plates
and count the colonies in both the plates.

Calculations:
After counting colonies in both the plates, calculate the
mean of colonies present in both the plates and this gives the
number of fungal colonies present in 1 ml of water sample.
Number of colonies =

No. of colonies in plate 1+ No. of

colonies in plate 2
Thus by pour plate method the total number of bacterial and
fungal colonies can be calculated in the water sample that is being
tested.
After calculating the number of bacterial and fungal
colonies, note them in the protocol for purified water, the bacterial

and fungal colonies should be present in the limit as specified

earlier, in specification for purified water.

WATER FOR INJECTION

Water for injection is water used for the preparation of
medicines for parenteral administration and water is used as
vehicle (water for injection in bulk) and dissolving and diluting
the substance or preparation for parenteral administration
(sterilized water for injection).
Raw water \ drinking water Softening and
Dechlorination

Ultrafilteration Reverse Osmosis

(0.5, 0.2)

U. V. Light Treatment Distillation

Water for Injection

Steam collect

WFI thus prepared is stored and

distributed in condition

designed to prevent growth of microorganism and to avoid any

other contamination.

Description of water for injection:

The USP 23 monograph states:

"Water for Injection (WFI) is water purified by distillation or

reverse osmosis."
WFI is produced by either distillation or 2-stage RO. It is usually
stored and distributed hot (at 80 degrees C) in order to meet
microbial quality requirements.
Multicolumn distillation unit operates on the principle of inter
stage heat exchange,
Thereby drastically reducing the consumption of heating energies
and cooling water. Distilling columns operate at different pressure
and temperature, making it possible to transfer the energy in the
process forward from column to column

The basic equipments consist of:

Vertical distilling columns (5 nos.).
Heat exchanger (condenser).
Electrical panel box.
Pre filter.
Distillate rejects.
Conductivity meter.
Dumping

Manufacturing of water for injection:

 The condenser is supplied with feed water i.e. purified water.

Feed water heated up during condensing of purified steam to
water for injection.
Preheated purified water passes through each column in the
form of water vapour through heat exchanging process, as
each column is circulated by steam with certain pressure.
While water vapour passes from one column to another, it
converted into steam.
The evaporated water rises upward and unevaporated feed
water, which is collected at the bottom, & forced to the next
column under pressure.
The rising vapours in the column are made to pass through a
precisely designed spiral guide, resulting in a high centrifugal
force.
This is eliminated effectively the entrapped water droplets
and only the pure vapour will be passed further to the heat
exchanger of the second column.
Thus the unevaporated portion of the feed water, pure vapour
of the first column is passed respectively to the tubes and
steam chest of the heat exchanger of the second column.
This process continues in subsequent columns. The purified
steam of 2ND , 3RD,4TH and 5TH column goes to the
condenser.

 In condenser, the purified steam gets condensed into water

for injection which is pyrogen free.
The unevaporated feed water in the last column is
automatically purged out through the purging hole; this
contains dissolved and undissolved impurities.
A conductivity meter sensor is provided in the WFI line to the
storage tank. The dumping value is also fitted in the line, which
automatically rejects the distilled water till the desired level of
conductivity is reached, and also rejects the distilled water if
conductivity goes beyond the set limit during generation phase.

Utilities associated with distillation

still:
Boiler steam.
Cooling water.
Power supply.
Compressed air.
Re-circulation loop: Water for injection is stored in thermo
insulated, stainless tank of capacity 1000 liters, which is provided
with a vent filter. There is a loop system for distribution of WFI to
point of use and back to storage.
Temperature of Water for Injection is maintained at 800c
throughout distribution system and in storage tank.

Specifications of water for injection:

Conductivity: Not more than 1.0 s/cm.
Total organic count: Not more than 500ppb.
Total bacterial count: Not more than 10 cfu/ 100ml.
Total fungal count: Nil cfu/100ml.
Endotoxin limit: less than 0.25 EU/ml.

1.

Total aerobic microbial count for water for

injection

Two types of aerobic count are tested in water for injection.

Total bacterial count
Total fungus count

Test procedure by membrane filtration method:

Equipments required.
Laminar airflow.
Filtration assembly.
Vacuum pump.
Sterile 0.45 membrane filter.

Materials required:

Sterile 0.1% w/v peptone.

70% IPA solution.
Sample for testing.
Soyabean casein digest agar plates.

Test procedure.
Keep all the sterile sampling devices i.e. sterile sampling
devices, sterile gloves in the hatch box of manufacturing
area, and enter in to the sterile area with sterile, lint free
gowning and only after wearing mask and sterile gloves.
Open the sampling valve of manufacturing tank before filter
housing. Disinfectant the sampling point with sterile 70%
IPA solution, open the sampling point valve and allow the
sample to flow for 10 seconds and by holding the base of
sterile sampling bottle, collect approximately not less than
200 ml bulk sample. Recap the sampling bottle.
Label the bottle properly with product name, batch number,
sampled by, date and time of sampling and quantity of the
solution sampled.
The sample is then taken to the microbiology laboratory for
further testing of the sample.
Take the bio load sample to the aseptic area in microbiology
laboratory and operate the Laminar airflow.

 Transfer all required sterile glasswares, media bottles and

filtration unit to the aseptic area of microbiology laboratory.
Clean outside surface of the sampling bottle with absorbent
cotton, which is moistened with 70% IPA solution.
Start the gas burner with the help of lighter and aseptically
connect the rubber tube of sterile manifold assembly to
receiver tank and receiver tank to rubber tube to vacuum line.
Aseptically assemble the filtration unit and place a sterile
0.45 membrane filter using sterile smooth tip forceps, in the
center of filter screen.
Without disturbing the filter, place the funnel on the top of
the filter holder base and transfer approximately to 100 ml of
sterile 0.1% peptone water to the membrane filter assembly
and filter it immediately.
Then add 100 ml of bulk sample to the membrane filter and
filter it immediately.
Wash the membrane filter three times with 0.1% peptone
water into the filtration funnel and filter under partial
vacuum.
After the completion of filter process, shut off the vacuum
with the help of vacuum control key.
Aseptically transfer the membrane filter to the surface of the
Petri plates containing Soyabean casein digest agar.

 For positive control, carry out the same procedure in

duplicate, except for sample use 100 ml peptone water
inoculated with 100 cfu bacterial colonies.
For negative control carry out the same procedure, except for
sample use 100ml sterile peptone water.
Invert and incubate the plates at 72 hours at 30 to 35c.
Count the number of colonies that are formed, calculate the
number of cfu per ml of the sample being examined.

Incubation:
Incubate all the plates at 30 to 35C for 2 days to allow growth
of bacteria, and then incubate at 22C for 3 days to allow the
growth of fungus colonies.

Observation:
After the completion of 5 days incubation period, count the
number of colonies in all four plates, i.e. two of bacteria and two of
fungus.

Test for specific microorganisms.

Conducted for the identification of following pathogens in the test
sample.
E. coli

 Salmonella
Staphylococcus aureus
Pseudomonas aeruginosa.

Procedure:
Aseptically take 1ml of water sample and directly inoculate
into 100 ml of sterile soyabean casein digest medium.
Incubate the tubes at 37c for 18-24 hours.
Carry out the positive control by aseptically transferring a
culture of E. coli in 100 ml of sterile soyabean casein digest
medium and negative control by keeping a blank tube of 100
ml sterile soyabean casein digest medium.
Observe the tubes for growth by means of turbidity. If growth
is observed in positive control and sample tube and absent in
negative tube, then proceed for identification for E. coli,
Salmonella,

Staphylococcus

aureus

and

aeruginosa..

Test procedure for detection of E. coli:

Primary test:

Pseudomonas

By means of inoculating loop, streak a loop full of enrichment

culture (obtained from Soyabean Casein digest broth of above test)
on the surface of MacConkey agar plates

Secondary test:
Simultaneously carried out the positive control by streaking a
growth of E.coli on the surface of MacConkey agar plates.
For negative control incubate the plate as it is without
inoculation.
Invert and incubate all the plates at 35 c to 37 c for 24
hours.
Upon examination, if none of the colonies are brick red in
colour and have a surrounding zone of precipitated bile, the
sample meets the requirements of the test for the absence of
E. coli.
If the colonies described above are found, transfer the suspect
colonies individually to the surface of Levine eosinmethylene blue agar medium
Simultaneously carried out the positive control by streaking a
growth of E. coli on the surface of MacConkey agar plates.
For negative control incubate the plates as it is without
inoculation
Invert and incubate all the plates at 35 to 37c for 24 hours

 Upon examination, if none of the colonies exhibit both a

characteristic metallic sheen under reflected light the sample
meets the requirements of the test for the absence of E. coli.

Test procedure for Salmonella:

Primary test:Aseptically

add 1.0ml of the enrichment

culture (obtained from SCDM of previous test) to each of two

tubes containing (a) 10 ml of sterile F broth and (b) tetrathionatebile-brilliant green broth and incubate at 35 to 37 c for 24 to 48
hours
From each of these two cultures subculture on at least two of the
following four agar media:
Bismuth sulphite agar:
Brilliant green agar:
Desoxycholate-citrate agar
Xylose-lysine-desoxycholate agar
Simultaneously carried out the positive control by streaking a
loop full growth of Salmonella colony on surface of one of
the above media used for testing. For negative control
incubate the agar plate with streaking or inoculation.
Invert and incubate al, the plates at 35 to 37c for 18 to 24
hours.

 Upon examination if none of the colonies confirms to the

description given in Table-1, the sample meets the
requirement of test for the absence of the genus, Salmonella.
If any colony confirms to the description given in table-1,
carried out the secondary test.
S.

Medium

No.
1.

Description of
colonies

Bismuth sulphite
agar.
Brilliant green agar.

Black or green colonies.

3.

Desoxycholate-citrate
agar.

Colourless, and opaque, with or

without black center.

4.

Xylose-lysinedesoxycholate agar

Red colonies with or without

black centers.

2.

Small, transparent and

Colourless, or opaque, pinkish or
white colonies (frequently
surrounded by a pink or red
zone.)

Secondary test
Subculture any colonies showing the characteristics given in
table-1, in Triple sugar-ion agar by first inoculating the
surface of the slope and then making a stab culture with the
same inoculating needle.

 At the same time inoculate a tube of urea broth. Incubate at

36 to 38 c for 18 to 24 hours.The formation of acid and gas
in the stab culture (with or without concomitant blackening)
and the absence of acidity from the surface growth in Triple
sugar iron agar, together with the absence of a red colour in
the urea broth, indicate the presence of salmonella. If acid but
no gas is produced in the stab culture, the identity of the
organisms should be confirmed by agglutination test

Test procedure for Staphylococcus aureus.

Transfer a prescribed quantity of sample in sterile flask
containing 100 ml of fluid soyabean casein digest medium,
shake and incubate at 35 c for 24 to 48 hours. Streak a
portion

of

enrichment

culture

obtained

on

the

surface of one of the following agar medium

Simultaneously carried out the positive control by
streaking a loopful culture of Staphylococcus aureus on
the surface of any of the above four agar media. For
negative control incubate the agar plate of any one of the
four above media without streaking.
Invert and incubate all the plates at 35 to 37 c for 18 to 24
hours.
If, upon examination, none of the plate contains colonies
having the characteristics listed in Table-2 for the media

used, the sample meets the requirements for freedom from

Staphylococcus aureus.
If colonies in any plate confirms to the description in
Table-2, carry out the Coagulase test.
S.N Medium

Colony

Gram

o..

characteristic

Stain

Black colonies
surrounded by yellow
zones.
Yellow colonies with
yellow zone.
Black, shiny colonies
surrounded by clear
zone of 2 to 5 mm.

Positive cocci
in clusters.

1.

Vogel-Johnson
agar

2.

Mannitol-salt
agar.
Baird-Parker
agar.

3.

Positive cocci
in clusters
Positive cocci
in clusters

Coagulase test:
Transfer suspected colonies from the agar surface or any of
the media listed above, to the individual tubes, each
containing 0.5 ml of mammalian, preferably rabbit or horse
plasma with or without additives.
Incubate at 37c and subsequently at suitable intervals up to
24 hours.
If no coagulation in any degree is observed, the sample meets
the

requirements of the test for the absence of

Staphylococcus

Test procedure for Pseudomonas aeruginosa:

Transfer a prescribed quantity of sample in a sterile flask
containing 100 ml of SCDM and incubate at 35 to 37c for 24
to 48 hours.
Streak a portion of the enrichment culture on the surface of
Cetramide agar
Simultaneously carry out the positive control by streaking a
loop full growth of Pseudomonas aeruginosa on the surface
of Cetramide agar. For negative control incubate the
Cetramide agar plate without inoculation. Invert and incubate
the plates at 35 to 37c for 18 to 24 hours.
If upon examination, none of the colonies having
characteristics listed in table-3 for the media used, the sample
meets the requirements for freedom from Pseudomonas
aeruginosa.
If any colonies confirming to the description in Table are
produced, carry out the Oxidase and Pigment test.

S.

Medium Colony

Fluores Oxid Gram

No

charact

cence

eristic

in

ase

Stain

UV

light
1.

Cetramide
Agar

Generally
greenish
colonies.

Greenish

Positive

Negative
rods.

2.

Pseudomonas
agar for
detection
fluorescein.

Generally
colourless to
yellowish
colonies.

Yellowish

Positive

Negative
rods.

3.

Pseudomonas Generally
agar for
greenish
detection
colonies
Pyocyanin

Blue

Positive

Negative
rods.

Pigment test :
Streak representative suspect colonies from the agar surface
of cetrimide agar on the surface of Pseudomonas agar
medium for detection of fluorescein and Pseudomonas agar
medium for detection of Pyocyanin.
Cover and invert the inoculated plates and incubate the plates
at 33 to 37c for less then 3 days.

 Examine the streaked surface under UV light and determine

whether confirms to the description in above table.

Oxidase test:
If growth of suspected colonies occurs, place 2 or 3 drops of a
freshly prepared 1% w/v solution of N, N, N1, N1-tetramethyl-4phenyldiamine dichloride on filter paper and smear with the
suspected colony. If there is a development of pink colour,
changing to purple, the sample meets the requirement for the
absence of Pseudomonas aeruginosa

DATA SHEET FOR PATHOGEN TESTING

(Escherichia coli)
1.Enrichment culture preparation.

Take 100ml of
sample, filter through membrane, and transfer the filter in 100ml of
nutrient broth and incubate at 370c for 24 hours.

2.Preparation:
Ste Mediu
p
m used

Amoun Inocula Temper Observation

t
of tion
ature
Result Positive
control
inocula period.
tion.

1.

MacConkey
broth.

1 ml E/C

48 hours

36-380c

Absen
t

Acid and gas

formation.

2..

MacConkey
broth

0.1 ml

24 hours

43-440c

Acid and gas

formation

3.

Peptone
0.1 ml
water (iodole
test.)

24 hours

43-440c

+ve

Positive control: +Ve

Remark:

Negative control: -Ve

Escherichia coli absent per 100 ml of sample.

DATA SHEET FOR PATHOGEN TESTING.

(Salmonella)
Preparation: As above
Ste Mediu
p
m
used

Amount Inoculat Tempera Observation

of
ion
ture
Result Positive
inoculati period
control
on

Selenite F 1 mlE/C
broth
&T.t.b.g.b
broth.
B.S. Agar Streaking

48 hours

36-380c

18-24 hours

36-380c

3.

X.I.D.
Agar

Streaking

18-24 hours

36-380c

4.

T.S.I.
Agar

Streak
&subculture

18-24 hours

36-380c

5.

Urea
broth

Inoculate

18-24 hours

36-380c

1.

2.

Positive control: +Ve

Absent Turbidity
present.
Black or
green
colonies.
Red with
or
without
black
centre
Acid, gas
w/wo
blackenin
g
Absence
of
red
colour.

Negative control: -Ve

Remarks :

Salmonella absent per gram of sample.

DATA SHEET FOR PATHOGEN TESTING

(Staphylococcus aureus)
1.Enrichment culture preparation.

Take 100ml of
sample, filter through membrane, and transfer the filter in 100ml of
nutrient broth and incubate at 370c for 24 hours
2. Preparation
.

Positive control: +ve

Remarks:

Negative control: -ve

Staphylococcus aureus absent in 100 ml of sample

DATA
SHEET
FOR
TESTING. (P.aeruginosa)

PATHOGEN

Preparation: As above
Ste
p

Medium
used

Amoun Inocula Temper Observation

t
of tion
ature
Result Positive
inocula period
control
tion

1.

Cetramide
agar.
Pseudomonas
agar medium
for detection
of fluorescein
Pseudomonas
agar medium
for detection
of Pyocyanin
Oxidase test

Streaking

24 hours.

370c

Streaking

3 days.

370c

Colourless
to
yellowish

Streaking

3 days

370c

Greenish

370c

+ve

2.

3.

4.

Spreading

Positive control: +Ve

Remarks :

Absent Greenish .

Negative control: -Ve

Pseudomonas aeruginosa absent in 100 ml of sample.

MICROBIAL LIMIT TEST

The Microbial Limit Tests are designed to perform the
qualitative and quantitative estimations of specific viable
microorganisms present in samples. It includes tests for total
viable count (bacteria and fungi) and Escherichia coli. The
most care must be taken in performing the tests, so that
microbial contamination from the outside can be avoided.
When test samples have antimicrobial activity or when they
include antimicrobial substances, these antimicrobial properties
must be eliminated by dilution, filtration, neutralization,
inactivation, or other appropriate means. The tests should be
conducted for samples prepared by mixing multiple portions
randomly chosen from individual ingredients or products.
When samples are diluted with fluid medium, the tests must be
conducted quickly. Due attention must be paid to the effective
quality control and the prevention of biohazard.

1. Total viable aerobic count

This test is to determine mesophilic bacteria and fungi which
grow under aerobic conditions. Psychrophilic, thermophilic,
basophilic, and anaerobic bacteria, and microorganisms which
require specific ingredients for growth may give a negative
result, even if they exist in a significant number. There are
four methods for this test :membrane filtration method, pour
plate method, spread plate method, and serial dilution method
(most probable number method). An appropriate method
should be taken from among these four, depending on
purposes. If automated methods are comparable or superior in
sensitivity and accuracy to the methods given here, they may

be used. Different media and incubation temperature are

required for the growth of bacteria and fungi (molds and
yeasts). The serial dilution method is applicable only to
bacteria.

Procedure
(1) Membrane Filtration Method
This method is applied to the sample which contains
antimicrobial substances. Use membrane filters of an
appropriate material with a pore size of 0.45 m or less.
Filters about 50 mm across are recommended, but other sizes
may be used. Sterilize the filters, filtration apparatus, media,
and other apparatus used. Usually, measure two test fluids of
10 ml each, pass each sample through a separate filter. Dilute
the pretreated test fluid if the bacteria concentration is high,
so that 10-100colonies can develop per filter. After filtration,
wash each filter three times or more with an appropriate
liquid such as phosphate buffer, sodium chloride-peptone
buffer,or fluid medium. The volume of the washings should
be about 100 ml each. If the filter used is not about 50 mm in
diameter, use an appropriate volume of washing,depending on
the size of the filter. If the sample includes lipid, polysorbate
80 or an appropriate emulsifier may be added to the
washings. After filtration, for bacteria detection, place the two
filters on a plate of soybean-casein digest agar medium, and
For fungi detection, add an antibiotic to the medium and place
them on a plate of one of Sabouraud glucose agar, potatodextrose agar, or GP agar media. Incubate the plates at least
for 5 days at 30-35C for bacteria detection and at 20-25C
for fungi detection, and count the number of colonies. If
counts obtained are considered to be reliable in shorter
incubation time than 5 days, these counts may be adopted for
Calculation of the viable count.

(2) Pour Plate Method

Use petri dishes 9-10 cm in diameter. Use at least 2 agar
media for each dilution.Take 1 ml of the test fluid or its
dilution into each petri dish aseptically, add to each dish 1520 ml of sterilized agar medium, previously melted and kept
below 45,and mix. For bacteria detection, use soybean-casein
digest agar medium and for fungi detection, use one of
Sabouraud glucose agar, potato-dextrose agar, and GP
agarmedia, to which antibiotic has previously been added.
After the agar solidifies, incubate at least for 5 days at 3035C for bacteria detection and at 20-25C for Fungi
detection. If a large number of colonies develop, calculate
viable counts based on counts obtained from plates with not
more than 300 colonies per plate for bacteria detection and
from plates with not more than 100 colonies per plate for
fungi detection. If counts are considered to be reliable in a
shorter incubation time than 5 days, these counts may be
adopted.

(3) Spread Plate Method

Place 0.05-0.2 ml of the test fluid on the solidified and dried
surface of the agar medium and spread it uniformly using a
spreader. Proceed under the same conditions as for the Pour
Plate Method, especially about Petri dishes, agar media,
incubation temperature and time, and calculation method.

(4) Serial

Dilution

Method (Most

Probable

Number Method)
Use 12 test tubes: 9 containing 9 ml of soybean-casein digest
medium each and 3 containing 10 ml of the same medium
each for control. Prepare dilutions using the 9 tubes. First, add
1 ml of the test fluid to each of three test tubes and mix to
make 10-times dilutions. Second, add 1 ml of each of the 10times dilutions to each of another three test tubes and mix to

make 100-times dilutions. Third, add 1 ml of each of the 100times dilutions to each of the remaining three test tubes and
mix to make 1,000-times dilutions. Incubate all 12 test tubes
for at least 5 days at 30 - 35C. No microbial growth should
be observed for the control test tubes. If the determination of
the result is difficult or if the result is not reliable, take a
0.1ml fluid from each of the 9 test tubes and place it to an
agar medium or fluid medium, incubate all media for 24-72
hours at 30-35C, and check them for the absence or presence
of microbial growth. Calculate the most probable number of
microorganisms per ml or gram of the
sample, using the table given below.
The number of Test Tubes in which microbial
growth is observed when the amount of the sample
given below ( per Test Tube) is added
0.1g or 0.1ml 0.01g or 0.01ml 1mg or 1l
3
3
3
3
3
2
3
3
1
3
3
0
3
2
3
3
2
2
3
2
1
3
2
0
1.

The
most
probable no. of
microorganisms
per gm or ml
1100
1100
500
200
290
210
150
90

Coliform test

This test is to determine Escherichia coli. Escherichia coli,

target for this test, becomes an important index to evaluate
microbial contamination in ingredients and intermediate
products as well as in the finished products. Escherichia coli
should not be present in each of them. Preparation of test
fluid Unless otherwise specified, proceed as directed in the
preparation of test fluid for total viable aerobic count. When a
fluid medium is used to dissolve or dilute the sample, use

lactose broth medium or BGLB unless otherwise specified.

Procedure Take 10 g or 10 ml of the sample, add lactose broth
medium to make 100 ml, and incubate for 24-72 hours at 30 35C. When the growth of microorganisms in the tube is
observed, shake the tube slightly, take a portion of the fluid
using an inoculating loop, streak it on MacConkey agar
medium, and incubate for 18-24 hours at 30-35C. Examine
the plate for suspicious colonies. If red-brick colonies of
Gram-negative rods surrounded by a reddish precipitation zone
are not found, the sample is determine to be negative. If
colonies meeting the above description are found, then transfer
the suspicious colonies individually on the surface of EMB
agar medium and incubate for 18-24 hours at 30-35C. Upon
examination, if no colonies exhibit a metallic sheen or a blue
black color under transmitted light, the sample is determined
to be negative. For suspected colonies on the plate, conduct
the IMViC tests (Indole production test, Methyl red reaction
test, Voges-Proskauer test, and Citrate utilization test). When the
result given in 1 or 2 in the table below for the four tests is
obtained, determine the colonies to be Escherichia coli. Rapid
detection kits for Escherichia coli may be used.
Indole
production test
1
2

Positive
Negative

Methyl red Voges

reaction test proskauer
test
Positive
Negative
Positive
Negative

Citrate
utilization
test
Negative
Negative

Citrate Utilization Test (Positive)

Citrate Utilization Test (Negative)

Indole Test (Positive)

Indole Test (Negative)

MICROBIOLOGICAL ASSAY OF
ANTIBIOTICS

INTRODUCTION:The microbiological assay is based upon a comparison of the

inhibition of growth of bacteria by measured concentrations of the
antibiotics to be examined with that produced by known
concentrations of a standard preparation of the antibiotic having a
known activity.
Any subtle change in the antibiotic molecules which may not be
detected by chemical methods will be revealed by a reduction in
the anti microbial activity and hence microbiological assay are
very useful for resolving doubts regarding possible loss of potency
of antibiotics and their preparations.
TEST ORGANISMS:-

Antibiotics
Ampicillin
Bacitracin
Chloramphinicol
Tetracycline
Neomycin
Amikacin
Gentamycin

Test organism
Micrococcus luteus
Micrococcus luteus
E coli
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus aureus
Staphylococcus epidermidis

INOCULUM PREPARATION:Incubate the bacterial culture on fresh slant for 24 hours. Wash the
incubated bacterial culture with sterilized water (8 to 1o ml).
Collect the washing and vortex to make the bacterial distribution
uniform throughout the inoculums.
Add this inoculum to the precooled (40 to 45C) liquefied media.
Immediately pore the inoculated medium into sterilized petridishes
to gave a depth of 3 to 4 mm. ensure that the layer of medium are
uniform in thickness by placing the dishes on a levelled surface
and then medium to solidify
.

Methods
Two general methods are generally employed:Method A:- Tuebidimetric (or tube assay) method
Method B:- Cylinder plate (or cup plate) method

Cup-Plate Method
Phosphate buffer preparation
Take 16.73 gm of K2HPO4 (dipotassium hydrogen phosphate) and
0.523 gm KH2PO4 (potassium dihydrogen phosphate) in 1000 ml
capacity beaker.
Add approximately 850 ml of water and stir well to dissolve ,
adjust the pH to 8.0 0.1 with 10 M potassium hydroxide.
Transfer the solution into a 1000 ml capacity volumetric flask and
mark with water.
10 M potassium hydroxide preparation:Weight about 56.11gm of potassium hydroxide in a beaker. Add
about 70 ml water , stir well to dissolve and cool. Transfer the
solution into a 100 ml volumetric flask and make the volume upto
the mark with water.
Standard preparation:Weight accurately neomycin sulphate working standard, equivalent
to about 72.4 mg of neomycin base and transfer to a 250 ml
volumetric flask, dissolve with sufficient base phosphate buffer pH
8.0 and make the volume 250 ml with phosphate buffer. Dilute 5ml
of this solution to 100 ml with phosphate buffer . Again dilute 2.5
ml of it to 100 ml with phosphate buffer (standard stock solution )
to make standard high (SH) and standard low (SH) dilutes
respectively.
Sample preparation :-

Weight the sample accurately to about 144.8 mg of neomycin base

and transfer to a 500 ml volumetric flask. Add about 250 ml of
phosphate buffer pH 8.0 and shake well. Dilute the 5 ml and
2.5ml of it to 100 ml witgh phosphate buffer ( test stock solution )
to make sample test high (TH) and test low (TL) dilution
respectively.

Procedure:Prepare minimum 5 petridishes. Make four cavities in each of the

petridishes with the help of a sterilized stainless steel borer having
outside diameter 8 0.1 mm and inside diameter 6 0.1 mm. Then
add 100 l of standard and sample dilution (high and low) in each
cavity of petridishes in such a manner that the highest
concentration of the standard and sample preparation are not
adjacent to each other.
After addition of sample and standard dilution in designed
cavities , keep the plates at room temperature for minimum 60
minutes. This is a period to minimize the diffusion due to variation
in the of application of sample and standard dilution in the cavities
of each petridish. Then incubate for about 18 hours at 36C to
37C .
After incubation , measure the diameters of the circular inhibition
zone s for standard high, standard low, test high and test low
dilutions. Average of the diameter of respective zones in all plates
is taken into account.
Calculations:Antibiotic % = Antilog (2.0 + a log.I)
Where:{(TH + TL) (SH + SL)}
a = -----------------------------------{(TH - TL) + (SH - SL)}

TH, TL, SH, SL indicate the sum of the inhibition zone

diameter with test high, low, standard high and standard low
dilutions respectively.
I = ratio of dilution for sample / standard.

Antibiotic Assay Result

Psedomonas aeruginosa

STERILITY TESTING
OBJECTIVE:

To lay down the procedure for performing the sterility testing of

sterile finished goods and sterile raw materials.
PRINCIPLE:

This test is based upon the principle that if bacteria and fungi are
placed in a medium which provides nutritive material and water, and
kept at favorable temperature, the organisms will grow and their
presence can be indicated by turbidity in the originally clear medium.
REQUIREMENTS:

 Conventional Method:

1. Sterile filtration Cups.

2. sterile Manifold.
3. Scissors.
4. Forceps.
5. Sterile 0.45u, 47mm edge hydrophobic membranes.
6. Vacuum pump.
7. Micropipettes, Sterile tips.
8. Suction Flask.
9. Suction tube.
10. Sterile Vent filters.
Closed Method:
1.
2.
3.
4.

Steritest Compaq system

Dilutors
Canisters
Disposable Syringe

REAGENTS:

1.
2.
3.
4.

Sterile Fluid Thioglycollate Medium

Sterile Soyabean Casein Digest Medium
0.1% Peptone water
Beta-lactamase(neutralizer)

PROCEDURE:

Articles to be tested:
Minimum no. of articles to be tested in relation to the no. of articles
in the Batch is described in table 1
No. of Articles in Batch
No. of Articles to be Tested
For Injections
More than 500 articles
2% or 20 articles which ever is less
For Antibiotic Solids Bulk and Blend Products

Aseptically remove a sufficient quantity of solids from the container mix to obtain a
composite sample not less than 10g of solids.

Volume of medium:
1. The volume of medium used in the test should not be less than the
volume indicated in table 2.
2. 100ml of Soya bean Casein Digest Medium and 100ml of fluid
thioglycollate medium is prepared in bottles for Closed System and
in test tubes for Conventional method. 0.1% of peptone in bottles

Table: 2 Quantities of articles for liquid Products.

Container content(ml) Minimum quantity taken from

Medium vol. in ml for each medium for

each container for each medium membrane filtration method

10 to less than 50

5ml

100

Table: 2 Quantities of articles for solid Products

Container content Minimum quantity taken from

Medium vol. in ml for each medium for

each container for each medium membrane filtration method

200-300mg
100mg
300-600mg
200mg
>600mg
200mg
Opening of Articles:

200mg
200mg
200mg

1. Care must be taken when opening an article so that the sample

to be tested for sterility is not contaminated by the micro
organisms present on the exterior of the container.
2. The exterior surfaces of the ampoules, vials, and bottles must
be cleansed with a suitable decontaminating agent and should
be placed in an environment that prevents recontamination of
the exterior surfaces.
Membrane Filtration Method (Conventional Method):
Before starting the test clean the LAF bench with 75%
filtered IPA.
Place the sterilized manifold on the LAF and connect the
tubing to the vacuum line through a collecting reservoir.
Place the required no. of filter holders on the manifold.
Put the sterilized membrane/ pre sterilized in between the
filter support with the help of a sterile forceps.
Antibiotic Solids for Injections
Pharmacy bulk packages.

For the sterile bulk material, reconstitute about 6gms of

material in 200ml sterile 0.1% Peptone water previously
inoculated with 0.6ml of Beta lactamase in a 500ml
conical flask.
Shake or swirl to dissolve it completely.

 Transfer the entire contents into the filtration funnel for

filtration.
For sterile WFI ampoules, decontaminate the exterior of
the ampoules using sterile 70% IPA, break the neck of
glass ampoules and transfer the contents into the
filtration funnel. In case of plastic ampoules, withdraw
the contents of the ampoules with the help of a sterile
syringe & needle and transfer into the filtration funnel.
For the testing of water for Injection, usage of
neutralizing agent is not required.
After the contents are filtered, rinse the membrane with
three 100ml portion of 0.1% Peptone contaning .8ml of
Beta lactamase.
Remove the upper part of the funnel, and cut the
membrane into two halves using sterile scissors and
forceps.
Aseptically transfer one half into Thioglycollate
medium and other half into Soya bean Casein digest
medium.
Inoculate each medium tube with the validated quality
of Beta lactamase enzyme. 0.6ml for Bulk Active
Pharmaceutical Ingredients and 2.0 ml for Sterile
Dosage Forms.

 Each time when the Sterility Test is performed, include

one tube of each medium as negative control, which has
been treated in the similar way as test samples except
the addition of product to confirm the sterility of the
medium.
Formulation:
1.From each of 20 containers aseptically transfer about 300
mg of solids into a sterile 200ml conical flask containing
0.6 ml of Beta lactamase, dissolve in 200ml of 0.1%
Peptone and mix or reconstitute.
2.Shake or swirl to dissolve the material completely.
Sterility by Closed System (Steritest - Compact) Millipore
1. Perform

the

testing

of

samples

as

per

the

SOP

no.VMRC/SOP/M/043 for the operation of sterility test.

2. fix the sterility canisters on the drain tray and tubing for peristaltic
pump holder.
3. Before filtering the sample, add 0.6ml of sterile Beta lactamase for
sterile API and 2.0 ml of Beta lactamase for sterile finished dosage
forms into both the FTGM and SCDM bottles.
4. Inoculate 0.6 ml/2.0 ml of Beta lactamase into the 0.1% peptone
solution bottles.
5. Before filtration, pre wet the membrane with 0.1% peptone water to
decrease the binding of the antibiotic with the membrane.
6. Filter the product solution in low speed to avoid spillage of
antibiotic solution on the wide area of canister walls.

7. After complete filtration, rinse the tubing, canister and membrane

with 300ml of 0.1% peptone water in 3 consecutive rinses to
remove antibiotic traces. (300 ml per each of two portions)
8. While giving the washings first fill the 100 ml of rinsing fluid in to
canister and filter it to remove the antibiotic traces on the canister
wall surface.
9. In second rinse, continuously filter the rising fluid to remove the
antibiotic traces from the membranes.
10. After the washing, procedures take up to 100ml mark with low
speed 25-30 rpm to avoid excessive aeration in the media.
11.Clamp the tubing around 4 inches distance from the canister cut the
tubing with sterile scissors and plug into vent filter.
12.Perform the negative control in same above manner with out
product.
13.The following information is to be written on canister.
Product
Batch no.
Date of testing
Media name/ lot no.
14. Transfer canisters from the sterility test for incubation.
Test for positive control:
1. Perform the positive control test in the controlled area of microbiology lab.
2. For performing the positive control for the same lot of media which is used
in the sterility test on particular day.
3. Inoculate the 10 100 cells of culture suspension into media bottles by
using a disposable syringe. Incubate the positive control media tube /

bottles along with test canisters / tubes to stimulate the same incubation
conditions.
4. Follow the table for the culture inoculation for positive control.
Organism of positive Test Media

Frequency

Incubation conditions

Daily
20 250C
Daily one organism 20 250C
20 250C
Alternatively
Clostridium sporogens
FTGM
Daily
30 350C
Staphylococcus aureus
FTGM
Daily one organism 30 350C
Pseudomonas aeruginosa FTGM
30 350C
Alternatively
Precautions to be taken before / during the test for sterility.
Bacillus subtilis
Candida albicans
Aspergilus niger

SCDM
SCDM
SCDM

1. The blower of the garment cubical should be always in

running mode.
2. Follow the gowning procedure as per the SOP correctly and
efficiently.
3. Always wear goggle during the test.
4. Use 70% filtered IPA frequently to disinfectant the gloves
during the test.
5. The sample reconstituted for testing should be immediately
filtered without any delay to avoid false negative results.
6. the forceps and the scissors used for cutting and inoculating
the membranes should not be hot while holding the membrane.
7. If the temperature or differential pressure is not as per the
acceptance criteria inform the maintenance department to take
necessary action and after it is resumed go for sterility testing.
8. Avoid excessive aeration of FTGM.

Incubation and observation:

1. Incubate the Fluid Thioglycollate medium tubes at 32.5 0C
and Soya bean Casein digest medium tubes at 22.50C for not
less than 14 days.
2. Observe the tubes on daily basis for any growth / turbidity
during the incubation period and at the end of incubation
period record the detail.
3. If turbidity, precipitate or other evidence of microbial
develops during incubation investigate the failure of the test
as per the SOP.
Interpretation of results:
The articles meet the requirements of the test for sterility
when no microbiological growth is observed.Observe result
compare with positive controls.

OBSERVATION
The sample of water for injection was tested for the following
microbiological tests ;i. Sterility test
ii. Microbia limit test
iii. Bacterial endotoxin test
After applyingThe abovetest on the given sample of water for
injection, following observation wererecorded;Sterility test;No turbidity was observed after fourteen days ofincubation of
filter membrane and incubation at 37c . Hence no growth of micro
organism is observed.

Microbial limit test;Test for E.coliNo acid and gas production was observed in mac conkeys broth.
Further, no growth of red or non mucoid colonies with metallic
sheen was observed on EMB plates. Hence the test for E.coli was
found to be negative.
Test for SalmonellaNo turbidity was observed in selenite F broth and tetrathionate bile
brilliant green broth. Further, no growths of black or green colour
colonies were observed on bismuth sulphite agar plates. No
growths of red with or without black centre colonies were observed
on Xylose lysine deoxycholate agar plates. Hence, the tests for
Salmonella were found to be negative.
Test for PseudomonasNo tubidity was observed in SCDM tubes and no formation of
greenish colour colonies were observed on cetrimide agar plates.
Hence, tests for Psudomonas were found to be negative.
Test for Staphylococcus aureusNo turbidity was observed in SCDM tubes. No growth of black,
shiny surrounded by clear zone colonies were observed on Baird
Parker Agar plates.

RESULTS
After performing sterility test,and microbial limit test,on the given
sample of water for injection, it was observed that the sample
being tested has no micro organisms or spores in it.
No turbiditywas observed in sterility test tubes, hence, the sample
being tested complies with the test of sterility.
No growth was observed on selective media plates hence the
sample being tested complies with MLT test and pathogen
i.e.E.coli, Salmonella, Pseudomonas and Staphylococcus aureus
are found to be absent in the sample being tested.

SUMMARY
After performing water chemical testing, microbial limit test,
bacterial endotoxin test, antibiotic assay and sterility test on the
given sample of water for injection, it was observed that the
sample being tested has no micro organisms or spores in it.
No growth was observed on selective media plates hence the
sample being tested complies with MLT test and pathogens i.e. E.
coli , Salmonella, Pseudomonas and Staphylococcus aureus are
found to be absent in the sample being tested. So, the sample of
WFI complies with the Microbial Limit Test.
No turbidity was observed in sterility test tubes till fourteen days,
hence, the sample being tested complies with the test of sterility.
Firm gel clot formation was observed in BET tubes; hence, the
sample being tested complies with the BET test.
The sample being tested shows negative results for all the
pathogens i.e. E.coli, salmonella, Pseudomonas and S aureus. It
also shows negative results for the test of pyrogens and endotoxins.
Negative test is also observed for aerobic and anaerobic microorganisms under sterility test.
Hence, it is concluded that the sample being tested complies with
the USP and IP standards and is considered to be sterile.

The given drug is found to be effective in checking the growth of

bacteria as clear zones are observed in antibiotic assay.
On comparing the results obtained with the standard table, it is
found that the given sample falls within the standard range and
hence complies with the standards of Parma industry.

APPENDIX
1) Alternative Thioglycollate Medium
L -Cystine
Sodium chloride
Dextrose
Yeast Extract (water soluble)
Pancreatic Digest of Casein
Sodium thioglycollate or thioglycolic Acid
Distilled water
The pH after sterilization is 7.10.2
2)BAIRD PARKER AGAR
Pancreatic digest of casein
Beef extract
Yeast extract
Lithium chloride
Agar
Glycine
Sodium pyruvate
Water
3) BISMUTH SULPHITE AGAR

0.5 gm
2.5 gm
5.5 gm
5.0 gm
15.0 gm
0.3 gm
1000 ml.

10.0 gm
05.0 gm
01.0 gm
05.0 gm
20.0 gm
12.0 gm
10.0 gm
1000 ml

SOLUTION (1):Beefextract
Peptone
Agar
Ferric (III) citrate
Brilliant green
Water
SOLUTION (2):Ammoniumbismuthcitrate
Sodium sulphate
Anhydrous disodium hydrogen phosphate
Dextrose monohydrate
Water
Mix, heat to boil, cool to room temperature, add
solution (2) to 10 volumes of solution (1).

06.0 gm
10 gm
24 gm
0.4 gm
10.0 mg
1000ml
03.0 gm
10.0 gm
5.0 gm
5.0 gm
1000 ml
1 volume of

4) BRILLIANT GREEN AGAR

Peptone
Yeast extract
Lactose
Sucrose
Sodium chloride
Phenol red
Brilliant green
Agar
Water

10.0 gm
03.0 gm
10.0 gm
10.0 gm
5.0 gm
0.08 gm
12.5 mg
12.0 gm
1000 ml

5) CETRIMIDE AGAR
Pancreatic digest of gelatin
Magnesium chloride
Potassium sulphate
Cetrimide
Agar
Glycerine
Water

20.0 gm
01.4 gm
10.0 gm
0.3 gm
13.6 gm
10.0 gm
1000 ml

6) DEOXYCHOLATE CITRATE AGAR

Beef extract
Peptone
Lactose
Trisodium citrate
Sodium thiosulphate
Ferric (III) citrate
Sodium deoxycholate
Neutral red
Agar
Water
7) EOSIN METHYLENE BLUE AGAR
Tryptone
Dipotassium phosphate
Lactose
Sucrose
Eosin Y
Methyleneblue
Agar
8) FLUID THIOGLYCOLLATE MEDIUM
L cystine
Sodium chloride
Glucose monohydrate
Granular agar
Yeast extract
Pancreatic digest of casein
Sodium thioglycollate
Resazurin (1.1% fresh solution)
Water
9) LACTOSE BROTH
Beef extract
Pancreatic digest of gelatin

05.0 gm
05.0 gm
10.0 gm
8.5 gm
5.4 gm
1.0 gm
5.0 gm
0.02 gm
12.0 gm
1000 ml
10.0 gm
2.0 gm
5.0 gm
5.0 gm
0.4 gm
0.065gm
13.5 gm
0.5 gm
2.5 gm
5.5 gm
0.75 gm
5.0 gm
15.0 gm
0.5 gm
1.0 ml
1000 ml
3.0 gm
5.0 gm

Lactose
Water
10) MacConkeys broth
Peptone
Sodium Chloride
Sodium taurocholate
Water
Bromocresol purple solution
Lactose
11) MANNITOL SALT AGAR
Pancreatic digest of casein
Peptic digest of animal tissue
Beef extract
D-mannitol
Sodium chloride
Agar
Water
12) Nutrient agar medium (NAM)
Peptone
Beef extract
Sodium chloride
Distilled water

5.0 gm
1000 ml
20 gm
5 gm
5 gm
1000 ml
10 ml
10 gm
5.0 gm
5.0 gm
1.0 gm
10.0 gm
75.0 gm
15.0 gm
1000 ml
5 gm / litre
3 gm / litre
5 gm / litre
1000ml

pH after sterilization is 6.9

13) NUTRIENT BROTH
Dextrose
Tryptone
Yeast extract
Monopotassium phosphate
Potassium chloride
Magnesium sulphate

50.0 gm
5.0 gm
4.0 gm
0.55 gm
0.425 gm
0.125 gm

Calciumchloride
Bromocresol green
Manganesesulphate
Ferricchloride
Water

0.125 gm
0.22 gm
0.0025gm
0.0025gm
1000 ml

14). Sabouraud dextrose agar (SDA)

Mycological peptone
Dextrose
Agar
pH after sterilization is 5.6 0.2

10 gm/ litre
40 gm / litre
15 gm / litre

15). Plate count agar (PCA)

Casein enzymic hydrolysate
Yeast extract
Dextrose
Agar
pH after sterilization is 7.0 0.2

5 gm / litre
2.5 gm / litre
1 gm / litre
15 gm / litre

16) SOYBEAN CASEIN DIGEST AGAR

Pancreatic digest of casein
Papaic digest of soyabean
Sodium chloride
Agar
Water
pH after sterilization
17) SOYABEAN CASEIN DIGEST MEDIUM
Pancreatic digest of casein
Papaic digest of soybean meal
Sodium chloride
Dipotassium hydrogen sulphate
Glucose monohydrate
Water

15.0 gm
0.50 gm
5.0 gm
15.0 gm
1000 ml
7.30.2
17.0 gm
3.0 gm
5.0 gm
2.5 gm
2.5 gm
1000 ml

18) Selenite F broth

Peptone
Lactose
Disodium hydrogen Orthophosphate
Sodium hydrogen selenite
Water
19) Tetrathionate Broth
Beef extract
Peptone
Yeast extract
Sodium chloride
Calcium carbonate
Sodium thiosulphate
Water
20) Triple Sugar Iron agar
Beef extract
Yeast extract
Peptone
Lactose
Sucrose
D- glucose monohydrate
Iron (II) sulphate
Sodium chloride
Sodium thiosulphate
Phenol red
Agar
Water
21) UREA BROTH
Potassium dihydrogen orthophosphate
Anhydrous disodium hydrogen orthophosphate
Urea
Yeast extract

5 gm
4 gm
10 gm
4 gm
1000 ml
0.9 gm
4.5 gm
1.8 gm
4.5 gm
25.0 gm
40.7 gm
1000 ml
3.0 gm
3.0 gm
20.0 gm
10.0 gm
10.0 gm
1.0 gm
0.2 gm
5.0 gm
0.3 gm
24 mg
13.0 mg
1000 ml
9.1 gm
9.5 gm
20.0 gm
0.1 gm

Phenolred
Water
22) VOGEL- JOHNSON AGAR
Pancreaticdigest of casein
Yeastextract
Mannitol
Dibasic potassium phosphate
Lithium chloride
Glycine
Agar
Phenol red
Water
23) XYLOSE LYSINE DEOXYCHOLATE AGAR
Xylose
L-Lysine
Lactose
Sucrose
Sodium chloride
Yeast extract
Phenol red
Agar
Sodium deoxycholate
Sodium thiosulphate
Ferric ammonium citrate
Water

0.01 gm
1000 ml
10.0 gm
5.0 gm
10.0 gm
5.0 gm
5.0 gm
10.0 gm
16.0 gm
25.0 mg
1000 ml
3.5 gm
5.0 gm
7.5 gm
7.5 gm
5.0 gm
3.0 gm
80.0 mg
13.5 gm
2.0 gm
6.8 gm
800 mg
1000ml

REFERENCE:1. Prescott, Harley, Klein, Microbiology

2. Dawson, M.E., Novitsky, T.J. and Gould, M.J.(1988).
Microbes, Endotoxins and Water Pharmaceutical
Engineering.
3. Novitsky, T.J. (1984), Monitoring and validation of high
purity water systems.
4. Indian Pharmacopeia (I.P.).
5. United States Pharmacopeia (U.S.P.).
6. British Pharmacopeia (B.P.).