PROJECT REPORT ON

QUALITY CONTROL OF MICROBIAL

QUALITY OF WATER
TRAINING PLACE:

INSTITUTE OF TECHNOLOGY-BHU,
VARANASI

DURATION:

17th JUNE TO 10th JULY

SUBMITTED TO:

Dr. RASHMI SHARMA (H.O.D)

(Dept. of Biotechnology)
HIMANSHU KUMAR

SUBMITTED BY:

B.E.(BIOTECHNOLOGY)
VII Semester
Roll. No. 07BT40(8428)

INSTITUTE OF ENGINEERING & TECHNOLOGY,

KHANDARI CAMPUS, AGRA.

ACKNOWLEDGEMENT

I am very thankful to DR. P. N. TIWARI. He has always been the sources of

inspiration and has enabled me to complete my project efficiently by his
valuable guidance, constructive criticism, innovation ideas and moral
support.
I also want to thank Mr. Manish Mishra for his valuable advice and guidance
throughout the training period. On the same note I also want to thank Mr.
Alok Sharma, Mr. Sandeep Sharma, Ms. Pooja for helping me in better
understanding of Microbial Quality of Water handing. They gave me
appropriate knowledge of understanding and identification of microorganism
in Q.C. and encouraging me to complete the training successfully.
I would also like to thank Dr. Sanjeev Sharma Director of the Institute of
Engineering and technology, Agra and Mrs. Rashmi Sharma H.O.D. of
Biotechnology and Training and placement coordinate Mr. Diwakar Tiwari
and all faculty members and friends for helping me.
At last special thanks to my family who directly and indirectly took a keen
interest in assembling my internship, without my family support its
impossible for me to complete this project.

(HIMANSHU KUMAR)

CONTENTS

MICROBIAL QUALITY OF WATER

GENERAL
TEST FOR TOTAL AEROBIC MICROBIAL COUNT
TEST FOR TOTAL COMBINED YEAST/MOLD COUNTS
ENRICHMENT FOR PATHOGENS
TEST FOR PATHOGENS
TEST FOR E.Coli.
TEST FOR SALMONELLA
TEST FOR PSEDOMONAS AERUGINOSA
TEST FOR STAPHYLOCOCCUS AUREUS
TEST FOR TOTAL VIABLE COUNTS IN WATER FOR
INJECTION
MICROBIAL LIMITS OF DM/PURIFIED WATER
REFERENCES
ABBREVIATIONS

DECLARATION

TITLE:- MICROBIAL QUALITY OF WATER.

Objective:- To lay down a procedure for monitoring of microbiological quality of

water.
E H S Precautions:- Use all safety protective equipment like hand gloves & safety
goggles during analysis.

Procedure:-

1. Sampling Procedure:1.1 Collect the sample daily as per the sampling schedule.

S.O.P ( Standard operating procedure)

1.2 For sampling purpose sterilize sample bottle or flask capacity to hold 250-ml
water. Properly classed with stopper or plug followed by derminum foil/butter
paper.
1.3 For UGT sample 5 drop of sodium Thio-sulphate is added in sampling bottle
and sterilized it for deactivation of unlorine.
1.4 Before collecting the sample drain out the water from tap line for about 10 to
15 liter.
1.5 Wear pre sterilized hand gloves & sanitize hand gloves with 70% IPA
(Isopropyl Alcohol) prior to sample collection.

1.6 Clean the outer surface of sampling bottle or flask with 70% IPA to avoid
external contamination.
1.7 Open the sterile bottle or flask by removing the lap/stopper or plug of flask in
such a way that bare hand should not touch to the inside of the plug of bottle or
flask.
1.8 Collect 250ml sample from the user point taking precaution to avoid
contamination.
1.9 User point is the point from where water is taken for actual use, for example
has pipe, which is either connected, to the reactor for charging or to the centrifuge.
1.10 Collect the sample and immediately clause the lap/stopper or plug. Cover the
bottle. Flask with aluminum foil/butter paper.
1.11 Label the sample bottle/ flask its identification and date of collection of
sample.
1.12 Bring the sample to the microbiology laboratory for analysis.
1.13 Analyze the sample with in two hour of collection, if the sample is not
analyzed within two hours then keep it at 2-8C and analyze them within four
hours.
1.14 Allow the sample to attain the room temperature before analysis if it is kept in
refrigerator.
2. General:2.1 As per specification, water is analyzed for total aerobic microbial count and
total combined yeast/ molds count.

2.2 As additional in house requirements, water is also tested for pathogens

(Eschericnia coli, Salmonella SP. Psedomonas aeruginase and stephylococus
aureus)
2.3 Filtration method is used for analysis of D.M water for total bacterial and
fungal count and for pathogens in water samples.
2.4 Pour plate method is used for analysis of Raw water / Portable for total
bacterial and fungal count and pathogens are tested through membrane filtration
method.
2.5 Filtration method is used for analysis of water for injection for total bacterial
and fungal count and for pathogens in water samples.
3. Test for total Aerobic microbial count:-

3.1 Take 1ml of the sample into a sterilized filtration assembly containing 0.45
membrane filter and filter using vacuum.
3.2 After filtration transfer the membrane filter with the sterile forceps on to
soybean casein digest agar for total aerobic microbial count.
3.3 Keep the Petri dish of soybean casein digest agar in incubator.

4. Test for total combined yeast/mold counts:-

4.1 Take 1ml of the sample into a sterilized filtration assembly containing 0.45
membrane filter and filter using vacuum.

4.2 After filtration transfer the membrane filter with the Sterile forceps on to
Sabouraud Dextrose agar for total combined yeast/mold count.
4.3 Keep the Petri dish of sabouraud dextrose agar in incubator at 20 to 25C for 57 days in inverted position.
4.4 At the competition of incubation period , count the no. of colonies developed.
4.5 Calculate the no. of CFU 1ml of the sample and report the results.

5. Enrichment for Pathogens:-

5.1 Filter 100ml of water sample by 0.22 or 0.45 micron membrane.

5.2 Transfer the membrane aseptically to 100 ml of or soybean casein digest media
or nutrient broth.
5.3 Incubate the tube at 30 -35C for 18-24 hours (Solution A)
5.4 Observe after incubation, if growth is present carry out the primary test.

6. Test for Pathogens:-

6.1 Primary Test of Escherichia Coli:6.1.1 Add 1ml from enrichment culture (Solution A) to 100ml Macconkey broth.
6.1.2 Incubate at 42 -44C for 24 48 hours.

6.1.3 If the content of the tube shows acid and gas formation, carry out the
confirmatory test. Acid production is indicated by change in colour of the broth
from purpose to yellow.

6.2 Secondary test:-

6.2.1 Subculture on a plate of macconkey agar and incubate at 30-35C for 18 to 72

hours. Growth of brick red, generally non-mucoid colonies of gram-negative rods,
sometimes surrounded by a precipitation Bile zone indicates the possible presence
of E. Coli.
6.2.2 Confirm the presence of E-Coli by inoculating the suspected colonies into
5ml of peptone water and incubating at 30-35C for 24 hours to test innate, add
0.5ml of kavacs reagent, shake well, and allow standing for one minute. If a red
color is produced in the reagent layer enable is present. Simultaneously if require
steak the colonies on EMB agar at 30-35C for 18-48 hours. Growth of blue black
colonies with metallic sheen confirms the presence of E. coli. The product passes
the test if such colonies are not seen or if the confirmatory biochemical tests are
negative.

Note:- Carry out a control test by sub culturing a 24 hours old broth culture of E
.Coli ATCC 8739/ MTCC 1687 on macconkey agar, EMB Agar and macconkey
broth. The test is invalid if the results dont indicate that the control contains
E.Coli.

Kovacs reagent :

P-dimethylaminobenzaldehyde

5ml

Amyl Alcohol

75ml/litres

HCL concentrated

25ml/litres

Or
Use kavacs reagent manufacture by Himedia

7.2 Primary test for salmonella:-

7.2.1 Shake the container and transfer 0.1ml of enriched culture (Solution A) to
10ml of Rappaport vassiliadis Enrichment broth and incubate 18 to 24 hours.
7.2.2 Secondary test:- Subculture on xylose lysine deoxycholate Agar or Bismuth
Sulphite agar or Brilliant green agar incubate at 30-35C for 18 to 48 hors. If any
colonies conforming to the description in Table 111 are produced carry out the
confirmatory test.
Table 111
Medium

Description of Colony

(1) Xylose lysine Deoxycholate

Well developed Red colonies,

with or without Black Centers.

(2)Bismuth sulphite agar

Black or green colonies

(3) Brilliant green agar

Small transparent colorless or

pink to white. Opaque (Surrounded by
pink to red zone)

7.2.3 Subculture any colonies showing characteristic given in Table 111 on triple
sugar iron agar using surface and deep inoculation. Incubate at 30-35C for 18 24
hours. The presence of salmonellae is provisionally confirmed if, in the deep
culture but not in the surface culture. There is a change of color from red to yellow
accompanied by gas formation with or without blackening. Precise confirmation
may be carried out by appropriate biochemical; & serological tests.
7.2.4 The preparation being examined passes the test, if in the primary test,
cultures of the type described do not appear or if, in the secondary test. The
confirmatory biochemical and servo tests are negative.

Note:- Carry out a control test by sub culturing a 24 hours old broth culture of
salmonella enteric SSP. Enteric Serotype abony NCTC 6017/MTCC 3858 on triple
sugar iron agar. The tests is invalid if the results do not indicate that the control
contains S. abony.

Interpretation for TSI Slants :-

Tube
1

Slant

Butt

Orange

Orange

Red

Red

Pink

Yellow

Gas

H2S

Inference

Remarks

Un-inoculated

Control
-

Slignt

Dextrase
fermentation
and H2S
production
(Slignt)

Yellow

Yellow

Dextrase and

Other

lactase/sucrase Enterobacteria
fermentation
4

Pink

Black

Dextrase

Characteristics

fermentation

of

and abundant

Salmonellae.

H2S
production

8. Primary Test for Psedomonal aeruginasa:8.1 Subculture on a plate of cetrimide agar by streaking agar by streaking from
solution A and incubate the plates at 30-35C for 18 to 72 hours. If no growth of
microorganism is detected, the preparation being examined passes the test.

8.2 Secondary test:- If growth of colonies of gram-negative rods occurs, proceed

further suspected colonies on Pseudomonas agar for flouresein and Pseudomonas
aga for Pyocyanim.

Mor phological characteristics of P. Aerugfinose on selective agar media

Selective media
1. Pseudomonas agar medium for detection of flouresein.
2. Pseudomonas agar medium for detection of Pyocyanim.
3. Cetrimide Agar

Characteristic Colonial Morphology:

1. Generally colorless to yellowish
2. Generally greenish
3. Generally greenish

Florescence in U. V. light:-

1. Yellowish
2. Blue
3. Greenish

8.3 oxidase test:- Smear the suspected colony on the oxidase test dise (N N
dimethyl p phenylene diamine oxalate). The test is positive if purple color is

produced within 5 to 10 seconds. Positive oxidase test and Gram negative bacilli
indicates the presence of P. aeruginase. If purple color is not produced within 5 top
10 seconds or produced later on indicates the absence of P. aeruginase.

9. Primary Test for Staphylococcus aureus:-

9.1 Streak the enriched culture on Vogel Johnson Agar or mannital salt agar or
baired Parker agar.
9.2 Cover and invert the dishes and incubate at 30-35C for 18 -72 hours.
9.3 If any colonies confirming to the description as given below, indicates the
presence of staphylococcus aureus.

Medium

Colony Characteristics

Gram Stain

Vogel Johnson Agar

Black coloies surrounded

Positive cocci in clusters

by yellow zone
Manital salt Agar

Yellow colonies with

Positive cocci in clusters

yellow zone
Baired Parker Agar

Black Shiny, surrounded

by clear zone

Positive cocci in clusters

9.4 If typical colonies observed, perform coagulase test.

9.5 Coagulase Test:-

9.5.1 Transfer suspected colonies to the tube containing 5ml mammalion Plasma/
rabbit/ horse with or without additives.
9.5.2 Incubate on water bath at 35C.
9.5.3 Examine the tubes at 3 hours and subsequently at suitable interval up to 24
hours. If no. coagulation in any degrees of sample meets the requirement of
absence of S. aureus.

10. Test for Total viable counts in water for injection:-

10.1 Filter about 100-100ml water for injection through sterile 0.22 or 0.45 micron
membrane filter separately for TAMC and TYMC.
10.2 Put the filter paper on freshly prepared and solidified soybean casein digest
agar plate for total Aerobic microbial count and sabouraud dextrose agar for total
combined yeast/ molds count.
10.3 Take precaution while transferring the filter paper that no air bubble that no
air bubble should be entrapped in between agar surface and filter paper.
10.4 incubate the plate in inverting position at 20 to 25C for 5-7 days for total
combined yeast / molds count and 30-35C for 3-5 days total aerobic microbial
count.

10.5 count no. of colonies which develop on surface preferatty on colony counter
under magnification.
10.6 calculate the No. of total colony farming unit/100ml and report the results.
10.7 Analysis for pathogens is done as per point no. 5 and 6.

11. Microbial limits of DM/ Purified water ( Release criteria):-

11.1 For total aerobic microbial count not more than 100 cfu/ml.
11.2 for total combined yeast/ molds count: NMT 10cfu/ml.
11.3 Pathogens : Absent
11.4 Refer the guide lines given below for avert & action limit.

For TBC

Loop system

Non Recirculation
system

Alert limit

Less than 40 cfu % ml

Less than 60 cfu/ml

Action limit

Less than 60 cfu / ml

Less than 80cfu/ml

12. Records and Trends analysis of Data:-

12.1 Record the samples for microbial quality of water in format no.
R/MB/62/03/03

12.2 Record the observations in format no. R/MB/157/00/10.

12.3 When observation of primary test for specified organisms (pathogens) are
secondary test and record the observations of format No. R/MB/158/00/10

WATER TREATMENT

Water is mostly used for industrial & municipal purpose. In order to ensure the
quality and quantity of water for these purposes, it is extremely important to
monitor water supply through out taking all the aspects into consideration. The
quality and quantity of water are important in the location of chemical plant. For
this purpose surface water as well as ground water may be used, but supply must
be adequate and continuous through out the year. The treatment of water to which
it is subjected depends on the purpose for which the treated water has to apply.
Head water contains dissolved salts of magnesium present as bicarbonates
sulphates and chlorides. We use the water treatment process to remove the
impurities like (bicarbonates sulphates and chlorides) of hard water. Working and
sanitation of equipments involved in water treatment are as follows:

Sources of water: Bore well is the source of the water the are four in number and
water is pumped out with the help of bore well pumps. Then the pumped out water
sent to plant by pipelines. The water pumped out is known as raw water, which is
stored and chlorinated and passes through sand filter, ACF.

1. Sand filter: It removes suspended particles present in water. It is cleaned

daily by reverse water flow & yearly sand charged.
2. ACF (Activated Carbon Filter): It removes chlorine added as a
disinfectant, from water. It is sanitized weekly & yearly it is replaced. These

filters have various alternative layers of media and carbon, which are
responsible for the removal of chlorine from water. There are 3 ACF filters.
3. Softener Filter: It removes hardness based on sodium base cationic
exchangers. It is charged by 33% brine solution. It is sanitized by
formaldehyde soln.(0.5%) and by reverse flow. Softener filters are three in
no. that works alternatively.

WATER TREATMENT

Water (Bore well)

Chlorine (6ppm)
Raw water storage tank
Sand filter
AFC (Activated Carbon Filter)
(Chlorine removes)
SF (Softener)
(.2% Nacl are used for charging)
R.O. System
R.O. water Storage tank
(addition of cl2 8ppm)
Micron Filter
UV Light
Product line

Flow diagram of water treatment

Difference between Treated Water and Raw Water

RAW

TREATED WATER

COLOR

No color

No color

ODOR

No odor

No odor

TASTE

No taste

No taste

TOTAL SOLIDS

>2000

<200

HARDNESS

CaCO3

230 ppm

Ca

230 ppm

Mg

28 ppm

Fe

0.2 ppm

<0.1 ppm

Silica

<10 ppm

<0.1 ppm

Sulphates and Chlorides

370 ppm

<20 ppm

Function of Equipment Involved in Water Treatment Plant

S.No.
1.

Equipment
Sand Filter

Function
It

filters

the

Maintenance
raw The filter is cleaned every month by cl 2

water with help of

water and sand is charge every year.

sand. It is fluidized Backwash is done in every 24 hours,

bed
2.

Activated

followed by rinsing

carbon It removes chlorine The filter is sanitized weekly by stream at

filter

85oC 3 with contact time of 1 hr and is

from water

replaced after once year.

3.

Softener

It removes hardness

Softener is cleaned every regeneration by

back wash.

4.

5.

Micro filter

Reverse osmosis

It removes micro size

Change

filters

every

month

weekly

particles

sanitation by 50 ppm chlorine.

TDS removal to mid

Cleaning every six month by membrane

to large size organic cleaning chemicals

molecules removal of
microorganism
6.

U.V. Light

It

removes

remaining

all 7500 working hrs or <3 microsimens

harmful

cemiwatt sec/cm2.

bacteria. Intensity 3-5

microsimens.

DRINKING WATER TREATMENT

Public Water Systems:

Public Water Systems (PWSs) come in all shapes and sizes, and no
two are exactly the same. They may be publicly or privately owned
and maintained. While their design may vary, they all share the same
goal - providing safe, reliable drinking water to the communities they
serve. To do this, most water systems must treat their water. The
types of treatment provided by a specific PWS vary depending on the
size of the system, whether they use ground water or surface water,
and the quality of the source water.
Tapping a Source of Water:
Large-scale water supply systems tend to rely on surface water
sources, while smaller systems tend to rely on ground water. Around
35 percent of the population served by community water systems
(CWSs) drink water that originates as ground water. Ground water is
usually pumped from wells ranging from shallow to deep (50 to
1,000 feet). The remaining 65 percent of the population served by
CWSs receive water taken primarily from surface water sources like
rivers, lakes, and reservoirs.

Treating Raw Water:

The amount and type of treatment applied by a PWS varies with the
source type and quality. Many ground water systems can satisfy all

Federal requirements without applying any treatment, while others

need to add chlorine or additional treatment. Because surface water
systems are exposed to direct wet weather runoff and to the
atmosphere and are therefore more easily contaminated, federal and
state regulations require that these systems treat their water.
Water suppliers use a variety of treatment processes to remove
contaminants from drinking water. These individual processes may be
arranged in a "treatment train" (a series of processes applied in
sequence). The most commonly used processes include filtration,
flocculation and sedimentation, and disinfection for surface water.
Some treatment trains also include ion exchange and adsorption.
Water utilities select a combination of treatment processes most
appropriate to treat the contaminants found in the raw water used by
the system.

TYPES OF TREATMENT
Flocculation/Sedimentation:
Flocculation refers to water treatment processes that combine or
coagulate small particles into larger particles, which settle out of the
water as sediment. Alum and iron salts or synthetic organic polymers
(used alone or in combination with metal salts) are generally used to
promote coagulation. Settling or sedimentation occurs naturally as
flocculated particles settle out of the water.
Filtration:
Many water treatment facilities use filtration to remove all particles
from the water. Those particles include clays and silts, natural
organic matter, precipitates from other treatment processes in the
facility, iron and manganese, and microorganisms. Filtration clarifies
water and enhances the effectiveness of disinfection.
IonExchange:
Ion exchange processes are used to remove inorganic contaminants if
they cannot be removed adequately by filtration or sedimentation. Ion
exchange can be used to treat hard water. It can also be used to
remove arsenic, chromium, excess fluoride, nitrates, radium, and
uranium.

Adsorption:
Organic contaminants, unwanted coloring, and taste-and-odorcausing compounds can stick to the surface of granular or powder
activated carbon and are thus removed from the drinking water.
Disinfection(chlorination/ozonation):
Water is often disinfected before it enters the distribution system to
ensure that potentially dangerous microbes are killed. Chlorine,
chloramines, or chlorine dioxide are most often used because they are
very effective disinfectants, not only at the treatment plant but also in
the pipes that distribute water to our homes and businesses. Ozone is
a powerful disinfectant, and ultraviolet radiation is an effective
disinfectant and treatment for relatively clean source waters, but
neither of these are effective in controlling biological contaminants in
the distribution pipes.

MONITORING WATER QUALITY

Water systems monitor for a wide variety of contaminants to verify
that the water they provide to the public meets all federal and state
standards. Currently, the nation's community water systems (CWSs)
and nontransient non-community water systems (NTNCWSs) must
monitor for more than 83 contaminants. The major classes of
contaminants include volatile organic compounds (VOCs), synthetic
organic

compounds

(SOCs),

inorganic

compounds

(IOCs),

radionuclides, and microbial organisms (including bacteria). Testing

for these contaminants takes place on varying schedules and at
different locations throughout the water system.
Transient non-community water systems may monitor less frequently
and for fewer contaminants than CWSs. Because these types of
systems serve an ever-changing population, it is most important for
them to monitor for contaminants such as microbiologicals and
nitrate that can cause an immediate, acute public health effect.
Water systems also monitor for a number of contaminants that are
currently not regulated. This monitoring data provides the basis for
identifying contaminants to be regulated in the future.

TREATMENT METHODS
Boiling:
In an emergency, boiling is the best way to disinfect water that is unsafe
because of the presence of protozoan parasites or bacteria.
If the water is cloudy, it should be filtered before boiling. Filters
designed for use when camping, coffee filters, towels (paper or cotton),
cheesecloth, or a cotton plug in a funnel are effective ways to filter
cloudy water.
Place the water in a clean container and bring it to a full boil and
continue boiling for at least 3 minutes (covering the container will help
reduce evaporation). If you are more than 5,000 feet above sea level,
you must increase the boiling time to at least 5 minutes (plus about a
minute for every additional 1,000 feet). Boiled water should be kept
covered while cooling. From Drinking Water for Emergency Use (pdf
file). You can also look at recommendations of the EPA.
The advantages of Boiling Water include:
Pathogens that might be lurking in your water will be killed if the
water is boiled long enough.
Boiling will also drive out some of the Volatile Organic
Compounds (VOCs) that might also be in the water. This method
works well to make water that is contaminated with living

organisms safe to drink, but because of the

inconvenience,

boiling is not routinely used to treat drinking water except in

emergencies.
The disadvantages of Boiling Water include:
Boiling should not be used when toxic metals, chemicals (lead,
mercury, asbestos, pesticides, solvents, etc.), or nitrates have
contaminated the water.
Boiling may concentrate any harmful contaminants that do not
vaporize as the relatively pure water vapor boils off.
Energy is needed to boil the water.

ABBREVIATIONS

NMT

Not more than

NLT

Not less than

Q.C.

Quality Control

IP

Indian Pharmacopeia

RS

Reference Standard

WS

Working Standard

LOD

Loss on Drying

BP

British Pharmacopoeia

USP

United States Pharmacopeia

REFERENCES

1. British Pharmacopeia
2. Indian Pharmacopeia
3. U.S. Pharmacopeia
4. Keith Wilson & John Walker
5. S.V.S. Rana
6. Albert L. Lehinger

DECLARATION

I HIMANSHU KUMAR , do hereby declare that the training

work

entitled

MICROBIAL QUALITY OF WATER AND

BIOLOGICAL TREATMENT OF DECOLOURIZATION OF

DISTILLARY EFFULENTS is an authenticated work carried out by
me at IT-BHU, VARANASI. under the guidance of Dr. P. N. TIWARI.
This work has not been submitted for similar purpose anywhere else
except to I.E.T.-KHANDARI CAMPUS, AGRA, affiliated to Dr. B. R.
Ambedkar University, Agra.

Date:
Place:

HIMANSHU KUMAR