2

LABORATORY MANUAL

ENVIRONMENTAL HYGIENE
VPH - 412

Prepared by
Dr. M. SEKAR
Dr. K.M. PALANIVEL
Dr. K.S. VENKATARAMAN
Dr. R. JAYAKUMAR

DEPARTMENT OF VETERINARY EPIDEMIOLOGY & PREVENTIVE MEDICINE

MADRAS VETERINARY COLLEGE
CHENNAI - 600 007
 CONTENTS

Sl Date Contents Page Signature

No. No.
1. Sampling for water Analysis 4
2. Physical analysis of water 7
3. Qualitative analysis of water for chemical 10
impurities
4. Qualitative chemical examination of water for 13
metallic impurities
5. Estimation of total hardness of water 16
6. Estimation of chlorides in water 18
7. Estimation of chlorine demand of water 20
8. Bacteriology of water 22
9. Enumeration of Escherichia coli in water 24
10.Properties of water 27
11. Air microbiology 31
12.Disinfection in animal house 35
13.Testing the efficiency of disinfectant solutions 38
14.Demonstration of water purification plant 40
15.Assignment 43
 CERTIFICATE

Certified that this laboratory manual represents the work done

byMr/Mrs/Miss......................................................................................................... I.D.
No..................................................for the course Environmental hygiene VPH 412 during
the year ......................... .............................

Marks awarded : /10 Course Teacher

Submitted to the Practical Examination in Veterinary Public Health IIheld

on ...............................

Internal ExaminerInternal ExaminerExternal Examiner

 EXERCISE - 1

SAMPLING OF WATER FOR ANALYSIS

Sampling is the act of collecting sample, which is a representative of the population from which
it is taken. Samples of water are collected for various types of analysis including physical, chemical,
microbial and sometimes radiological analysis. The sampling technique depends on the type of
analysis to be carried out.

Factors to be considered while sampling

1. Sample site: The point at which sample is to be drawn should be

decided appropriately as it largely reflects on the inference of the analysis.
Usually samples have to be collected from two or more points for
comparison of results.
2. Quantity of sample: In general, sample of about 250 ml is sufficient, but
more quantity may be required based on the type of analysis.
3. Sample size and For samples from a distribution system supplying
to cities, towns and other areas, the following guidelines may be followed.
sample interval:
Maximum interval between successive samples and minimum number of samples to be taken:
Population served Maximum interval between successive Minimum number of
samples samples to be taken from
whole distribution system,
each month
<20,000 1 month 1 sample/5000 population
20,000-50,0002 weeks 1 sample/5000 population
50,000-1,00,000 4 days 1 sample/5000 population
>1,00,000 1 day 1 sample/1000 population

4. Container:- The sampling container may have significant effect on the

results of water analysis. Eg. Plastic absorbs silver, oil or grease, glass is
unsuitable for borates and silicates as these can be contributed by glass
itself. Other factors like ease of handling, cleaning, sterilization,
disposable nature should also be considered while selecting a container.
5. Use of Preservatives: For microbiological assay if water is likely to contain chlorine, chloramine,
or other compounds thiosulphate solution (0.1 ml 3% solution or a small
crystal) should be added. If heavy metals are present (e.g. copper)
chelating agents like Ethylenediaminetetra-acetic acid (EDTA) can be
added.
6. Representative sample: The sample should be, as far as possible representative of the source.
For, this the container may be rinsed with the water from the source
before taking the sample. In deep lakes or stream samples may be
collected at different levels using suitable equipment.
Samples for physical and chemical analysis
These must be collected in clean glass stoppered bottle made of neutral glass of 2 litre capacity.
Bottle should be clean chemically, germ free or free of any organic material. Main objective is to check
the action of water on serving pipes and so it is important that water be allowed to stand in pipes
overnight and then collected. Stoppered bottles (Winchester quart bottle) should be used.
Before collection, rinse bottle thrice with sample water, following each time 1/3 rd full. Then fill it
up, stopper tightly, place a piece of cloth over the stopper, tie it.

Samples for bacteriological analysis

These samples should be collected in clean sterilised bottles, made of neutral glass with 200-250
ml capacity, provided with glass stopper, with an overlapping ring to avoid contamination. If water to be
sampled has chlorine, small quantity of sodium thiosulphate (0.1 ml 3% solution or a small crystal) to
be added to water sample. Sampling bottle should not be opened till it is used for filling
sample.Laboratory examination should be started in 24 hours. In hot tropics, keep water in ice till
analysed (within 48 hours). If not properly preserved, it is not suitable for bacterial examination.
For bacteriological examination the mouth of tap may be sterilised using a flame gun.

Collecting from different sources

1. Tap in regular use
Open the tap fully. Allow water to run for 2 minutes to flush stagnant water at the end of the pipe.
From taps not in regular use, water is collected after sterilising the tap by heating with a blow lamp, till it
is unbearably hot to touch. Cool it, allow water to run for some time and then collect. Fill bottle from a
gentle stream to avoid splashing. Sampling frequency for public water supply is based on number of
population served.
2. Collection from river, lake and reservoir
Grab sampling
It is the instantaneous collection of the required amount of water from appropriate source. In rivers,
sample should be collected away from bank and at some depth. For sampling, a sterile bottle with
string attached to neck is used. Another long string is tied to the end of sterilised string to lower bottle
into water. After filling, stopper bottle, cover with cloth and seal. Two samples are collected one at the
entry point and another at the exit point of the river.
The same method is used for well water.

I. Identification of sample

A standard proforma is used for this.

1. Labelling of sample bottle is as follows:
(i) Submitted for ..........
(ii) Submitted by ..........
(iii) Code No ..........
(iv) Source of sample ..........
(v) Reasons for examination ..........
(vi) Place of sampling ..........
(vii) Sample taken in presence of ..........
(viii) Signature of authority
(ix) Whether the water has been filtered, chlorinated or treated
(x) If water is from a well, give details of its depth, whether covered/uncovered,
recently dug etc.
(xi) If spring water, whether taken directly from spring or from collecting chamber.
(xii) If from a lake/reservoir, exact position and depth at which collected
(xiii) Does water become affected in appearance, odour or taste after heavy rains
(xiv) Mention possible sources of pollution in the area and distance from sampling unit.
(xv) Date and time at which sample taken and despatched.

Exercise

1. What is the source of your sample?

 EXERCISE - 2

PHYSICAL ANALYSIS OF WATER

Introduction

Physical analysis of water is employed to assess the physical properties. Any change in normal
physical properties such as pH, colour taste etc may by itself cause hazard to human health or may
give an indication of chemical or microbial hazards in water. The following are some important physical
properties that are usually analysed in drinking water.

Parameters of importance
1. pH:

pH may be defined as the negative logarithm of hydrogen ion concentration. Extreme pH can
cause irritation to eyes, skin, mucous membrane and rarely gastro intestional irritation. pH also
influences the aquatic life and corrosiveness of water.
Acceptable range of pH is 6.5-8.5. pH of water is measured by a pH paper strips and pH meter.

2. Colour:

Normally water is colourless or has a bluish tinge. Contributors of colour are

1. Organic impurities - green, greenish yellow
2. Iron and other metals - reddish brown
3. Decomposed organic matter - yellowish.
Water colour is graded by yellow units or Hazen scale unit ranging from 1-10

3. Odour:

In general water is odourless and its presence indicates contamination. The sample should be
taken in a test tube with stopper and slightly warm the water then remove the stopper and smell it.
 Some of the common contaminants and their characteristic odour are as follows.

Odour Contaminants
Earthy Minerals and clay
Vegetable Vegetable matter
Aromatic Volatile substance
Pungent Decomposing sewage
Ammonical Manure or faeces.

4. Taste:

Water is tasteless under normal condition. Dissolved oxygen and minerals impart palatability to
drinking water.
Some commonly described tastes are,
Flat - eg. rain water due to lack of oxygen and minerals.
Ferrogenous or metallic - due to iron and other metals.
Saline or brackish - due to salt (NaCl)
An earthy or musty taste is imparted by cyanobacteria, actinomycetes and few fungi.

5. Turbidity:

Turbidity is a measure of waters ability to scatter and absorb light. It is due to the presence of clay,
silt, colloidal particles, planktons and other microscopic organisms. Turbidity affects other parameters
such as colour, taste, odour, etc. It also affects the acceptability by consumers, increases chlorine
demand and protects microbes. Turbidity also increases the absorptive capacity of certain toxic
substances like 2,4-D, an organic herbicide.
Silica scale is used for grading turbidity from 1 to 10 or Nephlometric turbidity unit is also used.

6. Electrical conductivity:

Electrical conductivity determines the rate of corrosion. When two or more different metals are
used in distributing systems, galvanic cell is created hastening corrosion. Electrical conductivity is
proportional to dissolved minerals.
Type of water Electrical conductivity
Soft water 100 S/cm2
Hard water 2000 S/cm2
Sea water 50000 S/cm2
7. Temperature:

Temperature is an indicator of bacteriological status. It influences corrosion, dissolved oxygen and

affects the ability of organisms to act on pollutants. The normal temperature for water is 280 + 100c

8. Foaminess:

When large quantities of organic matter is present in water, foam develops on agitation.
Foaminess may be detected by comparison with distilled water.

Record your Result:

Exercise
1. Indicate the source of your sample?
2. Give details of the physical parameters analysed
3. List the result and outcome of your inference?
PARAMETER OBSERVATION INFERENCE

1. pH
2. Colour
3. Odour
4. Taste
5. Turbidity
6. Electrical conductivity
7. Temperature
8. Foaminess
 EXERCISE - 3

QUALITATIVE ANALYSIS OF WATER FOR

CHEMICAL IMPURITIES
Introduction:

Qualitative analysis is usually performed to determine the presence of specific chemical impurities.
Certain ions like nitrites, cyanide, etc could have serious implications on health, hence need to be
analysed in drinking water. The following are the chemical impurities that are usually tested in water.

Chemical Impurity Procedure for Reaction MPL Inference

to be analysed analysis expected
1. Ammonia 10 ml of sample Deep yellow 0.06
in a test tube, add or brown or ppm
few drops of black precipitate
Nesslers reagent

NH3 + alkaline K2[HgI4] NH2Hg2I3

Ammonia Nesslers Brown
reagent

2. Chloride 10ml of sample

in a test tube, White precipitate 250
add a few drops of ppm
silver nitrate

NaCl + AgNO3 AgCl + NaNO3

(White)

3. Sulphate 10 ml of sample White precipitate 250

add a few drops formation ppm
of HCl and a hastened by heat
few drops of
Barium Chloride
 SO42- + BaCl2 BaSO4 + 2Cl-

4. Nitrites 10 ml of sample, Formation of Not even

add a few drops of pink colour. in traces
sulphanilic acid and
-naphthalamine.
Shake well.

5. Nitrates 10 ml of sample After 3 minutes 1.5 ppm

(If nitrites are add a few drops of a pink colour
absent), sulphanilic acid is noticed
add few drops of
-naphthalamine
and pinch of
Zinc dust

NO3 NO2- + Sulphanilic + naphthalamine Azo dye (pink)

Nitrate Nitrite acid

6. Nitrates 5ml of sample Formation of 1.5

(If nitrites are add equal quantity pink colour ppm
present) of brucine solution.
Effervescence
settles down run con.
H2SO4 along
the sides
agitate gently.

7. Phosphate 10 ml of sample, Yellow Not even

add a few drops of precipitate in traces
ammonium molybdate soluble in
solution. Warm and ammonia solution
agitate gently and reappears

PO3-4 + (NH4)2MoO4 Ammonium

Phosphate Ammonium phospho
molybdate molybdate (yellow)
8. Fluoride 10 ml of sample formation of 1ppm
 add a few drops of White crystalline ferric chloride
precipitate

6NaF + FeCl3 Na[FeF6] + 3NaCl

(white)

9. Cyanide 10 ml of sample A greenish to Not even

add a few drops of blue colour is in traces
ferrous sulphate, boil developed
add HCl to obtain clear
solution, add few drops
of FeCl2

CN- + FeSO4 Ferrocyanide Clear Prussian

solution blue

Inference:
Zn.
 EXERCISE - 4

QUALITATIVE CHEMICAL EXAMINATIONOF WATER

FOR METALLIC IMPURITIES
Introduction:

Drinking water is usually contaminated with metallic impurities such as iron, copper, zinc, lead, etc.
The source for such impurities may be soil and other mineral sources or more commonly the pipelines
and distribution systems. Some of these impurities such as iron or copper may only cause mild
affections like gastrointestinal disturbances. Certain metals such as lead or arsenic may cause serious
health hazard.Thus it is essential to analyse water for metallic impurities. The following are some tests
routinely used for the identification of common metallic impurities.

Metallic Procedure Reaction Significance of Inference

impurities expected the impurity
to be if present
tested

1. Iron 5 ml of sampleAppearance Iron is a

in a test tube. of blue colour persistent
Add a few impurity. It imparts
drops of bitter taste,
potassium turbidity and
ferrocyanide, discoloration.
test solution Excess iron causes
constipation
and digestive
disturbances. Iron
helps growth of
iron bacteria Crenothrix
and Gallionella.
Leads to corrosion
of pipes. MPL 0.3 ppm

Fe3+ + K4[Fe(CN)6] KFe[Fe(CN)6] + 3K+

 Ferric ion Potassium Potassium
ferrocyanide ferric ferrocyanide
(Blue)
2. Copper 5 ml of sampleChocolate If consumed
add a few drops colour over a long
of potassium period can cause
ferrocyanide digestive disturbances.
solution Green staining of
clothes and bath.
Toxic to biological
species if
synergistically
present along with
zn MPL 3 ppm
2Cu2- + K4[Fe(CN)6] Cu2[Fe(CN)6] + 2k+
cupric ferrocyanide
(chocolate colour)
3. Lead 5 ml of Golden yellow Cummulative
sample add precipitate. poison. Causes
a few drops Boil to dissolve plumbism. Rain
of potassium the precipitate water, soft water
iodide cool with organic matter
solution under tap will corrode
water. lead pipes with
lead entering the water
MPL: 0.1 ppm
Pb2- + 2KI PbI2 + 2K-
potassium lead
iodide iodide
(golden yellow)
4. Zinc 5ml of sample,White Not desirable
add a few drops haziness/ in water.
of nitric acid, turbidity or Prolonged
boil to precipitate consumption will
concentrate depending on cause digestive
add a few the level of disturbances
drops of zinc MPL: 15 ppm
potassium present
ferro cyanide
 solution.
2Zn2- + K4[Fe(CN)6] Zn2[Fe(CN)6] + 2K-
Zinc ferrocyanide
(white)
5. Arsenic 5 ml of sample Silver nitrate It is a.
Gutzeit test in a test tube, crystals turn cummulative
add a few yellow and poison. It is used
granules of and then in sheep
zinc black due dip and weedicide
little con. to arsine from which it
H2SO4, gas from may have access
plug the test arsenic to water supply.
tube with MPL 0.05ppm
cotton, place a
filter paper over
this on which a
few crystals of
AgNO3 is kept.
b. Copper l0 ml of A steel
foil Test sample in a grey deposit
white china will form
dish add a pinch over the
of Na2CO3 and copper foil.
evaporate to
dryness on a
water bath.
Redissolve in 2-3
ml of distilled
water. Add
1 ml of HCl
and a few
pieces of Cu
foil, boil.
Copper + Arsenate Cupric arsenate
(grey)

Inference:
 EXERCISE - 5

ESTIMATION OF TOTALHARDNESS OF WATER

Introduction

Water hardness is a measure of the capacity of water to react with soap. Hard water requires
considerably more soap to produce lather. Hardness is contributed by a variety of polyvalent metallic
ions mainly carbonates and sulphates of calcium and magnesium. Other salts of iron, manganese,
zinc, etc. may also contribute to hardness. Hardness is most commonly expressed as mg / litre of
CaCO3 equivalent

Source:

Ployvalent metallic ions are contributed by rocks and seepage or run offs from soils. The sources
of calcium and magnesium are sedimentary rocks, limestone and chalk.

mg / L of CaCO3 equivalent Description of water

0-17 soft water
17-60 slightly hard
60-120 Moderately hard
120-180 Hard water
>180 very hard

Classification of hardness:
* Temporary / carbonate hardness: (Carbonates of calcium and magnesium ions)
* Permanent / Non - Carbonate hardness: (Sulphates of calcium and magnesium ions)

Estimation of total hardness (Temporary hardness + Permanent hardness)

Aim:
To estimate the total hardness of given water sample.

Materials Required
1. Burette
2. Conical flask
 3. Pipette
4. Solutions:
a. EDTA solution
4 gms of EDTA in 1 Litre of distilled water
b. Ammonia buffer
16.9 gms of ammonium chloride + 14.3 ml con. NH3OH + 1.15 gms of
mercuric chloride in 250 ml of distilled water
c. Erichrome indicator.
1 gm of erichrome in 1000 ml of absolute alcohol.

Procedure:
1. The EDTA solution is taken in the burette.
2. 25 ml of water sample is taken in a conical flask and 0.25 ml of ammonia buffer
is added to it.
3. To that, 1 or 2 drops of erichrome indicator is added.
4. The mixture in the conical flask is titrated against EDTA in the burette.
5. End point is the appearance of blue colour.

Test Sample

S.No. Volume Burette Reading (ml) Volume of EDTA

of sample (ml) Initial Final consumed (ml)
1. 25 ml water V
+0.25 ml
ammonia
buffer

Calculation

Total Hardness = Volume of EDTA consumed (V) x 20

Result:

Inference:
 EXERCISE - 6

ESTIMATION OF CHLORIDES IN WATER

Introduction:

Chlorides are major inorganic ions present in water. Chlorides in water may be formed in
combination with a variety of salts. They impart a saline taste especially the sodium salts. This is
detectable when the chloride level exceeds 250 ppm. But saline taste is less perceived with salts of
calcium and magnesium.

The effect of chlorides apart from taste is that they may have corrosive effect.

Sources: Agricultrual run off, septic tank effluents, industrial effluents and waste water treatment
plants.

Aim:

To estimate the chloride content of the given sample of water.

Principle:

Silver nitrate and chlorides react quantitatively to precipitate silver chloride. The end point of the
reaction is seen using potassium chromate indicator as a brick red colouration (reddish brown)

Materials Required

1. Conical flask
2. 50 ml burette
3. Measuring cylinder
4. 1 ml pipette
5. Solutions
a. 0.1N Silver nitrate solution
b. Pottasium chromate indicator
Procedure:

1. The burette is charged with silver nitrate solution.

2. 50 ml of water is taken in a conical flask and 1 ml of potassium chromate
indicator added.
3. The sample is titrated against silver nitrate and the end point is noted as
appearance of brick red colour.
4. The procedure is first performed in distilled water then with test sample of water.

Sl Quantity of Burette Reading Volume of

No. Distilled Water / AgNO3
Water Sample Taken (ml) Initial (ml) Final (ml) Utilised
1. 50 ml Distilled Water V1

2. 50 ml Water sample V2

Calculation:

Chloride content of Sample = (V2-V1) x 0.5 x 106

50 x 100

V1 (chromate factor) = Volume of AgNO3 used for distilled water

V2 = Volume of AgNO3 used for water sample
V2 _ V1 = Volume of AgNO3 used for Chlorides
0.5 = Each 0.1N AgNo3 will neutralise 0.5 mg of chloride

Result:

Inference:
 EXERCISE - 7

ESTIMATION OF CHLORINEDEMAND OF WATER

Chlorine demand of water is the amount of chlorine required to destroy bacterial organisms
present in water. To explain, it is the difference between the quantity of chlorine added to the water and
the residual chlorine remaining at the end of specific period of contact (1 hour). The minimum
recommended concentration of residual chlorine is 0.5 mg/L for 1 hr. For practical purpose, it is taken
as 0.5 ppm which ensures bacteria free water for domestic use. The total value of chlorine demand and
free residual chlorine of 0.5 mg/L constitutes the correct dose of chlorine to be added.

Principle
To detect the residual chlorine, orthotoludine test is employed. When the reagent orthotoludine is
added to water with chlorine, it turns yellow and the intensity varies with the concentration of chlorine.
Yellow colour is produced by both combined and free chlorine. But with the combined chlorine, reaction
is slow. So it is essential to take the reading, within 10 seconds after adding reagent. Colour produced
after a lapse of 15-20 minutes is due to action of free and combined chlorine.

Apparatus Required
Horrocks apparatus contains,
1. 6 white cups of 200 ml capacity
2. 2 metal scoops (2 g)
3. 7 glass rods
4. 6 standard colour vials
5. 6 glass tubes graduated at 10 ml
6. one comparator stand for comparison of colour
7. special dropper (marked 0.1ml)

Reagent
Orthotoludine, bleaching powder

Procedure
1. Pour water to be tested in all 5 cups upto the mark of 200 ml. Place cups in line one after another.
2. Add 1 scoop level of bleaching powder to 1st cup and stir well with glass rod. Wait for 15 minute.
3. With other scoop, add 1,2,3 and 4 scoop full of above bleaching water from 1st cup to successive
cups. Fill with water and stir with clean glass rods. Wait for 15 minutes.
4. Estimation of residual chlorine:
a. Place the 0 ppm marked standard colour vial into the central hole of back row of comparator stand,
i.e. row which is adjacent for glass.
b. Pour untreated water to two glass tubes so that they are almost full.
c. Place tubes in other holes of back row of comparator stand.
d. Place 2 drops (0.1 ml) of O-toluidine solution in another test tube and add chlorinated water from
2nd cup and keep in middle hole of front row of comparator stand. Match yellow colour developed
tubes by putting standard colour vials in remaining 2 holes of front row. Matching should be carried
out by holding comparator stand.
e. Note the value of residual chlorine in ppm in a standard vial which is a nearest match to nearest
tube. This is the residual chlorine of water under test.
5. Similarly estimate residual chlorine content of other 3 cups of water.
6. Find the cup which contain 0.4 ppm of residual chlorine. From this, by knowing volume of water in
reservoir, it is possible to estimate the amount of bleaching powder to be added.
7. If the residual chlorine is found to be >0.4 ppm, even in water of 2nd cup, then make a more dilute
solution in 6th cup by taking a few scoops of water from 5th cup and making it upto 200 ml.

Record your observation

Inference
 EXERCISE - 8

BACTERIOLOGY OF WATER
Introduction:
Drinking water supplies are commonly contaminated with microorganisms, many of which are
pathogenic to human beings. Hence regular examination of water to detect these micro organisms is
important to prevent water-borne diseases. Bacterial contamination in water can be detected by
culturing these organisms in bacteriological media. The identity of these can be established based on
growth in selective media, colonial characteristics, biochemical characteristics, morphology, etc. The
major sources of bacterial contamination are human and animal wastes contributing to pathogens like
E.coli, Salmonella, Yersinia and others.
As per WHO recommendation drinking water should not have any coliform / 100 ml of sample. The
EPA (environment Protection Agency) say that a minimum of 200 colonies / 100 ml of sample is
allowed.
Agent Selective Colony Biochemical Other
media characteristics characteristics characters
1. Escherichia MacConkey * Pink coloured colonies * I M Vi C test Gram - ve
coli agar (Indole, + ve Aerobic /
Nutrient agar * White or yellowish Methylred, + ve facultatively
white, moist glestening Voges-proskaur; - anaerobic
opaque and circular Citrate test - ve
Eosin - methylene * Blackish centre with * It ferments sugar Lactose
blue agar a metallic sheen produce gas fermenter
Litmus lactose * Red coloured and acid
agar colonies * It reduces nitrates
It does not
produce H2S

2. Salmonella Selenite F broth * Pink colonies of E-Coli * I M Vi C Gram - ve,

S. enterica 2-3 mm in diameter + + - + Pleomorphic,
S. bongori Deoxycholate * Smooth, round, convex * Ferment sugar, Non spore
citrate agar (dew drop like) produce acid forming,
(DCA) and gas
* Reduces aerobic /
nitrates facultatively
anaerobic
3. Klebsiella Mac Conkey agar *
Mucoid pink colour * Voges proskaur+ve Gram-ve,
 K.pneumoniae colonies * Methyl red -ve rod Shaped,
K. mobilis Blood agar * No haemolysis * Hydrolyse urea, non - motile,
catalase +ve very prominent
ploysaccharide
capsule
4. Proteus Blood agar On surface of solid * Non lactose Gram-ve,
P. vulgaris media the colonies fermenter pleomorphic,
spreads rapidly as * Indole variable motile,
a thin film-called * Citrate utilization non-spore
swarming * Produce H2S forming,
* positive to PPA non capsulative
(phenyl pyruvic acid)
5. Yersinia Blood agar Smooth Colonies I M VI C Gram-ve,
Y.entrocolitia Mac Conkey agar - + - -short ovoid,
* ferments sugar motile,
bipolar with
Giemsa,
non-spore
forming.
6. Leptospira Staurts medium, Sub surface Thin tightly
L. canicola Fletchers medium, coolonies coiled obligate
L.icterohae- 0.1-0.2% aerobes.
morrhagiae semi solid agar Flexuous type
L.hardjo medium, of motilty
1% agar medium Do not stain
with aniline dye
7. Shigella MacConkey agar, * Colourless mucoid * Methyl red + Gram - ve,
S.dysentri smooth colonies. * Reduce nitrate rod,
S.sonnei Deoxycholate * Red colonies to NO2 non motile,
citrate agar * Do not form H2S non capsulated
spore forming
8. Faecal Blood agar, * B-haemolytic Lactose Gram-ve, motile,
Streptococci MacConkey agar Smooth mucoid, opaque fermenter facultatively
S.faecalis glossy colonies anaerobic
 EXERCISE - 9

ENUMERATION OF E.COLI IN WATER

E.coli causes variety of diseases in animals and human beings. Water is an important source
for spread of E.coli. There are two types of coliforms, faecal and nonfaecal coliforms. The term coliform
refers to a facultatively anaerobic organism that ferments lactose to produce gas and is also Gram
negative and non spore forming.
E.coli and Enterobacter perfectly fits into this group and Klebsiella moderately fits into this
group. Presence of E.coli in water is an indication of faecal contamination.

I. Identification of coliforms:
(i) Presumptive test
(ii) Confirmatory test
(iii) Completed test

Lauryl Tryptose Mannitol Broth

Ingredient g/L
Tryptose 20
Mannitol 5
Sodium chloride 5
Dipotassium phosphate 2.75
Monopotassium phosphate 2.75
Sodium lauryl sulfate 0.1
L-Tryptophan 0.2
Final pH 6.8 + 0.2

(I) Presumptive Test: In presumptive test, only the presence of coliforms are identified.
a) Durhams Tube Test:
In this test lauryl tryptose mannitol broth is used.

Materials Required:
1. Test tube with durhams tube
2. 10 ml and 1 ml pipette
3. Lauryl tryptose mannitol broth
4. Incubator
5. Water sample
Procedure:
1. 5-8 ml of lauryl tryptose mannitol broth is taken and 1 ml of water sample is
added in a test tube.
2. Durhams tube is placed along with the broth.
3. The test tubes are incubated at 440C for 18-24 hours.

Interpretation:
1. In positive samples, E.coli present in the water utilizes the lactose and produces
acid and gas.
2. Acid production leads to change in colour of the medium.
3. Gas produced accumulates and the Durhams tube rises to the surface.

(II) Confirmatory Test

a) Inoculation in Eosin methylene blue agar:

The Gram negative lactose fermenters are confirmed by inoculating the water sample into
Eosin methylene blue agar. Methylene blue inhibits growth of Gram positive organisms. Coliforms grow
in EMB agar and produce dark centered colonies with greenish metallic sheen. Enterobacter does not
produce metallic sheen.

b) Enumeration by pour plate method:

Materials Required:
1. Petridish
2. 10 ml and 1 ml pipette
3. Deoxycholate lactose agar.
4. Water sample
5. Incubator
Deoxycholate lactose Agar:
Ingredient g/L
Peptone 10
Lactose 10
Sodium chloride 5
Sodium citrate 2
Sodium deoxycholate 0.5
Neutral red 0.03
Agar agar 15.0
pH 7.1 + 0.2
Procedure:
1. Place 1 ml of water sample using pipette in a petridish.
2. Pour 5-8 ml of DCA agar at a temperature of 44-460C into the petridish.
3. Thoroughly mix the sample and medium and allow to solidify.
4. Incubate at 370C for 18-24 hours.
Note: Entire work is to be carried out under sterile conditions.

Interpretation:
1. In positive samples E.coli produces pink colonies and Salmonella produces white
colonies.
2. As per WHO recommendation drinking water should not have any coliforms per
100 ml of water.
3. Environmental Protection Agency (EPA) says that a maximum of 200 colonies
per 100 ml water is allowed.

III Completed test

Test For Indole:

In this test 1% tryptone is used. It contains tryptophan.

Procedure:

1. 1% tryptone agar is taken in a test tube and 1 ml of water sample is added.

2. The tubes are incubated at 2 different temperatures (370C and 440C) for 24 hours.
3. Kovacs reagent is added to both tubes.

Interpretation:

1. In positive cases there will be a development of pink coloured ring

2. E.coli reacts with tryptophan and produces indole. This reacts with Kovacs
reagent to produce pink colour.
3. Pink colour production at 370C indicates presence of mildly pathogenic E.coli
4. Pink colour production at 440C indicates presence of highly pathogenic E.coli.

Result:

Inference:
 EXERCISE - 10

PROPERTIES OF AIR
Introduction

Pure air is never found in the immediate vicinity of animal houses because of the altrations in
the surroundings and the animals normal physiological process. The increase in carbon-di-oxide,
methane and reduction in oxygen is brought about by the physiological process and there is a change
in physical characters of the air by increasing its moisture and temperature.
Animals give out body heat constantly through convention, conduction, radiation and
evaporation to regulate their body temperature. The amount of heat produced is different for different
species and is influenced by environmental temperature.
Moisture is given out by animals continuously through respiratory route and by excretion as
urine. This moisture when projected into the atmosphere will increase the water vapour content or
humidity of air.
Odour present in the atmosphere arise from the animals and also through decomposition of
organic matter. The three important properties of air are:

a. Temperature
b. Moisture
c. Velocity

Measurement of Temperature:
Air temperature is recorded in 0F (Fahrenheit) or 0C (centigrade)
1. Room thermometer: This is an instrument with mercury or alcohol column mounted on wooden
frame. The bulb of the thermometer should be fully exposed to the influence of the surrounding air and
shielded against radiation. Air temperature should be recorded at 3 different places inside the animal
house. The thermometer shall be placed at the level of the head of the animal. Temperature should be
recorded at least twice (8am and 1pm), the average of which can be taken as the average day time air
temperature.

2. Maximum minimum thermometer:

The temperatures are recorded at one representative location of animal shed and the average of
the maximum and minimum temperatures is reported as mean air temperature of the day.
3. Thermograph:
It is an automatic self recording instrument for measuring the temperature continuously
throughout the day and night. It consists of a bimetallic strip made up of two metals invar and bronze,
which have different coefficient of expansion of heat. Change in temperature causes change in the
curvature of strip due to different coefficient of expansion. The curvature is magnified by the lever
system to the pen-point. The penpoint marks on the graph sheet, which is fixed on the drum driven by
the clock mehanism at the rate of one rotation / day or week.

Humidity:
Humidity is the amount of water vapour present in the air. Humidity is expressed as relative
humidity and absolute humidity.

Relative humidity: It is the ratio of moisture actually present in the air to the maximum amount it
would hold at saturation at the same temperature.

Dew point: It is the temperature at which the air would be saturated by the amount of moisture
present in it.

Measurement of humidity:

1. Masons wet and dry bulb thermometer:

This consists of two individual thermometers mounted side by side. A piece of muslin is
wrapped around the bulb of one of the thermometers. It is dipped in water. The water in the muslin
cloth evaporates depending on the temperature and humidity of the environment. Hence the wet bulb
records a lower temperature. The difference in temperature between wet and dry bulb thermometers
enables to determine the relative humidity from psychrometric table.

2. Sling / Whirling psychrometer:

It consists of a pair of thermometers mounted in a frame, which is provided with a handle by

which thermometers can be whirled rapidly. The bulb of the lower thermometer is kept wet using a
muslin cloth. The thermometer with wet muslin gives a lower reading than dry bulb. The difference in
temperature between wet and dry bulb thermometers enables to determine the relative humidity from
psychrometric table.

3. Hygrograph:

The sensitive element used for registering relative humidity is a bundle of hair fibres attached to
the marking pen through a lever. Hair is hygroscopic and hence gives an accurate estimate of the
moisture present.
Velocity:

The air velocity of 100 ft / min at 70 0F or 21.10C is comfortable for broilers. Higher air velocity
upto 300 ft / min will be desirable to give comfort at air temperature between 80 0F to 850F. High air
velocity will reduce humidity of the air, which is required under high temperature. Instruments used for
measuring air velocity are known as anemometers.

Apparatus used:
1. Wind vane
2. Anemometer - kata thermometer
High range 1300F - 1200F
Low range 1000F - 950F

Kata thermometer:

It is an alcohol thermometer consisting of a large bulb filled with blue colored alcohol.

Procedure:

1. Immerse the bulb in warm water till the alcohol rises upto 1300F in continuous
line and fill up half of the safety bulb.
2. Wipe the bulb with a dry cloth.
3. Fix it in a stand and stay away from the instrument with a stopwatch in hand.
4. Note time taken in second when alcohol cools from 1300F to 1200F.
5. Repeat 4 times and take average of last 3 readings.
6. Note the factor of the instrument
7. Record the temperature with room thermometer.
 8. Refer to kata chart to measure the air velocity.
9. Air velocity is measured in feet / min.

Example of An Observation

Location Room Dry bulb Wet bulb

1................ temperature Reading Reading

1.
2.
3.
4.
5.
Average

2................
1.
2.
3.
4.
5.
Average
3..................

1.
2.
3.
4.
 EXERCISE - 11

AIR MICROBIOLOGY
More than 500000 microbial species exist in nature but only a few hundred species cause disease.
Most microorganisms are closely associated with plants and animals in a stable beneficial relationship.
However, pathogenic species have profoundly negative impact on host organisms.

Air is often contaminated with biological pollutants such as bacteria, viruses, fungi and other forms.
While some of these are non pathogenic, many are infectious in nature and cause various diseases in
human and animals. Dust contamination is higher in pig and poultry houses than cattle housings.
Among various microbes in air 80% is represented by streptococci and staphylococci, 1% by fungi,
moulds, yeasts and 0.5% by E.coli type of bacteria.

Microbial Analysis of Air:

Sampling:

Air sampling must be carried out before identification of microorganisms. The following methods
are commonly employed.

1. Sedimentation method:

a. Direct sampling: The air source to be tested is decided and a sterile agar plate is kept open for
atleast 15 minutes. Potato dextrose agar is commonly used. For fungi sabouraud dextrose agar is
used. The plates are incubated for 5 days at 250C for fungi and 2 days at 35-370C for bacteria.

SABOURAUD DEXTROSE AGAR

Ingredients gm/lt
Mycological peptone 10
Dextrose 40
Agar 15
Final pH (at 250C) 5.6 + 2

For examination of fungi, place a drop of lactophenol cotton blue stain on a slide. Place a
cellophane tape over the fungal growth and stick it over the stain. Remove excess lactophenol, dry and
observe under microscope.
 For examination of bacteria, a loopful of colony is used to make a smear. This smear is stained
appropriately to identify bacteria.
b. Air sampler;
Principle:
The Air sampler operates on the impaction principle. The air under examination is sucked by the
impeller centrifugally and impacted against the agar medium strip. The impeller speed of 4100 rpm is
so adjusted that 280 litres of air is sampled every minute. The sampling volume corresponds to 40 liters
/ minute of separation volume. (i.e.) 1/7th of the sampling volume. The separation volume per unit of
time forms the basis for calculating the number of organisms per volume of air being sampled.

Materials required:

Air sampler system and sterile agar medium strips.

Components of air sampler:

The air sampler system consists of a controller unit with a sling and a hand held metal centrifugal
air sampling probe.

Controller unit:

This has a crystal controlled timer with time setting from 1-9 minutes. The controller unit has an
inbuilt battery and it should be charged for 14 hours. An AC mains adapter is also provided to supply
power to the handset unit.

Hand held probe:

This consists of the following:

1. Impeller (fan)
2. Impeller SS cup.
3. Impeller handle.
4. Socket for supply of power from control unit.

Procedure:

1. The impeller and the impeller cup are sterilized 1210C for 20 minutes.
2. The impeller handle is disinfected using alcohol.
3. The Agar medium is pulled out holding the edges only from the wrapper and inserted into the slot
in the metal cup without touching the medium so that only 2 cms stays out. The agar surface
should face the impeller.
4. The impeller handset is held in the area where the air sampling is to be carried out.
5. Press ON button. Red LED glows indicating the startup of the process.
6. Set time 1-9 minutes by pressing the buttons.
7. Press RESET button, Green LED glows indicating starting time of the process.
8. After SET time is elapsed the buzzer beeps, indicating the completion of the time cycle.
9. Holding the strip by its tab, the strip is pulled out and replaced in its original wrapper so that the
agar faces away from the sliding lid.
10. Then, the wrapper is sealed, marked for identification and incubated at 370C.
Calculation:
Colony forming unit / litre = Colonies on agar strip
(CFU) 40 x sampling time in minutes
1 minute corresponds to 40 litres of air separation volume.
CFU/m3 = Colonies on agar strip x 25
Sampling time in minutes

Application:

Monitoring the quality of ambient air for the presence of microorganisms is necessary to avoid
severe health hazards in hospitals etc. The GMP requirements could be fulfilled in pharmaceutical
manufacture. Similarly, in blood banks and surgical theatres as well as in tissue culture labs specified
monitoring of air facilitates the maintenance of necessary asceptic conditions.
The other environments that need monitoring are pathological labs, cosmetic manufacture,
fermentation industry, food processing plants, dairy industry, abattoirs and fisheries, medical research,
public health centres, dental clinics, electronic industry, vaccine manufacture, bio technology industry.

2. Liquid Impringement method:

Sterile distilled water or nutrient broth is taken in a dish or tube and exposed to air in the required
area for 15 min. The distilled water or broth is then inoculated onto agar plates which are in turn
incubated for 24 hours at 370C.

3. Filtration:
 Air may be filtered using various fillters such as paper, membrane, inorganic fibre filter, sand, etc.
Air is allowed to pass through the glass tube containing sand. A negative pressure is created inside the
tube. The sand is then inoculated into nutrient medium and observed for growth.

4. Electrostatic precipitation:

A petridish coated with nutrient medium is subjected to electric current of 2000V. This attracts the
suspended microbes to the plate. The plates are incubated and observed for growth.

5. Centrifugation method

Here a centrifugal force is created to deposit particles in air on the required medium. The agar
plates are then incubated and observed for growth.
 EXERCISE - 12

DISINFECTION IN ANIMAL HOUSE

Disinfection is the term applied for the destruction of microorganisms (potential pathogens) on the
surface of inanimate objects. Chemical disinfectants are most commonly used for animal housing.
Chemical disinfectants includes acids, alkalis, alcohols, aldehydes, halogens, phenols and quaternery
ammonium compounds.

Most disinfectants are liquids that require dilution which is particular for each. A specific contact
time is required for effective disinfection. Ethylene oxide and paraformaldehyde are the commonly used
gaseous disinfectants.

Ideal characteristics of chemical disinfectants

1. It should have broad spectrum of activity

2. It should not have toxic effects on livestock.
3. It should be soluble in water.
4. It should be non-corrosive
5. It should also be cheap and easily available.

Common Disinfectants

Group Individual Mode of Concen- Contact Pathogens

Agents ActiontrationTime (Min) Susceptible
1. Acids Citric acid, Alters the Many bacteria
Formic acid, pH of and fungi
Propionic microbial
acid, HCl, environment HCl - 2.5% anthrax
H2SO4, HNO3, spores in hides. Peracetic acid oxidising
agent 0.2%

2. Alkali Sodium 5% Anthrax spores,

hydroxide, pseudorabies virus
Calcium hydroxide 20% wide spectrum
Na2CO3 4% FMD virus
and Trisodium
phosphate
3. Alcohol Ethanol Denatures 70% 10 (lipid virus) Vegetative
proteins and bacterial cells
dissolves lipids and fungi.
Isopropanol Dehydrating 90% 10 Protozoa and
agent some viruses
4. Chlorine Chlorine Protein 250-1000 10 (lipo All vegetative
Compounds gas, Na and Ca oxidation ppm virus) cells Na
hypochlorite, and membrane 30 (bacteria) hypochlorite is
chlorine leakage effective
dioxide, against porcine Chloramine T,
parvovirus
Dichloramine T Broad spectrum
5. Iodine Lugols Iodine, Halogenation 0.5-2% 10 (lipo virus) Broad spectrum
Compounds tincture of tyrosine 30 (bacteria) of activity
of Iodine, in proteins
Iodophores
(Povidone
Iodine)
6. Peroxygen Hydrogen oxidizing 3-6% All vegetative
peroxide agent cells are killed

7. Phenols a) Phenol, coagulates 2% 10 (lipo virus) Against Gram

cresol, proteins and positive biphenyl, disrupts
bacteria, fungi and hexachlorophene, cell membrane
enveloped viruses. b) Biguanides 0.2% Active against
(chlorhexidine) mycobacteria

8. Quater nary Benzalkonium Membrane 1-2% 10 (lipo virus) Broad spectrum

ammonium chloride, damage of activity
compoundscetyl by action on
pyridinum phospholipids
chloride

9. Aldehyde Formaldehyde Reacts with Broad spectrum

gas, functional 3-8% 10 (lipo virus) of activity
formalin, groups of 38%
glutaraldehyde protein and 2% 10 (lipo virus)
nucleic acids

Disinfectant Selection Table:-

Compound Chlorine Iodine/ Chlorhe- Alcohol Oxidizing PhenolQAC
Aldehydes
 Iodophor xidine compound 0.01-5% 0.5-5% 0.05-
5% 70-95% 0.2-3% 0.2-3% 0.1-2% 1-2%
1. Bactericidal Good Good Very good Good Good Good Good Very Good
2. Virucidal Very good Good Very good Good Good Fair Fair Very good
3. Enveloped Yes Yes Yes Yes Yes Yes Yes Yes
4. Non enveloped Yes Yes No No Yes No No Yes
virus
5. Bacterial Fair Fair Poor Fair Fair/Good Poor Poor Good
spores
6. Fungicidal Good Good Fair to Fair Fair Good Fair Good
Good
7. Protozoal Fair at Poor Poor Poor Poor Poor Fair Good
Parasites high con.
8. Effective in Poor Fair Fair Fair Poor Good Poor Good
organic matter
9. Inactivated No No and No No No No Yes No
by soap Yes
10. Effective in Yes No Yes Yes Yes Yes No Yes
hard water
11. Contact time5-30 10-30 5-10 10-30 10-30 10-30 10-30 10-60
(minutes)
12. Residual Poor Poor Good Fair Poor Poor Fair Fair 2
activity
 EXERCISE - 13

TESTING THE EFFICACY OFDISINFECTANT

SOLUTIONS
1. Phenol coefficient test
Phenol coefficient is a measure of germicidal efficacy of an unknown disinfectant, when compared
under identical conditions, to the efficacy of 5% solution of phenol. Indian standards accept the Riedal
Walker (RW) and Staphylococcus aureus test for judging efficacy of disinfectants. RW test measures
the ratio between the highest dilution of unknown disinfectant killing Salmonella typhi MCTC 786 in 7
and 10 minutes, but not in 2 and 5 minutes, and highest dilution of phenol killing the same organism
in the same contact period (7 and 10 minutes). This test is found to have certain limitations as its
efficacy is influenced by presence of organic animal matter, number of organism, and choice of test
organism. As an alternative, the Chick Martin method can be used even in the presence of some
organic matter. Indian standards recommended use of Staphylococcus aureus MCTC 3750 in the
same manner, as prescribed by RW test.

Procedure
1. Determine inhibition concentration of unknown disinfectant. Test organism is exposed to graded
dilutions, of disinfectant as part of a ranging test. Highest dilution inhibiting growth of organism is
recorded (1d in 700).
2. Prepare 5 graded dilution of unknown disinfectant in sterile distilled water in such a manner that
highest dilution of disinfectant should be higher than inhibitory concentration (1 in 900). Place
tubes in water bath at 180C.
3. Prepare 5 graded dilutions of 5% stock solution of phenol in sterile distilled water in such a manner
that highest dilution shows growth of organisms in all a most contact periods and lowest shows no
growth in most or all contact periods.
Eg. No growth in 1 in 95 dilution and the 115 dilution shows all the growth. The 1 in 95 to 1 in 115
dilution is prepared by dissolving 1 g phenol in 95, 100,105,110 and 115 ml water. Fresh solution
should be used. Place tubes in water bath at 180C.
4. Prepare a set of 20 drop tubes each with 5 ml nutrient both, for testing unknown disinfectant and
another set of 20 identical tubes for testing phenol. Keep the growth of Staphylococcus aureus or
Salmonella typhi ready, which should be freshly cultured
5. To 5 ml of dilute solution of test disinfectant, beginning with lowest (1 to 500 ml), add 0.2 ml of a
24 hour growth of test organisms at zero time. Shake and place the inoculation tubes in water
bath, controlled at 180C. After 30 seconds, add 0.2 ml culture to 2nd dilution (1 in 600). The
culture is likewise inoculated in all disinfectant tubes at intervals of 30 seconds. Inoculation is
completed in 120sec.
6. After every 30 sec, transfer loopful of culture, beginning with first dilution of disinfectant to 5 ml
nutrient broth tube, till all broth tubes are inoculated. Then disinfectant exposed organisms from
each dilution is tested at 2 ,5,7 and 10minute intervals in broth tubes.
7. Carry out procedures as described in 5th and 6th steps for control disinfectant phenol.
8. Inoculate all tubes at 370C for 48 hours. Mix and observe tubes in 2 sets, showing growth or no
growth. Mark them + ve or -ve respectively.
9. Prepare phenol coefficient table. Calculate phenol coefficient of unknown disinfectant by dividing
dilution factor of test disinfectant showing growth at 2 to 5 minutes, but not at 7-10 minutes by
dilution factor of phenol, showing similar growth pattern. The obtained value will show how many
times, unknown disinfectant is superior or inferior to 5% phenol.

Eg. Phenol coefficient Table

Sl. Dilution CultureContact time of test culture

No. inoculation time in broth tube and disinfectant
2 5 7 10

1 Unknown disinfectant
a. 1 in 500 0 - - - -
b. 1 in 600 30 sec + - - -
c. 1 in 700 60 sec + + - -
d. 1 in 800 90 sec + + + -
e. 1 in 900 120 sec + + + +
2. 1% phenol
a. 1 in 95 0 - - - -
b. 1 in 100 30 sec + - - -
c. 1 in 105 60 sec + + - -
d. 1 in 110 90 sec + + + -
e. d1 in 115 120 sec + + + +

Phenol coefficient = *****************

 EXERCISE - 14

DEMONSTRATION OF WATERPURIFICATION PLANT

 EXERCISE - 15

ASSIGNMENT
Title