Transcript lab 8 PHT313

Bacteriological
Examination of water,
milk and air
Water
Milk
Air

Water intended for human
consumption should not contain
any pathogenic organisms.

Water is used for many
applications either at home for
cooking ,washing or drinking
or in industries such as food
and pharmaceuticals.

It is also important for
hospitals for example
haemodialysis unit

Testing of water samples
are done regularly to
make sure of its safety

Supplies of drinking water
contaminated with sewage
may cause diseases such as:
typhoid fever and cholera.

All sources of water should be
tested regularly.

Microorganisms which indicate
the fecal pollution in water are
usually common intestinal
commensal bacteria.

Escherichia coli:

The essential indicator of fecal pollution of human
/animal origin.
It is an important member of the coliform bacteria.


Coliforms are members of the enterobacteriaceae
family and they
1. grow in the presence of bile salts.
2. produce acid and gas from fermentation of lactose at 37°C.

It is the commonly-used bacterial indicator of sanitary
quality of food and water.
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Enterococcus faecalis:
less numerous than E.coli in
human feces, but more resistant
to chlorination.
Clostridium perfringens:
Less numerous in human feces
Its spores can survive in the
environment
Resist treatment processes than
most of the indicators.
Media used in bacteriological
examination of water
1. For coliforms:
MacConkey’s broth
Containing bromocresol purple as the pH
indicator.
To confirm the presence of E.coli :
EMB agar + IMVC

Enterococcus faecalis:
 Glucose

azide broth.
Clostridium perfringes:
 Differential
reinforced clostridial medium.
Methods Used in
Bacteriological Examination of
Water

Membrane Filtration Method

Determination of Most Probable Number
(MPN) by dilution method

Pour plate technique
Membrane Filtration Method
•
Using Millipore Filter Apparatus
MacConkey’s agar
Determination of MPN of
Coliforms by Dilution Method
50 ml water
sample
10 ml water
sample
1 ml water
sample
Water Sample
50 ml
DSMB
5 x 10 ml
DSMB
5 x 5 ml
SSMB

Positive tubes: showing production of
acid or gas.

Acid production: change color of tube
from purple to yellow

Gas production: detected in the
Durham’s tube.
Gas
Purple
Yellow
Determine no. of coliforms per 100 ml water sample
(MPN) using the standard probability table.
1
3
2
MPN = 14
i.e: No. of coliform bacilli per 100 ml water sample is 14 cells.
Viable Bacterial Count
Using 10 fold serial dilution method
1 ml water
Water sample
1 ml
1
1 ml
2
1/10 x 1/10
1/10
Melted NA
1 ml
1
1/100
3
1/100 x 1/10
1/1000
1 ml
2
9 ml Saline
1 ml
3
No. of colonies per plate
Y
Dilution
factor
1
2
3
X
X.y
10
x1
X1.y1
102
x2
X2.y2
x3
X3.y3
103
No. of cells per 1 ml =
X1.y1 + X2.y2 + X3.y3
3
Water
Milk
Air
Introduction:
Human infections may be caused by
theingestion of animal milk which
contains microorganisms derived from:
a.
b.
c.
Animal e.g. by contamination with its
feces
The environment
Milk handlers such as dairy workers

It is important to examine
milk for pathogens to ensure
that it is safe to be
consumed by man.

Milk is further used for
obtaining many milk
products like cheese ,cream
, butter and ice cream
 E.coli
 Streptoccus pyogenes
Mycobacterium bovis
Bacillus anthracis
 Salmonella sp.
 Brucella sp.
Using the pour plate method after preparation of 10
fold serial dilution from the milk sample with ringer
solution.


Permissible number of bacterial flora in
pasteurized milk is 5 x 104 cfu/ml
Permissible number of bacterial flora in long life
milk is 10 cfu/ml
Methylene Blue Reduction Test
To determine quality of the milk
Increasing the number of bacterial flora will reduce
the color of methylene blue more rapidly due to
increasing consumption of oxygen.
i.e.: The speed of reduction of methylene blue color is
directly proportional to the number of bacteria present
in milk sample.
Methylene Blue Reduction Test
Results:
The shorter the decolorization time, the higher
the number of bacterial flora present in milk,
and the poor quality of milk
Decolorization time
Result
30 min – 2 hrs
Poor quality
fair quality
2 – 6 hrs
6 – 8 hrs
Over 8 hrs
good quality
excellent quality
Test for coliforms
 Done
by inoculation of MacConkey’s broth
with 0.1 ml of milk sample.
 Examine
for the production of acid detected
by changing the color of the medium from
purple to yellow.
-ve
result
+ve result with gas
production
Water
Milk
Air
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Surgical theaters

Food preparations
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Drug materials

Cross infection and out
breaks in hospitals

Number of persons

Body movement

Disturbance of clothing
a. Settle plate:
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Petri dishes containing an agar medium are left open
for a measured period of time.
Large bacteria-carrying dust particles settle on the
medium.
The plates are incubated and a count of the colonies is
formed

Blood agar is suitable for
over all count

For detection of a
particular microorganism
suitable media is used .
Disadvantage of this method :
Despite its simplicity it measures only the
rate of deposition of large particles from
the air
b. Slit sampler

It draws in air from the environment at a fixed rate and
causes the suspended particles to fall on the surface of
the agar plate.
c. Air centrifuge



Centrifuging particles from the air on to a
culture medium.
The sampled air passed along a tube lined with
nutrient agar which was rotated on its long
axis.
After sampling the strip is removed from the
instrument and incubated then colonies can be
counted.
Notice:

No level of contamination however low
can be regarded as certainly safe.

Infection can be initiated by deposition of
a single infected particle at a favorable
site.

The probability of S. aureus initiated
infection is low in comparison with
Mycobacterium tuberculosis



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

Air examination
Settle plate
Water examination
Determination of MPN
Milk examination
Methylene blue reduction test