313 PHT
Lab. No. 8
Gram’s Stain
Gram’s +ve
Cocci
Gram’s -ve
Bacilli
Cocci
Bacilli
Pseudomonas

Aerobic, non-fermentative,
motile, oxidase-positive gramnegative bacilli.
 Most
Important Species
P.aeruginosa
 opportunistic
pathogen
 causes UTI, wound infections and
otitis media
Microscopical examination:
(morphology)
A) Gram’s Stain:
Gram –ve
Non-sporeforming bacilli ,
having single arrangement.
B) Examination of Motility:
Using the “Hanging Drop technique”
Pseudomonas is highly motile by means of polar
flagella.
Cultural characteristic:
It grows on simple media.
It usually produces exopigments.
1) Growth on nutrient agar:
Its growth on nutrient agar
showing greenish discolouration
due to exopigment production.
2) Growth on Cetrimide Agar:
Principle:

Cetrimide agar is a highly selective
medium for pseudomonas species due to
presences of cetrimide which inhibits the
growth of other bacteria.
 It
contains also MgCl2 & K2So4 to facilitate
production of the charactaristic green
pigment of pseudomonas.
Results:
Only Pseudomonas species can grow on
cetrimide agar showing growth of pale
colonies with diffusion of green
pigmentation.
3) Growth on MacConkey’s agar:
Principle:




MacConkey’s agar is a
selective and differential
medium
selective medium for
enteric gram –ve bacteria
(bile salt inhibit the growth
of non enteric bacteria).
Test sugar: lactose.
pH indicator: neutral red (
yellow in alkaline, pink in
acidic pH).
Biochemical reaction:
1)Oxidase test.
2) Nitrate Test.
3) Oxidation Fermentation (O/F) Test.
4) Growth on Triple Sugar Iron (TSI) agar.
1)Oxidase test:
Results:
+ve Test: Appearance of purple colour within few seconds.
No colour
purple colour
+ve test
Pseudomonas
-ve test
Enterobacteriaceae
2) Nitrate Test:
Principle:
Further reduction
Nitrate reductase
nitrite
Nitrate
Nirtogen (N2)
α-naphthyl amine (nit. A)
Sulphanilic acid (nit. B)
Red diazonium salt
Enterobacteriaceae
If no red colour!
Add zinc dust (reducing agent)
Procedure:
Nit.A
test m.o
Incubate
at 35oC
for 24 hrs
Nitrate broth
Red colour
Nit. B
No red colour
Add zinc dust
Results:
Red colour after addition of
nit.A & nit.B
Reduction of Nitrate
to nitrite
Enterobacteriaceae
Red colour after addition
of zinc dust
-ve reduction
No red colour after
addition of zinc dust
Further reduction
to Nitrogen
Pseudomonas
3) Oxidation Fermentation (O/F)
Test:
Sensitive O/F test
Results:
Positive Test:
O-/F-
O+/F+
O+/F-
Non
Fermentative
Oxidative
Saccharolytic
Enterobacteriaceae
Pseudomonas
O-/F+
4) Growth on Triple Sugar Iron
(TSI) agar:
Principle:
butt
slant
Results:
1. No Fermentation:
Butt: alkaline (red)
Slant: alkaline (red)
Results:
Butt: acidic (yellow)
acidic (yellow)
acidic (yellow)
alkaline (red)
Slant: acidic (yellow)
alkaline (red)
alkaline (red)
alkaline (red)
H2 S :
-ve
-ve
+ve
-ve
Pseudomonas aeruginosa
I'm also very
resistant to
most
antibiotics, so
it's very hard
to get rid me.
Bacteriological
Examination of water, milk
and air
Examination of water, milk and
air
Water
Milk
Air
Importance of water examination
for pathogens

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
in industries such as food and
pharmaceuticals.
or

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.
Most important indicators of fecal
pollution of water

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.

•

•
•
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
Results:
 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.
Most probable number of coliforms
by McCrady’s table
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
3
9 ml Saline
1/100 x 1/10
1/100
1/1000
1 ml
1 ml
2
3
Results:
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
Examination of water, milk and
air
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
Importance of milk examination for
pathogens

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
Pathogenic bacteria present in milk
 E.coli
 Streptoccus pyogenes
Mycobacterium bovis
Bacillus anthracis
 Salmonella sp.
 Brucella sp.
Determination of viable bacterial
count:
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
2 – 6 hrs
6 – 8 hrs
Poor quality
fair quality
good quality
Over 8 hrs
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
-ve
purple to yellow.
+ve result with gas
production
result
Examination of water, milk and
air
Water
Milk
Air
Importance of keeping the micro- organisms
count low in air

Surgical theaters

Food preparations

Drug materials

Cross infection and out
breaks in hospitals
Number on bacteria in air depends on

Number of persons

Body movement

Disturbance of clothing
Methods of examination of air
a. Settle plate:



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
Demonstration:
 Air
examination
 Settle plate
 Water examination
 Determination of MPN
 Milk examination
 Methylene blue reduction test