Download lab 7 PHT313

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).

Gram –ve bacteria are classified into:
Lactose fermenter
(Pink colonies)
Lactose non-fermenter
(pale colonies)
Results:
Pink colonies
Lactose fermenter
Pale colonies
Lactose nonfermenter
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)
2. Dextrose Fermentation:
a) Initial reaction:
(after 10 – 12 hrs)
Dextrose
acid
Butt: acidic (yellow)
Slant: acidic (yellow)
2. Dextrose Fermentation:
b) Delayed reaction:
(after 24 hrs)
Small amount of acid
Butt: acidic (yellow)
Slant: alkaline (red)
3. Lactose Fermentation:
Lactose
Large amount of acid
Butt: acidic (yellow)
Slant: acidic (yellow)
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 very
resistant to
most
antibiotics, so
it's very hard
to get rid me.
Thank you