Transcript +ve Test
Lab. No. 4 B
Gram’s Stain
Gram’s +ve
Cocci
Gram’s -ve
Bacilli
Cocci
Bacilli
Aerobic, non-fermentative,
motile, oxidase-positive gramnegative bacilli.
Most
Important Species
P.aeruginosa
opportunistic
pathogen
causes UTI, wound infections and
otitis media
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.
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.
Procedure:
1. Inoculate Cetrimide agar plate with the test
organism by streaking.
Flam & Cool
Flam & Cool
Flam & Cool
2. Incubate the plate at 35oC for 24 hrs.
3) Growth on MacConkey’s agar:
Principle:
MacConkey’s agar is a selective and
differential medium
selective medium for gram –ve bacteria
(bile salt & crystal violet inhibit the
growth of gram +ve 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)
Procedure:
1. Inoculate MacConkey’s agar plate with the
test organism by streaking.
Flam & Cool
Flam & Cool
Flam & Cool
2. Incubate the plate at 35oC for 24 hrs.
Results:
Pink colonies
Lactose fermenter
Pale colonies
Lactose nonfermenter
1)Oxidase test:
Principle:
Tetramethyl p-phenylene diamine
(oxidase reagent)
colourless
Cytochrome oxidase enzyme
Indophenol
(Purple colour)
Results:
+ve Test: Appearance of purple colour within 1-2 min.
No colour
purple colour
+ve test
-ve test
Results:
+ve Test: Appearance of purple colour within 1-2 min.
No colour
purple colour
+ve test
-ve test
2) Nitrate Test:
Principle:
zincreductase
dust
Nitrate
Further reduction
nitrite
Nitrate
Nirtogen (N2)
α-naphthyl amine (nit. A)
Sulphanilic acid (nit. B)
Red diazonium salt
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
Red colour after addition of
zinc dust
-ve reduction
No red colour after
addition of zinc dust
Further reduction
to Nitrogen
3) Oxidation Fermentation (O/F)
Test:
Principle:
O/F medium ( Hugh and Leifson Medium) is a
specifically formulated medium (sensitive O/F
medium) to detect weak acids produced from
saccharolytic Gram’s –ve bacteria.
i.e: Its composition differs from ordinary fermentation
medium to be more sensitive to detect the small
amount of weak acids produced by Gram’s –ve
bacteria.
Principle:
To be more sensitive this medium contains:
Higher conc. Of sugar to increase amount of
acid produced.
Lower amount of peptone to reduce formation
of alkaline amines which neutralize weak acids
formed.
Lower conc. Of agar making the medium
semisold to facilitate diffusion of acid throughout
the medium.
Procedure:
1 ml liquid paraffin
O
F
Results:
Positive Test:
O-/FNon Saccharolytic
O+/F+
Fermentative
O+/FOxidative
O-/F+
4) Growth on Triple Sugar Iron (TSI)
agar:
Principle:
This medium contains three types of sugars
(lactose, sucrose and dextrose).
The conc. of lactose and sucrose is 10 times
that of dextrose.
It also contains phenol red indicator.
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)
Principle:
This medium contains also ferrous sulfate as
an indicator for H2S production.
H2S + FeSo4
FeS
Black ppt. of ferrous sulfid
Procedure:
Results:
Butt:
acidic (yellow)
acidic (yellow)
acidic (yellow)
Slant:
acidic (yellow)
alkaline (red)
alkaline (red)
H2S Production:
-ve
-ve
+ve
Practicle Work
Gram’s stain (spot)
Growth on Cetrimide agar
Growth on MacConkey agar
Oxidase test.
Nitrate test.
O/F test.
TSI test.