Download +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.
