lab. no. 4. aerobic, non-fermentative, motile, oxidase-positive gram- negative bacilli. aerobic,...
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Lab. No. 4
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Gram’s Stain
Gram’s +ve Gram’s -ve
Cocci Bacilli Cocci Bacilli
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AerobicAerobic, , non-fermentativenon-fermentative, , motilemotile, , oxidase-positiveoxidase-positive gram-negative gram-negative bacilli.bacilli.
Most Important SpeciesMost Important Species P.aeruginosa P.aeruginosa opportunistic pathogen opportunistic pathogen causes UTI, wound infections and otitis causes UTI, wound infections and otitis
mediamedia
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A) Gram’s Stain:A) Gram’s Stain:Gram –ve Gram –ve
Non-sporeforming bacilli , Non-sporeforming bacilli ,
having single arrangementhaving single arrangement..
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B) Examination B) Examination of Motility:of Motility:
Using the Using the “Hanging “Hanging Drop technique”Drop technique”
Pseudomonas is Pseudomonas is highly motile by highly motile by means of polar means of polar flagella.flagella.
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It grows on It grows on simple media.simple media.
It usually produces It usually produces exopigments.exopigments.
1) Growth on nutrient agar:1) Growth on nutrient agar: Its growth on nutrient agar Its growth on nutrient agar
showing showing greenish discolourationgreenish discolouration
due to due to exopigmentexopigment production. production.
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Cetrimide agar is a Cetrimide agar is a highly selective highly selective mediummedium for pseudomonas species due for pseudomonas species due to presences of to presences of cetrimide cetrimide which which inhibits the growth of other bacteria.inhibits the growth of other bacteria.
It contains also It contains also MgClMgCl22 & & KK22SoSo44 to to facilitate production of the facilitate production of the charactaristic charactaristic green pigment green pigment of of pseudomonas. pseudomonas.
2) Growth on Cetrimide Agar:2) Growth on Cetrimide Agar:
Principle:
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Results:
Only Pseudomonas species can grow on cetrimide agar showing growth of pale colonies with diffusion of green pigmentation.
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Procedure:
1. Inoculate Cetrimide agar plate with the test organism by streaking.
2. Incubate the plate at 35oC for 24 hrs.
Flam & Cool
Flam & Cool
Flam & Cool
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MacConkey’s agar is a selective and differential medium
selective medium for selective medium for gram –ve bacteriagram –ve bacteria ( (bile saltbile salt & & crystal violetcrystal violet inhibit the growth of gram +ve bacteria). inhibit the growth of gram +ve bacteria).
Test sugar:Test sugar: lactose.lactose. pH indicator:pH indicator: neutral redneutral red ( yellow in alkaline, pink in ( yellow in alkaline, pink in
acidic pH).acidic pH).
3) Growth on MacConkey’s agar:3) Growth on MacConkey’s agar:
Principle:
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Gram –ve bacteriaGram –ve bacteria are classified into:are classified into:
Lactose fermenter (Pink colonies)
Lactose non-fermenter (pale colonies)
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Procedure:
1. Inoculate MacConkey’s agar plate with the test organism by streaking.
2. Incubate the plate at 35oC for 24 hrs.
Flam & Cool
Flam & Cool
Flam & Cool
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Results:
Pink colonies
Lactose fermenter
Pale coloniesLactose nonfermenter
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1)Oxidase test:1)Oxidase test:
Principle:Tetramethyl p-phenylene diamine
(oxidase reagent)colourless
Cytochrome oxidase enzyme
Indophenol(Purple colour)
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Results:
+ve Test: Appearance of purple colour within 1-2 min.
purple colour
+ve test
No colour
-ve test
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Results:
+ve Test: Appearance of purple colour within 1-2 min.
purple colour
+ve test
No colour
-ve test
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2) Nitrate Test:2) Nitrate Test:
Principle:
NitrateNitrate reductase
nitrite
α-naphthyl amine (nit. A)
Sulphanilic acid (nit. B)
Red diazonium salt
If no red colour!
Further reductionNirtogen (N2)
Add zinc dust (reducing agent)
zinc dust
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Procedure:
Nitrate broth
test m.o
Nit.A
Nit. B
Red colour
No red colour
Add zinc dust
Incubate at 35oC for 24
hrs
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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
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3) 3) Oxidation Fermentation (O/F) Test: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.
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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.
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Procedure:
O F
1 ml liquid paraffin
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Positive Test:
O-/F- O+/F+ O+/F- O-/F+
Results:
Fermentative OxidativeNon Saccharolytic
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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.
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Principle:
butt slant
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Results:
1. No Fermentation:1. No Fermentation:
Butt: alkaline (red) Slant: alkaline (red)
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2. Dextrose Fermentation:2. Dextrose Fermentation:
a) Initial reaction:
Dextrose
acid
(after 10 – 12 hrs)
Butt: acidic (yellow) Slant: acidic (yellow)
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2. Dextrose Fermentation:2. Dextrose Fermentation:
b) Delayed reaction:(after 24 hrs)
Peptone O
2
Alkaline amines
Butt: acidic (yellow) Slant: alkaline (red)
Small amount of acid
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3. Lactose Fermentation:3. Lactose Fermentation:
Lactose
Large amount of acid
Peptone O
2
Alkaline amines
Butt: acidic (yellow) Slant: acidic (yellow)
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Principle:
This medium contains also ferrous sulfate as an indicator for H2S production.
H2S + FeSo4 FeS
Black ppt. of ferrous sulfid
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Procedure:
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Results:
Butt:
Slant:
H2S Production:
acidic (yellow)
acidic (yellow)
-ve
acidic (yellow)
alkaline (red)
-ve
acidic (yellow)
alkaline (red)
+ve
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Practicle WorkPracticle Work
Gram’s stain (spot)Gram’s stain (spot) Growth on Cetrimide agarGrowth on Cetrimide agar Growth on MacConkey agar Growth on MacConkey agar Oxidase test.Oxidase test. Nitrate test.Nitrate test. O/F test.O/F test. TSI test.TSI test.
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