313 pht lab. no. 8
DESCRIPTION
313 PHT Lab. No. 8. Classification of Bacteria. Pseudomonas. Aerobic , non-fermentative , motile , oxidase-positive gram-negative bacilli. Most Important Species P.aeruginosa opportunistic pathogen causes UTI, wound infections and otitis media. Microscopical examination: - PowerPoint PPT PresentationTRANSCRIPT
313 PHT
Lab. No. 8
Gram’s Stain
Gram’s +ve Gram’s -ve
Cocci Bacilli Cocci Bacilli
AerobicAerobic, , non-fermentativenon-fermentative, , motilemotile, , oxidase-positiveoxidase-positive gram-negative bacilli.gram-negative bacilli.
Most Important SpeciesMost Important SpeciesP.aeruginosa P.aeruginosa
opportunistic pathogen opportunistic pathogen causes UTI, wound infections and causes UTI, wound infections and
otitis mediaotitis media
Pseudomonas
Microscopical examination:Microscopical examination:
(morphology)(morphology)
A) Gram’s Stain:A) Gram’s Stain:
Gram –ve Gram –ve
Non-sporeforming bacilli , Non-sporeforming bacilli ,
having single arrangementhaving single arrangement..
B) Examination of Motility:B) Examination of Motility:
Using the Using the “Hanging Drop technique”“Hanging Drop technique”
Pseudomonas is highly motile by means of Pseudomonas is highly motile by means of polar flagella.polar flagella.
Cultural Cultural characteristic:characteristic:
It grows on It grows on simple media.simple media.
It usually produces It usually produces exopigments.exopigments.
1) Growth on nutrient 1) Growth on nutrient agar:agar:
Its growth on nutrient agar Its growth on nutrient agar
showing showing greenish greenish discolourationdiscolouration
due to due to exopigmentexopigment production. production.
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:
Results:
Only Pseudomonas species can grow on cetrimide agar showing growth of pale colonies with diffusion of green pigmentation.
MacConkey’s agar is a selective and differential medium
selective medium for selective medium for enteric enteric gram –ve gram –ve bacteriabacteria ( (bile saltbile salt inhibit the growth of inhibit the growth of non enteric bacteria).non enteric bacteria).
Test sugar:Test sugar: lactose.lactose. pH indicator:pH indicator: neutral neutral
redred ( yellow in alkaline, ( yellow in alkaline, pink in acidic pH).pink in acidic pH).
3) Growth on MacConkey’s agar:3) Growth on MacConkey’s agar:
Principle:
Gram –ve bacteriaGram –ve bacteria are classified into:are classified into:
Lactose fermenter (Pink colonies)
Lactose non-fermenter (pale colonies)
Results:
Pink colonies
Lactose fermenter
Pale coloniesLactose nonfermenter
Biochemical reaction:Biochemical reaction:
1)Oxidase test.1)Oxidase test.
2) Nitrate Test.2) Nitrate Test.
3) Oxidation Fermentation (O/F) Test.3) Oxidation Fermentation (O/F) Test.4) Growth on Triple Sugar Iron (TSI)
agar.
Results:
+ve Test: Appearance of purple colour within few seconds.
purple colour
+ve testPseudomonas
No colour
-ve testEnterobacteriaceae
1)Oxidase test:1)Oxidase test:
2) Nitrate Test:2) Nitrate Test:
Principle:
NitrateNitrate reductase
nitrite
α-naphthyl amine (nit. A)
Sulphanilic acid (nit. B)
Red diazonium saltEnterobacteriaceaeEnterobacteriaceae
Further reduction
Nirtogen (N2)
Add zinc dust (reducing agent)
If no red colour!
Procedure:
Nitrate broth
test m.o
Nit.A
Nit. B
Red colour
No red colour
Add zinc dust
Incubate at 35oC for 24
hrs
Results:
Red colour after addition of nit.A & nit.B
Reduction of Nitrate to nitrite
EnterobacteriaceaEnterobacteriaceaee
Red colour after addition of zinc dust
-ve reduction
No red colour after addition of zinc dust
Further reduction to
NitrogenPseudomonas
3) Oxidation Fermentation 3) Oxidation Fermentation (O/F) Test:(O/F) Test:
Sensitive O/F testSensitive O/F test
Positive Test:
O-/F- O+/F+ O+/F- O-/F+
Results:
Fermentative
EnterobacteriaceEnterobacteriaceaeae
Oxidative
Pseudomonas
Non
Saccharolytic
Principle:
butt slant
4) Growth on Triple Sugar Iron(TSI) agar:
Results:
1. No Fermentation:1. No Fermentation:
Butt: alkaline (red) Slant: alkaline (red)
2. Dextrose Fermentation:2. Dextrose Fermentation:
a) Initial reaction:
Dextrose
acid
(after 10 – 12 hrs)
Butt: acidic (yellow) Slant: acidic (yellow)
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
3. Lactose Fermentation:3. Lactose Fermentation:
Lactose
Large amount of acid
Peptone O
2
Alkaline amines
Butt: acidic (yellow) Slant: acidic (yellow)
Results:
Butt:
Slant:
H2S :
acidic (yellow)
acidic (yellow)
-ve
acidic (yellow)
alkaline (red)
-ve
acidic (yellow)
alkaline (red)
+ve
alkaline (red)
alkaline (red)
-ve
Pseudomonas aeruginosa
I'm veryI'm very resistant to resistant to
most most antibioticsantibiotics, , so it's very so it's very hard to get hard to get
rid me. rid me.
Thank youThank you