non-fermentative gram-negative rodsfac.ksu.edu.sa/sites/default/files/6_32.pdf• gram negative,...
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Pseudomonas spp
NON-FERMENTATIVE GRAM-
NEGATIVE RODS
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Gram Stain
Gram-
Positive
Gram-
Negative
Cocci Bacilli Cocci Bacilli
Classification of Bacteria
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GRAM-NEGATITIVE BACILLI
Oxidase Test
Oxidase positive Oxidase Negative
O/F
O+/F-
Pseudomonadaceae
O+/F+
Vibrionaceae
O/F
O+/F+
Enterobacteriaceae Manal Al khulaifi
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CHARACTERS OF PSEUDOMONAS • Gram-negative bacilli belonging to Pseudomonadaceae
• Motile by means of a single polar flagellum.
• Non spore forming
• Capsulated "Polysaccharide capsule"
• Aerobic
• Breakdown glucose by oxidation i.e. Oxidative
• Oxidase and catalase positive
• It has very simple nutritional requirements i.e. non fastidious
• The most important pathogenic organism is Ps. aeruginosa
• Optimum temperature is 37 C, and it is able to grow at 42 C
• It is resistant to high concentrations of salts, dyes, weak antiseptics, and many
antibiotics
• Common inhabitants of soil, water, GIT
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• Ps. aeruginosa is opportunistic pathogen and
associated with a variety of infections
including:
• Urinary tract infections
• Wound and burn with blue green pus
• Respiratory system infections (Pneumonia)
• Eye infection and may lead to blindness
• Ear infection (external ear or otitis media)
• Meningitis
• A variety of systemic infections
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IDENTIFICATION OF PS. AERUGINOSA
• Laboratory diagnosis
• Specimen:
• Urine, pus, sputum, CSF, blood, skin swap
according to the type of infection
• Microscopical Examination
• Gram Stain: Gram-negative rods
• Motility Test:
• Hanging Drop Techniques
• Semisolid agar medium Motile
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Gram Stain of Pseudomonas
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CULTURAL CHARACTERISTICS • On Nutrient agar:
• Colonies are surrounded by bluish green coloration
• On selective media "Cetermide"
• Pigments are more obvious
• On Blood agar
• -hemolytic colonies
• On MacConkey agar
• Pale yellow colonies i.e. non lactose fermenters
• Ps. aeruginosa able to grow at 42 C for 3 days
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CULTURAL
CHARACTERISTICS
Ps. aeruginosa on Nutrient agar
Ps. aeruginosa on cetrimide agar
Pseudomonas on blood agar
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MOTILITY TEST :
direct microscopic observation ( hanging drop technique)
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MOTILITY TEST
Semi solid agar Manal Al khulaifi
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BIOCHEMICAL REACTIONS
• Oxidase positive
• Gelatinase positive
• Nitrate test
• O/F test
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OXIDASE TEST: PRINCIPAL
Oxidase Reagent
Cytochrome Oxidase
Indophenol
Play role in aerobic respiration Pseudomonas Vibrio
Purple color
Oxidize the reagent from
colorless to purple color
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Negative Positive
Method:
hold a piece of the oxidase test paper with forceps and
touch onto an area of heavy growth
Use loop or wood stick
Results
Color change to purple
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GELATIN LIQUIFACTION TEST: PRINCIPLE
Certain bacteria are capable of producing a proteolytic exoenzyme called gelatinase
Gelatinase hydrolyze the protein (solid) to amino acids (liquid)
At temperature below 25°C, gelatin will remain a gel, but if the temperature rises
about 25°C, the gelatin will be liquid.
Gelatin hydrolysis has been correlated with pathogenicity of some microorganisms
Pathogenic bacteria may breakdown tissue & spread to adjacent tissues
Nutrient gelatin
Gelatinase
Incubation at 37/overnight
Nutrient gelatin
Amino acids
Liquid at > 25 C
Gelatinase hydrolyze the
protein to aminoacids
Pseudomonas
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GELATINASE TEST:
PROCEDURE
Nutrient gelatin
Stab M.O.
Incubate at 37 C overnight
If tube remains solid
No change
E. coli
If tube liquefied at > 25 C
Ps. aeruginosa
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GELATIN LIQUIFACTION TEST
• Method
• Stab a nutrient gelatin tube with inoculums of
the tested organism
• Inoculated nutrient gelatin tube is incubated at
37°C for 24 h
• Result
• If a tube of gelatin liquefy indicates positive test
(Ps. aeruginosa)
• If a tube of gelatin remains solid indicates
negative test (E. coli)
Positive test
Negative test Manal Al khulaifi
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PRACTICAL WORK
☺ Gram stain
☺ Oxidase test
☺ Gelatinase test
☺ Motility test
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Vibrionaceae Vibrio
GRAM NEGATIVE RODS
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GENERAL CHARCTERS OF
VIBRIONACEAE • Gram negative, curved, comma shaped bacilli
• Motile by single polar flagella
• Non spore forming
• Non capsulated
• grow well in alkaline pH
• Facultative anaerobes
• Vibrios are capable of both respiratory & fermentative metabolism i.e. O+/F+.
• Oxidase and catalase positive
• Natural inhabitants of aquatic environment
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SPECIES OF VIBRIO
Vibrios
Vibrio cholerae
Cause Cholera
V. parahaemolyticus
cause
Gastroenteritis
Allied vibrios
Saprophytic
Classical type
V. cholerae
El-Tor-type
V. El-Tor
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SPECIES OF VIBRIO • V. cholerae is the causative agent of cholera
• V. cholerae divided serologically into 6 groups based on somatic O-antigens
• Vibrio parahaemolyticus is the cause of acute gastroenteritis following ingestion of contaminated
sea-food such as raw fish
• V. cholerae & V. parahaemolyticus, are pathogens of human, produce diarrhea
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IDENTIFICATION OF V. CHOLERAE • Specimen and microscopical examination:
•Rice watery stool or rectal swap collected in acute stage of disease
•Dark-field microscopy of stool specimen from patients with cholera reveal large numbers of
Vibrio (short, curved rods) with a characteristic motility that gives the appearance of shooting
stars
• Culture:
•Inoculation of rice water stool in enrichment media (alkaline peptone water, pH8.5), in which
the organisms multiply rapidly and tend to form pellicle at the surface of the medium after 6-8
h at 37 C.
•Subculture is made into Thiosulphate Citrate Bile Sucrose (TCBS) agar.
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TCBS medium is selective because
Also, contains bile salts
High conc. of thiosulfate and citrate & strong alkalinity of this medium (pH9)
TCBS medium is differential because
It contains sucrose
It contains bromothymol blue
Some species ferment sucrose & others not ferment
Sucrose fermenting Vibrio spp (V. cholerae) appears as
yellow colonies
Sucrose non fermenting Vibrio spp (V. parahemolyticus) appears as blue to green colonies
Alkaline pH: blue
Neutral pH: green
Acidic pH: yellow
Sucrose fermentation on TCBS is the gold standard in its identification
kills most intestinal commensals
Identification of V. cholerae Growth on TCBS
Principle
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IDENTIFICATION OF VIBRIO
DIFFERENTIATION BETWEEN SF & NSF BY GROWTH ON TCBS
• Method:
• TCBS agar is inoculated with tested organism recovered from alkaline peptone water using streak plate technique
• Incubate the plate in incubator at 37 C/24 hrs
• Results:
• SF organism appears as yellow colonies (V. cholerae)
• NSF organism appears as blue to green colonies (V. parahaemolyticus)
Flame & Cool
Flame & Cool
Flame & Cool
1 2
3
4 5
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REACTION ON TCBS
Yellow colonies of V. cholorae due sucrose fermentation and green
colonies of V. parahaemolyticus on TCBS Manal Al khulaifi
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ON MACKONCEY MEDIA
Noo lactose fermented
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IDENTIFICATION OF VIBRIO
CHOLREAE
Gram stain of Vibrio cholorae
Gram stain:
Gram negative short rods, comma shaped, motile
Electron Micrograph of V cholerae Rods with single polar flagella
Serology
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