Pseudomonas and nonfermenters (biochemical)بكتريا عملي
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Pseudomonas and Nonfermenters
Gram negative rods
(Biochemical reactions)
By
Dr. Nabil El Aila
Assistant Professor of Molecular Microbiology
Medical Technology Department
Al -Aqsa University
Gram Stain
Gram-
Positive
Gram-
Negative
Cocci Bacilli Cocci Bacilli
Classification of Bacteria
Gram-negatitive Bacilli
Oxidase Test
Oxidase positive Oxidase Negative
O/F
O+/F-
Pseudomonadaceae
O+/F+
Vibrionaceae
O/F
O+/F+
Enterobacteriaceae
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
• 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
• Ps. aeruginosa produce two types of soluble pigments:
– Pyoverdin or fluorscein: It is yellow-green pigment and
fluorescent
– Pyocyanin: It is a blue-green pigment and non-fluorescent
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
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
Cultural Characteristics
Gram Stain of Pseudomonas
Ps. aeruginosa on Nutrient agar
Ps. aeruginosa on cetrimide agar
Biochemical Reactions
• Oxidase positive
• Breakdown glucose oxdatively
• Nitrate Reductase negative
• Gelatinase positive
• Utilize Citrate
Oxidase Test: Principal
Oxidase Reagent
Cytochrome Oxidase
Indophenol
Play role in aerobic respiration Pseudomonas
Vibrio
Alternative substrate for Cytochrome
Tetramethyl-P-Pheneylenediamine
Colorless
Purple color
Oxidize the reagent from colorless to purple color
Negative Positive
Method:
hold a piece of the oxidase test paper with forceps and touch
onto an area of heavy growth
Use platinum loop (not used nichrome) or wood stick
Results
Color change to purple within:
10 seconds = positive
10 - 60 seconds = delayed positive
>60 seconds = negative
Oxidation/Fermentation (O/F) Test • Principle :
– To determine the ability of bacteria to breakdown glucose
oxidative or fermentative
– O/F medium ( Hugh and Leifson Medium) is formulated
to detect weak acids produced from saccharolytic M.O.
– O/F medium contains
• Sugar (glucose 1%)
• Low percentage of Agar and Peptone
• pH indicator (Bromothymol blue)
– Alkaline Blue
– Neutral Green
– Acidic Yellow
O/F Test: Principal
• O/F medium differs from carbohydrate fermentation medium to
be more sensitive to detect the small amount of weak acids
produced by M.O.
• O/F medium is more sensitive due to:
– Higher % of glucose to increase amount of acid produced
– Lower % of peptone to reduce formation of alkaline amines
which neutralize weak acids formed
– Lower % of agar making the medium semisolid to facilitate
diffusion of acid throughout the medium
O/F Test: Procedure
• Each organism is inoculated into two tubes of
glucose O/F medium
• Inoculation is carried out as a stab to within 1 cm of
the bottom of the tube
• One of which is covered with mineral oil to exclude
oxygen Incubate at 37
C for 24 hours.
O/F Test: Results
Non-Saccharolytic O-/F
Alcaligenes faecalis
Open & covered remain green
Oxidative O+/F-
Pseudomonas
Open turns yellow
Fermentative O+/F+
Enterobacteriaceae
Both turn yellow
Reaction 1 Reaction 3 Reaction 2
There are three types of reactions possible
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
Protein/Polypeptides
Solid
Gelatinase
Incubation at 37/overnight
Nutrient gelatin
Amino acids
Liquid at > 25 C
Gelatinase hydrolyze the protein to aminoacids
Pseudomonas
Gelatinase Test: Procedure
Nutrient gelatin
Stab M.O.
Incubate at 37 C overnight
If tube remains solid
No change
-ve
E. coli
If tube liquefied at > 25 C
+ve
Ps. aeruginosa
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
Nitrate Reductase Test • Principle
– To determine the ability of an organism to reduce nitrate to
nitrites or free nitrogen gas
• Method
– Inoculate a nitrate broth with tested M.O.
– incubate for 24 hrs at 37
C.
– After incubation, add 1 ml of sulphanilic acid and 1 ml of -
naphtylamine to nitrate broth tube
• Result
– The production of a red color occurs in the presence of nitrite
indicates the ability of the organism to reduce nitrate to nitrite.
– To broths showing a negative reaction add a few particles of
zinc. The appearance of a red color indicates that nitrate is still
present and hence has not been reduced by the organism. If the
solution does not change color the organism has reduced the
nitrate through nitrite to nitrogen gas.
Nitrate Reductase Test: Principal
Nitrate (NO3)
Nitrate reductase Nitrite (NO2)
α-naphthylamine Sulfanilic acid
Red diazonium salt
If no red color!
Further reduction
Nitrogen gas N2
Add zinc dust (reducing agent)
Nitrate Reductase
Nitrate Reductase Test: Procedure
Nitrate broth
M.O. 1m Sulfanilic acid
1m -naphthylamine
Red color
No red color Add zinc dust
Incubate at 37oC
for 24 hrs
Nitrate Reductase Test: Results
Red color after addition of
sulfanilic acid & -naphtylamine
Reduction of
Nitrate to nitrite
Red color after addition of zinc dust
-ve reduction
Nitrate unreduced
No red color after addition of zinc dust
Nitrate reduced into
nitrite and
further reduction to
Nitrogen
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