Non-Fermentative Gram-Negative
Rods
Pseudomonas spp
Classification of Bacteria
Gram Stain
GramPositive
Cocci
GramNegative
Bacilli
Cocci
Bacilli
Gram-negatitive Bacilli
Oxidase Test
Oxidase positive
Oxidase Negative
O/F
O/F
O+/FPseudomonadaceae
O+/F+
Vibrionaceae
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 nonfluorescent
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
Ps. aeruginosa on cetrimide agar
Gram Stain of Pseudomonas
Ps. aeruginosa on Nutrient agar
Biochemical Reactions
Oxidase positive
Breakdown glucose oxdatively
Nitrate Reductase negative
Gelatinase positive
Utilize Citrate
Oxidase Test: Principal
Alternative substrate
for Cytochrome
Oxidize the reagent from
colorless to purple color
Oxidase Reagent
Indophenol
Cytochrome Oxidase
Tetramethyl-PPheneylenediamine
Colorless
Pseudomonas
Vibrio
Purple color
Play role in aerobic respiration
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
Positive
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
There are three types of reactions possible
Reaction 1
Non-Saccharolytic O-/F
Alcaligenes faecalis
Open & covered remain green
Reaction 2
Oxidative O+/FPseudomonas
Open turns yellow
Reaction 3
Fermentative O+/F+
Enterobacteriaceae
Both turn yellow
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
Pseudomonas
Gelatinase
Incubation at 37/overnight
Nutrient gelatin
Protein/Polypeptides
Solid
Gelatinase hydrolyze the
protein to aminoacids
Nutrient gelatin
Amino acids
Liquid at > 25 C
Gelatinase Test: Procedure
Stab M.O.
If tube remains solid
No change
-ve
E. coli
Incubate at 37 C overnight
If tube liquefied at > 25 C
+ve
Nutrient gelatin
Ps. aeruginosa
Gelatin Liquifaction Test
Method
Positive test
– 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)
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
NitrateReductase
reductase
Further reduction
Nitrite
(NO2)
Nitrate
(NO3)
Sulfanilic acid
Nitrogen gas
N2
α-naphthylamine
Red diazonium salt
If no red color!
Add zinc dust (reducing agent)
Nitrate Reductase Test: Procedure
Red color
Nitrate broth
M.O.
1m Sulfanilic acid
Incubate
at 37oC
for 24 hrs
1m -naphthylamine
Add zinc dust
No red color
Nitrate Reductase Test: Results
Red color after
Red color
addition of
after addition
sulfanilic acid & of zinc dust
-naphtylamine
Reduction of
Nitrate to nitrite
-ve reduction
Nitrate
unreduced
No red color
after addition
of zinc dust
Nitrate reduced into
nitrite and
further reduction to
Nitrogen
Practical Work
☺ Gram stain
☺ Growth on Cetrimide agar
☺ Oxidase test
☺ O/F test
☺ Nitrate reductase test
☺ Gelatinase test
☺ Citrate Utilization Test
☺ (See under Enterobacteriacea)