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