Non-Fermentative Gram-Negative Non-Fermentative Gram-Negative RodsRods

Pseudomonas sppPseudomonas spp

Gram Stain

Gram-Positive

Gram-Negative

Cocci Bacilli Cocci Bacilli

Classification of Bacteria

Gram-negatitive BacilliGram-negatitive BacilliOxidase Test

Oxidase positive Oxidase Negative

O/F

O+/F-

Pseudomonadaceae

O+/F+

Vibrionaceae

O/F

O+/F+

Enterobacteriaceae

Characters of Characters of PseudomonasPseudomonasGram-negative bacilli belonging to Gram-negative bacilli belonging to PseudomonadaceaePseudomonadaceae

Motile by means of a single polar flagellum. Motile by means of a single polar flagellum.

Non spore formingNon spore forming

Capsulated "Polysaccharide capsule"Capsulated "Polysaccharide capsule"

AerobicAerobic

Breakdown glucose by oxidation i.e. Oxidative Breakdown glucose by oxidation i.e. Oxidative

Oxidase and catalase positiveOxidase and catalase positive

ItIt has very simple nutritional requirements i.e. non fastidioushas very simple nutritional requirements i.e. non fastidious

The most important pathogenic organism is The most important pathogenic organism is Ps. aeruginosaPs. aeruginosa

Optimum temperature is 37 C, and it is able to grow at 42 COptimum temperature is 37 C, and it is able to grow at 42 C

It is resistant to high concentrations of salts, dyes, weak It is resistant to high concentrations of salts, dyes, weak

antiseptics, and many antibioticsantiseptics, and many antibiotics

Common inhabitants of soil, water, GITCommon inhabitants of soil, water, GIT

Ps. aeruginosaPs. aeruginosa is opportunistic pathogen and associated is opportunistic pathogen and associated with a variety of infections including:with a variety of infections including:– Urinary tract infectionsUrinary tract infections– Wound and burn with blue green pusWound and burn with blue green pus– Respiratory system infections (Pneumonia)Respiratory system infections (Pneumonia)– Eye infection and may lead to blindnessEye infection and may lead to blindness– Ear infection (external ear or otitis media)Ear infection (external ear or otitis media)– MeningitisMeningitis– A variety of systemic infectionsA variety of systemic infections

Ps. aeruginosaPs. aeruginosa produce two types of soluble pigments: produce two types of soluble pigments:– Pyoverdin or fluorscein: Pyoverdin or fluorscein: It is yellow-green pigment and It is yellow-green pigment and

fluorescentfluorescent– PyocyaninPyocyanin: It is a blue-green pigment and non-: It is a blue-green pigment and non-

fluorescentfluorescent

Identification of Identification of Ps. aeruginosaPs. aeruginosa

Laboratory diagnosisLaboratory diagnosis

– Specimen:Specimen:

Urine, pus, sputum, CSF, blood, skin swap Urine, pus, sputum, CSF, blood, skin swap

according to the type of infectionaccording to the type of infection

– Microscopical ExaminationMicroscopical Examination

Gram Stain:Gram Stain: Gram-negative rods Gram-negative rods

Motility Test:Motility Test:

– Hanging Drop TechniquesHanging Drop Techniques

– Semisolid agar mediumSemisolid agar mediumMotile

Cultural CharacteristicsCultural CharacteristicsOn Nutrient agar:On Nutrient agar:

– Colonies are surrounded by bluish green colorationColonies are surrounded by bluish green coloration

On selective media "Cetermide"On selective media "Cetermide"

– Pigments are more obvious Pigments are more obvious

On Blood agarOn Blood agar

-hemolytic colonies-hemolytic colonies

On MacConkey agarOn MacConkey agar

– Pale yellow colonies i.e. non lactose fermentersPale yellow colonies i.e. non lactose fermenters

Ps. aeruginosaPs. aeruginosa able to grow at 42 C for 3 days able to grow at 42 C for 3 days

Cultural CharacteristicsCultural Characteristics

Gram Stain of Pseudomonas

Ps. aeruginosa on Nutrient agar

Ps. aeruginosa on cetrimide agar

Biochemical ReactionsBiochemical Reactions

Oxidase positive Oxidase positive

Breakdown glucose oxdatively Breakdown glucose oxdatively

Nitrate Reductase negativeNitrate Reductase negative

Gelatinase positiveGelatinase positive

Utilize CitrateUtilize Citrate

Oxidase Test:Oxidase Test: Principal Principal

Oxidase ReagentCytochrome Oxidase

Indophenol

Play role in aerobic respirationPseudomonasVibrio

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 stickResults Color change to purple within:

10 seconds = positive 10 - 60 seconds = delayed positive >60 seconds = negative

Oxidation/Fermentation (O/F) TestOxidation/Fermentation (O/F) TestPrinciple :Principle :

– To determine the ability of bacteria to breakdown To determine the ability of bacteria to breakdown glucose oxidative or fermentativeglucose 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: O/F Test: PrincipalPrincipal

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:O/F Test: Procedure Procedure

Each organism is inoculated into two Each organism is inoculated into two

tubes of glucose O/F medium tubes of glucose O/F medium

Inoculation is carried out as a stab to Inoculation is carried out as a stab to

within 1 cm of the bottom of the tubewithin 1 cm of the bottom of the tube

One of which is covered with One of which is covered with

mineral oil to exclude oxygen mineral oil to exclude oxygen

Incubate at 37°C for 24 hours.Incubate at 37°C for 24 hours.

O/F Test:O/F Test: Results Results

Non-Saccharolytic O-/FAlcaligenes faecalis

Open & covered remain green

Oxidative O+/F-Pseudomonas

Open turns yellow

Fermentative O+/F+Enterobacteriaceae

Both turn yellow

Reaction 1 Reaction 3Reaction 2

There are three types of reactions possible

Gelatin Liquifaction Test: Gelatin Liquifaction Test: PrinciplePrinciple Certain bacteria are capable of producing a proteolytic exoenzyme Certain bacteria are capable of producing a proteolytic exoenzyme

called gelatinasecalled gelatinase

Gelatinase hydrolyze the protein (solid) to amino acids (liquid)Gelatinase hydrolyze the protein (solid) to amino acids (liquid)

At temperature below 25°C, gelatin will remain a gel, but if the At temperature below 25°C, gelatin will remain a gel, but if the

temperature rises about 25°C, the gelatin will be liquid.temperature rises about 25°C, the gelatin will be liquid.

Gelatin hydrolysis has been correlated with pathogenicity of some Gelatin hydrolysis has been correlated with pathogenicity of some

microorganismsmicroorganisms

Pathogenic bacteria may breakdown tissue & spread to adjacent Pathogenic bacteria may breakdown tissue & spread to adjacent

tissuestissues

Nutrient gelatinProtein/Polypeptides

Solid

Gelatinase

Incubation at 37/overnight

Nutrient gelatinAmino acids

Liquid at > 25 C

Gelatinase hydrolyze the protein to aminoacids

Pseudomonas

Gelatinase Test:Gelatinase Test: ProcedureProcedure

Nutrient gelatin

Stab M.O.

Incubate at 37 C overnight

If tube remains solidNo change

-ve

E. coli

If tube liquefied at > 25 C

+ve

Ps. aeruginosa

Gelatin Liquifaction TestGelatin Liquifaction Test

MethodMethod

– Stab a Stab a nutrient gelatin nutrient gelatin tube with tube with

inoculums of the tested organisminoculums of the tested organism

– Inoculated nutrient gelatin tube is Inoculated nutrient gelatin tube is

incubated at 37°C for 24 hincubated at 37°C for 24 h

ResultResult

– If a tube of gelatin liquefy indicates If a tube of gelatin liquefy indicates

positive test (positive test (Ps. aeruginosaPs. aeruginosa))

– If a tube of gelatin remains solid If a tube of gelatin remains solid

indicates negative test (indicates negative test (E. coliE. coli))

Positive test

Negative test

Nitrate Reductase TestNitrate Reductase Test

PrinciplePrinciple – To determine the ability of an organism to reduce nitrate to To determine the ability of an organism to reduce nitrate to

nitrites or free nitrogen gas nitrites or free nitrogen gas

MethodMethod – Inoculate a nitrate broth with tested M.O.Inoculate a nitrate broth with tested M.O.– incubate for 24 hrs at 37°C.incubate for 24 hrs at 37°C.– After incubation, add 1 ml of sulphanilic acid and 1 ml of After incubation, add 1 ml of sulphanilic acid and 1 ml of --

naphtylamine to nitrate broth tube naphtylamine to nitrate broth tube

ResultResult – The production of a red color occurs in the presence of nitrite The production of a red color occurs in the presence of nitrite

indicates the ability of the organism to reduce nitrate to nitrite. indicates the ability of the organism to reduce nitrate to nitrite. – To broths showing a negative reaction add a few particles of To broths showing a negative reaction add a few particles of

zinc. The appearance of a red color indicates that nitrate is still zinc. The appearance of a red color indicates that nitrate is still present and hence has not been reduced by the organism. If the present and hence has not been reduced by the organism. If the solution does not change color the organism has reduced the solution does not change color the organism has reduced the nitrate through nitrite to nitrogen gas. nitrate through nitrite to nitrogen gas.

Nitrate Reductase Test:Nitrate Reductase Test: PrincipalPrincipal

Nitrate(NO3)

Nitrate reductaseNitrite(NO2)

α-naphthylamineSulfanilic acid

Red diazonium salt

If no red color!

Further reduction

Nitrogen gasN2

Add zinc dust (reducing agent)

Nitrate Reductase

Nitrate Reductase Test:Nitrate Reductase Test: ProcedureProcedure

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:Nitrate Reductase Test: ResultsResults

Red color after addition of

sulfanilic acid & -

naphtylamine

Reduction of Nitrate to

nitrite

Red color after

addition of zinc dust

-ve reductionNitrate

unreduced

No red color after addition of zinc dust

Nitrate reduced into nitrite and

further reduction to Nitrogen

Practical WorkPractical Work

☺ Gram stainGram stain

☺ Growth on Cetrimide agarGrowth on Cetrimide agar

☺ Oxidase testOxidase test

☺ O/F testO/F test

☺ Nitrate reductase testNitrate reductase test

☺ Gelatinase testGelatinase test

☺ Citrate Utilization Test Citrate Utilization Test ☺ (See under (See under EnterobacteriaceaEnterobacteriacea))