Practical Medical MicrobiologyPractical Medical MicrobiologyPHT382PHT382

ByBy

Dr. Mohamed Al-AgamyDr. Mohamed Al-AgamyAssistant Professor of MicrobiologyAssistant Professor of Microbiology

Department of Pharmaceutics & MicrobiologyDepartment of Pharmaceutics & Microbiology

College of PharmacyCollege of Pharmacy

King Saud UniversityKing Saud University

Medical MicrobiologyMedical Microbiology

Infectious Microorganisms

Bacteria"Bacteriology"

Fungi"Mycology"

Virus"Virology"

Medical BacteriologyMedical Bacteriology

Bacteria

I- Gram positive

II- Gram negative III- Acid fast

MycoplasmaChalamydiaRickettsia

Spirochetes

Classification of BacteriaClassification of Bacteria

Bacteria

Gram-Positive Gram-negative

Gram-Positive BacteriaGram-Positive Bacteria

I- Gram Positive bacteria

A- Gram positive cocci B- Gram positive rods

Spore-formingNon spore-formingCorynebacterium

AerobicBacillus anthracis

AnaerobicClostridium

Gram-Positive CocciGram-Positive Cocci

A- Gram-positive cocci

I- staphylococci II- streptococci

Species of Species of SatphylococciSatphylococci

Three species of staphyloccoci have medical Three species of staphyloccoci have medical importance:importance:– S. aureusS. aureus:: P Pathogenicathogenic & commensally found in & commensally found in

nose (nares)nose (nares) S. epidermidisS. epidermidis:: non pathogenicnon pathogenic & common & common

commensals in nares & skincommensals in nares & skin S. saprophyticus:S. saprophyticus: Cause UTI in female & Cause UTI in female &

occasionally occasionally commensallycommensally found skin found skin

StaphylococciStaphylococci

General characters:General characters:– Gram Positive CocciGram Positive Cocci– Grape-likeGrape-like– Non MotileNon Motile– Non Spore FormingNon Spore Forming– Non CapsulatedNon Capsulated– Non FastidiousNon Fastidious– Facultative AnaerobesFacultative Anaerobes– FermentativeFermentative– Catalase positiveCatalase positive

Characters of Characters of S. aureusS. aureus– Production of coagulaseProduction of coagulase– Production of phosphataseProduction of phosphatase– Production of DNaseProduction of DNase– Ferment MannitolFerment Mannitol– Gelatin liquefiedGelatin liquefied– ΒΒ-hemolysis on blood agar-hemolysis on blood agar– Acidification & clotting of Acidification & clotting of

litmus milklitmus milk

Gram stain of StaphylococcusGram stain of Staphylococcus

Virulence factors of Virulence factors of S. aureusS. aureus

Coagulase: Coagulase: – Converting fibrinogen into fibrinConverting fibrinogen into fibrin

Exofoliative toxin:Exofoliative toxin:– Desquamation of skin in case of exofoliative dermatitis in SSSSDesquamation of skin in case of exofoliative dermatitis in SSSS

TSST:TSST:– Fever, hypotension, & skin rash followed by desquamation of skinFever, hypotension, & skin rash followed by desquamation of skin

LeucocytesLeucocytes– Kills WBCsKills WBCs

Polysaccharide A and Protein APolysaccharide A and Protein A– Antiphagocytic and AdhesionAntiphagocytic and Adhesion

Enterotoxins (A,B,C,D, & E)Enterotoxins (A,B,C,D, & E)– Food poisoning (Diarrhea, and Vomiting)Food poisoning (Diarrhea, and Vomiting)

HyaluronidaseHyaluronidase– Destroy hyaluronic acid (constituent of connective tissues)Destroy hyaluronic acid (constituent of connective tissues)

,,,, and and Toxins Toxins– Destroy variety of cells (Polymorph)Destroy variety of cells (Polymorph)

Disease caused by Disease caused by S. aureusS. aureus

Localized suppurartive (Pyogenic) inflammation:Localized suppurartive (Pyogenic) inflammation:– Folliculitis Infection of hair folliclesFolliculitis Infection of hair follicles– Furuncle Infection of an obstructed hair follicleFuruncle Infection of an obstructed hair follicle– Carbuncle Larger abscessCarbuncle Larger abscess– Deep Lesions (Osteomyelitis, Endocarditis & Meningitis)Deep Lesions (Osteomyelitis, Endocarditis & Meningitis)

Toxigenic infectionToxigenic infection– Scalded Skin Syndrome (SSS)Scalded Skin Syndrome (SSS)– Toxic Shock SyndromeToxic Shock Syndrome

Food poisoningFood poisoning– Nausea, Vomiting, Diarrhea without Fever within 8 h after Nausea, Vomiting, Diarrhea without Fever within 8 h after

ingestion of toxins in the contaminated foodingestion of toxins in the contaminated food

Laboratory diagnosis of Laboratory diagnosis of StaphylococcusStaphylococcus

Specimen:Specimen:– Pus, Urine, Stool, Blood, CSFPus, Urine, Stool, Blood, CSF

Gram Stain:Gram Stain:– Gram Positive Cocci, arranged in clusterGram Positive Cocci, arranged in cluster

Culture:Culture:– Blood agarBlood agar (Non-Selective Media) (Non-Selective Media)

Coagulase Positive Staphylococci are Pigmented & hemolyticCoagulase Positive Staphylococci are Pigmented & hemolytic

Coagulase Negative Staphylococci are non-pigmented & non-Coagulase Negative Staphylococci are non-pigmented & non-hemolytichemolytic

MSAMSA is selective differential medium for staphylococci is selective differential medium for staphylococci– It contains: NaCl (7.5%), Mannitol, & Phenol RedIt contains: NaCl (7.5%), Mannitol, & Phenol Red– The cause of The cause of selectivityselectivity due to presence of high salt due to presence of high salt

concentrationconcentration– The cause of The cause of differentialdifferential because contains mannitol because contains mannitol

(sugar) and phenol red (pH indicators turns yellow in (sugar) and phenol red (pH indicators turns yellow in acidic pH and turns red in alkaline pH).acidic pH and turns red in alkaline pH).

Mannitol fermentation on MSA

Mannitol fermentedYellow colonies:

S. aureus

Mannitol nonfermenterRed colonies:

S. epidermidis& S. saprophyticus

The catalase test is distinguished streptococci from The catalase test is distinguished streptococci from staphylococcistaphylococci

flood culture with drops of 3% H2O2 flood culture with drops of 3% H2O2

Catalase-positive cultures bubble at onceCatalase-positive cultures bubble at once

The test should not be done on blood agar because The test should not be done on blood agar because blood itself will produce bubbles blood itself will produce bubbles

Catalase test

PositiveMicrocococcaceae

Staphylococci

NegativeStreptococcaceae

Streptococci

H2O2 H2O + O2 (gas, ↑)Staphylococci

Catalase

Catalase test

Coagulase TestCoagulase TestPrinciple:Principle:

This test used to differentiate between S. aureus (CPS) & other Staphylococcus species (CNS)

Coagulase test

Coagulase PositiveStaphylococus aureus

Coagulase-NegativeS. epidermidis & S. saprophyticus

Fibrinogen (Plasma)

Coagulase Fibrin )Clot(

Coagulase TestCoagulase Test

The tube coagulase test The tube coagulase test (Free):(Free):

Procedure:Procedure: – Mix 0.1 ml of culture + 0.5 ml of plasmaMix 0.1 ml of culture + 0.5 ml of plasma– Incubate at 37C for 4 hIncubate at 37C for 4 h– Observing the tube for clot formationObserving the tube for clot formation– Any degree of clotting constitutes a Any degree of clotting constitutes a

positive testpositive test

AdvantageAdvantage– More accurateMore accurate

DisadvantageDisadvantage– Time consumedTime consumed

S. aureus S. epidermidis

Coagulase TestCoagulase TestTwo Methods:Two Methods:– The slide MethodThe slide Method– Tube MethodTube Method

The slide coagulase test The slide coagulase test – Used to detect bound coagulase or clumping factorUsed to detect bound coagulase or clumping factor– Add one drop heavy bacterial suspension and one drop of plasma on Add one drop heavy bacterial suspension and one drop of plasma on

clean slideclean slide– Mixing well and observing for clumping within 10 secondsMixing well and observing for clumping within 10 seconds

AdvantageAdvantage– Rapid diagnosisRapid diagnosis

DisadvantageDisadvantage– Less accurate Less accurate

Deoxyribonuclease (DNAase) testDeoxyribonuclease (DNAase) test

Principle:Principle:– DNA is insoluble in acidDNA is insoluble in acid– DNA is hydrolyzed into oligonucleotides by the DNA is hydrolyzed into oligonucleotides by the

action of DNaseaction of DNase– Nucleotides soluble in acidNucleotides soluble in acid

DNase test

PositiveStaphylococus aureus

NegativeS. epidermidis & S. saprophyticus

DNase TestDNase Test

Procedure & result:Procedure & result:– Inoculate DNA agar with tested organism in circular motionInoculate DNA agar with tested organism in circular motion– Incubate at 37C for 24-48hIncubate at 37C for 24-48h– Observe DNase activity by adding 1N HCl to the agar surface, a zone of clearing Observe DNase activity by adding 1N HCl to the agar surface, a zone of clearing

indicates a positive testindicates a positive test– The zone represents the absence of DNAThe zone represents the absence of DNA– The medium around colonies not producing DNase remains opaque, which is a The medium around colonies not producing DNase remains opaque, which is a

reflection of the precipitation of DNA by the added acid.reflection of the precipitation of DNA by the added acid.

Novobiocin SensitivityNovobiocin Sensitivity

A simple disk diffusion test for estimating novobiocin susceptibility A simple disk diffusion test for estimating novobiocin susceptibility used to distinguish used to distinguish S. saprophyticusS. saprophyticus from other clinically species from other clinically speciesInoculated overnight culture on Mueller-Hinton agar Inoculated overnight culture on Mueller-Hinton agar Add novobiocin disk on inoculated plateAdd novobiocin disk on inoculated plateIncubate at 370C overnightIncubate at 370C overnight Novobiocin resistance is intrinsic to Novobiocin resistance is intrinsic to S. saprophyticusS. saprophyticus but but uncommon in other clinically important species.uncommon in other clinically important species.

Novobiocin test

SensitiveS. aureus

S. epidermidis

ResistantS. saprophyticus

Preparation of Smear and StainingPreparation of Smear and Staining

Preparation of smearPreparation of smear– Solid cultureSolid culture– Liquid cultureLiquid culture– Distribute culture in slideDistribute culture in slide– Air dryAir dry– Heat fixHeat fix– Ready to stainReady to stain

Gram StainGram Stain– Primary Dye (C.V.) Primary Dye (C.V.) – Mordant (iodine)Mordant (iodine)– Decolorizer (Alcohol)Decolorizer (Alcohol)– Counterstain (Safranin)Counterstain (Safranin)– All applied for 1 minAll applied for 1 min– After each step wash with After each step wash with

waterwater– Blot dryBlot dry– Add one drop of immersion oilAdd one drop of immersion oil– Examine under oil immersion Examine under oil immersion

lenslens

Practical WorkPractical Work

Gram stainGram stain

Catalase testCatalase test

Mannitol fermentation on MSAMannitol fermentation on MSA

Coagulase Test by Tube and Slide MethodCoagulase Test by Tube and Slide Method

DNAase TestDNAase Test