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Laboratory Diagnosis

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Implicating organisms include:

Bacteria

Parasites

Viruses

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Toxin producing:- Preformed toxin

- Toxin formed in vivo

Invasive

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Specimens Faeces, Rectal swab Others

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The use of a preservative,e.g buffered glycerol salineis recommended if there isdelay in submittingspecimen to the Laboratory

Stool iscollected in awide mouthcontainer

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Culture media forthe isolation ofSalmonella andShigella :MacConkeys AgarSalmonella and

Shigella Agar (SS)GN Broth

MacConkeys AgarDifferential willdifferentiatelactose and nonlactose fermenters

SalmonellaShigella (SS) AgarSelective Agarfor Salmonellaand Shigella

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A small amount of thestool is removed with aswab and plated to theculture media.Culture media areincubated

The swab is then placed in a GNbroth e.g. Selenite broth andincubated

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GN or Selenite brothincubated on day 1Is sub-cultured toMacConkeys and SSagar

This procedureincreases the chance ofisolating the pathogen

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MacConkey s agar showing Normal flora SS agar showing Normal flora

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Lactose fermentingcolonies (pink)

Non-Lactosefermenting coloni(pale )

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NoteSalmonella andShigella are nonlactose fermenters

Non- lactosefermentingcolonies areremoved andinoculated to :KliglersagarUrea

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A puregrowth ofnon lactosefermentingcolonies

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NoteThe media is so designed toshow a slant and a butt

Slant-for the detection ofLactosefermentation

Butt-for the detection ofglucose fermentationThe medium contains twosugars, Glucose andLactose along withcompounds to detect theproduction of H2S

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Organisms that producethe enzyme urease willform alkaline endproducts which willchange the colour of thismedia to pink

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Dextrose fermented, little or no gas, no H2S, urea negative:

Possible Shigella, Shigelloides

Dextrose fermented, gas H2S positive, urea negative:

Possible Salmonella, Arizona,

Citrobacter, Edwardsiella

Dextrose fermented, gas/no gas, little or no H2S, urea negative:

Possible Salmonella (e.g.Salmonella

typhii)

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Kligler and urea

Kligler Alkaline SlantH2S obscuring

Acid butt,Gas

Urea Negative

NoteSalmonella typhi will give similarreaction except there is a smallamount of H2S and no gas in the butt

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Kligler and urea

Kligler Alkaline Slant

Acid buttUrea Negative

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Further tests depend on resultsof kligler and ureae.g. 1. Slide agglutination- using O and H antisera for suspicious

Salmonella sp.- using O, H and Vi for suspiciousSalmonella typhi

- using species specific for Shigella spp.2. Further biochemical tests to confirm

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Kligler alkaline - slant-acid - butt with H2S and gas

Urea ve Lactose veCitrate +ve Sucrose veMotility +ve Dulcitol +veIndole ve Malonate -veLysine +ve Dextrose +ve

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Specimens:stool

Implicated food

Laboratory Investigation:- Gram stain from stool-gram positive cocci

- faecal leukocytes

- Culture : blood agar

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Colonies ofStaphylococcusaureus on blood agar

Gram stain of Staphylococcus aureus

Note!Serological tests using

specific antisera are used to

identify toxigenic strains

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MICROSCOPYMorphologically they may becurved or spiral shaped.GRAM REACTION:Organism is a gram negativecurved bacillus

On wet preparation, the organismhas a characteristic cork screwlike darting motility but are rigidrather than flexous

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ISOLATIONSpecimen is inoculated untoCampylobacter blood agar (CBA)IncubationCBA is placed in a microaerophilicatmosphere (90% Nitrogen, 5%Oxygen,5% carbon dioxide) and incubatedat 42oC for 48hrs.

Microaerophilic Usually generatedin a gas jar using amicroaerophilicgas sachet.

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CULTURAL CHARACTERISTICS:Colonies appear as water droplets ormelted paraffin and slightly gray.Theymay also appear tan or pink.After 24hours incubation colonies may appear tobe more solidified paraffin.Organism is :oxidase positiveCatalase positiveDoes not ferment glucoseReduce nitrates to nitritesIs susceptible to 30mcg Naladixic acidGrows in 1% glycine

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Gram positive sporulating bacilli

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Specimen of choice :stool rice water stool)

Definitive diagnosis :Demonstration of Vibriocholerain the patients stool.Culture Media Thiosulphate Citrate Bile- Salt(TCBS) agar.

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Definition:Gram negative rodsmotileaerobic and facultativeanaerobescatalase - negativeOxidase - positive

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A selective agarused for theisolation of Vibriocolera

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Colonialappearance ofyellow colonieson TCBS medium

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Cultural Characteristics :Oxidase testBiochemical testsSpecific antisera in slide agglutination testsFluorescence antibody (FA) tests

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V. cholera biotype V. cholera biotypeCholera (classical) El TorVP Reaction Negative PositiveSensitivity toPolymixin B Sensitive Resistant(50g disc)Sensitivity toMukerjee Sensitive ResistantIV phage

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V. cholera V.parahaemolyticusColonial appearance yellow colonies deep blue or greenon TCBS mediumGrowth in Peptone water no Growth Growthwith 7% NaCl conc.Acid from sucrose Positive Negative

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paired sera A fourfold rise or fall in vibriocidal titre isconsidered diagnostic.

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Escher ichia col i :

Colonial morphology: flat dry LF

Gram reaction: Gram negative

bacilli

Oxidase: negative

Kliglers: Acid/Acid GasUrea: negative

Citrate: negative

Motility: Motile

Indole: Positive

Lysine: Variable

flat dry lactose

fermenter (LF)

Note!

Serological tests using

specific antisera are used to

identify the various

pathogenic strains

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Specimens:Mainly stool/rectal swab

Blood

Laboratory investigations:Electron Microscopy (EM)

Isolation of Virus:

Tissue culture

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Include:RotavirusNorwalkCalicivirusesEnteric adenoviruses40 @ 41AstrovirusesHepatitis A

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Tissue culture cell line

Normal cell line Cell line showing CPE

Note!

Roundingof cells

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Enzyme-linked immunosorbent Assay (ELISA) TEST

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Giardia lamblia

In immunocompromised patients:

Cryptosporidium sp.

Isospora sp.

Micrsporidium sp

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Specimens: Stool

Blood

Small bowel fluid samples (e.g. entero string test)

Small bowel biopsy material

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Watery samples aremore likely tocontaintrophozoites of

iardia lamblia

Note theconsistencyof the stoolsample

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Laboratory investigations:1. Wet mount ( saline and/or iodine)

preparation (Giardia lamblia

trophozoites)

2. Modified ZN (Cryptosporidium sp. Isospora sp.

micrsporidium sp.

3. ELISA

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NoteOocysts are acid fast

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Oocyst of Isospora belli

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1.Salmonella typhi2. Salmonella paratyphi A

3.Salmonella parathphi B

4.Salmonella paratyphi C

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Blood cultures Urine samples

Stool samples

Bone marrow culture

Clotted blood samples for serology

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Submitted in all suspectedcases

1st-10thday of illness

Taken before treatment

The clot is cultures andserum used to perform

Widal serology testing

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Urine is concentrated and deposit used toinoculate appropriate media

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Stool will be positive in 2ndto 3rdweek ofinfection

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Bile aspirated by means of duodenal tube Valuable in the later stages of the illness and

in carrier detection

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CULTURE MEDIA USED

MacConkey

Salmonella and Shigella Agar (selective)

GN Broth( enrichment broth)

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NoteNon lactosefermenting colonies

RememberBiochemical reactions as

outlined in previous slides

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