PRACTICAL

Identification of unknown bacterium

Why species level identification required? To know which species is involved

in the particular condition To know the subspecies To further know the types

Immunotypes, serotypes, chemotypes etc

Epidemiological studies

Basic steps to follow

Get a pure cultureIf pure culture is

given proceedIf mixture is givenGet pure culture

Slope is givenNutrient agar Blood agar

Pure culture follow up-Day 1

Nutrient agar slant

Growth characteristics

Catalase, oxidaseMotility/after 4 hours from

PW/BHI

Purity, Pigment If pure, S/C (1) PW

(2) NA (3) BA (4) Mac

Gram stain

Pure culture follow up-Day 1

Blood agar slant

Growth characteristics

oxidaseoxidase Purity, Pigment If pure, S/C (1) BHIIf pure, S/C (1) BHI

(2) NA (3) BA (2) NA (3) BA (4) Mac(4) Mac

Gram stainGram stainMotility/after 4 hours fromMotility/after 4 hours from

PW/BHIPW/BHI

First day – First day – preliminarypreliminary findingsfindings Growth

characteristics Gram stain Catalase Oxidase Motility

Presumptive follow upGram Negative Rods

Oxidase negativeOxidase negativeCatalase positiveCatalase positiveMotile/ NonmotileMotile/ Nonmotile

Oxidase PositiveCatalase Positive

Motile

One of the members of enterobacteriaceae

Vibrio orPseudomonas etc

Members of enterobacteriaceae:oxidase Negative

Escherichia Klebsiella Citrobacter Salmonella Shigella Proteus etc

Non enterobacteriaceae

Oxidase positiveVibrios

Pseudomonasetc

Members of enterobacteriaceae:oxidase Negative(example)

LF (ONPG) NLF Escherichia

Salmonella Klebsiella Shigella Citrobacter Proteus

A gram negative rod on BA/Macconkey agarsNon hemolytic, moist, irregular, flat, centrally raised, 3-5 mm colonies,

Lactose fermenting, Flat,irregular centrally raised, moist 2-4 mm colonies

Tests to read on second day

•Check whether the culture is pure•Repeat catalase, oxidase,gram and motility to confirm previous day’s findings

Biochemical tests

Macconkey: LFGram: Gram Negative rodsCatalase: PositiveOxidase ; Negative

One of the members of Enterobacteriaceae

TSI: A/A, Gas +, H2S NegativeSIM: H2S,Neg, Indole: Positive., MotileMotility; (PW) MotileIt could be

ESCHERICHIA, CITROBACTER,OR ENTEROBACTERGlucose : Positive (Acid and Gas)Lactose : PositiveSucrose : Negative / PositiveMannitol : PositiveIndole : PositiveMR : PositiveVP : PositiveCitrate : NegativeUrease : Negative

LAO : +-+Melonate: -KCN ; _

So it is Escherichia coli

For further details: Contact Jasmn98