IDENTIFIKASI BAKTERI OLEHSUDRAJAT FMIPA UNMUL 2009

IDENTIFIKASI IDENTIFIKASI BAKTERIBAKTERI

OLEHOLEH

SUDRAJATSUDRAJATFMIPA UNMULFMIPA UNMUL

20092009

A. KARAKTERISTIK FENOTIF

Metabolic differences

• Colony morphology– Colonies can exhibit

macroscopic differences

• e.g., Colonies of streptococci generally form fairly small colonies

• e.g., Colonies of Serratia marcescens produce a pigment and are often red when incubated at 22oC

• e.g., Colonies of Pseudomonas aeruginosa often produce a soluble greenish pigment



• Culture characteristics– Selective and differential media can aid in the

identification of microbes• Selective media favors the growth of certain types

of microbes by inhibiting the growth of others• Differential media contains a substance that

certain bacteria change in a recognizable way


Metabolic differences• Culture characteristics

– MacConkey agar is both selective and differential

• Bile salts and dyes inhibit all but certain gram-negative rods

– “Selective”

• Acid produced by bacteria able to ferment lactose will turn a pH indicator red and form red colonies

– “Differential”



– Blood agar can be used to detect bacteria producing hemolysins

• e.g., Harmless Streptococcus species residing in the throat often cause alpha-hemolysis

– Greenish clearing around colonies

• e.g., “Strep throat”-causing Streptococcus pyogenes causes beta-hemolysis

– Clear zone around colonies



– Media lacking nitrogen can be used to detect nitrogen-fixing bacteria

• e.g., Azotobacter can be identified from soil samples incubated aerobically on such media



• Biochemical tests– Generally necessary for more conclusive

identification– Most rely on pH indicator or color change

when a compound is degraded


Metabolic differences• Biochemical tests

– Sugar fermentation• e.g., Lactose, sucrose,

glucose, etc.• Fermentation results in

acid production– pH indicator changes color– Pink yellow

• Inverted tube (Durham tube) collects any gas produced



• Biochemical tests– Urease detection

• Enzyme degrading urea– Urea CO2 & NH3

• pH indicator turns bright pink in alkaline conditions

• Helicobacter pylori can be detected using a breath test



• Biochemical tests– Urease detection

• Helicobacter pylori can be detected using a breath test

– Causative agent of most stomach ulcers– Culturing not necessary– Patient drinks solution containing 14C-labeled urea– 14C in expired are indicates presence of urease



• Biochemical tests– Catalase

• Commonly occurring enzyme– Possessed by most bacteria growing in the presence of

oxygen– Absent in lactic acid bacteria

» e.g., Streptococcus» Beta-hemolytic catalase-negative bacteria from a

throat culture may be Streptococcus pyogenes



• Biochemical tests– Catalase

• Simple assay– H2O2 H2O & O2

– O2 bubbles are visible



• Organisms are identified using a dichotomous key– Multiple biochemical and

other tests are typically required

• Multiple tests are generally run concurrently

– Avoids waiting for incubation time for each test



• Biochemical tests– Commercial modifications of traditional

biochemical tests• e.g., APITM system, EnterotubeTM

Metabolic Phenotypic Exam• cultural approaches

– required for positive diagnosis of infection– isolation and ID of pathogen– accuracy, reliability, and speed– specimen collection is important – commonly used for positive identification of most

prokaryotes

• methods used include– culture characteristics– biochemical reactions process

Metabolic Phenotypic Exam

• culture characteristics– organisms grown in a pure culture are

easier to identify due to the high number of organisms obtained

E. coli

Metabolic Phenotypic Exam• culture characteristics

– use of selective or differential media can provide additional information

• selective media inhibits the growth of organisms other than the one being sought

• differential media contains substances that particular bacteria change in a recognizable way

Metabolic Phenotypic ExamWhen identifying a suspected organism, a series of differential media is inoculated. After incubation, each medium is observed to see if specific end products of metabolism are present. This can be done by adding indicators to the medium that react specifically with the end product being tested, giving some form of visible reaction such as a color change. The results of these tests on the suspected microorganism are then compared to known results for that organism to confirm its identification.

Metabolic Phenotypic Exam

Some bacteria will produce the enzyme catalase. Catalase will break down hydrogen peroxide releasing oxygen, which is indicated by the bubbles that have formed.

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IDENTIFIKASI BAKTERI OLEHSUDRAJAT FMIPA UNMUL 2009