Bacterial Identification - Copy (2)

8/8/2019 Bacterial Identification - Copy (2)

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IDENTIFICATION OF

PROKARYOTES

IDENTIFICATION OF

PROKARYOTES

CHAPTER 10CHAPTER 10


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TAXONOMYTAXONOMY

The study and grouping of organisms

Three separate but interrelated areas

Identification

Process of characterizing organisms

Classification

Arrangement of organisms into groups

Nomenclature

Assignment of a specific name

The study and grouping of organisms

Three separate but interrelated areas

Identification

Process of characterizing organisms

Classification

Arrangement of organisms into groups

Nomenclature

Assignment of a specific name


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TAXONOMYTAXONOMY

Initial identification of microorganisms results in

their classification

Based on evolutionary relationships

Identification of microorganisms in particularenvironments remains important

e.g., Microbial contaminants can spoil food

e.g., Identification of microbes present in aclinical patient is important in determining

treatment

Initial identification of microorganisms results in

their classification

Based on evolutionary relationships

Identification of microorganisms in particularenvironments remains important

e.g., Microbial contaminants can spoil food

e.g., Identification of microbes present in aclinical patient is important in determining

treatment


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PROKARYOTE IDENTIFICATIONPROKARYOTE IDENTIFICATION

Various techniques are employed to characterizeand identify microorganisms

Phenotypic characteristics

Microscopic morphology Metabolic differences

Serology

Fatty acid analysis

Genotypic characteristics Nucleic acid probes

DNA amplification

rRNA sequencing

Various techniques are employed to characterizeand identify microorganisms

Phenotypic characteristics

Microscopic morphology Metabolic differences

Serology

Fatty acid analysis

Genotypic characteristics Nucleic acid probes

DNA amplification

rRNA sequencing


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PHENOTYPIC CHARACTERISTICSPHENOTYPIC CHARACTERISTICS

Microscopic morphology

Size and shape

Readily determined by microscopic examination

of a wet mount

Can determine whether the microbe is a

prokaryote, fungus, or protozoan

Microscop

ic morphology

Size and shape

Readily determined by microscopic examination

of a wet mount

Can determine whether the microbe is a

prokaryote, fungus, or protozoan


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Size and shape

Often sufficient for clinical

diagnosis e.g., Trichomonasvs. Candida in vaginal

secretions

e.g., Roundworm eggs instool

Size, shape, and otherfeatures often sufficient

for identification

Microscop

ic morphology

Size and shape

Often sufficient for clinical

diagnosis e.g., Trichomonasvs. Candida in vaginal

secretions

e.g., Roundworm eggs instool

Size, shape, and otherfeatures often sufficient

for identification


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Cell groupings

Cells adhering to one another following binary

fission often form characteristic arrangements

e.g.,Neisseria gonorrhoeae typically displays a

diplococcus arrangement

e.g., Most Streptococcus species form long chains e.g., Most Staphylococcus species form grapelike

clusters

e.g., Sarcina species for cubical packets


Cell groupings

Cells adhering to one another following binary

fission often form characteristic arrangements

e.g.,Neisseria gonorrhoeae typically displays a

diplococcus arrangement

e.g., Most Streptococcus species form long chains e.g., Most Staphylococcus species form grapelike

clusters



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Cell groupings

Cells adhering to one another

following binary fission oftenform characteristic arrangements

e.g., Neisseria gonorrhoeae

typically displays a diplococcus

arrangement

e.g., Most Streptococcus species form long chains

e.g., Most Staphylococcus species form grapelike clusters



Cell groupings

Cells adhering to one another

following binary fission oftenform characteristic arrangements

e.g., Neisseria gonorrhoeae

typically displays a diplococcus

arrangement

e.g., Most Streptococcus species form long chains

e.g., Most Staphylococcus species form grapelike clusters



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Gram stain

Differential stain

distinguishing betweengram-positive and

gram-negative bacteria

Narrows possible identities of an organism Excludes many possibilities

Generally insufficient alone for diagnosis

e.g., E. coli and Salmonella gram stains look alike


Gram stain

Differential stain

distinguishing betweengram-positive and

gram-negative bacteria

Narrows possible identities of an organism Excludes many possibilities

Generally insufficient alone for diagnosis

e.g., E. coli and Salmonella gram stains look alike


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Gram stain

Sometimes highly suggestive ofa particular microorganism

e.g., Gram-negative rods in urine E. coli UTI

e.g., Gram-positive encapsulateddiplococci and numerous white

blood cells in sputum Streptococcuspneumoniae

Sometimes enough for complete diagnosis

e.g., Gram-negative diplococci clustered in white bloodcells of male urethral secretions Neisseria gonorrhoeae


Gram stain

Sometimes highly suggestive ofa particular microorganism

e.g., Gram-negative rods in urine E. coli UTI

e.g., Gram-positive encapsulateddiplococci and numerous white

blood cells in sputum Streptococcuspneumoniae

Sometimes enough for complete diagnosis

e.g., Gram-negative diplococci clustered in white bloodcells of male urethral secretions Neisseria gonorrhoeae


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Special stains

Some microbes have

unique characteristicsthat can be detectedwith special staining

procedures

e.g., Filobasidiella (Cryptococcus) neoformans isone of a few types of capsule-forming yeast

Capsule stain on cerebrospinal fluid is diagnosticfor cryptococcal meningitis


Special stains

Some microbes have

unique characteristicsthat can be detectedwith special staining

procedures

e.g., Filobasidiella (Cryptococcus) neoformans isone of a few types of capsule-forming yeast

Capsule stain on cerebrospinal fluid is diagnosticfor cryptococcal meningitis


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Special stains

Some microbes have unique characteristics that

can be detected with special staining procedures e.g., Mycobacterium

species possess cellwalls with a high lipid

content Acid-fast stain on

sputum is diagnosticfor tuberculosis


Special stains

Some microbes have unique characteristics that

can be detected with special staining procedures e.g., Mycobacterium

species possess cellwalls with a high lipid

content Acid-fast stain on

sputum is diagnosticfor tuberculosis


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Metabolic differences

Colony morphology

Colonies can exhibit

macroscopicdifferences

e.g., Colonies of streptococci generally form fairly

small colonies e.g., Colonies ofSerratia marcescensproduce a

pigment and are often red when incubated at 22oC

e.g., Colonies ofPseudomonas aeruginosa often

produce a soluble greenish pigment


Colony morphology

Colonies can exhibit

macroscopicdifferences

e.g., Colonies of streptococci generally form fairly

small colonies e.g., Colonies ofSerratia marcescensproduce a

pigment and are often red when incubated at 22oC

e.g., Colonies ofPseudomonas aeruginosa often

produce a soluble greenish pigment


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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



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


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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



MacConkeyagar 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


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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-causingStreptococcuspyogenes causes

beta-hemolysis

Clear zone around colonies



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-causingStreptococcuspyogenes causes

beta-hemolysis

Clear zone around colonies


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Media lacking nitrogen can be used to detect

nitrogen-fixingbacteria

e.g., Azotobacter

can be identifiedfrom soil samples

incubated

aerobically on

such media



Media lacking nitrogen can be used to detect

nitrogen-fixingbacteria

e.g., Azotobacter

can be identifiedfrom soil samples

incubated

aerobically on

such media


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Biochemical tests

Generally necessary for more conclusive

identification

Most rely on pH indicator or color change when

a compound is degraded


Biochemical tests

Generally necessary for more conclusive

identification

Most rely on pH indicator or color change when

a compound is degraded


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Biochemical tests

Sugar fermentation

e.g., Lactose, sucrose,glucose, etc.

Fermentation results inacid production

pH indicator changes color Pink yellow

Inverted tube (Durham tube) collects any gasproduced


Biochemical tests

Sugar fermentation

e.g., Lactose, sucrose,glucose, etc.

Fermentation results inacid production

pH indicator changes color Pink yellow

Inverted tube (Durham tube) collects any gasproduced


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Biochemical tests

Urease detection

Enzyme degrading urea

Urea CO2 & NH3

pH indicator turns

bright pink in alkalineconditions

Helicobacter pylori can be detected using a breath

test


Biochemical tests

Urease detection

Enzyme degrading urea

Urea CO2 & NH3

pH indicator turns

bright pink in alkalineconditions

Helicobacter pylori can be detected using a breath

test


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Biochemical tests

Urease detection

Helicobacterpylori can be detected using a breathtest

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

Urease detection

Helicobacterpylori can be detected using a breathtest

Causative agent of most stomach ulcers

Culturing not necessary Patient drinks solution containing 14C-labeled urea

14C in expired are indicates presence of urease


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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 Streptococcuspyogenes


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 Streptococcuspyogenes


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Organisms are identifiedusing a dichotomous key

Multiple biochemical and

other tests are typicallyrequired

Multiple tests are

generally run concurrently

Avoids waiting forincubation time for each

test

Organisms are identifiedusing a dichotomous key

Multiple biochemical and

other tests are typicallyrequired

Multiple tests are

generally run concurrently

Avoids waiting forincubation time for each

test


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Biochemical tests

Commercial modifications of traditional

biochemical tests e.g., APITM system, EnterotubeTM


Biochemical tests

Commercial modifications of traditional

biochemical tests e.g., APITM system, EnterotubeTM


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Serology

Proteins and polysaccharides of some bacteriacan function as identifying markers

Generally molecules on surface structures e.g., Cell wall, glycocalyx, flagella, pili

Detection is based upon thespecific interaction between

antibodies and theseantigens

e.g., Rapid detection ofStreptococcuspyogenes

Serology

Proteins and polysaccharides of some bacteriacan function as identifying markers

Generally molecules on surface structures e.g., Cell wall, glycocalyx, flagella, pili

Detection is based upon thespecific interaction between

antibodies and theseantigens

e.g., Rapid detection ofStreptococcuspyogenes


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Fatty Acid Analysis

Bacteria differ in the type and relative quantity

of fatty acids that comprise their membranes

Can function as an identifying marker Gram-negative bacteria possess fatty acids in both

of their membranes

Gram-positive bacteria possess only a singlemembrane

FattyAcid Analysis

Bacteria differ in the type and relative quantity

of fatty acids that comprise their membranes

Can function as an identifying marker Gram-negative bacteria possess fatty acids in both

of their membranes

Gram-positive bacteria possess only a singlemembrane


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Fatty Acid Analysis

Cells are treated with NaOH and methanol

Fatty acids are released and converted into

methyl esters Methyl esters

analyzed via gas

chromatography Profile compared

to those of known

species

FattyAcid Analysis

Cells are treated with NaOH and methanol

Fatty acids are released and converted into

methyl esters Methyl esters

analyzed via gas

chromatography Profile compared

to those of known

species


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GENOTYPIC CHARACTERISTICSGENOTYPIC CHARACTERISTICS

Nucleic acid probes

Used to locate unique sequences

Single-stranded DNA (or RNA)

Generally labeled (radioactive or fluorescent)

Complementary to the sequence of interest

Observe and identify intact microorganisms

Fluorescence in situ hybridization (FISH)

Observe and identify samples

Generally preceded by DNA amplification

Nucleic acid probes

Used to locate unique sequences

Single-stranded DNA (or RNA)

Generally labeled (radioactive or fluorescent)

Complementary to the sequence of interest

Observe and identify intact microorganisms

Fluorescence in situ hybridization (FISH)

Observe and identify samples

Generally preceded by DNA amplification


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Polymerase chain reaction

Amplifies specific nucleotide sequences

DNA can be obtained from many sources

e.g., Body fluids, soil, food, water, etc.

Useful in detecting microbes present in extremely

small numbers

Useful in detecting microbes that are difficult toculture

Amplified DNA can be analyzed


Amplifies specific nucleotide sequences

DNA can be obtained from many sources

e.g., Body fluids, soil, food, water, etc.

Useful in detecting microbes present in extremely

small numbers

Useful in detecting microbes that are difficult toculture

Amplified DNA can be analyzed


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Procedure

DNA is isolated, then

denatured Complementary primers

are lengthened

DNA is doubled Repeat ~30 times


Procedure

DNA is isolated, then

denatured Complementary primers

are lengthened

DNA is doubled Repeat ~30 times


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Procedure

DNA is isolated, then denatured

Complementary primers are lengthened

DNA is

doubled

Repeat~30 times


Procedure

DNA is isolated, then denatured

Complementary primers are lengthened

DNA is

doubled

Repeat~30 times


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Sequencing ribosomal RNA genes

Three rRNAs present in 70S bacterial ribosomes

5S, 16S, and 23S

Evolutionarily highlyconserved genes

Variable regions are usedto identify an organism

Particularly useful inidentifying microbes thatare difficult to culture

Sequencing ribosomal RNA genes

Three rRNAs present in 70S bacterial ribosomes

5S, 16S, and 23S

Evolutionarily highlyconserved genes

Variable regions are usedto identify an organism

Particularly useful inidentifying microbes thatare difficult to culture


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Bacterial Identification - Copy (2)

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