DR.THAMINA SAYYED
REGISTRAR
MICROBIOLOGY
KKUH

 3 Domains 1978 Carl Woese
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1. Bacteria
• Unicellular prokaryotes with cell wall containing peptidoglycan
2. Archaea
• Unicellular prokaryotes with no peptodoglycan in cell wall
3. Eukarya
• Protista
• Fungi
• Plantae
• Animalia

Comparing Prokaryotic and Eukaryotic Cells

Taxonomic Classification Categories

 arranged in hierarchical order species is basic unit
Domain
Kingdom
Phylum or Division
Class
Order
Family
Genus
Species

Prokaryote Classification
 Technologies used to characterize
 and ID prokaryotes microscopic examination
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 culture characteristics biochemical testing nucleic acid analysis combination of the above is most accurate

Phenotypic & Genotypic classification

Phenotypic Characteristics for Identifying
Prokaryotes
 often does not require sophisticated equipment
 can easily be done anywhere


Microscopic Phenotypic Exam size and shape and arrangement
 enough information for diagnosis of certain infections
 Gram stain

 distinguishes between
Gram + and Gram – bacteria narrows the possibilities quickly

Microscopic Phenotypic Exam
 special stain
 allows for the distinction of microorganisms with unique characteristics
• capsule
• acid fast staining detects the waxy presence of
Mycobacterium tuberculosis
Capsule staining
Acid fast staining of
M. tuberculosis

Gram positive cell wall
 Consists of
 a thick, homogenous sheath of peptidoglycan
20-80 nm thick
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 tightly bound acidic polysaccharides, including teichoic acid and lipoteichoic acid cell membrane
 Retain crystal violet and stain purple
Gram negative cell wall
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Consists of
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 an outer membrane containing lipopolysaccharide (LPS) thin shell of peptidoglycan periplasmic space inner membrane
Lose crystal violet and stain pink from safranin counterstain
11

Gram Positive Gram Negative
12

The Gram Stain
Crystal violet
Gram's iodine
Counterstain with e.g. methyl red
Decolorise with acetone
Gram-positives appear purple
13 appear pink

Gram-positive cocci
Gram-positive rods
Gram-negative cocci
Gram-negative rods
15

Metabolic Phenotypic Exam
 cultural approaches
 required for positive diagnosis of infection
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 isolation and ID of pathogen accuracy, reliability, and speed
 methods used include

 culture characteristics biochemical reactions process

Serological Testing
Phenotypic Exam
 serological testing uses ELISA testing
 fast and easy to use

Classification of bacteria

Classification of medically significant bacteria
 I.Thick rigid walled cells
A. Free living extracellular
1.Gram positive a.Cocci
Staphylococcus - abcess
Streptococcus - puemonia,
Pharyngitis cellulitis b.Spore forming rods
Aerobic Bacillus - Anthrax
Anaerobic Clostridium - tetanus,gas gangrene botulism

c.Non spore forming rods (
GRAM POSTIVE CONTD)
1-Non filamentous Cornybacterium – Diphtheria
Listeria - meningitis
2.Filamentous Actinomycetes – Actinomycosis
Nocardia - Nocardiosis

 2.Gram negative
A.Cocci Neisseria -Gonorrhoea, meningitis
B.Rods
1.Facultative
a. Straight
1.Respiratory org. Haemophillus- meningitis
Bordatella-Whooping cough
Legionella- Pneumonia
2.
Zoonotic Brucella – Brucallosis
Francisella – Tularemia
Pasteurella –Cellulitis
Yersinia - Plague

 3.enteric & related (
GRAM NEGATIVE CONTD )
E.coli - UTI,Diarrhoea
Enterobacter – UTI
Serratia – Pneumonia
Klebsiella – Pneumonia.UTI
Salmonella – enterocolitis,typhoid fever
Shigella – Enterocolitis
Proteus – UTI b. Curved
Campylobacter – Entericolitis helicobacter – Gastritis,Peptic ulcer
Vibrio - Cholera

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(Gram negative)
C.Aerobic Pseudomonas – pneumonia,UTI
D. Anaerobic Bacteroids – peritonitis
3.ACID FAST
MYCOBACTERIUM Tuberculosis & Leprosy

 B . Non free living obligate intracellular parasites
1.Rickettsia
– Rocky mountain spotted fever
Typhus, Q fever
2.Chlamydia urethritis, trachoma. Psittacosis

Flexible thin walled
Spirochaetes Treponema – Syphilis
Borrelia – Lyme disease
Leptospira - leptospirosis
Wall- less cells
Mycoplasma pneumonia

Subtyping & Its applications
 To distinguishinguish between strains of different species
 Biotyping
 Serotyping
 Antimicrobial susceptibility system
 Bacteriophage typing
 Bacteriocin typing

Genotypic Characteristics for Identifying
Prokaryotes
 the use of genotypic testing has increased with the availability of technology
 genotypic testing is particularly useful in the case of organisms that are difficult to identify
 several techniques include
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 gene probes
PCR sequencing rRNA

 gene probes

 single stranded DNA that has been labeled with a identifiable tag, such as a fluorescent dye are complementary to target nucleotide sequences
• unique in DNA of pathogen


Genotypic Characteristics used in
Classifying Prokaryotes( non culture methods)
PCR: polymerase chain reaction
 used to detect small amounts of DNA present in a sample (blood, food, soil)
 the PCR chain reaction is used to amplify the amount of DNA present
 sequencing ribosomal RNA
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 of particular use for identifying prokaryotes impossible to grow in a culture focus is place on the 16S molecules of the RNA because of it’s size
• approximately 1500 nucleotides once the 16S molecule is sequenced, it can then be compared to the sequences of known organisms

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Genotypic Characteristics used in
Classifying Prokaryotes comparison of nucleotide sequences
 differences in DNA sequence can assist in determination of divergence of evolutionary path for organisms
DNA hybridization
 single strands of DNA anneal
16S ribonucleic acid
 comparing sequence of ribosomal RNA relatedness to other organisms can be determined using numerical taxonomy
 determined by the percentage of characteristics two organisms have in common