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Structures and

Identification of Bacteria

DR. THU ZAR HAN


ASSOCIATE PROFESSOR

September 29, 2014


Contents

 Scope and relevance of microbiology


 Microbial cell types with focus on the different bacterial cell
structures and functions (Prokaryotes and Eukaryotes)
 Medical importance of the differences between Mammalian
cell and Microbial cells, (Bacterial in particular)

 Classification of medically important bacteria based on


morphology, staining characteristics, oxygen requirement for
culture, DNA

 Methods of bacterial identification such as microscopy (types


and uses), staining methods, culture, biochemical and other
methods. September 29, 2014
Learning Outcomes

The student will be able to


1. classify microbes.
2. describe the features of different microbial cell
types .
3. compare and contrast structures and functions of a
bacterial cell with those of a mammalian cell.
4. Outline the methods and the principles involved in
the identification of bacteria (microorganism).

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What is Microbiology?

Microorganisms : 5 major groups


1. Bacterium / bacteria
2. Virus / viruses
3. Fungus / fungi
4. Protozoon/ protozoa
5. Helminth/s (worms)
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Biologic relationships of
pathogenic microorganisms
Type of cells Organisms

Prokaryotes Bacteria (Eubacteria)

Fungi
Eukaryotes
Protozoa

Helminths

Not cell Viruses

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Characteristics Prokaryotes Eukaryotes

Cell wall containing + -


peptidoglycan
Chromosome Single, circular multiple

Nucleus No nuclear Membrane+


membrane or nucleoli Nucleoli +
Membrane-bound _ +
organelles
Ribosomes 70S 80S

Replication Binary fission mitosis


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Prokaryotic vs. Eukaryotic cells

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Nomenclature of microorganisms

 Bacteria, fungi, protozoa and helminths named


according to the binomial Linnean system using
genus and species : scientific name
 Escherichia coli (E.coli) = E.coli
 Candida albicans (C.albicans)
 Entamoeba histolytica (E.histolytica)
 Ascaris lumbricoides (A.lumbricoides)

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Bacteria
 Single cell microbes
 Some - free living
 Some - obligate intracellular (Rickettsia,
Chlamydia)
 possess rigid cell wall - contain muramic acid
except Mycoplasma (wall-less bacteria)
 Nuclear apparatus – DNA
 Typical vs atypical (Mycoplasma, Chlamydia,
Rickettsia
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The Size, Shape & Arrangement of Bacteria

Size of Bacteria
 Average size of bacteria is 1 micrometer, μ (1 μm =
10-6 meter, 10-3 millimeter) : E.coli
 Range – 0.2 μ- 5 μ
 Smallest - Mycoplasma

All true bacteria can be seen by OLM except


Treponema species and Leptospira species ... Dark
ground microscope

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The Shape of Bacteria

Characteristic shape of bacterial species is maintained


by their rigid cell wall
• coccus (cocci) : spherical or oval
• bacillus (bacilli) : rod shape
• coccobacilli : bacilli which are so short
that they look like cocci
• vibrio : comma shape
• spirillum : non-flexous,spiral bacteria
• spirochaete : flexous, spiral bacteria
3 genera - Borrelia, Treponema, Leptospira
• actinomyces : filamentous bacteria with branching
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The Arrangement of Bacteria

Characteristic arrangement --- diagnostic value

 Cocci
o Staphylococcus : G (+) cocci, grape like clusters

o Streptococcus : G(+) cocci in chains

o Pneumococci : G(+) diplococci


o Neisseria : G(-) diplococci

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Bacilli
o Mycobacterium – AFB

o Corynebacterium diphtheriae : GPB with chinese


letter arrangement, metachromatic granules

o Yersinia pestis – GN coccobacilli with bipolar


staining, safety pin appearance

o Vibrio cholerae – GN comma shaped bacteria

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Shape of the bacteria

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Arrangement of the Bacteria

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A bacterial cell

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Essential Additional
structures structures

a. Cell wall a. Intracellular


• Plasmid
b. Cell membrane • Inclusion granules
• Transposons
c. Cytoplasm
b. Extracellular
d. Ribosome - RNA • Capsule & glycocalyx
• Flagella
e. Nuclear body - DNA • Fimbriae (pili)
• Bacterial spore

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1. Essential structures of bacteria

a. Cell wall – thick and multilayered


 Peptidoglycan (murein or mucopeptide) : peptide + glycan
(sugar); only found in bacterial cell wall:
maintain shape
CHO backbone (glycan)- alternating N-acetyl muramic acid
(NAM)and N-acetyl glucosamine (NAG)
Thicker and multilayer in Gram-positive,
thinner and single layer in gram-negative cell

This differences are reflected in their Gram reactivity


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Techoic acid and lipotechoic acid in GPB
Outer membrane in GNB
outer leaflet - LPS = lipid A + O polysaccharide
inner leaflet - phospholipid + protein
Porins – channels through both leaflets, eg. glucose move
Periplasmic space – between outer membrane
and cytoplasmic membrane
Target for antibacterial drugs : Penicillin, cephalosporin,
vancomycin
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Differences
Gram-positive Gram-negative
Peptidoglycan Thick and multiple Thin

Teichoic acid + -
Outer membrane - +

LPS - +
Lipid A - +
O Ag - +
Porin protein - +
Periplasmic space - +
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b. Cell (Cytoplasmic, Plasma) membrane

 Unit membrane - phospholipid bilayer, proteins, no


cholesterol in bacterial cell membrane
 Bipolar – hydrophilic (phosphate) head , hydrophobic tail
 is responsible
- for selective permeability and transport of
molecules into the cell
- energy generation by oxidative phophorylation
- synthesis of precursors of cell wall
- secretion of enzymes and toxins
 site of action of certain antibiotics such as polymyxin
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c .Cytoplasm

d . Bacterial Ribosomes - RNA


 sedimentation constant of 70s 30s
50s
 tens of thousands present in the bacterial cell
 ribosomes are strung together on strands of mRNA
to form polysomes- the site where mRNA is
translated in protein synthesis
 Aminoglycosides, Tetracyclines, Chloramphenicol &
Erythromycin interfere with protein synthesis at
ribosomal level
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e. Bacterial nucleus/nucleoid
single circular molecule of double stranded DNA

no nuclear membrane, no nucleolus, no spindle

contains a single chromosome ( DNA )- 1000 ~ 3000


genes

replication - binary fission (not by mitosis)

after replication -single molecule of DNA passed into


each daughter cell
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 Binary fission

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Essential Function
structure
Cell wall Peptidoglycan Rigid support, protects against osmotic
pressure
Lipid A Toxic component of endotoxin
Polysaccharides Antigen
& teichoic acid

Plasma Phospholipid Active transport of molecules into the cell,


membrane bilayer; sterol (-) energy generation by oxidative
phosphorylation, synthesis of precursors of
the cell wall, secretion of enzymes and
toxins
Ribosomes 70S in size, with Protein synthesis, site f action of antibiotics:
(RNA) 50S and 30S aminoglycoside, erythromycin, tetracycline
subunits and chloramphenicol
Nucleoid Single, circular Genetic material
(DNA) 2000 genes
No introns September 29, 2014
2. Additional Structures
2a. Intracellular
i . Bacterial Plasmid
o ds DNA inside cytoplasm (extra chromosomal)
o replicates autonomously ; persists for many
generations
o one copy is passed to each daughter cell
o contains genes that confer protective properties
- antibiotic resistance
- virulence factor (toxin, enzymes)
o Plasmid DNA/ gene transferred to other bacteria by
conjugation
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Conjugation : DNA transfer

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ii. Transposons
pieces of DNA move readily from one site to another
either within or between the DNAs of bacteria, plasmids
& bacteriophages (jumping gene)
Not capable of independent replication
can code for drug resistance enzymes, toxins

ii. Inclusion Granules


 nutrient reserves
 volutin granules, lipid granules, polysaccharide granules
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2b. Extracellular

i. Glycocalyx / Glycocalyces

 Gelatinous sticky substances made up of


polysaccharide, polypeptides that surround the
outside of bacteria

 slime layer : loose water soluble glycocalyx

 associated with adhesive properties of bacterial


cells, protect from desiccation

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ii Capsule
 composed of repeating subunits of glycocalyces that firmly
attached to the cell surface
 Antigens : polysaccharide, exception - capsule of Bacillus
anthracis - polyD glutamic acid
 Identification of capsular materials by specific antisera
 protects the bacteria from phagocytosis
Eg. Streptococcus pneumoniae
Bacillus anthracis
Klebsiella pneumoniae
 Capsular polysaccharides used as antigens in vaccines
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iii. Flagella / flagellum

 whip like appendages for locomotion


 Made up of proteins : flagellin -
antigens
 Polar (ends)
 peritrichous (surface of the cell)
Eg. Escherichia coli
Pseudomonas aeruginosa
Vibrio cholerae

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 Endoflagella / axial
filament :
spirochetes have
flagella at both ends
that spirally tightly
around the cell,
instead of
protruding into the
surrounding medium

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iv. Pili (Fimbriae)

 Sticky, hair like extend from cell surface composed mainly


of a protein – pilin

 two types
 oridinary pili for adhesion
 sex pili for attachment of donor & recipient bacteria in
plasmid mediated conjugation

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v. Bacterial Spores

 Formed in response to adverse conditions


 Spore formation – sporulation occurs when
nutrients are depleted
 Formed inside cell - contains DNA, small amount
of cytoplasm, LPS, cytoplasmic membrane, very
little amount of water and keratin coat ----
 Coat remarkable resistance to heat, radiation,
chemicals (medical importance)
 Sterilization achieved by autoclaving
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Spore formation

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 endospore - formed inside the vegetative cell
 free spore - without the vegetative portion

 helpful in identifying some species of bacteria


 Seen as colorless areas in cells stained by conventional
methods – gram stain
 weakly acid-fast
 commonly stained with malachite green or carbol fuchsin
 shape : spherical, oval
position : terminal, sub terminal, central
not buldging -narrower than the cell or
broader & bulging
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Spores

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Nonessential Functions
components
Capsule Protect against phagocytosis

Flagella Motility, attachment

Fimbriae or pili Attachment to cell surface, sex pilus (conjugation)

Glycocalyx Adherence to surfaces


(slime layer)
Spores Resist heat, dehydration and chemicals

Plasmid Contains a variety of genes for antibiotic resistance and


(extracellular ds toxins, enzymes, Transmissible/ non-transmissible
circular DNA)

Granules Nutrients in the cytoplasm

Transposons A piece of DNA can code for drug resistant enzymes,


toxins
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Classification of Medically Important Bacteria

 Based on morphology and biochemical


characteristics:
nature of cell wall, staining characteristics,
shape, ability to grow in the presence or
absence of oxygen, ability to form spores

 Base sequence of genomic DNA

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Classification of Medically Important Bacteria

Characteristics Genus

1. Rigid thick-walled cells

A. Free living (Extracellular bacteria)


Cocci Staphylococcus
Streptococcus
Bacilli Spore + aerobic Bacillus
anaerobic Clostridium
Gram-positive
Spore - Non-filamen Corynebacterium
tous Listeria
Filamentous Actinomyces
Nocardia
Cocci Neisseria
Bacilli Facultative Straight Escherichia
Salmonella
Shigella
Klebsiella
Proteus
Haemophilus
Gram-negative
Bordetella
Legionella
Yersinia
Curved Vibrio
Helicobacter
Aerobic Pseudomonas
Anaerobic Bacetroides
Acid fast Mycobacterium
B. Obligate intracellular parasites Rickettsia
Chlamydia
2. Flexible, thin-walled cells (Spirochaetes) Treponema
Borrelia
Leptospira
3. Wall-less cells Mycoplasma

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General approach to the diagnosis of
bacterial infection
Obtain a specimen from the infected site: pus, urine, stool, sputum,
CSF, blood
1. Microscopic examination (stained, unstained)
2. Culture the specimen on the appropriate bacteriological culture
media
Identification of organism by
 Biochemical tests (eg. sugar fermentation)

 Serological tests

 Perform antibiotic susceptibility test (C & S)

3. Antigen/antibody detection
4. Nucleic acid detection - DNA probe
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1. Staining and Microscopy

Stained slides are examined under microscopes to see


the morphology such as size, shape, arrangement,
staining reaction
Gram stain : Gram positive or negative
ZN stain : Acid-fast or non acid fast

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Type of stain Example Results Uses

Simple stain Crystal Uniform violet Reveal size, morphology,


violet arrangement of cell
Methylene Uniform blue
blue
Differential Gram G+ violet Differentiate G + from G
stain G - pink – bacteria

Ziehl AFB - pink Differentiate


Neelsen Non-acid fast – mycobacteria from other
blue/green bacteria
Special stain Negative Background - Reveals bacterial capsule
stain dark, cells
stained
Flagella Flagella - visible Determine number and
stain location ofSeptember
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Gram stain

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Gram stain procedure

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Gram stain ----classify bacteria

Gram-positive cocci Gram-positive rods

Gram-negative cocci Gram-negative rods

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Medically important bacteria that
cannot be seen in the Gram stain
Alternative microscpic
Organisms Reasons approach

Mycobacterium Too much lipid in cell Acid-fast stain/ ZN stain


wall so dye cannot
penetrate
Treponema Too thin to see DGI
Leptospira
Mycoplasma Wall-less -
Chlamydiae Intracellular,very Inclusion bodies in
small cytoplasm
Rickettsiae Intracellular Giemsa stain
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Ziehl-Neelsen stain (Acid fast
stain)

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Microscopes

1. Bright–field microscope:
Ordinary light microscope
2. Dark field microscope
3. Phase contrast microscope
4. Fluorescence microscope
5. Electron microscope:
TEM, SEM

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1. Bright field microscope

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Uses of OLM
Bacteriology
- to study morphology & structure of bacteria
- to find out the motility of the bacteria (live bacteria)
Entomology
to study insects: mosquitoes, flies, fleas, lice and mites
Immunology
to confirm agglutination and precipitation reactions
Parasitology
Protozoa ---- to identify trophozoites & cysts in
stool, protozoa in blood and tissues
worms ---- to identify ova, eggs and larvae, segments
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2. Dark Ground Microscopy

- special condenser - blocks


light rays directly passing
thro’ specimen
- Rays from sides of the
specimen ---entering the
objective lens
- The object, eg. bacteria,
appear brightly illuminated
against a dark background

A= bright field, B= dark29,ield)


September 2014
Dark-Ground Microscopy
Uses
Treponema pallidum &
syphilis

- Treponema pallidum
in syphilis
- Leptospira interrogans
in leptospirosis
-live organisms, motility

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3. Phase contrast Microscopy
 Uses a special optical system
 Contrast between cell and surrounding medium
 Can see living cells without staining
Uses:
- For studying internal details of living cells
- identify cilia and flagella
- the cytopathic effect of viruses on cell cultures

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4. Fluorescence Microscopy

- Use UV light (shorter


wave length than
visible light)
- Staining of cells or
bacteria with a
fluorescent dye
(auramine O, acridine
orange R, thioflavin S)
- bright objects against a
dark background Auramine stained TB bacilli
 Immunofluorescence :
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5. Electron Microscopy

 Uses a beam of electrons


 Wavelength of electrons – 100,000 times shorter than
that of ordinary light
 High resolution and great magnification
 Limit of resolution = 0.3 – 0.5 nm
 2 types:-
- transmission electron microscope (TEM)
- scanning electron microscope (SEM)

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2. Culture
Culture: cultivation of microorganisms
Culture media : nutrient preparation used for cultivation
of microorganisms
2 forms : agar and broth (fluid)
Inoculating specimen onto the medium inside the
Petridish
Incubating for appropriate temperature (37°C) for
overnight
Colonies : including texture, size, shape, pigment, speed of
growth,
Identification of organism by different biochemical tests
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Culture

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3a. Biochemical Tests

 Tests for Metabolism of carbohydrates and related


compounds (sugar fermentation)

 Tests for metabolism of proteins and amino acids

 Test for metabolism of lipids

 Tests for enzymes (coagulase, catalase, oxidase)

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Sugar fermentation
Acid and gas production

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Catalase test

Tube coagulase Test.


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3b. Serological Analysis
 Testing bacterial cultures with antibodies that are
known to be specific for a given species or genus of
bacterium.
 Serotypes

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4a. Antigen detection

 Identification of antigens of an organism an be used in


the diagnosis of specific infection
 known antisera used
Examples:
1. Enzyme Immunoassys (EIA) enzyme liked
immunosorbant assays (ELISA)
2. Latex agglutination test

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4b. Antibody detection

 Identification of serum antibodies with known antigen


 Examples
Serologic tests for syphilis
Widal test for typhoid fever

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5. Genetic (nucleic acid) Analysis

 Nucleic acid amplification test (PCR)


 Nucleic acid probe test
 Nucleic acid sequence analysis
highly specific, quite sensitive, much faster than
culture
Useful for the bacteria that are difficult to culture eg.
Chlamydia, Mycobacterium

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Methods and Principles of
Identification of Bacteria

1. Staining and microscopy


2. Culture
3. Biochemical and Serological identification
4. Antigen/antibody detection
5. Nucleic acid analysis

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References
1. Brooks, G.F., Carroll, K.C., Butel, J.S., Morse, S.A. and
Meitzner, T.A. (2013). Jawetz, Melnick and Adelberg's medical
microbiology. (26th ed.).United States of America: The
McGrew Hill Companies.

2. Levinson, W. (2012). Review of medical microbiology and


immunology. (12th ed.). United States of America: The
McGrew Hill Companies.

3. Harvey AH, Cornelissen CN and Fisher BD. (2013)


Microbiology, Lippincott Illustrated Reviews . (3ed ed.).
Lippincott Williams & Wilkins.
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Assignment

 Two( you tube ) videos uploaded in LMS


 Isolation of Bacteria & Identification of bacteria
 Enroll :Access code GNMC-DLRY
 Due : Oct 5 Sunday
 Home work

September 29, 2014