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.
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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

Single, circular

multiple

No nuclear
membrane or nucleoli

Membrane+
Nucleoli +

Ribosomes

70S

80S

Replication

Binary fission

mitosis

Chromosome
Nucleus
Membrane-bound
organelles

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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, (1 m =
10-6 meter, 10-3 millimeter) : E.coli
Range 0.2 - 5
Smallest - Mycoplasma
All true bacteria can be seen by OLM except
Treponema species and Leptospira species ... Dark
ground microscope

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September 29, 2014

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
o Neisseria

: G(+) diplococci
: 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

structures
a. Cell wall
b. Cell membrane
c. Cytoplasm
d. Ribosome - RNA
e. Nuclear body - DNA

Additional

structures

a. Intracellular
Plasmid
Inclusion granules
Transposons
b. Extracellular
Capsule & glycocalyx
Flagella
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
Peptidoglycan

Gram-positive
Gram-negative
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
structure
Cell wall

Function
Peptidoglycan

Rigid support, protects against osmotic

pressure

Lipid A

Toxic component of endotoxin

Polysaccharides
& teichoic acid

Antigen

Plasma
membrane

Phospholipid
bilayer; sterol (-)

Active transport of molecules into the cell,

energy generation by oxidative
phosphorylation, synthesis of precursors of
the cell wall, secretion of enzymes and
toxins

Ribosomes
(RNA)

70S in size, with

50S and 30S
subunits

Protein synthesis, site f action of antibiotics:

aminoglycoside, erythromycin, tetracycline
and chloramphenicol

Nucleoid
(DNA)

Single, circular
2000 genes
No introns

Genetic material
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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
components

Functions

Capsule

Protect against phagocytosis

Flagella

Motility, attachment

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

Glycocalyx
(slime layer)

Adherence to surfaces

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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September 29, 2014

Classification of Medically Important Bacteria

Characteristics

Genus

1. Rigid thick-walled cells

A. Free living (Extracellular bacteria)
Cocci

Staphylococcus
Streptococcus

Bacilli

Gram-positive

Spore +
Spore -

aerobic

Bacillus

anaerobic

Clostridium

Non-filamen

Corynebacterium

tous

Listeria

Filamentous

Actinomyces
Nocardia

Cocci
Bacilli

Neisseria
Facultative

Straight

Escherichia
Salmonella
Shigella
Klebsiella
Proteus
Haemophilus
Bordetella
Legionella
Yersinia

Gram-negative

Curved

Vibrio
Helicobacter

Aerobic

Pseudomonas

Anaerobic

Bacetroides

Acid fast
B. Obligate intracellular parasites

Mycobacterium
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

Simple stain

Differential
stain

Special stain

Results

Uses

Crystal
Uniform violet
violet
Methylene Uniform blue
blue
Gram
G+ violet
G - pink

Reveal size, morphology,

arrangement of cell

Ziehl
Neelsen

AFB - pink
Non-acid fast
blue/green

Differentiate
mycobacteria from other
bacteria

Negative
stain

Background Reveals bacterial capsule

dark, cells
stained
Flagella - visible Determine number and
location ofSeptember
flagella29, 2014

Flagella
stain

Differentiate G + from G
bacteria

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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-negative cocci

Gram-positive rods

Gram-negative rods

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Medically important bacteria that

cannot be seen in the Gram stain
Alternative microscpic
approach

Organisms

Reasons

Mycobacterium

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

wall so dye cannot
penetrate
Too thin to see
DGI

Treponema
Leptospira
Mycoplasma

Wall-less

Chlamydiae

Intracellular,very
small

Inclusion bodies in
cytoplasm

Rickettsiae

Intracellular

Giemsa stain
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Ziehl-Neelsen stain (Acid fast

stain)

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Microscopes
1. Brightfield 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

September
2014
A= bright field, B=
dark29,ield)

Dark-Ground Microscopy
Treponema pallidum &
syphilis

Uses

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

September 29, 2014

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
Immunofluorescence :

Auramine stained TB bacilli

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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 (37C) 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

September 29, 2014

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.
September 29, 2014

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