CLINICAL MICROBIOLOGY

PHT 416

Lecture # 1
 Objectives

1 - D ESC RIB E SO ME O F TH E D EST R UC TI VE A N D

B E N E FI C I A L AC TI ON S OF M I C RO B ES .

2 - RECO G N I ZE D I F FE RE N T TY P ES O F MI C RO B ES .

3 - I D E N TI F Y BAC TE R I A L STR U C TU R E .
 Introduction
4
Microbiology
 The science deals with the study of
microorganisms (M.O)
Microorganism
 Living organism that can not seen by naked eye
 Examples: bacteria, fungi, viruses, protozoa
Medical/Clinical microbiology
 The science deals with the study of M.O.
causing infection/disease
Prokaryotic & Eukaryotic Cells
 Cellular Evolution

Two theories:
1. Infolding theory
2. Endosymbiotic theory
History of microbiology
Bacteria
 Assigning Specific Names

 The binomial system of nomenclature

 The genera (genus) name followed by the species name

 Generic part is capitalized, species is lowercase

 Both are italicized or underlined if italics aren’t available

 Are “Latinized” and used worldwide.

 May be descriptive or honor a scientist.

Staphylococcus aureus
 Scientific Names

 1st into broad groups based on microscopic appearance, then

divided into species based on a range of different properties-often
biochemical reactions e.g. some may be able to metabolise a
sugar that others cannot.

Staphylococcus aureus
 Describes the clustered arrangement of the cells (staphylo-) and the
golden color of the colonies (aur-).

Escherichia coli
 Honors the discoverer, Theodor Escherich, and describes the
bacterium’s habitat–the large intestine or colon.
 Standardized naming

 Genus
 Category of biologic
classification
 Example: Staphylococcus

 Species of organism
 Represents a distinct type
of microorganisms
 Examples: Staphylococcus
aureus
Staphylococcus epidermidis
 Shape and Arrangement

Shape determined by its rigid cell wall

Typical bacterial cells are
 Spherical (cocci; coccus)
 Straight rods (bacilli; bacillus)
 Helically curved Rods (spirilla; spirillum)
Some cells are “Pleomorphic”
Staphylococcus Lactobacillus
Aquaspirillum Pleomorphic Arthrobacter
 Characteristic arrangements of cocci
Diplococci:
 Cells divide in one plane and remain attached
predominantly in pairs.
Streptococci:
 Cells divide in one plane and remain attached to form
chains.
Tetracocci:
 Cells divide in two planes and characteristically form groups
of four cells.
Staphylococci:
 Cells divide in three planes, in an irregular pattern,
producing “bunches” of cocci.
Sarcinae:
 Cells divide in three planes, in a regular pattern, producing a
cuboidal arrangement of cells
Pleomorphic
Corynebacteria

Monomorphic E.
coli
 Tools of Microbiology
1.Compound light Microscope

- live specimens , 1,000 mag. or less

2. Electron Microscope
- non-living specimens, > 1,000 X mag.
3. Incubator – keep microbes warm for growth
4. Staining- different techniques and procedures depend on the
suspected organism itself and the site of isolation of the microbe.
5. Microbial Culture- solid or liquid, aerobic or anaerobic. Mainly food
for the bacteria to help it to grow in a similar ecologic environment.
 Bacterial Cell Structures
& Functions

Bacterial Cell Structure& Functions
 Structure
External Structures

located outside of cell wall

Flagellum /-a

Axial filaments

Fimbria /-ae or Pilus /-I

Glycocalyx
Slime layer
 FLAGELLA
 Singular: Flagellum
 A long, slender, helical appendage from the cell body, composed
of microtubules, protruding through the cell wall
 In prokaryotes, responsible for swimming motility to propel the
cell by beating in a whip-like motion
 0.01 to 0.02 μm in diameter
 Location on the cell varies depending on the bacterial species
 At polar region(s): At one or both ends of the bacterium
 At lateral regions: Along the sides of the bacterium
 Flagella proteins are H antigens (e.g., E. coli O157:H7)
 Flagella

Outside cell wall

Made of chains of
flagellin
Attached to a protein
hook
Anchored to the wall
and membrane by the
basal body
Flagella Arrangement
https://www.youtube.com/watch?v=PBrxh3N8
CuA
 PILI (FIMBRIAE)

Pilus: Hollow, non-helical, filamentous appendages

that are thinner, shorter & and more numerous than
flagella
They are composed of protein subunits (pilin).
Not all for motility
Found on non-motile as well as motile species
Different types of pili
 F pilus (or sex pilus): Serves as the port of entry of genetic
material during bacterial mating
 For allowing pathogenic bacteria to attach to epithelial cells
lining the respiratory, intestinal, or genitourinary tracts, in
human infection.
https://www.youtube.com/watch?v=jfzReyQ5lew

https://www.youtube.com/watch?v=jfzReyQ5lew
Cell wall structure
 CELL WALL

Surrounding the cytoplasmic membrane

Highly rigid structure giving shape to the cell
Prevents the cell from expanding and eventually bursting because
of uptake of water (osmotic lysis)
Consists of Peptidoglycan (murein)  polymer of 2
monosaccharide subunits
 N-acetylglucosamine (NAG) and
 N-acetylmuramic acid (NAM)
Linked by polypeptides (forming peptide cross bridges) with
tetrapeptide side chain attached to NAM
Fully permeable to ions, amino acid, and sugars.
The structure components and functions of the cell wall distinguish
Gram-positive from Gram-negative bacteria.
 Functions of Peptidoglycan

1.Essential for the structure, for replication,

and for survival in the hostile conditions.

2.Interfere with phagocytosis and has

pyrogenic activity (induces fever).
 Gram-negative bacteria

More complex than those of Gram-positive bacteria.

A 8 nm-thick outer membrane surrounds a thin underlying layer
of peptidoglycan.
Rich in lipids (11 to 22% of cell wall dry weight).
Consists three major parts.
 (1) Outer membrane-Unique
 (2) Periplasmic space
 (3) Cytoplasmic membrane
 Major Components
 - Lipopolysaccharide (LPS) (Endotoxin)
 - Lipoprotein
Gram-Negative Outer Membrane

Figure 4.13c
 Gram + Gram –
Cell Wall Cell Wall
Thin peptidoglycan
Thick layer of
peptidoglycan No teichoic acids
teichoic acid on surface Outer membrane
 LPS contains:
 O - polysaccaride
 Lipid A and core polysaccharide
 Gram Stain

Method of differentiating bacteria.

Can be either Gram +ve or Gram –ve depending on how
they appear with the stain.
Can then be further grouped based on shape (rod=long thin
or coccus=round).
Thus we end up with 4 combinations:
G+ rod, G+ coccus, G- rod, G- coccus
Bacterial cell wall
 Gram Stain

G+ve G-ve
STAIN the slide with
crystal violet for 1-2 min.
Flood slide with Gram's
iodine for 1-2 min.
Decolourise by washing
the slide briefly with
alchohol (2-3 seconds).
Stain with safranin
counterstain for 2 min.
View under microscope
 Gram Stain

Gives an initial idea of the possible identity of the organism.

Can be done without growing the organism (i.e. rapid result)
Thus can be done on pus, joint fluid, sputum, CSF

Relevance of Gram reaction.

Gram +ve and Gram –ve organisms are susceptible to
different groups of antibiotics.
Cause different diseases
Differ in their ability to survive in the environment-cleaning,
infection control, outbreak management.
Gram Positive Cocci

Clusters: usually
characteristic of
Staphylococcus spp., such
as S. aureus

Chain or pairs: usually

characteristic of
Streptococcus spp., such as
S. pneumoniae
Gram Positive Roads

 Thick : usually characteristic

of Clostridium spp., such as
C. perfringens, C. difficile,
C. tetani

 Thin: e.g. Listeria spp.

Gram Negative Cocci

 Diplococci: usually characteristic

of Niesseria spp., such as N.
meningitidis or N. gonorrhoea.
Though In addition, Moraxella
spp. and Acinetobacter spp.are
often diplococcal in morphology.

 Coccobacilli: usually characteristic

of Acinetobacter spp., which can
be either Gram-positive or Gram-
negative, and is often called Gram-
variable.
Gram Negative Roads

 Thin rods: usually

characteristic of
enterobacteriaceae
(coliforms), such as E. Coli

 Coccobacilli: usually
characteristic of Haemophilus
spp., such as H. influenzae
GNR

 Curved: usually
characteristic of Vibrio
spp.or Campylobacter spp.,
such as V. cholerae, C. jejuni

 Thin needle shape: usually

characteristic of
Fusobacterium spp.
 Gram Stain Mechanism

 Gram stain is a powerful, easy test that allows clinicians to distinguish between the
two major classes of bacteria and to initiate therapy.
 Bacteria heat-fixed stained with Crystal violet  this stain is precipitated with
Gram’s iodine  washing with the acetone- or alcohol-based decolorizer A
counterstain, safranin, red
 Gram-positive bacteria, Purple, the stain gets trapped in a thick, cross-linked,
meshlike structure.
Gram-positive
 Alcohol dehydrates peptidoglycan
 CV-I crystals do not leave
Gram-negative
 Alcohol dissolves outer membrane and leaves holes in peptidoglycan.
 CV-I washes out
 Gram + Gram -
Outer - +
membrane
Cell wall Thicker Thinner
LPS - +
Endotoxin - +
Teichoic acid Often present -
Sporulation + -
Lysozyme Sensitive Resistant
Penicillin Sensitive Resistant
Capsule Sometimes Sometimes
Exotoxin Some Some
 Bacteria with No Cell Wall: Mycoplasmas

Instead, have cell

membrane which
incorporates cholesterol
compounds (sterols),
similar to eukaryotic cells
Cannot be detected by
This EM shows some typically
typical light microscopy pleomorphic mycoplasmas, in this case
M. hominis
 Acid-fast Cell Walls
Genus Mycobacterium and
Nocardia
mycolic acid (waxy lipid)
covers thin peptidoglycan
layer
Do not stain well with
Gram stain  use acid-fast
stain
Damage to the cell wall
 Internal Structures
 All cells have 3 main components:
 DNA (‘nucleoid”)
 surrounding membrane
(“cytoplasmic membrane”)
 cytoplasm, between the DNA and
the membrane
 where all metabolic reactions
occur
 especially protein synthesis,
which occurs on the ribosomes
The plasma membrane
 Functions of
the cytoplasmic membrane

a. control permeability
b. transport electrons and protons for cellular metabolism
c. contain enzymes to synthesize and transport cell wall substance
and for metabolism
d. secret hydrolytic enzymes
e. regulate cell division.
f. Damage to the membrane by alcohols, quaternary ammonium
(detergents), and polymyxin antibiotics causes leakage of cell
contents.
 Transport Across the Cell Membrane
 Basic rule: things
spontaneously move from
high concentration to low
concentration (downhill).
This process is called
diffusion.
 Getting many molecules
into the cell is simply a
matter of opening up a
protein channel of the
proper size and shape. The
molecules then move into
the cell by diffusing down
the concentration gradient.
Passive transport, or
facilitated diffusion.
Passive transport
Active transport
Cytoplasm
 Genetic material
Bacterial chromosomes:
 a single large circular double stranded DNA (up to 3500 genes)in a
discrete area known as the nucleoid with no histone proteins. The
only proteins associated with the bacterial chromosomes are the ones
for DNA replication, transcription etc.
Plasmids:
 Smaller, circular, extrachromosomal DNAs (5-100 genes)
 Most commonly found in gram-negative bacteria
 Not essential for cellular survival
 Provide a selective advantage: many confer resistance to one or more
antibiotics.
 Promote conjugation ; transfer of genetic material between bacteria
through cell-to-cell contact.
 Ribosomes
The prokaryotic ribosome is 70S in size, being composed
of a 50S (large) subunit and a and 30S (small) subunit.
The eukaryotic ribosome is 80S in size and is composed of
a 60S and a 40S subunit.
 Inclusion bodies

Not separate by a membrane but distinct.

Granules of various kinds:
 glycogen
 Metachromatic granules:
Composed of polyphosphate.


 Serves as a reserve source of phosphate.

 Poly-β-hydroxybutyrate. (PHB):
In aerobic bacteria


 Serves as a reserve carbon and energy source.

 Gas vacuoles:
 Bacteria living in aquatic habitats
 To provide buoyancy.
 SPORES

 Spore mRNA are transcribed and other mRNA are

turned off. Dipicolinic acid(DPA) is produced.
 Spore structure:
 Core: one copy of DNA and cytoplasmic contents
 Inner membrane and Spore wall
 Cortex: peptidoglycan layer
 Coat: Keratine-like protein which protect
 the spore.
 Exosporum
 • Formation of spores = Sporulation

• Germination = spores returns to vegetative state

Location important in classification
 Central, Subterminal, Terminal
Bacillus stearothermophilus -spores
 Used for quality control of heat sterilization equipment
Bacillus anthracis - spores
 Used in biological warfare
 Certain species of bacteria produce spores.
 Within the cell (endospores) or
 External to the cell (exospores).
 THANK YOU

Reference
Microbiology, an introduction, 11th edition, Gerard J. Tortora, Berdell R. Funke,
Christine L. Case
Chapters: 1, 2, 4).