UNIT 1: HISTORY OF MICROBIOLOGY
THE FIRST OBSERVATIONS
Lucretius (98-55 B.C.) and Girolamo Fracastoro
(1478-1553)
→ suggested that diseases were caused by “invisible
living creatures”
Francesco Stelluti (1577-1652)
→ made the earliest observations on bees and
weevils using a microscope supplied by Galileo
Lucretius Girolamo Francastoro Francesco Stelluti
Robert Hooke
→reported to the world that life's smallest structural
units were "little boxes," or "cells,"
→marked the beginning of the CELL THEORY--- all
living things are composed of cells Hooke's
Microscope (1665) Antonie van Leeuwenhoek
was inspired by this publication
Anton van Leeuwenhoek (1632- 1723)
→considered as the “first true microbiologist”
→first actually to observe live microorganisms
through the magnifying lenses of more than 400
microscopes he constructed
→wrote a series of letters to the Royal Society of
London describing the "animalcules“ (tiny living and
moving cells) he saw through his simple, singlelens
microscope (50x to 300x)
→made detailed drawings of "animalcules" in
rainwater, in his own feces, and in material scraped
from his teeth
✓Bacteria
✓Protozoa
✓Sperm cells
✓Blood cells
✓Microscopic worms
→ 3”-4” microscope
→ Required good lighting and patience
FPRE
SPONTANEOUS GENERATION
→ believed that some forms of life could arise
spontaneously from nonliving matter
→ toads, snakes, and mice could be born of moist
soil; flies could emerge from manure; and that
maggots, the larvae of flies, could arise from
decaying corpses
Aristotle (384-322 B. C.)
→ Mentioned that simple invertebrates could arise
from Spontaneous Generation
Francesco Redi (1626-1697)
→demonstrate that maggots did not arise
spontaneously from decaying meat (1668)
→results of his investigation invalidated the long-held
belief that life forms could arise from non-living things
John Needham (1731-1781)
→ observed that a boiled mutton broth eventually
became cloudy after pouring it into a flask that was
then sealed tightly
→ found that even after he heated nutrient fluids
(chicken broth and corn broth) before pouring them
into covered flasks, the cooled solutions were soon
teeming with microorganisms
→claimed that microbes developed spontaneously
from the fluids
→ Asserted that organic matter possessed a “vital
force” that could give rise to life
Lazzaro Spallanzani (1729-1799)
→ suggested that microorganisms from the air
probably had entered Needham's solutions after they
were boiled
→ proposed that air carried microorganisms to the
culture medium
→showed that nutrient fluids heated after being
sealed in a flask did not develop microbial growth
Anton Laurent Lavoisier (1743-1794)
→ showed the importance of oxygen to life
1
THEORY OF BIOGENESIS
Rudolf Virchow (1821-1902)
→ challenged the case for spontaneous generation
with the concept of Biogenesis: living cells can arise
only from pre-existing living cells
Theodor Schwann (1810-1882)
→Observed that no growth occurred in a flask that
contained a nutrient solution after allowing the air to
pass through a heated tube
Heinrich Schroder (1810-1885) and Theodore von
Dusch (1824-1890)
→ Noticed that no growth occurred after allowing the
air to pass through a sterile cotton wool placed on a
flask of heat-sterilized medium
Louis Pasteur (1822-1895)
→disproved the doctrine of spontaneous generation
→demonstrated that microorganisms are present
inthe air and can contaminate sterile solutions, but
that air itself does not create microbes
→showed that microorganisms can be present in
nonliving matter-on solids, in liquids, and in the air
→demonstrated conclusively that microbial life can
be destroyed by heat and that methods can be
devised to block the access of airborne
microorganisms to nutrient environments
→form the basis of Aseptic Techniques
Aseptic Techniques → techniques that prevent
contamination by unwanted microorganisms, which
are now the standard practice in laboratory and
many medical procedures
John Tyndall (1820-1893)
→Showed that dust carry germs that
contaminate a sterile broth
***Tyndallization → is a form of sterilization in the
19th century that uses moist heat for 3 consecutive
days to eradicate vegetative cells and endospores
Ferdinand Cohn (1828-1898)
→Discovered that there are bacteria that could
withstand a series of heating and boiling because of
heat resistant structures known as endospores
THE GOLDEN AGE OF MICROBIOLOGY
(1857-1914)
Fermentation and Pasteurization
Theodor Schwann
→ stated that yeast cells are responsible for the
conversion of sugars to alcohol
Pasteur
→found that microorganisms called yeasts convert
the sugars to alcohol in the absence
of air: FERMENTATION
→ Pasteur's solution to the spoilage problem was to
heat the beer and wine just enough to kill most of the
bacteria that caused the
spoilage: PASTEURIZATION
→ stated Souring and spoilage are caused by
different microorganisms called bacteria; in the
presence of air, bacteria change the alcohol in the
beverage into vinegar (acetic acid)
Germ Theory of Disease
***Microorganisms might have relationships with
plants and animals---specifically, that
microorganisms might cause disease
Agostino Bassi → had proved that another
silkworm disease was caused by a fungus
Ignaz Semmelweis (1816-1865)
→ demonstrated that physicians, who at the time did
not disinfect their hands, routinely transmitted
infections (puerperal, or child -birth, fever ) from one
obstetrical patient to another
→ demonstrated that routine handwashing can
could prevent the spread of disease
Joseph Lister (1827-1912)
→ introduced the system of antiseptic surgery in
Britain
→ applied the germ theory to medical procedures
FPRE
2
→began treating surgical wounds with a phenol
solution
→pioneered in promoting among surgeons
handwashing before and after an operation, the
wearing of gloves, sterilization of surgical
Instruments
Robert Koch (1843-1910)
→ First to show irrefutable proof that bacteria indeed
cause disease
→discovered Bacillus anthracis in the blood of
cattle that had died of anthrax (1876)
→Discovered Mycobacterium tuberculosis (1882)
→first to cultivate bacteria on boiled potatoes, gelatin,
meat extacts and protein
→ established a sequence of experimental steps for
directly relating a specific microbe to a specific
disease: Koch's Postulates
Koch’s Postulate
1. The microorganism must be present in every case
of the disease but absent from a healthy host
2. The suspected microorganism must be isolated
from a diseased host grows in a pure culture
3. The same disease must be present when the
isolated microorganism is inoculated into a healthy
host
4. The same organism must be isolated again from
the disease host
FPRE
Collaborators of Koch
Fanny Hesse (1850-1934)
→suggested the use of agar, a solidifying agent, in
the preparation of the culture media
Julius Richard Petri (1852-1921)
→developed the Petri Dish, which is a circular glass
or plastic plate for holding the culture media
Martins Beijerink (1851-1931) and Sergei
Winogradsky (1856-1953)
→developed the enrichment-culture technique and
the use of selective media
IMMUNOLOGY: ADVENT OF VACCINATION
Edward Jenner (1749-1823)
→ embarked on an experiment to find a way to
protect people from smallpox
→introduced the concept of vaccination
***Physicians in China→ immunized patients by
removing scales from drying pustules of a person
suffering from a mild case of smallpox, grinding the
scales to a fine powder, and inserting the powder
into the nose of the person to be protected
Louis Pasteur (1822-1895) and Pierre Paul Emile
Roux (1853-1933)
→Pasteur used the term vaccine for an attenuated
culture
→both made a series of experiments to produced
attenuated stains of bacteria
→prove that when attenuated strains are introduced
into healthy host, the latter remains protected and
healthy against the virulent agent
Charles Chamberland (1851-1908)
→created a porcelain bacterial filter and developed
the anthrax vaccine together with Pasteur
Emil von Behring (1854-1917)
→prepared antitoxins for diphtheria and tetanus
Elie Metchnikoff (1845-1916)
→first to described the immune system cells and he
process of phagocytosis
3
 THE BIRTH OF MODERN CHEMOTHERAPY: A FORTUNATE ACCIDENT
DREAMS OF A "MAGIC BULLET“ ANTIBIOTICS
Chemotherapy→ treatment of disease by using Alexander Fleming (1881-1955)
chemical substances
→ accidentally discovered Penicillin
→chemical treatment of non-infectious diseases,
→ mold was later identified as Penicillium notatum
such as cancer
(later renamed Penicillium chrysogenum
Antibiotics→ chemicals produced naturally by
bacteria and fungi to act against other
microorganisms
Synthetic drugs → chemotherapeutic agents
prepared from chemicals in the laboratory
THE FIRST SYNTHETIC DRUGS
Paul Ehrlich (1854-1915)
→ speculated about a bullet" that could hunt down
and destroy a pathogen without harming the infected
Howard Florey (1898-1968) and Ernst Chain
host
(1906-1979)
→ found a chemotherapeutic agent called Salvarsan
→Made the purification process for penicillin and
(Arsphenamine), an arsenic derivative effective
clinical trials to humans
against syphilis
Edward Abraham (1913-1999)
Selman Waksman (1888-1973)
→ First to propose the correct biochemical structure
→discovered streptomycin and neomycin antibiotibs
of Penicillin
→regarded as “Father of Antibiotics” by some
Historians

FPRE
4
UNIT 2: MICROBIAL TAXONOMY
Taxonomy i. Subspecies: taxonomic subgroups within a
species
→area of biologic science comprising three distinct
but highly interrelated disciplines: → species subdivided based on phenotypic the
following differences:
✓Classification
a. Biotype
✓nomenclature (naming)
→ considered the same species with the same
✓identification of organisms
characteristic genetic makeup that displays
→ orderly classification and grouping of organisms differential physiologic characteristics
into taxa (categories)
→based on biochemical test result differences
→based on similarities and differences in genotype
b. Serotype
and phenotype
→ based on serologic differences
Carl von Linne: laid down the basic rules for
taxonomic categories NOTE!!!
CLASSIFICATION Phage typing (based on susceptibility to specific
bacterial phages) has also been used for this
→ method for organizing microorganisms into groups purpose
or taxa based on similar morphologic, physiologic,
and genetic traits ➢Diagnostic microbiologists traditionally emphasize

→Hierarchical classification system consists of the placement and naming of bacterial species into three
following taxa designations:
(occasionally four or five) categories:
a. Domain: Bacteria and Archaebacteria
✓the family
b. Kingdom: contains similar divisions or phyla
✓a genus
c. Phylum: contains similar classes; equivalent to
✓a species
the Division taxa
➢Species definitions are distinguished using DNA
d. Class: contains similar orders
profiling, including a nearly complete 16S rRNA
e. Order: contains similar families
sequence in combination with phenotypic traits
f. Family: contains similar genera
NOMENCLATURE
→group of organisms that may contain multiple
genera and consists of organisms with a common → naming of microorganisms according to
attribute established rules and guidelines set forth in the
International Code of Nomenclature of Bacteria
Example:Enterobacteriaceae, Streptococcaceae (ICNB) or the Bacteriological Code (BC)
g. Genus: contains similar species Genus designation→ first letter is always
capitalized
→based on various genetic and phenotypic
characteristics shared among the species Species designation→ first letter is always lower
case
h. Species: specific epithet
→most basic of the taxonomic groups and can be
defined as a collection of bacterial strains that
sharecommon physiologic and genetic features and
differ notably from other microbial species
FPRE
5
➢Two components are used simultaneously and are
printed in italics or underlined in script Example:
Streptococcus pneumoniae, Streptococcus
pyogenes, Streptococcus agalactiae, and
Streptococcus bovis
✓When bacteria are referred to as a group, their
names are neither capitalized nor underlined (e.g.,
staphylococci)
IDENTIFICATION
→process by which a microorganism’s key features
are delineated
→Process of discovering and recording the traits of
organisms so that they may be placed in an overall
taxonomic scheme
→organism can then be assigned to the most
appropriate taxa (classification) and can be given
appropriate genus and species names
(nomenclature)
IDENTIFICATION METHODS
Genotypic Characteristics
→relate to an organism’s genetic makeup, including
the nature of the organism’s genes and constituent
nucleic acids
Examples: base sequencing of DNA or RNA and
DNA base composition ratio to measure the degree
of relatedness of two organisms
Phenotypic characteristics
→based on features beyond the genetic level and
include both readily observable characteristics and
characteristics that may require extensive analytic
procedures to be detected
Examples: macroscopic (colony morphology on
media) and microscopic (size, shape, arrangement
into groups or chains of organisms) morphology,
staining characteristics (gram-positive or gram-
negative), nutritional requirements, physiologic and
biochemical characteristics, and susceptibility or
resistance to antibiotics or chemicals
FPRE
Major Characteristics Used in Taxonomy
Classical Characteristics
→Useful in routine identification of phylogenetic
information
Example: morphology, physiology and metabolism,
ecology and genetic analysis
→ Phylogenetic and phyletic classification is based
on evolutionary relationships instead of general
resemblance
Molecular characteristics
→ Based on the study of nucleic acid composition
and proteins
Classification by Cellular Type:
Prokaryotes, Eukaryotes, and Archaeobacteria
➢Organisms fall into three distinct groups based on
type of cell organization and function: prokaryotes,
eukaryotes, and archaeobacteria
➢Taxonomists have placed all organisms into three
domains that have replaced some kingdoms:
Bacteria, Archaea, and Eukarya
➢Each of these domains is divided into kingdoms
based on the similarities of RNA, DNA, and protein
sequences
Prokaryotes: Archaea and Bacteria (Eubacteria)
Eukaryotes: fungi, algae, protozoa, animals, and
plants Archaea (Archaeobacteria)
→closely related to eukaryotic cells than to
prokaryotic cells
→found in microorganisms that grow under extreme
environmental conditions
→cell walls lack peptidoglycan but they mostly
contain a protein or glycoprotein wall structure—”S-
layer”
→can stain gram-positive and gram-negative
→Cellular structure include the cell wall, plasma
membrane, ribosomes and flagella
→Do not contain a nucleus and membrane-bound
organelles →Produce through binary fission,
fragmentation or budding Examples:
Methanospirillum, Halobacterium, Sulfolobus
6
UNIT 3: GENERAL CHARACTERISTIC OF
BACTERIA
PROKARYOTIC VS EUKARYOTIC CELLS: Chief Distinguishing Characteristics of
Prokaryotes
AN OVERVIEW
1. DNA is not enclosed within a membrane and is
✓Chemically similar
usually a singular circularly arranged
→both contain nucleic acids, proteins, lipids, and chromosome
carbohydrates
2. DNA is not associated with histones; other
✓Use the same kinds of chemical reactions to proteins are associated with the DNA.
metabolize food, build proteins, and store energy
3. Lack membrane-enclosed organelles
✓Difference: structure of cell walls and membranes,
4. Cell walls almost always contain the complex
and the absence of organelles (specialized cellular polysaccharide peptidoglycan
structures that have specific functions)
5. Usually divide by Binary Fission
Chief Distinguishing Characteristics of
→DNA is copied, and the cell splits into two cells
Eukaryotes
→involves fewer structures and processes than
1. DNA is found in the cell 's nucleus, which is
eukaryotic cell division
separated from the cytoplasm by a nuclear
membrane, and the DNA is found in multiple
chromosomes
2. DNA is consistently associated with chromosomal
proteins called histones and with nonhistone
3. Have a number of membrane-enclosed organelles
4. Cell walls, when present, are chemically simple
5. Cell division usually involves Mitosis

FPRE
7
BACTERIA
→unicellular organisms that lack a nuclear
membrane and true nucleus
→classified as prokaryotes (Greek: before kernel
[nucleus]), having no mitochondria, endoplasmic
reticulum (ER), or Golgi bodies
BACTERIAL MORPHOLOGY
✓Vary in size, morphology, and cellto-cell
arrangements and in the chemical composition
and structure of the cell wall
✓Bacterial cell wall differences provide the basis for
the Gram stain
A. BACTERIAL SIZE
✓Most clinically relevant bacterial species range in
size from 0.25 to 1 μm in width and 1 to 3 μm in
length
✓Bacterium is some hundred-fold larger than a virus,
and ten-fold smaller than a eukaryotic cell

***Variation of size and shape within a population

may also result from asymmetric growth of the cell
wall
B. BACTERIAL SHAPE
➢Common bacterial cellular morphologies
include:
a. Cocci → circular
b. Coccobacilli → ovoid
c. Bacillus → rod shaped
d. Fusiform → tapered, pointed ends
e. Curved
f. Spiral→ helical, like corkscrew
→Spirochetes vary in length and in the number of
helical turns (**not all helical bacteria are called
spirochetes)
g. Pleomorphic → no defined shape

FPRE
8
 C. Bacterial Arrangement
a. Pairs
b. Chains
c. Grape-like clusters
d. Group of four
e. Packets of eight
f. Palisades
g. Chinese characters

FPRE
9
 BACTERIAL CELL STRUCTURE
A. CELL ENVELOPE
→ outermost structure, comprises:
A. Outer membrane
✓in gram-negative bacteria only
B. Cell wall
→composed of the peptidoglycan macromolecule
(murein layer)
C. Periplasm
✓ in gram-negative bacteria only
D. Cytoplasmic or cell membrane
→encloses the cytoplasm

FPRE
10
 2. CELL WALL (MUREIN LAYER)
→referred to as the peptidoglycan, or murein layer
→gives the bacterial cell shape and strength to
withstand changes in environmental osmotic
pressures that would otherwise result in cell lysis
→protects against mechanical disruption of the cell
and offers some barrier to the passage of larger
substances
→synthesis and structure are often the primary
1. OUTER MEMBRANE targets for the development and design of several
→found only in gram-negative bacteria antimicrobial agents

→function as the cell’s initial barrier to the →structure is composed of

environment
→serve as primary permeability barriers to
hydrophilic and hydrophobic compounds and contain
essential enzymes and other proteins located in
theperiplasmic space
→bilayered structure composed of
Lipopolysaccharide
→ gives the surface of gram negative bacteria a net
negative charge
disaccharidepentapeptide subunitsN-acetyl-D-
glucosamine and N-acetyl-D-muramic acid
→alternating sugar components (moieties), with the
amino acid chain linked to N-acetylmuramic acid
molecules
→polymers of these subunits cross-link to one
another by means of peptide bridges to form
peptidoglycan sheets
→layers of these sheets are cross-linked with one

→plays a significant role in the ability of certain another, forming a multilayered, cross-linked
bacteria to cause disease structure of considerable strength

Porins __________________________________________
Referred to as the murein sacculus, or sack, this
→protein structures scattered throughout the
lipopolysaccharide macromolecules peptidoglycan structure surrounds the entire cell

→water-filled structures that control the passage of NOTE!!! Short peptides, each consisting of four
nutrients and other solutes, including antibiotics, amino acid residues, are attached to a carboxyl
through the outer membrane group on each NAM residue

→ number and types of porins vary with bacterial ✓Different types of cell wall structures traditionally
species have been categorized according to their staining
→influence the extent to which various substances characteristics

pass through the outer membranes of different ➢Major types of cell walls: gram-positive and gram
bacteria negative types

Murein Lipoproteins ✓Mycobacteria→ stain gram-positive, have a

→ facilitate the attachment of the outer membrane to
the next internal layer in the cell envelope, the cell
wall
modified cell wall called an ACID-FAST CELL WALL
✓Mycoplasmas → microorganisms that have no
cell wall

FPRE
11
a. Gram-Positive Cell Wall Outer membrane
→composed of a very thick protective peptidoglycan →Outside the peptidoglycan layer is an additional
(murein) layer outer membrane
→consists of glycan (polysaccharide) chains of →contains proteins, phospholipids, and
alternating N acetyl-d-glucosamine (NAG) and N- lipopolysaccharide (LPS)
acetyl-d-muramic acid (NAM)
LPS contains three regions:
→many antibiotics effective against gram-positive
a. O–specific polysaccharide= antigenic
organisms (e.g., penicillin) act by preventing
synthesis of peptidoglycan b. Core polysaccharide= ketodeoxyoctanoic acid
Gram-negative bacteria→ thinner layer of (KDO) and heptose
peptidoglycan and a different cell wall structure, are
less affected by these antibiotics b. Lipid A (also called endotoxin)= inner, major
constituents
❑Other components of the gram-positive cell wall
that penetrate to the exterior of the cell are: c. LPS Functions:
TEICHOIC ACID ✓Vital in evading the host defenses
→anchored to the peptidoglycan (N-acetylmuramic ✓Contribute to the negative charge of the
acid)
bacterial surface, which stabilizes the membrane
→glycerol or ribitol phosphate polymers combined structure
with various sugars, amino acids, and amino sugars
✓Considered as an endotoxin
LIPOTEICHOIC ACID
Lipid A moiety
→anchored to the PM
→consists of phosphorylated glucosamine
→linked to the next underlying layer, PM or cellular disaccharide units to which are attached a number of
membrane long-chain fatty acids
✓These two components are unique to the gram- →responsible for producing fever and shock
positive cell wall conditions in patients infected with gram-negative
✓Other antigenic polysaccharides may be present bacteria
on the surface of the peptidoglycan layer Outer membrane function:
Teichuronic acids ✓Acts as a barrier to hydrophobic compounds and
→ similar polymers, but the repeat units include harmful substances
sugar acids (eg, N-acetylmannosuronic or d- ✓ Acts as a sieve, allowing water-soluble molecules
glucosuronic acid) instead of phosphoric acids to enter through protein-lined channels called porins
→ synthesized in place of teichoic acids when ✓Provides attachment sites that enhance
phosphate is limiting attachment to host cells

b. Gram-Negative Cell Wall ✓Strong negative charge is an important factor in

→composed of two layers: evading phagocytosis

Inner peptidoglycan layer ✓Acts as a barriers to toxic substances that

prevents movement inside the cell
→ much thinner than in gram-positive cell walls

FPRE
12

3. PERIPLASMIC SPACE
→typically found only in gram-negative bacteria
→bounded by the internal surface of the outer
membrane and the external surface of the cellular
membrane encompassing the thin peptidoglycan
layer
→contains the murein layer, consists gellike matrix
containing nutrient-binding proteins that assist in the
capture of nutrients from the environment
→contains several enzymes involved in the
degradation of macromolecules and detoxification of
environmental solutes, including antibiotics that enter
through the outer membrane
NOTE!!!
Periplasmic space is absent in gram-positive
bacteria
c. Acid-Fast Cell Wall
→have a gram-positive cell wall structure
→contain a waxy layer of glycolipids and fatty acids
(mycolic acid) bound to the exterior of the cell wall
→More than 60% of the cell wall is lipid
FPRE
Mycolic acid
→ major lipid component
→strong “hydrophobic” molecule that forms a lipid
shell around the organism and affects its
permeability
→makes Mycobacterium spp. difficult to stain with
the Gram stain
NOTE!!! Mycobacterium and Nocardia
→stain a faint blue (gram-positive) color
→best stained with an acid-fast stain
d. Absence of Cell Wall
→lack a cell wall and contain STEROLS in their cell
membranes
→lack the rigidity of the cell wall
→seen in various shapes microscopically
Example: Mycoplasma and Ureaplasma
**Gram-positive and gram-negative cells can lose
their cell walls and grow as L-forms in media
supplemented with serum or sugar to prevent
osmotic rupture of the cell membrane
4. CYTOPLASMIC (INNER) MEMBRANE
→ present in both gram-positive and gramnegative
bacteria and is the deepest layer of the cell envelope
→consist of phospholipid bilayer, various proteins
(70%), including a number of enzymes vital to
cellular metabolism
→serves as an additional osmotic barrier
→Absence of sterols
Exceptions: Mycoplasma→ incorporate sterols
(e.g., cholesterol), into their membranes when
growing in sterol-containing media
Functions:
✓Transport of solutes into and out of the cell
✓Housing of enzymes involved in outer membrane
synthesis, cell wall synthesis, and the assembly and
secretion of extracytoplasmic and extracellular
substances
13
✓Generation of chemical energy (i.e., ATP)
✓Cell motility
✓Mediation of chromosomal segregation during
replication
✓Housing of molecular sensors that monitor
chemical and physical changes in the environment
✓Electron transport and oxidative phosphorylation in
aerobic species
✓Excretion of hydrolytic exoenzymes
Permeability and Transport
1. Passive Transport
→relies on diffusion, uses no energy, and operates
only when the solute is at higher concentration
outside than inside the cell
a. Simple diffusion→ accounts for the entry of very
few nutrients, including dissolved oxygen, carbon
dioxide, and water itself
b. Facilitated diffusion → selective and uses no
energy so the solute never achieves an internal
concentration greater than what exists outside the
cell (e.g., Glycerol) b. ABC transport
c. Channel proteins→ form selective channels that →uses ATP directly to transport solutes into the cell
facilitate the passage of specific molecules
Gram-negative: transport of many nutrients is
2. Active transport facilitated by specific binding proteins located in the
a. Ion-coupled transport periplasmic space

→move a molecule across the cell membrane at the
expense of a previously established ion gradient
such as protonmotive or sodiummotive force
→particularly common in aerobic organisms, which
have an easier time generating an ion-motive force
than do anaerobes
Three basic types:
Uniport→ catalyze the transport of a substrate
independent of any coupled ion
Symport→ simultaneous transport of two substrates
in the same direction by a single carrier
Antiport → simultaneous transport of two
likecharged compounds in opposite directions by a
common carrier (40% of the substrates transported
by E coli)
Gram-positive: binding proteins are attached to the
outer surface of the cell membrane
3. Group translocation
→vectorial metabolism
→not active transport because no concentration
gradient is involved
→Allows bacteria to use their energy resources
efficiently by coupling transport with metabolism
3. Special transport processes
Siderophores→ compounds that chelate Fe and
promote its transport as a soluble complex

FPRE
14
✓Some pathogenic bacteria use specific receptors Two kinds of Plasmid
that bind host transferrin and lactoferrin (as well as Large Plasmid
other iron containing host proteins)
→ responsible for the production of B-lactamase that
Cytoplasmic Structure provide resistance to B-lactam antibiotics (penicillin
and oxacillin)
A. Ribosomes
Small Plasmid
→site of protein biosynthesis and gives the
cytoplasm a granular structure → resistant to tetracyclines and chloramphenicol
→Consist of RNA and proteins 4. Inclusions Bodies
→70S in size and separates into two subunits, 50S →Serve as the energy source or food reserve of the
and 30S bacteria or as a reservoir of structural bulding blocks

Note!!! →Composed mainly of polysaccharides, they lessen

osmotic pressure
Streptomycin and Gentamicin→ attach to the 30S
Examples: glycogen, cyanophysin granules,poly-B-
subunit and interfere with protein synthesis hydroxybutyrate granules, carboxysomes
Erythromycin and Chloramphenicol→ interfere (cyanobacteria, nitrifying bacteria and thiobacilli), gas
with protein synthesis by attaching to the 50S subunit vacuoles (cyanobacteria, halobacterium and thiothrix)
and polyphosphate granules(volutin and
B. Genome metachromatic granules
→Consist of a single, circular chromosome Two common types of granules:
→lacks nuclear membrane and mitotic apparatus a. Glycogen
→Appears as diffused nucleoid or chromatin body → storage form of glucose
that is attached to a mesosome (sac-like structure)
b. Polyphosphate granules
Nucleoid= Feulgen positive
→storage form for inorganic phosphates
→Consists of a single continuous circular molecule
ranging in size from 0.58 to almost 10 million base →Source of phosphate for nucleic acid and
pair phospholipid synthesis

Exeptions: Borrelia burgdorferi and Streptomyces Examples:

coelicolor Babes-Ernst bodies (C. diphtheria) Bipolar

→Few bacteria have dissimilar chromosomes: Vibrio
cholera and Brucella melitensisC. Plasmid
→extrachromosomal, double-stranded element of
DNA that is associated with virulence
→Located in the cytoplasm and serve as a site for
the genes to code for antibiotic resistance and toxin
production
→Not essential for bacterial growth so a bacterial cell
may or may not contain a plasmid
→Sometimes disappears during cell division and it
can make bacteria (mostly Gram-neg) pathogenic
FPRE
bodies (Y. pestis) Much granules (M. tuberculosis)
Poly-B-hydroxybutyric acid (PHB)
→Lipid like compound consisting of chains of
Bhyroxybutyric acid units connected through ester
linkages
→Produced when the source of nitrogen, sulfur or
phosphorus is limited and there is excess carbon in
the medium
PHB and Glycogen
→ carbon source when protein and nucleic acid
synthesis are resumed
15
Sulfur granules
→ Hydrogen sulfide and thiosulfate5.
Endospores/Asexual Spores
→Small, dormant structures located inside the
bacterial cell
→Aid in the survival of bacteria against external
conditions
→Produced within vegetative cells of some Gram-
positive bacteria
→Composed of dipicolinic acid and calcium ions:
CALCIUM DIPICOLINATE
→Some locations could be a means
microscopically identifying bacteria
→Responsible for perpetuation, but not muliplication
Examples: Bacillus and Clostridium
Types of spores according to location:
a. Terminal spore= Clostridium tetani
b. Subterminal spore= Clostridium botulinum
c. Central spore= Bacillus anthracis
Properties of Endospores
1. Core
→ spore protoplast
→contains a complete nucleus (chromosome), all of
the components of the protein-synthesizing
apparatus, and an energy-generating system based
on glycolysis
2. Spore wall
→ innermost layer surrounding the inner spore
membrane
→contains normal peptidoglycan and becomes the
cell wall of the germinating vegetative cell
3. Cortex →thickest layer of the spore envelope
→ contains an unusual type of peptidoglycan, with
many fewer cross-links than are found in cell wall
Peptidoglycan
4. Coat→ composed of a keratin-like protein
containing many intramolecular disulfide bonds
of → Impermeability of this layer confers on spores
their relative resistance to antibacterial chemical
agents
5. Exosporium→ composed of proteins, lipids, and
carbohydrates
→consists of a paracrystalline basal layer and a
hairlike outer region
Example: B. anthracis and B. cereus
Cellular Appendages
→ play a role in the mediation of infection and in
laboratory identification, varies among bacterial
species and even among strains within the same
species
1. Glycocalyx
→ Outward complex of polysaccharide on
the bacterial surface and other cells
→Helps the bacteria to attach to the surface of the
solid objects or tissues
→Appears as a capsule or a slime layer
a. Capsule
→ organized and is firmly attached to the cell wall
→immediately exterior to the murein layer of gram
positive bacteria and the outer membrane of gram
negative bacteria
→Made up of polysaccharide polymers
Exception: Poly-D-glutamic acid capsules of
Bacillus anthracis and Bacillus licheniformis
FPRE
16
→ Protects the bacteria(virulence factor) from the
attacks of human defense system since it resist
phagocytosis and dessication
→capsules sometimes must be removed to detect
the somatic (cell wall) antigens present
→Capsule removal is accomplished by boiling a
suspension of the microorganism
→Does not ordinarily stain with use of common
laboratory stains, such as Gram or India ink
→ appears as a clear area (“halo”-like)
b. Slime Layer
→Unorganized material that is loosely attached to
the cell wall
→Made up of polysaccharide
→Can either inhibit phagocytosis or aid in the
adherence of the bacteria to the host tissue or
synthetic implants
→facilitates and maintains bacterial colonization of
biologic (e.g., teeth) and inanimate (e.g., prosthetic
heart valves) surfaces through the formation of
biofilms
Extracellular Polymeric Substance (EPS)
→ helps cells in a biofilm attach to their target
environment and to each other
→ protects the cells within it, facilitates
communication
among them, and enables the cells to survive by
attaching
to various surfaces in their natural environment
2. Flagella
→exterior protein filaments that rotate and cause
→complex structures, mostly composed of the
protein flagellin, intricately embedded in the cell
envelope
→thread-like appendages composed entirely of
protein, 12–30 nm in diameter
→plays an important role in survival and the ability of
certain bacteria to cause disease
→antigenic (H antigens), and some of the immune
responses to infection are directed against these
proteins
→Gliding motility: Capnocytophaga,
Cyanobacteria, Myxobacteria,
➢Flagellum is attached to the bacterial cell body by
a complex structure consisting:
Hook→ short curved structure that appears to act as
the universal joint between the motor in the basal
structure and the flagellum
Basal body→ bears a set of rings, one pair in gram
positive bacteria and two pairs in gram-negative
Bacteria
Filament
→ long outermost region
→constant in diameter and contains the globular
(roughly spherical) protein flagellin arranged in
several chains that intertwine and form a helix
around a hollow core
Motility → ability of an organism to move by itself
“Run" or “Swim”→ bacterium moves in one
direction for a length of time
"Runs“ → interrupted by periodic, abrupt, random
changes in direction called "tumbles" , then, a "run"
resumes
"Tumbles“→ caused by a reversal of flagellar
rotation
Proteus
→ can "swarm," or show rapid wavelike movement
across a solid culture medium

bacteria to be motile → endowed with many flagella

FPRE
17
 → bundles of fibrils that arise at the ends of the cell
beneath an outer sheath and spiral around the cell
→anchored at one end of the spirochete
→have a structure similar to that of flagella
→Rotation of the filaments produces a movement of
the outer sheath that propels the spirochetes in a
spiral motion
→Movement is similar to the way a corkscrew moves
through a cork

Note!!! SPIROCHETES
→ group of bacteria that have unique structure and
motility
→move by means of AXIAL FILAMENTS OR
ENDOFLAGELLA

Arrangement of the Flagella:

a. Atrichous→ without flagellum
b. Monotrichous→ single flagellum at one end
c. Amphitrichous→ single flagellum at both ends
d. Lophotrichous→ tuff or group of flagella on one
end or both ends
Note!!!
➢True motility and Brownian Movement are best
observed through the HANGING DROP METHOD
True motility→ bacteria seem to be going in a
definite direction
Brownian movement→ bacteria bounce back and
forth rapidly due to the bombardment of molecules of
water

Taxis→ movement of bacteria toward or away from

a particular stimulus
d. Peritrichous→ entire cell surface covered with
flagella Ways of demonstrating motility in the Lab:
✓Hanging Drop Method
✓SIM

Axial Filaments ✓Flagellar staining

✓Serologic test
FPRE
18
✓Fluorescent Antibody Technique(FAT) → fimbriae are the site of the main surface antigen---
M protein
✓Swarming Phenomenon
❑Lipoteichoic acid, associated with these fimbriae,
✓Darkfield Microscopy
→ responsible for the adherence of group A
3. Pili (Fimbria)
streptococci to epithelial cells of their hosts
→hairlike, proteinaceous structures that extend
❑N. gonorrhoeae
from the cell membrane into the external
→able to make pili of different antigenic types
environment; some may be up to 2 μm long
(antigenic variation)
→Hair-like microfibrils usually produced
by flagellated Gram-negative bacteria observable
by electron microscopy
→serve as adhesins that help bacteria attach to
animal host cell surfaces, often as the first step in
establishing infection
→composed of structural protein subunits--pilins
Twitching motility
→a pilus extends by the addition of subunits of pilin,
makes contact with a surface or another cell, and
then retracts (powerstroke) as the pilin subunits are
disassembled--- grappling hook model
→Results in short, jerky, intermittent movements
Example: Pseumdomons aeruginosa, Neisseria
gonorrheae, and some strains of E. coli
Common pili or Ordinary pili
→Play a role in bacterial adherence to surfaces thus
contributing to virulence

Sex pilus
→ serves as the conduit for the passage of DNA
from donor to recipient during conjugation
→present only in cells that produce a protein referred
to as the F factor .
F-positive cells initiate conjugation only with F-
negative cells, thereby limiting the conjugative
process to cells capable of transporting genetic
material through the hollow sex pilus

Note!!!
❑Streptococci

FPRE
19
UNIT 4: BACTERIAL METABOLISM
→biochemical reactions bacteria use to break down RESPIRATION
organic compounds and reactions they use to
→efficient energy-generating process
synthesize new bacterial parts from the resulting
carbon skeleton →molecular oxygen is the final electron acceptor
✓Diagnostic schemes analyse each unknown →Obligate aerobes and facultative anaerobes
microorganism for:
Note!!!
(1) utilization of various substrates as a carbon
source Certain anaerobes can carry out anaerobic
respiration, in which inorganic forms of oxygen, such
(2) production of specific end products from various as nitrate and sulfate, act as the final electron
substrates acceptors
(3) production of an acid or alkaline pH in the test Biochemical Pathways from Glucose to Pyruvic
Medium Acid
Energy Production ✓Three major biochemical pathways bacteria use
→Breakdown of chemical substrate through the to break down glucose to pyruvic acid are:
degradative process of catabolism coupled with 1. Embden-Meyerhof-Parnas (EMP) Glycolytic
oxidation-reduction reactions pathway

→Bacteria use biochemical pathways 2. Pentose Phosphate Pathway

to catabolize (break down) carbohydrates 3. Entner-Doudoroff Pathway

and produce energy by two mechanisms: 1. EMP Glycolytic Pathway

1. Fermentation → Major pathway in conversion of glucose

2. Respiration (oxidation) to pyruvate

FERMENTATION → Generates reducing power in the form of NADH2

→anaerobic process carried out by both obligate → Generates energy in the form of ATP

and facultative anaerobes → Anaerobic; does not require oxygen

→Electron acceptor is an organic compound Example: Enterobacteriaceae

→less efficient in energy generation---beginning 2. Pentose Phosphate (Phosphogluconate)

substrate is not completely reduced
→a mixture of end products (lactate, butyrate,
ethanol, and acetoin) accumulates in the medium--
identification of anaerobic bacteria
Application:
a. Voges-Proskauer (VP)
b. Methyl Red Tests
Pathway
→ Alternative to EMP pathway
→ Glucose to ribulose-5-phosphate, which is
rearranged into other 3-, 4-, 5-, 6-, and 7-carbon
sugars
→ Provides pentoses for nucleotide synthesis
→ Produces glyceraldehyde-3-phosphate, which can
be converted to pyruvate
→ Generates NADPH, which provides reducing
power for biosynthetic reactions

FPRE
20
→ May be used to generate ATP →Propionic acid is the major end product of
fermentations
Example: a. Lactobacilli→ Heterolactic fermenting
Example:
bacteria
a. Propionibacterium acnes
b. Brucella abortus→ lacks some of the enzymes
required in the EMP pathway b. some anaerobic non–spore-forming, gram positive
bacilli
3. Entner-Doudoroff Pathway
E. Mixed Acid Fermentation
→ glucose-6-phosphate (rather than glucose)
→produce a number of acids as end products—lactic,
to pyruvate and glyceraldehyde phosphate
acetic, succinic, and formic acids
→ Generates one NADPH per molecule of glucose
but uses one ATP →strong acid produced is the basis for the positive
reaction on the methyl red test
Example: Aerobic process used by:
Example: Members of:
a. Pseudomonas
a. Escherichia
b. Alcaligenes
b. Salmonella
c. Enterococcus faecalis
c. Shigella
d. Other bacteria lacking certain glycolytic
F. Butanediol Fermentation
Enzymes
→end products are acetoin (acetyl methyl carbinol)
Anaerobic Utilization of Pyruvic Acid
and 2,3- butanediol
(Fermentation)
→detection of acetoin is the basis for the positive VP
A. Alcoholic Fermentation
reaction
→major end product is ethanol
→Little acid is produced by this pathway
Example: yeasts
Example: Members of:
B. Homolactic Fermentation
a. Klebsiella
→end product is almost exclusively lactic acid
b. Enterobacter
Example:
c. Serratia
a. All members of the Streptococcus genus
***Organisms that have a positive VP reaction
b. members of the Lactobacillus genus usually have a negative reaction on the methyl red
test, and vice versa
C. Heterolactic Fermentation
H. Butyric Acid Fermentation
→in addition to lactic acid, the end products include
→Involves the conversion of pyruvate to butyric
carbon dioxide, alcohols, formic acid, and acetic acid
acid along with acetic acid, CO2 and Hydrogen
Example: Some lactobacilli Example:
a. Clostridium
b. Fusobacterium c. Eubacterium
D. Propionic Acid Fermentation Aerobic Utilization of Pyruvate (Oxidation)

FPRE
21
Krebs or Tricarboxylic acid (TCA) cycle
→most important pathway for the complete oxidation
of a substrate under aerobic conditions
→pyruvate is oxidized, carbon skeletons for
biosynthetic reactions are created, and the electrons
donated by pyruvate are passed through an electron
transport chain and used to generate energy in the
form of ATP
→results in the production of acid and the evolution
of carbon dioxide
Carbohydrate Utilization and Lactose
Fermentation
→use of various sugars (carbohydrates)
→fermentation is usually detected by acid production
and a concomitant change of color resulting from a
pH indicator present in the culture medium
***Glucose must not be present if the ability to
ferment another sugar is being tested

Classifying members of the

Enterobacteriaceae family→ ability to ferment
lactose
β-galactoside permease→ transport of lactose
across the cell wall into the bacterial cytoplasm
β-galactosidase→ break the galactoside bond,
releasing glucose, which can be fermented

***All organisms that can ferment lactose can also

ferment glucose

Energy Utilization
1. Biosynthesis of new cell components
2. Maintenance of the physical and chemical integrity
of the cell
3. Activity of the locomotor organelles
4. Transport of solutes across membranes
5. Heat production

FPRE
22
UNIT 5: BACTERIAL GENETICS
Frederick Miescher(1869) c. A nitrogen-containing base, or the “steps,”
→ discovered DNA either a purine or a pyrimidine
Phoebus A. T. Levine (1920s) RNA Molecule
→ discovered that DNA contained phosphates, five- →single-stranded and short, not double-stranded
carbon sugars (cyclic pentose), and
and long, and contains the sugar ribose, not
nitrogencontaining bases
deoxyribose
Rosalind Franklin
Three major types of RNA:
→ discovered the helical structure by x-ray
1. messenger RNA (mRNA)
crystallography
2. transfer RNA (tRNA)
James Watson and Francis Crick (1950s )
3. ribosomal RNA (rRNA)
→ described the three-dimensional structure of the
Gene → a DNA sequence that encodes for a specific
DNA molecule
product (RNA or protein)
GENETICS
Genome → all the genes in an organism
→ process of heredity and variation
Chromosomes → organized genome into discrete
***Ability to maintain viability, adapt, multiply, and elements
cause disease is determined by the organism’s
Bacterial Chromosome
genetic composition
→ contain a single, unpaired (i.e., haploid)
Three major aspects of microbial genetics:
chromosome
1. Structure and organization of genetic material
→ contains the genes essential for viability and
2. Replication and expression of genetic information exists as a doublestranded, closed, circular
macromolecule
3. Mechanisms by which genetic information is
altered and exchanged among bacteria Nonchromosomal Elements of the Genome

NUCLEIC ACID STRUCTURE AND Extrachromosomal Elements

ORGANIZATION
A. Plasmids
DNA Molecule
→double-stranded, closed, circular, autonomously
→double helical chain of nucleotides
replicating extrachroosomal genetic elements
→ helix is a double strand twisted together, referred
→ 1 to 2 kilobases up to 1 megabase or more
to as a “spiral staircase”
Genes encode for:
→information contained in DNA is determined
✓products that mediate plasmid replication and
primarily by the base sequence---
transfer between bacterial cells
Genetic Code
✓products that provide a specialized function, such
→involved in the production of RNA as determinants of antimicrobial resistance or a
unique metabolic process
Nucleotide is a complex combination of the following:
→ do not usually encode for products essential for
a. A phosphate group (PO4)
Viability
b. A cyclic five-carbon pentose sugar (deoxyribose)
FPRE
23
B. Transposable Elements
→ pieces of DNA that move from one genetic
element to another
→unable to replicate independently
→do not exist as separate entities in the bacterial
cell
Two types:
a. Simple Transposon or Insertion Sequence (IS)
→ contain the genes that encode information
required for movement from one site in the genome
to another
b. Composite Transposon
Expression of Genetic Information
→cassette (grouping of genes) flanked by insertion
Gene expression
sequences
→processing of information encoded in genetic
Internal gene embedded in the insertion sequence
elements (i.e., chromosomes, plasmids,
encodes for an accessory function (e.g.,
antimicrobial resistance) and transposons)
REPLICATION AND EXPRESSION OF GENETIC →results in the production of biochemical molecules,
INFORMATION including RNA molecules and proteins
Replication A. Transcription
→complex process mediated by various enzymes, → Synthesis of single-stranded RNA using one
strand of the DNA as a template
such as DNA polymerase and cofactors
→DNA base sequence of the gene is converted into
1. Unwinding of the chromosome’s supercoiled DNA
an mRNA molecule
2. Separation of the complementary strands of the
mRNA molecules are polycistronic
parental DNA
Polycistronic→ encode for several gene products
3. Synthesis of the new DNA strands
4. Termination of replication, releasing two identical B. Translation
chromosomes, one for each daughter cell →involves protein synthesis
➢Process generally takes approximately 20 to 40 →mRNA molecules is translated into specific amino
acid sequences that are responsible for protein
minutes in rapidly growing bacteria such as E. coli
structure and function

FPRE
24
 RecA→ protein playing a central role

3. Genetic Exchange
Three mechanisms:
A. Transformation
→recipient cell uptake of naked (free) DNA
released into the environment when another bacterial
cell (donor) dies and undergoes lysis
→DNA can be incorporated into the bacterial
genome by recombination
→development of antibiotic resistance and in the
dissemination of genes that encode factors essential
to an organism’s ability to cause disease
Competent→ cells that can take up naked DNA
Example: Streptococcus pneumoniae, Neisseria
gonorrhoeae, H. influenzae

GENETIC EXCHANGE AND DIVERSITY

Three basic mechanisms:
1. Mutation
B. Transduction
→alteration in the original nucleotide sequence of a
gene or genes within an organism’s genome → transfer of bacterial genes by a bacteriophage
(virusinfected bacterium) from one cell to another
→change in the organism’s genotype
→ viruses integrate their DNA into the bacterial cell’s
→induced by chemical or physical factors
chromosome, where viral DNA replication and
(mutagens) or by biologic factors
expression occur
2. Genetic Recombination
Generalized transduction→ bacterial DNA may be
→Homologous Recombination
randomly incorporated with viral DNA Specialized
→segment of DNA originating from one bacterial cell transduction → incorporated along with adjacent
(donor) enters a second bacterial cell (recipient) and viral DNA
is exchanged with a DNA segment of the recipient’s
Example: C. diphtheriae
genome
→pieces of DNA are usually have extensive
similarities in nucleotide sequences
→involves a number of binding proteins

FPRE
25
C. Conjugation
→transfer of genetic material from a donor bacterial
strain to a recipient strain
→Occurs between two living cells, involves cell-to-
cell contact, and requires mobilization of the donor
bacterium’s chromosome
→Plasmids and transposons---transferred by
conjugation
Example: E. coli→ contact is mediated by a sex pilus
→ Sex pilus establishes a conjugative bridge
that serves for DNA transfer from donor to recipient
cell

FPRE
26
UNIT 6: MICROSCOPY
→use of a microscope to magnify objects too small
to be visualized with the naked eye so that their
characteristics are readily observable
Applications:
1. Rapid preliminary organism identification
2. Rapid final identification of certain organisms
3. Detection of different organisms present in the
same specimen
4. Detection of organisms not easily cultivated in the
laboratory
5. Evaluation of patient specimens for the presence
of cells indicative of inflammation or contamination
6. Determination of an organism’s clinical
significance
7. Provide pre-culture information
8. Determine which tests and methods should be
used for identification and characterization of
cultivated organisms
9. Provide a method for investigating unusual or
unexpected laboratory test results
B. RESOLUTION
→extent to which detail in the magnified object is
maintained
→ability of the lenses to distinguish fine detail and
structure
→determined by numerical aperture and wavelength
of light
Resolving power
→closest distance between two objects that when
magnified still allows the two objects to be
distinguished from each other
Note!!! Shorter the wavelength of light used in the
instrument, the greater the resolution
Immersion Oil
→ specific optical and viscosity characteristics
designed for use in microscopy
→used to fill the space between the objective lens
and the glass slide onto which the specimen has
been affixed
→enhances resolution by preventing light rays from
dispersing and changing wavelength after passing
through the specimen

Note!!!
Refractive Index→ measure of the light-bending
ability of a medium
1000× magnification
→ required for optimal detection and characterization
of bacteria
BRIGHT-FIELD (LIGHT) MICROSCOPY
PRINCIPLES OF LIGHT MICROSCOPY
→visible light is passed through the specimen and
then through a series of lenses that bend the light in
a manner that results in magnification of the
organisms present in the specimen
A. MAGNIFICATION

FPRE
27

C. CONTRAST
→needed to make objects stand out from the
background
→achieved by staining techniques that highlight
organisms and allow them to be differentiated from
one another and from background material and
debris
Kohler Illumination
→ designed to provide maximum illumination and
resolution when observing images using a
microscope
PHASE-CONTRAST MICROSCOPY
→detailed examination of internal structures in living
microorganisms
→not necessary to fix or stain the specimen
Principle:
→based on the wave nature of light rays
→ light rays can be in phase (their peaks and
valleys match) or out of phase
Reinforcement (relative brightness)→ wave peak of
light rays from one source coincides with the wave
peak of light rays from another source
Interference (relative darkness)→ wave peak from
one light source coincides with the wave trough from
another light source
Set of light rays:
a. direct from light source
b. reflected or diffracted
from a particular
structure in the specimen
Diffraction→ scattering of light rays as they touch a
specimen's edge

Note!!! Two sets of light rays are brought together,

form an image of the specimen on the ocular lens,
containing areas that are relatively light and
through shades of gray, to black

FPRE
28
 Fluorescent Dyes
✓Acridine Orange, Auramine,
Fluorescein
Isothiocyanate (FITC)
→requires BLUE excitation light
Exciter filter: 450-490 wavelength
Barrier filter: 515 wavelength
✓Calcofluor White
FLUORESCENT MICROSCOPY
→requires a VIOLET excitation light
PRINCIPLE OF FLUORESCENT MICROSCOPY
Exciter filter: 355-425 wavelength
Fluors or Fluorochromes
Barrier filter: 460 wavelength
→raised to a higher energy level after absorbing
ultraviolet (excitation) light
→return to their normal, lower energy state, they
release excess energy in the form of visible
(fluorescent) light
Fluorescing objects appear brightly against a
dark background

A. FLUOROCHROMING
→ direct chemical interaction between the
fluorescent dye and a component of the bacterial cell
Advantage:
➢enhances contrast and amplifies the observer’s
ability to detect stained cells tenfold greater than light
microscopy
Examples:
✓Acridine orange stain
✓Auramine-rhodamine stain
Excitation filter
✓Calcofluor white stain
→passes light of the desired wavelength to excite
the fluorochrome
Barrier filter
→prevents the excitation wavelengths from
damaging the eyes of the observer

FPRE
29
1. ACRIDINE ORANGE Example:
→binds to nucleic acid FITC→ intense, APPLE GREEN fluorescence
→used to confirm the presence of bacteria in →most commonly used
blood cultures
→stains all nucleic acids---nonspecific
→bright orange fluorescence
→does not discriminate between gram-negative and
gram-positive bacteria
→used for detection of cell wall–deficient bacteria DARK-FIELD MICROSCOPY
grown in culture
→involves the alteration of microscopic technique
2. AURAMINE-RHODAMINE rather than the use of dyes or stains to achieve
→have affinity to waxy mycolic acids in the cell walls contrast
of mycobacteria →condenser does not allow light to pass directly
→non-specifically bind to nearly all mycobacteria through the specimen but directs the light to hit the
specimen at an oblique angle
→appear bright yellow or orange against a greenish
background →only light that hits objects will be deflected upward
into the objective lens for visualization
→used to enhance detection
→other light that passes through the specimen will
of mycobacteria directly in patient specimens miss the objective, making the background a dark
field
→initial characterization of cells grown in culture
→used to detect SPIROCHETES
3. CALCOFLUOR WHITE
Appear extremely bright against a black field
→bind in the cell walls of fungi
→directly detect fungi in clinical material
→observe subtle characteristics of fungi grown in
culture
→visualize some parasites such as microsporidia
B. IMMUNOFLUORESCENCE
→antibodies are conjugated to a fluorescent dye
ELECTRON MICROSCOPY
→dye-antibody conjugate detect, or “tag,” specific
→uses electrons instead of light to visualize small
microbial agents
objects
→microorganisms become readily detectable by
→ electrons are focused by electromagnetic fields
fluorescent microscopy and form an image on a fluorescent screen

→combines the amplified contrast provided by →magnifications in excess of 100,000×

fluorescence with the specificity of antibodyantigen
binding
→Legionella spp., Bordetella pertussis, and
Chlamydia trachomatis

FPRE
30
Two General Types
1. TRANSMISSION ELECTRON MICROSCOPE
(TEM)
→ passes the electron beam through objects and
allows visualization of internal structures
2. SCANNING ELECTRON MICROSCOPE (SEM)
→ uses electron beams to scan the surface of
objects and provides three-dimensional views of
surface structures

FPRE
31
 UNIT 7: STAINING
BACTERIAL STAINING: PREPARATION OF Basic Dyes cationic dyes with positively charged
SPECIMENS FOR LIGHT MICROSCOPY (pentavalent nitrogen) that adhere to the negatively
charged molecules
PURPOSE OF STAINING
Example: Crystal Violet, Methylene Blue
1. Observe and appreciate the appearance of
microorganism Malachite Green and Safranin
2. Differentiate one microorganism or group of Acidic dyes anionic dyes with negatively charged
microorganism from another groups
3. Identification of microorganisms and their special
structures
(carboxyl and phenolic) that bind to positively 2. DIFFERENTIAL STAINS react differently with
charged cell structures different kinds of bacteria and thus can be used to
distinguish them
Example: Eosin, Acid Fuchsin and Nigrosin
Example:
Note!!! Bacteria are slightly negatively charged at pH
7 Gram Stain

FIXING -kills the microorganisms and fixes them to Acid-Fast Stain

the slide preserves various parts of microbes in their
A. GRAM STAIN classifies bacteria into two large
natural state with only minimal distortion
groups:
Example:
a. Gram-positive
a. Heat-fixed
b. Gram-negative
b. Methanol Fixation- 95% Methanol for I minute
Counterstains -stains that have a contrasting color
preserves morphology of host cells, bacteria to the primary stain
especially useful for examining
Note!!!
bloody specimen material
Gram-positive cells retain the dye and remain
STAINING purple

Three kinds of staining techniques: Gram-negative cells do not retain the dye; they
are colorless until counterstained with a red dye
1. SIMPLE
MORDANT
2. DIFFERENTIAL
 chemically bond the alkaline dye to the bacterial
3. SPECIAL cell wall chemical added to the solution to
1. SIMPLE STAINS intensify the stain

single stain is used highlight the entire
microorganism so that cellular shapes and basic
structures are visible stain is applied to the fixed
smear for a certain length of time and then washed
off, dried and examined
Example: Methylene Blue, Carbolfuchsin,
Crystal Violet, Safranin
 increase the affinity of a stain for a biological
specimen
 coat a structure to make it thicker and easier to
see after it is stained with a dye
Example: GRAM’S IODINE

FPRE
32
 Note!!! Valuable information for the treatment of
disease Gram-positive bacteria tend to be killed
easily by PENICILLINS and CEPHALOSPORINS
Gram-negative bacteria generally more resistant
because the antibiotics cannot penetrate the
lipopolysaccharide layer

PRINCIPLE
***Based on the differential structure of the cellular
membranes and cell walls

Gram-Positive Organisms
 contain a highly cross-linked layer of
peptidoglycan that retains the primary dye-
Crystal Violet-following the application of the
mordant-iodine (I)
 iodine and crystal violet form a complex within
the peptidoglycan
 when decolorized---CV-I complex remains within
the cell
Appearance: Dark Purple to Deep Blue
Gram-Negative Organisms
 do not contain a thick cross-linked layer of
peptidoglycan
 CV-I complexes are not trapped within the
peptidoglycan
 decolorizer dehydrates the outer cellular
membrane, leaving holes in the membrane and
effectively washing or removing the CV-I
complex from the cells
 Secondary stain: Safranin

FPRE
33
Appearance: Pink to Deep Magenta waxy and resistant to staining with aqueous based
stains such as the Gram stain
Indirect Smear
Example: Mycobacteria spp, Nocardia
 Report the Gram stain organism’s cellular
Carbolfuchsin
shape, morphology, and Gram reaction
 primary stain
Note!!! QUALITY CONTROL
Heat or Tergitol
Gram-positive: Staphylococcus aureus
allow the stain to penetrate into the waxy
Gram-negative: Escherichia coli
surface of acid-fast microorganisms
General Rules of Gram Staining!!!
3% Acid Alcohol
1. All COCCI are GRAM-POSITIVE except for
Ethanol and Hydrochloric Acid
Neisseria, Veilonella and Branhamella (Moraxella)
 removed excess stain
2. All BACILLI are GRAM-NEGATIVE except for
Arcanobacterium, Actinomyctes, Bacillus, Methylene Blue or Malachite Green
Clostridium, Corynebacterium, Erysipelothrix,
 secondary stain
Eubactrium, Gordonia, Kuthria, Lactobacilli, Listeria,
Mycobacteria, Nocardia, Propionibacterium and Expected Results
Tsukamurella
Acid-Fast Organisms: PINK
3. All spirochetes are GRAM-NEGATIVE
Nonacid-Fast Oganisms: DARK BLUE
Reasons Why Gram-Positive Bacteria Becomes
***Background material should stain BLUE to BLUE
Gram-Negative GREEN
1. Removal of MgRNA by precipitation with bile salts
2. Autolysis, aging and temperature of incubation
result to loss of gram-positivity

***Antibiotic-treated bacterial cell have atypical

staining reaction

3. Acidic solution of Gram’s Iodine

4. Technical error Overdecolorization
Reasons Why Gram-Negative Bacteria Becomes
Gram-Positive
1. Incomplete decolorization
2. Thick smear
B. ACID-FAST STAIN
 binds strongly only to bacteria that have a waxy
material in their cell walls

Principle ***Acid-fast organism contain MYCOLIC

ACID in their outer membrane, making the cells

FPRE
34
 E. Auramine-Rhodamine Method selective for the
cell wall of AFB

Note!!!
 Mycolic acid renders the bacterial cell resistant
to decolorize-ACID-FAST
 Acid-Fastness is affected by colonial age,
medium for growth and UV light
 Ziehl-Neelsen method ideal for concentrated
smears partially acid-fast bacilli---Nocardia spp
 Acid-alcohol is composed of Hydrochloric Acid
and Ethanol
Ways to Facilitate Acid-Fast Staining 3. SPECIAL STAINS
1. Use of heating or steaming process for 5-7  used to color and isolate specific parts of
minutes to temporarily remove the mycolic acid, microorganisms
while the smear is flooded with stain
 endospores and flagella, and reveal the
2. Increasing the concentration of dye and phenol in presence of capsules
the staining reagent
A. CELL WALL STAIN
3. Prolonged contact of the specimen with the
primary stain Dyar Method

4. Addition of a wetting agent like TERGITOL B. INDIRECT/NEGATIVE STAINING

METHODS OF ACID-FAST STAINING  colorless bacteria against a colored background

A. Ziehl-Neelsen  excellent technique for studying bacterial

vacuoles and viral morphology
Hot Staining Method
NEGATIVE STAINING FOR CAPSULES
B. Kinyoun’s Method
 Demonstrating the presence of a capsule means
Cold Staining Method of determining the organism's virulence
C. Pappenheim Method Appearance: ***Bacteria as light colored bodies
Differentiate M. smegmatis from M. tuberculosis against a dark background

M. smegmatis: decolorized by the mixture of rosolic Cell surface repels acidic stain as a result of bacterial
acid and alcohol coloring it BLUE cells being negatively charged

M. tuberculosis: not decolorized and remains RED Example: INDIA INK OR NIGROSIN DYE

D. Baumgarten Method
 Differentiate M. tuberculosis from M. leprae
M. tuberculosis: does not readily take up the stain
and appears BLUE
M. leprae: easily stained by dilute alcoholic fuchsin
coloring it RED

FPRE
35
C. CAPSULAR STAINING SPORE STAIN
a. Hiss’s Copper Sulfate Method: capsules appear a. Dorner’s Method: spores stain red; bacterial cells
faint blue halos around dark blue to purple cells almost colorless against a dark gray background
b. Gin’s Method: capsules unstained with margin b. Wirtz and Conklin: spores are green; bodies of
delineated by ink; bacteria will be stained bacteria are red
c. Anthony’s Method: capsule is unstained against c. Acetic Acid Method
a purple background; cells are deeply stained
E. FLAGELLAR STAINING
d. Welch’s Method: capsules stains a pale violet
 tedious and delicate staining procedure
e. Muir’s Method: cells are stained red and the
 uses a mordant and the stain
capsule blue
CARBOLFUCHSIN to build up the diameters of
f. Tyler’s Method the flagella until they become visible under the
light microscope
g. Wadsworth’s Method
 Treating the cells with an unstable colloidal
h. MacNeal
suspension of TANNIC ACID SALTS cause a
i. Lawson heavy precipitate to form on the cell walls and
flagella
D. ENDOSPORE (SPORE) STAINING
 Diameter of flagella is increased to such an
 cannot be stained by ordinary methods because extent that subsequent staining with BASIC
the dyes do not penetrate the wall of the FUCHSIN makes the flagella visible in the light
endospore microscope
FLAGELLAR STAIN
a. Leifson Method: bacterial bodies blue; flagella
red
b. Gray’s Method
c. Fischer and Conn
d. Casares-Gil’s
e. Loefflers
f. Van Ermengen’s

Schaeffer-Fulton Endospore Stain -most
commonly used endospore stain
MALACHITE GREEN -primary stain

Note!!! Heat steam for about 5 minutes helps the

stain penetrate the endospore wall
SAFRANIN counterstain, stain portions of the cell
other than endospores
Appearance: Endospores appear GREEN
within Red or Pink cells

FPRE
36
Note!!!
Mordant TANNIC ACID : swells, coats and forms
precipitate with the flagella

FPRE
37
UNIT 8: MICROBIAL GROWTH AND NUTRITION
Requirements for microbial growth can be divided
into two main categories:
A. PHYSICAL
 temperature, pH, and osmotic pressure
B. CHEMICAL
 sources of carbon, nitrogen, sulfur, phosphorus,
oxygen, trace elements, and organic growth factors
PHYSICAL REQUIREMENTS
I. TEMPERATURE REQUIREMENT
Primary groups on the basis of their preferred
range of temperature: Minimum Growth Temperature lowest temperature
at which the species will grow
a. Psychrophiles/Cryophiles -cold-loving microbes
Optimum Growth Temperature temperature at
 Grow well 0°C to a maximum of 20°C
which the species grows best
Example: Listeria monocytogenes, Yersinia
Maximum Growth Temperature highest
enterolitica
temperature at which growth is possible
b. Psychrotrophs
Most organisms are MESOPHILIC 30°C (35-37°C)
 temperature optimum between 20°C and 30°C optimal for many free-living forms, and the body
but grow well at lower temperatures temperature of the host is optimal

 important cause of food spoilage
d. Mesophiles-moderate-temperature-
 loving microbes 20°C to 40°C (30°C-37°C)
 Most commonly encountered
bacteria in the clinical laboratory
e. Thermophiles-
 heat-loving microbes 50°C to 60°C
Example: Bacillus stearothermophilus
Thermal Death Time lowest or minimum time
required to kill organism under constant temperature
Thermal Death Point lowest temperature required
to kill microorganism in a constant time
pathogenic
Note!!!
 Diagnostic laboratory usually incubate cultures
for bacterial growth at 35°C
 Pseudomonas aeruginosa and
Campylobacter can grow at 35°C and 42°C

e. Hyperthermophilic- above the temperature of II. pH REQUIREMENT

boiling water
f. Extremophiles
 Prokaryotes that are able to survive in unusual
conditions like the absence of oxygen, increased
temperatures, and living below the earth’s surface
Most bacteria grow best in a narrow pH range near
neutrality, between pH 6.5 and 7.5
a. Neutralophiles
 grow best at a pH of 6.0–8.0
 maintain an internal pH of about 7.5 over an
external range of 5.5–8.5

FPRE
38
b. Acidophiles
 have optima as low as pH 3.0 (6.5-7)
 maintain an internal pH of about 6.5 over an
external range of 1.0–5.0
c. Alkaliphiles
 have optima as high as pH 10.5 (8.4-9)
 maintain an internal pH of about 9.5 over an
external range of 9.0–11.0
Facultative Halophiles
 do not require high salt concentrations but are
able to grow at salt concentrations up to 2%
b. Moisture
vital for bacterial growth and susceptibility testing
CHEMICAL REQUIREMENTS
Bacteria have three major nutritional needs for
growth:

Note!!! 1. Source of Carbon

 PEPTONES and AMINO ACIDS in media act as Carbon 50% of the dry weight of a bacterium
BUFFERS making cellular constituents

 Phosphate Salts exhibiting their buffering 2. Source of Nitrogen

effect in the pH growth range of most bacteria
Nitrogen 14% of the dry weight making proteins
 Culture Media for bacterial isolation are usually
3. Source of Energy
adjusted to a final pH between 7.0 to 7.5
ATP performing cellular functions
III. OSMOTIC PRESSURE REQUIREMENT
Phosphate nucleic acids and phospholipids of cell
Osmophilic
membranes
 Organisms requiring high osmotic pressures
Sulfur protein synthesis
Barophiles
Phosphate and Sulfur make up an additional
 Organisms that grow rapidly in high-pressure 4% of the weight
environment (600 to 1100 atm pressure)
Mineral ions: Na+, K+, Cl−, and Ca
Example: Shewanella, Colwellia, Photobacterium
I. Carbon Source
Plasmolysis
a. Heterothrophs (Organotrophs)
 shrinkage of the cell's cytoplasm
 require organic carbon for growth
OTHER PHYSICAL FACTORS
 Use reduced, preformed, organic molecules
a. SALT CONCENTRATION from other bacteria
Halophilic Chemoheterotrophs
 organisms requiring high salt concentrations  get most of their carbon from the source of their
energy- proteins, carbohydrates, and lipids
Example: Staphylococcus aureus Listeria
Monocytogenes ***includes all Vibrio species b. Autotrophs (Litotrophs)
except: V. mimicus and V. cholerae
 Use CO2 as the sole source of carbon
Extreme Halophiles/Obligate Halophiles
 do not require organic nutrients for growth
 high salt concentrations that they actually
Chemoautotrophs and Photoautotrophs
require them for growth
 derive their carbon from carbon dioxide

FPRE
39
Chemolithotrophs
 use an inorganic substrate such as hydrogen or
thiosulfate as a reductant and carbon dioxide as
a carbon source
II. Nitrogen Source
NH3
 sole nitrogen source
Nitrogen Fixation
 ability to assimilate N2 reductively via NH3
 requires a large amount of metabolic energy and
is readily inactivated by oxygen
Ammonification
 production of NH3 from the deamination of
amino acids
Assimilatory nitrate reduction and assimilatory
nitrite reduction
 ability to assimilate nitrate (NO3-) and nitrite
(NO2 -) reductively by conversion of these ions
into NH3
Denitrification
 conversion of NH3 to gaseous N2 under
anaerobic conditions
III. Phosphorus Source
Phosphate
component of ATP; nucleic acids; and such
coenzymes as NAD, NADP, and flavins assimilated
as free inorganic phosphate (Pi)
IV. Sulfur Source
 autotrophic bacteria can oxidize it to sulfate
 most microorganisms can use sulfate as a sulfur
source, reducing the sulfate to the level of
hydrogen sulfide (H2S)
 some microorganisms can assimilate
directly from the growth medium
 Sources: sulfate ion, hydrogen sulfide, sulfur-
containing amino acids

V. Mineral Sources
Magnesium Ion (Mg2+) and Ferrous Ion (Fe2+)
 enzyme function
Mg2+ and K+
 function and integrity of ribosomes
Ca2+
 constituent of gram-positive cell walls
----------------------------------------------------------------------
Other minerals: Mn2+, Mo2+, Co2+, Cu2+, and Zn2+
VI. Growth Factors
 organic compound that a cell must contain to
grow but that it is unable to synthesize
 Substances that are required by fastidious
bacteria for their growth and multiplication
Example: Amino Acids, Purines, Pyrimidines and
Vitamins, Hemoglobin, Pentose, Fatty Acids
Prototrophics
 do not require exogenous source of growth
factor since they synthesize their own
Auxotrophics
 require the addition of growth factor to culture
media for growth to occur
Note!!!
 All bacteria that inhabit the human body fall into
the heterotrophic or organotrophic group
 Fastidious bacteria require additional
substances such as vitamins, purines,
pyrimidines and haemoglobin for growth and
survival
 Saprophytes require dead organic substances
H2S
FPRE
40
OXYGEN REQUIREMENT b. Aerotolerant anaerobes (Facultative Aerobes)
1. Aerobes grow in the presence of atmospheric  can grow in its presence, but they do not use it
oxygen as a hydrogen acceptor
a. Obligate Aerobes  ferment carbohydrates to lactic acid
 Organisms that require oxygen Example: Lactobacilli, Propionibacterium acnes
 21% oxygen and 0.03% CO2 Anaerobes
 incubation in air or an aerobic incubator with  Reducing agents
10% carbon dioxide present satisfies their
Example: THIOGLYCOLATE
oxygen requirement
 Tubes of agar can be sealed with a layer of
Example: Bordetella, Brucella, Mycobacteria,
petrolatum and paraffin
Pseudomonas
 Culture vessel can be placed in a container from
b. Facultative Anaerobes
which the oxygen is removed by evacuation or
 can use oxygen when it is present but are able by chemical
to continue growth by using fermentation or
 Organism can be handled within an anaerobic
anaerobic respiration when oxygen is not
glove box
available
Example: Enterobacteriaceaec. Note!!!

c.Microaerophiles Obligate Aerobes and Facultative Anaerobes

 require a reduced level of oxygen to grow  contain Superoxide dismutase and Catalase

 2%-10% oxygen that counter the toxic effects of oxygen

 generated in culture jars or pouches using a

commercially available microaerophilic
atmosphere–generating system
5% O, 10% CO2, 85% N2
Example: Campylobacter spp. requires 5% to 6%
oxygen
Treponema pallidum
2. Anaerobes grow in the absence of
atmospheric oxygen
a. Obligate Anaerobes
 bacteria that are unable to use molecular oxygen
for energy-yielding reactions
 lack both superoxide dismutase and catalase
0% O, 5-10% CO2, 80-90% N2, 5-10% H2
Example: Clostridium, Bacteroides

FPRE
41
CARBON DIOXIDE REQUIREMENT
Capnophilic
 organisms grow best when the atmosphere is
enriched with extra carbon dioxide
15% O, 5% to 10% CO2 (CO2 incubator or bags);
3%CO2 (Candle jars)
Example: HACEK, Neisseria gonorrhoeae
----------------------------------------------------------------------
 Diagnostic microbiology laboratories often
maintain their aerobic incubators at a 5% to 10% A. LAG PHASE
carbon dioxide level Adjustment Phase or Physiologic Stage
 Most aerobic and facultative aerobic bacteria  period during which cells adapt to their new
need 0.03% CO2 environment
BACTERIAL GROWTH  little or no cell division
Generation Time/ Doubling time  intense metabolic activity involving synthesis of
time required for one cell to divide into two cells enzymes and various molecules

 20 minutes for a fast-growing bacterium such as B. LOG PHASE

E. coli Exponential Phase
 24 hours for a slowgrowing bacterium such as  cells begin to divide and enter a period of growth,
Mycobacterium tuberculosis or logarithmic increase

GROWTH CURVE  Cellular reproduction is most active

When the growth of a bacterial culture is plotted generation time reaches a constant minimum
during balanced growth, the resulting curve shows  cells are most active metabolically
four phases of growth:
 Phase in which microorganisms are utilized in
(1)Lag phase bacteria are preparing to divide physiological and biochemical testing
(2)Log phase bacteria numbers increase  Stage where the organism are most
logarithmically susceptible to antibiotics
(3) Stationary phase nutrients are becoming Continues until:
limited and the numbers of bacteria remain
1. Either one or more nutrients in the medium
constant become exhausted
(4)Death phase number of nonviable 2. Toxic metabolic products accumulate and inhibit
bacterial cells exceeds the number of viable cells growth

FPRE
42
C. STATIONARY PHASE
Phase of Equilibrium or Plateau Phase
 growth rate slows, the number of microbial
deaths balances the number of new cells, and
the population stabilizes
 metabolic activity of dividing cells slows down
 total cell count slowly increases, although the
viable count stays constant
Reason:
a. exhaustion of nutrients
b. accumulation of toxic products causes
c. harmful changes in pH and other factors
D. DEATH PHASE
Logarithmic Decline Phase
 number of deaths eventually exceeds the
number of new cells formed
 period when there is cessation of bacterial
growth
 continues until the population is diminished to a
tiny fraction of the number of cells in the
previous phase or until the population dies out
entirely
 lost of nutrients and increase in the amount of
toxic
 waste decrease in the number of viable
organism but not in the total bacterial count
FPRE
DETERMINATION OF CELL NUMBERS
1. Direct counting under the microscope
 estimate the number of bacteria present in a
specimen
does not distinguish between live and dead cells
2. Direct plate count
 dilutions of broth cultures on agar plates--colony
forming units per milliliter (CFU/mL)
 provides a count of viable cells only
 determining the bacterial cell count in urine
cultures
3. Density measurement
 correlated to CFU/mL of the culture
 prepare a standard inoculum for antimicrobial
Susceptibility testing
Direct Measurement of Microbial Growth
a. Plate Counts
 measures the number of viable cells
 often reported as colony-forming units
a. Serial Dilutions
b. Pour Plates and Spread Plates
Pour Plates
1.0 ml or 0.1 011 of dilutions of the bacterial
suspension is introduced into a Petri dish
Spread Plates
 0. I-ml inoculum is added to the surface of a
prepoured, solidified agar medium
 spread uniformly over the surface of the medium
with a specially shaped, sterilized glass o r metal
rod
43
 bacteria are left to grow in the tubes in a dilution
series
Direct Microscopic Count
 measured volume of a bacterial suspension is
placed within a defined area on a microscope
slide
 used to count the number of bacteria in milk
***Petroff-Hausser cell counter
used in direct microscopic counts

Estimating Bacterial Numbers by Indirect

Methods
a. Turbidity
 Spectrophotometer
McFarland Standards
Nephelometric Method

b. Filtration
b. Metabolic Activity
 water are passed through a thin membrane filter
whose pores are too small to allow bacteria to metabolic product is in direct proportion to the
pass number of bacteria present

 detection and enumeration of coliform bacteria c. Dry Weight

c. Most Probable Number (MPN) Method

 statistical estimating technique
 based on the fact that the greater the number of
bacteria in a sample, the more dilution is needed
to reduce the density to the point at which no

FPRE
44
UNIT 9: CULTURE AND CULTURE MEDIA
PRINCIPLES OF BACTERIAL CULTIVATION Types:
✓Grow and isolate all bacteria present in a clinical a. Pure (Axenic) Culture
specimen
→Composed of only one species
✓Determine which of the bacteria that grow are
b. Mixed Culture
most likely causing infection and which are likely
contaminants →Composed of more than one species
✓Obtain sufficient growth of clinically relevant c. Stock Culture
bacteria to allow identification, characterization, and
susceptibility testing →Composed of several species contained in a
separate culture medium---one specie per culture
TERMINOLOGIES medium
Cultivation →Grown in a large volume of broth and then divided
into small freezer vials---lengthen the shelf life of
→process of growing microorganisms in culture by
specimen to at least a year
taking bacteria from the infection site (in vivo
environment) by some means of specimen collection Agar
and growing them in the artificial environment of the
laboratory (in vitro environment) →Sulfated polymer made up of D-galactose, 3,6-
anhydro-Lgalactose, and D-glucoronic acid and
Culture Medium usually derived from red algae
→nutrient material prepared for the growth of →melt at 80°C-90°C (100°C) and solidify at 40°C-
microorganisms in a laboratory 50°C
→composed of mixture of nutrients: Carbon, Note!!!
Nitrogen, Sulfur, Phosphorus, Hydrogen, Oxygen
and Buffer 55°C-60°C

→Inhibitory agents →cooling temperature for distribution of culture

medium into Petri plates
→ facilitate isolation of desired organism while
suppressing the growth of other organism 20-25 ml

Inoculum → Amount of molten agar transferred to a sterile

plates
→microbes that are introduced into a culture medium
to initiate growth
CULTURE CLASSIFICATION OF CULTURE MEDIA
→microbes that grow and multiply in or on a culture I. According to Physical State or Consistency
medium
II. According to Composition
III. According to the Dispensing or Distribution
Method for the Medium
IV.According to Use

FPRE
45
I. According to Physical State or Consistency →useful for the isolation of medically significant
bacteria
a. Liquid Medium
Example: Nutrient Broth (NB) Medium, TSB and
→does not contain any amount of agar or solidifying
MAC Agar
substances
c. Tissue Culture Medium
→allows growth of aerobes, anaerobes and
facultative anaerobes →contains living tissues
Example: →used for obligate intracellular bacteria
✓Nutrient Broth Rickettsia and Chlamydia
✓Brain Heart Infusion (BHI) Example:
✓Trypticase Soy Broth (TSB) HeLa 229→ human cervical tissue, Chlamydia
✓Thioglycollate (THIO) McCoy and W 138→ fibroblasts, Chlamydia
b. Semi-solid Medium Embryonated Egg→ propagation of Rickettsia
→contains 0.5% to 1% agar III. According to the Dispensing or Distribution
Method for the Medium
→observed bacterial motility and detect indole and
sulfide production a. Plated Media
Example: Sulfide Indole Motility (SIM) Medium →distributed into sterile petri dish
c. Solid Medium b. Tubed Media
→contains 2% to 3% agar →distributed in sterile test tube
Example: Types:
✓Triple Sugar Iron (TSI) Agar 1. Slant
✓MacConkey (MAC) Agar 2. Butt-Slant
✓Blood Agar Plate (BAP) 3. Butt
✓Chocolate Agar Plate (CAP) Example: TSI, SIM, Simmon’s Citrate Agar (SCA),
Lysine Iron Agar (LIA)
II. According to Composition
a. Synthetic or Defined Medium
→exact chemical composition of the ingredients is
known (commercially prepared culture media)
→used for research purposes as either a liquid or
solid medium
→preferred for the isolation of cyanobacteria and
chemoorganotrophs
Example: BG-11 medium
b. Non-synthetic or Complex Medium
→precise composition of some or all of the nutritive
substances used is not known (Peptone, Meat and
Yeats Extracts)
FPRE
46
IV. According to Use
a. Simple Media, Supportive Media or General
Purpose Media
→routinely used in the laboratory and without
additional supplements
→support growth of most non-fastidious bacteria to
grow at natural rates, without providing advantage to
any particular bacteria
→usually composed of meat and soybean extracts
Example:
✓Nutrient Agar ✓Nutrient Broth ✓TSB
b. Enrichment Media
→enhance the growth of particular organisms
(pathogens) and suppress the growth of normal flora
present in specimen
→contain specific nutrients and without additional
supplements
→ incubated for a certain period and then sub
cultured to isolate the desired organism
→can also be used as a supplement to agar plates
to detect aerobes, anaerobes and microaerophlies
(THIOGLYCOLLATE)
Example:
✓Alkaline Peptone Water (APW)
→promote growth of Vibrio spp. before inoculation
into Thiosulfate-Citrate-Bile-Salts(TCBS) Agar
→adjusted to pH 8.5
✓Selenite F
→ isolation of Salmonella from feces, urine and
water Sample
✓Thioglycollate
→general support enrichment medium that promotes
the growth of almost all non-fastidious bacteria
Components:
▪ Dextrose, Vitamin K1, and Hemin have
been used to modify the basic thioglycollate formula
▪ 0.075% agar
FPRE
▪ Resazurin→ oxidation-reduction indicator
▪ Thioglycolic Acid→ reducing agent
✓Tetrathionate
→Selective enrichment broth for the isolation
of Salmonella and Proteus
****Bile Salt and Thiosulfate→ suppresses the
growth of other coliform bacilli
✓Gram-Negative Broth
→isolation of Salmonella and Shigella
→Enrichment and Selective medium
▪Sodium Citrate and Sodium Desoxycholate
(a bile salt) → inhibit gram-positive organisms
▪Mannitol→ primary carbon source
✓Lim Broth (Todd Hewitt with CNA)
→ Group B Streptococci
Thioglycollate Broth
c. Enriched Media
→media with additional supplements necessary for
growth of fastidious organisms
→Supplements: Blood, Vitamins, Serum, Peptone
and Yeast Extract
→solid type media
Example:
✓Blood Agar Plate (BAP)
→contains 5% defibrinated blood
→Differentiate haemolytic pattern of bacteria
Choices of blood: Sheep, Horse, Rabbit
47
 Example:
✓BAP
→ Hemolytic pattern of Streptococci
✓Eosin Methylene Blue (EMB)
→Lactose and Sucrose
→Eosin and Methylene Blue
✓Hektoen Enteric Agar (HEA)
✓MacConkey Agar
→differentiate Lactose Fermenter (pink colonies)
from Non-Lactose Fermenter (colorless colonies)
Components:
▪ Lactose
▪ Bile Salts
▪ Crystal Violet→ inhibit gram-positive bacteria and
fungi
▪ Neutral Red→ pH indicatorMacConkey Medium

✓Chocolate Agar Plate

→blood has been chemically-treated or heat-treated
(80°C) to lyse the RBC
→isolation of fastidious microorganisms:
***Neisseria gonorrhoeae and Haemophilus spp.

Note!!!!
✓Hemin
→ “X” factor
MacConkey Medium
✓Nicotinamide Adenine Dinucleotide (NAD)
***Neutral Red→ pH indicator
→“V” factor
e. Selective Media
d. Differential Media
→support the growth of one type or group of
→allow the visualization of metabolic differences microbes but not another
between groups of bacteria
→contain inhibitory substances such as
→distinguishes organisms growing together by their antimicrobials, dyes, or alcohol which inhibit the
diffrences in cultural characeristics growth of other organisms while promoting the
growth of the desired organism
→allow grouping of microbes based on different
characteristics demonstrated on the medium
FPRE
48
INHIBITORY AGENTS Hektoen Enteric (HE) Agar
1. Inhibit growth of Gram-Positive Microorganism
✓Crystal or Gentian Violet
✓Basic or Carbol Fuchsin
✓Bile Salts
✓Sodium Desoxycholate
2. Inhibit growth of Gram-Negative
Microorganisms
✓Potassium Tellurite
✓Sodium Azide Xylose-Lysine-Desoxycholate (XLD) Agar
3. Prevent Swarming of Proteus →Shigella spp. and Salmonella spp.
✓Alcohol Components:
✓Chloral Hydrate ▪Lysine, Lactose, Xylose and Sucrose
Example: ▪0.25% Sodium Desoxycholate
✓HEA → inhibits gram-positive bacteria
✓MAC Phenol Red→ pH indicator
✓Xylose Lysine Deoxycholate (XLD) Ferric Ammonium Citrate→ H2S indicator
✓Bismuth Sulfite Agar (BSA) ***Salmonella→ colonies are red with
✓Mannitol Salt Agar (MSA) black center
✓Thayer Martin Agar (TMA) Xylose-Lysine-Desoxycholate (XLD) Agar
✓Salmonella-Shigella Agar (SSA)
✓TCBS
Hektoen Enteric Agar (HEA)
→ Salmonella spp. and Shigella spp.
→bile salts and dyes (bromthymol blue and acid
fuchsin)
Bromthymol Blue→ pH indicator
Ferric Ammonium Citrate
→ H2S indicator
***Salmonella→ black precipitate

FPRE
49
MEDIA FOR GRAM-POSITIVE BACTERIA ▪ Trimethoprim Lactate***
a. Columbia CNA with Blood → inhibit Proteus spp
→three peptone sources and 5% defibrinated C. TRANGROW MEDIUM
sheep blood →Thayer-Martin with glucose, 2% agar,
Trimethoprim Lactate and CO2 incorporated in bottle
Colistin (C) and Nalidixic Acid (NA)
D. MARTIN-LEWIS AGAR
→suppress the growth of most gram-negative
organisms →substitute Anisomycin*** for Nystatin and higher
concentration of vancomycin
b. Phenylethyl Alcohol (PEA)
E. NEW YORK CITY MEDIUM
Agar
→ Modified Thayer-Martin with substitution of
→sheep blood agar supplemented with phenylethyl
alcohol to inhibit the growth of gram-negative Amphotericin B*** for Nystatin
bacteria
OTHER SELECTIVE MEDIA
Culture Media for Neisseria spp.
a. Gentamicin Blood Agar
A. THAYER-MARTIN
→ Streptococcus
→enriched Chocolate Agar with supplement B or
b. Bacitracin Chocolate Agar
Isovitale X
→ Haemophilus
Antibiotic Components:
c. Blood Agar Plate with Ampicillin
▪ Colistin
→ Aeromonas
→ inhibit gram-negative bacteria
d. Selective and Differential Media
▪ Vancomycin
→used in the primary isolation of enteric
→inhibit gram-positive bacteria,
GramNegative bacteria
▪ Nystatin
Example:
→ inhibit yeast
✓Endo Agar
B. MODIFIED THAYER-MARTIN AGAR
✓XLD Agar
→Neisseria gonorrhoeae and Neisseria meningitides
✓MAC Agar
→chocolatized blood + antibiotics
✓EMB Agar
Antibiotic components:
▪ Colistin
→ inhibit gram-negative bacteria
▪ Vancomycin
→inhibit gram-positive bacteria,
▪ Nystatin
→ inhibit yeast

FPRE
50
e.Special Media
→isolate bacteria with specific growth requirements
→specially prepared to support the growth of specific
microorganisms
Example:
✓Middlebrook 7H-10 Agar→ M. tuberculosis
✓Fletcher Medium→ Leptospira
✓“W”or Winsconsin Medium→Brucella
✓Bordet-Gengou Agar→ Bordetella pertussis
✓Thayer Martin→ Nesseria
✓MacBride→Listeria monocytogenes
✓Dieudonn’sMedium→ Vibrio cholerae
Lowenstein-Jensen
→protein rich medium composed of whole egg and
malachite green and supports the growth of
Mycobacteria
→Sterilization: Inspissation not Autoclaving
Thiosulfate-Citrate-Bile Salts-Sucrose
(TCBS) Agar
→selective for the isolation of Vibrio
→“Special Medium”
→Sterilization: Boiling not Autoclaving
Plating Media for Routine Bacteriology

FPRE
51
General Steps in Preparation of Culture Medium
TUBE METHOD
1. Weighing→ weigh the different ingredients then
place in clean, dry containers
✓Stabbing of medium is usually performed with
group A streptococci to create anaerobiosis and
promote subsurface hemolysis
✓Overlapping inoculation →used for antimicrobial
sensitivity

2. Dissolving→ add the exact amount of solvent to Test

the ingredients and then dissolve by heating
I. Inoculation of Tubed Media
3. Titration→ adjustment to the right pH : 7.2-7.4
a. Liquid Medium
4. Distribution→ distribute in test tubes
➢with the use of sterile Pasteur pipet
5. Sterilization
➢inoculate by shaking a previously heated wire loop
PLATED MEDIUM or needle

1.Weighing b. Slant Medium

2.Dissolving ➢With the use of a wire loop or needle, transfer the

inoculum to the bottom of the slant and streak in a
3.Titration zig-zag manner across the entire surface toward the
4.Sterilization mouth of the tubec.

5.Distribution Butt Medium

INOCULATION OF MEDIA ➢Just stab the medium with an inoculating needle

SPECIMEN CONSIDERATION d. Butt/Slant Medium

✓Sterile body fluids, pus, urine and sputum ➢Inoculate the butt first by stabbing the needle to
the bottom of the medium and then streak the
→inoculated directly into the selected media surface in a zig-zag manner
✓Specimens received on swabs toward the mouth of the tube
→can be inoculated directly into the culture media II. Inoculation of Plated Media
✓Specimens that require direct or “bedside” a. Streak Plate Technique
inoculations:
→isolate organisms in pure culture
Blood, Genital specimens, Corneal Scrapings,
Sterile Fluids like Synovial and Peritoneal Fluids b. Pour Plate
and Nasopharyngeal Swabs for isolation of →determine the approximate number of viable
Bordetella pertusis organisms in a liquid such as water, milk, urine or
INOCULATION TECHNIQUES broth culture

✓Streaking→ most common manner of inoculation c. Streak-Pour Plate

✓Specimen collected through a swab: gently roll the → for studying hemolysis
tip of the swab onto the upper portion of the plate;
inoculated area should be streaked by a sterile loop
afterwards
✓Placement of fluid specimen or swabs into a broth
or liquid culture media

FPRE
52

QUANTITATIVE ISOLATION
→Urine specimens
→use a calibrated loop to deliver a specified volume:
0.01 or 0.001 mL
→calibrated loop inserted into the urine and
transferred to the culture medium by making a single
streak down the center of the plate
→without flaming, the loop is streaked back and forth
through the original inoculum
Manner of Reporting (Grading) of
Growth on Plate
4+ = many, heavy growth; growth is up to the fourth
quadrant
3+ = moderate growth; growth is up to the third
quadrant
2+ = few or light growth; growth is in the second
quadrant
1+ = rare growth; growth is in the first quadrant only

FPRE
53
METHODS OF OBTAINING PURE CULTURE
✓Streak-plate method
✓Pour- plate method
✓Use of selective media and media containing
antibiotic
✓Animal inoculation test
COLONY MORPHOLOGY
✓Colony size
→usually measured in millimeters or describedvas
pinpoint, small, medium, large
✓Colony pigmentation
✓Colony shape
→includes form, elevation, and margin of the colony
✓Colony surface appearance
→glistening, opaque, dull, dry, transparent
✓Changes in agar media resulting from bacterial
growth
TYPES OF COLONY
→hemolytic pattern on blood agar, changes in color
A. Mucoid (M) Colony
of pH
→exhibits a water-like, glistening, confluent
indicators, pitting of the agar surface
appearance
✓Odor
→characteristic of organisms that form slimes or
→certain bacteria produce distinct odors that canbe welldeveloped capsules
helpful in preliminary identification
Examples: K. pneumoniae, S. pneumoniae, H.
influenzae
B. Smooth (S) Colony
→uniform texture and homogeneity
→asily emulsified in NSS
→haracteristic of freshly isolated wild type
microorganisms (virulent microorganisms)
• Examples: Salmonella, Shigella, E. coli, Serratia,
Proteus

FPRE
54
C. Rough (R) colony β-Hemolysis
→granulated and rough in appearance →complete clearing of erythrocytes in BAP around or
under the colonies because of the complete lysis of
→hard to emulsify in NSS
RBCs
→usually produced by mutant strains that lack
Example: Streptococcus pyogenes→ wide, deep,
surface proteins or polysaccharides indicating loss of
clear zone of β-hemolysis,
virulence
Streptococcus agalactiae and Listeria
Examples: Rough forms of enteric bacteria
monocytogenes →narrow, diffuse zone of β-
Exception: R forms of B. anthracis and human and
hemolysis close to the colony
bovine types of M. tuberculosis (more virulent)
b. Size
Gross Colony Characteristics Used to
→ described as large, medium, small, or pinpoint
Differentiate and Identify Microorganisms
→gram-positive bacteria produce smaller colonies
Presumptively
than gram-negative bacteria
a. Hemolysis
→Staphylococcus spp. are usually larger than
→observed in the media immediately surrounding or Streptococcus spp.
underneath the colony is a reaction caused by
LEFT BAP: small, white colonies are
enzymatic or toxin activity of bacteria
grampositive cocci
→presumptive identification of streptococci and
RIGHT BAP: large, gray, mucoid colonies are
enterococci enteric gram-negative Rods

Culture Media: Blood Agar (BAP)

α-Hemolysis
→partial lysing of erythrocytes in a BAP around and
under the colony that results in a green discoloration
of the medium
Example: Streptococcus pneumoniae and certain
viridans streptococci

FPRE
55
c. Form or Margin →raised, convex, flat, umbilicate (depressed center,
concave—an “innie”), or umbonate (raised or
→edge of the colonies
bulging center, convex—an “outie”)
→described as smooth, filamentous, rough or
Example:
rhizoid, or irregular
S. pneumoniae → umbilicate colonies (unless the
Example: Bacillus anthracis → “Medusa Heads”--
colonies are mucoid)
filamentous appearance
S. aureus→ convex colonies β-hemolytic
Proteus mirabilis and Proteus vulgaris→swarming
streptococci → flat colonies
phenomena
e. Density →transparent, translucent, or opaque
Swarming→ hazy blanket of growth on the surface
that extends well beyond the streak lines Example: ✓β-Hemolytic streptococci except group B
(S. agalactiae)→ translucent
Diphtheroids→ rough edges
✓S. agalactiae→ semiopaque
✓Staphylococci and other gram-positive bacteria→
opaque
✓Most gram-negative rods→ opaque
✓Bordetella pertussis→ shiny, similar to a half-
pearl, on blood-containing media

d. Elevation
→determined by tilting the culture plate and looking
at the side of the colony

FPRE
56
f. Color Example:
→white, gray, yellow, or buff S. aureus → creamy
Example: Nocardia spp.→ brittle, crumbly, and wrinkled,
resembling bread crumbs on a plate
✓Coagulase-negative staphylococci→ white
Diphtheroid→ dry and waxy
✓Enterococcus spp. → gray
h. Pigment
✓Certain Micrococcus spp. and Neisseria
(nonpathogenic) spp. → yellow or off-white Pseudomonas aeruginosa→ green
✓Diphtheroids→ buff Serratia marcescens→ brick-red (especially at
room temperature)
✓Most gram-negative rods → gray on BAP
Chromobacterium violaceum→ purple
Prevotella melaninogenica→ brown-black
(anaerobic)
Kluyvera spp.→ blue
Pseudomonas aeruginosa

Serratia marcescens

g. Consistency
→determined by touching the colony with a
sterileloop
→ brittle (splinters), creamy (butyrous), dry,
orwaxy

FPRE
57
i. Odor Erysipelothrix rhusiopathiae
S. aureus→ old sock---Mannitol Salt Agar ✓ BAP, tellurite, gelatin (testtube brush growth)
P. aeruginosa→ fruity or grapelike ✓ BHIA w/ 1% glucose, 5% CO2 at 35-37C
P. mirabilis→ putrid Nocardia
Haemophilus spp.→ musty basement, “mousy” or ✓ LJ, BHIA, SDA
“mouse nest” smell
Clostridium/C. perfringens
Nocardia spp.→ freshly plowed field
✓BAP = double zone hemolysis
OTHER MEDIA
✓Chopped meat glucose media = +gas
Staphylococcus aureus
✓Milk media = stormy ferm.
✓CNA ✓Chapman stone agar ✓Vogel-Johnson
C. tetani
medium ✓PEA, MSA
BAP = swarming, faint beta hemolysis
Streptococci
C. botulinum
✓ PEA, Todd-Hewitt broth, CAN
BAP = beta hemolysis
Neisseria
C. difficile
✓ Thayer-Martin ✓ Modified Thayer-Martin
BAP = yellow green w/ horse stable odor
✓ Transgrow medium ✓ Martin-Lewis
CCFA = yellow ground glass colonies
✓ New York City medium
Actinomyces israelii
Mycobacterium
BHIA = molar tooth colony/ breadcrumb-like,
✓ Dubos oleic acid medium
raspberry or smooth colony
✓ Middlebrook 7H10 or 7H11 ✓ Mitchison’s
ENTEROBACTERIACEAE
selective 7H11 ✓ Petragnani ✓ LJ ✓ Dorset egg
✓ MAC ✓ EMB ✓ Desoxycholate agars
✓ American Thoracic Society medium
✓ HEA ✓ XLD ✓ SSA ✓ Desoxycholate citrate
✓ Bactec 12B medium ✓ Middlebrook 7H9 broth
agars
**M. leprae – nonculturable
***Enrichment: Selenite F GN broth Tetrathionate
**M. fortuitum grows on MAC broth
Corynebacterium diphtheriae a. E. coli
✓Loeffler’s coagulated serum media ✓MAC, EMB, XLD ✓SMAC
✓Modified tinsdale b. Edwardsiella
✓Cystine tellurite media MAC = colorless
✓Pai’s coagulated egg media c. Shigella
Listeria monocytogenes ✓ EMB, MAC, SSA = colorless
✓ McBride agar ✓ PEA ✓ Cold enrichment ✓ XLD = red
technique
✓ HEA = green to blue green

FPRE
58
d. Salmonella Brucella
✓ EMB, MAC, SSA = colorless ✓ Castaneda biphasic medium
✓ XLD, BSA = black colonies w/ metallic silver ✓ W or Winsconsin medium
sheen ✓ Brilliant Green agar (BGA) ✓ HEA
✓ Trypticase soy agar
Enrichment: Selenite, GNB
Bordetella
e. Citrobacter
✓ Bordet-Gengou agar
KCN medium
✓ Jones-Kendrick charcoal agar ✓ Regan-Lowe
f. K. pnemoniae
✓ BCYE ✓ cold casein hydrolysate
EMB, MAC, XLD = +string’s test
✓ Casamino acid broth ✓ Modified Stainer-Scholte
f. Proteus agar with cyclodextrin and Cephalexin
BAP = burnt gun odor , swarming Kingella
g. Yersinia Y. enterocolitica= CIN BAP/CAP = “fried-egg” pitting appearance
Pseudomonas aeruginosa Vibrio cholerae
✓MHA ✓Pseudomonas P agar ✓Tech agar ✓APW, TCBS, ✓Gohar, Dieudonne’s, Monsur and
Aronson media
Eikenella
Campylobacter jejuni
CAP = corrodes, pearly sheen (mercury droplet),
bleach-like odor ✓ Butzler’s medium
Flavobacterium ✓ Skirrow’s medium (also Helicobacter)
BAP = yellow ✓ Campy Thio medium
Haemophilus ✓ Campy-BAP medium
✓ Satellite phenomenon, dewdrop like, bleachlike Leptospirae
✓ CAP ✓Fletcher’s medium
✓ Levinthal and Fildes enriched media ✓Noguchi’s
✓ Horseblood bacitracin by Klein and Blazevic ✓Stewart’s
Actinobacillus /actinomycetemcomitans ✓Ellinghausen, McCullough, Johnson and Harris
(EMJH) medium
CAP = dots and dashes of Morse code
Chlamydia trachomatis
Pasteurella
✓McCoy cells ✓C. pneumoniae ✓HeLa 229
musty or mushroom-like odor (BAP)
Mycoplasma
Francisella tularensis
✓SP-4 Mycoplasma medium ✓Edward-Hayflick
✓Glucose cysteine blood agar
agar ✓Shepard’s A-7B agar
✓Peptone cysteine agar
Rickettsia
✓Cystine heart agar
✓Chick embryo
✓Chocolate agar ✓Rarely on Thayer Martin

FPRE
59
Bartonella ANAEROBIC CULTURE METHODS
✓ BHIA A. Anaerobic Jars
Chromobacterium 1. Brewer Jar
✓ smell of ammonium cyanide, violet in BAP →Oxygen is removed by means of electrically
heated platinized catalyst with the electrical
Gardnerella vaginalis
connection outside the jar
✓ HBBT
2. Torbal Jar
Legionella
→uses a rubber O ring rather than Plasticine and a
✓ BCYE ✓ Feeley-Gorman medium catalyst active at room temperature thus requiring no

✓ MHA w/ hemin/isovitalex electrical heating

ANAEROBIC CULTIVATION 3. GasPak Jar

Ways to facilitate Anaerobic Cultivation →most convenient and widely used anaerobic jar

a. Special culture medium incorporated with →takes 30 to 45 minutes to obtain anaerobic

environment
Thioglycollate and Cystein (reducing agents)
Components
b. Boiling of culture medium
➢Hydrogen and CO2 generator envelop→
c. Anaerobic chamber system with a vacuum pump activated with water
and nitrogen gas to remove residual oxygen
➢Palladium-coated alumina pellets→ catalyst
d. Gas-pak jar conataining a palladium catalyst
➢Methylene Blue or Resazurin Indicator Strip
e. Small volumes: plastic bags, pouches containing
→ upon exposure to atmospheric oxygen turns blue
calcium carbonate and catalyst or pink
Specimen Collection Principle:
✓Aspirated with a sterile needle and syringe ➢With water added to the CO2 and H2 generator
✓Specimens are swabbed and immediately put into envelop and oxygen catalysed with H2 to water via
evacuated tube filled with oxygen-free CO2 or the pellets, anaerobiosis is achieved
nitrogen (gassed-out tube) containing an agar or Indication of Anaerobiosis
broth indicator system or other anaerobic transport
device: ✓Production of heat

➢Anaerobic Culturette ✓Moisture inside jar

➢Bio-Bag ✓Decolorization of indicator strip: WHITE or

COLORLESS
➢Anaerobic Pouch
Note!!!
➢GasPak Pouch
Failure to achieve anaerobic condition→ result of
✓Never used a dry swab or a regular aerobic
transport “poisoned” catalyst or a crack in the O ring, jar or lid

Media

FPRE
60
Jars utilizing the “Evacuation-Replacement” b. Palladium Pellets
System
→ remove residual oxygen by combining with H2 to
✓Air is removed by drawing a vacuum of 25 inches form water
of mercury
c. Silica Gel (Dessicant)
✓Jar is filled with oxygen-free gas such as nitrogen
→ absorbed water
between evacuations of air
d. Methylene Blue or Resazurin
✓Anaerobiosis is achieved more quickly with this
method but less convenient for routine use → oxygen reduction indicator
C. Role Tube Technique
→Pre-Reduced Anaerobically Sterilized (PRAS) agar
is distributed under anerobic conditions as thin layer
around the inner wall of test tubes
→tubes are rolled and cooled until the melted agar
forms the thin layer Inoculated in 2 ways:
Close Method by Hungate
→Syringe and needle are used through the rubber
seal
Open Method
→ remove the rubber stopper and insert a cannula
that has oxygen-free gas flowing from the tip
CULTURE MEDIA
1. Freshly made BAP
B. Glove Box or Anaerobic Chamber
2. Enrichment media (supplemented BHIA, special
→Enclosed sytem consist of large clear plastic, Brucella Blood Agar, Laked Blood Agar)
airtight bag or chamber filled with oxygen-free gas
mixture of nitrogen, hydrogen and CO2 3. Selective Media

→Allow materials to enter through an air lock Kanamycin-Vancomycin Blood Agar

→Anaerobiosis---maintained by Palladium catalyst → anaerobic gram(-) rods

and Hydrogen gas Kanamycin-Vancomycin-Laked Blood Agar
→Opertor uses gloves or sleeves that form airtight → Bacteroides melaninogenicus
seals around arms to manipulate items Neomycin-Vancomycin Blood Agar
Components → Fusobacterium and Veilonella
a. Nitrogen Gas Neomycin Blood Agar
→ filler for the remaining percentage of the → Clostridia and anaerobic
anaerobic straucture
gram(+) cocci
Nagler Agar (with egg yolk, Neomycin)

FPRE
61
→ Clostridia
4. Pre-Reduced Anaerobically Sterilized (PRAS)
media
5. Liquid Media
a. Mixture of Pyrogallol, KOH and Na2CO3
b. Cooked meat medium sealed with petrolatum
✓Petrolatum→ prevents entry of O2
✓Broth over meat
✓Chopped meat at the bottom of the tube→ provide
low redox potential
c. Thioglycolate Medium
→ only medium capable of supporting growth of 3
groups of microorganisms: Aerobes, Microaerophiles,
Anaerobes
Na Thioglycollate
→reducing substance, provides low redox potential
Resazurin Indicator
→narrow pink layer means that the medium is
reduced and can be used for anaerobic culture
→Pink layer extends to 1/3 of the medium: oxidized-
boiled or autoclave to restore the narrow layer
Note!!!
Thioglycolate Medium
→tored at room temperature in the dark because at
refrigerator temperature it absorbs more oxygen

FPRE
62
UNIT 10: CONTROL OF MICROORGANISMS
CONTROL OF MICROORGANISMS: ANTISEPTIC
DISINFECTION AND STERILIZATION
→chemical substance which opposes sepsis or
putrefaction either by killing microorganism or
preventing their growth
STERILIZATION vs DISINFECTION
→applied topically to living tissues
STERILIZATION
Example: Phisohex, Hexachlorophene, Tincture of
→ complete removal or destruction of all formsof
life, including bacterial spores Iodine/Povidone Alcohol (Iodophore)
→chemical or physical methods DECIMAL REDUCTION TIME (DRT/D/D)
DISINFECTION →time in minutes to reduce the bacterial population
or spores by 90% at a specified temperature
→process that eliminates a defined scope
Factors That Influence the Degree of Killing
of microorganisms
Microorganisms
→process of killing or removing microorganisms in
a. Types of organisms
inanimate surfaces thru the use of chemical agents
✓Bacterial spores→ resistant
→destroys pathogenic organisms, but not necessary
all microorganisms or spores ✓Acid-Fast Bacilli
→most are chemical substances ✓Nonenveloped viruses
TERMINOLOGIES ✓Vegetative bacteria
STERILE ✓Enveloped viruses
→free of life of every kind Note!!!
BACTERIOSTATIC Prions
→having the property of inhibiting bacterial growth or → most resistant to the actions of heat, chemicals,
multiplication and radiation
BACTERICIDAL →naked pieces of protein without nucleic acid
→having the property of killing or destroying bacteria →degenerative diseases of the nervous system
(transmissible spongiform encephalopathy—mad
→precipitates bacterial protein (H2SO4, HCl)
cow disease, CreutzfeldtJakob disease)
GERMICIDE OR DISINFECTANT
b. Number of organisms
→chemical substance used to kill infection producing
✓higher numbers of organisms require longer
microorganisms on surface but too toxic to be
exposure times
applied directly on tissues
c. Concentration of disinfecting agent
SEPTIC
d. Presence of organic material
→characterized by the presence of pathogenic
microbes in living tissue ✓blood, mucus, and pus
ASEPTIC f. Nature of surface to be disinfected
→characterized by the absence of pathogenic
microbes

FPRE
63
f. Contact time →sterilization method of choice for heat-stable
objects
✓Alcohol and Iodine preparations (Betadine) → at
least 1 to 2 minutes to kill microorganisms 1. BOILING
g. Temperature →100°C for 15-30 minutes (20 minutes minimum)
h. pH →surgical instruments, needles, hypodermic
syringes, rubber stoppers
i. Biofilms
→kills all vegetative organism but not all spores or
j. Compatibility of disinfectants and sterilants
viruses
2. AUTOCLAVING
→ Sterilize biohazardous trash and heat-stable
objects
→ all microorganisms (except for prions) and their
endospores are destroyed within approximately 15
minutes of exposure
Autoclave
→ chamber which is filled with hot Steam Under
Pressure
METHODS OF DISINFECTION AND Two common sterilization temperatures:
STERILIZATION
121°C (250°F), 15 psi for 15 minutes→ media,
liquids, and instruments
132°C (270°F), 15 psi for 30-60
minutes→infectious medical waste
BIOLOGIC INDICATOR: Geobacillus
stearothermophilus (B. stearothermophilus)
incubated at 56°C
Gravity Displacement Type
PHYSICAL METHODS → most commonly used steam sterilizer in the
I. HEAT microbiology laboratory

→most common method used for the elimination of

microorganisms
→most reliable and universally applicable method of
sterilization
A. Moist Heat
→kills microorganisms rapidly than dry heat
→Lethal effect is attributed toDENATURATION and
COAGULATION of protein and DEGRADATION of
nucleic acid
→fastest and simplest physical method of
sterilization
FPRE
64
3. FLOWING STEAM →Cooled very quickly in a vacuum chamber
→ Sterilizing heat-sensitive culture media containing Advantage: milk can be stored at RT for 2 months
carbohydrates without affecting its flavor
FRACTIONAL/INTERMITTENT STERILIZATION/ B. DRY HEAT
TYNDALLIZATION
→requires longer exposure times (1.5 to 3 hours)
→Materials are exposed for 30 minutes for 3 and higher temperatures than moist heat (160° to
successive days 180°C)
✓Day 1: Vegetative cells ✓Day 2: Sporulated cells →does not require water
✓Day 3: Remaining cells
→sterilization of glasswares, oil products or powders
→Destroys vegetative cells and spores after three
Lethal Effects: Protein denaturation, oxidative
consecutive days
damage and toxic effects of elevated levels of
→Instrument: ARNOLD STERILIZER--use flowing
Electrolytes
steam
1. FLAMING OR DIRECT HEATING
→100°C for 30 minutes4. INSPISSATION
Flaming with a Bunsen Burner
→Principle: thickening through evaporation
→flaming mouth of culture tubes or slides
→Sterilize high protein containing media that cannot
withstand high temperature Burning with a Bunsen Burner
Example: LJ, Loeffler’s and Dorset egg medium →wireloops, forceps and straight wire
→70-80°C for 2 hours for 3 consecutive days 2. HOT AIR OVEN
5. PASTEURIZATION/PARTIAL STERILIZATION →most widely used type of dry heat
→partially sterilizing organic solutions by heat →used for glasswares, certain metals and oils
without altering their chemical properties
→2 hours for 160-180°C kill organisms including all
→used to sterilize milk, dairy products, and alcoholic spore formers
beverages
QC: Bacillus subtilis var. niger (Bacillus
→eliminates food-borne pathogen and organisms atrophaeus) at
responsible for food spoilage
35-37°C
→cannot eliminate bacterial endospores
3. INCINERATION
3 types of Pasteurization
→Priciple: Burning of materials into ashes at 300-
1. Low Temperature Holding (LTH)/Batch Method 400°C
→destroys milk-borne pathogens →most common method of treating infectious waste
and infected laboratory animals
→63°C for 30 minutes
→destruction of sputum cups, garbage and used
2. High Temperature Short-Time(HTST)/Flash
dressings 870-980°C→ hazardous material
Pasteurization
4. CREMATION
→72°C for 15 seconds
→burning dead human bodies control the spread
→quick heating and immediate cooling
of communicable disease
3. Ultra-high Temperature
→140°C for 3 seconds
FPRE
65
II. LOW/COLD TEMPERATURE ✓Seitz (Compressed Asbestos)→98% effective
→considered BACTERIOSTATIC---reduces the rate ✓Membrane filter(Swinney)Millipore 0.22 um
of metabolism
→100% bacterial sterility
→exposure to 2°C for 72 hours kills the agent of
b. Membrane Filter (Circular Filter)
syphilis
→porous membranes (almost 0.1um thick)
FREEZING
→composed of cellulose acetate or polycarbonate
→not reliable method of sterilization
→sterilize pharmaceuticals, ophthalmic solutions,
→repeated freezing and thawing are much more
culture media, antibiotics and oil products
destructive than prolonged freezing
1. Liquid Filtration
→ preservation of bacterial culture via lyophilisation
or freeze-drying Lethal Effects: Protein denaturation, →pulling the solution through cellulose acetate or
toxic effects of cellulose nitrate membrane with a vacuum
increased intracellular electrolyte concentration 2. Air filtration
III. DESSICATION AND LYOPHILIZATION →uses HIGH-EFFICIENCY PARTICULATE AIR
filters
Dessication
→remove microorganisms larger than 0.3 μm from
→disruption of metabolism that involves removing of
isolation rooms, operating rooms, and biologic safety
water from microbes(BACTERIOSTATIC)
cabinets (BSCs)
Lyophilization
c. Filtration of bacteria, yeasts and molds
→changes in proteins and chemical reactions
→uses 0.45 and 0.80 μm pores of membrane filters
Note!!!
→0.2 to 0.45 um in diameter-remove most bacteria
➢Bacteria which remain active in dry environment
as well as fungi but not viruses
✓Neisseria gonorrhoeae→viable for 1 hour
→0.01 μm are capable of retaining small viruses
✓MTB→viable for several months
d. Critical Sterilizing 0.22-μm
✓Bacillus and Clostridium→viable for ten years
→critical sterilizing (parenteral solutions and alcohol)
IV. FILTRATION
→remove vegetative cells but not viruses
→method of choice for antibiotics solutions, toxic
V. RADIATION
chemicals,
Principle: when radiation passes through the cells,
radioisotopes, vaccines and carbohydrates, serum,
free hydrogen and hydroxyl radicals and some
plasma, urea(heat-sensitive solutions)
peroxidase are created which in turn cause different
→may be used with both liquid and air substances intracellular damage

Types of Filters BIOLOGIC INDICATOR: Bacillus pumilus

a. Depth Filters a. Ionizing radiation (Cold Sterilization)

→made up of fibrous or granular materials →gamma rays (1500-2500 radiation), electron

beams, X-rays
Example:
→short wavelength and high energy
✓Berkefield Filter→ Diatomaceous earth
✓Chamberland Filter→unglazed porcelain
FPRE
66
→ used for plastic syringes, catheters, sutures,
gloves, hormone solutions and antibiotics
→causes mutation in the DNA and produces
peroxidase
→destroys vegetative cells and endospores
b. Nonionizing Radiation
→damage to cellular DNA by producing Thymine
dimers
→ultraviolet rays (10 um to 400 um) in which 260 um
is the most lethal
→long wavelength(>1um) and low energy
→poor penetrability
→used to disinfect exposed surfaces, operating
rooms, nursery rooms
→control of airborne infection
c. Ultrasonic and Sonic Vibrations
→no practical value in sterilization and disinfection
since there are
many survivors after the treatment
CHEMICAL METHODS
→used mainly as disinfectants
Chemosterilizers
→chemical agents used to sterilize
Chemical agents exert their killing effect by the
following mechanisms:
✓Reaction with components of the cytoplasmic
membrane
✓Denaturation of cellular proteins
✓Reaction with the thiol (–SH) groups of enzymes
✓Damage of RNA and DNA
FPRE
I. Alcohol
→excellent in vitro bactericidal activity against most
grampositive and gram-negative bacteria
→ also kill Mycobacterium tuberculosis, various fungi,
and certain enveloped viruses
→ not sporicidal and have poor activity against
certain nonenveloped viruses
→ inactivated by the presence of organic material
→should be used in concentrations between 60%
and 90%
→denaturation of proteins and dissolution of lipid
membranes
→used as both antiseptic and
disinfectant(bactericidal and fungicidal)
→allowed to evaporate from the surface to achieve
complete antisepsis
→Isopropanol and EthanoL
II. ALDEHYDES/COLD/CHEMICAL STERILANTS
→inactivation of proteins and nucleic acids
→commonly used in sterilizing medical instruments
→8% formaldehyde and 2% gluteraldehyde
a. Formaldehyde
→used as formalin, a 37% aqueous solution or
formaldehyde gas
Formaldehyde gas→ often used to disinfectant
biosafety hoods, HEPA filters
→Mycobacteria:3%-8% formalin is used with a
contact time of at least 30 minutes
→irritability factor and its potential carcinogenicityb.
Glutaraldehyde
(Pseudomonocidal, Tuberculoidal, Fungicidal and
Virucidal)
→saturated five-carbon dialdehyde
→inactivation of DNA and RNA through alkylation of
sulfhydryl and amino groups
→broad-spectrum activity and rapid killing action
and remains active in the presence of organic matter
67
→extremely susceptible to pH changes because Iodophor
it is active only in an alkaline environment →combination of iodine and a neutral
polymer(Detergent) carrier that increases the
2% solution→ germicidal in approximately 10
solubility of the agent
minutes and sporicidal in 3 to 10 hours→does not
→less irritating, nonstaining, and more stable
penetrate organic material well when used as a
→used as antiseptics or disinfectants
sterilant
→slow and continuous release of free iodine
→sterilizer of choice for medical equipment that is
not heatstable and cannot be autoclaved as well as →Free iodine degrades microbial cell walls and
for material that cannot be sterilized with gas cytoplasm, denatures enzymes, and coagulates
chromosomal material
→ safe, high-level disinfectant for most plastic and
rubber items →Bactericidal action is due to the oxidative effects of
molecular iodine (I2) and hypoiodic acid (HOI)
→ effective against HIV and HBV with a minimum of
10 minutes’ exposure at a temperature between 20° Contact time: 30-60 seconds onto the skin prior to
C and 30° C blood collection
2% solution at 25° C to 30° C→ 100% b. Chlorine and Chlorine Compounds
tuberculocidal
→hypochlorite---liquid sodium hypochlorite
Cold Sterilants → sporicidal in a minimum of 10
(household bleach) and solid calcium hypochlorite
hours’ exposure at room temperature
→oxidative effects of hypochlorous acid, formed
III. HALOGENS
when chloride ions are dissolved in water
→destroys through oxidation process
→not used as sterilants because of the long
→Chlorine, Iodine, Fluorine, Bromine, Astatine exposure time required for sporicidal action
→tincture of iodine and iodophore---effective →inactivated by organic matter
antiseptics
→concentrated bleach solutions should not be used
1:10 NaOCl for disinfection—Corossive
→effective bleach →influenced by the pH of the surrounding medium
→freshly prepared every day with 10-30 minutes of 0.5% to 1% Sodium Hypochlorite
contact time---effective tuberculocide
→used for disinfection
Halozone
→stable for no longer than 30 days
→parasulfone dichloraminobenzoic acid
1 : 10 dilution of 5.25%
→contains Cl and is used to disinfect drinking water
→ recommended by CDC for cleaning up blood spill
a. Iodine Tincture
Note!!!
→alcohol and iodine solutions
Contact time: 3 minutes and longer if organic
→used mainly as antiseptics material is present and 10-30 minutes for
mycobacteria
→2%iodine in 70% alcohol
Disadvantage: ineffective use in the presence of
large amount of protein

FPRE
68
IV. Quaternary Ammonium Compounds →inactive against bacteria spores except at elevated
(Quats)/Detergents temperatures
→derived by substitution of the four-valence →binds to the skin and remains active for at least 6
ammonium ion with alkyl halides hours
→cationic, surface-active agents, or surfactants, that →pH-dependent: 5.5 - 7.0
work by reducing the surface tension of molecules in
a liquid Lipid enveloped viruses (herpesvirus, HIV,
respiratory viruses, influenza virus, cytomegalovirus)
→effectiveness is reduced by hard water and soap, → rapidly inactivated
and they are inactivated by excess organic matter
Nonenveloped viruses (rotavirus, adenovirus,
→disruption of the cellular membrane, resulting in enteroviruses)→ not inactivated
leakage of cell contents
b. Hexachlorophene
→not sporicidal or tuberculocidal
→effective against gram-positive bacteria
→disinfection of noncritical surfaces such as
benchtops and floors →chlorinated bisphenol

Pseudomonas aeruginosa → resistant to quaternary →interrupts bacterial electron transport, inhibits

ammonium compounds membrane-bound enzymes at low concentrations,
and ruptures bacterial membranes at high
Example: Zephiran (Benzalkonium Choride and concentrations
Ceepryn) and
Gram-positive bacteria: 3% hexachlorophene within
cetylpyridium chloride
15 to 30 seconds
V. PHENOLICS
Gram-negative bacteria: longer time
→molecules of phenol (carbolic acid) that have been
c. Chloroxylenol (Parachlorometaxylenol [PCMX])
chemically substituted, typically by halogens, alkyl,
phenyl, or benzyl groups →halogen substituted phenolic compound

→ortho-phenylphenol and ortho-benzyl- →cell wall disruption and enzyme inactivation(0.5%

parachlorophenol to 4%)

→not sporicidal →good activity against gram-positive bacteria, less

active against gram-negative bacteria M.
→stable, biodegradable, and relatively active in the tuberculosis, fungi, and viruses
presence of organic material
→unaffected by organic materials
→disruption of cell walls, resulting in precipitation of
proteins →neutralized by nonionic surfactants and
polyethylene glycol
→able to disrupt enzyme systems---lower
concentarion →intermediate-acting to slow-acting and has minimal
persistent effect of more than a few hours
→found in germicidal soaps
→low antimicrobial efficacy compared with iodines,
a. Chlorhexidine Gluconate iodophors, and CHG in reducing skin flora
→disrupts the microbial cell membrane and d. Triclosan
precipitates the cell contents
→diphenyl ether that disrupts the cell wall
→more effective against gram-positive than gram-
negative bacteria and has less activity against fungi →reaction time is intermediate; persistence is
and tubercle bacilli (0.5% to 4%) excellent

FPRE
69
→good activity against gram-positive bacteria, Hydrogen Peroxide and Periacetic Acid
gramnegative bacteria, and viruses
→shorter contact time
→fair activity against M. tuberculosis and poor
VIII. Acid and Alkaline Solutions
activity against fungi
→hydrolyzes and coagulate proteins
→not affected by organic matter but affected by pH
DISINFECTANT SCREENING TEST: PHENOL
and the presence of surfactants and emollients
COEFFICIENT
VI. Heavy Metals
Phenol Coefficient
→slowly bactericidal; bacteriostatic
→standard reference material for the evaluation of
→inactivating and precipitating cell protein disinfectants
Example: copper, arsenic, mercury, silver and zinc →highest dilution that kills the bacteria after a 10-
minute exposure
Note!!!
→potency or bactericidal power of a disinfectant is
1% Silver nitrate (1% eye drop solution) compared with phenol
→used as a prophylactic treatment to prevent Test organisms: Staphylococcus aureus and
gonococcal (Neisseria gonorrhoeae) conjunctivitis in Salmonella serotype typhi (20°C or 37°C)
Newborns

VII. Gas Highest dilution of disinfectant that will kill organisms

Factors: temperature, time, and relative humidity are at a given time
extremely important in determining the effectiveness Highest dilution of phenol that will kill organisms at a
of gas sterilization given time
Ethylene Oxide Result:
→ most commonly used for sterilization >1= disinfectant better than phenol
→explosive in its pure form; mixed with nitrogen or <1= phenol is much better,
carbon dioxide
Equal to 1=same efficiency as that of phenol
→relative humidity of 30% is optimal for the
destruction of spores Interpretation:

→alkylation of nucleic acids in the spore and Higher the PC value=more effective the disinfection
vegetative cell
End point
Recommended concentration: 450 to 700 mg of
→ lowest concentration that kills the test organisms
ethylene oxide/L of chamber space at 55° C to 60° C
in 10 minutes at 20°C
for 2 hours
VIII. Hydrogen Peroxide and Periacetic Acid
→primarily used as a sterilant in the pharmaceutical
and medical device
→againts all vegetative microorganisms and
bacterial and fungal spores
→used in a gaseous form as a sterilant primarily

FPRE
70
 UNIT 11: HOST-MICROORGANISM
INTERACTION
TERMINOLOGIES HANDWASHING---cornerstone of modern infection
control program
Infection
TYPES OF INFECTION ACCORDING TO HOST
→ growth and multiplication of microorganisms that
cause damage to the host DISTRIBUTION
→bodily invasion of pathogenic microorganisms that 1. Local infection→ signs and symptoms are
reproduce, multiply and then cause disease through confined in one area; wounds, boils, abcesses
local injury, toxin secreation or An-Ab reaction to the
2. Focal infection→starts as a focal infection before
host
spreading to other parts of the body
Types:
3. Systemic infection→spread throughout the body
a. Autogenous infection through the blood or lymph
→caused by microorganism from the microbiota of Bacteremia→presence of bacteria in blood; highest
the host concentration of bacteria in blood occurs before the
fever spikes
b. Iatrogenic infection
Septicemia→active multiplication of bacteria in
→ result of medical treatment or procedure
blood
c. Opportunistic infection
Pyremia→pus-producing organisms repeatedly
→affects immunocompromised host invade the bloodstream and become localized at
different parts of the body
d. Nosocomal infection
Toxemia→presence of toxins in the blood
→hospital-acquired infection
Classification of Disease According to
4 common types: UTI, Lung infection Occurrence
(Pneumonia), Surgical site infection, Blood 1. Sporadic→occurs occasionally
stream infection
2. Endemic→ a disease constantly present at some
Predisposing factors rate of occurrence in a particular location
a. Wide variety of microbes in the hospital 3. Epidemic→ a larger than normal number of
environment diseased or infected individuals in a particular
b. Immunocompromised patient location

c. Chain of transmission (direct or Indirect) 4. Outbreak→ a larger than normal number of

diseased or infected individuals that occurs over a
✓ Health worker to patient relatively short period
✓ Patient to patient 5. Pandemic→an epidemic that spans the world
✓ Use of fomites(catheters, needles, dressings, Carrier→a person who carries the etiologic agent
beds) but shows no apparent signs or symptoms of
infection or disease
✓ Airborne transmssion→TB: < 5um, Pertusis: >
5um Causal/Acute/Transient Carrier
✓ Vector-borne →harbors the microorganism temporarily for a few
days or weeks

FPRE
71
Chronic Carrier
→ remain infected for a relatively long time,
sometimes throughout its entire life (Typhoid Bacillus)
Convalescent Carrier
→recovered from infection but continuous to harbour
larger numbers of the pathogen
Vehicle/Fomite→ a non-living entity that is
contaminated with the etiologic agent and as such is
the mode of transmission for that agent
Vector→ a living entity (animal, insect, or plant) that
transmits the etiologic agent
Host→ an animal or plant that harbors or nourishes
another organism

Active carrier Parasite→ an organism which is dependent on

another organism for food and shelter
→overt clinical case of the disease
Surveillance→ any type of epidemiologic
PHASES OF INFECTIOUS DISEASES investigation that involves data collection for
Incubation Period characterizing circumstances surrounding the
incidence or prevalence of a particular disease or
→time between the exposure to a pathogenic infection
organism and the onset of symptoms
Morbidity→the state of disease and its associated
Prodromal Period effects on the host
→appearance of signs and symptoms Mortality→death resulting from diseaseStrain
typing→ laboratory-based characterization of
Clinical or illness Period
etiologic agents designed to establish their
→peak of characteristic signs and symptoms relatedness to one another during a particular
outbreak or epidemic
Decline Period
Reservoir→ origin of the etiologic agent or location
→signs and symptoms begin to subside as the host’s from which they disseminate (e.g., water, food,
condition improves insects, animals, other humans)
Convalescence or the Period of recovery Common source→ the etiologic agent responsible
→host is recuperating towards full recovery for an epidemic or outbreak originates from a single
source
Causative/Etiologic Agent→ a microorganism
or reservoir
responsible for causing infection or infectious
disease HOST-MICROBE RELATIONSHIP

Pathogen→ organism capable of producing disease Symbiosis→association of two organisms living in

Virulence→ a quantitative measure of the degree of
pathogenecity of a particular microorganism
Nonpathogenic→ microorganism that does not
cause disease; may be part of the normal flora
Opportunstic pathogen→ an agent capable of
causing disease only when the host's resistance is
impaired (PAE, Stenotrophomonas maltophilia)
Mode of transmission→ means by which etiologic
agents are brought in contact with the human host
(e.g. infected blood, contaminated water, insect bite)
close proximity
Mutualism→refers to a mutually beneficial
relationship between two species
Commensalism→ a relationship wherein the
parasite derives benefits from the host without
causing injury or harm to the host
Parasitism →a relationship whereby one organism
derives benefits at the expense of another
Pathogenicity→ability of the organism to produce
disease

FPRE
72
Pathogenicity Island→large groups of genes that MICROBIAL FLORA
are associated with pathogenicity and are located on
Normal, Usual, or Indigenous Flora
the bacterial chromosome
→microorganisms that are commonly found on or in
Invasiveness→ the ability of the organism to enter
body sites of healthy persons
host tissues, multiply, and spread faster
Resident Microbial Flora
Toxigenity→ability of the organism to produce
toxins →microorganisms that colonize an area for months
or years
Toxoid→non-poisonous forms of toxins which can
be used for vaccination Transient Flora
Preparation: →microorganisms that are present at a site
temporarily represent
✓By aging
Presence depends on:
✓By exposure to heat
✓physiologic factors of temperature
✓By exposure to 50% alcohol, formaldehyde,
and dilute acids ✓moisture
General stages of microbial-host interaction ✓presence of certain nutrients and inhibitory
substances
Role!!!
➢Provide a first line of defense against microbial
pathogens
✓competition for receptors or binding sites on
host cells
✓competition for nutrients
✓mutual inhibition by metabolic or toxic
products
✓mutual inhibition by antibiotic materials or
bacteriocins
➢Assist in digestion and absorption of nutrients; also
synthesis of Vitamin K
➢Play a role in toxin-degradation
➢Contribute to maturation of the immune system

FPRE
73
Composition of Microbial Flora at Different Body Oropharynx
Sites
a. Usual Flora of the Skin

b. Usual Flora of the Mouth

c. Usual Flora of the Gastrointestinal Tract
***Streptococcus→ predominant genus

d. Usual Flora of the Genitourinary Tract

c. Usual Flora of the Respiratory Tract

Viridans Streptococci→ mouth, nasopharynx, and
oropharynx
✓Streptococcus mitis
✓Streptococcus mutans
✓Streptococcus milleri
✓Streptococcus sanguis
Moraxella catarrhalis
Neisseria spp
Diphtheroids
S. aureus→ anterior nares
Nose and Nasopharynx

FPRE
74
PATHOGENESIS OF INFECTION →interfering with the binding of the host’s antibodies
to the surface of the organism
Pathogenicity
→ binds to the Fc portion of IgG preventing
→ ability of a microbe to produce disease in a
opsonization and phagocytosis by turning the
susceptible individual
antibody around on the surface
True pathogens
CELL WALL PROTEINS
→are organisms recognized to cause disease in
M protein
healthy immunocompetent individuals
→heat resistant and acid resistant protein, mediates
Example: Yersinia pestis Bacillus anthracis
attachment to host epithelial cell and helps resist
Opportunistic pathogens phagocytosis; overcome by antibodies produced
against the M protein
→cause disease if the host is immunocompromised
Fimbriae and Outer membrane protein
Example: E.coli
→N. gonorrhoeae
Virulence
Mycolic acid
→relative ability of a microorganism to cause
disease or the degree of pathogenicity →M. tuberculosis; resist digestion during
phagocytosis; the bacteria can even multiply inside
→measured by the numbers of microorganisms macrophages
necessary to cause infection in the host
Antigenic variation
Microbial Virulence Factors
→N. gonorrhoeae
✓inhibiting phagocytosis
✓Facilitating adhesion to host cells or tissues
✓enhancing intracellular survival after phagocytosis
✓damaging tissue through the
✓production of toxins and extracellular enzymes
a. Ability to Resist Phagocytosis
1. Capsule
→highly virulent
→mask the cell surface structures that are
recognized by receptors on the surface of the
phagocytic cell
→inhibits the activation of complement by masking
structures to which complement proteins bind
Example: S. pneumoniae H. influenzae N.
meningitidis, K. pneumonia, S. typhi, P.
aeruginosa, B. anthracis, Y. pestis
2. Protein A
→found in the cell wall of Staphylococcus aureus

FPRE
75
3. Hemolysins
c. Ability to Survive Intracellularly and Proliferate
→produced by Streptococci
✓Prevent fusion of phagosomes and lysosomes
→lyse red blood cells and induce toxic effects on
WBC ✓Resistance to the effects of the lysosomal contents
4. Leukocidins ✓Escape from the phagosome into the cytoplasm
→realesed by pathogenic staphylococci Meningococci
→cause lysosomal discharge into cell cytoplasm → use lactoferrin as a source of iron
***Panton-Valentine H. influenzae, N. gonorrhoeae, and N.
meningitides
→ Staphylococcal leukocidin
→produce an IgA protease that degrades the IgA
→lethal to leukocytes and contributes to the
found at mucosal surfaces
invasiveness of the organism
Borrelia spp.
b. Surface Structures That Promote Adhesion to
Host Cells and Tissues Adhesins →circumvent host antibodies by shifting key
→cell surface structures that mediate attachment cell surface antigens
Main Adhesins in Bacteria: Chlamydia, Mycobacterium, Brucella, and Listeria
1. Fimbriae (pili) →able to multiply intracellularly
→enable bacteria to adhere to host cell surfaces, Invasion
offering resistance by attachment to target cells,
increasing the organism’s colonizing ability → ability to penetrate and grow in tissues

2. Surface Polysaccharides Dissemination

→ disease or organisms spread to distant sites
Example: Salmonella spp.

FPRE
76
Clostridium perfringens → highly invasive
organism that may not disseminate
d. Ability to Produce Extracellular Toxins and
Enzymes Toxins
→poisonous substances produced by organisms that
interact with host cells, disrupting normal metabolism
and causing harm
Proteases and Hyaluronidases
→soluble substances that liquefy the hyaluronic acid
of the connective tissue matrix, helping to spread
bacteria in tissues, promoting the dissemination of
infection
ENZYMES
Collagenase →breaks
down collagen, which forms the connective tissue of
muscles and other body organs and tissues
Hyaluronidase →hydrolyzes hyaluronic acid, a type
of polysaccharide that holds together certain cells of
the body, particularly cells of the connective tissue
helping the organism spread from its initial site of
infection
Example: S. pyogenes (cellulitis), C. perfringens (gas
gangrene)
Coagulase →produced by S. aureus and
accelerates the conversion of fibrinogen to a fibrin
clot (the clot may protect the bacteria from
phagocytosis by walling off the infected area and by
coating the organisms with a layer of fibrin ultimately
resulting to evasion of the bacteria from other
defenses of the host)
Kinases
Streptokinase
Staphylokinase
Immunoglobulin A protease (IgA protease)
→ destroy IgA antibodies found on secretions
Leukocidin
→destroy neutrophilic leukocytes and macrophages
FPRE
EXOTOXINS
→composed of two subunits: nontoxic (binds the
toxin to the host cells) and toxic
→produced by both gram-negative and gram positive
bacteria
→secreted by the organism into the extracellular
environment, or they are released on lysis of the
organism
→mediate direct spread of the microorganisms
through the matrix of connective tissues and can
cause cell and tissue damage
Toxin Gene
→encoded by phages, plasmids, or transposons
→most toxic substances
→good antigens and induce the production of
antibodies called ANTITOXINS
→When treated with formaldehyde (or acid or heat),
the exotoxin polypeptides are converted into
TOXOIDS, which are used in protective vaccines
Examples:
✓Diphtheria toxin ✓Tetanospamin
✓Botulism toxin ✓Heat labile enterotoxin by E.
coli, Vibrio, Bacillus ✓Verotoxin
✓Erythrogenic toxin ✓Three toxins of B.
anthracis (EF, PA, LF) ✓TSST-1
3 principal types on the basis of their structure
and function:
1. A-B Toxin
→Consists of two domains or subunits, one
responsible for binding to the cell and entry into the
cell and the other possessing the toxic activity
2. Membrane-Disrupting Toxins
→cause lysis of host cells by disrupting their plasma
membrane; some form protein channels in the
plasma membrane (S. aureus); others disrupt the
phospholipid portion of the membrane (C.
perfringens)
Example: Leukocidins
Hemolysins (e.g. Streptolysins)
77
3. Superantigens
→antigens that provoke a very intense immune
response; nonspecifically stimulate the proliferation
of immune cells called T cells, which in turn secrete
enormous amounts of cytokines
Note!!!
✓Severe neutropenia
✓Activates macrophages, activates complement,
and has an adjuvant effect with protein antigens
✓stimulates interferon production and causes
changes in carbohydrates, lipids, iron, and sensitivity
to epinephrine

Excessively high levels of cytokines released by T _____________________→ test to detect

cells enter the bloodstream and give rise to a number endotoxins in drugs, medical devices, and body
of symptoms, including fever, nausea, vomiting, fluids
diarrhea, and sometimes shock and even death
POSITIVE RESULT:
Example:
HOST RESISTANCE
Diphtheria toxin
Physical Barriers
→inhibits protein synthesis and affects the heart,
a. Healthy, Intact Skin
nerve tissue, and liver
→ primary mechanical barrier to infection
Botulinum toxin
→has substantial numbers of microbial flora that
→neurotoxin that blocks nerve impulse transmission,
contribute to a low pH, compete for nutrients, and
causing flaccid paralysis, especially in infants—
produce bactericidal substancesaddition
FLOPPY BABY
→low pH resulting from long-chain fatty acids
Streptococcus pyogenes and S. aureus
secreted by sebaceous glands ensures that relatively
→produce EXFOLIATIN which causes rash and few organisms can survive and prosper in the acid
massive skin peeling or exfoliation environment

ENDOTOXINS Leptospira spp., Francisella tularensis,

Treponema spp.
→composed of the LPS portion of the outer
membrane on the cell wall of gram-negative bacteria →capable of penetrating normal, healthy skin
→do not have enzyme activity b. Stricture at the urethral opening, flushing action of

→secreted in only very small amounts urination and the thick mucus plug in the cervical
openig
→do not have specificity in their activity on host cells
b. Cleansing Mechanisms
→not very potent
✓Desquamation of the skin surface
→not destroyed by heating
✓Tears contain IgA and lysozyme
→less antigenic and induce antibody production in a
poor manner ✓Respiratory Tract- mucus traps particles and
microbes and sweeps them to the oropharynx
→no toxoid have been produced from enough
endotoxin ✓Trachea is lined with ciliary epithelium containing
cilia that sweep particles and organisms upward
Effects of Endotoxin toward the oropharynx
✓Stimulates the fever centers in the hypothalamus ✓“Cough-sneeze” reflex contributes to the removal
(1 hour after exposure) of potentially infected agents
✓Hypotension (30 minutes after exposure)→Shock
✓Initiates coagulation→DIC
FPRE
78
✓GIT is protected by mucous secretions and PMN
peristalsis that prevent the organisms from attaching
→has receptors on the cell membrane for some
to the intestinal epithelium
complement components that stimulate cell motion,
✓Genitourinary tract is cleansed by voiding urine
the metabolic burst, and secretion of the lysosome
✓Acidity of the vagina tends to inhibit transient contents into a phagosome
organisms from colonizing
→end-stage cell and has a circulating half-life of 2 to
c. Antimicrobial Substances 7 hours
Lysozyme →may migrate to the tissues---half-life is less than 1
week
→low-molecular-weight (approximately 20,000 D)
enzyme that hydrolyzes the peptidoglycan layer of Macrophages
bacterial cell walls
→circulate as monocytes for 1 to 2 days and then
→found in serum, tissue fluids, tears, breast migrate through the blood vessel walls into the
milk,saliva, and sweat tissues and reside in specific tissues
Secretory IgA as part of the MONONUCLEAR PHAGOCYTE
SYSTEM
→found in mucous secretions of the respiratory,
→widely distributed in the body and play a central
genital, and digestive tracts
role in specific immunity and nonspecific
→serve as opsonins, fix complement and neutralize phagocytosis
the infecting organism
β-lysins
→low-molecular-weight cationic proteins in serum
→lethal against gram-positive bacteria and are
released from platelets during coagulation
Interferon
→inhibits proliferation of viruses
d. Indigenous Microbial Flora
→compete with pathogens for nutrients and space 1. Chemotaxis
Bacteriocins
DIAPEDESIS
→substances that inhibit the growth of closely
related bacteria → movement of the neutrophils between the
endothelial cells of the blood vessels into the tissues
e. Phagocytosis
CHEMOTAXIS
→process by which phagocytes engulf and dispose
of microorganisms and cell debris →directed migration of PMNs into the area of
Infection
Lysosomes
→myeloperoxidase, proteases, cathepsin, lactoferrin,
lysozyme, and elastase---necessary for the
killing and digestion of the engulfed particles

FPRE
79

2. Attachment
→facilitated by the binding of specific antibodies to
the microorganism
✓Neutrophil membrane receptors for the Fc portion
of IgG1, IgG3, and the C3b component of
complement
OPSONIZATION
→coating of the bacterium with antibody or
complement components results in enhanced
phagocytosis by the PMN
Can be accomplished by three different types of
responses:
(1) IgG1 or IgG3 binds to the organism
(2) antibody response is insufficient for opsonization,
but complement is fixed on the surface of the
organism
(3) Alternative complement pathway is activated by
the
endotoxin or polysaccharides of the organism
3. Ingestion
✓Cell membrane of the phagocytic cell invaginates
and surrounds the attached particle
✓Particle is taken into the cytoplasm and enclosed
within a vacuole called a PHAGOSOME
✓Phagosome fuses with lysosomes---
PHAGOLYSOSOME
✓Lysosomes release their contents into
phagosome— DEGRANULATION
Enzymes include: proteases, lipases, RNase,
DNase, peroxidase, and acid phosphatase
4. Killing
Metabolic or Respiratory Burst
→phagocytosis of a particle triggers a significant
increase in the metabolic activity of the neutrophil or
macrophage
→increases in glycolysis, the hexose
monophosphate shunt pathway, oxygen use, and
production of lactic acid and hydrogen Peroxide
Inflammation
→body’s response to injury or foreign body
→hallmark of inflammation: accumulation of large
numbers of phagocytic cells
→leukocytes release mediators or cause other cell
types to release which cause erythema as a result of
greater blood flow, edema from an increase in
vascular permeability, and continued phagocyte
accumulation, resulting in pus
CARDINAL SIGNS OF INFLAMMATION:
SWELLING, REDNESS, HEAT, PAIN, LOS
OF FUNCTION
Chemical Mediatiors of Inflammation:
✓Histamine ✓Kinins ✓Leukotrienes
✓Prostaglandins ✓Acute phase reactants (e.g.,
CRP, Serum amyloid A, antitrypsin, fibrinogen)
✓Cytokines (IL-1, IL-6, TNF-α, IFN-γ, IL2)
IMMUNE RESPONSES
Immunity
→mechanism whereby the body is able to protect
itself from invasion by diseasecausing organisms
Immune system
→consists of numerous cells and protein molecules
that are responsible for recognizing and removing
these foreign substances Divided into two broad
categories:
Innate or Natural immunity→ little or no specificity
the Adaptive or Specific → highly Specialized
FPRE
80
 → major constituents of the adaptive or specific
immune response
Immunologic Memory
→ able to remember each time it encounters a
particular foreign antigen
A. HUMORAL IMMUNE RESPONSE
→Antibody mediated
Immunoglobulin G (IgG)

INNATE, OR NATURAL, NONSPECIFIC IMMUNITY

→immediate response to the pathogen that does not
confer longlasting protective immunity
1. Physical and chemical barriers: skin and mucous
membranes
2. Blood proteins that act as mediators of infection:
→70% to 75% of the total serum immunoglobulin
Cytokines, Complement
pool
3. Cellular mechanism capable of phagocytosis such
→halflife in serum: 3 to 4 weeks
as neutrophils
→cross the maternal placenta to the fetus
and macrophages and other leukocytes such as
natural killer cells → passive immunity for newborns, neutralization of
viruses and exotoxin; responds best to protein
antigens, mainly involved in secondary (anamnestic)
immune response
ADAPTIVE, OR SPECIFIC IMMUNITY
Immunoglobulin M (IgM)
→capable of being specific for distinct molecules,
responding in particular ways to different types of
foreign substances and developing memory, which
allows for a more vigorous response to repeated
exposures to the same foreign invader
→can be Humoral or Cellular Immune Response
Lymphocytes and Antibodies

FPRE
81
 →major role in allergic response
Immunoglobulin D (IgD)
→little is known; may serve as a B-cell receptor or
play a role in autoallergic diseases
Primary and Secondary Antibody Responses
Primary immune response
→ relatively rapid appearance of IgM antibodies
→10% to 15% of serum immunoglobulins
Secondary or Anamnestic immune response
→half-life in serum: 5 days
→rapid increase in IgG antibody associated with
→ cannot cross the placenta
higher levels, a prolonged elevation, and a more
→consists of five basic subunits—each composed of
two heavy chains and two light chains (similar to an gradual decline
IgG molecule) and linked to another polypeptide
chain (J chain) by disulfide bonds
→endotoxin neutralization, bacterial agglutination,
complementmediated bacteriolysis, strong
opsonization ability; responds best to polysaccharide
antigens, mainly involved in primary immune
Response
Immunoglobulin A (IgA)
→15% to 20%
→predominant immunoglobulin class in certain body
secretions, such as saliva, tears, and intestinal
secretions B. CELL-MEDIATED IMMUNE RESPONSE
→ prevention of bacterial and viral invasion of →based on the action of specific kinds of T-
mucous membranes through interference with lymphocytes that directly attack the cells that are
adherence of microorganism to site; found in tears, infected with virus, parasites, cancer cells or
milk, saliva, and respiratory and GI secretions transplanted cells
Serum IgA occurs T Lymphocyte
→ two subunits (each similar to an IgG molecule) → primary effector cell in cell-mediated immunity
linked together by a J chain
Lymphokines
Secretory IgA
→low-molecular-weight proteins resulting from
→ contains a secretory component that stabilizes the antigen binding, activation, cell division, and
moleculeImmunoglobulin E (IgE) differentiation of the T cell
→increase during infection by numerous parasites
and may play a role in eliminating these infectious
agents from the host
→Total serum IgE levels may increase during
parasitic infection

FPRE
82
Mechanisms by Which Microbes May Overcome ROUTES OF TRANSMISSION
Host Defenses
1. Airborne Transmission
a. Bringing about tolerance
Respiratory Spread
Tolerance
→ common
→ inability to induce an immune response to a
→aerosolized by coughing, sneezing, and talking
microbial antigen
→TB, Brucellosis, Tularemia, Legionellosis and
b. Immunosuppression
Plague—inhalation of infectious particles in liquid
c. Change in the appropriate target for the
droplet
immune response
→some infectious agent may be transmitted by dust
d. Antigenic variation
particles
Example: Borrelia recurrentisTransmission
Droplet Nuclei
✓Most pathogen are acquired from external
→residue from the evaporation of fluid from larger
sources
droplets and are light enough to remain airborne for
✓Pathogens usually exit the infected patient most long periods
frequently from the respiratory tract and 2. Transmission by Food and Water
gastrointestinal tract; hence, transmission to the
→infection occurs via the fecal-oral route
new host usually occurs via airborne respiratory
droplets or fecal contamination of food and water Enterotoxigenic E. coli (ETEC)
✓Organisms can also be transmitted through: sexual →common cause of TRAVELER’S DIARRHEA and
other intestinal problems
contact, urine, skin contact, blood transfusions,
contaminated needles, or insect bites Vibrio cholera
✓Four (4) important portals of entry for pathogenic →enterotoxin that causes the outpouring of fluid from
the cells into the lumen of the intestine
organisms: GIT, GUT, respiratory tract,
integumentary Preformed toxin: Clostridium botulinum, Bacillus
system cereus, and S. aureus

✓Mucous membranes of the GIT, GUT, repiratory Other intestinal pathogens: C. difficile, Shigella spp.,
tract and conjunctiva (dses: conjunctivitis, trachoma, Aeromonas hydrophila, Campylobacter jejuni,
ophthalmia neonatorum) and Salmonella spp
✓Parenteral route: punctures, injections, bites, cuts, 3. Close Contact
wounds, surgery, and splitting of the skin or mucus
membrane due to swelling or drying →passage of organisms by salivary, skin, and genital
contact
✓“preferred portals of entry”; some organisms have
many portals of entry (Yersinia and B. anthracis) 4. Cuts and Bites
→infection by the mouth flora
Pasteurella multocida
→dog-bite and cat-bite infections

FPRE
83
5. Arthropods
→tick, flea, or mite bite
→relapsing fever, plague, Rocky Mountain spotted
fever, Lyme disease, typhus
6. Zoonoses
→depends on contact with animals or animal
products
→arthropod vectors (plague), contact with secretions
(brucellosis), and contact with animal carcasses and
products (tularemia, listeriosis)
Numbers of Invading Microbes
✓ID50 (Infectious Dose for 50% of a sample
population)
→compare relative virulence under experimental
conditions; it is not an absolute value
B. anthracis
• Cutaneous = 10-50 endospores
• Inhalation = 10k-20k endospores
• Gastrointestinal = 250k-1M endospores
• V. cholerae = 108 cells (decreased upon
neutralization of stomach acidity or administration of
bicarbonates)
✓LD50 (Lethal Dose for 50% of a sample
population)
→potency of a toxin
• Botulinum toxin = 0.03 ng/kg
• Shiga toxin = 250 ng/kg
• Staphylococcal enterotoxin = 1350 ng/kg

FPRE
84
UNIT 12: SPECIMEN TRANSPORT-COLLECTION
SPECIMEN COLLECTION, TRANSPORT AND
PROCESSING
GENERAL GUIDELINES FOR SPECIMEN
COLLECTION
1. Collected during the acute or early phase of an
illness
2. Select the correct anatomic site
3. Collect using the proper technique and supplies
with minimal contamination from normal biota
4. Collect the appropriate quantity of specimen
5. Collected before the administration of any
antimicrobial agent
Note!!!
If taken after initiation of antibacterial therapy,
counteractive measures such as adding
penicillinase or diluting the specimen should be
done
6. Package in a container or transport medium
designed to maintain the viability and avoid hazards
7. Label the specimen accurately (specific anatomic
site and patient)
8. Transport promptly or make provisions to store the
specimen that will not degrade the suspected
organism
9. Swabs are generally poor specimens(except:nares
and throat specimens) compared with tissue or
needle aspirates submitted on 2 swabs: DIRECT
SMEAR AND CULTURE
10. Lesions, wounds and abcesses collected from
advancing margin of the lesion preferably by
ASPIRATION place in sterile tube
REJECTION OF UNACCEPTABLE SPECIMENS
1. Unidentified or improperly labelled specimen
2. Information on the label does not match the
information on the requisition
3. Specimen transported at the improper temperature
FPRE
4. Specimen has not been transported in the
proper medium/container
5. Quantity of specimen is insufficient for testing
6. Specimen is leaking
7. Grossly contaminated specimen
8. Transport time exceeds 2 hours post collection or
the specimen is not preserved
9. Received in a fixative (formalin) kills
any microorganism present
10. Received for anaerobic culture from a site known
to have anaerobes as part of the normal flora (vagina,
mouth)
11. Specimen is dried (dry swabs for culture)
12. Not the proper specimen for the test (material
smeared on a slide for culture)
13. Processing the specimen would produce
information of questionable medical value (Foley
catheter tip)
14. If duplicate specimen is received only one
must be processed
Note!!!
All specimens not processed refrigerated for
3 days before being discarded
15. More than one specimen from the same source
was submitted from the same patient on the same
day; blood cultures are an exception
16. One swab was submitted with multiple requests
for various organisms
17. Gram stain of expectorated sputum reveals <25
WBCs and >10 epithelial cells per LPF and mixed
bacterial flora
85
COLLECTION PROCEDURES than 2 hours use special preservatives or holding
media
Collected in a sterile containers
 Specimen containes should be leak-proof
Stool specimens can be collected in clean,
leakproof containers  Specimen containers should be placed in a
sealable, leak-proof plastic bags
Swabs
 Patient specimens or culture isolates must be
upper respiratory tract, external ear, eye, and
triple packaged before being shipped
genital tract
 Specimen bags should be marked with a
Not recommended: insufficient quantity, easily
biohazard symbol
contaminated, and can become dried out
 All specimens should be transported to the
tips of swabs may contain COTTON, DACRON, laboratory immediately after collection
OR CALCIUM ALGINATE
Cotton-tipped swabs have excessive fatty
acids which may be toxic to certain bacteria
LABELING AND REQUISITIONS
Patient Identification:
Name, Identification number, Room number,
Physician, Culture site, Date of collection and Time
of collection
Requisition Form:
Patient’s name, age (or date of birth) and
gender, room number or location, Physician’s name
and address, Specific anatomic site, Date and time
of specimen collection, Clinical diagnosis or relevant
patient history,
Antimicrobial agents (if the patient is receiving) and
Name of individual transcribing orders
SPECIMEN TRANSPORT
 Transported to the laboratory ideally within 30
minutes of collection, preferably within 2
hours
 Anaerobic Bacteria transport should not take
more than 10 minutes Kinds of Swabs According to Tip Used

 CSF transport should be done immediately a. Cotton-tipped Swab

and preferably within 15 minutes
 most commonly used, may contain fatty acids
 Presence of oxygen (anaerobic bacteria), that may be detrimental to microbial growth
changes in temperature (Neisseria meningitidis),
b. Calcium Alginate-tipped Swab
or changes in pH (Shigella) prevent recovery
 excellent substitute, dissolves in certain
 Transportation of specimens delayed for more
solutions that are compatible with bacterial
preservation
FPRE
86
 never be used in Herpes Virus interferes with the
extraction reagents used in direct antigen
detection may be toxic to some chlamydia
species
c. Polyester-tipped Swab (DACRON or RAYON)
 used for delayed recovery of Group
streptococci from throat culture
 incorporation of small amount of silica gel in a
glass tube containing polyester swab viability of
Group A streptococci os maintained for 3 days
Kinds of Swabs According to Applicator Used
a. Wooden Applicator Stick commonly used
b. Nichrome or Thin Aluminum Wire
HANDLING OF SPECIMENS
 refrigeration at 4°C-6°C
a. Swabs from wounds(except anaerobic culture),
urogenital tract, throat and rectum and samples of
feces or sputum refrigerated for 2-3 hours
b. Urine specimen can be refrigerated for at least 24
hours
c. CSF from patients with suspected meningitis
should be examined at once
d. Specimens for viral isolation should be refrigerated
immediately but never frozen
SPECIMEN PRESERVATION
Urine
BORIC ACID
maintain the bacterial population in the urine at room
temperature for 24 hours
Stool
 can be refrigerated
 delay is longer than 2 hours added to CARY
BLAIR TRANSPORT MEDIA
***Stools for Clostridium difficile toxin assay
 collected without a preservative and can be
refrigerated delay longer than 48 hours: frozen
at −70°C
TRANSPORT or HOLDING MEDIUM
 designed to prolong the survival of
microorganisms when a significant delay
between collection and culturing cannot be
avoided but do not allow their multiplication
A  STUART’S MEDIUM**
 AMIES MEDIUM**
 CARY and BLAIR MEDIUM
 TRANSGROW MEDIUM
 JOHN E. MARTIN BIOLOGICAL
ENVIRONMENTAL CHAMBER (JEMBEC)
 contains selective agar and a carbon dioxide
(CO2)–generating tablet N. gonorrhoeae
Note!!!
**CHARCOAL added to absorb fatty acids that
could kill fastidious (fragile) organisms such as
Neisseria gonorrhoeae or Bordetella pertussis
ANTICOAGULANTS
 used to prevent clotting of specimens such as
blood, bone marrow, and synovial fluid
 synovial and peritoneal fluids can also be
inoculated into a blood culture broth bottles
1. 0.025% SODIUM POLYANETHOL SULFONATE
(SPS)
 used for blood culture media because Neisseria
spp. and some anaerobic bacteria are
particularly sensitive to higher concentrations
 ratio of specimen to SPS is so important
Adult: 10-30 ml
Children: 5 to 10 ml
Reasons for using 0.025% SPS as the preffered
anticoagulant for blood culture
a. Inhibit phagocytosis and complement activation
b. Neutralizes aminoglycosides antibiotics
c. Neutralize the bactericidal effect of plasma
d. Precipitates fibrinogen, β-lipoproteins, β-1-C
globulin
FPRE
87
SPS may inhibit the growth of the following d. Feezer Temperature (-20°C or -70°C)
bacteria
 serum for serologic studies: frozen up to one
 Neisseria gonorrhoea week at -20°C
 Neisseria meningitidis  Tissues or specimens that are to be stored for a
long time should be kept at -70°C
 Gardnerella vaginalis
 Fecal specimens for studying C. difficile toxin
 Streptobacillus moniliformis
at -70°C if the storage will be longer than 3 days
 Peptostreptococcus anaerobius
Note!!!
SPECIMEN PRIORITY
***Inhibitory effect can be neutralized by 1% gelatin
Level 1 specimens
2. Heparin  critical or invasive
 viral cultures and for isolation of Mycobacterium  potentially life-threatening illness and are from
spp. from blood, may inhibit growth of gram- an invasive source
positive bacteria and yeast
 require immediate processing
3. Citrate and Ethylenediaminetetraacetic Acid
(EDTA) Level 2 specimens
- should not be used for microbiologySPECIMEN  unprotected and may quickly degrade or have
STORAGE overgrowth of contaminating flora
a. Refrigerator Temperature (4°C) Level 3 specimens

 Urine, stool, viral specimens, sputa, swabs, and  require quantitation

foreign devices (catheters) and viral specimens
 delay may adversely affect the accuracy of
 stool specimen not transported immediately can quantitation
be refrigerated, but if delay >2 hours:placed in a
Level 4 specimens
Clary Blair transport medium
 arrive in the laboratory in holding or transport
 stool for the isolation of Clostridium difficile
media
toxin— stored up to 3 days
 Specimens for acid-fast bacilli that need to be
 specimens suspected of containing anaerobic
digested and decontaminated can be
bacteria should never be stored in refrigerator
refrigerated and processed once per day
b. Incubator Temperature (35°C)
 Acid-fast, viral, and fungal specimens are
 CSF should always be kept at this temperature usually batched for processing at one time
(6 hours)
 CSF samples intended for Gram staining, culture
and sensitivity tests
c. Ambient (Room) Temperature (22°C-25°C)
 specimens for anaerobic culture, sterile body
fluids, genital specimens and swabs (ear and
eye)

FPRE
88
 Cytocentrifugation preferred for this type
of specimen (bronchoalveolar lavage fluids)
DIRECT MICROSCOPIC EXAMINATION
Purpose:
1. Quality of the specimen can be assessed
 sputum can be rejected that represent saliva and
not lower respiratory tract secretions by
quantitation of WHITE BLOOD CELLS OR
SQUAMOUS EPITHELIAL CELLS present in
the specimen Sputum samples accepted for
cultivation:
<10 Epithelial Cells and >25 Pus Cells per field
->BARTLETT’S CRITERIA
 2. Give the microbiology technologist and the
physician an indication of the infectious process
SPECIMEN PROCESSING
involved
Gross Examination of Specimen
3. routine culture workup can be guided by the
 Areas with blood or mucus should be located results of the smear
and sampled for culture and direct examination
4. Can dictate the need for nonroutine or additional
 Stool should be examined for evidence of testing
barium (chalky white color)
Methods of Direct Examination
Preparation of Smears
1. Gram Staining
 should not be prepared from a swab after it has
2. Acid Fast Staining-Ziehl-Neelsen, Kinyoun and
been used to inoculate culture media
Fluorescent Method
 prepared by rolling the swab back and forth over
PROCESSING OF SPECIMENS
contiguous areas of the glass slide to deposit a
thin layer of sample material a. Homogenization tissue grinding
 single swab: culture should be prioritized b. Concentration (centrifugation or filtration)
Smears from Thick Liquids or Semisolids large volumes of sterile peritoneal or pleural fluids
 swab is immersed in the specimen for several c. Decontamination for isolation of bacteria
seconds and used to prepare a thin spread of
material on the glass slide Note!!!

Smears from Thick, Granular, or Mucoid Swab specimens vortexed in 0.5 to 1 ml of saline or
Materials broth for 10 to 20 seconds to dislodge material from
fibers Biological fluids more than 1 ml can be
 Opaque material must be thinly spread centrifuged for 20 minutes at 3,000 rpm
 Granules within the material must be crushed
Smears from Thin Fluids
urine, cerebrospinal fluid (CSF), and transudates
should be dropped but not spread on the slide

FPRE
89
 SPECIMENS USED FOR BACTERIOLOGICAL (aerobic-THIO)-- > recommended for opyimal
STUDIES recovery of pathogen
1. BLOOD  Blood culture should be incubated at 35°C and
examined visually daily (subcultures done at
 Venipuncture site should be cleansed with 70%
interval) for 7 days reporting as negative or no
alcohol then with 2% iodine or iodophore(30-60
growth
seconds) allowed to dry at least 1 minute
 Patient suspected of having endocarditis,
 Most advantageous time to draw blood cultures
brucellosis and fungemia, incubation should be
will be just before the anticipated rise of
prolonged for 2-4 weeks
temperature
Growth is usually indicated by the following:
Bacterial endocarditis organisms
are released in the bloodstream at constant  Hemolysis of red cell
rate---timing is not important
 Gas bubbles in the medium
 Minimum collection of 2 up to 4 sets taken in a
 Turbidity
24-48 hours
 Colony formation
 2 sets should be drawn from the same arm
Newer(faster) Methods of Blood Culture
 Aerobic and anaerobic culture bottles are
used  Lysis-Centrifugation (Isolator)
 Fever of unknown origin 4 separate blood  Self-contained Subculture(Septi-Check and
culture, 2 drawn on each of 2 days Vacutainer Agar Slant) modification of the
biphasic blood culture medium
 0.025%-0.05% SPS best anticoagulant for
blood  Detection of microorganisms through changes in
electrical impedence (BACTOMETER)
Blood Culture
 Detection of CO2 end product
Basic culture media contain nutrient broth and
metabolism(BACTEC System)
SPS
2. CEREBROSPINAL FLUID (CSF)
Examples:
a. Collection and Handling
 TSB
 Collection is by aseptically inserting a needle
 BHIA
into the subarachnoid space at the level of the
 Supplemental Peptone lumbar spine
 THIO  3 TO 4 TUBES are usually filled; tubed 3 and
 Columbia Broth 4 is used for differential count while the other tubes
are for microbial and chemical studies
 Brucella Broth
Tube 1 Chemical and Serologic Tests
 Castaneda Medium biphasic medium-agar
slant with broth Tube 2 Microbiology
Penicillinase -may be added to inactivate Tube 3 Cell Count
penicillins and other Beta-lactam antibiotic
 Specimen for microbial examination placed in
1:10 ideal dilution factor a sterile screw-capped and air-tight tubes
 Use of 2 different bottles of blood culture media,  CSF should be transported immediately without
one vented (aerobic-TSB) and other unvented refrigeration

FPRE
90
 If delayed, incubated no longer than 1 hour or Note!!!
left at room temperature
10 ml required volume (microbiology and
Specimens for viral studies ,refrigerated up chemistry)
to 24 hours or frozen at -70°C if longer delay is
6 hours at 35°C storage, bacteria associated with
anticipated
meningitis are fastidious and susceptible to cold or
b. Processing drying
 Specimens are centrifuged and decanted, using Gram Stain can be performed by
the sediments for smear (Gram Stain, India Ink) cytocentrifugation
and culture (H. influenzae, N. meningitidis, S.
pneumonia and Cryptococcus neoformans) 3. THROAT AND NASOPHARYNGEAL
SPECIMENS
-Supernatant, test the presence of antigens
or for chemistry examination A. Throat Swab

Examples of Serologic Tests: ***Alpha Streptococcus most abundant normal

flora
a. Counter Current Immune Electrophoresis
bacterial antigen ***S. pyogenes (Group A Beta Hemolytic
Streptococcus) most common pathogen
b. Coagglutination Test PHADEBACT
Staphylococcal Protein A coated with antiserum Should be diagnose with the following precautions:

c. Latex Agglutination Test 1. Posterior pharynx, tonsils and any areas of

purulence, inflammation and ulceration cultured
Culture Media
2. Swab inoculated to Soybean-Casein Digest
a. Enrichment Broth TSB of THIO(37°C for at Agar containing 5% sheep blood
least 5 days)
3. Bacitracin Disk Test -placed directly into BAP
b. CAP Gram-negative cocci with a purified subculture

c. BAP Gram-positive cocci 4. Incubated at 35°C for 18-24 hours with

reincubation of 1 day if no growth, before being
d. EMB, MAC Gram-negative bacilli ***Plated discarded as negative Patient throat should be
media should be incubated at 37°C in 5-10% CO2 swab from the uvula to the tonsil on both sides of the
for at leats 72 hours throat
e. Fungal media (non-blood containing media, BHIA, Adequate for the recovery of:
Mysosel) 30°C for 4 weeks
 Adenovirus
f. Tissue culture viruses
 Herpes Virus
 C. diphteriae
 Chlamydia
 Mycoplasma
 Yeast
 Haemophilus spp.
 Swab is moistened with Stuart’s and Amies
medium for collection of specimen

FPRE
91
B. Nasopharyngeal Swab g. Bronchoalveolar Lavage (BAL)
 recommended for the isolation and detection of h. Protected Catheter Bronchial Brushing
carriers
Culture
 specimen of choice for the recovery of B.
pertusis  Cultured on MAC, BAP, CAP

 recovery of Respiratory Syncytial Virus, Anaerobic culture

Parainfluenza Virus and Viruses causing  obtained by percutaneous aspiration
rhinitis (transtracheal aspiration) and protected
bronchial brush
C. diphtheria cultured on Loeffler’s Agar slant
and Cystein-Tellurite Agar Plate  not done on expectorated culture, bronchial
washing and lavage, tracheal aspirates and
B. Pertussis-Charcoal Cephalexin Medium or
tracheostomy or endotracheal tube aspirates
BordetGengou Agar
Gram’s and Acid Fast Stain
Neisseria meningitidis and Neisseria
gonorrhoeae -Modified Thayer-Marin or Martin-  done on direct smear
Lewis Agar
5. GASTROINTESTINAL TRACT
 Swab moistened with Stuart’s or Amies medium
used for collection of specimen a. Stool

 Flexible swab is inserted through the nose into Collection and Handling
the posterior nasopharynx and rotated for 5x  Walnut-sized specimen in a clean waxed
 4. SPUTUM cardboard or plastic container

Collection  Delivered to the laboratory within 1 hour:

delay >2 hours placed in Transport medium
-Patient should rinse his/her mouth with sterile water
Shigella
First morning specimen- preferred for AFB
microscopy  inoculated at bedside: If not, used Glycerol
Transport Medium (equal parts of glycerol and
2 to 3 consecutive early-morning sputum- 0.033 M phosphate buffer)
submitted for AFB
Viral culture
Methods:
 refrigerated if not inoculated to media within 2
 Deep Cough (expectorated sputum) hours
 Aerosol-Induced (induced sputum) C. difficile cytotoxin
-pediatric patientsOther means of obtaining  refrigerated for a maximum of 2 hours
sputum:
Staining of Smears
a. Gastric Aspiration AFB
Gram Staining -not routinely done, however may
b. Transtracheal Aspiration help in the detection of:
c. Transthroracic Needle Biopsy a. Many clumps of gram(+) cocci
d. Bronchoscopy with Aspiration or Biopsy  Staphylococcal infection
e. Thoracentesis b. Many thin, comma-shaped gram(-) bacilli
f. Open Lung Biopsy  Campylobacter and Vibrio

FPRE
92
c. Large numbers of large and thin gram(+) bacilli b. Gastric Aspirate
 C. difficile  collected early in the morning before the patient
rises from bed or takes his/her first meal
Acid-Fast Staining
 best specimen for infants
 detect Crypstosporidium spp. and
Mycobacteria  used for examination of AFB
Culture  must be neutralized within one hour of collection
a. General supportive media yeast spp., c. Gastric Biopsy
staphylococci, enterococci and gram(-) bacilli
 recommended for the detection of and isolation
b. Moderately selective media of Helicobacter pylori
MAC, EMB----Enterobacteriaceae, Vibrios and other d. Rectal Swab
pathogen
 substituted as a specimen for bacterial and viral
HEA, XLD, SSA------inhibit growth of most culture
Enterobacteriaceae, allowing Salmonella and
 placed in Cary-Blair or Stuart’s Transport
Shigella to be detected
Medium
c. Highly selective agar
2.5 cm swab--- inserted through the anal
Brilliant Green, Bismuth Sulfite---Salmonella spp. sphincter
Campy-Blood Agar---Campylobacter spp. Methylene Blue---utilized to observe fecal
leukocoytes
TCBS---Vibrio cholera
6. URINE
CIN---Yersinia enterocolitica
Collection
Cycloserine Cefoxitin Egg Fructose Agar (CCFA)
 Clean-catch midstream urine
---C. difficile
 First-morning urine preferred specimen
Note!!!
Area should be cleaned first with soap and water
Incubated at 35-37°C and examined at 24 and 48
hours for colonies except:  Suprapubic Aspiration

Campy-BAP incubated at microaerophilic  Catheterization

atmosphere at 42°C and examined 24 and 48 hours
a. Straight Catheter
(C. jejuni C. coli)
---first 15 ml of the urine should be voided or
TCBS incubated at 35-37°C for 48 hours
expelled before
CIN Agar incubated at room temperature for 48
collecting the ensuing urinary stream as specimen
hours
b. Indwelling Catheter(Foley Catheter)
CCFA incubated anaerobically for 48 hours
---catheter collection port should be cleaned before
d. Enrichment broth
starting
Selenite F, Hajna Gram Negative, Tetrathionate
---5ml to 10ml of urine should be aspirated with
 enhanced recovery of Salmonella, Shigella and needle and syringe
Campylobacter
 incubated at 35°C for 12-18 hours

FPRE
93
Handling  S. saprophyticus cause UTI with colony counts
in a lower range of less than 1x105 colonies/mL
 Cultured within 2 hours after collection
7. ABSCESS (LESION, WOUND PUSTULE,
 May be stored as long as 24 hours under
ULCER)
refrigeration (4°C)
 preferable to use needle and syringe
Gram Stain of Uncentrifuged Specimen
 needle aspiration enhances recovery of
 simplest and most rapid method for detecting
anaerobic bacteria
significant bacteriuria
Superficial Abscess
 presence of 1 organism/OIF (examining 20 fields)
correlates with significant bacteriuria  aerobic swab moistened with Stuart’s or Amies
(>105CFU/ml) medium may be used
Culture  swab along the leading edge of the wound
a. 5% Sheep BAP Deep Abscess
b. MAC  aspirated from the wall of the abscess or the
advancing margin of the lesion
c. Columbia Colistin-Nalidixic Agar (CNA)
 aspirated specimen should be transferred to a
d. PEA
sterile tube or transport vial
Colony Count
8. BODY FLUIDS
a. Pour Plate Method
 Amniotic, Abdominal, Ascites/Peritoneal, Bile,
b. Calibrated Loop Method (0.01 or 0.001ml) Synovial, Pericardial, Pleural)

Interpretation  skin should be disinfected before aspirating the

sample
a. Colony count >105CFU/ml (100, 000 CFU/ml) for
 soncentrating through centrifugation or filtration
1 or 2 bacterial species clinically significant of may be required prior to smearing and
infection cultivation
b. Colony count > 105 CFU/ml for 3 or more bacterial 9. BONE
spp. contamination
specimen may require homogenization
c. Colony count >103 /ml of a bacterial specie from
midstream urine of a male clinically significant 10. EAR

d. Colony count 102 − 103 /ml of 1 or 2 bacterial spp. Inner ear

from females with lower urinary tract symptoms
clean ear canal with mild soap, aspirate fluid with
clinically significant
needle if eardrum intact; use swab if eardrum
e. Colony count < 5,000 CFU/ml contamination ruptured

f. Presence of organism in any quantity obtained by Outer ear

suprapubic aspiration significant
remove debris or crust from ear canal with saline-
Note!!! moistened swab; rotate swab in outer canal

 Most common cause of UTIs: 11. FOREIGN BODIES

Enterobacteriaceae (E. coli); others – Klebsiella,
a. Intrauterine Device (IUD)
Proteus, Enterobacter, Enterococcus faecalis,
Pseudomonas aeruginosa ---usually cultured for the detection of Actinomyces
spp.
FPRE
94
b. Catheters and Prosthetic Valves
---segment of catheter (5cm to 7 cm) is rolled four
times across the agar using sterile forceps (Maki Roll
Technique)
---More than 15 colonies are required to perform
identification and susceptibility test Whole Foley
catheter should not be cultured
12. GENITAL TRACT
Cervix and Vagina
---remove mucus before collection; do not use
lubricant on speculum; swab endocervical canal or
vaginal mucosa
Urethra
---flexible swab inserted 2-4 cm into urethra for 2-3 s
or collect discharge
Prostate gland
---secretions on the swab or in the tube should be
collected
13. TISSUE
---disinfect skin; do not allow tissue to dry out; if
necessary, moisten with sterile saline
CRITICAL (PANIC) VALUES
1. Positive blood cultures
2. Positive CSF Gram stain or culture
3. Streptococcus pyogenes (group A
Streptococcus) in a surgical wound
4. Gram stain suggestive of gas gangrene
5. Positive acid-fast stain
6. Positive antibiotic-resistant bacteria
(e.g., Vancomycin-resistant S. aureus)
7. Positive for Legionella, Francisella and
Brucella

FPRE
95
UNIT 13: ANTIMICROBIALS AND ANTIBIOTICS
ANTIMICROBIALS Narrow-Spectrum
 chemical substances produced by  effective against limited number of pathogens
microorganisms with the capacity to inhibit
 bacitracin,clindamycin, dapsone, erythromycin,
(bacteriostatic) or kill (bactericidal) other
isoniazid, penicillin, polymyxin B and
microorganisms
vancomycin
 can also be synthesized by means of chemical
Broad-Spectrum
procedures that are independent from microbial
activity  destroys different kinds of organisms
Source  ampicillin, cephalosporins, chloramphenicol,
ciprofloxacin, rifampicin, sulphonamides,
trimethoprim and tetracyclin
Classification of Antibacterial Drugs
1. Natural Drugs
 produced by bacteria and fungi
Examples: amphotericin B, erythromycin, kanamycin,
neomycin, nystatin, rifampicin, streptomycin,
tetracyclin, vancomycin, bacitracin, gentamicin,
polymyxin, griseofulvin, penicillin and cephalosporin
2. Semi-synthetic Drugs
 modified natural drugs with added chemical
group
Examples: ampicillin, carbenicillin and methicillin
3. Synthetic Drugs
 chemically produced drugs
Examples: sulphonamides, trimethoprim,
chloramphenicol,
ciprofloxacin and dapson
MODE OF ACTION OF ANTIBACTERIAL AGENTS
A. Inhibitors of Cell Wall Synthesis
 most selective with a high therapeutic index
 inhibit the activity of the transpeptidase enzymes
BACTERIOCIDAL AGENTS in which cell growth stops and death of the cell
 Antimicrobial agents that inhibit bacterial growth often follows Examples:
but generally do not kill the organism β-Lactam (Beta-Lactam)
BACTERICIDAL AGENTS  inhibits transpeptidation and cell wall synthesis
 agents that usually kill target organisms  penicillins, cephalosporins, carbapenems and
monobactams

FPRE
96
Vancomycin Metronidazole
 inhibits translocation and elongation of  disrupts DNA and effective against anaerobic
peptidoglycan layer bacteria
Bacitracin D. Cell Membrane Inhibitors
 inhibits synthesis of peptidoglycan precursors Polymyxin B and E
Isoniazid  for gram(-) bacteria (e.g., Pseudomonas
aeruginosa) and as topical antibiotic
 acts only on growing cells and can either be
bactericidal or bacteriostatic E. Essential Metabolite Inhibitors
Cephalosporins Sulfamethoxazole (SMZ)
 cephalothin, cefoxitin, ceftriaxone, cephalexin,  inhibits folic acid metabolism and has a high
cefixime and cefoperazone therapeutic index
Penicillinase-resistant penicillin drugs Trimethoprim (TMP)
 methicillin, nafcillin and oxacilin  blocks tetrahydrofolate synthesis
Carbenicillin Dapsone
B. Protein Synthesis Inhibitors  interferes with folic acid synthesis
 binds with 30S subunit that results in the Isoniazid
misreading of mRNA and thus interferes with
 inhibits cord factor synthesis (mycolic acid)
aminoacyl-tRNA binding
 also binds with 50S subunit that results in the
inhibition of peptidyl transferase and peptide
chain elongation
Examples:
Binds to 30S---aminoglycosides(gentamicin,
kanamycin, neomycin, streptomycin, tobramycin),
tetracyclins, spectinomycin and mioglycosides
Binds to 50S---chloramphenicol, erythromycin,
lincomycin and clindamycin
Linezolid---blocks the initial step in protein synthesis
C. Nucleic Acid Synthesis Inhibitors
Rifampicin
 inhibits RNA polymerase
Quinolone
 interferes with DNA gyrase and topoisomerase
IVand highly effective for enteric bacteria (e.g,
E.coli)
 ciprofloxacin, norfloxacin and levofloxacin

FPRE
97
UNIT 14: ANTIMICROBIAL SUSCEPTIBILITY
TESTING
Antimicrobial Susceptibility Testing (AST)
 procedures used to produce antimicrobial
susceptibility profiles and detect resistance to
therapeutic agents
 determine which antimicrobial agents might be
effective in treating infections caused by the
bacteria
Purpose
 Determine the capacity of the bacterial isolate to
cause disease
 Identify susceptibility patterns of the bacterial
isolate to the antimicrobial agents
 Ascertain the availability of the standard
susceptibility methods
TERMINOLOGIES
Breakpoint (Cutoff)
concentration of antimicrobial agent that coincides
with a susceptible or intermediate MIC breakpoint for
a particular drug
Minimum Bactericidal Concentration
lowest concentration of the antimicrobial agent that is
needed to kill the bacterial growth
Minimum Inhibitory Concentration
lowest concentration of the antimicrobial agent that
inhibits the bacterial growth
 may indicate contamination, improper incubation,
improper concentration of antimicrobials and
presence of unusual resistant isolates
Tolerance
 ability of organism to be inhibited by a drug
which is normally bactericidal
 MBC:MIC ratio of >32
Trailing Growth
heavy bacterial growth at lower concentrations that is
followed by one or more wells that show greatly
reduced growth in the form of a small button or light
haze
STANDARDIZED COMPONENTS OF
ANTIMICROBIAL SUSCEPTIBILITY TESTING
• Bacterial inoculum size
• Growth medium (Mueller-Hinton base)
 pH
 Cation concentration
 Blood and serum supplements
 Thymidine content
• Incubation atmosphere
• Incubation temperature
• Incubation duration
• Antimicrobial concentrations
LIMITATIONS OF STANDARDIZATION
 Antibiotic diffusion into tissues and host cells

Minimum Lethal Concentration  Serum protein binding of antimicrobial agents

lowest concentration of the antimicrobial agent that  Drug interactions and interference
kills the bacterial growth when subcultured in a fresh
 Status of patient defense and immune systems
medium
 Multiple simultaneous illnesses
Paradoxic (Eagle) Effect
 Virulence and pathogenicity of infecting
decreased bactericidal activity at a higher drug
bacterium
concentration
 Site and severity of infection
Skipped Wells
 growth at higher concentrations and no growth
at one or more of the lower concentrations

FPRE
98
TESTING METHODS  compared by examining turbidity against a dark
Background
PRINCIPLES
Wickerham Card
3 General Methods
1. Directly measure the activity of one or more
antimicrobial agents against a bacterial isolate
2. Directly detect the presence of a specific
resistance mechanism in a bacterial isolate
3. Measure complex antimicrobial-organism
interactions
A. METHODS THAT DIRECTLY MEASURE
ANTIMICROBIAL ACTIVITY
1. Conventional susceptibility testing methods Mueller-Hinton Agar

 Broth Dilution  standard susceptibility medium for non-

fastidious bacteria
 Agar Dilution
 recommended because it has reduced
 Disk Diffusion concentrations of sulphonamides, trimethoprim
and tetracycline inhibitors
2. Commercial susceptibility testing systems
3. Special screens and indicator tests Composition: Beef infusion, nucleic acids, vitamins,
casein hydrolysate (neutralizes fatty acids) and agar
1. Conventional Testing Methods
MH Broth with 2% NaCl
GENERAL CONSIDERATIONS
 improve detection of oxacillin-resistant
INOCULUM PREPARATION staphylococci
 Two important requirements  Susceptibility testing of staphylococci against
methicillin, oxacillin or nafcillin
1. Use of a Pure Culture
MH Broth with 5% lysed Horse or Sheep Blood
 select 4 or 5 colonies (same morphology),
inoculate into broth medium(TSB), and allow to  test susceptibility of streptococci, Neisseria
achieve active growth (midlogarithmic phase) meningitides and other fastidious organisms
 four to five colonies 16 to 24 hours of age may
be selected from an agar plate and suspended
in broth or 0.9% saline solution to achieve a
turbid suspension
2. Use of a Standard-sized Inoculum
0.5% McFarland Turbidity Standards (Barium
Sulfate Suspension)
 mix 99.5ml 1% sulfuric acid and 0.5ml 1.175%
barium chloride
 equivalent to 1.5 x CFU/ml

FPRE
99
SELECTION OF ANTIMICROBIAL AGENTS FOR Note!!!
TESTING
MH BROTH
Antimicrobial Battery or Panel
 most commonly used medium
 antimicrobial agents chosen for testing against a
 contains little or no cations (Ca and Mg)
particular bacterial isolate
 Standard Inoculum size: 1.5 x CFU/ml
 Criteria for antimicrobial battery content and use
 Organism identification or group Two General Categories:

 Acquired resistance patterns common to local Broth Microdilution

microbial flora  total broth volume is 0.05 to 0.1 mL
 Antimicrobial susceptibility testing method use  Standard inoculum size for anaerobic bacteria:
 Site of infection 1.5 x CFU/ml

 Availability of antimicrobial agents in the  Incubation: 16 to 24 hours at 35°C and 48 hours

formulary at 35°C for anaerobes

Testing profiles are considered for each of the Advantage: testing different antimicrobials
common organism groupings: (10 to 15) at the same time against a single isolate

 Enterobacteriaceae Disadvantage: inability to produce penicillin

MIC that is consistently inside the resistant range for
 Pseudomonas aeruginosa and Acinetobacter staphylococci (low-level Blactamase producers)
spp.
Broth Macrodilution
 Staphylococcus spp.
 broth volumes are usually 1 mL or greater
 Enterococcus spp.
 rarely used method
 Streptococcus spp. (not including S.
pneumoniae)  Incubation: 16 to 24 hours at 35°C

 Streptococcus pneumoniae Advantage: testing fastidious bacteria and

identifying MBC endpoints
 Haemophilus influenzae
Disadvantage: impracticability
 Neisseria gonorrhoeae
Microtiter tray used for broth microdilution
BROTH OR TUBE DILUTION METHOD testing
 involves challenging organism of interest with
antimicrobial agents in a liquid environment
 agent is tested using a range of concentration
expressed as ug/ml
 best method but time consuming
 determine the MIC and MLC
 preferred method is serial twofold-dilution

FPRE
100

Reading and Interpretation of Results
Examined for bacterial growth
 Each tray should include: growth control and a
sterility control
 Bacterial growth, manifested as light to
heavy turbidity or a button of growth on the
well bottom
 Determine the MIC lack of visual turbidity
Determination of MBC or MLC
 0.1 ml of inoculum from each tubes that
exhibited no visible growth is subcultured to solid
agar
 Number of colonies is counted and compared to
the number of CFU/ml the original inoculum
 Lowest concentration that allowed <0.1% of
theoriginal inoculum to survive or the lowest
concentration that is bactericidal or lethal to at
least 99% of the original inoculum---MBC or
MLC
A microdilution, or microtiter, plate used for
testing for minimal inhibitory concentration (MIC) of
antibiotics.
FPRE
Definitions of Susceptibility Testing Interpretive
Categories
SUSCEPTIBLE
 antimicrobial agent in question may be an
appropriate choice for treating the infection
caused by the organism
 bacterial resistance is absent or at a clinically
insignificant level
INTERMEDIATE
 potential utility in body sites where it may be
concentrated (e.g., the urinary tract) or if high
concentrations of the drug are used
 possible effectiveness against the isolate, but
possibly less so than against a susceptible
isolate
 interpretive safety margin to prevent relatively
small changes in test results from leading to
major swings in interpretive category (e.g.,
resistant to susceptible or vice versa)
Resistant
 antimicrobial agent in question may not be an
appropriate choice for treatment, either because
the organism is not inhibited with serum-
achievable levels of the drug or because the test
result highly correlates with a resistance
mechanism that indicates questionable
successful treatment
AGAR DILUTION METHOD
 antimicrobial concentrations and organisms to
be tested are brought together on an agar based
medium
 allows examination of one or more bacterial
isolates per plate
 reference method for testing anaerobes and N.
gonorrhoeae
 plates are inoculated with Steers-Foltz
replicator( deliver 0.001ml of bacterial
suspension)
101
Note!!! Notes!!!
 Standard inoculum size: 1 × 104 CFU/ml  Standard medium: MHA
 Medium medium: MHA  Standard inoculum size: 1.5 x CFU/ml
 Medium for anaerobes: Brucella-Laked  Long-term disk storage: −20° C or below in a
non–frost-free freezer
Sheep(Wadsworth Method)
 Working supply: 2° C to 8° C for at least 1 week
Note!!!
 Standard inoculum of bacteria is spot-inoculated
onto a 100-mm plate
 Incubation: 35°C for 48 hours
 Shelf life: 1 week
 Determines MIC but does not determine MBC or
MLC
 Fastidious bacteria: 5% defibrinated
sheep’sblood is added to MHA

Note!!!
 Surface of the plate is swabbed in three
directions (60° between steaking overlapping
streaking)
DISK DIFFUSION METHOD  Surface of the medium allowed to dry for 3 to
 aka KIRBY-BAUER TEST 5 minutes but not longer than 15 minutes

 detected by challenging bacterial isolates with  Within 15 minutes of inoculation, antimicrobial

antibiotic disks placed on the surface of an agar
plate that has been seeded with a lawn of
bacteria
 limited to aerobic and facultatively anaerobic
bacteria
 qualitative method that provides greatest
flexibility and costeffectiveness
disks are applied and inverted for incubation
 Incubation: 35°C for 16 to 18 hours to a
maximum of 24 hours
 No 2 disks should be closer than 24 mm from
the disk center and no closer than 10 to 15 mm
from the edge of the plate and not more than 8
disks in a 90mm plate

 allows to test any 12 antimicrobial agents on a  Dark background and reflected light used to
150-mm MHA plate examine a disk diffusion plate

 Principle: larger the zone of inhibition

(mm)=lower MIC; ZOI is inversely related to MIC

FPRE
102
Kirby-Bauer Method. (a) A multiple antibiotic disk
dispenser and (b) disk diffusion test results.

Interpretation of Results
a. Susceptible
-presence of zone of inhibition around the antibiotic
disk
b. Intermediate
Reading of Results -falls into a range of susceptibility in which the MIC
approaches exceeds the antimicrobial agent level
 plate is examined to confirm that a confluent
that can be achieved
lawn of growth has been obtained
-less clinical response compared with a susceptible
 If growth is satisfactory, diameter of each zone is
strain
measured using a CALIPER OR RULER
-narrow toxic to therapeutic ratio
 Examined 2 to 3 inches above a black, non-
reflective surface and measured from the back c. Resistant
side of the plate
-no zone of inhibition or the inhibition did not meet
 Transmitted light will improve accuracy with the criteria for susceptible or intermediate
penicillinase-resistant penicillins, linezolid, and
vancomycin Cause of False Resistance

 Mixed cultures require purification and repeat  Use of heavy inocula or undiluted specimen
testing  Late examination of test plates after zones had
 Media that contain blood examined with the lid become overgrown prolonged incubation
removed  Use of disc with inadequate drug concentration
 Tiny colonies at the zone edge and swarm of  prolonged storage
growth into the zone (Proteus spp.) are ignored;
obvious zone is measured  Failure to immediately refrigerate the disc after
used
 Resulting haze of growth should be ignored for
disk interpretation sulfonamides and  Use of wrong bacterial isolate
trimethoprim and trimethoprimsulfamethoxazole
Factors Affecting Zone of Inhibition
1. Amount of inoculum or test organisms
Too light=false susceptibility
Too heavy=false resistance
2. Thickness of the susceptibility agar plate
4mm
Too thick=zone size is smaller
Mixed Culture
Too thin=zone size is larger

FPRE
103
3. Growth rate of the test organism
 Temperature >35°C may lead to false detection
of MRSA
 Inceased CO2(5% to 7%) N. meningitides and
S. pneumonia
 Lower temperature larger zone of inhibition
4. pH of the medium 7.2-7.4
 Incubation in CO2 decreased pH
 High pH=decreased activity of tetracyclin
 Low pH= diminished activity of aminoglycosides
and erythromycin
5. Number of antibiotic disk per plate
6. Concentration of divalent cations (Calcium
and Magnesium)
 Affect testing of AMINOGLYCOSIDES and
TETRACYCLINES against P. aeruginosa
 Increase cations=diminished activity
Disk Elution Method
 Antimicrobial impregnated filter paper disk are
added into the broth or agar medium
 antimicrobial agent diffuses throughout the broth
or agar medium (elute) to provide a desired
concentration of the antimicrobial in the medium
 bases of some rapid, semi-automated system
(Autobac MS-2 or Avantage) used for aerobic
and facultatively anaerobic bacteria
 Used for anerobic bacteria and Mycobacteria
FPRE
2. Commercial Susceptibility Testing Methods
 Same for all commercial methods, but the
principles and practices vary with respect to:
 Format in which bacteria and antimicrobial
agents are brought together
 Extent of automation for inoculation, incubation,
interpretation, and reporting
 Method used for detection of bacterial growth
inhibition
 Speed with which results are produced
 Accuracy
A. Broth Microdilution Methods
 provide microdilution panels already prepared
and formatted
 Reading ---semiautomated reading devices
B. Agar Dilution Derivations
 growth patterns on a plate containing an
antibiotic gradient applied by the Spiral Gradient
instrument
C. Diffusion in Agar Derivations
E-test
 dilution test based on the diffusion of a
continuous varying concentration of antimicrobial
agent
 plastic strip with one side containing the
antimicrobial agent concentration gradient and
other contains a numeric scale that indicates the
drug concentration
 alternative susceptibility for fastidious bacteria: S.
pneumonia, H. influenzae and anaerobes
104
 MIC is read at point where the ellipse intersect 2. MicroScan WalkAway
strip
 system uses the broth microdilution panel format
manually inoculated with a multiprong device
 growth patterns are automatically read and
interpreted
 bacterial growth may be detected using
spectrophotometry (overnight incubation) or
fluorometry(3.5 to 5.5 hours)
3. Phoenix System
D. Automated Antimicrobial Susceptibility Test
Systems  convenient, manual, gravity-based inoculation
process
1. Vitek 2 AST
 growth is monitored in automated fashion based
 facilitates standardized susceptibility testing with
on a redox indicator system with results
validated results and recognition of antimicrobial
available in 8 to 12 hours
resistance mechanism in 6 to 8 hours
Supplemental Testing Methods
 automatically introduced inoculum by a filling
tube into a miniaturized, plastic, 64-well, closed a. Oxaxillin Agar Screen
card containing specified concentrations of
antibiotics Purpose: Detection of staphylococcal resistance to
penicillinaseresistant penicillins (e.g., oxacillin,
 optical readings are performed every 15 minutes methicillin, or nafcillin)
 MIC and antimicrobial testing result are identified Conditions:
using Advance Expert Sytem (AES)
Medium: Mueller-Hinton agar plus 6 μg
oxacillin/mL plus 4% NaCl
Inoculum: Swab or spot from 1.5 × 108 standard
suspension
Incubation: 30°-35°C 24 hr, up to 48 hr for non–
Staphylococcus aureus
Interpretation: Growth = Resistance
No growth = Susceptible
b. Vancomycin Agar Screen
Purpose: Detection of enterococcal resistance to
vancomycin
Conditions:
Medium: Brain-heart infusion agar plus 6 μg
vancomycin/mL Inoculum: Spot of 105 - 106 CFU
Incubation: 35°C 24 hr
Interpretation:
Growth = Resistance
No growth = Susceptible
FPRE
105
c. Aminoglycoside Screen
Purpose: Detection of acquired enterococcal high-
level resistance to aminoglycosides that would
compromise synergy with a cell wall–active agent
(e.g., ampicillin or vancomycin)
Conditions:
Medium: Brain-heart infusion broth: 500 μg/mL
gentamicin; 1000 μg/mL streptomycin Agar: 500
μg/mL gentamicin; 2000 μg/mL streptomycin
A positive D test indicates that the presence of
Inoculum: Broth; 5 ×105 CFU/mL agar; 106 macrolide-inducible resistance to clindamycin
CFU/spot
Predictor Antimicrobial Agents
Incubation: 35°C, 24 hr; 48 hr for streptomycin,
only if no growth at 24 hr Predictor Drugs- most sensitive indicators of certain
resistance mechanisms
Interpretation:
Staphylococcal resistance to oxacillin
Growth = Resistance
--determine and report resistance to beta-lactams
No growth = Susceptible
Enterococcal high-level gentamicin resistance
d. Oxacillin Disk Screen
--predicts resistance to nearly all other currently
Purpose: Detection of Streptococcus pneumoniae available aminoglycosides
resistance to penicillin
Enterococcal resistance to ampicillin
Conditions:
--predicts resistance to all penicillin derivatives
Medium: Mueller-Hinton agar plus 5% sheep
blood plus 1 μg oxacillin disk 2. METHODS THAT DIRECTLY DETECT SPECIFIC
RESISTANCE MECHANISMS
Inoculum: as for disk diffusion
A. Phenotypic Methods
Incubation: 5%-7% CO2 35° C; 20-24 hr
a. BETA-LACTAMASE DETECTION
Interpretation:
Chromogenic Cephalosporinase Test
Inhibition zone ≤20 mm: penicillin susceptible
---β-lactamases opens the β-lactam ring
Inhibition zone <20 mm: penicillin resistant,
intermediate, or susceptible; further testing by MIC Chromogenic cephalosporin colored
method is needed product

e. D Test Example: Cefinase disk Substrate: Nitrocefin

Purpose: Differentiate clindamycin resistance Applications:

among S. aureus resulting from efflux (msrA gene or
 N. gonorrhoeae resistance to penicillin
MLSB resistance)
 H. influenzae resistance to ampicillin
Conditions: Approximation of clindamycin and
erythromycin disk to look for blunting of clindamycin  Staphylococcal resistance to penicillin
zone
b. Chloramphenicol Acetyltransferase Detection
Interpretation: Blunting of clindamycin zone to give
Chloramphenicol modification by
“D” pattern, indicating inducible clindamycin
chloramphenicol acetyltransferase (CAT)
resistance
FPRE
106
---one mechanism by which bacteria may express
resistance to this agent
B. Genotypic Methods
---molecular methods involving NUCLEIC ACID
HYBRIDIZATION AND AMPLIFICATION for the
study and detection of antimicrobial resistance
---Can be used to evaluate phenotype-based
methods
3. SPECIAL METHODS FOR COMPLEX
ANTIMICROBIAL/ORGANISM INTERACTIONS
---designed to measure bactericidal activity or
measure the antibacterial effect of combination
therapy with antimicrobial agents
A. BACTERICIDAL TESTS
---designed to determine the ability of antimicrobial
agents to kill bacteria
Methods:
 Minimal bactericidal concentration(MBC)
testing
 Time-kill studies
 Serum-cidal testing
a. Minimal Bactericidal Concentration
---in-vitro determination of the amount of
antimicrobial agent required to kill as well as inhibit
bacteria
Note!!!
Standard inoculum: 5 ×105 CFU/mL
Final dilution: 1:1000 (0.01 ml of the original tube in
10 ml)
Volume spread per plate: 0.1 ml
Dilution factor: 1:10,000 (104 )
(+) Result: 99.9% reduction in the CFU/ml
b. Time-Kill Studies
---involves exposing a bacterial isolate to a
concentration of antibiotic in a broth medium and
measuring the rate of killing over a specified period
---1000-fold decrease in number of viable bacteria
after a 24-hour period, compared with the number of
FPRE
bacteria in the original inoculum, is interpreted as
bactericidal activity
c. Serum Bactericidal (Schlichter Test)
---analogous to the MIC-MBC test except the
medium used is the patient’s serum
---determines the activity of one or more
antimicrobials agents present in the serum
Two serum samples:
Trough specimen collected just before the patient
is to receive the next antimicrobial dose
Peak specimen collected when the serum
antimicrobial concentration is highest
Final inoculum size: 5 ×105 CFU/mL
Procedure: aliquot of each dilution is plated on
sheep’s BAP and incubated
Result: the highest dilution that inhibits visible
growth in the serum-static titer
Interpretation: serum dilution resulting in 99%
reduction in CFU/ml compared with the original
inoculums is the serumcidal titer
Serum-static Titer --highest dilution that inhibits
visibly detectable growth
Serum Bactericidal or Lethal Titer --highest
dilution of patient’s serum required to kill a
microorganism
Serum Bactericidal Level --lowest dilution of
patient’s serum that kills a standard inoculum
B. Tests for Activity of Antimicrobial
Combinations
SYNERGY TESTING
---determines the effectiveness of combined
antimicrobials against a single bacterial isolate
---performed using a broth dilution checkerboard
method or time-kill assay
---two antimicrobial agents are test in each well or
tubes
Results: Killing the organism by 100-fold or more
than the most active antibiotic tested singly after 24
hours of incubation
107
Interpretation: Antagonism is seen when the two
antibiotics appear less potent than the most active
single antimicrobial
Three outcome categories :
Synergy activity of the antimicrobial combination is
substantially greater than the activity of the single
most active drug alone
Indifference activity of the combination is no better
or worse than the single most active drug alone
Antagonism activity of the combination is
substantially less than the activity of the single most
active drug alone

Synergism between two different antibiotics

FPRE
108
 UNIT 15: BIOCHEMICAL TEST RESPIRATION
BACTERIAL METABOLISM →efficient energy-generating process
→biochemical reactions bacteria use to break down →molecular oxygen is the final electron acceptor
organic compounds and reactions they use to
synthesize new bacterial parts from the resulting →Obligate aerobes and facultative anaerobes
carbon skeleton Note!!!
✓Diagnostic schemes analyse each unknown Certain anaerobes can carry out anaerobic
microorganism for: respiration, in which inorganic forms of oxygen, such
(1) utilization of various substrates as a carbon as nitrate and sulfate, act as the final electron
source acceptors

(2) production of specific end products from various Biochemical Pathways from Glucose to Pyruvic
substrates Acid

(3) production of an acid or alkaline pH in the test ✓Three major biochemical pathways bacteria use to
medium break down glucose to pyruvic acid are:

Energy Production 1. Embden-Meyerhof-Parnas (EMP) Glycolytic

pathway
→Breakdown of chemical substrate through the
degradative process of catabolism coupled with 2. Pentose Phosphate Pathway
oxidation-reduction reactions 3. Entner-Doudoroff Pathway
→Bacteria use biochemical pathways to catabolize 1. EMP Glycolytic Pathway
(break down) carbohydrates and produce energy by
two mechanisms: →major pathway in conversion of glucose to
pyruvate
1. Fermentation
→generates reducing power in the form of NADH2
2. Respiration (oxidation)
→generates energy in the form of ATP
FERMENTATION
→anaerobic; does not require oxygen
→anaerobic process carried out by both obligate
and facultative anaerobes Example: Enterobacteriaceae

→Electron acceptor is an organic compound

→less efficient in energy generation---beginning
substrate is not completely reduced
→a mixture of end products (lactate,
butyrate, ethanol, and acetoin) accumulates in the
medium--- identification of anaerobic bacteria
Application:
a. Voges-Proskauer (VP)
b. Methyl Red Tests

FPRE
109
2. Pentose Phosphate (Phosphogluconate)
Pathway
Anaerobic Utilization of Pyruvic Acid
→alternative to EMP pathway (Fermentation)
→glucose to ribulose-5-phosphate, which is A. Alcoholic Fermentation
rearranged into other 3-, 4-, 5-, 6-, and 7-carbon
→major end product is ethanol
sugars
Example: yeasts
→provides pentoses for nucleotide synthesis
B. Homolactic Fermentation
→produces glyceraldehyde-3-phosphate, which can
be converted to pyruvate →end product is almost exclusively lactic acid
→generates NADPH, which provides reducing power Example:
for biosynthetic reactions
a. All members of the Streptococcus genus
→may be used to generate ATP
b. members of the Lactobacillus genus
Example:
C. Heterolactic Fermentation
a. Lactobacilli→ Heterolactic fermenting bacteria
→in addition to lactic acid, the end products include
b. Brucella abortus→ lacks some of the carbon dioxide, alcohols, formic acid, and acetic acid
enzymes required in the EMP pathway Example: Some lactobacilli
3. Entner-Doudoroff Pathway D. Propionic Acid Fermentation
→glucose-6-phosphate (rather than glucose) →Propionic acid is the major end product of
fermentations
to pyruvate and glyceraldehyde phosphate
Example:
→generates one NADPH per molecule of glucose
but uses one ATP a. Propionibacterium acnes
Example: Aerobic process used by: b. some anaerobic non–spore-forming, grampositive
bacilli
a. Pseudomonas
E. Mixed Acid Fermentation
b. Alcaligenes
→produce a number of acids as end products—lactic,
c. Enterococcus faecalis
acetic, succinic, and formic acids
d. Other bacteria lacking certain glycolytic enzymes
→strong acid produced is the basis for the positive
reaction on the methyl red test
Example: Members of:
a. Escherichia
b. Salmonella
c. Shigella

FPRE
110
F. Butanediol Fermentation ***Glucose must not be present if the ability to
ferment another sugar is being tested
→end products are acetoin (acetyl methyl carbinol)
and 2,3- butanediol Classifying members of the Enterobacteriaceae
family→ ability to ferment lactose
→detection of acetoin is the basis for the positive VP
reaction β-galactoside permease→ transport of lactose
across the cell wall into the bacterial cytoplasm
→Little acid is produced by this pathway
β-galactosidase→ break the galactoside bond,
Example: Members of:
releasing glucose, which can be fermented
a. Klebsiella,
***All organisms that can ferment lactose can also
b. Enterobacter ferment glucose

c. Serratia Energy Utilization

***Organisms that have a positive VP reaction 1. Biosynthesis of new cell components

usually have a negative reaction on the methyl red
2. Maintenance of the physical and chemical integrity
test, and vice versa
of the cell
H. Butyric Acid Fermentation
3. Activity of the locomotor organelles
→Involves the conversion of pyruvate to butyric acid
4. Transport of solutes across membranes
along with acetic acid, CO2 and Hydrogen
5. Heat production
Example:
a. Clostridium
b. Fusobacterium
c. Eubacterium
Aerobic Utilization of Pyruvate (Oxidation)
Krebs or Tricarboxylic acid (TCA) cycle
→most important pathway for the complete oxidation
of a substrate under aerobic conditions
→pyruvate is oxidized, carbon skeletons for
biosynthetic reactions are created, and the electrons
donated by pyruvate are passed through an electron
transport chain and used to generate energy in the
form of ATP
→results in the production of acid and the evolution
of carbon dioxide
Carbohydrate Utilization and Lactose
Fermentation
→use of various sugars (carbohydrates)
→fermentation is usually detected by acid production
and a concomitant change of color resulting from a
pH indicator present in the culture medium

FPRE
111
ACETAMIDE UTILIZATION
➢ability to use acetamide as the sole source of
carbon
➢produce acylamidase, which deaminates
acetamide to release ammonia resulting in an
alkaline pH
Result:
✓Positive: BLUE COLOR
✓Negative: No color change BACITRACIN (TaxoA) SUSCEPTIBILITY

QC: ➢presumptive identification and differentiation of

✓Positive: Pseudomonas aeruginosa Streptococcus pyogenes

✓Negative: Escherichia coli ➢distinguish staphylococci species (resistant) from

micrococci
(susceptible)
Result:
✓Positive: ZOI>10 mm
✓Negative: No zone of inhibition
QC:
✓Positive: Streptococcus pyogenes
Micrococcus luteus

ACETATE UTILIZATION ✓Negative: Streptococcus agalactiae

➢ability to use acetate (sodium acetate) as the sole Staphylococcus aureus

source of carbon
➢differentiate Shigella sp. from Escherichia coli
Result:
✓Positive: Medium becomes alkalinized = BLUE
✓Negative: No growth or no indicator change to blue
QC:
✓Positive: Escherichia coli
✓Negative: Shigella sonnei

FPRE
112
BILE ESCULIN TEST BUTYRATE DISK
➢differentiates ENTEROCOCCI and GROUP D ➢detect BUTYRATE ESTERASE
STREPTOCOCCI from non–group D viridans ➢identification of Moraxella (Branhamella)
streptococci catarrhalis
➢growth in the presence of 4% bile and to ➢bromochlorindolyl butyrate → indoxyl + O2 →
hydrolyze esculin to esculetin which reacts with Indigo
Fe3+ to form dark brown to black precipitate
Result:
Result:
✓Positive: Development of a BLUE COLOR (5-
✓Positive: GROWTH and BLACKENING of the minute incubation)
agar slant
✓Negative: No color change
✓Negative: Growth and no blackening of medium
QC:
QC:
✓Positive: Moraxella catarrhalis
✓Positive: Enterococcus faecalis
✓Negative: Neisseria gonorrhoeae
✓Negative: Escherichia coli

BILE SOLUBILITY TEST

➢differentiates Streptococcus pneumonia CHRISTIE, ATKINS, AND MUNCH-PETERSON
(positive–soluble) from alpha-hemolytic (CAMP) TEST
streptococci (negative–insoluble)
➢differentiate Streptococcus agalactiae (positive)
➢Bile/Bile salt (sodium desoxycholate) rapidly lyses
from other streptococcal species
pneumococcal colonies
➢CAMP factor acts synergistically with the beta-lysin
➢Lysis depends on amidase→ autolytic enzyme
of S. aureus to cause enhanced lysis of RBC
Result:
Result
✓Positive: Colony disintegrate
✓Positive: Arrowhead-shaped zone of beta-
✓Negative: Intact colonies hemolysis

QC: ✓Negative: No enhancement of hemolysis

✓Positive: Streptococcus pneumonia QC

✓Negative: Enterococcus faecalis ✓Positive: Streptococcus agalactiae

✓Negative: Streptococcus pyogenes

FPRE
113
 CATALASE TEST

CAMP TEST
CAMP Positive Organisms:
1. Streptococcus agalactiae
2. Listeria monocytogenes
3. Proprionibacterium acnes CETRIMIDE AGAR
Reverse CAMP Positive Organisms: ➢isolate and purify Pseudomonas aeruginosa from
1. Clostridium perfringens contaminated specimens

2. Corynebacterium pseudotuberculosis ➢ability of an organism to grow in the presence of

cetrimide
3. Corynebacterium ulcerans
Result:
4. Corynebacterium urealyticum
✓Positive: Growth, variation in color of colonies
CATALASE TEST
✓Negative: No growth
✓differentiates catalase-positive MICROCOCCAL
and STAPHYLOCOCCAL spp. from catalase- QC:
negative STREPTOCOCCAL spp. ✓Positive: Pseudomonas aeruginosa
✓H2O2 → H2O + O = EFFERVESCENCE ✓Negative: Escherichia coli
Result:
✓Positive: Copious bubbles are produced
✓Negative: No or few bubbles are produced
QC:
✓Positive: Staphylococcus aureus
✓Negative: Streptococcus pyogenes

FPRE
114
CITRATE UTILIZATION ✓Negative: No clot
➢ability to use SODIUM CITRATE (sole carbon QC:
source) and inorganic ammonium salts (sole nitrogen
✓Positive: Staphylococcus aureus
source)
✓Negative: Staphylococcus epidermidis
➢citrate-permease converts citrate to pyruvate
➢ammonium phosphate → ammonia and
ammonium hydroxide→ alkaline pH →
BROMTHYMOL BLUE indicator from green to blue
Result:
✓Positive: Growth on the medium = BLUE
✓Negative: Absence of growth
QC:
✓Positive: Enterobacter aerogenes
✓Negative: Escherichia coli
DECARBOXYLASE TESTS (MOELLER’S
METHOD)
➢differentiate decarboxylase producing
Enterobacteriaceae from other gram-negative rods
➢Amino acid → Amine = alkaline pH →from
orange to purple
➢pH indicator: BROMCRESOL PURPLE
Result:
✓Positive: Alkaline (PURPLE) color change
✓Negative: No color change or acid (yellow)

COAGULASE TEST QC:

➢differentiate S. aureus (+) from coagulase- ✓Positive:

negative staphylococci ▪ Lysine—Klebsiella pneumoniae
Two Forms of Coagulase ▪ Ornithine—Enterobacter aerogenes
1. Bound Coagulase→ clumping factor ▪ Arginine—Enterobacter cloacae
2. Free Coagulase→ extracellular protein enzyme ✓Negative:
Result: ▪ Lysine—Enterobacter cloacae
1. Slide Test ▪ Ornithine—Klebsiella pneumoniae
✓Positive: Clumping in 10 seconds or less ▪Arginine—Klebsiella pneumoniae
✓Negative: No clumping
2. Tube Test
✓Positive: Clot of any size
FPRE
115
 Result:
✓Positive: BLACKENED MEDIUM
✓Negative: No blackening
QC:
✓Positive: Enterococcus faecalis
✓Negative: Escherichia coli

DNA HYDROLYSIS (DNASE TEST AGAR)

➢distinguish Serratia sp. (+) from Enterobacter sp.,
Staphylococcus aureus (+) from other species, and
Moraxella catarrhalis (+) from Neisseria sp.
➢ability to hydrolyze DNA (DNAase)
➢DNA–methyl green complex→ from green to
colorless zone
FERMENTATION MEDIA
Result
➢ability to ferment carbohydrates ANDRADE’S
✓Positive: COLORLESS around the test organism
FORMULA
✓Negative: Medium remains green
→ differentiate enteric bacteria from coryneforms
QC
Result:
✓Positive: Staphylococcus aureus
✓ Positive: Indicator change to PINK
✓Negative: Escherichia coli
✓ Negative: Growth, but no change in color
QC:
✓ Positive, with gas: Escherichia coli
✓ Positive, no gas: Shigella flexneri
BROMOCRESOL PURPLE → distinguish
enterococci from streptococci
Result:
✓ Positive: Indicator change to YELLOW
ESCULIN HYDROLYSIS
✓ Negative: Growth, but no change in color
➢presumptive identification and differentiation of
QC:
Enterobacteriaceae
✓ Positive, with gas: Escherichia coli
➢Esculin is hydrolyzed to esculetin, which reacts
with Fe3+ and forms a dark brown to black ✓ Negative, no gas: Moraxella osloensis
precipitate

FPRE
116
 Result:
✓Positive: Partial or total liquefaction at 4°C within
14 days
✓Negative: Complete solidification
QC:
✓Positive: Bacillus subtilis
✓Negative: Escherichia coli
FLAGELLA STAIN (WET MOUNT TECHNIQUE)
➢presence and arrangement of flagella
QC:
✓Peritrichous: Escherichia coli
✓Polar: Pseudomonas aeruginosa
✓Negative: Klebsiella pneumonia
GROWTH at 42°C
➢differentiate a pyocyanogenic pseudomonads from
other Pseudomonas sp.
➢ability of an organism to grow at 42°C
Result
✓Positive: Good growth at both 35°and 42°C
MOTILITY ✓Negative: No growth at 42°C but good growth at
35°C
Rapid darting/ shooting star = Vibrio
(monotrichous) QC
Darting = Campylobacter ✓Positive: Pseudomonas aeruginosa
Twitching = Kingella, Bartonella ✓Negative: Pseudomonas fluorescens
Tumbling (end over end) = Listeria HIPPURATE HYDROLYSIS
Gliding = Capnocytophaga, Mycoplasma ➢HIPPURICASE hydrolyzes hippuric acid→
pneumoniae GLYCINE AND BENZOIC ACID
Corkscrew (axial filaments) = Spirochetes ➢Glycine is deaminated by ninhydrin, which is
reduced during the process
Spinning = Leptospira
➢end products of the ninhydrin oxidation react to
GELATIN HYDROLYSIS
form a PURPLECOLORED PRODUCT
➢gelatinases hydrolyze gelatin
Result
➢presumptive identification of Staphylococcus sp.,
✓Positive: Deep purple color
Enterobacteriaceae, and some gram-positive bacilli
✓Negative: Colorless or slightly yellow pink color

FPRE
117
QC
✓Positive: Streptococcus agalactiae
✓Negative: Streptococcus pyogenes
HIPPURATE HYDROLYSIS
1. Streptococcus agalactiae
2. Listeria monocytogenes
3. Campylobacter jejuni subsp. jejuni
4. Gardnerella vaginalis
INDOLE PRODUCTION
➢Tryptophan → INDOLE + KOVAC’S REAGENT
(dimethylaminebenzaldehyde and hydrochloride) or
EHRLICH’S REAGENT = RED COLOR
Result
✓Positive: PINK- TO WINE-COLORED RING
✓Negative: No color change
QC:
A. KOVAC’S METHOD
✓ Positive: Escherichia coli
✓ Negative: Klebsiella pneumoniae
B. EHRLICH’S METHOD
✓ Positive: Haemophilus influenzae
✓ Negative: Haemophilus parainfluenzae
C. EHRLICH’S METHOD (ANAEROBIC)
✓ Positive: Porphyromonas asaccharolytica
✓ Negative: Bacteroides fragilis
FPRE
LEUCINE AMINOPEPTIDASE (LAP) TEST
➢presumptive identification of CATALASE-
NEGATIVE GRAMPOSITIVE COCCI
➢Hydrolysis of Leucine beta-naphthylamide by
leucine aminopeptidase
→ beta-naphthylamine + cinnamaldehyde reagent =
RED COLOR
Result
✓Positive: Development of a red color within 1
minute
✓Negative: No color change or development of a
slight yellow color
QC
✓Positive: Enterococcus faecalis
✓Negative: Aerococcus viridans
LITMUS MILK MEDIUM
➢ability to metabolize litmus milk: fermentation,
reduction, clot formation, digestion, and the formation
of gas
Result:
➢Fermentation of lactose → litmus turns pink
➢Sufficient acid: casein is coagulated→solidifying
the milk
➢hydrolyze casein→ straw colored
118
➢reduce litmus→ colorless in the bottom of the
tube
QC:
➢Fermentation: Clostridium perfringens
➢Acid: Lactobacillus acidophilus
➢Peptonization: Pseudomonas aeruginosa
LITMUS MILK MEDIUM

LYSINE IRON AGAR (LIA)

➢LIA: lysine, peptones, a small amount of glucose,
ferric ammonium citrate, and sodium thiosulfate
➢GLUCOSE FERMENTED→ BUTT BECOMES
ACIDIC (YELLOW)
➢LYSINE DECARBOXYLASE PRESENT
→CADAVERINE → ALKALINE STATE (PURPLE)
➢OXIDATIVE DEAMINATION of lysine and in the
presence of FERRIC AMMONIUM CITRATE and
FLAVIN MONONUCLEOTIDE, forms BURGUNDY
COLOR on the slant
➢pH indicator: Bromocresol Purple
▪ yellow at or below pH 5.2
▪ purple at or above pH 6.8

FPRE
119
LYSINE IRON AGAR (LIA)
Result
✓Alkaline slant/alkaline butt (K/K)—lysine
decarboxylation and no fermentation of glucose
✓Alkaline slant/acid butt (K/A)—glucose METHYL RED/VOGES-PROSKAUER (MR-VP)
fermentation TESTS

✓Red slant/acid butt (R/A)—lysine deamination and
glucose
QC
✓Alkaline slant and butt: H2S positive: Citrobacter
freundii
✓Alkaline slant and butt: Escherichia coli
✓Alkaline slant and butt: H2S positive: Salmonella
typhimurium
✓Red slant, acid butt: Proteus mirabilis
LYSINE IRON AGAR (LIA)
➢ability to produce and maintain stable acid end
products from glucose fermentation, overcome the
buffering capacity of the system, and to determine
the ability to produce 2,3-BUTANEDIOL or
ACETOIN from
glucose fermentation
METHYL RED
→detects mixed acid fermentation that lowers the pH
→red at pH 4.4 and yellow at pH 6.2

VP
→detects the ability to convert the acid products
to acetoin and 2,3- butanediol→ alpha-naphthol is
added, followed by potassium hydroxide
(KOH)→RED

QC
MR positive/VP negative: Escherichia coli
MR negative:/VP positive: Enterobacter aerogenes
Medium: MR-VP medium or Clurk and Lubs
Dextrose Broth

FPRE
120
MICRODASE TEST (MODIFIED OXIDASE) MRS Broth
➢differentiate STAPHYLOCOCCUS from ➢determine gas formation during glucose
MICROCOCCUS SPP. by detection of the enzyme fermentation
oxidase
➢Lactobacillus spp. and Leuconostoc sp.
➢oxidase reacts with the oxidase reagent and produce gas
cytochrome C to form the indophenol
➢Selective medium: SODIUM ACETATE and
Result: AMMONIUM CITRATE
✓Positive: Development of BLUE TO PURPLE- Result:
BLUE COLOR
✓Positive: Leuconostoc sp. - Growth, gas
✓Negative: No color change production indicated by a bubble in the Durham tube
QC: ✓Positive: Lactobacillus spp. - Growth, no gas
production
✓Positive: Micrococcus luteus
✓Negative: No growth
✓Negative: Staphylococcus aureus
QC
✓Positive: Lactobacillus lactis
✓Negative: Escherichia coli

MOTILITY TESTING
➢diffuse zone of growth extending out from the line
of inoculation
Result:
✓Positive: Spread out from the site of inoculation
✓Negative: Remain at the site of inoculation
QC:
✓Positive: Escherichia coli
✓Negative: Staphylococcus aureus

FPRE
121
4-Methylumbelliferyl-β-D-Glucuronide (MUG) Test QC:
➢presumptively identify various genera of ✓Positive: NO3+, no gas: Escherichia coli
Enterobacteriaceae and verotoxin-producing E. coli
✓Positive: NO3+, gas: Pseudomonas aeruginosa
(neg)
✓Negative: Acinetobacter baumannii
➢β-d-glucuronidase hydrolyzes 4-
methylumbelliferyl-β-d-glucuronide to 4-
methylumbelliferyl which fluoresces blue under long
wavelength UV
Result
✓Positive: Electric blue fluorescence
✓Negative: Lack of fluorescence
QC
✓Positive: Escherichia coli
✓Negative: Klebsiella pneumoniae

NITRATE REDUCTION
➢nitrate reductase converts the nitrate (NO3) to
nitrite (NO2)
➢reduction is determined
by adding SULFANILIC ACID and
ALPHANAPHTHYLAMINE
➢sulfanilic acid and nitrite react to form a diazonium
salt→ couples with the alphanaphthylamine to
produce a red, water-soluble azo dye
➢Zinc is added to validate colorless result for the
presence of untreated nitrate
Result:
✓Positive: RED
✓Negative: No color change

FPRE
122
NITRITE REDUCTION
➢reducing nitrite to nitrogen do not turn color and do
produce gas in the nitrate reduction test
Result:
✓Positive: No color change to red 2 minutes; gas
production in Durham tube
OPTOCHIN (Taxo P) SUSCEPTIBILITY TEST
✓Negative: Broth becomes red ; no gas production
is observed ➢aka ETHYL HYDROCUPREINE
HYDROCHLORIDE
QC:
➢lyses Streptococcus pneumoniae (positive test),
✓Positive: Proteus mirabilis but alpha-streptococci are resistant (negative test)
✓Negative: Acinetobacter baumannii ➢zone of 14 to 16 mm is considered susceptible
➢presumptive identification of Streptococcus
pneumoniae
Result
✓Positive: ZOI ≥ 14 mm in diameter, with 6-mm
disk
✓Negative: No zone of inhibition
QC
✓Positive: Streptococcus pneumoniae
✓Negative: Streptococcus pyogenes
o-Nitrophenyl-β-D-Galactopyranoside (ONPG)
Test
➢ability to produce Β-GALACTOSIDASE which
hydrolyzes substrate ONPG to form orthonitrophenol
(yellow product)
➢distinguishes late LLF from NLF of
Enterobacteriaceae
➢Indicates presence of β-galactosidase
OXIDASE TEST (KOVAC’S METHOD)
Result:
➢presence of cytochrome oxidase which oxidize
✓Positive: YELLOW (presence of β-galactosidase)
TETRAMETHYL-p-PHENYLENEDIAMINE
✓Negative: Colorless DIHYDROCHLORIDE to indophenol (DARK
QC: PURPLE)

✓Positive: Shigella sonnei ➢identification of oxidase-negative

Enterobacteriaceae, differentiating them from other
✓Negative: Salmonella typhimurium Gram (-) bacilli

FPRE
123
Result:
✓Positive: Development of a dark purple color within
10
✓Negative: Absence of color
QC:
✓Positive: Pseudomonas aeruginosa
✓Negative: Escherichia coli

Oxidase Test for Neisseria

Reagent: 1% dimethyl-p-phenylenediamine
hydrochloride
(+): colonies become purple black

Indophenol Method Carbohydrate Fermentation Medium

Reagent: p-aminodimethylaniline hydrochloride A. Base Medium→ sugar-free
(or Oxalate) and alpha-naphthol
a. Peptone water
(+): Intense Blue color within 2 minutes
b. Trypticase
c. Tryptone Broth
Oxidation/Fermentation (of) Medium (CDC
Method) B. pH Indicators

➢ability to oxidize or ferment specific carbohydrates a. Bromcresol Purple → Purple (alk) to Yellow(acid)
at pH 6.3
➢glucose,xylose, mannitol, lactose, sucrose, and
maltose b. Andrade’s Acid Fuchsin→ Pale Yellow (alk) to
Reddish Pink (acid) at pH 5.5
Hugh and Leifson’s formula
c. Phenol Red → Red (alk) to Yellow at pH 7.9
→low peptone-to-carbohydrate ratio and a limiting
amount of carbohydrate.
→reduced peptone limits the formation of alkaline
amines that may mask acid production resulting from
oxidative metabolism

FPRE
124
Other Methods
1. Rusell’s Double Sugar (RDS)
→conatins glucose and lactose with Andrade’ Acid
Fuchsin
2. Smith Fermentation TubeResult:
✓Positive: Acid production (A) indicated by the color
indicator changing to yellow
✓Weak-positive (Aw): Weak acid formation
✓Negative: Red or alkaline (K) color in the deep
✓No change (NC) or neutral (N)
QC:
L-PYRROLIDONYL ARYLAMIDASE (PYR) TEST
✓Fermenter (Glucose): Escherichia coli
➢presumptive identification of Streptococcus
✓Oxidizer (Glucose): Pseudomonas aeruginosa pyogenes and enterococci by the presence of L-
pyrrolidonyl arylamidase
PHENYLALANINE DEAMINASE AGAR
➢L-pyrrolidonyl- β-naphthylamide → β-
➢ability to oxidatively deaminate phenylalanine to naphthylamine + N,Nmethylaminocinnamaldehyde
phenylpyruvic acid reagent → BRIGHT RED PRECIPITATE
➢Morganella, Proteus, and Providencia can be Result
differentiated from other members of the
Enterobacteriaceae family ✓Positive: Bright red color within 5 minutes

➢Phenylpyruvic acid → detected by adding a few ✓Negative: No color change or an orange color
drops of 10% FERRIC CHLORIDE→ green colored
QC
complex
✓Positive: Enterococcus faecalis
Result:
Streptococcus pyogenes
✓Positive: GREEN COLOR on slant
✓Negative: Streptococcus agalactiae
✓Negative: Slant remains original color
QC:
✓Positive: Proteus mirabilis
✓Negative: Escherichia coli
MEDIUM: PHENYLALANINE AGAR

FPRE
125
PYRUVATE BROTH ✓Positive: Enterococcus faecalis
➢ability of an organism to utilize pyruvate ✓Negative: Streptococcus bovis
➢differentiation between Enterococcus faecalis (+)
and Enterococcus faecium (-)
➢Pyruvic acid→ added to the broth to determine
whether the microorganism is able to use pyruvate
➢Bromthymol blue indicator changes from blue to
yellow
Result:
✓Positive: Indicator changes from green to yellow
SPOT INDOLE TEST
✓Negative: No color change
➢determine the presence of tryptophanase
QC:
➢Tryptophanase breaks down tryptophan →indole,
✓Positive: Enterococcus faecalis detected by its combination with certain aldehydes to
✓Negative: Streptococcus bovis form a colored compound
➢Indole + Cinnamaldehyde = blue-green
compound
Result:
✓Positive: Development of a blue color within 20
seconds
✓Negative: No color development or slightly pink
color
QC:
SALT TOLERANCE TEST ✓Positive: Escherichia coli
➢ability of an organism to grow in high ✓Negative: Klebsiella pneumoniae
concentrations of salt
➢differentiate enterococci (+) from nonenterococci (-)
➢Medium: Brain-Heart Infusion Broth containing
6.5% NaCl
➢Indicator: Bromcresol Purple
Result:
✓Positive: Visible turbidity in the broth, with or
without a color change from purple
to yellow
✓Negative: No turbidity and no color change

QC:

FPRE
126
TRIPLE SUGAR IRON AGAR (TSI)
➢determines whether Gram (-) rod ferments glucose
and lactose or sucrose and forms hydrogen sulfide
(H2S)
UREASE TEST (CHRISTENSEN’S METHOD)
➢differentiate members of the Enterobacteriaceae
➢ability to produce urease, which hydrolyzes urea to
family from other gram (-) rods
ammonia and CO2
➢Composition: 10 parts Lactose, 10 parts sucrose,
➢Proteus sp. → rapidly hydrolyze urea
1 part glucose and peptone
➢ammonia alkalinizes the medium
➢Phenol Red→ pH indicator
➢pH Indicator: PHENOL RED → from light orange
➢Ferrous Ammonium Sulfate→ H2S Indicator
at pH 6.8 to magenta (pink) at pH 8.1
➢CO2 and hydrogen gas (H2) → presence of
Medium: CHRISTENSEN’S UREA AGAR or
bubbles or cracks or by separation of the agar
BROTH
Result:
Result:
✓Alkaline slant/ Alkaline butt (K/K)→ glucose,
✓Positive: Change in color of slant from light orange
lactose, and sucrose nonutilizer
to magenta
✓Alkaline slant/Acid butt (K/A)→ glucose
✓Negative: No color change
fermentation only
QC:
✓Acid slant/Acid butt (A/A)→ glucose, sucrose,
and/or lactose fermenter ✓Positive: Proteus vulgaris
✓Black Precipitate→ production of ferrous sulfide ✓Weak positive: Klebsiella pneumonia
and H2S gas (H2S+)
✓Negative: Escherichia coli
✓Bubbles or cracks→ gas production
QC:
✓ gas production: Escherichia coli
✓K/A, +/− gas production, H2S+: Salmonella
typhimurium
✓K/K: Pseudomonas aeruginosa
X and V FACTOR TEST
✓K/A, H2S+: Proteus mirabilis
➢differentiation Haemophilus species
✓K/A: Shigella flexnerA/A, (+) G, 2S
Result:
✓Positive:
▪ Growth around the XV disk → requirement for both
factors
▪ Growth around the V disk, no growth around the X
disk, and light growth around the XV disk shows a V
factor requirement
K/A, (+) G,
✓Negative: Growth over the entire surface of the
(-) H2S agar indicates no requirement for either X or V factor

FPRE
127
QC:
✓Haemophilus influenza→ halo of growth around
the XV disk, no growth on the rest of the agar
surface
✓Haemophilus parainfluenzae→ halo of growth
around the XV and V disks
✓Haemophilus ducreyi → halo of growth around the
XV and X disks
X factor→ heat stable substance known as HEMIN
or HEMATIN
V factor→ heat labile substance known as NAD or
COENZYME I
→supplied by yeast, potato extract and bacteria
(Staphylococci, Pneumococci and Neisseria)

FPRE
128
 UNIT 16:CATALASE- POSITIVE, GRAM-
POSITIVE COCCI
GENERA AND SPECIES
 Staphylococcus aureus
COAGULASE-NEGATIVE STAPHYLOCOCCI
 Staphylococcus epidermidis
 Staphylococcus haemolyticus
 Staphylococcus saprophyticus
 Staphylococcus lugdunensis
 Staphylococcus schleiferi
COAGULASE-NEGATIVE STAPHYLOCOCCI
 Staphylococcus capitis
 Staphylococcus caprae
 Staphylococcus warneri
 Staphylococcus hominis
 Staphylococcus auricularis
 Staphylococcus cohnii
 Staphylococcus xylosus
 Staphylococcus simulans

 Micrococcus spp. and related genera
 Alloiococcus
FPRE
GENERAL CHARACTERISTICS: Staphylococcus
---catalase-producing gram (+) cocci that belong
to the family Staphylococcaceae
---aerobic or facultative anaerobic except S.
aureus subsp. anaerobius and S. sacchrolyticus
which are obligate anaerobes
---nonmotile, non–spore-forming
---spherical cells (0.5 to 1.5 μm) that appear singly,
in pairs, and in clusters
---normal inhabitants of skin, mucous membranes
and intestines
BAP: colonies = medium sized (4 to 8 mm) and
appear cream-colored, white or rarely light gold, and
“butterylooking”, other spp. may have gray colonies;
some may be β- hemolytic (S. aureus)
Differential Test Between Staphylococci and
Micrococci
Staphylococcus aureus
---true coagulase positive and most virulent species
of staphylococci
---grow well on most routine media like NA and TSB
---on solid media, round, smooth, opaque and
butyrous
---on BAP, colonies have golden yellow color and
β- haemolytic
---cultivated by adding 7.5 to 10% NaCl---
HALOPHILIC
---responsible for various skin, wound and deep
tissue infection
129
 3. STAPHYLOKINASE (FIBRINOLYSIN)
--fibrinolytic activities by dissolving fibrin clot
4. LIPASE (Fat-splitting Enzyme)
--produced by both coagulase (+) and coagulase (-)
VIRULENCE FACTORS staphylococci

A. ANTIGENIC STRUCTURE --act on lipids present on the surface of the skin,

particularly fats and oil secreted by the sebaceous
Teichoic Acid--contain ribitol teichoic acid in cell glands
wall
--important in the formation of furuncles, carbuncles
Peptidoglycan--together with teichoic acid, it and boils
protects the bacteria from lysis and probably aids in
adherence 5. DEOXYRIBONUCLEASE (DNASE) and
PHOSPHATASE
Protein A--group specific antigen unique to S.
aureus --lowers viscosity of exudates giving the pathogen
more mobility
--prevents antibody-mediated phagocytosis by
PMN—competes for the Fc portion --destroys DNA

Clumping Factor--component on cell wall 6. PROTEASE

responsible for clumping of the whole staphylococci 7. GELATINASE
in the presence of plasma
8. β-LACTAMASE
Capsular Polysaccharide--protects fro
phagocytosis --breaks down Penicillin and β-lactam drugs

B. ENZYMES C. TOXINS

1. COAGULASE (STAPHYLOCOAGULASE) 1. CYTOLYTIC TOXINS: Hemolysins and

Leukocidins
--coagulates fibrinogen in the plasma
4 Types Hemolysins
--promotes fibrin layer formation around the
staphylococcal abscess protecting the bacteria from a. α-Hemolysin
phagocytosis
--damage RBC, platelets and macrophages and
2 Types: cause severe tissue damage

 Cell-bound Coagulase or Clumping Factor - --Predominant hemolysin

-bound to the cell wall and clots human, rabbit or
b. β-Hemolysin (Sphingomyelinase C)
pig plasma
--acts on sphingomyelin in the plasma membrane of
 Unbound or Free Coagulase--extracellular
RBC
enzymes not bound to the cell wall and cause
clot formation when bacterial cells are incubated --aka“hot-cold” lysine : enhanced hemolytic activity
with plasma on incubation at 37° C (heat labile) and subsequent
exposure to cold (4° C)
2. HYALURONIDASE (SPREADING FACTOR)
--exhibited in the CAMP test and lethal and
--hydrolyzes hyaluronic acid present in the
dermonecrotic
intracellular ground substance, permitting the spread
of infection

FPRE
130
c. δ-Hemolysin --SUPERANTIGEN stimulating T-cell proliferation
and production of a large amount of cytokines
--less toxic to cells than either α-hemolysin or β-
hemolysin --low concentrations= leakage by endothelial cells;
higher concentrations= cytotoxic
--produced by all S. aureus strain that cause RBC
injury in culture and produce edematous lesions 4. EXFOLIATIVE TOXIN
d. γ-Hemolysin --aka EPIDERMOLYTIC TOXIN A and B or
EXFOLIATIN serotypes A and B
--associated with Panton-Valentine leukocidin
(PVL) --Serine protease that divides the intrcellular bridges
of the epidermis and causes excessive sloughing of
Staphylococcal Leukocidin/ Panton-Valentine
the epidermis (stratum granulosum)
leukocidin
--causes STAPHYLOCOCCAL SCALDED SKIN
--exotoxin lethal to polymorphonuclear leukocytes
SYNDROME referred to as RITTER’S DISEASE
--Pore forming exotoxin that suppress phagocytosis
--implicated in BULLOUS IMPETIGO
and associated with severe cutaneous infections and
necrotizing pneumonia RELATED INFECTIONS AND DISEASES
--associated with community-acquired 1. Cutaneous Infections
staphylococcal infections
a. Folliculitis
2. ENTEROTOXINS
mild inflammation of a hair follicle or oil gland;
--heat-stable exotoxin: 100° C for 30 minutes infected area is raised and red
--resistant to hydrolysis by gastric and jejunal b. Furuncles (Boils)
enzymes
focal suppurative lesions which has resulted from an
--act as neurotoxins that stimulate vomiting through infection (folliculitis) that extend into subcutaneous
the vagus nerve tissue; large, raised, superficial abscesses
--produced by 30% to 50% of S. aureus isolates c. Carbuncles
Enterotoxins A, B, and D larger, more invasive lesions develop from multiple
furuncles, which can progress into deeper tissues;
--Staphylococcal food poisoning
present with fever and chills, indicating systemic
Enterotoxins B and C and sometimes G and I -- infection
TSS
Superficial folliculitis in which raised, domed
Enterotoxin B pustules form around hair follicles

--Staphylococcal Pseudomembranous
--Enterocolitis (contaminated milk products)
3. TOXIC SHOCK SYNDROME TOXIN-1(TSST-1)
--aka ENTEROTOXIN F or PYROGENIC
EXOTOXIN C
--menstruating-associated TSS= TSS associated
with tampon use
--chromosomal-mediated toxin

FPRE
131
Furuncle arises when a large abscess forms around
a hair follicle.

Lesions of impetigo. This disease is characterized

by isolated
pustules that become crusted.
A carbuncle consists of a multilocular abscess
around several hair follicles

Toxic Shock Syndrome. The photograph shows the

peeling skin often associated with TSS.

d. Bullous Impetigo
--larger pustules surrounded by a small zone of
erythema
--highly contagious infection that spread by direct
contact, fomites, or autoinoculation

Impetigo --superficial cutaneous infection commonly

seen in newborns and young children characterized
by the formation of encrusted pustules surrounded
by red border
e. Scalded Skin Syndrome
--bullous exfoliative dermatitis that occurs primarily in
newborns and previously healthy young children
Lesions of scalded skin syndrome. Staphylococci
produce a toxin that causes the skin to peel off in
sheets. It is especially likely to occur in children
under age 2.
f. Toxic Epidermal Necrolysis (TEN)

--localized skin lesion: few blisters, pemphigus
neonatorum, Ritter disease; generalized form:
cutaneous erythema, profuse peeling of the
epidermis
--clinical manifestation with multiple causes;
symptoms are due to hypersensitivity reaction
2. Toxic Shock Syndrome
--rare but potentially fatal, multisystem disease
characterized by sudden onset of fever, chills,
vomiting, diarrhea, muscle aches, and rash, which
can quickly progress to hypotension and shock

FPRE
132
3. Food Poisoning
--Enterotoxins A (78%), D (38%), and B (10%)
--intoxication resulting from ingestion of a toxin
formed outside the body
--symptoms appear rapidly (2 to 8 hours after
ingestion) and resolve within 24 to 48 hours: nausea,
vomiting, abdominal pain,
A i
and severe cramping, diarrhea
LABORATORY DIAGNOSIS
Note!!!
Specimen: Pus, Purulent Fluids, Sputum, Urine,
FOOD POISONING: perfuse and watery diarrhea Blood
due to water and electrolyte loss
1. Gram Stain: Gram(+) cocci in irregular clusters
• Food won’t appear or taste tainted
2. Culture Media
• Death: Intoxication rather than infection
 BAP, PEA, MSA, CNA, Chapman Stone Agar,
• Reheating the food – kills the bacteria, but not Vogel-Johnson Medium
inactivate the heat stable toxin
 Columbia Colistin–Nalidixic acid (CNA)
ENTEROCOLITIS: Enterotoxin A and (leukocidins) purulent exudates
LukE and LukP
 MSA and PEA heavily contaminated
4. Staphylococcal Bacteremia specimen

--leads to secondary pneumonia and endocarditis  CHROM Agar selective-differential for

observed among IV drug users MRSA

5. Staphylococcal Osteomyelitis 3. Biochemical Tests

--secondary to bacteremia a. Catalase Test

6. Staphylococcal Pneumonia Reagent: 3% H2O2 (Aerobic Catalase Test) 15%

H2O2 (Anaerobic
--secondary to influenza virus infection
Catalase Test)
--multiple abscesses and focal lesions in the
pulmonary parenchyma Staphylococcus: catalase + ; Streptococcus:
catalase -
7. Septic Arthritis
b. Coagulase Test
--frequent in children and occur in patients with a
--best single criterion of recognition and
history of rheumatoid arthritis or IV drug abuse pathogenicity of S. aureus
8. Acute Bacterial Endocarditis Reagent: Rabbit plasma with EDTA
9. UTI Methods
i. Slide Method
--rapid screening test
--detects cell-bound coagulase or clumping factor
Other Slide Coagualse Positive: S. lugdunensis
and S. schleiferi
FPRE
133
ii. Tube Method
--sensitive but definitive; confirm all slide negative
results
--detects extracellular or free coagulase
Other Tube Cuagulase Positive: S. hyicus, S.
intermedius, S. lutrae, S. delphini and S.
schleiferi subsp. Coagulans
d. Growth on Tellurite Glycine Agar
--S. aureus reduce tellurite producing JET-BLACK
COLONIES
--Other Staphylococci---inhibited; if growth occurs
gray colonies are seen
e. Polymyxin B Sensitivity
S. aureus = Resistant ; Other Staphylococci=
Suceptible
f. Lysostaphin Sensitivity Test (2ug/ml)
S. aureus = Sensitive ; Micrococci = Resistant
g. Voges-Proskauer (VP) Test
--differentiate S. aureus (+) from S. intermedius (-)
Culture Medium: VP Broth with 5% glucose
Reagent: α-naphthol and KOH

Note!!! Other VP Positive: S. lugdunensis, S.

 Tube Method: incubate for 1-4 hours at 35°C-
37°C; if no clot forms after 4 hours reincubate at
room temperature for additional 20 hours
 Result should be read within 4 hours to prevent
False (-)
Reaction FIBRINOLYSIN which lyses the clot
formed
haemolyticus, and S. schleiferi
h. Deoxyribonuclease (Dnase) Test
Culture Medium: DNA-Methyl Green Agar
T. aureus = Dnase Positive
Tellurite Glycine Agar

 Coagulase plasma with Citrate not suitable

 False (+) Result
= Pseudomonas and Enterococci use citrate and
release calcium forming clot in the absence of
coagulase

c. Mannitol Fermentaion Test

Jet Black colonies of S. aureus
--S. aureus can ferment mannitol and can tolerate
high salt concentrations (7.5-10%)
Culture medium: MSA
pH indicator: Phenol Red
Positive Result: yellow-colored colonies surrounded
by a yellow halo

FPRE
134
i. Pyrrolidonyl Arylamidase (PYR) Test Real-time PCR----identifying both MRSA and MSSA
--differentiates coagulase(+) staphylococci by slide Qualitative Nucleic Acid Hybridization Assays
method
--staphylococci from prepared smears in blood
Substrate: Pyroglutamyl-β-naphthylamide cultures
(Lpyrrolidonyl-β-naphthylamide; PYR)
--identification of mecA gene
Reagent: p-dimethylaminocinnamaldehyde
Specimen: Anterior Nares Swabs
End Product: L-pyrrolidone and β-naphthylamine
Advantage: rapid detection test for MRSA
Result: Cherry Red
Staphylococcus epidermidis
(+) PYR = S. lugdunensis, S. intermedius S.
--indigenous microbiota of the skin
schleiferi, S. haemolyticus
--contaminant of medical instruments, catheters,
(-) PYR = S. aureu
CSF shunts and prosthetic heart valve implants
j. Rapid Methods of Identification (implanted medical devices), hip prostheses
Particle Agglutination Test Disease: Stitch abcess, Health care-acquired UTIs,
Endocarditis, Bacteremia
 Staphyloslide----use sensitized sheep RBC
Poly-γ-DL-Glutamic Acid (PGA)---adherence of S.
 Staphaurex
epidermidis
 BACTiStaph
LABORATORY DIAGNOSIS
 Staphylochrome
BAP: gray to white, opaque, small to medium-sized
 Sero-STAT pi heads and non-hemolytic colonies

 Bacto Staph Latex Biochemical Test: Coagulase (-), CNA (+),

 Accu-Staph DNase(-), Mannitol Fermentation (-)

 Hemostaph  Susceptibility with 5-ug NOVOBIOCIN

(16mm-27mm)
 Staphylatex

--plasma-coated carrier particles (latex)

--plasma detects both clumping factor
(with fibrinogen) and protein A in the cell wall of S.
aureus (with IgG)

Staphylococcus saprophyticus
k. Molecular Methods

FPRE
135
--present on the normal skin and in the periurethral Other Coagulase-Negative Staphylococci
and urethral flora
 S. warneri, S. capitis, S. simulans, S. hominis,
--adheres effectively to the epithelial cells lining the and S. schleiferi
urogenital tract
--endocarditis, septicemia, and wound infections
Disease: common cause of UTI in young sexually
S. haemolyticus
active women
-- wounds, bacteremia, endocarditis, and UTIs
--urine culture <10,000 CFU/ml = significant
--medium-sized colonies, with moderate or weak
LABORATORY DIAGNOSIS
hemolysis
BAP: white, opaque, slightly larger than pin-heads,
nd variable pigment production
nonhemolytic colonies although some strains
produce yellow pigments NOVOBIOCIN SUSCEPTIBLE CoNS
Biochemical Test: Coagulase (-), Dnase (-)  S. epidermidis
Mannitol Fermentation (-)  S. capitis
 Resistance to NOVONIOCIN (5ug; 6mm-  S. haemolyticus
12mm) and NALIDIXIC ACID
 S. hominis subsp. hominis
 Absence of Phosphatase production
 S. lugdunensis
NOVOBIOCIN SUCEPTIBILITY TEST
 S. saccharolyticus
 S. warneri
NOVOBIOCIN RESISTANT CoNS
 S. saprophyticus
 S. cohnii
 S. kloosii
 S. xylosus

S. Saphrophyticus
Staphylococcus lugdunensis
--clumping factor (+), tube coagulase (-)
--contain mecA gene that encodes
oxacillin resistance
--more aggressive than other CoNS in
inefectivity
Disease: infective endocarditis,
septicemia, meningitis, skin and soft
tissue infections, UTIs, and septic shock

ALGORITHM FOR IDENTIFICATION

FPRE
136
RESISTANT GENES PRODUCED BY b. CHROMOGENIC TEST
STAPHYLOCOCCI
Chromogenic Selective Differential Media:
a. Erythromycin Ribosomal Methylase (ERM)
MRSA Select, Spectra MRSA,
Gene
CHROMagar MRSA
--Class of enzyme inactivating genes
CEFOXITIN ---inhibit non-MRSA
--Codes for the methylation of the 23s rRNA
Result: Changes in color of MRSA colonies within
>Results in resistance to erythromycin
24 to 48 hours using CHROM Agar against colonies
>Inducible or constitutive resistance to clindamycin of non-MRSA
--May not be detected in routine susceptibility testing CHROM agar
--Confer cross-resistance to macrolides MRSA Mauve-colored colonies
(erythromycin) and streptogramins (quinupristin)
b. Methionine Sulfoxide Reductase (MSR) A Gene
--Codes for efflux mechanism---resistance
to erythromycin but susceptibility to clindamycin
Methicillin-Resistant Staphylococci aureus
(MRSA)
--type of S. aureus that is resistant
c. Latex Agglutination
to methicillin, nafcillin, and oxacillin
--detect altered PBPs
--acquired after prolonged stay in the hospital, close
contact with carriers, effects of broad spectrum --alternative method for testing and confirmation of
antibiotic treatments and exposure to nasal oxacillin resistance
secretions
-- performed on both CoNS and S. aureus
--VANCOMYCIN = treatment of choice for MRSA
d. Molecular Nucleic Acid Probes or PCR
Types: Amplification
Community-Associated MRSA (CA-MRSA), >“gold standard” for MRSA detection
Health Care–Associated Community-Onset
(HACO-MRSA), Hospital-Associated (HA-MRSA) Vancomycin-Resistant Staphylococci

>mecA--codes for altered Penicillin-Binding Screening: Vancomycin Agar Plate

Protein (PBP) = PBP2a or PBP2′ Macrolide Resistance

LABORATORY DIAGNOSIS >MODIFIED DOUBLE DISK DIFFUSION TEST (D-

zone test)= discrepant macrolide test results
a. OXACILLIN-SALT AGAR PLATE (erythromycin resistant and clindamycin susceptible)
--used to screen for MRSA in clinical samples
--differentiate MRSA from hyperproducers
of β-lactamase, or Borderline Oxacillin-Resistant
Staphylococcus aureus (BORSA)

FPRE
137
 A. Screening Test for MRSA: Oxacillin Screen
Plate Culture Media: MHA with 4% NaCl and 6
ug/mL oxacillin
>spot inoculated with cotton swab and incubated for
24 hours at 35°C
Microdilution Testing
>oxacilin in cation-supplemented MH broth
containing 2% NaCl
15ug Erythromycin disk and 2 ug clindamycin
Result:
placed 15mm to 26 mm apart on a MHA and BAP
Resistant- growth of more than one colony
BETA-LACTAMASE TEST
Susceptible- no growth on the agar plate
A. Cephalosporinase Test
CoNS:
>uses cephalosporin or cefinase disk
Resistant- 24 mm zone of inhibition
Substrate: nitrocefin
Susceptible- >25 mm zone of inhibition
(+) result: deep pink or red color within 10
minutes (60 minutes for Staphylococci) Disadvantage: Does not reliably detect oxacillin-
resistant CoNS
B. Acidimetric Method
B. Cefoxitin Disk Diffusion (30 ug)
Reagent: citrate-buffered penicillin
--preferred method for detection of oxacillin-
pH indicator: phenol red
resistance for both S. aureus and S. lugdunensis
(+) result: red yellow (penicilloic acid =
--improves detection of MRSA
decrease pH)
--serves to induce greater PBP2a in mecA-containing
C. Iodometric Method
strains
Reagent: citrate-buffered penicillin and starch iodine
>Test reagent to detect resistance- both MIC and
complex
diffusion method
(+) result: colorless solution- penicilloic acid
Interpretation:
reduces iodine and prevents it to combine with starch
Resistant- < 21 mm
(-) result: purple (no color change)
Susceptible- > 22 mm
ANTIMICROBIAL TESTING
C. Macro E Test
Treatment : methicillin, oxacillin, nafcillin, cloxacillin,
and dicloxacillin (penicillinase-resistant penicllin --detection of heteroresistant VISA because the test
drugs) uses a higher concentration of organisms (1 x 108
bacteria/ mL)
Oxacillin---most commonly used
D. Vancomycin Agar Screen Plate
Cutaneous infections: oral oxaillin or dicloxacillin, if
allergic, erythromycin may be substituted --S. aureus should be screened with 6-ug/mL
vancomycin incorporated into BHIA
Systemic: parenteral nafcilllin or oxacillin, if allergic,
--Broth microdilution test
vancomycin or cephalosporin may be used
MRSA vancomcin alone or in a combination with
rifampicin
FPRE
138
--best method for detection of either Vancomycin-
resistant S. aureus (VRSA) or VISA
Result: any growth or colony

Confirmatory test for oxacillin resistance: Broth

dilution and E-test

FPRE
139
 UNIT 17:CATALASE-NEGATIVE, GRAM-
NEGATIVE COCCI
STREPTOCOCCUS and ENTEROCOCCUS
Beta-Hemolytic Streptococci
 Streptococcus pyogenes
 Streptococcus agalactiae
Groups C, F, and G betahemolytic Streptococci,
Streptococcus pneumoniae,
streptococci
 Streptococcus mutans
 Streptococcus salivarius
 Streptococcus mitis
 Streptococcus bovis
 Streptococcus urinalis
 Streptococcus anginosus
 (Streptococcus milleri )
Nutritionally variant streptococci
 Abiotrophia defective
 Granulicatella adiacens
 Granulicatella balaenopterae
 Granulicatella elegans
Enterococci
 Enterococcus faecalis
 Enterococcus faecium
 E. durans
 E. mundtii
 E. dispar
 E. gallinarum
 E. avium
 E. hirae
 E. raffinosus
 E. casseliflavus
Other Catalase-Negative, Gram-Positive Cocci
 Leuconostoc spp.
 Lactococcus spp.
 Globicatella sp.
 Pediococcus spp.
 Aerococcus spp.
 Gemella spp.
 Helcococcus sp.
Viridans
 Alloiococcous otitidis
 Dolosicoccus paucivorans
 Facklamia
 Dolosigranulum pigrum
 Ignavigranum ruoffiae
 Tetragenococcus
GENERAL CHARACTERISTICS
--belong to the family Streptococcaceae
--spherical to ovoid, catalase(-), gram (+) cocci
arranged in pairs or chains when grown in liquid
media
--non-spore-former and generally non-motile
except for the rare motile strains of group D
streptococci
--facultative anaerobes but some strains require
added CO2 (***microaerophilic strains = Viridans
streptococci)
--generally non-encapsulated except for some strains
of groups A, B, C and D
--commonly found as part of normal human flora
--growth enhanced by blood (BAP), serum or glucose
BAP: grayish, pinpoint, circular, and translucent
to slightly opaque colonies while some have mucoid
colonies
Biochemical Tests: (-) catalase, oxidase and gas
production
FPRE
140
Gram stain of Streptococcus from Solid medium
Gram stain of Streptococcus from Liquid medium
β-Hemolytic streptococcal colonies
α-Hemolytic streptococcal colonies
CLASSIFICATION OF STREPTOCOCCI
FPRE
I. ACADEMIC OR BERGEY’S CLASSIFICATION
--based on temperature requirement
A. PYOGENIC GROUP
--neither grow on 10C nor 45C, grow only at 37C
--produce pus; mostly β-hemolytic
Species: S. pyogenes, group C and G
streptococci (largecolony forming isolates)
B. VIRIDANS GROUP
--grow both at 45C and 37C but not at 10C
--not part of the Lancefield group: resist Lancefield
Precipitation Test
--α-hemolytic or non-hemolytic
--normal biota in URT in humans
--some may have A, C, G, or N antigen
Species: S. salivarius, S. mutans (dental
plaque), S. mitis, S. sanguis, S. anginosus
C. LACTIC GROUP
--grow on 10C and 37C but not at 45C
--non-hemolytic with Lancefield N antigen
--found in dairy products
Species: S. lactis normal coagulation and
souring of milk
D. ENTEROCOCCUS GROUP
--grow at 10C, 45C , and 37C; can withstand
temperature above 60oC
--normal of human intestine
Species: E. faecalis (part of normal fecal flora)
II. SMITH & BROWN’S CLASSIFICATION
-- based on hemolytic patterns on BAP
A. ALPHA-HEMOLYTIC STREPTOCOCCI
--partial/incomplete hemolysis of RBC around
colonies
--Culture: greenish or brownish discoloration
around colony
141
 Species: S. pneumoniae (green streptococci),
some Enterococci spp., and Streptococcus bovis
complex spp.
B. BETA-HEMOLYTIC STREPTOCOCCI
--complete hemolysis of RBC around colony
Culture: clear zone around colony
Species: S. pyogenes, S. agalactiae, S.
dysagalactiae subsp. equisimilis and S. anginosus
group and some enterococci spp
C. GAMMA-HEMOLYTIC STREPTOCOCCI
--exhibit NO hemolysis of RBC around colony
Culture: RBC surrounding colony are not affected
(no change)
Species: Enterococci and Streptococcus bovis
complex

GROUP A STREPTOCOCCI:
Note!!! Streptococcus pyogenes
Alpha-prime (α′) or wide zone small area of intact --not considered as a part of indigenous flora =
RBCs around colony surrounded by a wider zone of pathogenic
complete hemolysis
--acquired through contaminated droplets released
 Hemolysis is enhanced by stabbing the
inoculating loop into the agar several times through coughing and sneezing

 Plates are always examined for hemolysis by --resistant to drying and can be recovered from
holding them in front of light source swabs several hours after the collection

III. LANCEFIELD CLASSIFICATION --colonizes throat and skin on humans

--Rebecca Lancefield VIRULENCE FACTORS

--based on antigenic characteristics of GROUP- 1.M PROTEIN

SPECIFIC C SUBSTANCE, a cell wall major virulence factor = resist phagocytosis and
polysaccharide adherence of the bacterial cell to mucosal cells
--extraction of C carbohydrate from the --attached to peptidoglycan of cell wall and extends
streptococcal cell wall by dilute acid and heating the
suspension for 10 mins to cell surface

--mostly significant in classifying and identifying M1 serotype = most common in pharyngitis

betahemolytic streptococci
mediates post-streptococcal diseases:
Rheumatic fever class 1 M
Acute glomerulonephritis class I or II
--bind beta globulin factor H = regulatory protein
involved in the degradation of C3b

FPRE
142
--binds to fibrinogen blocking complement alternate
pathway activation
2. PROTEIN F
aka FIBRONECTIN-BINDING PROTEIN
-- adhesion molecule that mediates epithelial cell
attachment
3. LIPOTEICHOIC ACID
--adhesion molecule present in the cell wall that
is responsible for the adherence into respiratory
epithelial cells
4. HYALURONIC ACID CAPSULE
--weakly immunogenic
B. STREPTOLYSIN S (SLS)
--prevents opsonized phagocytosis by PMN or
macrophages --oxygen stable; nonimmunogenic

--mask bacterial antigens --surface hemolysis seen around colonies that have
been incubated aerobically
5. STREPTOKINASE
--causes lysis of RBC, WBC and platelets in the
--causes lysis of fibrin clots presence of room air
--binds plasminogen and activates the production of --inhibited by nonspecific inhibitor that is frequently
plasmin
present in sera of humans and animals
--allows bacteria to move from clotted area (spread
infecion)
Application: treatment of pulmonary emboli,
coronary artery, and venous thromboses
6. HEMOLYSINS
A. STREPTOLYSIN O (SLO)
7. DEOXYRIBONUCLEASE (DNASE)
--oxygen labile; highly antigenic = induce antibody
response --aka STREPTODORNASE (streptococcal
deoxyribonuclease)
--responsible for subsurface hemolysis on BAP
incubated anaerobically --lowers viscosity of exudates, giving pathogens
more mobility
--causes lysis of RBC, WBC, platelets, tissue cells;
Four types: A, B (most common), C, and D
--inhibited by the cholesterol in skin lipids
*antigenic enzyme
*absence of protective antibodies associated with
skin infection Antibodies can be detected following infections
Serologic Test: ANTI-STREPTOLYSIN O (ASO) (normal limit = 100 units), especially after skin
TEST infections
8. HYALURONIDASE
--aka SPREADING FACTOR
FPRE
143
--solubilizes the ground substance of mammalian RELATED INFECTIONS AND DISEASES
connective tissues (hyaluronic acid)
1. BACTERIAL PHARYNGITIS OR TONSILLITIS
--antigenic and specific for each bacterial or tissue (STREP THROAT)
source
Incubation period: 1 to 4 days
9. C5a PEPTIDASE
MOT: spread by droplets and close contact
--serine protease capable of inactivating the
Diagnosis: culture of specimen (throat swabs) or
chemotactic factor for neutrophils and monocytes
direct antigen detection
(C5a)
--highly virulent strains can cause sharp outbreaks of
10. Streptococcal Pyrogenic Exotoxins (SPEs)
sore throats and scarlet fever in schools and camps
--formerly called ERYTHROGENIC TOXINS
--infants and small children = tendency to extend to
--cause a red spreading rash = SCARLET FEVER the middle ear and mastoid
--heat labile and rarely found in group C and G
--act as SUPERANTIGENS activating macrophages
and Thelper cells
*induce release of powerful immune mediators :
IL-1, IL-2, IL-6, TNF-alpha, TNF-beta, interferons,
and cytokines-----shock and organ failure
--associated with STREPTOCOCCAL TOXIC
SHOCK SYNDROME 2. PYODERMAL INFECTIONS

Four Exotoxin Types: SpeA, SpeB, SpeC, and a. Impetigo

SpeF
--localized skin disease that begins as small vesicles
Exotoxin B (cysteine protease) that progress to weeping lesions that crust over after
degrades protein mediates rashes that are caused several days
by scarlet fever
--usually seen in young children (2 to 5 years)
b. Erysipelas
Streptococcal Diseases.
--acute spreading skin lesion that is intensely
Some of the more
erythematous with a plainly demarcated but irregular
prominent diseases
edge
associated with group A
--rare infection of the skin and subcutaneous tissues
streptococcal infections, and
observed frequently in elderly patients
the body sites affected
Lesions of Erysipelasc.

FPRE
144
Cellulitis 4. STREPTOCOCCAL TOXIC SHOCK SYNDROME
--diffuse, spreading infection of subcutaneous skin --characterized by a precipitous drop in blood
tissue characterized by defined area of redness pressure, failure of multiple organs, and a very high
(erythema) and the accumulation of fluid (edema) fever
--follows infection associated with mild trauma, burns, --caused by an invasive strep A that produces one or
wounds, or surgical incisions more of the streptococcal pyrogenic exotoxins
--may lead to gangrene --initial streptococcal infection includes pharyngitis,
peritonitis, cellulitis, wound infections
Differentiated from erysipelas by two clinical
findings: SpeA --play a major role in the pathogenesis of the
disease = superantigens
*lesion is not raised
M1 and M3 most common strains associated with
*line between the involved and uninvolved tissue is
streptococcal TSS
indistinct
5. SCARLET FEVER (SCARLATINA)
3. NECROTIZING FASCIITIS
--punctate exanthem overlying a diffuse erythema
aka GALLOPING GANGRENE, FLESH-EATING
that appears initially on neck and upper chest, 1 to 2
BACTERIA SYNDROME, SUPPURATIVE
days following strep throat rash disappears over the
FASCIITIS, HOSPITAL GANGRENE, or
next 5 to 7 days and is followed by desquamation
NECROTIZING ERYSIPELAS
--communicable and spread by inhalation of
--invasive infection characterized by rapidly
infectious respiratory droplets
progressing inflammation and necrosis of the skin,
subcutaneous fat, and fascia --results from a throat infection with a strain of S.
pyogenes that carries lysgenic bacteriophage (T12)-
Exotoxin A acts as a superantigen, causing the
cause by release of streptococcal pyrogenic
immune system to contribute to the damage
exotoxins
Types:
Cardinal Signs: diffuse red rash on the upper chest
Type 1 NF polymicrobial infection from which and spreads to the trunk and extremities, and
aerobic and anaerobic bacteria are recovered “strawberry-colored” tongue

Type 2 NF GAS
Type 3 NF gas gangrene or clostridial
myonecrosis

TEST FOR SCARLET FEVER

a. DICK’S TEST
--susceptibility test for scarlet fever
Test arm: 0.1 ml of Dick’s toxin (Eryhtrogenic
Toxin)
Control arm: : 0.1 ml of Dick’s toxoid
Read reaction after 24 hours

FPRE
145
 (+) reaction: Eythema or redness in the test LABORATORY DIAGNOSIS
site
Specimen: Pharynx and Tonsillar Swabs (Throat
Interpretation: Susceptible to scarlet fever Swabs)
b. SCHULTZ- CHARLTON TEST 1. Culture Medium
--based on neutralization of eryhtrogenic toxins when BAP: colonies are transparent to translucent, convex
anti-toxin is injected on the skin of patient with scarlet or domed entire, circular, shiny and surrounded by
fever wide zone of β- hemolysis
--diagnose whether rashes of patient is due to scarlet 2. Bacitracin Susceptibility Test/Taxo A (0.02-0.04
fever or not U)
(+) reaction: “BLANCHING PHENOMENON” – --presumptive identification of S. pyogenes (S)
fading of the --screening for GAS in throat cultures
Rashes --groups C and G are also susceptible
6. POSTSTREPTOCOCCAL SEQUELAE Result: Susceptibility or any zone of inhibition
a.RHEUMATIC FEVER S. Pyogenes (S)
--follows S. pyogenes pharyngitis S. agalactiae R
--autoimmune disease characterized by fever and
inflammation of the heart, joints, blood vessels, and
subcutaneous tissues
--mediated by antibodies produced against S.
pyogenes M protein that cross-react with human
heart tissue
--most serious result: chronic, progressive damage to
the heart valves
--most common cause of permanent heart valve 2. Pyrrolidonyl-α-Naphthylamide Hydrolysis Test
damage in children
--detects L-pyrrolidonyl arylamidase
b. ACUTE GLOMERULONEPHRITIS
--more specific for S. pyogenes than bacitracin
--aka BRIGHT’S DISEASE
--S. pyogenes is the only species of Streptococcus
-- inflammatory disease of the renal glomeruli that is PYRpositive
--develops 1–4 weeks after S pyogenes skin Other PYR(+) : Enterococcus, Aerococcus, and
infection (pyoderma, impetigo) or respiratory Gemella
infection
(+) result: Cherry Red color
--deposition of antigen-antibody complexes,
possibly involving the streptococcal M protein
Type III hypersensitivity reaction

FPRE
146
3. Sulfamethoxazole and Trimethoprim Test VIRULENCE FACTORS
--Group A and B streptococci is resistant to SXT CAPSULE
(positive result)
--important virulence factor
--Group C is susceptible to SXT (negative result)
--prevents phagocytosis but is ineffective after
--interfering respiratory microbiota will be inhibited by opsonization
SXT
SIALIC ACID most significant
(+) result: Resistance component of the capsule and critical
virulence determinant
4. Serologic Test: ASO TEST
AVIRULENT FACTORS
--Serum is added with measured amount of SLO
reagent and incubated  Hemolysin
--Reagent RBC are added indicator  CAMP factor
--Enough antibody is present: SLO neutralized  Neuraminidase
and no hemolysis occurs
 Dnase
Titer ---reciprocal of the highest dilution
 Hyaluronidase
demonstrating no hemolysis expressed in TODD
units  Protease
5. Anti-DNase B Testing RELATED INFECTIONS AND DISEASES
--Anti-DNase B Antibodies ---neutralize reagent 1. Most common etiologic agent of NEONATAL
DNase B, preventing it from depolymerizing DNA SEPSIS and MENINGITIS
--- DNase measured by its effect on a DNA methyl- 2. Pneumonia
green conjugate
3. Postpartum Infection ENDOMETRITIS
GROUP B STREPTOCOCCI:
4. Osteomyelitis
Streptococcus agalactiae
5. UTI
--have group B–specific antigen, acid-stable
polysaccharide located in cell wall 6. Puerperal Infection

--normal flora of female genital tract and lower 7. Endocarditis (Tricuspid Valve Endocarditis)
gastrointestinal tract 8. Skin infection
MOT 9. Important etiologic agent of bovine mastitis
*Endogenous strain: gaining access to sterile site(s) Neonatal GBS Disease
probable
Early-onset infection (<7 days old) pneumonia
*Direct contact: person to person from mother and sepsis
in utero or during delivery; or nosocomial Late-onset infection (7 days old - 3 months old)
transmission by unwashed hands of mother or health meningitis and sepsis
care personnel
*Causes infection of fetus during passage through
the colonized birth canal and premature rupture of
mother’s membrane

FPRE
147
LABORATORY DIAGNOSIS 2. CHRISTIE, ATKINS, AND MUNCH-PETERSEN
(CAMP) TEST
Specimen: Vaginal and Rectal swabs
--presumptive identification of GBS: S. agalactiae (+)
1. Culture Medium
--CAMP FACTOR act synergistically with β-
BAP: grayish white, mucoid, more translucent to
hemolysin produced by S. aureus to cause
opaque, soft, smooth colonies surrounded by smaller
enhanced lysis of RBC
zone of β- hemolysis
--GBS are streaked perpendicular to a streak of S.
Selective Broth
aureus on sheep blood agar
a. Lim Broth Todd-Hewitt broth with Colistin and
(+) result: Arrowhead-shaped hemolysis
Nalidixic acid = TRANSPORT MEDIUM
RAPID CAMP TEST OR SPOT CAMP TEST
b. TransVag broth Gentamicin and Nalidixic acid
--place a drop of extracted β-lysin on area of
c. StrepB Carrot Broth orange or red pigment (6 confluent growth of suspected GBS
hours incubation)
* incubation: 35° C for 20 minutes
***Granada Agar selective agar for vaginal or
(+) result: enhanced hemolysis
rectal swabs yellow to orange colonies

3. HIPPURATE HYDROLYSIS TEST

--S. agalactiae (+) has hippuricase (hippurate
hydrolase) that hydrolyzes sodium hippurate to form
sodium benzoate and glycine
--addition of ninhydrin * ammonia from deamination
of glycine as well as the reduced form of ninhydrin,
hydrindantin
--Ammonia + ninhydrin and hydrindantin =
PURPLECOLORED COMPLEX

FPRE
148
4. SEROTYPING Group C and G Streptococci
--Coagglutination or Latex Agglutination --similar types of acute infections described for S.
pyogenes and S. agalactiae, but usually involve
Treatment: PENICILLIN
compromised patients
S. agalactiae
Group G streptococci occur in patients with
GROUPS C AND G STREPTOCOCCI underlying malignancies

--recovered from the upper respiratory tract, vagina Group C organisms occasionally have been
and skin associated with acute pharyngitis

--also posses M protein ANTIMICROBIAL SUSCEPTIBILITY TESTS

--Group C streptococci are animal pathogens and the *Group C streptococci ---SUCEPTIBLE to
main source of STREPTOKINASE Bacitracin and SXT

--species are S. dysagalactiae subsp. equisimilis *Group G streptococci may be bacitracin resistant or
and S. equi subsp. Zooepidemicus susceptible

Types of Group C and G Streptococci VIRIDANS STREPTOCOCCI

1. Large Colony-forming Isolates --aka α-prime streptococci that lack Lancefield

--isolates with groups A, C, G and L antigens belong
to the pyogenic streptococci
--large-colony–forming β-hemolytic isolates with
group C and G antigens belong to S. dysgalactiae
subsp. equisimilis
group antigens
--normal microbiota of the upper respiratory tract, the
female genital tract, and the gastrointestinal tract
--fastidious, with some strains requiring CO2 for
growth

2. Small-Colony–forming Isolates --most common cause of SUBACUTE BACTERIAL

ENDOCARDITIS (SBE), condition associated with a
small-colony–forming β-hemolytic isolates with group transient bacteremia
C and G antigens belong to the S. anginosus group
MOT: gain access to sterile site; most notably results
Group C Streptococci from dental manipulations
--all species (S. equi, S. equisimilis, S.
zooepidemicus) are β-hemolytic except S.
dysagalctiae which may be α- hemolytic or non-
hemolytic
--differentiated by carbohydrate fermentation
RELATED INFECTIONS AND DISEASES
--animal pathogens but may infect humans
S. Equi--causes disease in horse Note!!!
S. equisimilis  S. anginosus group can possess Lancefield
group A, C, F, G, or N antigen and in some
--may cause pharyngitis, puerperal sepsis,
instances may not be groupable
endocarditis, bacteremia, osteomyelitis, brain abcess,
post-operative wounf infection and pneumonia in  S. bovis group and the enterococci possess the
humans group D antigen
--source of streptokinase used in Thrombolytic 
Therapy
FPRE
149
VIRULENCE FACTORS *primary contributor to dental caries and also
associated with bacteremia
Polysaccharide Capsule and Cytolysin
*Most commonly isolated spp. of viridans
-- identified in some members of anginosus group
streptococci
Extracellular Dextran and Adhesin (cell surface–
LABORATORY DIAGNOSIS
associated proteins)
Specimen: Blood, Gingival Scrappings, Pus
*adherence and colonization of these organisms in
Secretions
endocarditis
1. Culture Medium
***LOW VIRULENCE oropharyngeal
commensals and opportunistic pathogens BAP: small

RELATED INFECTIONS AN DISEASES and are surrounded by a zone of α-hemolysis;

 Major etiologic agent of Subacute Bacterial some isolates are β-hemolytic or nonhemolytic
Endocartitis
S. anginosus in pure culture or in high
 Major etiologic agent of Dental concentration = sweet odor of honeysuckle or
Carries(plaque) -------S. mutans butterscotch

 Fulminant cardiovascular collapse or meningitis 2. Biochemical Test

 Gingivitis
 Sinusitis
 Cellulitis and Wound Infection
 Abscesses, osteomyelitis, and empyema a.Leucine Aminopeptidase (LAP) Test
 Biliary or Intra-abdominal infections peptidase that hydrolyzes peptide bonds adjacent to
S. anginosus group a free amino group

*abscess formation in the oropharynx, brain, and Substrate: Leucine-β-naphthylamide

peritoneal cavity End-product: β-naphthylamine
S. constellatus subsp. pharyngis ------ Reagent:
pharyngitis Paradimethylaminocinnamaldehyde(DMACA)
S. mitis group (+) result: Red color
*bacterial endocarditis in native valves and prosthetic (+) LAP: Viridans Streptococci, Streptococcus
valve infections pyogenes, Streptococcus agalactiae,
S. salivarius Streptococcus pneumoniae, Enterococcus, and
Pediococcus
* bacteremia, endocarditis, and meningitis
(-) LAP: Aerococcus and Leuconostoc spp
S. bovis group
.b. Voges-Proskauer Test
*bacteremia, septicemia, and endocarditis
--distinguish small-colony–forming β-hemolytic
S. gallolyticus subsp. gallolyticus ------- anginosus group containing groups A or C antigens
presence has high correlation with gastrointestinal from large-colony–forming pyogenic strains
carcinoma
(+) VP: S. anginosus, S. bovis, and S. mutans
S. mutans groups

FPRE
150
c. β-D-Glucuronidase
--detects action of β-D-glucuronidase
Result:
 Large-colony–forming β-hemolytic groups C and
G streptococci (+)
 Small-colony–forming β-hemolytic anginosus
group(-)
Note!!!
All members are PYR (-) and LAP (+)

Diagnostic test for S. bovis group (Group D

Streptococci)
a. Bile Esculin Test
Reagent: Esculin and 40% Bile Salt
(+) result = Blackening of the agar
(+): Group D streptococci and Enterococcus spp.
(-): S. pyogenes and Viridans Streptococci
b. Salt Tolerance = 6.5% NaCl ENTEROCOCCUS

(+): Enterococci --previously classified as group D streptococci

(-): Non-enterococci = S. bovis --all species produce the cell wall–associated group
D antigen
c. PYR Test
--natural inhabitants of the intestinal tracts of humans
(+): Enterococcus (-): S. bovis and animals
d. Penicillin Test --most are nonhemolytic or α-hemolytic, some are β-
Susceptible: S. bovis hemolytic
--ability to grow under extreme conditions—presence
of bile or 6.5% NaCl or at 45° C or alkaline pH
--not highly pathogenic but frequent causes of
nosocomial infection
Species: E. faecalis, E. faecium, E, avium, E.
gallinarum, E. durans, E. raffinosus
VIRULENCE FACTORS
--can grow in extreme conditions
--resistant to multiple antimicrobial agents

FPRE
151
E. faecalis 7. Motility
*Extracellular surface adhesin proteins,
extracellular serine protease, and gelatinase --
---colonization and adherence to heart valves and
renal epithelial cells
*Cytolysin
---two-subunit toxin
---similar to bacteriocins produced by gram (+)
bacteria and is expressed by a quorumsensing
mechanism
RELATED INFECTIONS AND DISEASES
2. Biochemical Test
a. Urinary Tract Infections (UTIs) most common
a. Bile Esculin Test (+)
b. Endocarditis -elderly patients with prosthetic
b. PYR test (+)
valves or valvular heart disease
c. LAP test (+)
c. Bacteremia
d. Growth in 6.5% NaCl (+)
d. Intraabdominal or Pelvic Wound Infection
e. Acid Production (+) utilize an acid producing
e. CNS and Respiratory Tract infections rare
carbohydrates and methyl-α-D-glucopyranoside
LABORATORY DIAGNOSIS
f. Penicillin = Resistant
Specimen: Blood, Urine, or Wound
g. Vancomycin = Resistant
1. Culture Medium
h. 100-μg efrotomycin acid disk = Resistant
 TSB or BHI with 5% sheep blood
 Grow well at 35° C in the presence of CO2
Selective Media
 Bile Esculin azide, CAN, PEA, Cephalexin-
Aztreonam-Arabinose Agar----contaminated
specimens
E. faecalis identified by its ability to grow in the
presence of TELLURITE

Identified based on their:

Note!!!
1. Ability to produce acid in carbohydrate broth
 E. faecalis requires cyanocobalamin as
2. Ability to hydrolyze arginine growth factor
3. Tolerance of 0.04% tellurite  Other organisms Bile Esculin (+) and 6.5%
NaCl: Leuconostoc, Pediococcus,
4. Utilization of pyruvate
Globicatella, S. urinalis andLactococcus
5. Ability to produce acid from methyl-α-D-
glucopyranoside
6. Growth around 100-μg efrotomycin acid disk

FPRE
152
3. MOLECULAR TYPING METHODS 7. Hemolysin
 Pulsed-field gel electrophoresis RELATED INFECTIONS AND DISEASES
 Contour-clamped homogeneous electric-field 1. Lobar Pneumonia
electrophoresis
 characterized by the presence of voluminous
 Ribotyping fluid which hastens in the spread of bacteria in
the lungs
 PCR-based typing methods
 sudden onset with chills, dyspnea, and cough
ANTIMICROBIAL RESISTANCE
 most common cause of bacterial pneumonia
 Intrinsic or acquired resistance to
in elderly and immunocompromised
aminoglycosides, β-lactams, and glycopeptides
individual
 Vancomycin-resistant
Microscopy of sputum: large number of S.
Streptococcus pneumoniae pneumonia cells and WBC; absence of
oropharyngeal microbiota
aka PNEUMOCOCCUS and DIPLOCOCCUS
2. Meningitis
 encapsulated, characteristically lancet-shaped
which occurs singly, on pairs and short chains  follows other S. pneumonia otitis media or
pneumonia
 contains antigen referred to as C substance
which reacts with CRP  most common cause of meningitis in adults

 facultative anaerobe requiring an increase CO2 3. Otitis Media

tension (Candle Jar)
 most common isolate in children under 3 years
VIRULENCE FACTORS
old with recurrent otitis media
1. Polysaccharide Capsule
4. Endocarditis, Peritonitis, and Bacteremia
2. Adherance
5. Secondary Atypical Hemolytic Uremic
3. Enzymes Syndrome

a. Neuraminidase degrades surface structures of LABORATORY DIAGNOSIS

host tissue
Specimen: Sputum, Swabs, Pus, CSF and Blood
b. Protease facilitate bacterial colonization on
***RUST-TINGED SPUTUM may indicate S.
mucosal surfaces
pneumonia
by eliminating Ig
1. Gram-Stain
c. Hyaluronidase
Gram(+) cocci in pairs = DIPLOCOCCI
4. Pneumolysin O oxygen-sensitive toxin that is
cytolytic for cells cells are slightly pointed = LANCET SHAPE
5. Autolysin facilitate the release of pneumolysin 2. Culture Medium
O and other toxic proteins or inflammatory substance
BHIA, TSA with 5% sheep’s RBC and CAP
from cells
6. C-substance component of cell wall which is
teichoic acid that reacts with CRP resulting in the
activation of some nonspecific host immune
response

FPRE
153
Note!!! c. Neufeld-Quellang Reaction/ Capsular Swelling
Test
BAP:
 most useful, specific and rapid method for the
 Young colonies: circular, glistening, dome
identification of S.
shaped, wet, mucoid
pneumonia (+) and allows serotyping of isolates
 As the colonies become older, AUTOLYTIC
CHANGES result in a collapse of each colony’s  performed by mixing on a slide loopful of
center, giving it umbilicate or doughnot emulsified sputum or CSF with a loopful of
appearance, Dimple-shaped (CHECKER or ANTICAPSULAR SERUM and METHYLENE
NAILHEAD COLONIES) BLUE
 Colonies incubated aerobically produce α- (+) reaction: capsule appears swollen due to
hemolysis. change in refractive index which in turn due to
serologic reaction (OIL IMMERSION OBJECTIVE)
 Colonies incubated anaerobically produce β-
hemolysis due to oxygen-labile PNEUMOLYSIN d. Francis Test
O
 SKIN TEST for determining the presence of
ANTIBODIES against pneumococci

3. BIOCHEMICAL TEST e. Mouse Virulence Test

a. Optochin Susceptibility Test/ TAXO P  Based on the sensitivity of mouse to even small
inoculum of pneumococci
 presumptive identification of S. pneumoniae
 Sputum containing pneumococci is injected
 Optochin (ETHYLHYDROCUPREIN intraperitoneally to a mouse which eventually
HYDROCHLORIDE) disk is added to the surface dies within 16-48 hours
of an SBA plate inoculated with an α-hemolytic
Streptococcus  Heart blood of the mouse contain pure culture of
pneumococci
Result:
Serologic Test
ZOI >14 mm with a 6-mm disk = SUSCEPTIBLE
Coagglutination Test
ZOI >16 mm with a 10-mm disk =
SUSCEPTIBLE *uses particle-bound antibody to enhance the
visibility of the agglutination reaction between
b. Bile Solubility Test Antigen and Antibody
 based on the presence of autocatalytic enzyme
AMIDASE in S. pneumoniae
 Under the influence of BILE SALT OR
DETERGENT, bacteria’s cell wall lyses during
cell division
Reagent: SODIUM DEOXYCHOLATE
Result: Tests
S. pneumoniae = solution becomes CLEAR (+)
Other α-hemolytic = solution remains CLOUDY(-)
Negative control: Suspensions made in saline

FPRE
154
 Related Infections:
Bacteremia, endocarditis, otitis media,
osteomyelitis, endophthalmitis after
cataract extraction, brain abscess,
chronic sinusitis, septic arthritis,
meningitis, and breast implant–
associated infections
Microscopy: Gram-variable and
Pleomorphic forms
Species:
Granulicatellaadiacens,
Granulicatella elegans, and
Granulicatella balaenopterae,
Abiotrophia defective, and
ALGORITHM FOR IDENTIFICATIOS Abiotrophia adjacens

SEROLOGIC TEST FOR STREPTOCOCCI Biochemical Characteristics

a. Lancefield Precipitin Test  α-galactosidase

 confirmatory test wherein cell wall antigens are  β-galactosidase

extracted either physically by heating or  β-glucuronidase,
chemical or enzymatic extraction of cell
suspension grown overnight in Todd-Hewitt  Hippurate hydrolysis
Broth
 Arginine hydrolysis
b. Direct Fluorescent Antibody Test
 Acid production from trehalose and starch
c. Coagglutiantion (Phadebact)
STREPTOCOCCUS-LIKE ORGANISMS
d. Enzyme-linked Immunosorbent Assay (ELISA)
 Aerococcus, Gemella, Lactococcus,
e. Latex Agglutination Test
Leuconostoc, and Pediococcus = resemble
f. ASO Titer for Group A Streptococcal Infection viridans streptococci
Abiotrophia and Granulicatella  infections similar to enterococci and streptococci
 formerly known as the NUTRITIONALLY  Vancomycin-resistant = Leuconostoc and
VARIANT STREPTOCOCCI Pediococcus; Aerococcus = susceptible
 aka pyridoxal-dependent or vitamin B6- Aerococcus
dependent, thiol-dependent and symbiotic
 common airborne bacteria
streptococci
 resemble viridans streptococci on culture but
 grow as “satellite colonies” around other
microscopically similar to staphylococci (tetrads
bacteria and require sulfhydryl compounds for
or clusters)
growth
 growth in 6.5% NaCl; weak catalase or
 part of the human oral and gastrointestinal
pseudocatalase
microbiota

FPRE
155
Aerococcus viridans Biochemical Test:
bacteremia and endocarditis; bile esculin(+) and (-) catalase , (-) PYR, and (-) LAP ; (+) Bile Esculin;
PYR(+) growth 6.5% NaCl; and production of gas from
glucose
Aerococcus urinae
Pediococcus
UTI, endocarditis, lymphadenitis, and peritonitis; bile
esculin(-) and PYR(-)  Gram(+) cocci in pairs, tetrads, and clusters that
can grow at 45° C
Gemella
 bacteremia, abscess formation, and meningitis
 similar colony morphology and habitat to viridans
streptococci Species:
 α-hemolytic or nonhemolytic, gram-negative Pediococcus acidilactici, Pediococcus damnosus,
cocci in pairs, tetrads, clusters, or short chains Pediococcus dextrinicus, Pediococcus parvulus, and
Pediococcus pentasaceus
 endocarditis, wounds, and abscesses
Biochemical Test:
Species: Gemella haemolysins
(+) bile esculin, (+) LAP, and (-), do not produce gas
Lactococcus
from glucose, some grow in 6.5% NaCl
 Gram(+) cocci occur singly, in pairs, or in chains
Globicatella sanguinis
and physiologically similar to enterococci
 sepsis, meningitis, bacteremia, and UTIs
 α-hemolytic or nonhemolytic
 α-hemolytic, PYR(+), LAP(-), and vancomycin
 previously classified as group N streptococci
susceptible
 UTI and endocarditis
Helcococcus kunzii wound infections
 Does not produce acid from carbohydrates
Alloiococcus otitidis
Leuconostoc
 otitis media in children
 Similar biochemical characteristics with
 nonhemolytic
enterococci and viridans streptococci
 but α-hemolysis after prolonged incubation
 found on plant surfaces and vegetables, and in
milk products  PYR- and LAP-(+); grow slowly in 6.5% NaCl
 meningitis, bacteremia, UTIs, and pulmonary
infections
Species:
Leuconostoc citreum, Leuconostoc cremoris,
Leuconostoc dextranicum, Leuconostoc lactis,
Leuconostoc mesenteroides, and Leuconostoc
pseudomesenteroides
Microscopy: Irregular coccoid

FPRE
156