BACTERIOLOGY LECTURE

LECTURE 1: INTRODUCTION AND HISTORY

Prof. Lyslie Jean Vasquez-Baylon, RMT
AUG 15,2021
For updates and corrections → @mar4rii on Twitter

MICROBIOLOGY ● Veterinary
● Mikros = very small ○ Spread & control of diseases in animals
● Bios = life ● Agricultural
● Logos = Study of ○ The role of microbes in plants & livestock
● The study of very small living organism seen only with ● Sanitary
the use of a microscope ○ Processing & disposal of garbage & sewage
● Microorganism or Microbes waste; purification and processing of water
○ Minute living things that are usually too small to ● Industrial
be seen the unaided eye ○ Production of beer, wine, alcohol, vitamins,
○ Ex: Bacteria, Fungi, Protozoa, Viruses antibiotics, etc.
● Microbial Physiology & Genetics
What comes to your mind when you hear the word bacteria or ○ Genetic manipulation
virus? ● Environmental
● Infection ○ Cycling & recycling of elements by microbial
● Disease environmental & geochemical processes

Pathogens almost swept the whole of human race →
EPIDEMICS
● Plague - Yersinia pestis
● Cholera - Vibrio cholerae
● Smallpox - Variola virus
Why study Microbiology?
● A well developed knowledge of clinical microbiology is
critical to us because we are the ones identifying the
presence of bacteria, parasites, and etc.
● In studying microbiology, you will know more about the
dynamics of infectious diseases along with technical
developments available for diagnosing, treating, and
controlling diseases.
HISTORICAL DEVELOPMENTS OF BACTERIOLOGY
Robert Hooke (1665, England)
● Observed a thin slice of cork through a crude microscope
● He wrote the historical significant book, “Micrographia:
or Some Physiological Description of Minute Bodies
Made by Magnifying Glasses with Observations and
Inquiries Thereupon”
○ Contains observations through various lenses
and one of the first books to illustrate plants,
insects, and other microbes.
● Used an improved version of Compound microscope and
its uses
○ Lacked the resolution but would have allowed a
clear observation of microbes
● First observation of individual cells
● His discovery marked the beginning of the cell theory
○ “All living things are composed of cells”

Anton van Leeuwenhoek (1673-1723, Delft, Holland)

● Dutch merchant, amateur scientist
● First to observe live microorganism (animalcules)
● Single-lens microscope
○ His samples include rainwater, his own feces,
and materials scraped from his teeth
● Made a series of letters to the Royal Society of London
● Beneficial aspects describing the microbes both in words and drawings.
○ Normal microbiota - found inside our body ● Until his death in 1723, he revealed the microscopic
○ Decomposers world to scientists and is regarded as one of the first to
○ Industrial (food & beverages) provide accurate descriptions of protozoa, fungi, and
○ Producers of Oxygen (algae & Cyanobacteria) bacteria
○ Food chain
○ Genetic engineering; Pharmaceuticals; and The Transition Period
antibiotics used to treat infectious diseases ● 1700s Focus:
● Pathogenic organisms ○ Plant & animal life
○ Microorganisms such as bacteria that are ○ Attempts for categorization of microbes
capable of causing diseases Carolus Linnaeus (Swedish botanist)
● Opportunistic pathogens ● Developed the Binomial nomenclature (Systema
○ Harmless microorganisms that can become Naturae”, 1735 ; the genus & specific epithet
pathogenic once the host’s resistance is ○ Nomenclature - naming of microorganism
imapired according to established rules and guidelines
○ Provides accepted labels by which organisms
Scopes of Microbiology are universally recognized
● General ○ Binomial nomenclature is very important, so
○ study & classification of microbes that whatever microorganisms is discussed in
● Medical bacteriology in the Ph, is the same
○ Pathogens, diseases they cause & the body’s microorganism studied in other countries
defenses against diseases

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 ○ Binomial nomenclature is named as such John Needham (1745 - 1748)
because binomial = 2-way system of naming ● Put boiled nutrient broth into covered flasks
■ Every organism is assigned a genus
and a species of latin or greek
derivation
● Binomial nomenclature
○ Genus - first letter is capitalized
○ Species - first letter is lowercase
- Printed in italics or underlined in script
- Ex. Streptococcus pneumoniae

While scientists discovered and observed ○ The cool solutions were soon teeming of
microorganisms, the rest of the scientific community became microorganisms
interested in their origin. Many scientists believed in ○ He claimed that microbes developed
spontaneous generation, while some doubted this theory and spontaneously from the fluids
believed in biogenesis instead.
CONDITIONS RESULTS
ABIOGENESIS VS. BIOGENESIS
● Spontaneous Generation/ Abiogenesis Nutrient broth heated, Microbial growth
○ That living organism arise from nonliving cooled then placed in sealed
matter flask
○ A “vital force forms life
○ Ex, Flies could be born of manure or feces Lazzaro Spallanzani (1765)
○ Ex. 2 Maggots could arise from decaying ● Showed that fluids heated in sealed flasks did not
food and corpses contain microbes
● Suggested that microorganisms from the air probably
Biogenesis entered Needham’s solution after they were boiled
● The alternative hypothesis: that the living organisms
arise from preexisting life

Abiogenesis/ Spontaneous Generation

● Aristotle’s hypothesis:

\
● Jan Baptiste van Helmont’s Recipe for mice:
○ He had 2 sets of flasks, one was left opened
and the other was sealed
○ Both flasks were boiled
○ Fluid heated in sealed flasks did not contain
microbes
Evidence Pro and Con ■ He claimed that vital force needed
Francesco Redi (an italian physician, 1668) for spontaneous generation had
● 1st real experiment to dispute abiogenesis been destroyed by heat and was
● He showed that flies and maggots did not arise from kept out by the seals
decaying meat as others believe, if the meat is
covered to prevent the entry of flies Rudolf Virchow/ Theodor Schwann/ Matthias Schleiden
(1839 - 1858)
● Cell theory: all living things are composed of cells
● Rudolf Virchow (1858)
○ Proposed the theory of Biogenesis
○ Challenged the case of spontaneous
generation with biogenesis
● Louis Pasteur resolved the issue in 1864
CONDITIONS RESULTS
Louis Pasteur (1864)
1st: 3 sealed jars No maggots ● Observed Needham’s and Spallanzani’s experiments
- Decaying meat was kept isolated and began to ask questions such as:
from flies, which could not lay ○ Is there a “life force” in air that can cause
their eggs microbes to develop by spontaneous
2nd: 3 open jars Maggots appeared after flies generation?
- Flies laid their eggs on the meat ○ Is there a means of allowing air to enter a
3rd: 3 jars No maggots and no flies inside (only at container but not the bacteria that are
covered w/ fine the top of the net) present in it?
net Experiment 1
CONDITIONS RESULTS
● As a result of Francesco Redi’s experiment, scientists
and people began to doubt Aristotle’s theory, they Nutrient (beef) broth placed Unsealed: Contaminated
began to doubt spontaneous generation. in short-necked flasks, then with microbes
● The case for Spontaneous Generation strengthened sealed/unsealed Sealed: Free of
again in 1745 microorganisms

2

● From these results, Pasteur concluded that microbes
in the air were responsible for contaminating
non-living matter
Experiment 2
CONDITIONS RESULTS
Broth placed in open-ended, long-necked No signs of life
flasks and bent the necks into S-shaped
curves; boiled and cooled
● Pasteur’s unique design allowed air to pass into the
flask but the curved neck trapped any airborne
microorganisms that might contaminate broth
● Conclusion:
○ Microbial life can be destroyed by heat and
that methods can be devised to block the
access of airborne microorganisms to
nutrient environments
○ There is no such life force in air, and
organisms do not arise by spontaneous
generation in this manner
○ “Life is a germ and a germ is Life. Never will
the doctrine of spontaneous generation
recover from the mortal blow of this simple
experiment.
The Golden Age of Microbiology
● Includes
○ Louis Pasteur’s Work
○ The Germ Theory of Disease
○ Vaccination
○ Antimicrobial Drugs
Louis Pasteurs’ Work
● Microbes are responsible for fermentation and
spoilage of food
○ Fermentation- conversion of sugar to alcohol
in the absence of air using microorganisms
such as yeast
○ The souring of food and spoilage is caused
by different microorganisms
■ In the presence of air, bacteria
change the alcohol into vinegar
(acetic acid)
● Spoilage bacteria could be killed through
pasteurization
○ Pasteurization- the application of high heat
for a short time without destroying the
proteins
The Germ Theory of Disease
● Postulated by Louis Pasteur
○ Microorganisms are the cause of infectious
diseases
○ Attempt to prove the Germ Theory of
Disease was unsuccessful
● Diseases were thought to be caused by demons, evil
spirits, and the wrath of God
● Hard for people to believe that diseases were caused
by microbes
● People who made contributions in linking
microorganisms to diseases
○ Agostino Bassi (1835)
■ Discovered a silkworm disease
caused by a fungus
○ Louis Pasteur (1865)
■ Discovered another silkworm
disease caused by a protozoan
○ Ignaz Semmelweis (1840s)
■ Advocated handwashing to prevent
spread of puerperal fever (fever
cause by uterine infection following
childbirth)
○ Joseph Lister (1860s)
■ Used phenol (carbolic acid) to kill
bacteria to prevent surgical wound
infections
○ Robert Koch (1876)
■ First proved that bacteria actually
caused disease
■ Provided experimental steps
(Koch’s Postulates) used to prove
that a specific microbe causes a
specific disease
■ Discovered rod-shaped bacteria,
Bacillus anthracis, the causative
agent of Anthrax
■ He was Pasteur’s young rival in
discovering the cause of Anthrax
○ Microbial etiology of important diseases
established by Koch:
■ Cholera- Vibrio cholerae
■ Tubercolosis- Mycobacterium
tuberculosis
■ Anthrax- Bacilllus anthracis
Take note: Robert Koch cultivated bacillus anthracis on
nutrient broth, then injected pure culture of the bacilli into the
healthy animals. When the animals died, the co-isolated the
bacteria in their blood and compared them to the bacillus
anthracis that was originally isolated.
Robert Koch found out that the 2 sets of bacteria have the
same characteristics and showed that the bacilli invariably
caused anthrax
Koch’s Postulates: Experimental steps that directly relates a
specific microbe to a specific disease
Exceptions to Koch’s Postulates
1. Many healthy people carry pathogens but do not
exhibit symptoms of the disease.
2. Some microbes are very difficult or impossible to grow
on artificial media.
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 3. To induce a disease from a pure culture, the
experimental animal must be susceptible to the
pathogen.
4. Certain diseases develop only when an opportunistic
pathogen invades a weakened host.
Vaccination
● May 4, 1796: Edward Jenner : First vaccine
○ found a way to protect people from smallpox
● Sarah Nelms: a dairy maid, who had fresh cowpox
lesions from her hands and arms from which Jenner
collected scrapings from her blisters
● Found a way to protect people from smallpox
● He inoculated a person (James Phipps: 8-year-old)
with cowpox virus resulting to protection from
smallpox
● The protection is called immunity
● Vaccination from vacca (cow)
○ This process provides protection from
diseases
Antimicrobial Drugs
The Birth of Modern Chemotherapy
● Chemotherapy
○ Treatment of noninfectious diseases (such
as cancers) using chemical substances
● Chemotherapeutic agents can be synthetic drugs
(chemicals) or natural (antibiotics).
○ Synthetic = prepared in the lab
○ Antibiotics = naturally produced by bacteria
and fungi to act against other
microorganisms
During the early stages of chemotherapy, the only known
chemical in the Europe’s medical arsenal was an extract from
the bark of the south american tree QUININE
● Quinine (tree bark) = for malaria
● 1910: Paul Ehrlich
○ developed salvarsan (arsenic derivative
effective against syphilis)
● 1928: Alexander Fleming
○ discovered penicillin, the first antibiotic
■ Penicillin: from Penicillium notatum
(a mold) later renamed as
Penicillium chrysogenum
■ Mold juice was capable of killing a
wide range of bacteria such as
Streptococcus, Meningococcus,
and Diphtheria bacillus
● 1930s: Sulfonamides were synthesized
● 1940s: Penicillin was tested clinically & mass
produced
MODERN DEVELOPMENTS IN MICROBIOLOGY
● Bacteriology
● Mycology
● Parasitology
● Phycology
● Virology
● Genomics
○ The study of an organism’s genes
○ provided new tools to classify microbes.
● Immunology
○ Rebecca Lancefield (1933) proposed the
use of immunology to identify bacteria
according to serotypes
■ group of related microbes
distinguished by a common set of
antigens.
■ Vaccines and interferon are
investigated for viral diseases
○ Paul Berg (1960s) introduced Recombinant
DNA
○ Recombinant DNA- made from two (2)
different sources
■ Done through insertion of animal
DNA into bacterial DNA and then
the bacteria will produce animal
protein.
■ Recombinant DNA Technology
● Uses genes in microbes,
plants, and animals
manipulated for practical
applications
● Production of human
blood-clotting factor by E.
coli to aid hemophiliacs
:
MICROBES AND HUMAN WELFARE
Bioremediation
● Bacteria degrade organic matter in sewage & detoxify
pollutants.
○ Ex: oil and mercury.
● example: Pseudomonas and Bacillus
Biological Insecticide
● ‘Microbes that are pathogenic to insects but not to
humans or animals
● Microbes as alternatives to chemical pesticides
○ Ex: Bacillus thuringiensis
● control pests such as alfafa caterpillars, bollworms,
corn borers, cabbage worms, tobacco budworms and
fruit tree leaf rollers
Modern Biotechnology
● Practical application of microbiology.
● Commercial use of microorganisms to produce some
common foods and chemicals
○ Genetic engineering
■ Biotechnological technique
■ May use bacteria or fungi to
produce a variety of proteins such
as vaccines and enzymes.
○ Gene therapy
■ In replacing a defective gene in
human cells, this one uses a
harmless virus to carry the missing
gene or new gene into host cells
and then the gene is picked up into
the chromosomes.
○ GMOs
MICROBES AND HUMAN DISEASE
● Flora, microflora – bacteria once classified as plants
● Normal Microbiota – new name
○ Normally present in the human body
○ They prevent growth of pathogens.
○ They produce growth factors (folic acid), vit.
K.
● Resistance – ability to ward off diseases factors: skin,
stomach acid, antimicrobial chemicals.
● Infectious Disease – when a pathogen overcomes
the host’s resistance, disease results
Emerging Infectious Diseases (EIDS)
BACTERIAL
● Outbreaks of previously unknown diseases
● Known diseases that are rapidly increasing in
incidence or geographic range in the last 2 decades
● Persistence of infectious diseases that cannot be
controlled
● Include HIV infections, Lyme disease, Escherichia coli
O157:H7 (E. coli), hantavirus, dengue fever, West Nile
virus, Zika virus, SARS and COVID19.
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 Reemerging diseases: ● Target: Alveolar epithelial type II cells
● Reappear after they have been on a significant ● More contagious but less severe than SARS and
decline MERS
● May happen because of a breakdown in public health
measures for diseases that were once under control “Microbiology is like sand; it is blown about by time and is
● Can also happen when new strains of known constantly shifting”
disease-causing organisms appear
● Human behavior affects reemergence, i.e. overuse of
antibiotics.
● Include malaria, tuberculosis, cholera, pertussis,
influenza, pneumococcal disease, and gonorrhea
Emerging infectious disease
Invasive group A Streptococcus
● Rapidly growing bacteria
● cause extensive tissue damage.
● Flesh eating bacteria
Anthrax
● Bacillus anthracis
● Veterinarians and agricultural workers (at risk).
Escherichia coli O157: H7
● Enterotoxigenic E. coli
● Leading cause of bloody diarrhea worldwide.
SUPERBUGS
● Microorganisms that have become resistant to
antibiotics that are usually used to treat the infections.
Carbapenem-resistant Klebsiella pneumoniae (CRKP)
● pneumonia, meningitis, UTI, wound & blood
infections; LA-2011
● Colistin
○ 50 yr old drug
○ Last resort for the treatment of CRKP
Methicillin-resistant S. aureus (MRSA)
● boils & abscesses, pneumonia or flu-like symptoms
● Vancomycin
○ Reference standard for the treatment of
systemic infections caused by MRSA
both are transmitted via contact
Affects elderly, immune-compromised

VIRAL
West Nile encephalitis
● West Nile Virus
● First diagnosed in the West Nile region of Uganda in
1937.
● Appeared in New York City in 1999.
Bovine Spongiform Encephalopathy
● Prions
○ Abnormally or misfolded proteins
● Also causes Creutzfeldt-Jakob disease (CJD)
● New-variant CJD in humans related to cattle fed
sheep
Ebola hemorrhagic fever
● Ebola virus
○ causes fever, hemorrhaging, and blood
clotting -First identified near Ebola River,
Congo
Hantavirus pulmonary syndrome
● Hantavirus
● a cause hemorrhagic fever
● a .S.,1995: a disease with respiratory symptoms was
seen
● Hantavirus Sin Nombre virus (U.S., carried by rats)
Acquired immunodeficiency syndrome (AIDS)
● Human immunodeficiency virus (HIV)
● STD; pandemic identified in 1981
NOW: SARS-Cov2
● SARS-Cov2 infection: COVID 19
● Typical symptoms
○ Fever, dry cough, fatigue, phlegm
production, shortness of breath, muscle pain,
sore throat, and Headache
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 BACTERIOLOGY

LECTURE 2: BACTERIAL STRUCTURES

Prof. Lyslie Jean Vasquez-Baylon, RMT
August 18, 2021
For updates and corrections → @mar4rii on Twitter

DIVISIONS OF MICROBIOLOGY ■ Mycelia - composed of long

1. Virology - study of viruses filaments called hyphae that
● Viruses: branch an intertwine
○ small intact infectious agents
○ Complex molecules
○ Mostly seen with an electron 4. Phycology – scientific study of algae
microscope ● Alga - singular
○ can only reproduce inside a cell they ● Algae
have infected ○ Mainly aquatic (fresh/salt water),
○ Consist of a core of RNA or DNA contain chlorophyll, carry out
surrounded by protein, sometimes an photosynthesis
envelope of glycoprotein ○ Some species produce neurotoxins
■ Can only have 1 type: RNA which can concentrate in fish or
or DNA shellfish and cause poisoning when
2. Parasitology – study of protozoans and helminths eaten by humans
● Protozoans: 5. Bacteriology – study of bacteria
○ Unicellular eukaryotic organisms, ● Bacterium - singular
enclosed in a membrane, and contain ● Bacteria:
visible organelles ○ Unicellular, Prokaryotic
○ Ex. Amoeba - E. histolytica; ○ Liver freely in the environment
Flagellates - Trypanosoma; ciliates - ○ Contain both RNA and DNA
Paramecium ○ Some bacteria lack cell walls, do not
● Helminths: cause disease and are beneficial or
○ Multicellular parasites, worms pathogenic
(tapeworms, roundworms, flatworms), ○ Multiply by binary fission
also have a microscopic stage in their ○ Eubacteria (true bacteria);
life cycle. Archaebacteria (ancient bacteria) –
● Protozoa: Singular: Protozoan different cell walls
○ Single-celled eukaryotes
○ Similar to animals in nutrient needs
and cellular structure
○ Live freely in water; some live in
animal hosts
○ Asexual (most) and sexual
reproduction
○ Most are capable of locomotion by
■ Pseudopodia – cell
extensions that flow in
direction of travel
■ Cilia – numerous, short,
hairlike protrusions that
propel organisms through
environment
■ Flagella – extensions of a
cell that are fewer, longer,
and more whiplike than cilia
Example: Entamoeba
histolytica
3. Mycology – study of fungi (fungus - singular)
● Fungi (yeasts, molds):
○ Unicellular or multicellular, thick cell
wall
○ Develop from spores or fragments of
hyphae - Arthropods and parasites could be the largest and can be
● Yeast vs. Molds seen using the naked eye
○ Yeast = unicellular; often oval; larger - Followed by algae, bacteria , viruses, and prions
than bacteria - Prions = abnormally folded proteins or
○ Molds = multicellular; most typical misfolded proteins
fungi; usually found in bread or fruits;
form visible mass called mycelia

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 PROKARYOTES VS EUKARYOTES

Characteristics Prokaryotes Eukaryotes

Size of cell Typically 0.2-2.0 m m in diameter Typically 10-100 m m in diameter
Nucleus No nuclear membrane or nucleoli (nucleoid) True nucleus, consisting of nuclear membrane & nucleoli

Membrane-enclosed Present; examples include lysosomes, Golgi complex,

Absent
organelles endoplasmic reticulum, mitochondria & chloroplasts

Flagella Consist of two protein building blocks Complex; consist of multiple microtubules

Glycocalyx /Capsule Present as a capsule or slime layer Present in some cells that lack a cell wall

Usually present; chemically complex (typical

Cell wall When present, chemically simple
bacterial cell wall includes peptidoglycan)

Plasma membrane No carbohydrates and generally lacks sterols Sterols and carbohydrates that serve as receptors present

Cytoplasm No cytoskeleton or cytoplasmic streaming Cytoskeleton; cytoplasmic streaming

Ribosomes Smaller size (70S) Larger size (80S); smaller size (70S) in organelles
Chromosome (DNA)
Single circular chromosome; lacks histones Multiple linear chromosomes with histones
arrangement

No meiosis; transfer of DNA fragments only

Sexual reproduction Involves meiosis
(conjugation)

BACTERIAL MORPHOLOGY

● Both contain plasma membrane, cytoplasm. DNA, and

ribosomes ● Bacterial cells generally appear in one of several shapes
● Organelles are absent in the prokaryotic cell as seen on this slide
● Eukaryotic cell (parasites & fungi) has membrane ● Morphology:
enclosed organelles ○ Rods or cocci
○ ER = which processes and transport proteins ○ Curved or spiral
○ Golgi body = modify and transport substances ○ filamentous
○ Mitochondria = generates ATP
○ Lysosome = suicide sac of teh cell Size
○ Nucleus = control center of the cell ● 0.2 - 2 microns in width or diameter
● Up to 1-10 microns in length
● Prokaryotic cells do not
contain membrane bound Shape
organelles
● All functions take place in
the cytoplasm or
cytoplasmic membrane
● Cell wall (peptidoglycan)
○ only present in
bacterial cell
○ Important in
classifying and ● Bacilli = rod-shape (singular: bacillus)
identifying ● Cocci = spherical (singular: coccus)
microorganisms ● Spiral = corkscrew or curved
● Pleomorphic = bacteria that are variable in shape

Arrangements
● Determined by the orientation and degree of
attachment of a bacteria at the time of cell division
● Arrangement of bacteria can be in:
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 ○ Singly ○ Vibrio - spiral bacteria that look like curved
○ In pairs rods
○ Groups as tetrads, clusters on in chains ○ Spirillum - helical in shape, corkscrew but
fairly rigid bodies
○ Spirochete - spirals that are helical and
flexible
■ Ex. Treponema pallidum
● Note:
○ To determine the size, shape and
arrangement of bacteria
■ Stain: ex. Gram stain

● For cocci, division in one plane may result to single STRUCTURES OF A BACTERIAL CELL
and pairing ● General Structure of a Prokaryote
○ Cocci may occur in pairs and are called
diplococci
○ Cocci in chains are known as streptococci
○ Division in 2 planes allow bacteria to remain
in groups
■ Ex. groups of 4 are known as tetrad
■ Cube-like groups of 8 are called as
sarcinae
■ Grape-like clusters named as
staphylococci
○ Ex. of staphylococci - Staphylococcus
aureus

Structures External to the Cell Wall

1. Glycocalyx
2. Flagella
3. Axial Filaments
4. Fimbriae and Pili
5. Cell Wall

The Bacterial Surface Coating → Glycocalyx

● Means “sugarcoat”
● Surrounds bacterial cells and made up of
polysaccharides, polypeptide, or both
● Coating of molecules external to the cell wall
● Made of sugars and/or proteins
● Functions
○ Attachment
○ Inhibits killing by white blood cells
○ receptor
● For bacilli, they divide only across their short axis
○ Fewer arrangements or groupings
○ Most bacilli appear as single rods
○ Bacilli that appear in pairs are diplobacilli
○ Bacilli that occur in chains are named
streptobacilli
○ Other may look like cocci, that they are
called coccobacillus

● 2 types:
1. Capsule
- highly
organized
- tightly
attached to the
cell wall
2. Slime layer
- unorganized
- loosely
attached to the
cell wall
● In certain bacteria, the
capsule is very
important because:
○ It contributes to virulence
● For the spirals, bacteria that are spiral have one or
■ Virulence - a degree to which a
more twists, we have 3:
pathogen causes disease

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 ○ Capsule protects pathogens from
phagocytosis and prevents it from being
destroyed
○ Ex. of bacteria with capsule
■ Bacillus anthracis
■ Streptococcus pneumoniae
■ Klebsiella
The Flagella
● Some prokaryotes have flagella (singular - flagellum)
● Long, filamentous appendages that propel bacteria
● 3 parts
○ Filament
■ Long, thin, helical structure composed
of proteins (protein: flagellin)
■ Outermost region
○ Hook
■ Curved sheath
■ Where the filament is attached to
○ Basal body
■ Stack of rings firmly anchored in cell
wall
■ Anchors the flagellum to the cell wall
and plasma membrane
● Rotates 360°
● 1-2 or many distributed over entire cell
● Functions in motility
- Flagella may be in different arrangements
● Flagellar Arrangements
1. Atrichous - bacteria with no flagella
2. Peritrichous - flagella dispersed over surface of
cell, slowest
- Flagella may also be polar: at one or both poles/ ends of
the cell
1. Monotrichous - single flagellum at one end
2. Lophotrichous - small bunches arising from one
end of cell; tuft of flagella located on both
3. Amphitrichous - flagella at both ends of cell
● Picture 1 (Upper left) - flagella is dispersed over the
surface of the cell - peritrichous
● Picture 2 (Lower left) - tuft of flagella found only on one
end - lophotrichous
● Picture 3 (Upper right) - flagellum at one end -
monotrichous
● Picture 4 (lower right) - flagella is found at both ends -
amphitrichous
Internal Flagella → Axial filaments
● Polar vs. Peritrichous
● Take note that each prokaryotic flagellum
moves the cell by rotating from the basal bodies
● Rotation can either be clockwise or counter
clockwise around its long axis
● Counter clockwise rotation aligns the flagella
into a single rotating bundle causing the
bacterium to swim in a straight line
● Clockwise rotation breaks the flagella bundle
apart such that each flagellum points in a
different direction
○ Causes the bacterium to tumble in
place
lCHEMOTAXIS
● The cell’s motility may be used by the bacteria to move to
a favorable environment. This movement toward or away
from a particular stimulus is known as TAXIS.
● Chemotaxis- the movement in response to a chemical
stimulus.
● Important to bacteria
○ Example: Due to chemotaxis, bacteria may
move toward a chemical stimulus such as
oxygen, ribose, and galactose for their own
benefit
○ In response to stimuli, information is passed to
the flagella and the n the bacteria starts
moving.
○ If ppositive,
● Positive Stimulus- Attractant
○ Bacteria movies toward the stimulus with many
runs and few tumbles
● Negative Stimulus- Repellent
○ Bacteria moves away from it
● A.k.a periplasmic
● Internal flagella
● Endoflagella
○ Bundles of fibrils that arise at the ends of the
cell, beneath an outer sheath, and spiral around
the cell
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 ● Enclosed between the cell wall and cell membrane of
spirochetes
● Motility
● Means of locomotion for spirochetes like Treponema
pallidum and Borrelia burgdorferi
○ Allows spirochetes to move in a spiral motion
like how corkscrew move through a cork
Appendages for Attachment → Fimbriae (Fimbria)
● Fine hair-like bristles form the cell surface
● Function in adhesion to other cells and surfaces
● Help the bacteria to adhere to epithelial surfaces in the
body;
● Involved in biofilms and othe aggregation
○ Example: Neisseria gonorrhoeae (Gonorrhea)
uses fimbriae to colonize mucus membranes
to cause disease
○ E. coli O157H7
■ Adhere to the lining of the small
intestine and cause severe water
diarrhea
○ The absence of fimbriae of a pathogen due to
mutation will not allow colonization, thus
disease won’t occur
Appendages for Mating → Pili
● Rigid, tubular structure
● Usually longer than fimbriae
● Made of pilin protein
● Found only in Gram-Negative cells
● Functions:
○ Joins bacterial cells for DNA transfer
(conjugation); (sex pilus)
■ Conjugation: one bacterial cell can
transfer one bacterial cell to another
bacterial cell
○ Adhesion
○ For motility
■ Twitching motility (Pseudomonas,
Neisseria, E. coli)
- Pilus extends to make
contact with another
surface of the cell followed
by retraction that causes a
jerky, intermittent movement
■ Gliding motility
● Myxobacteria
The Cell Envelope
● External covering outside the cytoplasm
● Composed of two basic layers
○ Cell wall and cell membrane
● Maintains cell integrity
The Structural Cell Wall→ Peptidoglycan
● a.k.a Murein Layer
● Maintains the shape of the cell, protects the cell, and
serves as a point of anchorage of the flagella
● Macromelcule composed of a repeating framework of
long glycan chains cross-linked by short peptide
fragments
● Provides strong, flexible, support to keep bacteria from
bursting or collapsing because of changes in osmotic
pressure or when the water pressure inside the cell is
greater than the outer environment of the cell, it causes
cell lysis.
● Has unique structure composed of disaccharide,
pentapeptide subunits
● Disaccharide portion/alternating sugar portion
○ NAG: N-acetylglucosamine
○ NAM: N-acetylmuramic acid
■ NAG and NAM are linked and
alternating, forming a
backbone/glycan portion of the cell
wall
■ Polypeptide link adjacent rows
GROUPS BASED ON CELL WALL COMPOSITION
● Four groups:
○ Gram-Positive Cells
○ Gram-Negative Cells
○ Bacteria without cell walls
○ Bacteria with chemically unique cell walls
A. Gram-Positive Cell Wall
● Consists of:
○ A thick, homogenous sheath of peptidoglycan,
20-80 nm thick
○ Tightly-bound acidic polysaccharides
■ Teichoic acid
- Linked to the peptidoglycan layer
- Made up of alcohol and phosphate
- Negative charges from phosphate
content blinds and regulates
cation movement in and out of the
cell
- Helps in the identification of
Gram-positive bacteria in the lab
using Gram-staining
■ Lipoteichoic acid
- Spans the peptidoglycan layer
and linked to the plasma
membrane
○ Cell membrane
● Retain Crystal Violet and stain purple
● Gram-Positive = Purple
B. Gram-Negative Cell Wall
● Consists of
○ an outer membrane containing
lipopolysaccharide (LPS)
■ Functions as the cell’s initial barrier to
the environment and is composed of
LPS
■ LPS: gives the surface of a
gram-negative bacteria a net negative
charge
○ thin shell of peptidoglycan
○ periplasmic space
■ A gel-like fluid between the outer
membrane and plasma membrane
■ Take note: gram-negative cell walls
do not contain decomic acid. Since
the gram-negative cell walls have a
thin peptidoglycan the cell is more
susceptible to mechanical breakage
○ inner membrane
● Lose crystal violet and stain red from safranin
counterstain
● Protective structure while providing some flexibility and
sensitivity to lysis
● LPS
○ Endotoxin that may become toxic when
released during infections
○ may function as receptors and blocking immune
response
○ contains porin proteins in upper layer
5
 ■ Regulates molecules entering and
leaving cell
○ 3 Components:
1. Lipid A
- Lipid portion of the
lipopolysaccharide
- Located at the top layer of
the outer membrane
- Released whenever the
gram-negative cell dies
- Functions as endotoxin and
responsible for symptoms
associated with
gram-negative infection
2. Core
- Attached to lipid A and
provides stability
3. O polysaccharide
- Extends outward and
functions as an antigen
- Used to distinguish species
of gram negative bacteria

The Gram Stain

● Developed by Hans Christian Gram
● Method of staining used in the laboratory to distinguish
and classify bacteria into two large groups: (a)
Gram-positive bacteria and (b) the Gram-negative
bacteria
● Differential stain
○ Gram-negative
■ lose crystal violet and stain red from
safranin counterstain
○ Gram-positive
■ retain crystal violet and stain purple
● Important basis of bacterial classification and
identification
● Practical aid in diagnosing infection and guiding drug
treatment

6
 ● Primary Stain - Crystal Violet ● not essential to bacterial growth & metabolism
● Mordant - Gram’s iodine ● may encode antibiotic resistance, tolerance to toxic
● Decolorizer - Alcohol (ethyl alcohol) metals, enzymes & toxins
● Counterstain - Safranin ● used in genetic engineering- readily manipulated &
transferred from cell to cell
The primary stain stains both gram (-) and (+) purple. ● Contain extra chromosomal DNA which are not really
↓ connected to the main bacterial chromosome so they
Gram’s iodine
replicate independently.
↓
● Can be transferred from one bacterium to another
When the decolorizer is applied, the peptidoglycan of gram
negative is dissolved causing the crystal violet iodine to diffuse Site of Protein Synthesis→ Ribosomes
↓ ● prokaryotic differ from eukaryotic ribosomes in size &
Gram positive retains purple and gram negative is colorless number of proteins
↓ ● site of protein synthesis
After application of safranin, gram (+) is purple and gram (-) is red ● all cells have ribosomes
Storage Bodies→ Inclusions & Granules
C. Atypical Cell Walls ● Reserve deposits or nutrients accumulated over time
● Without cell walls or very little wall material and used when needed.
● Mycobacterium and Nocardia ● intracellular storage bodies / reserve deposits
○ Contains high concentration of hydrophobic ● vary in size, number & content
waxy lipids mycolic acid ● Examples:
■ Unable to take off the color of the ○ Glycogen
dyes used in gram staining thus, we ■ Polysaccharide granules appears
use another method of staining: reddish brown
acid fast staining ■ Starch = blue when iodine is
● Some bacterial groups lack typical cell wall structure applied
● Gram-positive cell wall structure with lipid mycolic ○ Lipid inclusions which contain
acid . Poly-b-hydroxybutyrate acid
○ pathogenicity ■ Lipid inclusions are present in
○ high degree of resistance to certain Bacillus, mycobacterium and
chemicals and dyes spirillum.
○ basis for acid-fast stain ○ gas vesicles for floating
Some have no cell wall ○ sulfur
● Mycoplasma ○ polyphosphate granules
● cell wall is stabilized by sterols ○ Metachromatic granules
● Pleomorphic ■ Also known as Volutin
■ Appears red when stained with
Structures Internal to the Cell Wall methylene blue
1. Plasma (Cytoplasmic) Membrane ■ Present in Corynebacterium
2. Cytoplasm diphtheriae
3. The Nucleoid ● Causes diphtheria
4. Plasmids ■ Represents inorganic phosphate
5. Ribosomes which are actually reserves for the
6. Inclusions synthesis of ATP
7. Endospores Endospores
Plasma (Cytoplasmic) Membrane ● Specialized, resting, dormant cells
● Also called as the inner membrane ● produced by some G+ genera
● Thin structure lying inside the cell wall and enclosing ○ Clostridium, Bacillus & Sporosarcina
the cytoplasm of the cell ● Highly durable dehydrated cells with thick walls
● Composition: phospholipids and proteins additional layers
● Phospholipid bilayer ○ Formed internal to the bacterial cell
○ Arrange in such a way that it contains a membrane
hydrophilic polar head composed of ● When they are release, they can survive extremes
phosphate and glycerol and hydrophobic environment
nonpolar tails composed of fatty acid. ● have a 2-phase life cycle
● Serves as a selective barrier for materials going in ○ vegetative cell
and out of the cell ○ endospore
Cytoplasm ● Sporulation
● Substance of the cell inside the plasma membrane ○ formation of endospores
● dense gelatinous solution of sugars, amino acids, & ● Germination
salts ○ return to vegetative growth
● 70-80% water serves as solvent for materials used in ● withstand extremes in heat, drying, freezing, radiation
all cell functions & chemicals
Nucleoid: Chromosome ● resistance linked to high levels of calcium & certain
acids
● single, circular, double-stranded DNA molecule
● longevity verges on immortality
● contains all the genetic information required by a cell
○ 25 to 250 million years
● DNA is tightly coiled around a protein
● pressurized steam at 120oC for 20-30 minutes will
○ dense area called the nucleoid
destroy
Plasmids
● small circular, .
● free or integrated into the chromosome
● duplicated and passed on to offspring
7
Spore septum begin to isolate newly replicated DNA in a small
portion of the cytoplasm
↓
Plasma membrane starts to surround the DNA cytoplasm and
the membrane is isolated
↓
Spores septum surrounds isolated portion forming the
forespore
↓
Peptidoglycan layer form between membrane
Spore coat forms
↓
Endospore is freed from the cell

● Whatever endospore is released from the cell, this

can survive for thousands of years regardless of
conditions because it can withstand extremes.
● When the condition is favorable, the endospore
produces fresh vegetative cell .

8
 BACTERIOLOGY LEC

LECTURE 3: BACTERIAL GROWTH AND

GENETICS
Prof. Hinay
Aug 23, 2021
For updates and corrections → @mar4rii on Twitter

PHYSIOLOGICAL AND NUTRITIONAL

GROWTH REQUIREMENTS OF BACTERIA
● Physiology
○ study of vital life processes of organisms & how these processes normally function in living organisms
● Nutritional Requirements
○ substances required for energy generation and cellular biosynthesis.
○ chemicals & elements of the environment that are utilized for bacterial growth
○ In the laboratory, bacteria are grown in culture media, which are design to provide all the essential nutrients in solution for
bacterial growth
The Needed ELEMENTS for growth
● Major Elements:
○ C, O2 , N, H, P, S, K, Mg, Ca, Fe
● These elements are found in the form of water, inorganic ions, small molecules, and macromolecules which, serves either a structural
or functional role in the cells

Carbon ● 50% of its dry weight

● Main constituent of cellular materials
Oxygen ● 20%
● Electron acceptor in aerobic respiration
Nitrogen ● 14% of bacterium’s dry weight
● Constituent of amino acids, nucleic acids, nucleotide, and coenzymes
Hydrogen ● 8%
● Main constituent of organic compound and cell water
Phosphorous ● Also a constituent of nucleic acids, nucleotides, and phospholipids in the cell wall
of bacteria
● They are found attached to the lipopolysaccharides and teichoic acids
Sulfur ● 1%
● Main cellular inorganic cation and cofactor for certain enzymes
Magnesium ● 0.5%
● Inorganic cellular
● Acts as a cofactor for certain enzymatic reactions
Calcium ● 0.5%
● Acts as a cofactor for certain enzymatic reactions
● A component of endospores
●
Iron ● 0.2%
● A component of cytochromes and certain non-heme iron proteins
● A cofactor for certain enzymatic reactions

● Trace Elements:
○ Mn, Co, Zn, Cu, Mo
● metal ions that act as co-factors for essential enzymatic reactions
● Required in such small amounts & not necessarily added to culture media
● Present as contaminants of the water or other media components

1
 TYPES OF ORGANISMS BASED ON PHYSIOLOGIC REQUIREMENTS:

NUTRITIONAL TYPE ENERGY SOURCE

Phototroph Light
Chemotroph Chemical

a. Chemolithotrophs Inorganic chemicals

b. Chemoorganotrophs Organic chemicals

Autotroph CO2
Heterotroph Organic compounds

● Phototrophs
○ Derive energy by photophosphorylation
● Chemotrophs
○ Derive energy by oxidative phosphorylation

Metabolic Diversity Among Organisms

NUTRITIONAL ENERGY SOURCE CARBON EXAMPLE

TYPE SOURCE

Photoautotroph Light CO2 Oxygenic:

Cyanobacteria, plants.
Anoxygenic: Green,
purple bacteria.

Photoheterotroph Light Organic Green, purple nonsulfur

compounds bacteria

Chemoautotroph Chemical CO2 Fe-oxidizing,S, H,

nitrifying bacteria

Chemoheterotroph Chemical Organic Most bacteria,

compounds fermentative bacteria,
animals, protozoa, fungi.

GROWTH FACTORS
● Some chemoheterotrophs may require small amounts of certain organic compounds for growth because they are:
○ essential substances that the organism is unable to synthesize from available nutrients
● required in small amounts by cells because they fulfill specific roles in biosynthesis.
● The need for a growth factor results from either a blocked or missing metabolic pathway in the cells

CATEGORIES:
1. Purines , pyrimidines
● Required for synthesis of nucleic acids like DNA and RNA
2. Amino acids
● Required for synthesis of proteins
3. Vitamins
● These are needed as coenzyme and functional groups of certain enzymes
● Some bacteria ex. E coli does not require any growth factors.
● They can synthesize all essential purines, pyrimidines, amino acid, and vitamins starting with their carbon source as a part of their
own intermediary metabolism
● But other bacteria like lactobacillus, require all these categories in order to grow
● The compounds must be added in advance to culture media that are used to grow these bacteria
● The growth factors are not metabolized directly as sources of carbon or energy rather they are assimilated by cells to fulfill their
specific role in metabolism

Fastidious organisms tend to require a variety of growth factors

EX. Haemophilus influenzae

The Requirements for Growth:

→ Prokaryotes exist in nature under an enormous range of physical conditions such as:
● Oxygen concentration
● Hydrogen ion concentration (pH)
● Temperature

2
 1. Oxygen Requirement
→ oxygen is a universal component of cells, always provided in large amounts by water
→ however, prokaryotes display a wide range of responses to molecular oxygen

A. Aerobes
● Obligate Aerobes
○ Bacteria that require oxygen for growth
○ Use oxygen as a final electron acceptor in aerobic respiration
● Facultative Anaerobe
○ Can switch from aerobic to anaerobic type of metabolism
○ In the presence of oxygen, they switch to aerobic respiration
B. Anaerobes
● Obligate Anaerobes
○ Do not need/ use oxygen as a nutrient
○ Oxygen is toxic for them
■ Either kills or inhibits their growth
○ May live by fermentation, anaerobic respiration, bacterial photosynthesis or the novel process of methanogenesis
● Facultative Aerobes
○ Can switch from anaerobic to aerobic type of metabolism
○ Under anaerobic conditions, they grow by fermentation or anaerobic respiration
● Aerotolerant Anaerobes
○ Bacteria with an exclusively anaerobic or fermentative type of metabolism
○ Insensitive to the presence of oxygen
○ Lives in fermentation alone whether or not oxygen is present in their environment
C. Microaerophiles
● Require oxygen to survive but only at a lower concentration (about 20% con. of oxygen)
● Many microaerophiles are also capnophiles
D. Capnophiles
● Require an elevated concentration of carbon dioxide
● Easily cultivated in a candle jar (a lit candle is introduced before sealing, flame burns until extinguished by oxygen
deprivation creating a carbon dioxide - rich and oxygen - poor atmosphere)

● Inoculated an unknown organism into a liquid broth

● After 24 hours, you observe the growth..

Obligate aerobes Facultative anaerobes Obligate anaerobes Aerotolerant anaerobes Microaerophiles

Signs of growth appears at the Homogenous turbidity in the Signs of precipitation or Homogenous growth inside the Growth is seen in the
surface level of broth medium medium turbidity at the bottom of the medium sub-surface level of the
medium broth

The enzymes: superoxide dismutase, catalase, and peroxidase

Fig. The action of superoxide dismutase, catalase and peroxidase. These enzymes detoxify oxygen radicals that are inevitably generated by living systems in the
presence of O2. The distribution of these enzymes in cells determines their ability to exist in the presence of O2.

3
 ● How come aerobes and aerotolerant anaerobes survive the lethal accumulation of superoxide? and how come obligate anaerobes
can’t survive in the presence of these oxygen radicals?
○ They have enzymes that protect them
○ Nearly all organisms which can lead to the presence of oxygen contain superoxide dismutase to form superoxide into
oxygen and hydrogen peroxide
○ Nearly all bacteria contain catalysts that can breakdown hydrogen peroxide into water molecules
● On the other hand, aerotolerant bacteria like streptococci has peroxidase that can derive electrons from NADH and reduce peroxide
to water

Group Superoxide Catalase Peroxidase

dismutase

Obligate aerobes,
most facultative
anaerobes (e.g.
enterics, e.coli, + + -
enterobacter,
klebsiella)

Most aerotolerant
anaerobes (e.g.
streptococci)
+ - +
Obligate
anaerobes (e.g.
clostridia, - - -
Bacteroides)
○ Above 45C (45C - 70C)
○ Hyperthermophiles/ extreme thermophiles - (80C or higher)

Obligate anaerobes lack superoxide dismutase and catalase and/or peroxidase, and therefore undergo lethal oxidations by various
oxygen radicals when they are exposed to oxygen

2. Thermal Requirement
● Psychrophiles (cold loving)
○ 0-20 C
○ Psychrotrophs/ facultative psychrophiles
■ 0 degrees, optimum T in mesophile range
■ Food spoilage (refrigerator T)
→ grow slower inside the refrigerator
○ Psychroduric can endure very cold or freezing T
■ adaptive to cool environment by having largely unsaturated fatty acids in their plasma membranes
● Mesophiles (optimum T near 37C)
○ Where most pathogenic organisms belong
○ 20-45C
○ Most pathogenic organisms
● Thermophiles (hot loving)
○
■ Archaea
■ Even as high as 150C

3. Water
● Solvent in which the molecules of life are dissolved
○ Availability of water is therefore a critical factor that affects the growth of all cells
○ The availability of water for a cell depends upon its presence in the atmosphere = called relative humidity
○ presence in solution or substance = water activity
● Source: equilibrium relative humidity = water activity (AW)
○ Water activity is affected by the presence of solutes such as salts or sugars that are dissolved in water
● The higher the solute concentration, the lower is the A W and vice versa
● MOISTURE = Humidophiles

Water activity (AW):

Pure H2O = 1.0 (100% of water)
Microorganisms = 1.0 - 0.7
Human blood = 0.99

Water and Salinity

● Salt: only common solute in nature that occurs over a wide concentration range
○ Some microorganisms are named based on their growth response to salt
● Halophiles (osmophiles): usually found in the archea group

4
 ○ Mild halophiles = 1-6% salt
○ Moderate halophiles = 6-15% salt
○ Extreme halophiles = 15-30% salt
○ Bacteria that grow at moderate salt concentrations even though they grow best in the absence of sodium chloride
○ Vibrio cholerae
○ The term osmophile are usually reserved for organisms that are able to live in environments that are high in sugar
● Halotolerant (osmotolerant)
● Xerophiles
○ Able to live in very dry environments due to lack of water
○ The concept of lowering water activity in order to prevent bacterial growth is the basis of the preservation of food by drying
in sunlight, or by evaporation, or by addition of a high concentration of food or sugar

Movement across membranes:

● Simple diffusion
○ Movement of a solute from an area of high concentration to an area of lo concentration
● Facilitative diffusion
○ Solute combines with transporter protein in membrane
○ Needs ATP
High water concentration inside or outside can also have an effect on its growth or survival due to osmosis and osmotic pressure
● Osmosis
○ Movement of H2O fro an area of high H2O concentration to anarea of low H2O concentration
● Osmotic pressure
○ Pressure required to stop H2O movement across the membran

● If we want to grow bacteria, they should be placed in anistonic solution

● Inn a hypotonic solution, extracellular water enters the cell that may caise the cell to burst
● In a hyper tonic solution moves ooout of the celll causing it to shrink

Osmotic Pressure and Salinity

● Hypertonic environments cause plasmolyss
● Extreme halophiles need ↑ osmmottic pressure
○ Bacteria that can grow in saltwater
● Halotolerant organisms tolerate ↑ osmotic pressure

4. pH Requirement
● The acidity or alkalinity of a solution
○ Most bacteria = pH 6.5-7.5 (7.2-7.6)
○ Molds & yeasts = pH 5-g
● Acidophile (pH 2-5)
○ Bacteria that can grow well below optimum pH
● Neutrophile (pH 7)
○ Grow best in optimum pH
● Alkaliphile (pH 8-9)
○ Bacteria that can grow best onn alkaline conditions
● The pH or hydrogen ion concentration of natural environments varies from 0.5 in the most acidic solis to about 10.5 in the most
allkaline lakes
● Most free-living prokaryotes can grow over a raage of three pH units, a thousan-fold cahnge in hydrogen
● The range of pH over which an organism grows is defined by three cardinal points
○ The minimum pH which the organism cannot grow
○ The maximum pH which the organism cannot grow
○ The optimum pH at which an organism grows best
● For most bacteria, there is an orderly increase in growth rate between the minimum and optimum and corresponding orderly decrease
in growth rate between the optimum and the maximum pH reflecting the general effect of changing hydrogen ions on therats oof
enzymatic reaction
● In the use of culture meia, one must always consider the optimum pH for growth of a desired organism and incorporate buffers in
order to maintain the pH of the medium changing the leuie of bactteriaal waste productsthat accumulate during growth
● Many pathogenic bacteria exhibit a relatively narrow range of pH over which they will grow
● Most diagnostic media for the growth and iidentfication of pathogens have a pH near 7

5
Requirements for Bacterial Growth:
● Major & trace elements
● Carbon & energy sources
● Grwth fffactors
● O2, CO2
● Temperature
● Water/Moisture
● pH requirement

BACTERIAL GROWTH CURVE

● Growth: an orderly increasse in the quanttity of cellular constituents
● Binary Fission: asexual reproduction
○ Increase in cell mass and number of ribosomes
○ Duplication of bacterial chromosome
○ Cell wall & plasma membrane synthesis
○ Chromosome partitioning & septum information
○ Cell division

Each cell increases in size and divides into two cells

There is orderly increase in cellular structures and components
↓
Replication and segregation of bacterial DNA and formation of a septum or cell wall
which divides the cell into two progeny cells
↓
The process is coordinated by the bacterial membrane by means of the mesosomes
↓
The DNA molecule is attached to a point of the membrane where it is replicated
The two DNA remains attached at points side by side on the membrane while new
membrane material is synthesized between the two points
↓
These draws the DNA molecules in opposite directions while new cell wall and
menmbrane are laid on as septum between the two chromosomal components
When septum formation is complete, the cells split into two progeny cells

Generation Time
● The time interval required for a bacterial cell to divide or for a population of bacterial cells to double
● May be as short as 15 mins or as long as several days
● Will vary when bacteria are grown artificially under standard nutritional conditions for when they grow inside a host.
○ Example: the generation time of E. coli in the laboratory is 15-20mins but 12-24hrs in the intestinal tract
● doubling time
● the time it takes for an organism to double its number
● time required for a cell to divide

The increase in a bacterial population is by geometric progression

If a single bacterium reproduces every 20 minutes, how many would there be in 2 hours?

6
What if generation time is unknown?
● G = t/n
● G = 120mins/6
● G = 20 mins

● G (generation time) = (time, in minutes or hours)/n (number of generations)

● T = time interval in hours or minutes
● N = number of generations (number of times the cell population doubles during the time interval)

Try!

A pastry chef accidentally inoculated a cream pie with six Staphylococcus aureus cells. If S. aureus has a generation time of 60 minutes,
how many cells would be in the cream pie after 7 hrs?

Bacterial Growth Curve

● When a fresh medium is inoculated with a given number of cells, the population growth is monitored over a period of time and plotting
this data will yield a typical bacterial growth curve
● When bacteria are grown in a test tube, the population of cells almost always exhibits these growth dynamics
● In the first phase, cells initially adjust to the new medium, lag phase, until they can start dividing regularly by the process of binary
fission - Exponential phase.
● When their growth becomes limited, the cells stop dividing - stationary phase
● Eventually, they show loss of viability - death phase

Microbial growth:
- The increase in number of cells, not cell size

THE FOUR PHASES

A. Lag phase
- adjustment period
- Immediately after inoculation of the cells into a fresh medium, the population remains temporarily unchanged. although there
is no apparent cell division occurring, the cells may be growing in volume or mass synthesizing enzymes, proteins, rna, etc,
and increasing in metabolic activity
- The length of the lag phase is apparently dependent on a variety of factors including the size of the inoculum time necessary
to recover from physical damage or shock and transfer time required for synthesis of essential coenzymes or division factors
and time required for enzymes that are necessary to metabolize the substrate present in the medium.
B. Log phase
- cellular reproduction stage
- exponential phase
- Is a pattern of balanced growth where all the cells are dividing regularly by binary fission
- Growing by geometric progression
- The cells divide at a constant rate, depending upon the composition of the closed medium and the conditions of incubation
C. Stationary/Plateau phase
- Pop density high
- microbes dividing = microbes dying
- Population growth is limited by one of three factors
7
 1. Exhaustion of available nutrients
2. Accumulation of inhibitory metabolites or end products that may be toxic
3. Lack of biological space
- Viable cells are being counted
- Spore forming bacteria have to induce or unmask the activity of dozens of genes that may be involved in sporulation process
D. Death phase
- Dead/dying microbes > new cells
- decline continues until there is complete cessation of reproduction
- Note that we are counting bacteria by turbulent measurements or microscopic counts, the death phase cannot be observed

What causes exponential growth to stop?

● Exhaustion of nutrients
● Waste Product Accumulation
● Toxin Production
● Harmful pH change

Culture Media
● Provides the appropriate biochemical and biophysical environment for microbial growth
● Inoculum – introduced
● Culture - growing
○ Pure Culture – 1 species
○ Colony – macroscopic visible masses of growth arising from a single cell

TYPES OF CULTURE MEDIA

According to consistency:
● Liquids media
○ No agar
○ Used for growth of pure batch cultures

● Semi - solid media

○ 0.3-0.5% agar

● Solid media
○ 1-5% agar
○ Used widely for the isolation of pure cultures
○ For estimating viable bacterial populations and variety of other

● Agar: solidifying agent

○ a hydrocolor derived from red algae
○ Used because of its unique physical properties
○ Melts in 100 degrees and remains liquid until 40 degrees (the temperature at which it gels)
○ Because it cannot be metabolized by most bacteria, the medium component is relatively inert (holds nutrients that are in an
aqueous solution)

According to Composition:
● Synthetic medium / chemically defined
○ Use: studying the minimal nutritional requirements of microorganisms, enrichment cultures, physiological studies.
○ Exact chemical composition is known
● Non-synthetic medium / complex media
○ undefined & defined (minimal media)
■ Defined media are usually composed of pure biochemicals of
■ Complex media usually contain complex materials of biological origin
■ Undefined media is a minimal medium since it provides only the exact nutrients
○ Contains: blood, milk, yeast or beef extracts
■ Exact chemical composition is undetermined
○ provides a full range of growth factors needed by an organism for cultivation of bacterial pathogens & fastidious bacteria.
■ The use of defined media, requires the investigator to know the exact nutritional requirement of the organism in
question

According to Function/Purpose of Medium:

General purpose Nutrient Agar

8
 Enriched plate Blood Agar Plate (BAP),
Chocolate Agar Plate (CAP)
Enrichment broth TSB, Selenite F
Selective Eosin Methylene Blue (EMB),
MacConkey
Differential EMB, Mac, BAP
Anaerobic growth Thayer-Martin
Specimen transport Stuart’s
Assay Antibiotic medium
Enumeration Potato Dextrose Agar (PDA),
Blood agar (BA)
● Nutrient Agar - Can grow almost all kinds of bacteria
● Enriched plate - If bacteria requires blood or blood components for growth
○ Chocolate Agar Plate (CAP)
■ If the blood is heated, it’s color will turn a chocolate brown color
● Enrichment broth
○ Selenite F broth
■ For the growth of Salmonella or Shigella bacteria in a stool specimen
○ Tryptic soy broth
■ Enhance growth of bacteria in body fluids specimen
● Selective culture media
○ Why selective? Only gram negative bacteria can grow on this media due to the addition of dyes and bile salts (which the
gram positive bacteria are very sensitive to)
○ EMB and MacConkey agar can distinguish lactose and non-lactose fermenters
○ In function, it is also called as Differential media
● In food microbiology, they do a lot of anaerobic culture and use Thioglycollate broth (or cooked meat broth)
● When you want to diagnose a gonorrheal disease, specimens are inoculated into special medium like the Thayer-Martin medium
○ Can only grow Glyceria species and related species
○ Prevent the growth of other microbes

A particular culture medium can function both as selective and differential

Selective Media & Differential Media

● Suppress unwanted microbes
● Encourage desired microbes
● Differentials metabolic activities of the gram-negative bacilli
● Example: Eosin Methylene Blue and Salmonella Shigella Agar

● Top pic: green metallic sheen is seen in E.coli

○ Differentiate E. coli from the rest of the enteric bacteria
○ EMB inhibits the growth of other gram positive bacteria
○ Differential: the pink or red-colored colonies indicate lactose fermenters (if non-lactose fermenters = colorless)
● Bottom pic: black dots seen in S. typhi
○ Only select species that are not gram positive or other coliform bacteria
○ Black dots mean that the organism can produce Hydrogen sulfide

Enriched and Differential Media

● Blood enriched
○ Differential in purpose it can showcase 3 types of hemolytic pattern
○ gamma hemolytic- will not exhibit any hemolytic pattern
○ Alpha hemolytic bacteria- greenish discoloration surrounding the colonies.
■ It means there's only partial hemolysis of the RBC

9
 - Pic above: complete hemolysis by S. aureus.
- Beta hemolytic pattern
● Aids in distinguishing colonies of different microbes.

Enrichment media
● Encourages growth of desired microbe (salmonella) from fecal specimens.
● Inhibits coliforms and fecal streptococci.
● Selenite F
○ Very good enrichment medium

Streak Plate

● We streak for the purpose to isolate the organisms.

● Left: Tertiary: first, second, third streak
● Right: first- mixed growth
● Second: closely positioned growth
● Third: colonies- isolation
○ These isolated colonies belong to a single class of species.
● You can already estimate the growth.
○ Since the growth reached the tertiary streak then we can say that it is a heavy growth.
Preserving Bacterial Cultures
● Deep-freezing: -50°to -95°C
● Lyophilization: Frozen (-54° to -72°C) & dehydrated in a vacuum.

Anaerobic Culture methods

● Not all bacteria can grow in the presence of oxygen
● They're actually killed in the presence of oxygen.

● If you don't have this chamber u really need to work very fast and place them in a gas pack system (?).
● Palladium catalyst
○ Enhance the action of the sachet containing sodium bicarbonate and sodium borohydride.
○ This will mix with oxygen present in this jar.
● There is an indicator if there is still oxygen or not- color indicator.

Carbon dioxide & Anaerobic culture methods

10
 ● There are bacteria that are microaerophilic and at the same time capnophilic.
● They require low oxygen tension but high carbon dioxide concentration in the environment.
● Ex: Strep. Pneumoniae

Types of Colonies
- We are describing the colonies based on the consistency.
● Mucoid colony
○ Slimy, sticky
○ They tend to produce capsules
● Smooth colony
● Rough colony

Measurement of Cell Numbers

● When we talk about bacterial growth, we don't really mean growth in terms of size but in terms of numbers.
1. Direct microscopic counts
a. Counting chambers- u will have to count it by using the microscope
2. Electronic counting chambers.
3. Indirect viable cell counts.

11
 BACTERIOLOGY

LECTURE 3: BACTERIAL GENETICS

Prof. Hinay
Aug 23, 2021
For updates and corrections → @mar4rii on Twitter

OVERVIEW ● The chromosome is looped and folded and attached at

● Why study genetics? one or several points to the plasma membrane
○ As scientist, we are interested in how traits are ● DNA = double stranded
expressed within a cell and how traits ○ Double helix with chain of nucleotides from 5’ to
determine the characteristics of an organism 3’ and are antiparallel
○ there are thousands of genes necessary for an ○ There is complementary base pairing
organism to grow and flourish ■ A-T = Adenine to Thymine
● Genes = made up of DNA segments ■ G-C = Guanine to Cytosine
○ The traits and characteristics of organisms are ○ Between these nitrogenous bases, are the
encoded in these dna segments deoxyribose and phosphate group bonds
● DNA = (Deoxyribonucleic acid)
● The genetic material of all cellular organisms from
bacteria to humans
● Basic genetic phenomena (the same for all life forms)
○ gene mutation
○ gene replication - dna is obtained either from
another cell in the same generation or from a
parent cell during cell division or binary fission
○ gene recombination
● The is always gene expression as DNA is the blueprint
for a cell’s proteins, enzymes, and others
● At times, spontaneous mutations can occur in gene
replication
● DNA can be expressed within a cell or transferred to
another cell through recombination and replication

● The chromosome
○ Bacteria = has only 1 chromosome
○ single, long piece of circular, double-stranded
DNA
○ Contains 2000 to 4000 genes

● Escherichia coli - prototypic microorganism for the past

50 years
● E. coli - first bacterium in which the entire genome has
been decoded
● The DNA of E.coli has about 4.6 million base pairs and is
about 1 millimeter long
● 1 thousand time longer than the entire cell
● However, the chromosome take sup only about ten
percent of the cells volume because the DNA is twisted
or coiled
BACTERIAL GENOME
● Has a single circular chromosome consisting of a single
circular molecule of DNA with associated proteins
● The totality of its genetic material
● Consists of a single chromosome
○ carries all of the essential genes and
■ Essential = needed for growth
○ 1 or more varieties of plasmid (generally carry
nonessential genes) ● Bacterial replication follows the semiconservative
■ Non-essential - not needed for growth replication fashion
and metabolism ○ One strand is the original or the conserved
strand and the other one is a new strand

1
 ● Small segments of DNA that can move or be transposed
from one region of DNA molecule to another
● 700-40,000 base pairs long
● The simplest transposons are actually enzymes called
insertion sequences and they contain only a gene that
codes for the enzyme transposase
○ Catalyzes the cutting and resealing of DNA that
occurs in transposition and recognition sites
● the repository for many antibiotic resistance genes
● responsible for the ability of some plasmids to integrate
into the chromosome

“Mobile Genetic Elements”

Barbara McClintock Nobel Prize Winner in Physiology or
Medicine (1983)
● Discovered transposons in corn but they occur in
organisms and have been studied most thoroughly in
microorganisms. They move from one side to another
side on the same chromosome, or to another
chromosome or plasmid
● Transposons may insert themselves within genes and
(Example of genetic math of E.coli) they can actually inactivate certain genes in the process
● Numbers inside = number of minutes it takes to transfer ● Transposition occurs relatively rarely
the genes during mating between to cells ● The frequency of transposition is comparable to the
● Colored boxes = indicate the number of kilo base pair spontaneous mutation rate that occurs in bacteria and
● 1 kbp = 1000 base pairs that is 10th to the 5th, 10th to the 7th per generation
● All transposons contain the information for their own
Plasmids transposition
● Extra chromosomal gene
● Small DNA circles
● range from 1.5 kb pairs to 120 kb pairs (1/10)
● Replicate independently (1 or more) from the cell’s
chromosome
● 1.5% of the size of the bacterial chromosome
● However, plasmids differ from bacterial chromosomes in
that the genes they carry are not essential for the growth
of cell under normal conditions
● Genes for toxins, proteins that promote transfer,
enzymes
○ Code for toxins ex:
■ Exfoliating toxin = Staphylococcus
aureus
■ Neurotoxin = Clostridium tetani
■ Toxin of bacillus anthracis
● Resistance (R) factors, useful markers in the
identification of bacteria
○ Researchers discovered that certain bacteria
have acquire resistance through the spread of
genes from one organism to another Bacteriophage
○ Plasmids that mediated this transfer are the R
factors
■ Carry genes that confer upon their
host cell resistance to antibiotics,
heavy metals, or cellular toxins
■ Plasmids are also responsible for
conjugation (transmissible between
cells during conjugation)
■ F factor - structure/ factor that is ● A virus that replicates inside a bacterial cell
responsible for conjugation ● Either has an RNA or DNA genome and they are
● F factor (conjugative plasmid) carries genes for sex pili encapsulated in a protective protein code
○ Transfer of plasmid to another cell ● Bacteriophage usually attach to a receptor site that is
○ They can insert into the chromosome or into part of the cell wall of the bacterial host. In other cases, it
another plasmid, or replicate the entire plasmid is part of the fimbriae or flagella
and do the process of conjugation (transfer the ● Since it kills bacteria, scientists are taking interest in the
genes through the sex pilus on to another cell) potential to use these viruses to treat diseases due to
● Important tool in genetic engineering their narrow host range and their ability to kill their host
○ What genes are responsible in the transposing cells - phage therapy
of one to another, inserting one gene to the ● Consists of a nucleic acid encapsulated in a protective
chromosome and to another plasmid? The protein coat
Transposons ● DNA or RNA, single/double stranded (3000-200,000
● Transposons bases)
Transposons
● mobile DNA sequences, “jumping genes”
● move between plasmids & between plasmids & the
chromosome

2
 Corynebacterium diphtheriae exotoxin found in the DNA of Beta
phage
➢ Majority of the commensals of the respiratory tract are
TYPES OF PHAGES
actually non-pathogenic Corynebacterium diphtheriae
● Virulent phage (T-phages)
➢ But after being lysogenized by the Beta phage, it can
○ (T-phages: T1-T7)
then produce exotoxins (encoded in the DNA of the beta
○ Infection results in the death of the cell by lysis
phage)
○ 1 phage produces 100 progeny in 20 minutes
➢ There is a stable association between the viral gene and
● Temperate phage (phage lambda)
the host’s cell genome but it can also be destabilize
○ (Bacteriophage Lambda)
○ Choose between a lytic & lysogenic pathway of
Plaque Assay
development
● Used for isolation and enumeration of phage particles
● Lysogenic bacteria
● The specimen suspected for phages is introduced into a
few millimeters of soft agar along with some bacterial
Bacteriophage Replication: Lytic & Lysogenic host cells
● This soft agar mixture is laid over a hard agar base or
seeded agar overnight
● After the period of incubation, the phages lyse the
bacteria cells in their vicinity resulting in zones of clearing
on the plate, which is known as PLAQUES
● Each plaque represents the single patch particle in the
original sample

- Bacteriophages start their life cycle when they absorb to a

permissive host???
- After injecting their genome into the host’s cell, they
reproduce a set of early proteins and a few copies of their
genome Recall
- On this stage, a decision about lysis vs lysogenic is made ● Bacterial genome
- Usually, in poor growth conditions of the host cell, a phage ● Plasmids and transposons
chooses the lysogenic pathway ● Bacteriophages
○ Because the number of progeny it can produce in such ○ Virulent and temperate phage
cell is usually low ○ Lytic and lysogenic infections
○ When lysogenic is chosen, the phage integrates its
genetic material with the host’s cells How do bacteria transfer gene?
○ It may be done by physical incorporation of the phage
into the host’s genome or the prophage may be GENETIC TRANSFER
integrated as stably-maintained plasmid
- When a prophage is induced, it starts to produce viral proteins Vertical Gene Transfer
and copies the virus’ genome using bacterial resources and ● Mother cell to offspring
biosynthetic apparatus ● Binary fission
- So, progeny virus particles are formed and after completing
the cycle, release during host cell lysis Horizontal Gene Transfer
● Transformation
● Lysogenic bacteria ● Conjugation
○ Carry a prophage (lysogenized infection) ● Transduction
○ Stable association but destabilized by various
treatments (exposure to UV → repression lifted CHANGE = SURVIVE
→ follows the lytic type of replication) ● Survival of their species
○ Lysogenic conversion (acquisition by bacteria ● Exchange of genetic material allows for the sharing of
of properties due to the presence of a genes that code for PROTEINS such as those that
prophage) provide:
○ Antibiotic resistance

3
 ○ Exotoxin ● Then, the daughter cell lysis and releases phage
○ Enzymes particles containing bacterial DNA
○ Other virulent factors (pili, flagella & capsules) ● A phage carrying bacterial DNA infects a new host cell
● Recombination can occur producing a recombinant cell
3 Distinct Mechanisms: with a genotype different from both the donor and
1. Conjugation recipient cells
● F positive plasmid ● In generalized transduction, any bacterial DNA can be
● cell-to-cell contact transferred from one cell to another
● Donor and recipient cells
● Requires the sex pili
2. Transduction
● Via a phage vector
● Generalized and specialized
3. Transformation
● Means of naked DNA (genetic material)
● Competence = the ability of a cell to be
transformed

CONJUGATION
● During the process, genetic material is transferred into
another bacterium through the mating bridge
● Pili are often incorrectly believed to transfer DNA, but
these structures are simply used to bring mating bacteria
close enough to form a mating bridge
● To form a pili, an F plasmid is required
○ Consists of 28 genes which are mostly required
for the production of the virus
○ F positive denotes cell that contain the F In specialized transduction, bacterium undergoes lysogenic
plasmid while F negative cells do not pathway.
○ F plasmid are considered also as episome ● Prophage is integrated between gene A and B.
which may become integrated into the main ● Occasionally, prophage DNA exits incorrectly, taking
chromosome adjoining bacterial DNA with it
● When the F genes became integrated into the ● Assembled in a phage capsid
chromosome, the cell is said to be HFR (High Frequency ● Phage particles carry bacterial DNA (here, gene A) along
Recombination) with phage DNA
● In infecting other bacterial cells, gene A can now be
transferred.
● In the lipid (?) pathway, general transduction by (?) can
occur
● In the lysogenic pathway, is specialized transduction.

TRANSFORMATION

TRANSDUCTION

Generalized transduction
● A phage infects a donor bacteria cell
● The phage DNA and proteins are made and the
bacterium chromosome is broken into pieces
● Occasionally, during phage assembly, pieces of bacterial
DNA are packaged in a phage capsid

4
 ● When there are (?) DNA (?) with closely related bacterial
cells, transformation can occur. GENE REGULATION
● Fragments of DNA can be taken up by the competent
cells and the DNA fragments can now be integrated into
the recipient cells chromosome resulting to a transformed
cell.

A. Negative control (repression)

● Initial experiment on transformation was performed by
B. Positive control (catabolite activation)
Frederick Griffith in England in 1988 when he was
a. Regulate the transcription of mRNA and
working with 2 strains of Strep. Pneumonia
consequently the synthesis of enzymes.
● Griffith was interested in determining whether injections
b. The process that turn on the transcription of a
of heat killed the bacteria of encapsulated strain could be
gene is INDUCTION.
used to vaccinate mice against pneumonia.
c. Inducer - a substance that acts to induce
● As he expected, injections of living encapsulated bacteria
transcription of a gene
killed the mouse
d. Inducible enzymes - enzymes that are
● Injection of NON or dead encapsulated bacteria did kill
synthesized in the presence of inducers
the mouse.
e. Genes required for lactose metabolism in E.coli
● When encapsulated bacteria were mixed live non
are well known examples of an inducible
encapsulated bacteria and injected into the mice, many
system.
of the mice died.
f. One of these genes code for the enzyme beta
● In the blood of the dead mice, Griffith found living
galactosidase which splits the substrate lactose
encapsulated bacteria
into two simple sugars: glucose and galactose.
● Hereditary material from the dead bacteria had entered
g. If E coli is placed in intramedium in which no
the live cells and changed them genetically so that their
lactose is present, organisms contain almost no
progene(?) were encapsulated and therefore virulent.
beta galactosidase. However, when lactose is
Examples:
added to the medium, bacterial cells produce a
● Corynebacterium diphtheriae
large quantity of enzyme.
● Streptococcus pneumoniae
h. Lactose→ related compound allolactose which
● Clostridium botulinum
is the inducer for these genes.
● Vibrio cholerae
i. Presence of lactose thus indirectly induces cells
to synthesize more enzymes.
GENETIC VARIATION
j. Repression- the regulatory mechanism that
A. Mutation
inhibits gene expression and decreases the
● Stable, some unstable
synthesis of enzymes.
● Any change in the structure of genetic material → any
i. Usually a response to over
change in the base sequence of DNA
abundance of the end product of a
https://www.youtube.com/watch?v=zjR6L38yReE
metabolic pathway.
B. Mobile genetic elements
ii. Causes a decrease in the way of the
● Transposons = DNA segments that have the ability to
synthesis of the enzyme leading to
move from place to place on the chromosome and into
the formation of that product.
and out of plasmids
iii. Mediated by regulatory proteins called
● Replicative and nonreplicative
repressors which block the RNA
● Probably responsible for most of the genetic variability &
polymerase to initiate transcription
the spread of antibiotic resistance genes
from the repressed genes.
C. Mechanisms of acquired antibiotic resistance
C. Modifications of RNA polymerase specificity
1. Decreased uptake (or increased influx) of antibiotic or
Induction
EFFLUX
a. Efflux- the pumping out of the drugs across the
cell surface
2. Alteration of the target site for antibiotics.
a. Ex: alteration of penicillin binding protein which
is a binding target site of penicillin.
3. Acquisition of the ability to destroy or modify the antibiotic
a. Drug inactivation or modification
i. Ex: enzymatic deactivation of
penicillin G in some penicillin resistant
bacteria were the production of beta
lactamases
https://www.youtube.com/watch?v=_K_D1Qz3Zu

5
 BACTERIOLOGY
LECTURE 4: SPECIMEN COLLECTION
Prof. Joanne Krystine Tago, RMT
Aug 23, 2021
For updates and corrections → @mar4rii on Twitter
● 3 phases:
○ Preanalytical phase
■ Specimen collection
● Patient identification,
preparation, specimen
collection, labelling.
Transportation, storage
○ Analytical phase
○ Postanalytical phase
● Proper specimen collection is essential to provide
accurate and appropriate results
● Failure to properly collect specimens for culture may
result in the failure to isolate the organism
● Failure for doing the processes correctly can lead to
incorrect treatment for patients
● The recovery of contaminants or normal microbiota may
lead to inappropriate or unnecessary antibiotic treatment
GENERAL SPECIMEN COLLECTION GUIDELINES
● The laboratory must establish a criteria for specimen
collection and for injecting specimens that do not need to
meet the requirements
● Ensure that during the pre-analytical phase, everything is
done properly so that the results will be precise
● Ex. A patient presents characteristic signs and
symptoms of his condition to his physician
○ Clinician will provide a presumptive diagnosis
and would request specific tests
■ Type of specimen that we would
collect would depend on the types of
tests that were requested
● The samples that will be collected through the different
methods of identifying the microbial agent present in it
○ Microbial morphology
○ Culture = provide us the genes of the organism
○ Biochemical test= provide the specie
○ Serological tests
○ Genomics = to identify the organisms
● After the tests, we are able to come up with a definitive
diagnosis/causative agent
● Furthermore, we can perform antimicrobial susceptibility
testing to be able to identify antimicrobial drugs that will
be best used to control or kill the organisms
● All information will be gathered to utilize by the clinician
to come up with the ideal treatment for the patient
● Appropriate specimen management is critical to ensure
laboratory effectiveness and to achieve an acceptable
turnaround time of results
● Obtain specimen before treatment;
○ Specimen for culture must be collected properly
prior to the initiation of any antimicrobial
therapy
■ To ensure optimal conditions for the
recovery of the pathogens in the
specimen
■ Because this antibiotics may inhibit
growth of pathogens and may result
to a negative culture even if the
patient has the infection
■ However, there are times where
immediate treatment is needed
● The clinician should note the
name/identify the antibiotics
prescribed for the patient
prior to collection
● Collect material from the appropriate site and avoid
contamination.
○ Sometimes it is not practical to collect samples
at the site of infection
■ ex. Bacterial endocarditis
● Require invasive type of
procedure
● Make use of blood samples
to culture
○ Contamination
■ Avoid mixing samples
■ Avoid areas rich in normal microbiota
(may overgrow the pathogens -> lead
to inaccurate findings)
1
 ■ Materials used should be sterile
■ Site for collection has been cleaned
properly
● 70% alcohol
● Antiseptic (povidone iodine
or chlorhexidine)
● Obtain material during the acute stage of the illness and
ensure correct timing of specimen collection;
○ Collect ASAP or after the onset symptoms for
proper recovery of the etiologic agent of
infection
■ Enteric pathogens associated w/
gastroenteritis = efficiently culture
when the patient is in the acute
diarrheal stage
■ Causative agent may be recovered
from various sites at different times
during the course of the illness
● Ex. Salmonella typhi =
typhoid fever
● First indeed the bloodstream
so it can be isolated from
the blood
○ 1st week = wise to
collect blood
samples
○ 2-3 week = highest
yield is in the urine
and feces
● As organism start to
traverse into the GIT via the
bloodstream
○ 5th week = san
collect serum
samples to use to
determine
antibody titer of
the px against the
infection
● Collect using sterile collection method;
○ Ensure to apply aseptic technique during
collection
■ 70% alcohol
■ Antiseptic ● Nowadays, most labs have computerized accessioning
■ 2% chlorhexidine and 70% alcohol systems that would provide the accessioning number for
○ Check containers for cracks and ill-fitting each specimen (barcones, qr codes)
covers, broken seals bc it might lead to leakage ○ Sticker type (has proper manner)
○ Recommended to make use of disposable or ○ Accessioning number should be found near the
one-time use instruments cap (right side up) (vertical)
● Collect sufficient quantity ○ Proper coloring for the type of test needed
○ To be able to perform all procedures needed ■ red/green - hematology
○ If the specimen volume is inadequate, it will be ■ Gold - bacte
marked “Q and S” = quantity not sufficient
■ Not thrown = hold on to sample and
ask clinician if anything as to be done
on the sample, if non u can discard
and ask repeat collection
■ Some are easily collected and some
would require paperwork and
incidence reports if a recollection is
needed ex. Invasive procedures (CSF,
synovial, biopsy)
○ Important to communicate with the physician

SPECIMEN LABELLING
● Label
○ Patient’s full name
○ Identification number of the patient
○ the source of specimen
■ Where
○ date and time of collection ● Labels should NOT be wrinkled
○ Collector’s signature ○ Cannot be read properly by the machine
■ Help in tracing if there are problems ● Check ink
due to improper collection ● Should not be wet or stained
● Information should be similar in the container and ● Upside down
request form ● Wrong color of tube used for collection
2
 SPECIMEN TRANSPORT GUIDELINES ● Secondary receptacle is placed inside a rigid
● All specimen must be sent to the laboratory on the day of outer packaging
collection with as little delay as possible. ○ A box made of rigid carton
○ ASAP!!!! ○ You can find the infectious substance
● Bacteria are vulnerable to delay in processing label
Delay In Transport & Processing ○ Proper shipping name
● REFRIGERATION ○ UN Number
○ Ex: URINE → refrigerate (24 hrs) ○ UN package certification mark
● TRANSPORT MEDIUM ○ Shipper or consignee identification
○ maintain viability of bacteria and slows down all
3 processes Shipping Labels
■ Growing
■ Reproducing
■ Dying
○ Nutritionally poor; enough to allow sufficient
survival of organisms
○ Not for prolonged storage

Transport Of Specimen (Referral Testing)

● Volume: must not exceed 40 ml
○ bacteria & fungi → tubed solid media
● Packing: leak-proof, tightly sealed containers with
Biohazard label
○ DOH Manual on Packaging & Transport of
● Who it is from, who is the recipient
Laboratory Specimen for Referral
● Address, cell phone numbers
○ IATA’s Dangerous Goods Shipping Guidelines
● Content
■ IATA = International air transport
● UN Number - 2814
association
○ Usually are known infectious agents
● Paperworks: itemized content
○ Identified
○ Second package
○ Category A
○ Outer covering
● Category B - 3373
■ Where did the sample come from?
○ Patient samples
■ Contact person
○ Suspected to be positive for a certain infectious
■ Who is going to receive it?
agent
■ What type of specimen
● UN Certification Mark
● Infectious substance label
Packaging Infectious Substances for Shipping
● Triple packaging system
CRITICAL TIME DELIVERY OF SPECIMEN IN THE
MICROBIOLOGY LABORATORY
SPECIMEN DELIVERY TIME
● Respiratory 1 hour
● Gastrointestinal 1 hour
● Blood culture 1 hour
● Anaerobic 30 mins
● CSF Immediately
● Other fluids (synovial, Immediately
peritoneal)
● Wound, skin & soft 30 mins
tissue
→ swabs dry out easily
● Urine 1 hour
● Fungal 1 hour
● Mycobacterial 1 hour
● Chlamydia 1 hour
→ Except for CSF, body fluids specimens should be delivered
within 30 mins - 1 hr to the lab
● Triple because the primary container is the vial
or cultured tube CRITERIA FOR SPECIMEN REJECTION
● If there's liquid or solid media, it's usually 1. Unlabelled specimen
protected by an absorbent packing material ● Incomplete, unmatched, no label
● It prevents the specimen form moving around ● Labels and information on the request form
or banging the walls of the second container does not match = no culture
● Immobilized by absorbent packing material 2. Specimen exceeding the allowable time for submission
● Together with the absorbent packing material, it 3. Specimens inoculated on agar plates that have dried out
is inserted in a secondary vessel that has a cap or are outdated
○ Itemized list of what's inside 4. Inappropriate transport or storage of specimen
○ Can be made of tin or other material ● Samples placed in syringes
● Primary receptacle can be glass, metal or ○ There are samples that are allowed to
plastic be delivered in a syringe (ex.
○ Ideal to use plastic tubes with screw Peritoneal fluid, synovial fluid)
caps ● If to deliver other samples like urine, etc. in a
syringe = not processed

3
 5. Improper container or damaged, leaking container CLINICAL SPECIMEN
6. Insufficient sample volume ● Blood
7. Obvious contamination of sample ● Respiratory Tract
● Dirty request form ○ Upper
8. Any specimen received in formalin ○ Lower
9. Incorrectly collected sample ● Urine
● Cerebrospinal fluid
Rejected Specimen: Anaerobic Culture ● Gastrointestinal tract
1. Gastric washings ● Genital tract
2. Voided urine ● Wound and abscesses
● Urine could be gathered using suprapubic
aspirate 1. Blood
3. Stool ● Collection and inoculation of blood in a culture medium
● If it's for the recovery of Clostridium difficile, it with the aim of growing pathogenic organisms for
could be accepted diagnostic purposes
4. Oropharyngeal ● Blood culture
● Except if sample submitted is deep tissue ○ Determines bacteremia, fungemia and
samples that were obtained during surgery or septicemia
biopsy ○ Bacteremia - presence of bacteria in the
5. Sputum bloodstream
● Contaminated already ○ Fungemia - presence of fungal elements in the
6. Swabs of ileostomy or colostomy sites bloodstream
7. Superficial skin specimen ○ Septicemia - presence of organisms that
● Accepts deep skin layer samples produced toxins
● Purpose:
○ Confirms the infectious etiology
○ Identify the etiologic agent
○ Guide antimicrobial therapy
● Factors that affect recovery of etiologic agent in the blood
○ Specimen collection
■ Time of collection, contamination, vol.
of blood, blood and (?) ratio, and the
number of blood cultures that we
need to collect
○ Culture medium
■ Type of culture media used, inhibitory
agents that may be present that may
affect the growth
○ Incubation period
■ Temp, oxygen, carbon dioxide
■ Fastidious organisms that require
● These can be accepted for aerobic cultures
additional nutrients or special
○ In these processes the organism has been
nutrients to grow successfully in an
exposed to air
artificial environment
● Study about the contaminants commonly found in blood
UNIVERSAL PRECAUTIONS
entitled “Updated Review of Blood Culture
● Consider all human samples infectious
Contamination” by Keri K. Hall and Jason A. Lyman
● Must be followed when handling all specimens
● Frequent blood culture contaminants but also true
● Appropriate barriers (PPE)
pathogens: (disease causing organisms)
○ Gloves
○ Coagulase-negative staphylococci
○ Lab coat
○ viridans Streptococci
○ Masks
○ Corynebacteria
○ Goggles
○ Bacillus species
○ Impermeable gowns or aprons
○ Propionibacterium species
○ For full PPE. face shield added
● Volume of blood:
○ Enhanced PPE, full suit, airmask
○ 1 set = 2 tubes
■ Laboratories with Biosafety Level IV
■ Aerobic (blue cap) and anaerobic (red
■ Process highly infectious agents, ex.
cap)
Ebola virus
○ 20 - 60 mL (adult)
■ Hazmat suits, oxygen tanks
■ Adults have very low colony-forming
● Biological safety cabinet
units in their blood
■ Very low number of organisms can be
isolated in adult patients
■ The more blood samples, the more
chances of isolating the organism
○ 5 - 10 mL (pediatric patient)
■ 1- 2 mL (neonates)
■ 2 - 3 mL (1 month – 2 years old)
■ 3 – 5 mL (children below 10 y.o)

4
 Samples for Rejection
● Clotted specimens
● Specimen collected using only alcohol as antiseptic
● For adults: less than 20mL of blood sample

Blood to Broth Ratio

● Recommend - 1:10 ratio
○ For traditional manual culture
● Dilute the antibacterial and inhibitory substances in the
blood
● Commercial Continuously Monitoring Blood CUlture
System may use smaller blood to broth ratio of less than
1:5

Media Used Isolate agents causing:

Glucose broth
Bile broth
Trypticase Soy broth (TSB)
● Number of blood culture sets
Brain Heart Infusion broth
Thioglycolate broth
Columbia or Brucella Broth
Endocarditis
Enteric fever
● Nutrient broth
● Not used for Neisseria and
Streptococcus pneumoniae
because these organisms
will be inhibited
Common enrichment media
Anaerobes
Brucella

Sodium Polyanethol Sulfonate (0.025 to 0.03%)

● Anticoagulant and Anticomplementary action
○ Neutralizes bactericidal effect of
serum
○ 2 sets = 4 bottles (2 aerobic, 2 anaerobic) ○ Prevents phagocytosis
○ 3 stes = 6 bottles (3 aerobic, 3 anaerobic) ○ Inactivates certain antimicrobial
○ Technically, we should collect blood samples agents
when px has a fever or before the px’s ■ Gentamycin, galamycin,
temperature spikes but it is difficult to time streptomycin, and polymixin
○ Look for feverish symptoms instead b
○ After collection, blood is incubated and checked ● Downside of SPS
every 24-48 hours ○ Toxic to certain fastidious organisms
■ Neisseria
■ Mycoplasma
■ Petostreptococcus
■ Streptobacillus moniliformis
○ 1-2% Gelatin
○ Penicillinase
■ Inactivates penicillin
○ Resin / charcoal
■ Found in the bottom of
culture bottles
○ Osmotic stabilizer
● BacT/ALERT FA = charcoal additive
● BacT/ALERT FA+ and FN+ = resin/ polymeric resins

Incubation: Temperature and Length

● Temperature: 35- 37°C
● Subculture on BAP & CA: after 14-17 hours, 3rd /5th
/7tth day
● 3-5 days is sufficient
● Not more than 7 days; > 5days → contamination
● Sensitivity of Blood Culture Sets ● 7 days is useful for:
○ The higher the volume of blood sample ○ Fungemia
culture, the higher the chances of isolating the ○ Bacteremia (HACEK group, Brucella,
organisms in the blood Legionella)
■ HACEK: Haemophilus species,
Aggregatibacter species,
Cardiobacterium hominis, Eikenella
corrodens, and Kingella species
■ Slow-growing bacteria
○ Mycobacterial culture > 4 weeks

BLOOD CULTURE METHODS

Traditional/Conventional Systems
5
 ● The purpose of studying traditional methods is to be able
to continue lab work when the machines are broken
down
● Manual Examination Bacterial Growth
○ Turbidity
○ Hemolysis
○ Gas production
○ Pellicle formation or discrete colonies on the
surface
Commercially Developed System
Biphasic Septicheck System
○ Two-in-one system: solid media and a broth
media
Oxoid Signal Broth Displacement System
○ Has a cylindrical signal device attached to the
main bottle
○ When organisms are present in the main bottle,
gas will produce and will cause some of the
blood mixture to go up into the cylinder
○ The presence of blood in the cylinder indicates
positive blood culture
● Haemophilus influenzae type B
● Neisseria gonorrheae
Asymptomatic carriers :
● S. aureus
● S. pneumoniae
● M. catarrhalis
● B. pertussis
● N. meningitidis
Throat Swabbing
- With the use of tongue depressor and sterile swab
- Recommended swabs used should be calcium alginate
and not cotton swabs since cotton swabs are known to
produce fatty acids that might affect the viability of
organisms
- To collect, simply rub the swab across the consular area
and the posterior pharynx targeting inflamed areas
- Try not to touch the roof of the mouth as well as the inner
cheeks and the tongue
MEDIA for URT specimen:
● S. pyogenes: Swab ➞ BAP
● H. influenzae: CAP or BAP w/ Staphylococcus
○ Reacts with staphylococcus
● N. meningitidis: CAP/Thayer Martin
● B. pertussis: Charcoal Cephalexin

Nasopharyngeal Secretion
- Aspirate is more superior but swab is easier to do
Swab use:
● 2 Sterile swabs
● Ca alginate swabs
Aspiration:
● mucus extractor connected an NGT
Automated Detection System
● Three most commonly used systems are: B. Lower Respiratory Tract
○ BACTEC 9240/ 9120/ 9050 ● Sputum
■ Detects positive bacterial growth by ● Endotracheal aspirate
detecting carbon dioxide through ● Bronchoalveolar Lavage
using spectral light ● Bronchoscopy secretions
■ Same as BAC T/alert except that it ○ Collected with the use of a
uses fluorescent, rather than spectral bronchoscope
light to detect changes in the conc of
CO2 PATHOGENS OF THE LRT:
○ BAC T/Alert ● S. pneumoniae
■ Organism grow ➞ CO2 is liberated ➞ ● Enterobacteriaceae
sensed by CO2 sensitive chemicals➞ ○ E. coli
The CO2 lowers the pH of medium ➞ ○ Klebsiella spp
produce a color change in the sensor ○ Serratia species
➞ ALERT (Positive) ● P. aeruginosa
○ TREK ESP culture system ● S. aureus
● Alerts when the culture is positive ● Legionella spp.
● Gram stain & subculture follows ● Mycobacterium spp.
● Mycoplasma spp.
Advantages & Disadvantages of Continuous Monitoring
Systems: Collection & Transport of Sputum
● Advantages: ● Use a dry, wide-mouth bottle (screw cap)
○ Decrease the laboratory work ● Collect sample early morning (purulent)
○ Decrease the pseudo bacteremia due to ● Transport ASAP
decrease in sampling and handling ● may be refrigerated but examined w/in 2-3hrs
○ Increase in the speed of detection and rate of
recovery
● Disadvantages:
○ Requires an instrument
○ Limited selection of medium
○ Expensive

2. RESPIRATORY TRACT SPECIMENS - To check whether the samples are ideal, they should oir
A. Upper Respiratory Tract can be:
● Throat Swab - Mucoid
● Nasopharyngeal swab/aspirate - Purulent (best)
- Blood stained
PATHOGENS OF THE URT : - Discard samples with saliva
● S. pyogenes
● C. diphtheriae

6
 - There are some sputum samples that appear to be ○ Early morning
watery like induced sputum samples (should be indicated ● Processing:
in the request form so it will not be reject) ○ Within 1 hour after collection
Suitability of Sputum for Culture
● Bartlett’s Sputum Classification PROCESSING OF URINE
○ Allows us to grade sputum specimens ● Gram Staining of uncentrifuged urine
● < 10 epithelial cells and > 25 pus cells ○ rapid UTI screening
○ numerous squamous cells (vaginal/urethral
contamination)
● PROCEDURE FOR CULTURE
○ Inoculate sample on Blood Agar Plate (BAP),
Eosin Methylene Blue (EMB), Mac with a 1 uL
loop (calibrated)
■ 1 uL = 0.001 mL
■ 10 uL = 0.01 mL
■ 1 mL = 1000 uL
○ Incubate overnight
■ (+) growth (count colonies)
■ (-) re-incubate for 24 hrs

Computation for CFU/mL

● What to do with the counted colonies?
○ Use it for Colony Forming Unit (CFU) per mL

PROCEDURE:
● Make a direct smear (GS & AFS)
● If it’s positive for Acid fast bacilli proceed to
○ Do digestion & concentration technique
■ N-acetyl-L-cysteine-NaOH ➢ 1 uL= 0.001 mL = 1000 uL
(NALC-NaOH) ➢ 10 uL = 0.01 mL = 100 uL
➢ Used to digest the mucus
■ dissolve fats & mucus to free bacteria Example: 1 ul loop yields 25 colonies? 145 colonies? What is the
● Sometimes we’ll get a negative AFS because the mucus colony count?
is too thick and it has trapped the organisms in it
● Culture 25 colonies X (use 1 uL) 1000 = 25,000 CFU/mL
● Does this indicate infection?
3. URINE ○ NO, because it does not exceed 100,000
bacterial CFU/mL

145 colonies X 1000 = 145,000 CFU/mL

● Does this indicate infection?
○ YES, because it falls above 100,000 bacterial
CFU/mL

POUR PLATE METHOD

● prepare 1:1000 dilution of urine w/ sterile H2O (or broth)
○ You can heat only three tubes
● transfer 1mL of solʼn to Petri dish
● add agar & mix well
● incubate for 18 – 24 hrs
● count colonies & multiply by 1000
● Purpose: Diagnosis of UTI
● report result in bacteria/mL
● Method of collection:
○ Clean-catch midstream
■ For voided urine
■ Midstream
- Let go of the first few drops
(may contain hydro
concentrated substance)and
last drops (diluted)
- only catch the middle
portion
■ Clean-catch
- The avoidance of areas rich
in microbiota
- Do not allow the mouth of
the bottle to touch areas of
the skin
○ Suprapubic aspiration
■ gathered directly in the urinary Prepare tree tubes if you wish to do a 1:1000 dilution
bladder ↓
■ Usually done for infants or young Make sure that the tubes are sterile and contain 9 mL distilled
children water or broth
○ Cystoscopy or catheterization ↓
● Collection Time

7
 After, put in 1 mLof the original inoculum (urine) in the first tube
(1;10 dilution)
↓
Mix the first tube well and transfer 1 mL to the proceeding tubes
↓
Transfer 1 mL from the tube containing 1:1000 dilution into a clean
petri dish
↓
Pour to 10 to 12 mL of nutrient agar (melted agar - allow it to cool
first before pouring to not kill the organisms)
↓
Mix it well and incubate for 18 to 24 hours at 35 degree Celsius
↓
CSF Processing
Count the colonies and multiply by 1000 (report result in
bacteria/mL)

Remember:
● Values > 1.0 x 10^5 CFU/mL = INFECTION
● Values of 1.0 x 10^3 – 1.0 x 10^5 colonies/mL =
contaminated or convalescence from UTI
● If 10 uL loop = colonies counted x 100
● E.coli = 90% of UTI cases
■ Other organisms that can cause UTI
include enterobacteriaceae:
■ Klebsiella proteus
■ Enterobacter
■ Enterococcus faecalis
■ Pseudomonas aeruginosa
■ Staphylococcus saprophyticus
■ yeast
● Macroscopic Examination
4. CEREBROSPINAL FLUID ○ Volume (3 - 5 mL)
● Samples should be collected before treatment.* ○ Color
● Should be collected from patients with suspected ○ Appearance
virulent meningitis even if they have already undergone ● Normal CSF
antibiotic treatment ○ Clear and colorless
● Avoid delay because of high mortality rate & rapid ● Abnormal CSF
proliferation of microbes is associated w/ meningitis ○ Xanthochromic - there’s yellowish pigment due
● MAJOR PATHOGENS to the presence of bilirubin (a by-product of red
○ Streptococcus pneumoniae cell degradation)
○ Haemophilus influenzae type b ○ Bloody
○ Neisseria meningitidis ○ cloudy
PROCEDURE:
Cerebrospinal Fluid ● Centrifuge CSF
● Collection: lumbar puncture (between the 3rd or 4th / 4th ○ Separate the supernatant and sediment
or 5th lumbar vertebrae) ○ Sediments will be used for culture.
● Volume : 0.5 - 5.0 mL ○ Supernatant will be reserved for rapid antigen
○ Adults: 3 tubes testing
■ 1st tube = Chem / Sero (for protein ● Culture CSF on recommended media:
and glucose test) ○ Trypticase Soy Broth / thioglycollate
■ 2nd tube = Microbiology (GS and ○ BAP for Gram (+) cocci
culture) ○ CAP for Gram (-) cocci
● If there are more than enough ○ EMB/Mac for Gram (-) bacilli
sample and if you have multiple ● Rapid Antigen Testing
microbiology tests to perform ○ supernatant
- Set aside 1 mL for bacterial ● Make a smear for GS and India ink
culture, 2 mL for fungal ○ sediment
culture, and the rest for
mycobacterial culture
- Any extra tube can be
extracted goes to
microbiology
- If there is only one tube
collected, it is again
reserved for microbial
culture
- priority
■ 3rd tube = Hematology (cell count and
differential count)

Deliver the CSF as quickly as we can; if the specimen is delivered

in the lab for <1 hour
↓
Centrifuge at 2000 rpm/ 20 mins

8
 ↓ a. Subculture- transfer a portion of culture to
Separate supernatant and sediment another EMB and Mac.
↓ ↓ 4. No growth: inoculate culture from enrichment media into
EMB or Mac.
Supernatant sediment
a. If by chance that there were no colonies
Serologic testing (latex agglutination for gram staining
appeared in EMB and Mac, u can inoculate
and culturing.
again on a fresh set of EMB and Mac and get
_____________________________________________________
ur inoculum from the enrichment media.
If the sample has reached the lab more than an hour after
Stool Culture : 3rd day
collection
● Note patterns of biochemical reactions
↓ ○ Cultures that appeared positive on the 2nd day.
First is to inoculate it in a trans-isolate (T-I) media before it dries ● If suggestive of Salmonella, Shigella, Vibrio, DO
out SEROLOGIC TYPING
↓ ○ From no growth in the 2nd day: If growth occurs
Incubate overnight 35o C in CO2 after doing step 3 (2nd day), DO biochemical
↓ test
The next day, subculture to chocolate agar and blood agar ○ Incubate overnight and perform step 1 & 2 of
FOR DELAYS IN CSF PROCESSING day 3
CSF for bacterial culture: ■ If there was still no growth after 2nd
● Incubate @ 37° C for not > 12 hrs (6) OR; up to the 3rd day, it would be best to
● Stand @ room T° not > 1 hour recollect the sample.
● DO NOT REFRIGERATE!
○ Some orgs. are sensitive to low T°
EXUDATES TRANSUDATES
○ N. meningitidis & H.influenzae
CSF for viral culture:
● Temperature doesn't matter ● Wounds ● Fluids
● Refrigerate immediately ● Boils ● Synovial
● If held for more than 24 hrs, freeze specimen at –700C ● Abscesses ● Pleural
STOOL ● Ulcers ● Pericardial
● Ideal specimen for gastroenteritis ● Granules ● Peritonial
● Freshly collected stool (early stage of disease) ● Rash ● Hydrocele
● Rectal swab may be used ● Eye discharge
● Gastric aspirate ● Ear discharge
○ May be processed especially for the isolation of ● Endocervical
acid fast bacilli ● Urethral
● Gastric biopsy→ Helicobacter pylori ● Anorectal
Amount: 1-2g ● discharge
Container: Clean, wide mouth with lid ● wounds
Transport time : 2 hours after collection; 24 hrs @ 4ºC
Transport medium : Cary Blair ● Transudates
PROCEDURE: ○ Fluid build up caused by systemic condition that
● If the sample is stool, we can easily inoculate it into your alter pressure in blood vessels causing the fluid
culture media or we can place look full into the selenite to leave in the vascular system
broth. ● Exudates
● Put rectal swab in enrichment broth (Selenite broth) ○ Fluid build up caused by tissue leakage due to
○ Put the swab directly. inflammation or cellular damage.
● GS is NOT usually done but helps in identifying possible
etiologic agents
○ Not usually done because we expect that there Lab test Transudate Exudate
would be lots of organisms
■ Gram + cocci in clusters appearance Clear, pale yellow Turbid, bloody
■ Gram – comma-shaped bacilli
■ Gram + bacilli in large numbers Fluid total protein 30 g/L or less > 30 g/L
■ Gram – bacilli
Stool Culture : 1st day Fluid/ serum <0.5 >0.5
● Inoculate the specimen and incubate it overnight. protein
● MEDIA:
○ Differential (EMB, Mac)
fluid / serum LD <0.6 >0.6
■ Would differentiate form lactose
fermenters from non lactose
fermenters Fluid <0.67 x ULN serum >0.67 x ULN serum
○ Selective (SSA, HEA, XLD)
■ To selectively grow shigella and cholesterol < 1.2 mmol/l 1.2 mmol/l or
salmonella. greater
■ Both diff. Adn selection media are
solid Specific gravity <1.015 >1. 015
○ Enrichment (Selenite F, APW)
Transudates is always on the lower limit; exudates is on the higher
■ Liquid
limit
Stool Culture : 2nd day
COLLECTION
1. Check enrichment tube: +/- turbidity
DISCHARGES / FLUIDS : depends on the type lesions
2. Check differential media for LFs & NLFs
● Dry wound – moisten swab w/ NSS before collecting
a. LF- produced colored colonies in EMB and
● Skin lesion – remove crust of pustule/ vesicle cap then
Mac. Pink to purple colonies
gently swab lesion
b. NLF’s - do not produce colored colonies
NOTE:
3. With growth: Subculture in another set of tube & plates
and do biochemical tests.
9
 ● Superficial wounds→ collected along the edge of the
wound after cleaning with sterile saline
● Deep wound→ needle aspiration
● Endocervical : use swab
● Urethra : use swab or scrape mucosa of anterior urethra
● Anorectal: insert swab about 4.5 cm. into the anal canal
● Irrigation, Intravenous or Intra-arterial Catheters
○ Use of endoscopic procedures

● Tissue (scrapings,etc)
EXUDATES / TRANSUDATES: Collection container
● Aspirates: Sterile vial
● Ulcerative lesions:
○ biopsy in sterile vial w/o preservatives
● Irrigation, Intravenous
○ Sterile vial
● Intra-arterial Catheter tips
○ Sterile vial
● Swabs: 2 pcs. in a sterile tube (do not allow to dry)
● Fluids: syringe with sterile rubber stopper
● Corneal scraping: direct inoculation
● Transport time :
○ ASAP (30 mins)
● Delay in transport :
○ Refrigerate
○ If swabs, place in TSB or Thioglycollate
○ Amies or Stuart Transport Medium
○ SBA slant
GENITO-URINARY SPECIMEN
SPECIMEN
● Cervical (female)
● Urethral (male)
● Rectal (may be paired with throat swabs)
PURPOSE
For the determination of:
● STDs
● Vaginitis
● Urethritis
● Childbirth infections
Possible Pathogens In Anogenital Specimen:
● T. pallidum
● N. gonorrheae
● C. trachomatis
● G. vaginalis
● C. albicans
● HSV
● N. gonorrhoeae:
○ GS: G (-) diplococci
○ CAP: enriched medium + CO2
○ MTM: selective medium to inhibit NMB

● If u are going to transport N. gonorrhoeae, always keep it

in this position otherwise CO2 will escape from the
container.
10
SUMMARY

11
12
13
14
 BACTERIOLOGY LEC

LECTURE 5: METHODS OF IDENTIFICATION

PROF. JOANNE KRYSTINE TAGO, RMT
AUG 28, 2021
For updates and corrections → @mar4rii on Twitter

METHODOLOGIES ■ Denatures anything that is

● Microscopy & Staining protein (ex. Proteolytic
○ Allows us to observe the morphology, viability, enzymes) which would lead
and motility of microorganisms to inhibition of autolysis
○ Provide us with the general groupings of their ○ METHANOL FIXATION
shape and whether they are gram + or gram - ■ Prevent lysis of rbc
● Culture Methods ■ Avoiding damage to all host
○ allows us to see the macroscopic cells
characteristics of the organisms ■ Results to a cleaner
○ Type of colonies and environments needed for background
survival ■ Strongly recommended for
○ May provide the genera/genus of an organism all clinical material esp urine
● Biochemical Tests ■ Quite toxic and not
○ demonstrates the enzyme system in each recommended to be used in
organism routine testing
○ Specific tests of biochemical tests for a ● PURPOSE:
particular group of organisms ○ Kills & preserves the organism
● Serological Tests ○ Anchors the smear to the slide
○ done to identify bacterial serotypes
○ If the results of the first 3 mehos would lead to II. STAINING
an organism with several serotypes, it would ● A process of adding stains or dyes for enhanced
require serological testing visualization of the microbes
● Molecular Microbial Staining Methods:
○ DNA sequencing brought in newer techniques 1. Direct
to identify microbes more precisely ○ techniques which allow the stain to come in
○ Does not require to culture microbes contact with the organism to be identified or
○ Useful for non-culturable organisms and for objectively viewed
identifying viruses as well ○ Directly color the organism and leave the
○ Provides a more robust toolset in identifying background colorless
infectious agents ■ Simple
● Animal Inoculation ● Use only 1 color or stain
○ Not used in the clinical lab; used in research lab (Crystal violet or methylene
○ done to investigate the effects of the inoculated blue)
material ● Not commonly performed in
lab
I. MICROSCOPIC METHOD ■ Special
SMEAR PREPARATION ● Used to demonstrate the
Smear - a thin film of microbial solution on a glass slide presence of special
Microbes can be viewed in the ff. states: structures that cannot be
A. LIVING STATE regularly seen in gram stain
● Visualize size, shape & arrangement slides (ex. Flagella, spores,
● Check motility capsule)
● Examples: ● Not commonly performed in
○ WET MOUNTS lab
■ Plated media - Need to add ■ Differential
ionized water/ sterile ● Use of several stains to
distilled water show contrast and
■ Liquid media - No need to differentiation of organisms
add that may be found in a
○ HANGING DROP METHODS single smear
■ The drop hangs on the ● Most common staining
coverslip because of the technique in lab
presence of paraffin wax ● Gram staining, acid fast
■ Edge of the droplet = initial method
viewing 2. Indirect
■ Organisms are stuck on the ○ The background is stained, leaving the
edge of the droplet organism to be identified colorless
B. B. FIXED STATE ○ Helpful because the light or colorless organism
- Organisms are no longer viable bcs they are appear highlighted against the dark background
subjected to heat or chemical
● Methods Of Fixation:
○ HEAT FIXATION
■ Preserves the overall
morphology but not the
internal structures SPECIAL STAINING
1
 SPECIAL STAINING
Capsule Welch, Anthony’s, Gin;s, Hiss, Tyler’s
Muir’s, India Ink
Cell Wall Dyar
Metachromatic granules Albert’s Ljubinsky, LAMB, Niesser’s
Flagella Leifson’s, Fischer-Conn, Gray’s
Endospore Dorner’s Schaeffer & Fulton/ Wirtz -
Conklin, Glacial acetic acid

Modification of Gram Staining

Hucker’s Burke’s
Modification Modification
Primary Stain Crystal Violet Crystal Violet
Mordant Gram’s Iodine Gram’s Iodine
Decolorizer Acetone-Alcohol Sodium bicarbonate
+ Ether-acetone
Counterstain Safranin Safranin
● Initially, Hans Christian Gram used methyl violet as
primary stain, acetone as decolorizer and dilute Carbol
Fuchsin as counterstain
● (a) Negative Staining - demonstrate capsules,
background is fully colored while the structure to be General Rules:
viewed is left colorless; colorless against a colored ● All cocci are gram (+) except Niesseria, Branhamella,
background Moraxella, and Veilonella
● All bacilli are gram (-) except Mycobacterium,
DIFFERENTIAL STAINING Corynebacterium, Lactobacillus, Listeria, Clostridium,
→ most widely used staining technique in the microbiology lab Bacillus, Erysipelothrix and Nocardia
A. Gram Staining ● All spiral bacteria are gram (-) when stained
● Divide bacterias into 2 separate groups:
○ Gram positive THEORIES OF GRAM STAINING
○ Gram negative
GRAM (+) GRAM (-)
● Based on the thickness and chemical
composition of the cell wall MgRNA Theory MgRNA + CV - I 2 MgRNA is absent
● Hans Christian Gram - danish microbiologist complex = insoluble
○ Identify if a bacteria have a compound
peptidoglycan wall Benian Theory Less permeable cell Permeable cell walls
○ Dye was introduced to bacteria walls
○ if bacteria has a peptidoglycan cell Stearn &Stearn ↓isoelectric pt making ↑ isoelectric pt
wall, it absorbs the dye and turns Theory them acidic; bind well making them basic
purple; thick peptidoglycan -- positive with CV (basic dye)
○ Those who didnt absorb the dye have Lipid Content ↓lipid content but ↑ ↑lipid content; no
thin or no peptidoglycan cell wall -- teichoic acid teichoic acid
negative
● MgRNA Theory
○ A compound of magnesium ribonucleate and a
Chemical Gram + Gram - basic protein concentrated at cell wall helps
Crystal violet Purple Purple gram positive bacteria to retain primary dye
Gram’s Iodine Purple Purple ○ However, this particular compound is not found
Acetone alcohol Purple Colorless on the cell walls or the cell membrane of gram
negative organisms
Safranin Purple Red
○ If MgRNA is present, it binds wall with crystal
● Crystal violet - primary stain violet creating an insoluble compound
● Gram’s Iodine - mordant ● Benian Theory
○ Forms large crystals with the dye, gets trapped ○ Some substances are not easily absorbed into
and locked with crystal violet, teichoic acid and the thick peptidoglycan layer specially when
MgRNA another substance is trapped in it
● Acetone alcohol dehydrates the peptidoglycan wall of ○ Gram negative organisms cell walls are
gram positive cells making the pores closed and restricts sensitive to acetone alcohol rendering it to be
the absorbance of other materials into the cell wall permeable; pores are produced making it
○ For gram negative, it will create pores in the easier to be penetrated
lipopolysaccharide layer, removing the primary ● Stearn & Stearn Theory
stain leading to a colorless cell wall ○ Gram positive organisms are said to have a low
● Safranin - counterstain isoelectric point making them acidic, making it
○ Gram negative will absorb the color of safranin bind to basic dyes like crystal violet
○ Gram negative is said to have high isoelectric
points making them basic, which doesnt bind
well with basic dyes
● Lipid Theory
○ In the cell wall of gram negative bacteria, theres
the lipopolysaccharide (outer membrane) which
has lipids, but doesnt have teichoic acid
2
 ■ Lipids are easily penetrated by the ● The crystal violet stain is susceptible to washing out with
action of acetone alcohol, allowing water
materials to come in and come out ● Do not use a more the 5-second water rinse at any
the cell walls stage of the procedure
○ Gram positive cell walls dont have that much ● As counterstain is also a basic dye, it is possible to
lipids, but they do have teichoic acid which replace the Crystal violet iodine complex in the
binds perfectly well with crystal iodine, allowing gram-positive cell wall especially when we overexpose it
it to get stuck in the peptidoglycan layer to Safranin
● It’s either 30-60 seconds should be the contact time of
ERRORS IN GRAM STAINING the slide with Safranin Red
Gram (+) becomes Gram (-) Gram (-) becomes Gram (+)
Gram (-) become Gram (+)
Acidic Grams I2, insufficient I2 Inadequate decolorization Inadequate decolorization
Aging, dying, autolysis, ● Most common reason
overheating ● If you’re still practicing, you can prepare several smears
Removal of MgRNA 1/ and check and follow the decolorization time
precipitation from bile salts in
media Thick Smears
Thick smears ● You will not be able to remove the crystal violet if thick
Low concentration of crystal
violet smears
overdecolorization
Non-Stain System to Determine Gram Stain Reaction
Excessive washing, excessing L-alanine, 4 - nitroanilite (LANA)
counterstaining ● Turns yellow if Gram (-)
● Aminopeptidase activity (released in the presence of
Gram (+) becomes Gram (-) L-Alanine) → causes the release of 4 nitroaniline from
Acidic Gram’s I2, Insufficient I2 the reagent → yellow
● Acidic Gram’s Iodine would destroy the peptidoglycan ● Reagent: L-alanine
layers. ○ Used to make a suspension with the bacteria
● There would be no Crystal Violet Iodine complex ● In a small tube get 1mL of L-Alanine and add a loopful
because it’s too acidic and damages the peptidoglycan of the organism then observe
so the Crystal Violet would not have anything to latch on
to and it will not be retained in the cell wall 3% KOH String Test
● Insufficient Iodine exposure would also lead to the ● The formation of string-like material indicates G (-)
removal of the crystal violet organisms
● The amount of mordant available is important for the ● Use a drop of 3% KOH and put it on a glass slide then
formation of the Crystal Violet Iodine complex add a loopful of cells from a single colony and mix
● The concentration of Gram’s Iodine should be just right ● If the mixture is viscous within 60 seconds, it means that
● The lower the concentration, the easier the Crystal Violet it is KOH positive
is removed from the cell wall ● If a string-like material is developed after 1 minute of
mixing it means that the organisms are gram (-)
Aging, Dying, Autolysis, Overheating ● Dilute alkali (KOH) → Lysis → release of cellular DNA →
● Cell walls are no longer intact viscous (suspension)
● The peptidoglycan layer may have been damaged
● During heat fixation, remember not to do it excessively B. Acid Fast Staining
● Simply let the slide pass along the flame moving back ● Mycolic acid- responsible for the acid fastness of
and forth mycobacteria
● Do not leave slide on top of the flame and destroy cell ○ Long carbon atom chains
morphology ● Second most common differential staining method in the
base lab
Removal of MgRNA w/ precipitation from bile salts in media ● Some bacteria have the ability to retain the primary stain
● Bile salts in culture media are also bad for the and resist the acid decolorization process
magnesium RNA fin the cell walls of gram-positive
organisms General rule
● When this is removed, it binds well with the Crystal violet ● All organisms are non-acid fast EXCEPT:
iodine complex ○ Mycobacterium- longest chain of mycolic acid
● When it is removed or precipitated from bile salts, there ○ Nocardia spp. - short cell wall-bound mycolic
will be a lack of something for the Crystal violet iodine acid
complex to latch on to ○ Corynebacterium spp.- shortest chain of
mycolic acid
Low concentration of Crystal Violet ■ Even in gram staining,
● Up to 2% of crystal Violet can be used successfully corynebacterium won’t demonstrate
● Standard soln is around 0.3% which is good provided acid fastness because they have sort
that you won’t decolorize more than 10 seconds chains
● Paul Ehrlich in 1882 & later improved by Ziehl and
Overdecolorzation Neelsen
● If you are not yet familiar with the decolorization Chemicals AFB NON-AFB
process, you may use 95% ethanol to decolorize since it Carbolfuchsin Red Red
decolorizes slowly
● Those who are experienced can freely use acetone
Heat Red Red
alcohol Acid alcohol Red Colorless
Methylene Blue Red rods in a Blue Blue organisms
Excessive washing, excessive counterstaining Background
● In the course of staining, different chemicals are applied ● Carbolfuchsin is used as the primary stain
and you need to wash your slide with water in between ● If the cell wall has mycolic acid, it binds well with

3
 carbolfuchsin ● M. tb (red)
● Heat allows the pores of the microbial cell wall to open
up to allow penetration of carbolfuchsin 4. Baumgarten’s
● In the presence of acid-fast bacilli, the background is ● diluted alcoholic fuchsin = primary stain
blue but acid-fast bacilli will retain their color ● Differentiates blue M. tb & red M. leprae
● Non-AFB organisms will stain blue along with the
background CULTURE METHOD
Culture Media
Ways to facilitate Acid Fast Staining ● provides the appropriate biochemical and biophysical
● Using steam environment for microbial growth
○ Opens pores of microbial cell wall allowing the ● Inoculum= specimens being introduced into the media
entry of carbolfuchsin into the cell wall ● Culture= growing in the culture media
● Increase concentration of phenol and basic fuchsin ○ Types:
○ When the concentration is higher, there is a ■ Pure - refers to a group of organisms
greater chance that more carbolfuchsin will be that is basically similar
able to enter into cell wall ■ Mixed - composed of different types
● Prolonged contact time of microorganisms
○ Instead of applying 1-minute rule, you can ■ Stock - pure culture of known
increase the time by 3-5 minutes to ensure that organisms that are kept in the
the carbolfuchsin is being transported into the laboratory for reference purposes;
cell walls of the organisms used as a negative or positive control
● Adding wetting agent (tergitol) COLONY
○ There is no need for heating - Macroscopic or visible masses of microbial growth that
○ Also called as surfactants you see arising from a culture media
■ Chemical substances that increase Types:
the spreading and penetrating ● Mucoid colony - slimy, water, or glistening
properties of a liquid ● Smooth colony - uniformed texture which allows them to
■ Done by lowering the surface tension be easily emulsified with NSS
■ When territory is added into ● Rough colony - wrinkled, dry looking, granulated, not
carbolfuchsin the surface tension of easily emulsifed with NSS
carbolfuchsin is lower allowing it to
easily flow into the cell wall of AF TYPES OF CULTURE MEDIA
organisms Based on:
● Physical Form / Consistency
TYPES OF ACID FAST STAINING ● Composition
● Function / Purpose
1. Ziehl - Neelsen Method ● Distribution
● Uses heat (hot sustain)
● Red AFB I. PHYSICAL FORM
● Blue Non-AFB A. LIQUID MEDIA
● Most commonly used ● No agar
Prepare a smear from specimen or culture and fix the smear (heat ● water– based
fix or formaldehyde vapour) ● broth, milk, infusion
↓ ● (+) growth
Cover the smear with Primary stain Carbol Fuschsin ○ dispersed
↓ ○ cloudy
Heat(act as Mordant) the slide from below intermittently 2-3 times ○ turbidity
up to 5 mins till vapours are seen ○ particulate appearance (floating materials)
↓
Wash off the stain B. SEMI-SOLID MEDIA
↓ ● with solidifying agent
Decolorize with acid( H2SO4) or alcohol ○ 0.3-0.5% Agar
↓ ● detects bacterial motility
Wash immediatley with tap water Examples:
↓ ● Motility test medium
Pour counter stain (Methylene Blue). Wait for 60 seconds. ● SIM = detects H2S & indole production
↓ ○ Used for biochemical testing
Wash off the stain ○ Indole production = pink
↓ ○ H2S production = blackening
Examine under OIO ■ if dispersed = motile
2. Kinyoun Method
● Uses wetting agents C. SOLID MEDIA
○ Facilitate absorption or penetration of
● allows growth of discrete colonies
the primary stain into the microbial
● 1-5% Agar
cell wall
● Red AFB
LIQUEFIABLE
● Green/blue non-AFB (Malachite green/
● You can prepare it earlier in an Erlenmeyer
Methelyne blue)
flask
● Called the cold stain process
● NA, MacConkey, EMB
● Carbolfuchsin is still the primary stain
NON – LIQUEFIABLE
● Middlebrook 7H11
3. Pappenheim’s
● Lowenstein – Jensen media
● resolic acid in alcohol = decolorizer
○ Both are used for the cultivation of TB
● Differentiates M. tb from M. lacticala and M.
○ Since TB is fastidious, these culture
smegmatis
media contains nutrients and
● M. lacticala and M. smegmatis (blue)
4
 additives that are proteinaceous ● Various approaches in making a medium selective
○ Proteins are sensitive to heat includes the addition of:
therefore, we do not reheat culture ○ Antibiotics
media such as this ○ Alterations of pH
○ Dyes
II. COMPOSITION ○ Chemicals
A. SYNTHETIC MEDIA ○ Combination of any of these
● chemically defined ● Permits preliminary identification of genus or spp.
● All those in bottles in powder ● Examples
○ Mannitol Salt Agar = 7.5% NaCl
B. NON-SYNTHETIC MEDIA ■ Both a selective and differential media
● complex media ■ Selective - presence of high salt
● at least one of the ingredients is not chemically defined concentration which inhibits those
● for cultivation of bacterial pathogens & fastidious bacteria who cannot survive the presence of
● Culture media that contain blood, milk, yeast, heart blood high salt conc
components, broth ○ Hektoen Enteric Agar = Bile salts
○ Inhibits growth of most gram-positive
III. FUNCTION/PURPOSE organisms
General Purpose Nutrient agar
Medium Selective Agent Uses
Enriched BAP, CAP
Mueller Tellurite Potassium tellurite Isolation of C.
Enrichment TSB, Selenite
diphtheriae
Selective EMB, MacConkey
Enterococcus Na azide Tetrazolium Isolation of fecal
Differential - by virtue of colonial EMB, Mac, BAP
faecalis broth Enterococci
characteristics
Phenylethanol agar Phenylethanol chloride Isolation of
Aerobic Growth Thayer-Martin
Staphylococcus and
Specimen Transport Stuart Streptococcus
Assay MHA Tomato juice agar Tomato juice, acid Isolation of
Enumeration PDA Lactobacilli
MacConkey Bile, crystal violet Isolation of Gram (-)
A. GENERAL PURPOSE MEDIA enteric
● BASAL MEDIA SSA Bile, citrate, brilliant Isolation of
○ supports most non-fastidious bacteria green dye Salmonella &
○ for primary isolation of microorganism Shigella
○ Designed to grow a broad spectrum of Lowenstein - Jensen Malachite green dye Isolation &
microbes agar maintenance of
○ Examples: Sabouraud’s agar pH 5.6 Isolation of fungi
■ Nutrient agar
■ Nutrient broth
E. DIFFERENTIAL
● Different bacteria can be recognized on the basis of their
B. ENRICHED
colony color
● SOLID
● Various approaches includes:
● Addition: blood, seru, egg yolk, vitamins, AA
○ Incorporation of dyes
● Supports growth of fastidious organisms
○ Metabolic substrates
○ Blood Agar Plate (BAP)
● Those bacteria that can grow on diff. media can utilize
■ When blood is added to blood agar
those additives and appear differently colored colonies
base, the basal media is slightly warm
● Differential/Indicator media - Indicators are usually added
■ Blood is intact
to differentiate one organism to another
○ Chocolate Agar Plate (CAP)
● Allows growth of more than one microorganism of
■ Blood is added while the basal media
interest, but it can distinguish one from another
is hot
● Displays visible differences among microbes
■ Blood is cooked in the process,
● Examples
turning blood into chocolate
○ MacConkey agar
○ Thayer-Martin
■ Neutral red - indicates lactose
fermentation
C. ENRICHMENT ● Lactose fermenters - pink
● LIQUID ● Non-lactose fermenters -
● Promotes Growth of certain organisms by providing it colorless
with essential nutrients ○ Spirit blue agar
● Sometimes contain certain inhibitory substances to ■ Spirit blue dye - indicates fat
prevent the growth of normal competitors, like normal hydrolysis
microbiota
● Examples: Blood agar plate is also Differential Media
○ Selenite F ● Make it easy to distinguish colonies of different microbes
○ BHI ● Differentiates the hemolytic reactions of some
○ Thioglycollate microorganisms

D. SELECTIVE
● Contains inhibitory agents that prevents/suppress growth
of unwanted microbes
● Designed to inhibit unwanted commensals ,
contaminating bacteria and help recover pathogens from
a mixture of organisms
● Technically are agar plates by the addition of certain
inhibitory agents that don't affect the pathogen of interest

5
 and vancomycin
○ Robertson Cooked Meat = bullock heart meat
■ Commonly used to grow Clostridium
species
■ Bullock - castrated bull or young bull

Anaerobic Culture Methods

● Anaerobic chamber
○ Where we inoculate anaerobic culture media
○ We need to observe the proper environment to
allow the anaerobes to grow successfully in an
○ Beta-hemolysis artificial environment
■ Identified with a presence of a clear ○ Similar to Biosafety cabinet but instead of
zone of hemolysis around the glass, there is a thick plastic sheet that covers
colorless the opening of the chamber
■ Organisms are able to breakdown the ○ There are glove ports where you will insert your
RBCs hands and work
○ Alpha-hemolysis ○ After inoculation, the plates are placed in an
■ Hemolytic reactions because of the anaerobic system and then open the anaerobic
change in color chamber
■ Greenish discoloration that is visible,
partial hemolysis or destruction of Gaspak Anaerobic Culture Method
RBCs
○ Gamma-hemolysis
■ No hemolytic reaction

Medium Substance for Differentiates

differentiation between
BAP Intact RBCSs Types of hemolysis
Mannitol Salt Agar Mannitol, phenol red , Species of
(MSA) 7.5% NaCl Staphylococcus
Hektoen Enteric Bromthymol blue, Salmonella, Shigella,
Agar (HEA) salicin, lactose, other LFs and NLFs
sucrose, sodium
thiosulfate, ferric
ammonium citrate, bile
Spirit Blue Agar Spirit blue dye, oil Fat-utilizing bacteria
(SBA) from those that do
not
Urea Broth Urea, phenol red Urea-hydrolyzing
bacteria
Sulfide Indole Thiosulfate, Fe H2S gas production
Motility (SIM)
Triple Sugar Iron Triple sugars, Fe, Sugar fermentation
(TSI) Agar phenol red dye & H2S production ● Contains:
Xylose Lysine Lactose, sucrose, Allows differentiation ○ Palladium catalyst pellets
Deoxycholate (XLD) phenol red, sodium of xylose ○ Envelopes that are supposed to be open to
agar fermentation, lysine produce carbon dioxide
decarboxylation & ○ Sodium bicarbonate and sodium borohydride
H2S production which supply CO2
○ Indicator systems - so that we can check if it is
F. Anaerobic Growth still anaerobic
● Anaerobic bacteria requires ○ Petri dishes - placed in an inverted manner
○ Low oxygen content because moisture usually builds up on the cove
○ Reduced oxidation-reduction potential ■ If we have incubated it in right side
○ Extra nutrients up, with the cover on top, and the
● Media for anaerobes may be supplemented with: agar-filled portion at the bottom, the
○ Hemin moisture build up will be found on the
○ Vitamin K cover
● Reduced by chemical or physical means ■ So, everytime the system moves,
○ Boiling in the water bath expels dissolved moisture will fall on the lower portion
oxygen which will contain the agar, thus we
○ Then, sealed with sterile liquid paraffin need to incubate the plates in an
● Examples inverted manner
○ Anaerobic Phenylethyl Alcohol Blood Agar
(PEA)
■ Enriched selective media
■ Widely used for inhibition of
facultative gram negative rods and
swarming of certain Clostridium
species and Proteus species from
clinical specimen
○ Anaerobic Kanamycin and Vancomycin (KV)
Thioglycollate broth
■ Contains the antibiotics - kanamycin ● If the organism should be transfered, you can place them

6
 in an anaerobic pack which contains gas generator
BUFFERED GLYCEROL SALINE
● Good for mailing fecal specimens
● High ph to favor fecal pathogens
○ Pathogens grown in highly alkaline
environments.
● Not for transport of fecal specimens suspected to be
Campylobacter sp (+)
● Designed for stool samples ONLY

Functional type
H. ASSAY MEDIA
● for the assay of vitamins, amino acids and antimicrobial
● If you don't have gaspak, you can use a large jar that will agents.
allow petri dishes to fit inside it ● to monitor the effects of the administration of certain
● Place a candle on a glass slide, light it up, close the jar, antimicrobial drugs.
and the candle inside will remain lighted so long as ● Example:
there’s oxygen in the environment ○ Mueller Hinton Agar
● If it has consumed the oxygen, the light will be off and ● Usually placed in a large petri dish to allow zone of
signifies that the system is already anaerobic inhibition to spread.

G. TRANSPORT MEDIA I. ENUMERATION MEDIA

● Maintains viability of bacteria and slows rate of ● Used by industrial and environmental microbiologists to
reproduction count the number of organisms in milk, water, food, soil
● We should be familiar of the time limit - 2 hours and other samples.
● Purpose ● Used for testing food samples for the presence of
○ Prevents drying/desiccation of specimen microorganisms
○ Inhibit overgrowth of unwanted bacteria ○ Ex: Potato-dextrose agar
■ Nutrient-poor Measurement of Cell Numbers
1. Direct microscopic counts
STUART’S (1954) a. Possible using special slides known as
● Maintains original state of specimen, especially Neisseria counting chambers
gonorrhoea 2. Electronic counting chambers
● Prevents organisms from being exposed to heat, cold, a. Counts numbers and measures size distribution
drying, of cells
● With charcoal-impregnated swabs and Glycerophosphate b. Widely used
in medium 3. Indirect viable cell counts
○ Charcoal is added to neutralize inhibitory a. Aka plate count
factors, but may result to difficulty in gram b. If plated in a suitable medium, each viable units
staining interpretation grows enforce a colony, each colony that can
● We need to follow the 2-hours limit, because some gram be counted is called colony forming units (CFU)
negative bacteria may utilize the glycerophosphate in the c. Number of CFU is related to the viable number
medium and overgrow, which can kill the organisms we of bacteria in the sample.
need to isolate Distribution:
TUBED

AMIES (1965)
● Modified Stuart’s medium
● Replace glycerophosphate with balanced salt solution ● Butt (left)
● Charcoal is incorporated into medium ○ Aka agar deep
○ Instead of impregnating the swabs with ● Slant (mid)
charcoal, it is added into the media ○ Used widely for transportation
○ Thus, much better than stuart’s ○ Stock cultures
● Slant/Butt (right)
○ Widely used in biochemical testing.

PLATED
● Placed in a petri dish

CARY BLAIR (1964)

● pH increased from 7.4 to 8.4
● No charcoal
● Recommended for fecal specimens suspicious for
campylobacter sp
● Similar to Stuarts but it is modified for fecal specimens

7
 METHODS OF OBTAINING PURE CULTURE

a. Streak plate

● Easiest and widely used

● Best isolation of pure colonies on solid media.

b. Pour plate

Quadrant streaking
● U only touch your loop in the sample once
● Practice aseptic techniques: sterilize ur wipe loop
● Gather your inoculum and streak on the first quadrant
● Heat
● Gather another loop of organisms (get organism from the
c. Selective method first streak)
● Simply using selective media ● Allow it to cool a little and allow it to touch on the
shoulders of the first streaking and drag the streaking to
d. Animal inoculation the next quadrant
● We don't perform in clin lab ● Heat, sterilize your loop
● Widely performed in reference lab or research lab ● Continue with the third quadrant
○ Allow it to touch a portion of streak second
INOCULATION OF CULTURE MEDIA quadrant and drag it towards the third quadrant.
● After inoculation, you incubate.
A. TUBED MEDIA
● The next day, check for colonies.
● Liquid = mix a loopful of microbe
● Semi – solid = stab media w/ needle
Preserving Bacterial Cultures
● Solid = stabbing or streaking or both
● Deep-freezing:
When u stab, do not touch or push the needle up to the bottom of
○ -50°to -95°C
the tube.
● Lyophilization:
○ Frozen (-54° to -72°C)
B. PLATED MEDIA
○ then vacuum-dehydrated

● Quadrant
○ Most common way of inoculating on plated
media
○ We divide surface of the media into quadrants
● In Mueller Hinton agar, we inoculate on the entire surface
area to produce a “matt” of bacterial growth.

8
 BACTERIOLOGY LEC
LECTURE 6: METHODS OF BACTERIAL
IDENTIFICATION II: BIOCHEMICAL TESTS AND ETC.
PROF. ALBAIRA KOUDAI, RMT
September 6, 2021
For updates and corrections → @mar4rii on Twitter
BIOCHEMICAL TEST PART I
● Forms the precise basis for bacterial species
identification
○ In doing biochemical tests, u will be able to
determine the species of the organism
○ Per organism, u will have a set/array of
biochem tests to perform
● Designed to demonstrate the enzymatic system within a
bacterial cell
○ Enzymes are what will allow your organisms to
perform all these biochemical reactions
○ Enzyme + substrate = products
○ These products are what we will be detecting
I. CATALSE TEST
● Differentiates Staphylococcus(+) species from
Streptococcus(-) species
● Principle:
● If an organism such as Staphylococcus possess
catalase, then the catalase of that organism will break
the hydrogen peroxide down forming oxygen and water
● How will u know if oxygen and water were produced from
the breakdown of hydrogen peroxide?
○ Manifested as effervescence
■ Effervescence - bubbling
● If the organism such as Streptococcus is catalase
negative, then it cannot break down hydrogen peroxide.
There should be no effervescence
● Method:
○ Fish–out & add a colony
○ Place a drop of 3% H2O2 onto the slide
○ Observe for bubble formation (positive)
○ Lack of or weak bubble formation (negative)
● What if the bubble formation is weak?
○ Most of the time, it is considered as negative
○ A true catalase reaction is really effervescent
QUALITY CONTROL:
● Positive: Staphylococcus aureus
● Negative: Streptococcus pyogenes
II. OXIDASE TEST (KOVAC’s METHOD)
● To determine the presence of bacterial cytochrome C
oxidase using the oxidation of the substrate 1%
tetramethyl-para-phenylenediamine dihydrochloride to
indophenol, a dark purple-colored end product
○ cytochrome C oxidase - enzyme found in the
electron transport chain of aerobic organisms
○ Test for those bacteria that are oxidative by
nature that makes use of oxygen for them to
survive
○ If an organism has cytochrome C oxidase, then
this enzyme can oxidase the substrate which is
1% tetramethyl-para-phenylenediamine
dihydrochloride. This will result to the
production of indophenol - dark purple
● Detects the ability of some organisms to oxidize aromatic
amines in the presence of air
● Principle:
● Oxidase REAGENTS:
○ PADAM (dimethyl compound) Pink - Black =
para-aminodimethyl aniline monohydrochloride
○ TMPDD = 1% Blue – Dark Purple
tetramethyl-para-phenylenediamine
dihydrochloride
● Procedure can vary
○ Get filter paper and soak it with the oxidase
reagent and idikit sa colony that u are trying to
identify
QUALITY CONTROL:
● Positive: Pseudomonas
aeruginosa
○ Oxidative gram
negative na
bacilli
● Negative: Escherichia coli
○ More on the
fermentative side
(process without
oxygen)
Neisseria gonorrhoeae (is one of
the famous organisms known to
be oxidase positive; a diplococci;
aerobic)
and Campylobacter jejuni are
also positive.
Always remember NOP; Neisseria Oxidase Pseudomonas =
Positive
1
Slant and Butt

● Pyruvic acid can enter any of the available fermentation

● Reactions that takes place in the slant are requiring pathway it just depends on the organism alin ang
oxygen; aerobic process. If the reaction happens in the capable sila of performing
butt; anaerobic. Slant has rich oxygen supply bcs ● After the pyruvic acid has been fermented by any of the
exposed. While butt nasa ilalim so low oxygen supply. fermentation pathway, it will always produce
fermentation end product are specific and dependent on
Manner of Inoculation the type of pathway performed
● Slant - only have to streak for it ● Fermentation pathway results on the production of acid
● Slant and Butt portion - stab with the inoculating needle
up to the 3/4th before the bottom kunin and ilabas tapos i III. CARBOHYDRATE FERMENTATION TEST
streak (stab and streak) ○ Sugar = source of energy
● Deep - stab only ■ Glucose
■ Lactose
■ Sucrose

Culture Media Composition

TSI (Triple Sugar Iron) Glucose, lactose, sucrose,

iron

KIA (kligler’s Iron agar) Glucose, lactose, iron

RDA (Russel’s Double Agar) Glucose, lactose,

A. TRIPLE SUGAR IRON

● Just like people, bacteria also make use of
carbohydrates, sugar or glucose as their main source of
energy but aside from glucose they can also use other
sugar.
● Just like us they can make use of glucose, the difference
is that when you talk about the metabolism of bacteria
the way they metabolize glucose the glycolytic process
that they do to the glucose molecule is more anaerobic
and fermentative.
● In human beings glucose; aerobic mechanism. In
bacteria they do glycolysis, their glycolytic pathway
continuous in a anaerobic manner or fermentative
● Fermentative is common on gram negative bacilli under
the family Enterobacteriaceae, process the pyruvate that
you produce from glycolysis of glucose ginagawa nila
most of the time, they transport or metabolize it further
via fermentation
● Determine whether a gram-negative rod utilizes glucose
and lactose or sucrose fermentatively and forms
hydrogen sulfide (H2S)
○ TSI most of the time is used to identify
members of the gram negative rod;
Enterobacteriaceae
○ TSI can give 3 results
■ if an organism can ferment sugar
■ if an organism produce hydrogen
sulfide
■ If an organism can produce gas as it
ferments the different sugar
● Indicators used:
○ Indicators detect pH changes; in that indicators
change in color together with a change of ph
○ Phenol Red -
■ Acidic - yellow
■ Alkaline - red
■ Neutral - red-orange
○ If sugar is fermented, dapat mag yellow siya
● Sodium thiosulfate - provides sulfur
● Ferrous Sulfate or Ferric Ammonium Citrate
○ Serves as hydrogen sulfide indicator
○ (+) blackening of the medium (ferrous sulfide
and H2S gas)
○ Hydrogen sulfate originally is a colorless gas
● Gas formation = bubbles or cracks in the gar or by
separation of the agar from the sides or bottom of the

2
 tube (CO2 and H2 gas) ○ Most of the time acidic siya
○ When an organism ferments sugar aside from ● 7th tube: Red slant, yellow butt, + gas, +H2S
producing acid as a by-product, an organism ○ Alkaline yung slant, yellow yung butt
may produce also gas ○ Assumption: basta may hydrogen sulfide

TSI REACTIONS: (Slant over Butt)

K = Alkaline; A= acidic

= acid with gas; NC = no change

Slant/Butt
Color Sugar Fermented
Reaction

K/K; K/NC RED/RED No sugar fermented

K/A RED/YELLOW Glucose is fermented

A/A YELLOW/YELLOW 2-3 sugars fermented

● Aside from sugars, may peptone yan siya which is
another source of energy/nutrient for your organism and
almost all organisms can utilize peptone for them to live
● Kung may peptone tas pinatubo mo yung organism sa
TSI, they could actually eat/metabolize peptone
● Peptone metabolism byproduct= ammonia
○ Ammonia is an alkalinizing agent
○ Whenever ammonia is present, alkaline talaga
yung environment
● Ito yung nangyayari sa TSI if walng sugar fermentation,
but there was metabolism of peptone, there is liberation
of ammonia, the ammonia alkalnizes your TSI, it will be
red
● So that’s why you see some TSI are red after incubation
meaning walang fermentation ng sugar but there was
metabolism of peptone
Lactose/sucrose is
A/K YELLOW/RED
fermented
● For you to assess TSI it should always slant over butt
● You can only produce gas if there was fermentation
● If K/K or K/NC
○ Slant is alkaline, butt is alkaline
○ Slant is alkaline, and no change sa butt
■ Most of the time alkaline parin yung
butt
● There are sugars in your TSI as the source of energy.
But, there is also peptone which the organisms can
readily metabolize
● Ammonia is produced
○ By nature is alkaline
● Yellow because phenol red in acid is yellow
● Fermentation of glucose is assessed by looking at the
butt
● Lactose and sucrose is assessed by looking at the slant

● First tube: cracks with gas

● 2nd Tube: Control
○ Original color ng TSI pagkagawa mo,
pagkaluto mo
● 3rd Tube: Red slant, Red butt, No gas, No H2S
○ Pag red red ang siya bothe meaaning wala PROCEDURE:
talagang fermentation ng any sugar kasi wla Get from your colonies using your needle
siya nag acidify ↓
● 4th tube: Red slant, yellow butt, no gas, no H2S Stab and streak
○ There was a sugar na naferment ↓
○ No gas kasi walanng cracks Incubate 35-37 degrees celsius for 18-24 hours
○ No H2S kasi walng blackening (overgrowing after 24hrs = TSI invalid)
● 5th tube: Yellow slant, yellow butt, + gas, No H2S
○ There must be a fermented sugar Possible Results:
○ Na separate ang medium meaning may gas 1. Control - Original Color
● 6th tube: Yellow slant, yellow butt, + gas, + H2S 2. A/A
○ If yellow ang slant and black ang butt 3. A/A GAS+ (medium is lifted due to gas production)
○ If that happens the assumption is na-acidify 4. K/A H2S+ (red slant = no fermented sucrose or lactose;
yan siya but natabunan ng hydrogen sulfide blackening = H2S+; yellow = glucose is fermented)
yung color ng butt so hindi mo alam if the butt is
red (allkaline) or yellow (acidic)
3
TSI - Carbohydrate Fermentation Test allowing it react to pDAB
● Quality Control ■ More sensitive and better than
Kovac’s
A/A Gas+ Escherichia coli ○ Spot Indole Test – 1%
p-dimethylaminocinnamaldehyde
K/A +/-Gas, H2S+ Salmonella typhimurium ■ 20 seconds
■ Kovac’s and Ehrlich’s can take 1-2
K/NC or K/K Pseudomonas aeruginosa days because you have to allow your
organism to break tryptophan down
K/A H2S+ Proteus mirabilis ■ Spot - can generate results very fast
■ Important because Kovac’s and
K/A Shigella flexneri Erhlichs results can take up to 1 to 2
days

Positive for Hydrogen Sulfide in TSI:

SPACED
● S = salmonella
● P = proteus
● A = arizona/arizonae (subsp of salmonella)
● C = citrobacter
● ED = (??)

B. IMViC TEST ● Incolaute with organisms. Incubate at 35 C for 18 to 24

- Used to differentiate members of hours
Enterobacteriaceae but more on the lactose
fermenter members of Enterobacteriaceae QUALITY CONTROL QUALITY CONTROL
- EKE (lactose fermenters) A. KOVAC’S method B. ERHLICH’S method
- E: escherichia coli Positive: Escherichia coli Positive: Elizabethkingia
- K: klebsiella meningoseptica or Haemophilus
- E: enterobacter influenzae
● Tests: Negative: Klebsiella pneumoniae Negative: Haemophilus
○ Indole parainfluenzae
○ Methyl red
○ Voges - Proskauer Test
○ Citrate
● Media Used:
○ Indole(Tryptophan 1%) Broth or Sulfur
Indole Motility (SIM)MRVP or Clark &
Lubs Dextrose Broth, Simmon’s
Citrate Slant

i. Indole Test
● Aim: To determine the ability of the microbe to degrade
amino acid tryptophan
● Will check if the organism has the enzyme:
tryptophanase
● Media:
○ Contains amino acid tryptophan
● You can only produce indole if you can breakdown
● SIM
tryptophan using the tryptophanase enzyme
○ Can give you results for sulfide or sulfur by
● To detect indole: ADD pDAB (color developer)
looking at the blackening, indole, and motility
○ p-dimethylaminobenzaldehyde
○ Turbidity means that the organisms swamped
● pDAB + indole = quinoidal red-violet compound
and moved from the point of inoculation =
motile
○ Immotile = organisms will stay at one place and
won't move if you stab it; no turbidity
● S. pyogenes - indole negative; sulfide negative; non
motile
● S. typhimurium - hydrogen sulfide positive
● E. coli - indole positive

ii. Methyl Red/Voges-Proskauer Test (MRVP)

● Methyl Red: Determine the ability of an organism to
produce and maintain stable acid end products from
glucose fermentation, to overcome the buffering capacity
of the system
● METHODS:
○ Checks for which pathway
○ Kovac’s – addition of p –
○ Positive: if the organism is able to produce a lot
dimethylaminobenzaldehyde
of stable acid from glucose fermentation; mixed
■ Most commonly used since it is a
acid pathway
safer method compared to Erhlich’s
● Voges-Proskauer: Determine the ability of some
○ Ehrlich’s – addition of (extractants) absolute
organisms to produce neutral end products
ETOH/ether/xylene/chloroform, add pDAB
(acetyl-methyl carbinol or acetoin from glucose
reagent
fermentation)
■ Purpose: extracts indole first before
4
 ○ Positive: if the organism is able to produce
acetoin as an immediate product from glucose
fermentation) QUALITY CONTROL QUALITY CONTROL
● Rationale: performed in concurrent but will separate
along the way and have different uses
● An organism can only be positive for either or one of the
two test

METHYL RED TEST

● PRINCIPLE:

● (+) if pH = 4.4 or below [RED] METHYL RED VOGUES-PROSKAUER

○ Because the environment is acidic
● (-) if pH = exceeds 4.4 [NO COLOR CHANGE] = Yellow
at 6.2
● METHODS:
○ MRVP medium = (+) deep red color
○ Buffered Peptone Glucose Broth = (+) bright red
■ **hold for 5 days before reporting
negative
● If the organism can produce these acids from
fermentation of glucose, then it is positive for methyl red
test:
○ Lactic
○ acetic
○ Formic
○ succinic acid
● How will you know if the 4 acids are produced?
○ Add the indicator - methyl red
Positive: Escherichia coli Positive: Enterobacter
aerogenes
Negative: Enterobacter aerogenes Negative: Escherichia coli
NOTE: new name Enterobacter aerogenes = klebsiella aerogenes
iii. Citrate Test
PRINCIPLE:
● Will detect if an organism can utilize the compound or the
molecule citrate as its only source of carbon.
● If an organism can utilize citrate as its only source of
carbon then it can grow in your citrate medium.
● Can also tell us if an organism can extract nitrogen from
inorganic ammonium source.

VOGES – PROSKAUER TEST

● PRINCIPLE:

● MEDIUM:
● TEST RESULT: ○ Simmon’s Citrate slant.
○ Positive: Cherry Red (Red-Burgundy) layer on ● pH INDICATOR:
top of the broth in 10-15 minutes ○ Bromthymol Blue
○ Negative: Yellow Color ■ (+) prussian blue [alkaline] (pH 7.6)
■ (-) yellow [acid pH]
■ green [neutral pH]
● If the organism has the enzyme citrase then it can use
citrate as its only source of carbon.
● If na metabolize yun ng organism using citrase enzyme,
citrate→ oxaloacetic acid→ pyruvic acid + acetic acid +
excess carbon dioxide(by product).
● U produce a lot of carbon dioxide
○ Carbon dioxide will eventually react with
sodium forming sodium carbonate.

● Sodium carbonate
○ End product when an organism is able to utilize
citrate
Barritt’s method for gram-negative rods ○ Anything that has carbonate = alkaline
● Solution A (5% alpha-naphthol) ● Ammonium hydroxide
○ Nag rereact sa diacetyl. ○ Contributes to the alkalinity of a positive test
○ Color developer medium.
● Solution B (40% KOH in 0.3% creatine) ○ Ammonium hydroxide + Sodium carbonate =
○ Converts acetone to diacetyl alkalinize the test medium
● Citrate original color
○ Green

RESULT:
Turns medium alkaline

Positive: Blue with growth

Negative: Green without growth

5
 Salmonella species & Shigella species
○ Proteus(rapid urease positive)
■ Paspas mag display ug positive result
PRINCIPLE:

● MEDIUM:
○ Christensen’s urea agar
● pH INDICATOR:
○ phenol red
■ Acid ph - yellow
■ Alkaline ph - shade of red
○ From light orange at pH 6.8 to magenta at pH
8.1
○ POSITIVE: magenta color

Escherichia coli: NEGATIVE

Salmonella typhimurium: POSITIVE
QUALITY CONTROL
● Positive : Enterobacter aerogenes
● Negative: Escherichia coli

Strong Urease production

Uninoculated Tube
Negative Urease production
Left: Positive
Mid: original color of Christensen's urea medium
Right: Nega

Left: negative Result

Mid: positive
Right: positive Positive: shade of red or magenta
● Presence of Growth. Negative: remain in color or yellowish or anything not
Which is more important? Growth or color change? magenta-ish and red-sh ganern
● GROWTH
○ Even if hindi nag change ng color ang citrate
but u see colonies growing in the slant =
positive QUALITY CONTROL:
○ Nabuhay ang organism; they were able to ● Positive: Proteus vulgaris
utilize citrate as its only source of carbon in the ○ Less than 24 hrs
test medium. ● Weak: Klebsiella pneumoniae
○ Kulang ang alkalinization. ○ Considered positive but the reaction is weak
IMViC Tests Results and slow (+24 hrs)
Thus: ● Negative: Escherichia coli
● Escherichia coli gives
○ ++--
● Enterobacter and Klebsiella give the reverse
○ --++
● Positive for both MRViC
○ hafnia alvei
○ proteus mirabilis

C. UREA HYDROLYSIS
● Christensen’s Method
○ Makes use of tube medium
○ Stuarts medium = broth
■ Both contains urea in its medium
● Determine the ability of an organism to produce the
● enzyme urease.
○ If u grow urea in those 2 medium, the urease
hydrolyzes the urea forming ammonia and
carbon dioxide (alkaline)
● Hydrolysis of urea produces ammonia and carbon
dioxide (alkaline)
● Differentiates Proteus(rapid urease positive) from

6
 Biochemical Test Part 2

D. PHENYLALANINE DEAMINASE TEST ○ H2S indicator: Ferrous ammonium citrate

● Detects if an organism possesses the enzyme ○ Sodium thiosulfate (source of inorganic sulfur)
phenylalanine deaminase ● LIA allows u to visualize 5 reactions
○ Phenylalanine = amino acid ○ Check if an organism ferments glucose =
○ Deaminase = enzyme that deaminates byproduct is acid
■ Deaminates - capable of removing the ■ pH indicator assumes the color of
amino portion yellow
● Principle: ■ Which part of LIA gets yellow?

E. Lysine Iron Agar Test (LIA)

● 2 Tests
● Principle:

● Amino acid is already incorporated in the culture medium

If the organism has phenylalanine deaminase, the
organism removes the amino portion of the
phenylalanine amino acid
Once removed, the by-product formed is phenylpyruvic
acid Amination of amino acid lysine
● How can we detect the production of phenylpyruvic acid?
○ Make use of a color developer, 10% Ferric If an organism has lysine deaminase it can remove the amino
chloride portion of lysine and result to many by products which react to
○ After addition of the reagent, it will result to the flavin mononucleotide which reacts with ferrous ammonium citrate
formation of dark green color = Red or Burgundy ; (slant portion)
● Reagent:
○ 10% Ferric chloride If an organism has the enzyme lysine decarboxylase; it can
● QUALITY CONTROL: remove the carboxyl portion of lysine and the by product is
○ Positive: Proteus mirabilis cadaverine (alkaline substance) = purple (butt portion)
○ Negative: Escherichia coli
○ Also positive:
■ Proteus, Providencia, Morganella ● Media: Lysine Iron Agar
(PPM) ● pH indicator: Bromcresol Purple (5.2/6.8)
● Genus that is always ○ Glucose is fermented if the butt portion is color
positive for any deaminase yellow (butt portion)
test ● Pyridoxal phosphate (coenzyme)
● All members of ● H2S Indicator: Ferrous ammonium citrate
Enterobacteriaceae ○ Blackening of medium
○ Not as good as TSI
● Sodium thiosulfate (source of inorganic sulfur)

LIA allows to visualize gas production; if there is a fermentation

process there is also gas production. Most common in TSI instead
of LIA which produces small or no gas at all.

Positive: Green Negative: Remains original color

E. LYSINE IRON AGAR

● 2 tests
● Principle:

● Pinaka left: original color ng LIA

○ Originally purple
● If glucose fermentation has transpired, nag yeyellow
yung butt
● If walang lysine decarboxylation, yellow lang
● If merong lysine decarboxylation na override ng
cadaverine yung acidity nito so nagiging alkaline to ulit
and so Bromcresol Purple will turn purple
● Media:
● 2nd tube: slant is purple meaning deaminase negative
○ Lysine Iron Agar
● 3rd tube: deaminase positive kasi red siya , and
○ Lysine = amino acid
decarboxylase negative kasi yellow
○ Has sugar
● You don’t have to tell if glucose has been fermented or
● pH Indicator: Bromcresol purple (5.2/6.8)
not in a LIA result form
■ Acidic pH (5.2 below) = yellow
● Ang ilagay nalang doon is if decarb (-) or decarb (-)
■ Alkaline pH (6.8 above) = purple
○ Pyridoxal phosphate (coenzyme)

7

● You can apply the writing system in TSI
● C: control tube
○ Originally colored purple
● Slant portion for deamination and butt portion for
deamination
● TT1: deaminase (-), Decarb (-)
○ Purple ang slant
○ Walang cadaverine sa butt
○ A typical organism with that reaction is
Citrobacter SEROLOGICAL TESTING
○ Although sometimes Citrobacter will have
hydrogen sulfide, remember SPACed
mnemonics
● TT2: deaminase (+), decarb (-)
○ Typical reaction of (PPM)
■ Proteus, Providencia, Morganella
○ Basta nag (+) sa deaminase, regardless of the
technique, it can only be any of the three
● TT3: deaminase (+), decarb (+)
○ Decarb (+) kasi purple naa siya
○ Acidity was override because of cadaverine
○ Bromcresol Purple nagging purple na because ANIMAL INOCULATION
alkaline na ang pH because of cadaverine
○ Typical reaction for E.coli and Enterobacter
● TT4: deaminase (-), decarb (+), H2S (+)
○ The assumption in case na dili klaro ang butt
kay natabuna sa hydrogen sulfide will be
decarb (+)
○ since H2S (+) na you have to inndiate, H2S (+)
○ A typical reaction for Salmonella and
Edwardsiella
LIA Quality Control
● Alkaline slant and butt: Escherichia coli
● Alkaline slant and butt: H2S positive: Salmonella
typhimurium
● Red slant, acid butt: Proteus mirabilis
● Most Commonly Used Tests:
○ TSI – STAB STREAK
○ LIA – STAB STAB STREAK
○ CITRATE – STREAK
○ UREASE – STREAK
○ SIM – STAB ¾
COMMERCIAL IDENTIFICATION SYSTEMS FOR VARIOUS
ORGANISMS
Get a colony from your unknown organism
↓
Mix with sterile saline to create a suspension
↓
Drop to tubes
↓
Incubate
↓
Observe for positive reactions
● Seven digit code: represents an identity of an organism
● Involves antigen and antibody reaction in vitro using the
patients serum
● No detecting of the actual organism but just the
immunologic reactions
● Examples:
○ ASO - infections from group A streptococci
○ RPR - for syphilis caused by T. pallidum
○ Widal Test - for presumptive diagnosis of
enteric fever or aka typhoid fever caused by
salmonella typhi
● makes use of some types of animal as live medium for
cultivation
● When do you use animal inoculation?
○ If the target organism does not grow in an
artificial culture medium
● EXAMPLES:
○ armadillo and mouse’s foot pads for the
isolation of Mycobacterium leprae
8
 MOLECULAR TECHNIQUES FOR MICROBIAL 2. Amplification
IDENTIFICATION AND CHARACTERIZATION ● “Pinaparami” ang genetic material

1. Hybridization
● Ex. Fluorescence In-Situ Hybridization (FISH)

Types:
● PCR based
○ Get a few sample of genetic material of the
organism that you want to cultivate
○ Then, subject it to repeated cycles of
denaturation , annealing, and extension
○ After a certain time of the PCR cycle, for just a
few strands, you have already created billion
copies of that original strand
○ And use those copies in identifying an organism
by looking at their genetic sequences
● Non PCR based
○ Does not make use of the polymerase chain
reaction
○ Ex:
■ isothermal amplification
■ Probe amplification

3. Sequencing and enzymatic digestion of nucleic acids

● The organism A is known (ex. S. aureus) and

organism B is what you are trying to identify
● We compare the genetic material of organism A
and B by doing hybridization
● You can only create bonds and hybridized if the
strands are complementary (if same species)
● Sequencing
Hydrolyze so that the complementary strands separate ○ Determination of the exact genetic sequence or
↓ nucleic sequence of a certain gene or a set of
Once separated, allow one strand of orgaims A and another strand genes.
of organism B to create bonds and pair with each other ● May use both hybridization and amplification techniques.
↓ ● In bacteria, we sequence 16S rRNA gene PCR
If they are able to create hydrogen bonds in between them, it amplification of variable region.
means that they are complementary ○ Does not evolve rapidly; remains constant.
↓ ● Procedure (refer to pic above)
If they are complementary, it means that they must be the same Sample collection
organism ↓
Extract DNA or RNA of the organism
↓
Extract 16S rRNA
↓
Determine the nucleotide sequence
↓
Compare nucleotide sequence to a known database
● If walang kapareha sa database, possibility is the
unknown bacteria that u have is a new organism.
RECAP:
● Microscopy and staining
● Culture methods
● Biochemical methods
● Serological testing
● Animal inoculation
● Molecular

9
 BACTERIOLOGY LEC

LECTURE 7: Physical and Chemical Methods of

Microbial Growth Control
Prof. Albaira Koudai, RMT
September 6, 2021
For updates and corrections → @mar4rii on Twitter

Control of Microbial Growth: Introduction ○ An agent that inhibits the growth of bacteria,but
● Early civilizations practiced salting, smoking, pickling, does not necessarily kill them.
drying, use of spices in cooking and exposure of food ○ Suffix stasis: To stop or steady.
and clothing to sunlight to control microbial growth. ● Germicide:
● In mid 1800s Semmelweis and Lister helped developed ○ An agent that kills certain microorganisms.
aseptic techniques to prevent contamination of surgical ○ Bactericide: An agent that kills bacteria. Most
wounds. Before then: do not kill endospores
○ Nosocomial infections caused death in 10% of ○ Virucide: An agent that inactivates viruses.
surgeries. Fungicide: An agent that kills fungi.
○ Up to 25% mothers delivering in hospitals died ○ Sporicide: An agent that kills bacterial
due to infection endospores or fungal spores.
○ Ignaz Semmelweis = handwashing Control of Microbial Growth: Rate of Microbial Death
○ Lister = use of carbolic acid to clean and ● When bacterial populations are heated or treated with
decontaminate antimicrobial chemicals, they usually die at a constant
rate.
Control of Microbial Growth:
Definitions
● Decontamination
○ All encompassing term used to describe a
process of inactivating or reducing
contaminants to an acceptable level
● Sterilization
○ Killing or removing all forms of microbial life
(including endospores) in a material or an
object.
○ Destruction of all forms of microbial life
including endospores
● Commercial Sterilization: Heat treatment that kills Several factors influence the effectiveness of antimicrobial
endospores of Clostridium botulinum, the causative treatment
agent of botulism, in canned food. 1. Number of Microbes
○ Does not kill endospores of thermophiles (not ● The higher number of contaminating microbe,
pathogens that may grow at temperatures the harder to kill
above 45C.) 2. Type of Microbes/nature of microbes
● Disinfection ● Organisms have different levels of resistance
○ Reducing the number of pathogenic 3. Environmental influences
microorganisms to the point where they no ● Presence of organic material (blood, feces,
longer cause diseases. saliva) tends to inhibit antimicrobials, pH etc.
○ Usually involves the removal of vegetative or 4. Time of Exposure
non-endospore forming pathogens. ● Chemical antimicrobials and radiation
○ Generally, disinfection is a less lethal process treatments are more effective at longer times
than sterilization ● In heat treatments, longer exposure
○ May use physical or chemical methods. compensates for temperatures
○ Disinfectant: Applied to inanimate objects
○ Antiseptic: Applied to living tissue (antisepsis).
○ Degerming: Mechanical removal of most
microbes in a limited area.
■ Example: alcohol swab on skin
○ Sanitization: reduction of the number of
microorganisms to safe hygienic level
■ E.g: Hot soap & water.
● Sepsis
○ Comes from Greek for decay or putrid.
Indicates bacterial contamination
● Asepsis
○ Absence of significant contamination
● Aseptic techniques
○ prevent contamination of surgical instruments,
medical personnel, and the patient during
surgery; prevent bacterial contamination in food
industry.
○ Ex. heating inoculating loop
● Bacteriostatic Agent:

1
 Used in canning industry
Order of resistance against biocidal process
1. Moist Heat: kills microorganisms by coagulating their
Microorganisms Examples proteins; much more effective than dry heat.
○ Boiling: heat to 100C or more at sea level. Kills
Prions CJD, BSE, Scrapie vegetative forms of bacterial pathogens, almost
all viruses, and fungi and their spores within
Bacterial spores Geobacillus, Clostridium 10minutes or less.
○ Endospores and some viruses are not
Protozoal oocysts Cryptosporidium destroyed this quickly. However brief boiling will
kill most pathogens
Helminth eggs Ascaris, Enterobius i. Hepatitis virus: can survive up to
30minutes of boiling.
ii. Endospores: can survive up to
Mycobacteria Mycobacterium
20hours or more of boiling
tuberculosis
Moist heat (continued):
● Reliable sterilization with moist heat requires
Small non-enveloped Poliovirus, Parvovirus temperatures above that of boiling water
viruses
● Autoclave: Chamber which is filled with hot
Protozoal cysts Giardia, Acanthamoeba steam under pressure.
○ Temperature of steam reaches 121C
Fungal spores Aspergillus, Penicillium at 15psi, 15minutes
■ For infectious medical
Gram-negative bacteria Pseudomonas, wastes, 132C for
Escherichia 30-60minutes
○ Most effective when organisms
Vegetative fungi and algae Aspergillus, Candida contact steam directly or are
contained in a small volume of liquid
Vegetative helminths and Ascaris, Cryptosporidium ■ All organisms and
protozoa endospores are killed within
15minutes
○ Require more time to reach center of
Large non-enveloped Adenovirus, Rotavirus solid or large volumes of liquid
viruses ○ For biohazardous trash and
heat-stable objects
Gram-positive bacteria Staphylo/Strepto/Enteroc
occus

Enveloped viruses HIV, HBV, HSV

● Prions = most resistant; faulty protein that can cause

disease
○ Ex. Creutzfeldt-Jakob disease (CJD), Bovine
spongiform encephalopathy (BSE), Scrapie
● Viruses can be enveloped or naked
○ Enveloped = has envelope coating
○ Naked = no envelope
○ When a virus is enveloped, mas madali patayin
Control of Microbial Growth: Rate of Microbial Death
Several factors influence the effectiveness of antimicrobial
treatment.
1. Number of Microbes
2. Type of Microbes
3. Environmental influences
a. Presence of organic material (blood, feces,
saliva tends to inhibit antimicrobials, ph etc.
4. Time of Exposure
a. Chemical antimicrobials and radiation
treatments are more effective at longer times
b. In heat treatments, longer exposure
compensates for lower temperature
Physical Methods of Microbial Control:
Heat:
● Kills microorganisms by denaturing their enzymes and
other proteins. Heat resistance varies widely among
microbes
● Thermal Death Point (TDP): Lowest temperature at
which all of the microbes in a liquid suspension will be
killed in ten minutes.
● Thermal Death Time (TDT); Minimal length of time in
which all bacteria will be killed at a given temperature.
○ Constant is temperature
● Decimal Reduction Time (DRT): Time in minutes at which
90% of bacteria at a given temperature will be killed.
● Pasteurization: Prevents the spoilage of
beverages. Used to reduce microbes
responsible for spoilage of beer, milk, wine,
juices, etc.
○ Denature the organisms but not the
protein of the milk; if protein is
destroyed the milk cannot be
consumed anymore
○ Classic method of pasteurization: milk
is exposed to 63C for 30 minutes
○ High temperature short time
pasteurization (HTST): used today.
Milk is exposed to 72C for 15 seconds
○ Ultra High Temperature
Pasteurization (UHT): Milk is treated
at 140C for 3 seconds and then cold
very quickly in a vacuum chamber.
○ Advantage: Milk can be stored at
room temperature for several months
2. Dry Heat: Kills oxidation effects
○ Oxidizes your organisms especially the outer
layer of your microorganisms like the cell wall.

2
 By oxidizing the cell wall you are killing the
microorganism as well
○ Direct Flaming: used to sterilize inoculating
loops and needles. Heat metal until it has a red
glow
○ Incineration: an effective way to sterilize
disposable items such as dressings and
biological waste
i. 870°C-980°C (high heat)
ii. Literally burning to ash. In this
process, toxic air emissions and the
presence of heavy metals may be
produced and because of this, the
use of incineration is limited
○ Hot Air Sterilization: Place objects in an oven.
Requires 2 hours at up to 160-180°C for
sterilization
i. Dry heat is much less effective than
moist heat and transfers heat to a
cool body less effectively than moist
heat so mas mabagal ang killing and
you have to compensate through
time
ii. Hot air sterilization is most commonly
applied in glasswares and any
heat-resistant material which are
subjected to drying
○ Filtration: removal of microbes by the passage
of a liquid or gas through a screen like material
with small pores
● Vaccines, enzymes,
antibiotics, and some culture
media
ii. High-Efficiency Particulate Filters
(HEPA)
○ Used in operating rooms and burn
units to remove bacteria 0.3 um size
from air
○ It can remove organisms from the air
as long as the bacteria is 0.3 um
bacteria in size
○ In the lab, the application of HEPA is
and biosafety cabinet
○ Almost all processes that we do in
bacte should be done inside the
biosafety cabinet
○ Because when you process bacteria
inside, your organisms will always
have the tendency to contaminate air
because they are lightweight
○ So if you process organisms inside
the biosafety cabinet, malimit niyo
lang siya inside
○ Biosafety cabinet has HEPA filter
inside
○ Contaminated air will enter HEPA and
HEPA will remove contaminating
bacteria sa air, HEPA will now
produce air na wala na ang organism
○ Once the air is circulated outside, the
air is HEPA filtered already
i. Membrane filters
○ Are constructed out of a wide range of
synthetic materials including:
● Cellulose acetate
● Cellulose nitrate (collodion)
● Polyamide (nylon)
● Polycarbonate
● Polypropylene
● Polytetrafluoroethylene
(Teflon)
○ Used in industry and research.
Different sizes
● 0.22 and 0.45um pores:
used to filter most bacteria.
Don’t retain spirochetes,
mycoplasmas and viruses
● 0.01um pores: retain all the
viruses and some large
proteins
○ Can be used when filtering liquid
○ Low Temperature: Effect depends on microbe
and treatment applied
i. Refrigeration:
● Temperature form 0 to 7°C
● Bacteriostatic effect
● Reduces metabolic rate of
most microbes so they
cannot reproduce or
produce toxins
● Toxin is a byproduct of their
metabolism and growth
Freezing
● Temperatures below 0C
● Flash Freezing: Does not kill most microbes
● Slow Freezing: More harmful because ice crystals
disrupt cell structure.
● Over a third of vegetative bacteria may survive 1 year.
● Most parasites are killed by a few days of freezing.
Desiccation:
● In the absence of water, microbes cannot grow or
reproduce, but some may remain viable for years.
● Susceptibility to desiccation varies widely:
○ Neisseria gonorrhoeae: Only survives about 1
hour.
○ Mycobacterium tuberculosis: May survive
several months.
○ Clostridium spp. and Bacillus spp: May
survive decades.
○ Viruses are fairly resistant because they do not
use water that much
Osmotic Pressure
● The use of high concentrations of high concentration of
salts and sugars in foods is used to increase the osmotic
pressure and create a hypertonic environment.
Plasmolysis:
● As water leaves the cell, plasma membrane shrinks away
from cell wall. Cell may not die, but usually stops
growing.
● Yeasts and molds: More resistant to high osmotic
pressures. - because of the added layers in their cell wall
● Staphylococci spp. that live on skin are fairly resistant
to high osmotic pressure.
Forms of Radiation
Radiation: Three types of radiation kill microbes:
1. Ionizing Radiation:
● gamma rays, X rays, electron beams, or higher
energy rays.
● Have short wavelengths (less than 1
nanometer).
● The shorter the wavelength, the higher the
frequency
○ Dislodge electrons from atoms and
form ions.
3
 ○ Cause mutations in DNA and produce
peroxides (toxic to cells)
○ Sterilize pharmaceuticals and
disposable medical supplies.
● Disadvantages:
○ Penetrates human tissues.
○ May cause genetic mutations in
humans.
2. Ultraviolet light (Nonionizing Radiation):
● The wavelength is longer than 1 nanometer.
● Damages DNA by producing thymine dimers
○ Trigger mutations
● Used to disinfect operating rooms, nurseries,
cafeterias, biosafety cabinet light
● Disadvantages:
○ Damages skin, eyes.
○ Doesn’t penetrate paper, glass, and
cloth.
3. Microwave Radiation:
● Wavelength ranges from 1 millimeter to 1 meter
● Heat is absorbed by water molecules
● May kill vegetative cells in moist foods
○ There is water thus transfer of heat is
more effective
● Bacterial endospores, which do not contain
water, are not damaged by microwave
radiation.
● Solid foods are unevenly penetrated by
microwaves.
● Trichinosis outbreaks have been associated
with pork cooked in microwaves.
Chemical Methods of Microbial Control Types of Disinfectants
1. Phenols and Phenolics:
● Phenol (carbolic acid) Rarely used today because it is a
skin irritant and has strong odor.
○ Used in some throat sprays and lozenges.
○ Acts as local anesthetic.
● Phenolics are chemical derivatives of phenol
○ Cresols: Derived from coal tar o-phenylphenol
– lysol
○ Bisphenols (hexachlorophene - pHisoHex):
■ Effective against gram posiutive.
■ Used in nurseries.
■ Excessive use in infants may cause
neurological damage.
○ Triclosan (dichlorophenoxy phenol) –
antibacterial (broad spectrum)
● Destroy plasma membranes and denature proteins.
● Advantages: Stable, persist for long times after applied,
and remain active in the presence of organic compounds
1. Halogens: Effective alone or in compounds.
A. Iodine: alters protein synthesis and alters cell membranes
● Tincture of iodine (alcohol solution) was one of the first
antiseptics used.
○ Combines with amino acid tyrosine in proteins
and denatures proteins.
○ Stains skin and clothes, somewhat irritating.
● Iodophors: iodine plus organic (may be solubilizing
agents) from which iodine is slowly released, take
several minutes to act. Used as skin antiseptic in surgery.
Not effective against bacterial endospores
○ Betadine
■ Povidone - organic compound
○ Isodine
2. Halogens
: Effective alone or in compounds.
B. Chlorine:
● When mixed in water forms hypochlorous acid:
● Cl2 + ____ ------> H+ + Cl- + HOCl
● Used to disinfect drinking water, pools, and
sewage.
● Disadvantage: Chlorine is easily inactivated by
organic materials.
○ Vit C - can neutralize or inactivate
chlorine
● Sodium hypochlorite (NaOCl): Is active
ingredient of bleach
● Chlorine dioxide: disinfection of water and
surfaces
● Chloramines: Consist of chlorine and ammonia.
Water disinfection. Less effective as
germicides.
3. Alcohols
● Kill bacteria, fungi, but not endospores or naked viruses
● Act by denaturing proteins and disrupting cell
membranes
● Volatile
○ Evaporate, leaving no residue.
○ An advantage because it is less irritating
● Used to mechanically wipe microbes off skin before
injections or blood drawing.
● Not good for open wounds, because it causes proteins to
coagulate; does not promote healing of the wound
○ Ethanol: Drinking alcohol. Optimum concentration
is 70%
■ Not lower, not higher than 70%
■ 30% water is needed for the
coagulation process to proceed to the
denaturing effect
■ If absolute alcohol, it evaporates in a
faster time
○ Isopropanol: Rubbing alcohol. Better disinfectant
than ethanol
■ Less irritating to living tissues and
retains moisture
■ Can be antiseptic and disinfectant
4. Heavy Metals: combine with Sulfhydryl groups
● Denature proteins of microbes
● Include copper, selenium, mercury, silver, and zinc.
● Oligodynamic action: Very tiny are effective.
○ Oligodynamic action - ability of small amounts of any
metal to exert a lethal effect to bacterial cells
A. Silver:
● 1% silver nitrate used to protect
infants against gonorrheal eye
infections until recently.
● If a mother is infected with gonorrhea
and will be giving birth naturally, once
the baby passes through the vagina,
the organism can be transferred from
the vaginal walls to the eye of the
baby
● The baby may have gonococcal eye
infections
● Now, we use actual antibiotics since
silver is metal
B. Mercury
● Organic mercury compounds like
merthiolate and mercurochrome are
used to disinfect skin wounds.
● Inorganic mercuric chloride –
bacteriostatic
C. Copper
● Copper sulfate is used to kill algae in
pools and fish tanks.
○ The reason why the
swimming pool is light blue
in color
D. Selenium
● Kills fungi and their spores. Used for
fungal infections.
● Also used in dandruff shampoos
○ Since dandruff is caused
by fungus
E. Zinc
● Zinc chloride is used in mouthwashes.
● Zinc oxide is used as an antifungal
4
 agent in paints. ● Used in acne medications.
D. Peracetic Acid:
Surface-active agents – soaps and detergents ● One of the most effective liquid sporicides available.
5. Quaternary Ammonium Compounds (Quats): ● Sterilant :
● Widely used surface active agents; cationic detergents ○ Kills bacteria and fungi in less than 5 minutes.
(positively charged detergents). ○ Kills endospores and viruses within 30 minutes.
● Effective against gram positive bacteria, less effective ● Used widely in disinfection of food and medical
against gram-negative bacteria. instruments because it does not leave toxic residues.
● Also destroy fungi, amoebas, and enveloped viruses but Efficiency of Different Chemical Antimicrobial Agents
not endo and myco
● Zephiran (Topical) and Cepacol (Throat Lozenges)
● Pseudomonas strains that are resistant and can grow in
presence of Quats are a big concern in hospitals.
● Advantages: Strong antimicrobial action,
colorless,odorless, stable, and nontoxic.
● Disadvantages: Form foam. Organic matter interferes
with effectiveness.
○ Organic compounds - may neutralize them.
● Neutralized by soaps and anionic agents.
Types of Disinfectants
Aldehydes:
● Include some of the most effective antimicrobials.
● Inactivate proteins by forming covalent crosslinks with
several functional groups.
A. Formaldehyde gas:
● Excellent disinfectant.
● Commonly used as formalin, a 37% aqueous solution.
● Formalin was used extensively to preserve biological
specimens and inactivate viruses and bacteria in
vaccines.
○ Formalin - chemicals to inactivate microbes.
● Irritates mucous membranes, strong odor.
● Also used in mortuaries for embalming.:
B. Glutaraldehyde:
● Less irritating and more effective than formaldehyde
● One of the few chemical disinfectants that is a Sterilizing
agent.
● A 2% solution of glutaraldehyde (Cidex) is:
○ Bactericidal, tuberculocidal, and virucidal in 10
minutes.
○ Sporocidal in 3 to 10 hours.
● Commonly used to disinfect hospital instruments.
● Also used in mortuaries for embalming.
Gaseous Sterilizers:
● Chemicals that sterilize in a chamber similar to an
autoclave
● Denature proteins, by replacing functional groups with
alkyl groups.
A. Ethylene Oxide:
● Kills all microbes and endospores, but requires exposure
of 4 to 18 hours.
● toxic and explosive in pure form.
● Highly penetrating. EtO gas is carcinogenic,explosive
and mutagenic.
○ ETO - limit usage.
● Most hospitals have ethylene oxide chambers to sterilize
mattresses and large equipment.
Peroxygens (Oxidizing Agents):
● oxidize cellular components of treated microbes.
● Disrupt membranes and proteins.
A. Ozone:
● Used along with chlorine to disinfect water.
● More effective killing agent than chlorine, but less stable
and more expensive.
● Highly reactive form of oxygen.
○ Potentially irritating.
● Made by exposing oxygen to electricity or UV light.
B. Hydrogen Peroxide:
● Used as an antiseptic.
● Not good for open wounds because quickly broken down
by catalase present in human cells.
● Effective in disinfection of inanimate objects.
● Sporicidal at higher temperatures.
● Used by food industry and to disinfect contact lenses.
C. Benzoyl Peroxide:

5
 WEEK 5: ANTIMICROBIAL SUSCEPTIBILITY TESTING
INHIBITORS OF CELL MEMBRANE FUNCTION
ANTIMICROBIAL ACTIVITY • Lipopeptides
Classifications of antibacterial agents
• Bactericidal INHIBITORS OF PROTEIN SYNTHESIS
o Agent that kills certain microorganisms
o Ex. Cell wall active agents • Chloramphenicol
▪ Concentration-dependent killing o Binds to 50S subunit, inhibits peptide bond
▪ Time-dependent killing formation
• Bacteriostatic • Tetracycline
o Agent that inhibits growth of bacteria o Interfere with attachment of tRNA to mRNA-
▪ Doesn’t necessarily kill the organism ribosome complex by binding reversibly to 30S
o Ex. Drugs that inhibit protein synthesis ribosomal subunit
• Aminoglycosides
Bactericidal Bacteriostatic o Gentamicin, streptomycin (30S)
• Aminoglycosides • Chloramphenicol ▪ Interfere with the initial step of
• Beta-lactams • Erythromycin and other
protein synthesis → changing the
• Vancomycin macrolides
shape of the 30S → causing genetic
• Daptomycin • Clindamycin
code on the mRNA to be read
• Teicoplanin • Sulfonamides
incorrectly
• Quinolones (e.g., • Trimethoprim
▪ Gentamicin is produced by the
ciprofloxacin, • Tetracyclines
levofloxacin) • Tigecycline fermentation of Micromonospora
• Rifampin • Linezolid purpurea or M. echinospora
• Metronidazole • Quinupristin/dalfopristin ▪ Streptomycin source is the
• Broad Spectrum actinomycin Streptomyces griseus
o Drugs that are active against a wide range of • Macrolides
microorganisms o Erythromycin
▪ Gram + & Gram - ▪ Binds to 50S → prevents translocation-
• Narrow Spectrum movement of ribosome along mRNA
o Drugs with limited spectrum of action ▪ Bacteriostatic antibiotic drug produced
▪ Penicillin G – only active against Gram by a strain of Saccharopolyspora
+ bacteria erythraea
• Lincosamide
o Clindamycin
ACTIONS OF ANTIMICROBIAL DRUGS ▪ Binds to 50S ribosomal subunit
• Inhibits cell wall synthesis ▪ Natural antibiotic produced by the
actinobacterium Streptomyces
• Inhibits cell membrane function
lincolnensis
• Inhibits protein synthesis
• Oxazolidinones
• Inhibits nucleic acid synthesis
o Linezolid
• Inhibitors of other metabolic processes
▪ Binds with ribosomal RNA of 50S
subunit
ANTIBACTERIAL ANTIBIOTICS

INHIBITORS OF CELL WALL SYNTHESIS INHIBITORS OF NUCLEIC ACID SYNTHESIS

• Beta-Lactams • Fluroquinolones, Metronidazole
o Penicillins o Fluoroquinolones will block DNA synthesis by
o Cephalosporins binding with the topoisomerase II and
▪ Come from the fungus Acremonium topoisomerase IV
o Monobactams ▪ Ciprofloxacin
▪ Come from Chromobacterium ▪ Ofloxacin
violaceum ▪ Levofloxacin
o Carbapanems o Metronidazole has direct interactions between
• Fosfomycin the activated drug and the DNA. It will result to
• Glycopeptides and Lipoglycopeptides the breakage of the DNA strand
• Rifampin
o Binds to the DNA – dependent RNA polymerase
• Penicillin
& inhibit synthesis of RNA
o From Penicillium notatum
o Act by inhibiting transpeptidases (enzymes that
catalyze cross-linking steps in peptidoglycan INJURY TO THE PLASMA MEMBRANE
synthesis)
• Changes in the plasma membrane → loss of important
• Isoniazid (INH)
metabolites
o Also known as isonictonic acid hydrazide
• Polymyxin B and E (Colistin)
o Inhibits mycolic acid synthesis
o Comes from the bacterium Bacillus polymyxa
▪ Mycobacterium tuberculosis
o Disrupts bacterial cell membrane
 o Topical with bacitracin & neomycin in Over-
the-counter preparations
▪ Bacitracin comes from Bacillus subtilis
(Tracy strain)
INHIBITORS OF OTHER METABOLIC PROCESSES
• Sulfonamides: dihydropteroate synthase
o Inhibits production of Dihydropteroic acid
o Example: Sulfamethoxazole
• Trimethoprim: dihydrofolate reductase
o Inhibits production of Tetrahydrofolic acid
• Targets folic acid pathways
• Differs in enzyme inhibited
• Inhibit specific metabolic steps necessary for bacterial
growth
• Trimethoprim-sulfamethoxazole
EFFECTS OF COMBINATIONS OF DRUGS
• Synergy
o The activity of the antimicrobial combination is
substantially greater than the activity of the
single most active drug alone
• Indifference
o The activity of the combination is no better or
worse than the single most active drug alone
• Antagonism
o The activity of the combination is substantially
less than the activity of the single most active
drug alone
▪ Usually happens when two different
doctors prescribe the drugs
REASONS FOR USE OF MULTIPLE THERAPY
• Polymicrobial infections – different antimicrobial profile
• Achieving more rapid bactericidal activity than use of a
single agent
• No single agent is lethal
• Minimizing the risk of resistant organisms emerging
during therapy
ANTIMICROBIAL SUSCEPTIBILITY TESTING
• PRINCIPLE
o It measures the ability of an antibiotic or other
antimicrobial agent to inhibit bacterial growth
in vitro
• PRIMARY GOAL
o To determine whether the bacterial isolate is
capable of expressing resistance to the
antimicrobial agents selected for treatment
• PURPOSE
o To guide the clinician in selecting the
appropriate antimicrobial agent
o To gather epidemiological data on microbial
resistance
STANDARDIZATION
• Purposes
o To optimize bacterial growth conditions
o To optimize conditions for maintaining
antimicrobial integrity and activity
o To maintain reproducibility and consistency in
the resistance profile
• Factors
o Bacterial inoculum size
o Growth medium: Mueller-Hinton (MH) base
▪ pH
▪ Composition: cation concentration,
blood and serum supplements,
Thymidine content
▪ Thickness
o Incubation atmosphere
o Incubation temperature (35°-37°)
o Incubation duration (18-24 hours or 24 above)
o Antimicrobial concentrations
• Limits of Standardization
o Antibiotic diffusion into tissues and host cells
o Serum protein binding of antimicrobial agents
o Drug interactions and interference
o Status of patient defense and immune systems
o Multiple simultaneous illnesses
o Virulence and pathogenicity
o Site and severity of infection
• General Considerations
o Inoculum Preparation – key to any
antimicrobial susceptibility testing methods
▪ 2 important requirements
• Pure culture
• Standard-sized inoculum
▪ The pure inoculum can be obtained by
different methods
• Selecting 4-5 colonies of the
same morphology
• Inoculating them into a broth
medium or 0.9% saline
solution.
• Allowing the culture to
achieve active growth
o We determine active
growth by turbidity
▪ For most organisms, inoculum
preparation requires 3-5 hrs of
preparation, but it still depends on the
organism
 ▪ Currently, the laboratories use BBL
Prompt Inoculation System to allow
direct standardization of an inoculum
of a rapidly growing bacteria without
turbidity adjustment or pre-incubation
Three General Methods
▪ Assessment of turbidity may vary from
one person to another, that’s why we
need the second requirement of
inoculum preparation, which is the use
of standard-sized inoculum.
▪ The use of standard-sized inoculum is
accomplished by comparing the
turbidity of the organism suspension
with the turbidity standard
• McFarland Turbidity
Standard
o Prepared by mixing
1% H2SO4 and
1.175% BaCl
o The 0.5 McFarland
Standard,
commercially
available, provides
an optical density
comparable to the
density of the
bacterial suspension
of 1.5 x 108 CFU/mL
o Selection of Antimicrobial Agents for Testing
▪ Antimicrobial battery or panel
• Is where the antimicrobial
agents for testing against a
particular bacterial isolate are
chosen
▪ List of antimicrobial agents from CLSI
Criteria for Antimicrobial Battery Content and Use
Organism Identification or Group
Antimicrobials to which the organism is intrinsically resistant are
routinely excluded from the test battery (e.g., vancomycin versus
gram-negative bacilli). Similarly, certain antimicrobials were
developed specifically for use against particular organisms, but not
against others (e.g., ceftazidime for use against Pseudomonas
aeruginosa but not against Staphylococcus aureus); such agents
should be included only in the appropriate battery
Acquired Resistance Patterns Common to Local Microbial Flora
If resistance to a particular agent is common, the utility of the agent
may be sufficiently limited, and routine testing is not warranted.
More potent antimicrobials are then included in the test battery.
Conversely, more potent agents may not need to be in the test
battery if susceptibility to less potent agents is highly prevalent.
Antimicrobial Susceptibility Testing Method Used
Depending on the testing method, some agents do not reliably detect
resistance and should not be included in the battery
Site of Infection
Some antimicrobial agents, such as nitrofurantoin, achieved effective
levels only in the urinary tract and should not be included in batteries
tested against bacterial isolates from other body sites (i.e., the agent
must be able to achieved anatomic approximation)
Availability of Antimicrobial Agents in the Formulary
Antimicrobial test batteries are selected for their ability to detect
bacterial resistance to agents used by the medical staff and accessible
in the pharmacy
TESTING METHODS
• Depends on clinical need, accuracy, convenience, and
other factors
I. Direct measurement of activity of one or more
antimicrobial agents against a bacterial isolate
II. Direct detection of the presence of a specific resistance
mechanism in a bacterial isolate
III. Special methods that measure complex antimicrobial-
organism interactions
I – Direct Measurement of Antimicrobial Activity
• You test the infecting bacterium with the antimicrobial
agents of interest in the same in vitro environment
• It determines the effect of the drug’s presence on the
bacterial growth or viability
• Measures levels of effect on bacterial growth and the
organism’s resistance or susceptibility to each agent
• I.1: Conventional susceptibility methods
o I.1a: Broth dilution
o I.1b: Agar dilution
o I.1c: Disk diffusion
• I.2: Commercial susceptibility testing systems
o I.2a: Broth Microdilution Methods
o I.2b: Agar Dilution Derivations
o I.2c: Diffusion in Agar Derivations (Etest)
o I.2d: Automated Antimicrobial Susceptibility
Test Systems
• I.3: Special screens and indicator tests
Conventional Susceptibility Methods
I.1a: BROTH DILUTION
• Each antimicrobial agent is tested using a range of
concentration commonly expressed in micrograms of
active drug per mL
• Involves challenging the organism of interest with
antimicrobial agents in a liquid environment
• Testing: µg/mL
o MIC: lowest antimicrobial concentration that
completely inhibits visible bacterial growth
o MBC: lowest antimicrobial concentration that
kills bacteria
• There are changes/differences in the concentrations
used. It depends on the criteria such as
o Safest therapeutic concentration that is
possible in a patient serum
o Based on the level of drug required to reliably
detect a particular resistance mechanism
• Concentration range often varies from one drug to the
other based on the organism, source, and therapeutic
concentration possible on the patient serum
o Example: When you use the drug cefepime for
Streptococcus pneumoniae in CSF, it uses a
maximum concentration of 2 µg/mL. But, for
non-meningits isolates, we used 4 µg/mL
instead of 2.
• Two general categories
o Microdilution
▪ Total volume: 0.05 to 0.1 mL
o Macrodilution
▪ Total volume 1 mL or greater

• A single microtiter tray used
in microdilution, which allows the
medtech to perform testing of
several antibiotics or doubling
dilution of antimicrobial agents at
several different concentration or
smaller volume
• The need for multiple large
test tubes for microdilution method makes it more
cumbersome and labor-intensive
o Rarely used in most clinical laboratories and
subsequent discussion about broth dilution
focuses on the microdilution approach
• For macrobroth dilution susceptibility testing, we use
Mueller-Hinton broth with calcium and magnesium
added.
o For testing S. aureus, with methicillin, oxacillin,
and nafcillin, the medium should also contain
2% sodium chloride
• You need to prepare 13 tubes and
o Pipet 1 mL of MH broth into tube 2 until tube
10.
o Next, you add 2 mL MH broth in tube 12.
o Pipet 1 mL of working antibiotic solutions into
tubes 1, 2, and 13.
o Perform serial dilution (2-fold) from tubes 2 to
10, then discard.
o Add 1 mL of standardized inoculum to tubes 1
until tube 11
• After incubation at 35°C for 18-24 hrs, take note of
tubes 11, 12, and 13 to validate your test. Tube 11 only
contains the inoculum (inoculum control). Tube 12 only
contains the MH broth (broth control). Tube 13 only
contains the antibiotic solution (antibiotic control)
• Examine tubes for visual turbidity
o Tube 11 – positive for growth through turbidity
o Tube 12 – negative for growth
o Tube 13 – negative for growth
• After incubation, the first tube showing no visible
growth is considered MIC. Then subculture all tubes
showing no visible growth in culture media. After that,
incubate again for 18-24 hours. Take note of the first
plate showing the lowest antibiotic concentration, which
is considered the MBC, results to 99% killing.
I.1b: AGAR DILUTION
• Antimicrobial concentrations and organisms are bought
together in an agar-based medium
• Uniformly diluted antibiotic
concentration are incorporated in
Mueller-Hinton
• Surface of each plate: inoculated
with 1 x 104 CFU
I.1c: DISK DIFFUSION
• Limitations of dilution methods arise due to more
antimicrobial agents created. Testing of several
antimicrobial agents in one plate is possible in this
method
• Detects antimicrobial resistance by
challenging bacterial isolates with
antibiotic disks
• Antibiotic paper discs are placed on agar
medium seeded with the test organism
• When disk containing unknown concentration of an
antimicrobial agent are placed on the surface of the
freshly inoculated plate, the agent immediately diffuses
into the agar and establishes a concentration gradient
around the paper disc
• Zone of inhibition: measured in millimeters
• Also known as Kirby-Bauer Technique
o Performed by inoculating a standardized
suspension of bacteria on a Mueller-Hinton
agar
1. Inoculum preparation
• From a primary isolation media (ex. BAP),
you transfer 4-5 colonies into
approximately 5 mL of TSB (Trypticase Soy
Broth) by touching the top of the colony
using a sterilized cold wire loop or
disposable loop. In other laboratories, they
use saline solution
• Once colonies are transferred, compare
your TSB with your McFarland turbidity
standard. If still less turbid, you may
incubate the TSB for 35°C for 2-8 hrs until
the turbidity is achieved
2. Inoculation of agar plates
• The plate surface is inoculated using a
swab that has been submerged in a
bacterial suspension standardized to match
0.5 McFarland Turbidity standard.
• After matching, submerge the swab in the
bacterial suspension and swab it in 3
directions on the surface of the Mueller-
Hinton agar plate
o Rotate the plate 60°
o Ensure even & complete
distribution of inoculum
3. Application of antibiotic disks to agar surface
• Allow the inoculum to dry for 3-15
minutes.
• The disks with a known concentration of an
antimicrobial agent is placed on a surface
of a freshly inoculated plate. The agent
begins to diffuse into the agar and
establishes a concentration gradient
around the paper disc
4. Incubation of plates (inverted position)
• Upon incubation, the bacteria grow on the
surface of the plates
• It is inverted to prevent the accumulation
of moisture on the agar surface which
would interfere the interpretation
5. Reading & interpretation of results
• After incubation, the diameter around the
zone of inhibition around each disk is
measured in millimeters
• Factors
o pH of the media
▪ Should be 7.2-7.4
▪ Too acidic pH (low pH) can lose the
potency of certain drugs
(aminoglycosides, quinolones,
microlides), while some certain drugs
show excess activity (tetracycline)
▪ Too high pH, reverse reactions would
occur
o Thickness of the agar
▪ 4 mm
 o Growth rate of the microbe swarming haze is ignored and the zones are
o Amount of inoculum measured at the point where growth is
▪ Standardized at 1.5 x 108 CFU/mL inhibited
o Suitability of the medium used
o Diffusability of the antibiotic • Kirby-Bauer
o Discrepancy in inoculum and incubation

• Appropriate Media for Sensitivity Testing

o Mueller-Hinton agar (MH)
▪ Best for routine susceptibility testing
of non-fastidious bacteria
▪ Used for organisms such as
Enterobacteriaceae, Pseudomonas
aeruginosa, Enterococci
o MH with 2% NaCl – Staphylococcus o Susceptible
o MH with 5% sheep blood ▪ Agent may be an appropriate choice
▪ S. pneumoniae for the treatment of infection caused
▪ β-hemolytic Streptococcus by the organism
o HTM (Haemophilus Test Medium) – H. ▪ Bacterial resistance is absent at
infleunzae clinically significant level
o GC agar with PolyVtex o Intermediate
▪ N. gonorrheae ▪ Potential utility of the antimicrobial
agent in buddy sites where it may be
• Disk Application concentrated or if high concnetrations
o Apply disks within 15 are used
minutes after inoculation of ▪ Possible effectiveness of antimicrobial
agar surface agent against the isolate but less than
o Proper spacing of antibiotic susceptible
disks o Resistant
o Not be crowded because it may produce ▪ Indicates that the agent is not or may
overlapping zone of inhibitions not be an appropriate choice of
o Once the antibiotic disk is placed on the agar, treatment
the agent immediately diffuses (touch move) • The organism is not inhibited
o Use a sterile forcep to place the antibiotic disk with serum achievable levels
of the drug or
• Incubation • The test result correlates with
o Routine susceptibility testing of common resistance
aerobic organisms requires 18-24 hours Organism Antimicrob Zone diameter Equivalent MIC
incubation period @ 37°C ial agent Breakpoint
▪ For Staphylococcus aureus and (disk (µg/mL)
content)
Enterococcus requires a full 24 hours
R I S R S
incubation S. aureus 1 µg ≤ 10 11-12 ≥ 13 ≥4 ≤2
o For fastidious organisms: 35°C with 5-7% CO2 CNS 1 µg ≤ 17 ≥ 18 ≥ 0.5 ≤ 0.25
▪ Haemophilus influenzae o Advantages
▪ Neisseria gonorrhoeae ▪ Convenience & user friendly
▪ Streptococcus pneumoniae ▪ Up to 12 antimicrobial agents can be
tested
• Reading & Interpretation of Results ▪ Minimal use of extra materials &
o Before results are read, the plate is examined devices
for confluent lawn of growth ▪ MOST COMMONLY USED METHOD
o If growth between inhibitory zones around o Disadvantages
each disk is poor and non-confluent, the test ▪ Lack of interpretative criteria
should be repeated ▪ Inability to provide more precise data
▪ It means that there is insufficient regarding the level of an organism’s
inoculum resistance or susceptibility as can be
o Measure the zone of inhibition around each provided by dilution methods
disk using a caliper (ruler if none)
o Record in mm
o Compare results using NCCLS interpretative • Agar Overlay Disk Diffusion Method
chart
▪ National Committee for Clinical
Laboratory Standards
o Report as susceptible, intermediate or
resistant
o Some motile organisms swarm over the surface
of the plate, it complicates clear interpretation
of the zone boundaries. In these areas, the
 Commercial Susceptibility Testing Systems
I.2a: BROTH MICRODILUTION METHODS
• Microdilution panels already prepared and formatted
• Commercial panels:
o Designed to receive the standard inoculum
o Incubated using conditions and durations used
in conventional methods
• Require overnight incubation
• Uses CLSI interpretative criteria
• Reading of panels: semi-automated reading devices
I.2b: AGAR DILUTION DERIVATIONS
• Uses instrument to apply the antimicrobial agent to the
surface of an already-prepared agar plate
• Pattern: Concentric spiral
o From the center of the plate, the instrument
deposits the highest concentration of
antibiotic. The drug application proceeds into
the periphery
• Diffusion of drug: concentration gradient (high at
center) to (low at periphery)
• Distance is measured from edge (+ growth) of the plate
to the center (inhibited growth) of the plate
• The distance measured is calculated used to calculate
the MIC
I.2c: DIFFUSION IN AGAR DERIVATIONS
• Etest (Epsilometer test)
o A quantitative technique for
determining antimicrobial
susceptibility of both non-
fastidious and aerobic
bacteria
o The test combines convenience of this diffusion
pattern with the determination of MIC
o Principle: predefined antibiotic gradient on a
plastic strip to generate MIC value
o Detect and confirm low levels of resistance
o Uses rectangular plastic strips
▪ With predetermined gradient of
antibiotic concentration that
correspond to MIC dilution
▪ One side: contains antimicrobial agent
concentration gradient
▪ Other side: numeric scale that
indicates drug concentration
o Inoculation
▪ Mueller-Hinton plates are inoculated
as for this diffusion.
▪ Inoculum suspension is inoculated on
the entire agar surface using a sterile
cotton swab (using the same step in
Kirby-Bauer technique)
o Application of Strips
▪ When the inoculated agar surface is
completely dry, the Etest strips are
placed on the inoculum lawn using
applicator or forceps
▪ Several strips may be placed radially
on the same plate so that multiple
antimicrobial agents can be tested
against a single isolate
▪ After placing the strip on the agar
surface, there is an immediate release
of antibiotics from the plastic carrier
surface into the agar surface (touch
move)
o Reading
▪ Bacterial growth becomes visible on
the plate with symmetrical inhibition
ellipse along the strips
▪ The MIC value from the scale in terms
of µg/mL is seen on the inhibition
ellipse edge that intersects the e-strip
▪
o Interpretation
▪ The MIC is read at the point of
intersection between the ellipse edge
and the etest strip.
▪ The same MIC interpretative criteria
used for dilution methods as provided
by CLSI guidelines are used and are
interpreted as susceptible,
intermediate, or resistant
I.2d: AUTOMATED SYSTEMS
o Vitek 2
o MicroScan WalkAway System by Beckman
Coulter
o Phoenix System by BD Microbiology Systems
• These systems may vary with the extent of automation
of inoculum preparation, in the way they inoculate the
bacterial suspension, in methods used to detect growth,
and the algorithms for interpretation.
• II – Direct Detection of Specific Resistance Mechanisms
o II.1: Phenotypic methods
▪ II.1a: Beta-Lactamase Detection
• Play a key role in bacterial
resistance to beta-lactam
agents such as Penicillin,
cephalosporin, and
monobactams
• When the presence or
absence is established, the
resistance profile is
generated, meaning that
there is no more need to test
several different
antimicrobial agent
▪ II.2b: Chloramphenicol
Acetyltransferase (CAT) Detection
o II.2: Genotypic methods
• III – Special Methods
o These tests are designed to measure
antibacterial effect of combination therapy
with antimicrobial agent
o III.1 Bactericidal Tests: determines ability of
antimicrobial agents to kill bacteria
▪ III.1a: Minimal Bactericidal
Concentration (MBC)
 • Assessed after incubation and
determination of MIC by an
aliquot from each tube/well
in dilution series
demonstrating inhibition of
visible bacterial growth is
subcultured to an enriched
medium (sheep blood agar)
• After overnight incubation,
the plates are examined, and
CFUs are determined
▪ III.1b: Time-Kill Studies
• Exposes a bacterial isolate to
a concentration of antibiotic
in broth medium and
measure the rate of killing
over a specified time of
period
▪ III.1c: Serum Bactericidal Tests
(Schlichter Test)
• Analogous to MIC-MBC test,
except the test medium used
is the patient’s serum
containing the therapeutic
antimicrobial agent that the
patient has been receiving
• 2 serum samples are
collected (Uses TROUGH
specimen and PEAK
specimen)
o First sample is
collected just before
or within 30 minutes
the patient is to
receive the next
antimicrobial dose
(TROUGH specimen)
o The other sample is
collected after
microbial agent is
given. This sample
contains the highest
serum antimicrobial
concentration (PEAK
specimen)
o III.2: Tests for Activity of Antimicrobial
Combinations
Specific Testing Procedures for Organisms of Medical Interest
METHICILLIN-RESISTANT S. aureus (MRSA)
• S. aureus strains are treated with penicillin. However,
there are strains that are resistant to it, so they are
treated with penicillinase table penicillin such as
oxacillin and methicillin.
• MRSA infection is caused by a strain of Staphylococcus
bacteria that’s become resistant to oxacillin and
methicillin
• Staphylococcal resistance to oxacillin/methicillin occurs
when Staph produces an altered penicillin-binding
protein, PBP2a, which is encoded by the mecA gene
o The variant penicillin-binding protein binds
beta-lactams with lower avidity, which results
in resistance in resistance to this class of
antimicrobial agents
• 2 types of MRSA
o Health care-associated MRSA (HA-MRSA)
▪ Associated with invasive procedures or
devices, such as surgeries, intravenous
tubing or artificial joints
▪ Occur in people who have been in
hospitals, nursing homes and dialysis
centers, and other healthcare worker
setting
o Community-associated MRSA (CA-MRSA)
▪ Often begins as a painful skin boil. It’s
spread by skin-to-skin contact
▪ At-risk populations include groups
such as high school wrestlers, child
care workers and people who live in
crowded conditions
• Tests for MRSA
o Clinical and Laboratory Standards Institute
(CLSI) recommends:
▪ Broth microdilution testing
▪ Cefoxitin disk screen test
▪ Latex agglutination test for PBP2a
▪ Plate containing 6 µg/mL of oxacillin in
Mueller-Hinton agar supplemented
with 4% NaCl as alternative methods
of testing for MRSA
▪ Chromogenic agars that can be used
for MRSA detection
▪ Nucleic acid amplification tests, ex.
Polymerase chain reaction (PCR)
• To detect mecA gene, is the
most common gene that
mediates oxacillin resistance
in staphylococci
• Method
o Cefoxitin disk screen test
o ≤ 21 mm
Test Inoculum
↓
Inoculation on MH (with 2% NaCl)
↓
Application of Cefoxitin Disk (30 mcg)
↓
Incubation at 35°C for full 24 hours
↓
Reading and Interpretation of results
• Breakpoints for testing susceptibility of staphylococci
to oxacillin
Interpretative Criteria (in µg/mL) for Oxacillin MIC Tests
Susceptible Intermediate Resistant
S. aureus ≤ 2 µg/mL N/A ≥ 4 µg/mL
CoNS ≤ 0.25 µg/mL N/A ≥ 0.5 µg/mL
Interpretative Criteria (in µg/mL) for Cefoxitin MIC Tests
Susceptible Intermediate Resistant
S. aureus ≤ 4 µg/mL N/A ≥ 8 µg/mL
CoNS N/A N/A N/A
Interpretative Criteria (in mm) for Cefoxitin Disk Diffusion Test
Susceptible Intermediate Resistant
S. aureus ≥ 22 mm N/A ≤ 21 mm
CoNS ≥ 25 mm N/A ≤ 24 mm
o Oxacillin disk diffusion testing is not reliable for
detecting oxacillin/methicillin resistance.
 Cefoxitin should be used as a surrogate for disk
diffusion testing

• Why are oxacillin and cefoxitin tested instead of

methicillin?
o Methicillin is no longer commercially available
in the United States
o Oxacillin maintains its activity during storage
better than methicillin
▪ More likely to detect heteroresistant
strains
▪ Cefoxitin is an even better inducer of
the mecA gene
▪ Tests using cefoxitin give more
reproducible and accurate results than
tests with oxacillin
• Points to remember (for MRSA & MRS)
o Penicillins, cephems, carbapenems & other β-
lactams are not effective clinically
o Reported as RESISTANT or
o Do not report it as SUSCEPTIBLE
• Methicillin-Resistant S. aureus
o Pathogenic
o Transmissible
o Vancomycin (severe MRSA)
 Neisseria gonorrhoeae Gonorrhea 3-8 days
Week 5: Microbial Mechanisms of Pathogenicity & Principles of

Genitourinary
Treponema pallidum Syphilis 9-90 days
Disease and Epidemiology Chlamydia trachomatis Nongonococcal urethritis 1-3 weeks

Tract
Herpes simplex virus type 2 Herpes virus infections 4-10 days
Human Immunodeficiency virus (HIV) AIDS 10 years
Candida albicans Candidiasis 2-5 days
Clostridium perfringens Gas gangrene 1-5 days
MICROBIAL MECHANISMS OF PATHOGENICITY

Parenteral Route
Clostridium tetani Tetanus 3-21 days
Rickettsia rickettsii Rocky Mountain spotted fever 3-12 days

Skin or
PATHOGENICITY Hepatitis B virus (Hepadnavirus) Hepatitis B 6 weeks – 6
months
• The ability of an organism to cause disease Rabiesvirus (Lyssavirus) Rabies 10 days -1yr
Plasmodium spp. (protozoan) Malaria 2 weeks
• Determined by its virulence factors
VIRULENCE • Microbial Virulence Factors
• Measure or degree of pathogenicity of an organism o Virulence Factors:
• Severity of a pathogen’s infectivity ▪ Provide microorganisms with the
capacity to avoid host defenses and
damage host cells, tissues and organs
• Both pathogenicity and virulence reflect the degree to in a number of ways
which an organism is capable of causing disease ▪ Cell structure (used for attachment
and adherence)
• Portals of Entry
o Mucous membranes (lining of RT, GIT, GUT, and ATTACHMENT & ADHERENCE
conjunctiva)
• First step of infection and disease development
▪ RT – easiest and most frequently
portal entry microorganisms that • Achieving Attachment & Adherence to Host Cell
cause common cold, pneumonia, Surfaces
tuberculosis, influenza, measles, and o Pili
small pox ▪ N. gonorrhoeae – has sex pilus that
▪ GIT – ingestion of contaminated food allow it to bind to cervical cells and
and water which causes diseases such mucous cells to cause gonorrhea
as polio, hepatitis A, typhoid fever, o Adherence proteins
amoebic dysentery, shigellosis, cholera o Biofilms
▪ GUT – contracted sexually and may ▪ Accumulation of microorganisms
penetrate through broken mucous embedded in a polysaccharide matrix
membrane which causes diseases such ▪ Used by pathogens to adhere to
as STIs, HIV, genital warts, chlamydia, implants and prosthetic device
herpes, syphilis, gonorrhea ▪ Staphylococcus aureus, Pseudomonas
o Skin aeruginosa
▪ Larges organ of the body in terms of o Various protein adhesins
surface area and weight • Adhesins / Ligands bind to receptors on host cells:
▪ Microorganisms penetrate through o Glycocalyx: Streptococcus mutans
hair follicles, sweat gland, and broken ▪ Causes tooth decay
skin o Fimbriae: Escherichia coli
o Parenteral route o M protein: Streptococcus pyogenes
▪ Microorganisms are deposited directly o Opa protein: Neisseria gonorrhoeae
unto the tissues beneath the skin or o Tapered end: Treponema pallidum
under mucous membrane • Capsules & organs of locomotion also contribute to
▪ Needle stick injury, punctures, microbial pathogenicity
injections, animal bites, cuts, wounds, o Capsules enable bacteria to be more virulent
surgery, and splitting of skin because macrophage and neutrophils are
• Portals of Exit unable to phagocytize the capsulated bacteria
o RT (cough, sneeze)
o GIT (feces, saliva) ENZYMES
o GUT (urine, vaginal secretions)
o Skin • Invasion is accomplished through disrupting the skin and
o Blood (arthropods, needles/syringes) mucosal surfaces by mechanisms using enzymes
• Coagulase: Coagulate the fibrinogen in the blood
o Fibrinogen is a plasma protein produced by the
Portal Pathogen Disease Incubation
of Entry Period liver converted by coagulases into fibrin to
Streptococcus pneumoniae Pneumococcal pneumonia Variable form a fibrin blood clot, which protect the
Mycobacterium tuberculosis Tuberculosis Variable
Bordetella pertussis Whooping cough (pertussis) 12-20 days bacterium from phagocytosis and isolate it
Respiratory Tract

Influenza virus (Influenzavirus) Influenza 18-36 hours

Measles virus (Morbillivirus) Measles (rubeola) 11-14 days from other host defenses
Rubella virus (Rubivirus)
Epstein-Barr virus
German measles (rubella)
Infectious mononucleosis
2-3 weeks
2-6 weeks
• Kinases: Digest fibrin clots to isolate the infection
(Lymphocryptovirus) o Fibrinolysin
Varicella-zoster virus (Varicellovirus) Chickenpox (varicella) (primary 14-16 days
infection) • Hyaluronidase: Hydrolyses hyaluronic acid
Histoplasma capsulatum (fungus) Histoplasmosis 5-18 days
Shigella spp. Shigellosis (bacillary dysentery) 1-2 days o Helps microorganism spread from the site of
Brucella spp. Brucellosis (undulant fever) 6-14 days infection
Gastrointestinal

Vibrio cholerae Cholera 1-3 days

• Collagenase: Hydrolyzes collagen
Tract

Salmonella enterica Salmonellosis 7-22 hours

Salmonella typhi Typhoid fever 14 days
Hepatitis A virus (Hepatovirus) Hepatitis A 15-50 days o Produced by several species of Clostridium and
Mumps virus (Rubulavirus) Mumps 2-3 weeks
Trichinella spiralis (helminth) Trichinellosis 2-28 days
facilitates the spread of infection
 • IgA proteases: Destroy IgA antibodies • Types of Exotoxins
o Adhere to mucosal surfaces o Type I: Cell surface active toxins
• Siderophores: Take Fe from host’s Fe-binding proteins ▪ Cause intense immune response due
o Iron is required in the bacterial growth to release of cytokines from host cells
▪ Superantigens – bacterial proteins,
non-specifically stimulate the
• Antigenic variation: Alter surface proteins proliferation of T cells, which are
o Neisseria gonorrhoeae has several copies OPA stimulated to released enormous
encoding gene amounts of cytokines. High levels of
cytokines in the blood will result to
Penetration to host cell different s/s including fever, nausea,
• Host cell cytoskeleton: provides actual mechanism of vomiting, diarrhea, shock, death.
bacterial entry o Type II: Membrane-disrupting toxins
▪ Disrupts phospholipid bilayer by
• Acting: utilized by some microbes to penetrate host cells
forming channels
• Invasins: Salmonella strains & E. coli
▪ Cause lysis of host cell’s plasma
o Attachment triggers signals in the host cell that
membrane to create protein channels
activate factors resulting to entry of bacteria
those used by Staphylococcus aureus
o Invasins are surface proteins produced by
▪ Some disrupt phospholipid portion of
microorganism such as Salmonella and E. coli
the membrane such as Clostridium
which rearrange nearby actin filaments of
perfringens
cytoskeleton
▪ Hemolysins, cytolysins
o Salmonella typhirium: Invasins cause the host
o Type III: Intracellular toxins
cell’s plasma membrane to appear and create
▪ A-B toxins (cholera toxin, diphtheria
membrane ruffling, which resembles a splash of
toxin)
drop of a liquid hitting a solid surface. The
• A part – Active component;
microbe will sink into the ruffle and are being
enzyme
engulfed by the host cell
• B part – binding component
▪ Active & binding components alters
TOXINS cell function
• Toxin: Substances that contribute to pathogenicity • Examples of Exotoxins
o Biochemically active substances released by o Neurotoxin
microorganisms to have a particular effect on ▪ Act on the nerves or motor end plates
host cells to cause paralysis
o Used by microorganisms to establish infections ▪ Ex. Tetanus toxin (Clostridium tetani),
and multiply within the host Botulinum toxin (Clostridium
o Can cause disease even in the absence of the botulinum)
pathogen that produced them o Enterotoxin
• Toxigenicity: Ability to produce toxin ▪ Act on the GIT and causes diarrhea
• Toxemia: Presence of toxin in host’s blood • Infectious diarrhea
• Toxoid: Inactivated toxin used in a vaccine o Bacteria colonize
o Chemically modified toxin from a pathogenic and bind to the GIT
microorganism continuously
o No longer toxic but is still antigenic releasing their
enterotoxins locally
• Antitoxin: Antibodies against a specific toxin
o Chloeragen (Vibrio
cholerae), Heat
2 TYPES stable toxins (E.
EXOTOXIN coli), Shiga toxins
(Shigella spp.)
o Proteins that are released by both gram-
• Food poisoning
positive and gram-negative bacteria
o The bacteria grow
o Released by all major gram-positive bacteria
and release
o Produced inside most gram-positive bacteria as
enterotoxin in the
part of their growth and metabolism. They are
food and is ingested
then secreted or released following lysis into
which causes
the surrounding medium
diarrhea and
o Even small amounts are quite harmful because
vomiting for less
of its enzymatic nature
than 24 hours
• Naming of Exotoxin
o Bacillus cereus and
o Cell attacked
Staphylococcus
▪ Neurotoxin, Cardiotoxin, Hepatotoxin,
aureus
Leukotoxin, Enterotoxin, Cytotoxin
o Pyrogenic exotoxin
o Disease associated
▪ Stimulate the release of cytokines and
▪ Diphtheria toxin, Tetanus toxin
can cause rash, fever, toxic shock
o Specific bacterium associated
syndrome
▪ Vibrio enterotoxin,
 ▪ Ex. Staphylococcus aureus (TSS toxin), Property Exotoxin Endotoxin
Bacterial Source Mostly from gram-positive Gram-negative bacteria
Streptococcus pyogenes bacteria
(Streptococcus pyrogenic toxin) Relation to Metabolic producing of growing Present in LPS of outer
Microorganism cell membrane of cell wall and
o Tissue-invasive exotoxin released with destruction of
▪ Allows bacteria to destroy and tunnel cell or during cell division
Chemistry Proteins, usually with two parts Lipid portion (Lipid A) of LPS of
through tissues (A-B) outer membrane
▪ Ex. Enzymes that destroy DNA Pharmacology Specific for a particular cell General, such as fever,
(Effect on Body) structure or function in the host weaknesses, aches, and shock;
(collagen, fibrin, RBCs, WBCs) (mainly affects cell functions, all produce the same effects
▪ Ex. Hemolysins, Streptokinase, nerves, and GIT)
Heat Stability Unstable; can usually be Stable; can withstand
Hyaluronidase (S. pyogenes), Lipases, destroyed at 60°-80°C (except autoclaving (121°C for 1 hour)
Penicillinases, Leucocidin, and staphylococcal enterotoxin)
Toxicity (Ability to High Low
Exfoliatin (S. aureus) Cause Disease)
o Miscellaneous exotoxin Fever-Producing No Yes
Immunology Can be converted to toxoids to Not easily neutralized by
▪ Include Anthrax toxin, Diphtheria (Relation to immunize against toxin; antitoxin, therefore, effective
toxin, Pertussis toxin, and Toxin A & Antibodies) neutralized by antitoxin toxoids cannot be made to
immunize against them
Toxin B Lethal Dose Small Considerably large
• In A-B Toxin, the bacterium Representative
Diseases
Gas gangrene, tetanus,
botulinum, diphtheria, scarlet
Typhoid fever, urinary tract
infections, and meningococcal
produces and releases fever meningitis
exotoxins. The binding
component of the exotoxin PRINCIPLES OF DISEASE
attaches to the host cell
receptor to allow it to enter • Pathology: Study of disease
the cell through endocytosis. • Etiology: Study of the cause of disease
Once it is inside, the A • Pathogenesis: Development of disease
component alter cell function o Comes from the Greek words “Pathos” – which
by inhibiting protein means disease and “Genesis” – which means
synthesis. The B component is creation
released from the host cell • Pathogens: Disease-causing microorganisms
• Host: Shelters and supports the
growth of pathogens
ENDOTOXIN • Infection: Pathogenic colonization of
• A component of the cellular the body
structure of the gram-negative • Disease: An abnormal state in which
bacteria. This is the lipid the body is not normally functioning
portion (Lipid A; major part) • Symptom: A change in body function that is felt by a
• General toxin common to all patient due to a disease
gram-negative bacteria which o Indicators as described by patients
are released when the bacteria o Headache, fatigue, nausea
die, destroyed, or when they multiply • Sign: A measurable or observable bodily change as a
• Stimulates macrophage to release cytokines in very high result of disease
amounts, which can result to several symptoms and can o Increase in temperature, development of rash,
even induce miscarriage swelling
• Results to activation of blood clotting proteins causing • Syndrome: A group of signs and symptoms that
blood clots accompany a disease
• Effects of endotoxins being released
o Disruption of clotting, forming clots to form
CLASSIFYING INFECTIOUS DISEASES
(Disseminated Intravascular Coagulation)
o Fever • Communicable disease: Spread from one host to
o Complement Activation another
o Circulatory changes • Contagious disease: Easily spread from one host to
another
• Noncommunicable disease: Not transmitted from one
host to another

OCCURRENCE OF DISEASE
• Macrophage ingests a gram-negative bacterium and is
degraded in a vacuole, releasing endotoxins that induces
the production of interleukin-1. IL-1 is then released into
the blood stream and will travel throughout the body to
reach into the brain. Once in the brain, it induces the
hypothalamus to produce prostaglandins, which reset
the body’s temperature to a higher one, producing fever
• Incidence: Fraction of a population that contracts a
disease during a specific time
• Prevalence: Fraction of a population having a specific
disease at a given time
• Sporadic disease: Disease that occurs occasionally in a
population
• Endemic disease: Disease constantly present in a
population
• Epidemic disease: Disease acquired by many hosts in a
given area in a short time
• Pandemic disease: Worldwide epidemic
SEVERITY OR DURATION OF A DISEASE
• Acute disease: Symptoms develop rapidly
• Chronic disease: Disease develops slowly
o Progression of disease can occur over a period
of years
• Subacute disease: Symptoms between acute and
chronic
• Latent disease: Disease with a period of no symptoms
when the patient is infective
o Must occur before the disease becomes
acute/chronic and severe.

CONTINUAL SOURCES OF INFECTION (RESERVOIRS)

STAGES OF DISEASE
• Human: AIDS, gonorrhea
• Animal: Rabies, Lyme disease (Zoonoses)
• Nonliving (soil): Botulism, tetanus

TRANSMISSION OF DISEASE
• Contact
o Direct: Close association with infected host
▪ Horizontal or Vertical
o Indirect: Spread by fomites
o Droplet: Via airborne droplets
• Incubation Period: Interval between the initial infection
• Vehicle: By an inanimate object
and the first appearance of any signs or symptoms
• Vectors: Arthropods (fleas, ticks & mosquitoes)
• Prodromal Period: Relatively short period that follows
o Mechanical: (Arthropod carries pathogen on
the period of incubation in some disease.
feet)
o Characteristics: early, mild symptoms
o Biological: (Pathogen reproduces in vector)
• Period of Illness: Disease is most severe where most
o Lyme disease and Rocky Mountain Spotted fever
signs and symptoms occur
• Period of Decline: Signs and symptoms subside
• Period of Convalescence: Body returns to prediseased NORMAL MICROBIOTA
state
• Transient microbiota
• Normal microbiota
HOST-MICROORGANISM INTERACTIONS • Opportunistic microbiota
Encounter Colonization Invasions Outcome
and entry and entry
Pathogen Pathogen Pathogen invades Pathogen completes cycle:
encounters multiplies and deeper tissues and - Leaves host Symbiotic Relationship
and breaches host disseminates, - Destroys host
colonizes
host
surface
defenses
encounters
inflammatory and
- Remains in latent stage
- Is destroyed by the host
• Commensalism: One organism benefit and the other is
surface immune response unaffected
CORRESPONDING INFECTION-DISEASE STAGES
Incubation Prodromal Clinical stage Stage of decline Convalescent • Mutualism: Both organism benefits
stage stage stage • Parasitism: One organism benefits at the expense of the
No signs First signs and Peak of Condition of host Full recovery
and symptoms, characteristic signs deteriorates or of surviving others
symptoms pathogen may and symptoms of signs and host or chronic
be highly infection or disease symptoms begin infection
communicable to subside as develops, or
host condition death MICROORGANISMS AND THE HOST
improves
• Normal microbiota
EXTENT OF HOST INVOLVEMENT o Microorganisms that establish permanent
colonies inside or on the body without
• Local infection: Pathogens limited to a small area of the producing disease
body • Transient microbiota
• Systemic infection: An infection throughout the body o Microbes that are present for various periods
• Focal infection: Systemic infection that began as a local then disappear
infection
• Bacteremia: Bacteria in the blood
• Septicemia: Growth of bacteria in blood MICROBIAL ANTAGONISM
• Toxemia: Toxins in blood • Normal microbiota preventing the pathogens from
• Primary infection: Acute infection that causes initial causing disease
illness • Probiotics are live microbes applied to or ingested into
• Secondary infection: Opportunistic infection after a the body, intended to exert a beneficial effect
primary infection
• Subclinical disease: No noticeable signs and symptoms
(inapparent infection)
OPPORTUNISTIC MICROORGANISM EPIDEMIOLOGY
• Do not cause disease under normal conditions • Epidemiology
• Cause disease under special conditions o Science that studies when and where disease
occur and how they are transmitted in
populations
REPRESENTATIVE NORMAL MICROBIOTA BY BODY REGION
Region Principal components Comments • Descriptive Epidemiology
Propionibacterium • Most of the microbes in direct contact o Entails collecting all data that describe the
Staphylococcus with skin do not become residents
Corynebacterium because secretions from sweat and oil occurrence of the disease under study
Micrococcus glands have antimicrobial properties
• Analytical Epidemiology
Skin Acinetobacter • Keratin is a resistant barrier, and the
Brevibacterium low pH of the skin inhibits many o Analyzes a particular disease to determines it
microbes
Pityrosporum (fungus) probable cause
Candida (fungus) • The skin also has a relatively low
Malassezia (fungus) moisture content • Experimental Epidemiology
Staphylococcus epidermidis • The conjunctiva, a continuation of the o Begins with a hypothesis about a particular
Staphylococcus aureus skin or mucous membrane, contains
Diphtheroids basically the same microbiota found disease; experiments to test the hypothesis are
Eyes
(Conjunctiva)
Propionibacterium on the skin then conducted with a group of people
Corynebacterium • Tears and blinking also eliminate some
Streptococci microbes or inhibit others from • Case Reporting
Micrococcus colonizing o Provides data on incidence and prevalence to
Staphylococcus aureus • Although some normal microbiota are
S. epidermidis potential pathogens, their ability to
local, state, and national health officials
Nose and Aerobic diphtheroids cause disease is reduced by microbial
Throat S. epidermidis antagonism
(Upper S. aureus • Nasal secretions kill or inhibit many
Respiratory Diphtheroids microbes, and mucus and ciliary action
System Streptococcus pneumoniae remove many microbes
Haemophilus
Neisseria in the throat
Streptococcus • Abundant moisture, warmth, and the
Lactobacillus constant presence of food make the
Actinomyces mouth an ideal environment that
Bacteroides supports very large and diverse
Veillonella microbial populations on the tongue,
Neisseria cheeks, teeth, and gums
Mouth • However, biting, chewing, tongue
Haemophilis
Fusobacterium movements, and salivary flow dislodge
Treponema microbes. Saliva contains several
antimicrobial substances
Staphylococcus
Corynebacterium
Candida (fungus)
Escherichia coli • The large intestine contains the largest
Bacteroides numbers of resident microbiota in the
Fusobacterium body because of its available moisture
Lactobacillus and nutrients
Enterococcus • Mucus and periodic shedding of the
Large Bifidobacterium lining prevent many microbes from
Intestine Enterobacter attaching to the lining of the GIT, and
Citrobacter the mucosa produces several
Proteus antimicrobial chemicals
Klebsiella • Diarrhea also flushes out some of the
normal microbiota
Candida (fungus)
Coliform
Staphylococcus • The lower urethra in both sexes has a
Micrococcus resident population; the vagina has its
Enterococcus acid-tolerant population of microbes
Lactobacillus because of the nature of its secretions
Urinary and Bacteroides • Mucus and periodic shedding of the
Reproductive Aerobic Diphtheroids lining prevent microbes from attaching
Systems Pseudomonas to the lining; urine flow mechanically
Klebsiella removes microbes, and the pH of
Urethra Proteus urine and urea are antimicrobial
Lactobacilli • Cilia and mucus expel microbes from
Vagina the cervix of the uterus into the
Streptococcus
vagina, and the acidity of the vagina
Clostridium
inhibits or kills microbes
Candida albicans (fungus)
Trichomonas vaginalis
(protozoan)

Beneficial Effects of Normal Microbiota

• Prevent growth of pathogens by occupying niches that
pathogens might occupy
• Produce growth factors such as folic acid and vitamin K
• Probiotics
CLINICAL BACTERIOLOGY (MLS 411 LEC)
PATHOGENESIS AND EPIDEMIOLOGY
• Etiology- the cause of disease
• Pathogenesis- development of disease
• Structural and functional-
• Infection- invasion or colonization by the
microorganism
• Disease- occurs when the infection results
to any damage from results of health
Microbial Mechanisms of Pathogenicity
• Both pathogenicity and virulence reflect
the degree to which a microorganism is
capable of causing disease
• Pathogenicity- the ability of the organism
to cause a specific disease. It is
determined by its virulence factors.
• Virulence- measure or degree of
pathogenicity of an organism. Severity of
the pathogen’s infectivity.
I. Portals of Entry:
o Mucous membrane- lining of the
respiratory tract, gastrointestinal
tract, genetic urinary tract, and
conjunctiva.
▪ Respiratory tract is the
easiest and most frequently
travelled portal of entry for
infectious microorganisms:
common cold, pneumonia,
tuberculosis, influenza, etc
▪ Gastrointestinal tract- used
by microorganisms to infect
a host through ingestion of
contaminated food and
water: polio, hepa A,
typhoid fever, etc
▪ GUT- sexually transmitted
infections, HIV, herpes,
syphilis, etc.
o Skin- largest organ, microorganism
penetrate through har follicles,
sweat glands, and broken skin.
o Parenteral route- microorganisms
are deposited directly into the
tissues beneath the skin or into
mucous membrane: needlestick
injury, puncture, injections, animal
bites, cuts, wounds, etc.
III. Portals of Exit:
o RT (Cough, sneeze)
o GIT (Feces, saliva)
o GUT (Urine, vaginal secretions)
o Skin
o Blood (arthropods, needles/syringes)
Microbial Virulence Factors
• Virulence Factors
o provide microorganisms with the
capacity to avoid host defenses
and damage host cells, tissues and
organs in a number of ways
o characteristics that enable
pathogens to cause disease
III. Attachment & Adherence
Achieving Attachment & Adherence to
Host Cell Surfaces
o sex pili- N. gonorrhea has sex pilus
to allow it to bind to cervical cells
and buccal cells to cause
gonorrhea
o adherence proteins
o biofilms- accumulation of
microorganisms embedded in a
polysaccharide matrix to implant
and prostatic. Bacteria such as S.
aureus, P. aeruginosa
o various protein adhesins
Adhesins / Ligands bind to receptors on host cells:
• glycocalyx- Streptococcus mutants may
cause tooth decay and uses glycocalyx to
attach to tooth surface
• Fimbriae- E. coli
• M protein- Streptococcus pyogenes
• Opa protein- N. gonorrhea
• Tapered end- Treponema pallidum
• Capsules- protective walls that surrounds
cell membranes of gram + and gram –
bacteria. It enables the bacteria to be
more virulent because macrophage and
neutrophils are unable to phacytized the
encapsulated bacteria & organs of
locomotion also contribute to microbial
pathogenicity
• After attachment, invasion, it is
accomplished by disruption of the skin and
mucosal surfaces by several mechanisms
using enzyme which aids in bacterial
virulence
 IV. Enzymes Rearrange nearby actin filaments
o Coagulase- coagulate blood of the cytoskeleton
▪ Can coagulate fibrinogen o Ex: salmonella typhimurium.
in the blood. Fibrinogen is a Invasions cause the host cell
plasma protein produce by plasma membrane to appear and
the liver converted by create membrane ruffling
coagulases into fibrin or o Some microorganisms used actin
threads that form a blood to propel themselves to host cell
clot. The fibrin clot formed cytoplasm
protect the bacterium from V. Toxins
phagocytosis • Toxin- substances that contribute to
o Kinases- digest fibrin clots pathogenicity
▪ Fibrinolysin o Biochemically active
▪ Kinases digest fibrin clots substances release by
formed by the body to microorganisms to have a
isolate infection particular effect on host
o Hyaluronidase- hydrolyses cells
hyaluronic acid or holds together o Established infections and
certain cells of the body such as multiply within the host
connective tissue • Toxigenicity- ability to produce
▪ Helps microorganisms to toxin
spread from its initial site of • Toxemia- presence of toxin in the
infection hosts blood
o Collagenase- hydrolyzes collagen • Toxoid- inactivated toxin used in a
▪ Produce by several species vaccine
of clostridium and other o Chemically modified toxin
microorganism from a pathogenic
▪ This facilitates the spread of microorganism
infection • Antitoxin- antibodies against a
o IgA proteases- destroy antibodies specific toxin
▪ Used to adhere to mucosal • 2 types of toxin
surfaces o Exotoxin
o Siderophores- take iron from the ▪ Are proteins that are
hosts iron binding proteins released by both
▪ Iron is required in the gram + and gram –
bacterial growth bacteria
o Antigenic variation- alter surface ▪ Released by all
proteins so that they cannot be major gram +
inactivated or destroyed by bacteria as part of
antibodies their growth and
▪ Changes their surface metabolism. They
antigen are secreted or
▪ N. gonorrhea- has several releases in the
copies of the opa surrounding medium
encoding gene ▪ Naming of exotoxin
Penetration to host cell • Cell they
• Host cell cytoskeleton: provides actual attacked-
mechanism of bacterial entry neurotoxin,
• Actin: utilized by some microbes to cardiotoxin,
penetrate host cells hepatotoxin,
• Invasion: Salmonella strains and E. coli leukotoxin,
o Attachment triggers signal in the • Disease
host cell that activates factors associated-
resulting to entry of bacteria Bacteria
o Invasins are surface proteins toxin such as
produced by microorganism such diphtheria
as salmonella and e. coli. toxin,
 tetanus
toxin,
• Specific
bacterium
associated-
V.
enterotoxin
Type I: Cell surface active toxins
• Cause intense immune response due to
release of cytokines from host cells
• Superantigens- bacterial protein non-
specifically stimulate the proliferation of
immune cell such as t cell, and t cells are
stimulated to release enormous amounts
of chemicals called cytokines. High levels
of cytokines in the blood will result to fever,
nausea, vomiting, diarrhea, etc.
Type II: Membrane disrupting toxins
• Disrupts phospholipid bilayer by forming
channels
• Hemolysins, cytolysins
• Causes lysis of the hosts cells plasma
membrane to create protein channel; S.
aureus. Some disrupts the phospholipid
membrane; Clostridium perfringens
Type III : Intracellular toxins
• A-B toxin (cholera toxin, diphtheria
toxin)
• active & binding components alters
cell fxn
o A part: active component
(enzyme)
o B- part: binding component
Examples of exotoxins
1. neurotoxin- act on the nerves or motor end
plates to cause paralysis
Ex: tetanus toxin for C. tetani and
botulinium toxin for C. botulinium
2. enterotoxin- act on the GIT thus causing
diarrhea
Ex: infectious diarrhea- bacteria colonize
and bind to the GIT continuously releasing
their enterotoxins locally such as
choleragen for V. cholerae and heat
labile and heat stable for E. coli as well as
shiga toxin for shigella.
Food poisoning- bacteria grow in the food
and release enterotoxin in the food. The
enterotoxin is ingested resulting to diarrhea
and vomiting for less than 24 hrs: Bacillus
cereus as well as S. aureus
3. pyrogenic exotoxin- stimulates the release of
cytokines and can cause rash, fever, and toxic
shock syndrome
Ex: S. aureus producing the toxic shock
syndrome toxin and S. pyogenes producing
streptococcus pyrogenic toxin
4. tissue invasive exotoxin- allows the bacteria to
destroy and tunnel through tissue such as enzyme
that destroy DNA, collagen, fibrin, rbc, and wbc
Ex: hemolysins, Streptokinase,
hyaluronidase which are produce by
Strep. pyogenes. Lipases, penicillinase,
leucocidin are produce by
staphylococcus aureus
5. Miscellaneous exotoxin- anthrax toxin,
diphtheria toxin as well as pertussis toxin and toxin
A and B.
A-B toxin- the bacterium produces and release
exotoxin
↓
Binding component of the exotoxin attaches the
host cell receptor
↓
A-B toxin enters the host cell by endocytosis
↓
A-B exotoxin enclosed in pinched-off portion of
plasma membrane during pinocytosis
↓
A-B components separate and A component
alters cell function by inhibiting protein synthesis.
The B component is released from the host cell
Endotoxin
• The component of the cellular structure of
the gram – bacteria which is actually the
lipid portion or lipid A or major part. They
are released when the bacteria die or
when they are destroyed or multiplies
• Stimulates macrophage to release
cytokines in very high amounts thus the
release of endotoxin will result to several
symptoms and induce miscarriage
• Results to activation of blood clotting
proteins resulting to blood clots
o Clots: DIC-disseminated
intravascular coagulation
o Fever
o Activation of complement
o Circulatory changes
 Occurrence of Disease
• Incidence- Fraction of a population that
contracts a disease during a specific time.
• Prevalence- Fraction of a population
having a specific disease at a given time.
• Sporadic disease- Disease that occurs
occasionally in a population
• Endemic- Disease constantly present in a
population
• Epidemic- Disease acquired by many hosts
in a given area in a short time
• Pandemic- Worldwide epidemic
• Shows how gram negative bacterium
produce fever Severity or Duration of a Disease
• Acute disease- Symptoms develop rapidly
• Chronic- disease develops slowly
• Subacute- disease Symptoms between
acute and chronic
• Latent- Disease with a period of no
symptoms when the patient is infective

• Exotoxin is protein in nature while

endotoxin are lipid in nature

Principles of Disease
• Pathology- Study of disease
• Etiology- Study of the cause of disease
• Pathogenesis- Development of disease
whether it is acute, chronic, or recurrent.
Pathos- disease, genesis- creation
• Infection- Pathogenic colonization of
the body
• Disease- An abnormal state in w/c the
body is not normally functioning

• Symptom- A change in body function that

is felt by a patient due to a disease.
• When the pathogen attaches to the host,
Indicators: headache, fatigue, nausea
the corresponding infection disease stage
• Sign- A measurable or observable bodily
wherein there are no sign and symptoms
changes as a result of disease. Increase in
• When the pathogen multiplies and
temperature, development of rash, and
reaches the host surface defenses, the
swelling
patient is in prodromal stage where first
• Syndrome- A group of S/S that
signs and symptoms appear
accompany a dse.
• When the pathogen invades deeper
Classifying Infectious Diseases
tissues and disseminate inside the hosts
• Communicable disease- spread from one
body, the patient experiences the clinical
host to another.
stage or the period of illness where most
• Contagious disease- easily spread from
signs and symptoms appear
one host to another
• As the pathogen leaves the host or being
• Non communicable disease- not
destroyed by the host, the patient will
transmitted from one host to another
 experience the stage of decline and • Biological (Pathogen reproduces in
convalescent stage vector)
Extent of Host Involvement
• Local infection- Pathogens limited to a NORMAL MICROBIOTA
small area of the body • Transient microbiota
• Systemic infection- An infxn throughout the • Normal microbiota
body • Opportunistic microbiota
• Focal infection- Systemic infxn that began o Symbiotic relationship
as a local infection ▪ Commensalism- one
• Bacteremia- Bacteria in the blood organism benefits and the
• Septicemia- Growth of bacteria in blood other is unaffected
• Toxemia- Toxins in blood ▪ Mutualism- both organisms’
• Primary infection- Acute infxn that causes benefit
the initial illness ▪ Parasitism- one organism
• Secondary infection- Opportunistic infxn benefits at the expense of
after a primary infection the other
• Subclinical disease- No noticeable S/S Microorganism and the Host
symptoms (inapparent infection)

• Normal microbiota
o microorganisms that establish
permanent colonies inside or on
the body without producing
• Reservoir is the origin of the etiologic
disease
agent or the location from which it
o normally present in the body
disseminates: humans, animals, food,
o E. coli on large intestine
water, air, and soil
o Staphylococcus- skin, nostrils
o Human: AIDS, gonorrhea
• Transient microbiota
o Animal: Rabies, Lyme disease
o microbes that are present for
(Zoonoses)
various periods then disappear
o Nonliving (Soil): Botulism, tetanus
Microbial antagonism
• Through different modes of transmission,
• normal microbiota preventing the
the microorganism is brought in contact
pathogens from causing disease
with the human hosts: agents such as
• probiotics are live microbes applied to or
vectors and vehicles
ingested into the body, intended to exert
Transmission of Disease
a beneficial effect.
A. Contact
Opportunistic microorganism
• Direct: close association with infected host
• do not cause disease under normal
o Horizontal- human to human
conditions
o Vertical- mother to newborn
• cause disease under special conditions
o Lime disease and rocky mountain
• only cause infection when one or more
fever
hosts defense mechanism is disrupted or
• Indirect: Spread by fomites
malfunctioned
o Vehicle- non living contaminated
• fungal infection
entity
• Droplet: via airborne droplets
B. Vehicle
• By an inanimate object
C. Vectors
• Arthropods (fleas, ticks, & mosquitoes)
• Mechanical (Arthropod carries pathogen
on feet)
BENEFICIAL EFFECTS of the NORMAL MICROBIOTA:
• prevent growth of pathogens by
occupying niches that pathogens might
occupy
o they occupy areas that pathogen
might occupy
• produce growth factors such as folic acid
and vitamin K
o may produce substances that are
detrimental for pathogens
• Probiotics

Epidemiology
• Science that studies when and where
diseases occur and how they are
transmitted in populations
• Descriptive Epidemiology
o Entails collecting all data that
describe the occurrence of the
disease under study
• Analytical epidemiology
o Analyzes a particular disease to
determine its probable cause
• Experimental Epidemiology
o Begins with a hypothesis about a
particular disease; experiments to
test the hypothesis are then
conducted with a group of people
• Case reporting
o Provides data on incidence and
prevalence to local, state, and
national health officials