(Pha6114 Lec) 1ST Shifting Reviewer

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PHARMACEUTICAL MICROBIOLOGY AND PARASITOLOGY

BY: Daphne Nava & Sofia Larrauri
the Theory of Biogenesis - life can
WEEK 1 arise only from preexisting life, and,
therefore, that cells can arise only
Historical Notes in Microbiology from preexisting cells.
● Cell theory conclusion:
CELL ○ M. SCHLEIDEN - “Plants
● ROBERT HOOKE - an English consist of cells.”
physicist named who published a ○ T. SCHWANN - “Animals
book, entitled Micrographia, consist of cells”
containing descriptions of objects ○ R. VIRCHOW “Cells came
he had observed using a compound from preexisting cells.”
light microscope that he had made.
DISCOVERY OF ENDOSPORES
● JOHN TYNDALL - (1876 & 1877)
British physicist who, while
performing spontaneous generation
experiments, concluded that certain
bacteria exist in two forms:
a. Heat-labile - form that is
readily killed by simple
boiling
b. Heat-stable - form that is
● These objects included:
not killed by simple boiling
a. Molds
● Tyndallization - fractional
b. Rusts
sterilization technique developed by
c. Fleas
J. TYNDALL which involves: boiling
d. Lice
-> incubating -> reboiling -> repeat
e. fossilized plants and animals
several times
f. sections of cork
➔ Successfully killed both
● “Cells” - Hooke referred to as the
heat-labile and heat-stable
small empty chambers in the
➔ The bacteria that emerge
structure of cork because they
from the spores during the
reminded him of the bare rooms
incubation steps are
(called cells) in a monastery.
subsequently killed during
the boiling steps.
CELL THEORY
● FERDINAND COHN - (1877)
● MATTHIAS SCHLEIDEN and
German botanist who described the
THEODOR SCHWANN - (around
microscopic appearance of the two
1838-1839) German botanist and
forms of the “hay bacillus,” which he
zoologist, respectively, concluded
named Bacillus subtilis.
that all plant and animal tissues
➔ “Spores” - He referred to the
were composed of cells -> became
small refractile bodies within
known as M. SCHLEIDEN the cell
the bacterial cells that
theory.
observed the conversion of
● RUDOLF VIRCHOW - (1858)
German pathologist who proposed
[D.N. & S.L.] | #2APHRPhCutie 1

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spores into actively growing differed from the rRNA of
cells. eukaryotic organisms.
➔ Cohn also concluded that
when they were in the spore
phase, the bacteria were
heat-resistant.
● Today:
a. Endospores - bacterial
spores
b. Vegetative cells - active,
metabolizing, growing CHARLES DARWIN AND
bacterial cells MICROBIOLOGY
● The experiments of Tyndall and ● CHARLES DARWIN - (1859) In his
Cohn supported Louis Pasteur’s book entitled “On the Origin of
conclusions regarding spontaneous Species,” the British naturalist wrote
generation and dealt the final death that all species of life have
blow to that theory. descended over time from common
ancestors.
THREE-DOMAIN CLASSIFICATION ➔ He proposed the scientific
● Formulated by CARL WOESE theory that this branching
● Although this system of pattern of evolution (the “tree
classification was not widely of life”) resulted from a
accepted at first, this has become process that he called
the classification system most natural selection.
favored by microbiologists. ➔ Darwin is considered by
many to be one of the most
CARL WOESE influential figures in human
● CARL WOESE - (During the 1970s) history.
a molecular biologist, along with his NAMING OF BACTERIA
colleagues at the University of ● Sometimes, bacteria and other
Illinois, shook up the scientific microorganisms are named for the
community by developing a system person who discovered the
of classifying organisms that was organism.
based on the sequences of ● Example:
nucleotide bases in their ribosomal a. BLACK DEATH: Yersinia
RNA (rRNA) molecules. pestis
➔ They demonstrated that - (1894) discovered by
prokaryotic organisms can ALEXANDRE EMILE JEAN
be divided into two major YERSIN, a French
groups (referred to as bacteriologist of Swiss
domains), based on descent, who worked for
differences in their rRNA many years at various
sequences, and that the Pasteur Institutes in Vietnam
rRNA from these two groups

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shell of a foraminiferan — a type of
protozoan — and two species of
microscopic fungi
➔ Some scientists consider
these to be the first written
descriptions of
microorganisms and feel that
Hooke (rather than
Leeuwenhoek) should be
given credit for discovering
microbes.
ORIGIN OF GRAM STAIN

b. Bordetella (JULES BORDET) ● HANS CHRISTIAN GRAHAM -
c. Escherichia (THEODORE (1880s) Danish physician who, while
ESCHERICH) working in a laboratory in the
d. Neisseria (ALBERT LUDWIG morgue of a Berlin hospital,
NEISSER) developed what was to become the
e. Salmonella (DANIEL ELMER most important of all bacterial
SALMON) staining procedures.
● M.R.J SALTON - (1963) the
EARLY MICROSCOPES mechanism of Gram differentiation
● HANS JANSEN - (bet. 1590 and was explained
1595) an optician in Middleburg,
Holland, often given credit for
developing the first compound
microscope
➔ Although his son,
ZACHARIAS, was only a
young boy at the time, he
apparently later took over
production of the Jansen
CULTURING BACTERIA IN THE
microscopes.
LABORATORY
● Jansen microscopes - contained
● FERDINAND COHN (1872),
two lenses and achieved
JOSEPH SCHROETER (1875), and
magnifications of only 33 to 39
OSCAR BREFELD (1875) - made
● MARCELLO MALPIGHI (Italy) &
the earliest successful attempts to
ROBERT HOOKE (England) - used
culture microorganisms in a
the compound microscopes having
laboratory setting
a three-lens system both of whom
● ROBERT KOCH - (1881) described
published papers between 1660 and
his culture techniques:
1665 describing their microscopic
➔ Initially, he used slices of boiled
findings
potatoes on which to culture
● HOOKE - (1665) In his book entitled
bacteria, but he later developed
Micrographia, described a fossilized

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both liquid and solid forms of from a cowpox lesion and then
artificial media. exposed them to smallpox.
➔ Gelatin was initially used as a ➔ He thus proved that immunity to
solidifying agent in Koch’s culture cowpox, a very mild disease,
media. However, gelatin was too provided protection against
soft. smallpox.
➔ FANNY HESSE - wife of Dr. ➔ This procedure of injecting
Walther Hesse, one of Koch’s cellular material became known
assistants — suggested the use of as vaccination, from vacca, the
agar (stiffer than the gelatin) Latin word for “cow.”
instead. ● LOUIS PASTEUR - vaccines against
● RICHARD JULIUS PETRI - (1887) anthrax, cholera, and rabies
Another of Koch’s assistants, (developed in the late 1800s)
invented glass Petri dishes for use ➔ first used the terms “immune”
as containers for solid culture media and “immunity”
and bacterial cultures. ➔ applied principle of attenuation to
● JOSEPH LISTER - (1878) became the prevention of rabies, a fatal
the first person to obtain a pure disease at that time
culture of a bacterium ❖ Attenuation, or change, -
(Streptococcus lactis) in a liquid may occur through heat,
medium aging, or chemical means,
● LOUIS PASTEUR & ROBERT and it remains the basis
KOCH - As a result of their ability to for many of the
obtain pure cultures of bacteria in immunizations that are
their laboratories, they made used today.
significant contributions to the ● EMIL BEHRING & KITASATO
Germ Theory of Disease -- which SHIBASABURO - (1890) Most likely,
states that microorganisms known immunology got its start when they
as pathogens or "germs" can lead to discovered antibodies while
disease. developing a diphtheria antitoxin.
● ELIE METCHNIKOFF - At about the
ORIGINS OF IMMUNOLOGY same time (1890), discovered
● EDWARD JENNER - smallpox phagocytes and introduced the
vaccine (first administered in 1796) cellular theory of immunity.
➔ English country doctor who ➔ Phagocytosis - process of
discovered a remarkable engulfing foreign particles
relationship between exposure to
cowpox and immunity to
smallpox.
➔ After observing the fact that
milkmaids who were exposed to
cowpox had apparent immunity
to smallpox, he deliberately
injected individuals with material

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● LOUIS PASTEUR - developed
successful vaccines to prevent: a.
a. cholera in chickens,
b. anthrax in sheep and cattle, and
c. rabies in dogs and humans
➔ In July 1885, while he was
developing a vaccine that would
prevent rabies in dogs, he faced
an ethical decision. A 9-year-old
boy, named JOSEPH MEISTER,
had been bitten 14 times on the
legs and hands by a rabid dog.
➔ Meister’s mother begged
Pasteur to use his vaccine to
save her son. Pasteur was a
chemist, not a physician, and
● In 1910, main elements of clinical thus was not authorized to
immunology (allergy, autoimmunity, treat humans. Also, his
and transplantation immunity) had experimental vaccine had never
been described, and been administered to a human
immunochemistry had become a being.
quantitative science. ➔ Nonetheless, 2 days after the
● Major advances in immunology boy had been bitten, Pasteur
began to take shape in the late injected Meister with the vaccine
1950s, when the focus shifted from in an attempt to save the boy’s
serology (investigating antigens and life.
antibodies in serum) to cells. ➔ The boy survived, and Pasteur
● The use of vaccines to prevent realized that he had developed a
disease may date as far back as the rabies vaccine that could be
11th century, when the Chinese administered to a person after
used a powder prepared from dried he or she had been infected with
smallpox scabs to immunize people the rabies virus.
-- either by introducing the powder
into a person’s skin or by having ANTISEPSIS & ASEPSIS
him/her inhale the powder. ● JOSEPH LISTER (1827–1912), a
➔ This method of preventing British surgeon, made significant
smallpox—using actual smallpox contributions in the areas of
scabs—was known as the antisepsis (against infection) and
Chinese method. asepsis (without infection).
● Because JENNER was the first ➔ During the 1860s, he
person to publish (in 1798) the instituted the practice of
successful results of vaccination, he using phenol (carbolic acid)
is generally given credit for as an antiseptic to reduce
originating the concept. microbial contamination of
open surgical wounds.

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➔ Phenol - very corrosive thus, ➔ Christopher Colombus went
it is not used anymore to the King & Queen of Spain
➔ Later contributions by Lister to ask for funding to build
included: ships. He went to an island
a. aseptic techniques as where blankets
steam sterilization of contaminated with smallpox
surgical instruments; were given to locals. The
b. the use of sterile crew of Columbus had
masks, gloves, and intercourse with the locals
gowns by members which infected them with
of the surgical team; syphilis.
and ➔ Smallpox is the disease that
c. the use of sterile was from the old world
drapes and gauze (Europe) to the new world
sponges in the ➔ Syphilis is the disease that
operating room was from the new world to
the old world (Europe)
CHEMOTHERAPY ● Rosaniline - Ehrlich also found
● PAUL EHRLICH - The true useful for treating African
beginning of modern chemotherapy trypanosomiasis
came in the late 1800s when a
German chemist began his search HANDWASHING
for chemicals (referred to as “magic ● IGNAZ PHILIPP SEMMELWEIS
bullets”) that would destroy (1818–1865) - referred to as the
bacteria, yet would not damage “Father of Handwashing,” the
normal body cells. “Father of Hand Disinfection,” and
➔ By 1909, he had tested more the “Father of Hospital
than 600 chemicals, without Epidemiology.”
success. ➔ He instituted a policy that
➔ Finally, in that year, he stated that “all students or
discovered an arsenic doctors who enter the wards
compound that proved for the purpose of making an
effective in treating syphilis. examination must wash their
This was the 606th hands thoroughly in a
compound Ehrlich had tried, solution of chlorinated lime
thus he called it “Compound that will be placed in
606.” The technical name for convenient basins near the
Compound 606 is entrance of the wards.”
arsphenamine, and the ● OLIVER WENDELL HOLMES
trade name was Salvarsan. (1809– 1894) - an American
● Until the availability of penicillin in physician, had concluded some
the early 1940s, Salvarsan and a years earlier that puerperal fever
related compound— Neosalvarsan was spread by healthcare workers’
were used to treat syphilis. hands.
● Syphilis

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➔ He gave the name
DISCOVERY AND THERAPEUTIC USE OF “penicillin” to the inhibitory
BACTERIOPHAGES substance being produced
by the mold.
● SIR HOWARD WALTER FLOREY &
ERNST BORIS CHAIN - two
biochemists during World War II
who purified penicillin and
demonstrated its effectiveness in
the treatment of various bacterial
infections.
● By 1942, the US drug industry was
able to produce sufficient penicillin
● FREDERICK TWORT (1915) & for human use, and the search for
FELIX D’HERELLE (1917) - other antibiotics began.
discovered bacteriophages were ● GERHARD DOMAGK - chemist
discovered independently who, earlier in 1935, discovered that
● D’HERELLE - along with his the red dye, Prontosil, was effective
collaborators, he coined the term against streptococcal infections in
“bacteriophage” (phagein is Greek mice.
for “devour”), and first used ➔ Further research
bacteriophages therapeutically. demonstrated that Prontosil
● During the next 20 or so years, there was degraded or broken
were hundreds of published down in the body into
reports—many of which were sulfanilamide, and that
controversial— concerning the use sulfanilamide was the
of bacteriophages to treat bacterial effective agent.
infections in humans and animals. ● SELMAN WAKSMAN - In 1944, he
● Interest in phage therapy started to and his colleagues isolated
decline around the time that streptomycin (the first
antibiotics were discovered. antituberculosis drug) and
● However, the emergence of subsequently discovered antibiotics
multidrug-resistant bacteria has such as:
rekindled interest in phage therapy. a. Chloramphenicol,
b. tetracycline, and
FIRST ANTIBIOTICS c. erythromycin in soil samples
● ALEXANDER FLEMING - a Scottish ➔ It was WAKSMAN who first
bacteriologist accidentally used the term “antibiotic.”
discovered the first antibiotic in
1928 when he noticed that growth “TYPHOID MARY” AN INFAMOUS
of contaminant Penicillium notatum CARRIER
mold colonies on his culture plates ● MARY MALLON - was a domestic
was inhibiting the growth of employee, a cook—who worked in
Staphylococcus bacteria. the New York City area in the early
1900s.

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➔ After several typhoid fever BROAD STREET PUMP
outbreaks were traced to her, ● JOHN SNOW - a British physician in
she was offered the choice the mid-19th century designed and
of having her gallbladder conducted an epidemiologic
(where the salmonella investigation of a cholera outbreak
inhabits) removed surgically in London.
or being jailed. She opted for ➔ “Father of Epidemiology”
the latter and spent several ● He carefully compared households
years in jail. affected by cholera with households
➔ She was released from jail that were unaffected and concluded
after promising never to cook that the primary difference between
professionally again. them was their source of drinking
However, the lure of the water.
kitchen was too great. She ● At first, because of the Black Death,
changed her name and they thought cholera was because
resumed her profession in of rats again so they ordered all rats
various hotels, restaurants, to be killed.
and hospitals. ● At one point in his investigation, he
➔ As in the past, “everywhere ordered the removal of the handle of
that Mary went, typhoid fever the Broad Street water pump, thus
was sure to follow.” She was helping to end an epidemic that had
again arrested and spent her killed more than 500 people.
remaining years quarantined ● He concluded that cholera was
in a New York City hospital. spread via fecally contaminated
She died in 1938 at the age water.
of 70. SMALLPOX
● No treatment, vaccination only
● The WHO was able to eradicate
BLACK DEATH: Yersinia pestis smallpox, worldwide, by a
● At first, they thought it was airborne combination of isolation of infected
but it was actually from rat fleas. persons and vaccination of others in
● During the Middle Ages, plague was the community.
referred to as the black death - ● The last known case of naturally
because of the darkened, bruised acquired smallpox in the world
appearance of the corpses. occurred in Somalia in October
➔ The blackened skin and foul 1977.
smell were the result of cell ● In May 1980, the WHO announced
necrosis and hemorrhaging the global eradication of smallpox.
into the skin. ● Smallpox virus is currently stored in
● The last major plague epidemic in several laboratories , including those
Europe occurred in 1721. at the CDC and a comparable
● Plague still occurs, but the facility in Russia.
availability of insecticides and ● Smallpox virus - a potential
antibiotics has greatly reduced the biological warfare (BW) and
incidence of this dreadful disease. bioterrorism agent

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compared with a mortality rate of
BIOLOGICAL WARFARE (BW) AGENTS about 0.1% for modern-day flu
● The use of pathogens as biological epidemics.
warfare (BW) agents dates back ● Whereas most victims of
thousands of years. modern-day flu epidemics are very
● Ancient Romans threw carrion young, very old, and
(decaying dead bodies) into wells to immunosuppressed people, the
contaminate the drinking water of 1918– 1919 pandemic killed
their enemies. primarily healthy young adults.
● In the Middle Ages, the bodies of ● Scientists have recovered the virus
plague victims were catapulted over (designated A/H1N1) that caused
city walls in an attempt to infect the the pandemic from frozen tissue
inhabitants of the cities. samples and are currently studying
● Early North American explorers it.
provided Native Americans with ● It spread further because of World
blankets and handkerchiefs that War I when American troops went
were contaminated with smallpox back to the US after the war.
and measles viruses.
● Category A - most contagious and THE GREAT POTATO FAMINE
with highest incidence of mortality ● The mould killed off Ireland’s potato
rate (compared to other categories) crops in 1845, 1846, and 1848,
causing more than 1 million people
to die of starvation and illnesses
resulting from malnutrition.
● ANTOINE DE BARY - not until
1861 that he proved that it was a
fungus that had caused the blight.
THE MOSQUITO - A PERSISTENT AND

TUBERCULOSIS
DEADLY FOE
● Tuberculosis has been referred to as
● “No animal on earth has touched so
possibly the first born of the Mother
directly and profoundly the lives of
of Pestilence and Disease and is
so many human beings.”
known to have had few if any peers
● For all of history, and all over the
in causing incapacity and death of
globe, she has been a nuisance, a
people and domestic animals.
pain, and an angel of death.
● The mosquito has killed great
WORLD’S INFLUENZA PANDEMIC
leaders, decimated armies, and
● The 1819-1919 Spanish flu
decided the fate of nations.
pandemic (also known as the swine
flu pandemic) killed an estimated 20
AIDS
to 100 million people, worldwide -
● It was not until 1983 that the virus
between 2.5% and 5% of the world
that causes AIDS - now called
population.
human immunodeficiency virus or
● The pandemic killed between 2%
HIV was discovered.
and 20% of those infected

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● By the end of 2009, a total of ➔ In that paper, they
619,400 Americans (more than announced their discovery
those who had died in World Wars I that DNA, not proteins as
and II combined) had died of AIDS. had earlier been suspected,
is the molecule that contains
genetic information (i.e., that
COVID-19
DNA is the hereditary
● Severe acute respiratory syndrome molecule).
coronavirus 2 (SARS-CoV-2) is the ● ALFRED HERSHEY & MARTHA
etiologic agent of the current rapidly CASE - provided the additional
growing outbreak of coronavirus evidence that DNA is the molecule
disease (COVID-19), originating from that contains genetic information in
the city of Wuhan, Hubei Province, 1952.
China. ➔ Their work involved a
● Coronavirus disease 2019 bacteriophage that infects E.
(COVID-19) is caused by a new coli.
● In 1969, Hershey shared a Nobel
coronavirus first identified in Wuhan,
Prize with MAX DELBRUCK &
China, in December 2019. SALVADOR LURIA for their
● Although most people who have discoveries involving the genetic
COVID-19 have mild symptoms, structure and replication of
COVID-19 can also cause severe bacteriophages.
illness and even death. ● In the early 1950s, an American
● Some groups, including older adults named JAMES WATSON and an
and people who have certain Englishman named FRANCIS
underlying medical conditions, are CRICK published two extremely
at increased risk of severe illness. important papers.
● On February 11, 2020, the World ➔ The first (published in 1953)
proposed a double-stranded,
Health Organization announced an
helical structure for DNA (a
official name for the disease that is “double helix”).
causing the 2019 novel coronavirus ➔ The second (published in
outbreak. The new name of this 1954) proposed a method by
disease is coronavirus disease which a DNA molecule could
2019, abbreviated as COVID-19. In copy (replicate) itself exactly,
COVID-19, ‘CO’ stands for ‘corona,’ so that identical genetic
‘VI’ for ‘virus,’ and ‘D’ for disease. information could be passed
Formerly, this disease was referred on to each daughter cell.
to as “2019 novel coronavirus” or
“2019-nCoV.”
THE DISCOVERY OF THE HEREDITARY

MOLECULE
● OSWALD T. AVERY - In 1944, he
and his colleagues at the Rockefeller
Institute wrote one of the most
important papers ever published in
biology.
[D.N. & S.L.] | #2APHRPhCutie

10
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● Whereas GRIFFITH’S experiments
were conducted in vivo, AVERY’S
experiments were conducted in
vitro.
● Experiments conducted in 1952 by
ALFRED HERSHEY & MARTHA
CHASE using E. coli and
bacteriophages, confirmed that DNA
carried the genetic code.
================================
You’ve reached the end of this reviewer.
May you never discredit how far you’ve
come. You got this, future RPh! ♡
- Daph & Sof ♡
● The idea for the double-helical
structure was based on an x-ray
diffraction photograph of crystallized
DNA that WATSON had seen in the
London laboratory of MAURICE
WILKINS.
➔ The now famous photograph
had been produced by
ROSALIND FRANKLIN, an
x-ray crystallographer.
● WATSON, CRICK, & WILKINS
received a Nobel Prize in Chemistry
in 1962 for their contributions to our
understanding of DNA.
➔ FRANKLIN did not share the
prize because she had died
before 1962; the Nobel Prize
is not awarded
posthumously.
● Transformation was first
demonstrated in 1928 by the British
physician FREDERICK GRIFFITH
and his colleagues, performing
experiments with S. pneumoniae
and mice.
● It was not until 1944 that OSWALD
AVERY, COLIN MACLEOD, &
MACLYN MCCARTHY who also
experimented with S. pneumoniae,
first demonstrated that DNA was the
molecule that contained genetic
information.

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shown to be effective, safe,
WEEK 2 and of good quality.
Chapter 1: Introduction to Microbiology ➢ Pharmaceutical microbiology
Benefits of Studying Microbiology must encompass the
1. Improvement of life expectancy is subjects of sterilization and
due to better nutrition and preservation against
sanitation. microbial spoilage, and a
➢ Microorganisms are of great pharmacist with
significance to foods for the responsibility for the safe,
following reasons: hygienic manufacture and
- Microorganisms can use of medicines must know
cause spoilage of where microorganisms arise
foods in the environment, example,
- Microorganisms are the sources of microbial
used to manufacture contamination, and the
a wide variety of food factors that predispose to, or
products prevent, product spoilage.
- Microbial diseases
can be transmitted by The Husband-and-Wife Team Behind the
foods Leading Vaccine to Solve COVID-19
2. Improvement of healthcare and The German company
greater availability of effective BioNTech, founded by two
medicines scientists, has teamed up
3. Development of vaccines with Pfizer on a vaccine that
4. Disinfection through use of was found to be more than
chemicals as antiseptics, 90 percent effective.
disinfectants, and preservatives. Two years ago, Dr. Ugur
5. Benefits of using different Sahin took the stage at a conference in
microorganism in pharmacy Berlin and made a bold prediction.
➢ The growth of the Speaking to a roomful of infectious disease
pharmaceutical industry in experts, he said his company might be able
recent decades has been to use its so-called messenger RNA
paralleled by rising standards technology to rapidly develop a vaccine in
for product quality and more the event of a global pandemic. At that
rigorous regulation of time, Dr. Sahin and his company, BioNTech,
manufacturing procedures. were little known outside the small world of
➢ In order to receive a European biotechnology start-ups.
manufacturing license, a BioNTech, which Dr. Sahin founded with his
modern medicine must be wife Dr. Oziem Tureci, was mostly focused

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on cancer treatments. It had never brought - Simpler than viruses, being
a product to market. COVID-19 did not infectious particles comprising
exist yet, but his words proved prophetic. single stranded RNA without any
associated protein
Microorganisms - Plant pathogen
- Microorganisms differ enormously in - No known human pathogen
terms of their shape, size, and Prions
appearance in their genetic - Unique as infectious agents in that
metabolic characteristics. they contain no nucleic acid
➢ All of these properties are - Atypical form of mammalian protein
used in classifying that can interact with a normal
microorganisms into the protein molecule and cause it to
major groups with which undergo a conformational change
many people are familiar so that is, in turn, become a prion
examples are, bateria, fungi, and ceases its normal function
protozoa, and viruses. - Agents responsible for transmissible
spongiform encephalopathies eg
Viruses Creutzfeldt-Jakob disease (CJD)
- Do not have cellular structure and bovine spongiform
- Incapable of independent replication encephalopathy
as they do not contain the enzymes - Simplest and most recently
necessary to copy their own nucleic recognized agents of infectious
acid. disease, and are important in a
- Intracellular parasite and are pharmaceutical context owing to
reproduced using the metabolic their extreme resistance to
capabilities of the host cell conventional sterilizing agents like
- Observed in different shape (helical, steam, gamma radiation, and
linear, or spherical), size (20 to disinfectants
400nm) and nucleic acid Prokaryotes
composition (single-stranded, or - Bacteria and Archaea
double-stranded, linear or circular - No true nucleus; possess single
RNA or DNA) chromosome that is not separated
- Smaller than bacteria, and they from the other cell contents by the
cannot be seen with normal light membrane
microscope - Normally haploid (possess only one
- Viewed using electron microscope copy of the set of genes in the cell)
Viroids and reproduce asexually
- Also known as virusoids

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Eukaryotes
chloroplast
- All other cellular microorganisms s
examples are fungi, protozoa, and
algae Cell Sterols Sterol
- True cell nucleus in which the membrane present absent
compositio
chromosomes are separated from
n
the cytoplasm by the nuclear
membrane Cell wall Cell wall Walls
- Diploid (possess two copies of the compositio (when usually
set of genes in the cell), and n present) contain
usually peptidoglyc
reproduce sexually
contain ans
Distinguishing Features of Prokaryotes
cellulose or
and Eukaryotes chitin but
not
peptidoglyc
Characteris Eukaryotes Prokaryote
ans
tics s
Ribosomes Cytoplasmic Ribosomes
Size Normally > Typically 1
ribosomes are smaller,
10um to 5um
are 80s usually 70s
Location of Within a In the
Flagella Structurally Structurally
chromoso true nucleus cytoplasm,
complex simple
mes separated usually
from the attached to Pili Absent Present
cytoplasm the cell
by a nuclear membrane Fimbriae Cilia Present
membrane
Storage Poly-B-hydr Poly-B-hydr
Nuclear Exhibits Mitosis and compound oxybutyrate oxybutyrate
division mitosis and meiosis are s absent present
meiosis absent
Nucleolus Present Absent Bacteria

- Essentially unicellular, although
Reproducti Asexual or Normally
some species arise as sheathed
on sexual asexual
chains of cells
reproductio reproductio
n n - Possess the properties listed under
prokaryotes
Chromoso >1 1 - Exhibit great diversity of form,
me number habitat, metabolism, pathogenicity,
Mitochondr May be Absent and other characteristics
ia and present

PHA6114 LEC 2APH
- Bacteria of interest in pharmacy are eye, thus excluding toadstools and
the EUBACTERIA mushrooms.
Archaea - Tangled mass (mycelium) of
- No pharmaceutical importance but filaments or threads (hyphae) which
formerly considered largely to vary between 1 and 50um wide.
comprise organisms capable of Protozoa
living in extreme environment - Eukaryotic, predominantly
(extreme temperature, salinity, pH) unicellular microorganisms that are
or organisms exhibiting specialized regarded as animals rather than
modes of metabolism plants
Eubacteria - Free-living motile organisms that
- Typically rod-shaped (bacillus), occur in water and soil
spherical (cocci), curved or spiral - Not normally found as contaminants
cells of approximately 0.5-5mm of raw materials or manufactured
- Divided into 2 groups designated as medicines
gram-positive and gram-negative Naming of Microorganism
bacteria Genus and Species
Fungi - Plural = genera
- Structurally complex and varied in - Genus - written with an uppercase
appearance than bacteria, ane being initial letter
eukaryotes - Species - written with a lowercase
- Most fungi are saprophytes with initial letter
relatively few having pathogenic - Italicized or underline
potential, but their abil form spores - May be abbreviated by shortening
that are resistant to drying makes the name of the genus provided that
them important as contaminants of the shortened form is unambiguous
pharmaceutical raw materials, (not open to more than one
particularly materials of vegetable interpolation)
origin - Example:
- Non-photosynthesizing plants; Genus: Pseudomonas
covers both yeasts and molds Species: aeruginosa
- Yeasts are normally unicellular Pseudomonas aeruginosa
organisms that are larger than Pseudomonas aeruginosa
bacteria (5 to 10um) and divide P. aeruginosa
either by process of binary fusion or P. aeruginosa
budding
- Mold is an imprecise term used to
describe fungi that do not form
fruiting bodies visible to the naked

PHA6114 LEC 2APH
Terms - Microbial Metabolism
● Chemoheterotrophs - obtain
Carbon, nitrogen, and energy by
breaking down organic compounds
● Catabolic reactions - chemical
reactions by which the energy is
liberated by digestion of food
materials
● Anabolic reactions - use of
liberated energy to make complex
cellular polymers, proteins, ● With 2 to 3% agar
carbohydrates, and nucleic acids
● Oxidation - removal or loss of
electron Semi-solid media
● Redox reaction - oxidation and ● With 0.5 to 1% agar
reduction ● Example: motility medium
● Glycolysis - conversion of glucose,
through a series of reactions to
pyruvic acid
● Fermentation - releasing more
energy from sugar
Terms - Microbial Cultivation
● Culture media
● Cultivation methods
Culture media
● Planktonic and sessile (biofilm)
● Carbohydrates are used in the form of
growth
starch or sugars, but glucose (dextrose)
is the only sugar regularly employed as
Culture Media
a nutrient.
Liquid or Solid
● Microorganisms differ in terms of their
1. Liquid media (broth) - no solidifying
ability to ferment various sugars, and
agent
their fermentation patterns may be used
2. Solid media - designated for the
as an aid in identification.
growth of anaerobic organisms. Usually
● Sodium chloride may be incorporated in
contain non-toxic reducing agents
culture media to adjust osmotic
pressure, and occasionally buffers are
added to neutralize acids that result
from sugar metabolism.
● Routine culture media may be enriched
by the addition of materials like milk,

PHA6114 LEC 2APH
blood or serum, and organisms that
need such supplements in order to
grow are described as ‘exacting’ in their
nutritional requirements.
In contrast to medium ingredients designed

to support microbial growth, there are many
materials commonly added to selective or
diagnostic media whose function is to
restrict the growth of certain types of
microorganism while permitting or
enhancing the growth of others. ● Solid media designed for the growth of
● Examples include antibacterial anaerobic organisms usually contain
antibiotics added to fungal media to non-toxic reducing agents, e.g. sodium
suppress bacterial contaminants, and thioglycolate or sulfur-containing amino
bile to suppress organisms from acids; these compounds create redox
anatomical sites other than the potentials of -200mV or less and so
gastrointestinal tract. diminish or eliminate the inhibitory effects
of oxygen or oxidizing molecules on
anaerobic growth.
● The inclusion of such compounds is less
important in liquid media where a
sufficiently low redox potential may be
achieved simply by boiling; this expels
dissolved oxygen, which in unstirred
liquids only slowly resaturates the upper
few millimeters of liquid.
● Redox indicators like methylene blue or
resazurin may be incorporated in
anaerobic media to confirm that a
An enrichment medium is one designed to sufficiently low redox potential has been
permit a particular type of organism to grow achieved
while restricting others, so the one that
grows increases in relative numbers and is Media for yeasts and molds often have a
‘enriched’ in a mixed culture. lower pH (5.5-6.0) than bacterial culture
media (7.0-7.4).
● Lactic acid may be used to impart a low
pH because it is not, itself, inhibitory to
fungi at the concentrations used

PHA6114 LEC 2APH
● Some fungal media that are intended for value; turbidity is, therefore, an indirect
use with specimens that may also means of monitoring culture growth
contain bacteria may be supplemented
with antibacterial antibiotics, examples
are chloramphenicol or tetracyclines
When growing on solid media in petri

dishes (often referred to as ‘plates’)
individual bacterial cells can give rise to
colonies following overnight incubation
under optimal conditions.
● Colony is simply a collection of cells
Cultivation Methods arising by multiplication of a single
● Most bacteria and some yeasts divide by original cell or a small cluster of
a process of binary fission whereby the them (called a colony-forming unit
cell enlarges or elongates, then forms a or CFU).
cross-wall (septum) that separates the cell ● The term ‘colony’ does not, strictly
into two more or less equal speaking, imply any particular
compartments each containing a copy of number of cells, but it is usually
the genetic material taken to mean a number sufficiently
➔ Growth continues until one or more large to be visible by eye.
nutrients is exhausted, or toxic
metabolites (often organic acids) Colonies vary between bacterial species,
accumulate and inhibit enzyme and their shapes, sizes, opacities, surface
systems. markings and pigmentation may all be
characteristic of the species in question, so
Starting from a single cell, many bacteria these properties may be an aid in
can achieve concentrations of the order of identification procedures.
109 cells mL-1 or more following overnight
incubation in common liquid media.
At concentrations below about 107, cells

mL-1 culture media are clear, but the liquid
becomes progressively more cloudy (turbid)
as the concentration increases above this

PHA6114 LEC 2APH
Anaerobic organisms may be grown on their appearance when growing on
Petri dishes provided that they are common foods.
incubated in an anaerobic jar. ➔ The mold colony consists of a
● Such jars are usually made of rigid mycelium that may be loosely
plastic with airtight lids, and Petri dishes or densely entangled
are placed in them together with a depending on the species,
low-temperature catalyst often with the central area (the
● The catalyst, consisting of oldest, most mature region of
palladium-coated pellets or wire, the colony) showing
causes the oxygen inside the jar to be pigmentation associated with
combined with hydrogen that is spore production.
generated by the addition of water to ➔ The periphery of the colony is
sodium borohydride; this is usually that part which is actively
contained in a foil sachet that is also growing and it is usually
placed in the jar; alternatively, oxygen non-pigmented.
may be removed by combination with
ascorbic acid Planktonic and Sessile (Biofilm) growth
● After its removal, an anaerobic ● Freely suspended cells
atmosphere is achieved and this is ● Bacteria attached to a substrate in this
monitored by an oxidation - reduction way are described as sessile, and are
(redox) indicator; methylene blue or said to exhibit the biofilm or micro-
resazurin can be used. colony mode of growth.
● Planktonic cells are routinely used for
Anaerobic jar almost all the testing procedures that
have been designed to assess the
activity of antimicrobial chemicals and
processes, but the recognition that
planktonic growth is not the natural
state for many organisms prompted
investigations of the relative
susceptibilities of planktonic- and
biofilm-grown cells to antibiotics,
disinfectants, and decontamination or
Cultivation methods sterilization procedures.
● Yeast colonies often look similar to ● In many cases it has been found that
those of bacteria, although they may be planktonic and sessile bacteria exhibit
larger and more frequently colored markedly different susceptibilities to
● The appearance of molds growing on these lethal agents, and this has
solid microbiological media is similar to prompted a reappraisal of the

PHA6114 LEC 2APH
appropriateness of some of the ● Total viable count (TVC) - used in
procedures used. most pharmacopoeias and by many
regulatory agencies to mean a viable
Enumeration of Microorganisms count that records all different
● In a pharmaceutical context, there species or types of microorganism
are several situations where it is that may be present in a sample
necessary to measure the number of (bacteria + fungi)
microbial cells in a culture, sample,
or specimen: Traditional methods of enumerating cells
a. Measuring the levels of ● Viable Count
microbial contamination in a a. Pour plate (counting colonies
raw material or manufactured in agar)
medicine b. Surface spread or surface
b. Evaluating the effects of an drop (Miles Misra) methods
antimicrobial chemical or (counting colonies on agar
decontamination process surface)
c. Using microorganisms in the c. Membrane filter methods
manufacture of therapeutic (colonies growing on
agents membranes on agar
d. Assessing the nutrient surfaces)
capability of a growth d. Most probable number, MPN
medium (counts based on the
● In some cases, it is necessary to proportion of liquid growing
know the total number of microbial after receiving low inocula)
cells present, example, both living
and dead: Rapid methods (indirect viable counts) of
➔ Dead and living cells in enumerating cells
vaccine manufacture may a. Epifluorescence
both produce an immune - Uses dyes that give
response characteristic fluorescence
➔ Dead and living cells in only in living cells; often
pyrogen testing may both couples to image analysis
induce fever when injected b. ATP methods
● Total count - a counting procedure - Measure ATP production in
enumerating both living and dead living cells using
cells bioluminescence
● Viable count - far more common; c. Impedance
records living cells along - Measures changes in
resistance, capacitance, or

PHA6114 LEC 2APH
impedance in growing
cultures
d. Manometric methods
- Measure oxygen
consumption or CO2
production by growing
cultures ● These procedures are suitable for
samples that are expected to
Enumeration of Microorganisms contain concentrations exceeding
● After incubation, the resultant approximately 100 CFU ml-1 so that
colonies are counted and the total is the number of colonies arising on
multiplied by dilution factor (if any) the plate is sufficiently large to be
to give any concentration in the statistically reliable. If there are no
original sample. clear indications of the order of
➔ In a surface spread magnitude of the concentration in
technique, the sample the sample, it is necessary to plate
(usually 0.1-0.25 mL) is out the sample at each of two, three
spread over the surface of or more (decimal, i.e. 10-fold)
agar which has previously dilutions so as to obtain Petri dishes
been dried to permit with conveniently countable
absorption of added liquid numbers of colonies (usually taken
to be 30-300 colonies).
Most probable number (MPN) counts

● MPN may be used when the
anticipated count is relatively low, i.e
➔ Miles Misra (surface drop) -
from <1 up to 100 microorganisms
similar to surface spread in
mL-1
principle but several
● The procedure involves inoculating
individual drops of culture
multiple tubes of culture medium
are allowed to spreadover
(usually 3 or 5) with 3 different
discrete areas of about 1 cm
volumes, e.g. three tubes each
diameter on the agar surface
inoculated with 0.1 mL, three with
0.001 mL.
● If the concentration in the sample is
in the range indicated above, there
should be a proportion of the tubes
receiving inocula in which no

10
PHA6114 LEC 2APH
microorganisms are present; these for molds, and indirect measures of
will remain sterile after incubation, biomass like assays of insoluble
while others that received inocula nitrogen, protein or nucleic acids are
actually containing one or more CFU possible for all cell types, but rarely
show signs of growth. used outside the research
● The proportions of positive tubes laboratory.
are recorded for each sample ● The traditional methods of viable
volume and the results are counting all suffer from the same
compared with standard tables limitations:
showing MPN of organisms per mL a. Relatively labor intensive
(or per 100 mL) of the original b. Not easy to automate
sample. c. Slow, because they require
● 3 trials - microbiological tests an incubation period for
● 2 trials - assay, Id, etc. colonies to develop or liquid
● The procedure is more commonly cultures to become turbid
used in the water, food and dairy d. May require relatively large
industries than in the volumes of culture media,
pharmaceutical industry. many petri dishes and a lot
● Turbidity measurements are the of incubator space
most common means of estimating ● Epifluorescent techniques - Use
the total number of bacteria present fluorescent dyes that either exhibit
in a sample. different colors in living and dead
● Measuring the turbidity using a cells (e.g. acridine orange) or appear
spectrophotometer or colorimeter colorless outside the cell but
and reading the concentration from become fluorescent when absorbed
a calibration plot is a simple means and subjected to cellular
of standardizing cell suspensions for metabolism (e.g. fluorescein
use as inocula in antibiotic assays or diacetate)
other tests of antimicrobial ➔ Living cells generate
chemicals. adenosine triphosphate (ATP)
● Dry weight determinations on known that can readily be detected
volumes of culture are an alternative by enzyme assays, e.g.
means of estimating fungal luciferin emits light when
biomass. exposed to firefly luciferase
in the presence of ATP; light
Enumeration of microorganisms emission can be measured
● Direct microscopic counting may be and related to bacterial
an appropriate method for bacteria, concentration.
yeasts, and fungal spores but not

PHA6114 LEC 2APH
➔ The resistance, capacitance possesses, regardless of whether
or impedance of a culture they are expressed or not.
medium changes as a result ➔ For example, many bacteria
of bacterial or yeast growth possess the genes that code
and metabolism, and these for β-lactamases; these
electrical properties vary in enzymes hydrolyse and
proportion to cell inactivate β-lactam antibiotics
concentration. (e.g. penicillins)
➔ Manometric techniques are ● In many organisms, β-lactamases are
appropriate for monitoring only produced in response to
growth of organisms that presence of the antibiotic
consume or produce ➔ This form of non-genetic
adaptation is termed
significant quantities of gas
phenotypic adaptation, and
during their metabolism, e.g.
there are many situations in
yeasts or molds producing
which bacteria adopt a
carbon dioxide as a result of
phenotypic change to counter
fermentation
environmental stress.
➔ But microorganisms may also
Microbial genetics use an alternative strategy of
● The nature of the genetic material genetic adaptation, by which
possessed by a microbial cell and they acquire new genes either
the manner in which that genetic by mutation or by conjugation;
material may be transferred to other subsequently, a process of
cells depends largely upon whether selection ensures that the
the organism is a prokaryote or a mutant organisms that are
eukaryote. better suited to the new
● Microorganisms may adapt rapidly environment become
to new environments and devise numerically dominant.
strategies to avoid or negate ● In bacteria, mutation is an
stressful or potentially harmful important mechanism by which
circumstances. resistance to antibiotic and other
● Their ability to survive adverse microbial chemicals is achieved,
conditions may result from the although the receipt of entirely new
organism using genes it already genes directly from other bacteria is
possesses, or by acquisition of new also clinically very important.
genetic information. ➔ On average, a mutant rises
● Genotype - describes the genetic once in every 100,000 to
composition of an organism, it refers every 10 million cell
to the genes that the organism divisions.

PHA6114 LEC 2APH
➔ Although these figures might Bacteria
suggest that mutation is a ● Many of the medically and
relatively rare event, the pharmaceutically important bacteria
speed with which are pathogens, and some of these
microorganisms can multiply pathogens are of long-standing
means, for example, that notoriety as a result of their ability to
mutants exhibiting increased resist the activity of antibiotics and
antibiotic resistance can biocides (disinfectants, antiseptics,
arise quite quickly during the and preservatives).
course of therapy. ● In addition to these long-established
resistant microorganisms, other
Pharmaceutical importance of the major bacteria have given more recent
categories of microorganisms cause for concern including
methicillin-resistant
Viruses Staphylococcus aureus (MRSA),
● The importance of viruses derives vancomycin-resistant enterococci
exclusively from their pathogenic (VSE), and multiply resistant
potential, and because of their lack Mycobacterium tuberculosis.
of intrinsic metabolism, they are not ● The ability of bacteria to grow on
susceptible to antibiotics. diverse substrates ensures that their
● Viral infections are among the most potential as agents of spoilage in
dangerous and difficult to cure, and manufactured medicines and raw
of all the categories of materials is well-recognized, and the
microorganisms, only viruses ability of many species to survive
appear in (the most serious) Hazard drying means that they survive well
Category 4 as classified by the in dust and so become important as
Advisory Committee on Dangerous contaminants of manufactured
Pathogens medicines.
● Viruses are incapable of growing on Fungi
manufactured medicines or raw ● Fungi are able to form spores that
materials, so they do not cause survive dying, so they too arise
product spoilage, and they have no commonly as contaminants of
synthetic capabilities that can be manufactured medicines.
exploited in medicines manufacture. ● However, the degree of resistance
● Viruses are relatively easy to destroy presented by the spores is usually
by heat, radiation or toxic less than that exhibited by bacteria,
chemicals, so they do not represent and fungi do not represent
a problem from this perspective. sterilization problems.

PHA6114 LEC 2APH
● Fungi do not generally create a ● Organisms that readily form spores -
significant infection hazard either; Bacillus and Clostridium species of
relatively few fungal species are bacteria and most fungi - can be
considered major pathogens for stored for months or even years in
animals that possess a fully this way provided that the culture
functional immune system. medium does not evaporate to
dryness, but non-sporing organisms
Protozoa vary substantially in their survival
● Protozoa are of significance largely capacities.
owing to the pathogenic potential of ● Gram-positive bacteria generally
a few species. tend to survive better than
● Because protozoa do not possess Gram-negative ones: species like
cell walls, they do not survive well Pseudomonas aeruginosa.
drying well (unless in the form of ● The dual aims of a culture
cysts), so they are not a problem in preservation procedure therefore are
the manufacturing environment - to maintain the viability of the
and even the encysted forms do not highest possible percentage of cells
display resistance to sterilizing and to minimize the risk of selecting
processes to match that of bacterial atypical mutants.
spores. ● The most common procedures for
● It should be noted that protozoal long-term storage are by freezing at
infections are not currently a major -800C (or lower) in refrigerators, by
problem to human health in storage in liquid nitrogen at -1960C
temperate climates although they in special vessels or by
are more troublesome in veterinary freeze-drying (also called
medicine and in the tropics. lyophilization).
● There are concerns that the ● Cryoprotectant chemicals --
geographical ranges of protozoal compounds like glycerol or
infections such as malaria may dimethylsulfoxide are incorporated
extend substantially if current fears at a concentration of about 10% v/v
about global warming translate into in the liquid culture of the organism
reality. in order to minimize both the
formation of damaging ice crystals
Preservation of microorganisms and osmotic stresses that can
● Many bacteria and fungi can accelerate cell death during freezing
conveniently be stored for a few and thawing.
days, or possibly weeks, in the form ● Reference cultures, those with
of liquid cultures in tubes, or as well-defined biosynthetic
colonies in Petri dishes. capabilities or resistance properties,

14
PHA6114 LEC 2APH
can be obtained in a freeze-dried
form from internationally accessible
culture collections like the
American Type Culture Collection
(cultures having the designation
ATCC before a reference number) or
the UK National Collection of
Industrial and Marine Bacteria
(NICMB)
● Pharmacopoeias and regulatory

agencies are requiring tests that
employ microorganisms to be
conducted with cultures or test
suspensions of cells that are no
more than five subcultures from the
reference material obtained from the
designated culture collection.

PHA6114 LEC 2APH
BY: Daphne Nava, Sofia Larrauri, & Jana Samonte
WEEK 3 Bacterial cell components
Chapter 2: Bacteriology Cell wall
● defines the shape of bacteria

● PEPTIDOGLYCAN or MUREIN
LAYER consists of glycan
(polysaccharide chains of alternating
N-ACETYL GLUCOSAMINE (NAG)
Prokaryote Eukaryote
AND N-ACETYL-D-MURAMIC ACID
Nuclear No nuclear Classic (NAM)
body membrane, membrane-boun ● Some components responsible for
nucleoid d nucleus pathogenicity:
Cell Usually by Mitosis a. M protein – Streptococcus
division binary fission pyogenes
b. Mycolic acid –
Cell wall Eubacteria – Animals, Mycobacterium sp.
Peptidoglycan protozoans – no
● Gram stain reaction
CW CW Plants, fungi
➔ Gram-positive
Archaebacteria – with CW
–Res. - thicker peptidoglycan
peptidoglycan - contains teichoic acid
Cytoplasmi Phospholipid Phospholipid

c bilayer without bilayer with
membrane CHO and CHO and sterol
sterol component
component
Cell Absent Present

organelles
Site of Cytoplasmic Mitochondria

energy membrane
production
➔ Gram-negative
Site of Free ribosomes Rough ER - Thinner peptidoglycan
protein - outer membrane is bilayered
synthesis structure composes of
lipopolysaccharide
1. Surface ‘O’
or somatic antigen
[D.N., S.L., & J.S] | #2APHRPhCutie 1

PHA6114 LEC 2APH
2. Core looking for WBCs, epithelial cells,

polysaccharides debris, and larger organisms such
as fungi or parasites
3. Lipid A - Next, the smear should be
endotoxin examined using the oil immersion
(1,000x magnification lens)
- Periplasmic space
● Gram stain reaction
- Periplasmic
a. Gram-positive - purple
flagella/endoflagella/axial
- Formation of crystal
filaments
violet - iodine -
teichoic acid MgRNA
complex which is
insoluble to alcohol
b. Gram-negative - pink, red
- Lipids are highly
soluble to alcohol
- Decolorization - most critical step
in gram staining
- All cocci are gram positive except
● Gram staining Neisseria, Moraxella, and Veillonella
- All bacilli are gram negative
except Bacillus, Clostridium,
Reagents Gram + Gram -
Mycobacterium, Corynebacterium,
1O stain Crystal Stains Stains Listeria, Ersipelothrix, Lactobacillus,
violet purple purple Propionobacterium, Nocardia and
Actinomyces
Mordant Gram’s Remains Remains
- Spiral organisms are hard to stain;
iodine purple purple
those that are stainable react as
Decolorizer Alcohol Remains Becomes gram negative
and/or purple colorless - Mycoplasma, Ureaplasma - gram
acetone negative
Counterstai Safranin Remains Stains ● Gram positive cocci (aerobes)
n purple pink a. Micrococcus
b. Staphylococcus
- Once stained, the smear is c. Streptococcus
examined using the 10x objective ● Gram positive cocci (anerobes)
(100 magnification). The a. Peptococcus
microbiologist should scan the slide b. Peptostreptococcus

PHA6114 LEC 2APH
● Gram-positive in clusters
c. Sarcina
● Gram negative cocci (aerobes)
a. Neisseria
b. Branhamella
● Gram negative cocci
(anaerobes)
a. Veillonella
● Gram positive bacilli (aerobes) ● Gram-positive cocci in pairs
a. Bacillus (diplococci)
b. Corynebacterium
c. Erysipelothrix
d. Listeria
e. Mycobacterium
f. Nocardia
● Gram positive bacilli
(anaerobes) ● Gram-positive cocci in chains
a. Actinomyces
b. Clostridium
c. Propionobacterium
● Gram negative bacilli (aerobes)
a. Acinetobacter
b. Aeromonas
c. Alcaligenes
d. Bordetella ● Gram-negative cocci (pink/red)
e. Brucella
f. Enterobacteriaceae
g. Francisella
h. Legionella
i. Pasteurella
j. Pseudomonas
● Gram negative bacilli
(anaerobes)
a. Fusobacterium
● Gram-negative bacilli
b. Bacteroides
(rod-shaped)

PHA6114 LEC 2APH
● Acidfast staining
PARTS INTERNAL TO CELL WALL
Ziehl-N Kinyou Acid-fa Non ● Cytoplasmic membrane
eesen n st acid-fa - Selectively permeable
st - Site of energy production
1ostain Carbolf Carbolf Stains Stains ● Mesosomes
uchsin uchsin red red - Point of attachment for
chromosome
morda Steam Tergitol Remain Remain ● Inclusions - nutrient storage
nt s red s red
➔ MUCH GRANULES -
decolo HCl, HCl, Remain Becom contains lipids, MTB
rizer alcohol alcohol s red es ➔ BABES-ERNST/METACHR
colorles OMATIC/VOLUTIN
s GRANULES - accumulation
of polymerized
counte Methyle Methyle Remain Stains
rstain ne blue ne blue s red blue polyphosphates
(Corynebacterium
diphtheria)
● Bipolar bodies
● Mycobacteria
➔ Yersinia pestis
- Gram-positive bacilli
- safety pin
- On actual gram-staining:
appearance on
gram-ghost or gram neutral
Wayson stain
- Cell with mycolic acid -- hard
- Bipolar bodies
to stain, but once stained, it
resists decolorization ACID
FAST

PHA6114 LEC 2APH
➔ Clostridium tetani spores

- Round, terminally located
- Tackhead, lollipop bacillus
PARTS EXTERNAL TO CELL WALL

● Capsule
- Organized material firmly
attached to the cell wall
● Endospore
- Increase virulence by
- Resting cell, highly resistant
preventing phagocytosis
to desiccation, heat and
- Antigenic; on the basis of
chemical agents
serotyping by QUELLUNG
- Composition: DIPICOLINIC
REACTION
ACID/CALCIUM
- Colonies often slimy
DIPICOLINATE
● Slime layer
➔ Bacterial genera w/spores:
- Unorganized material not
a. Bacillus sp.
firmly attached to the cell
- Gram positive bacilli,
wall
sporulate aerobically
- Staphylococcus epidermidis
- Catalase (+)
● Pili (Syn. FIMBRIAE)
b. Clostridium sp.
a. ordinary/common/somatic
- Gram positive bacilli,
pili - adherence of bacteria
sporulate
to host cell
anaerobically
b. Sex/fertility pili - bacterial
- Catalase (-)
conjugation
➔ Clostridial spores
● Flagella
- Oval, subterminally located

PHA6114 LEC 2APH
➔ Atrichous - no flagellum
➔ Monotrichous - flagellum on
one pole
a. Tumbling motility: Listeria

b. Darting motility:
Campylobacter
➔ Amphitrichous - single c. Gliding motility:
flagellum on each pole Capnocytophaga
➔ Semisolid medium
- uses 0.5 to 1% agar
- Motility best seen at 250C
➔ Stains for flagella
a. Leifson
➔ Lophotrichous - tuft of b. Gray
flagella at one or both sides c. Fisher & Conn
➔ Peritrichous - flagella all

over the organism
Bacterial virulence factors
● Pathogenicity - ability to cause
diseases
● Virulence - degree of pathogenicity
1. Adherence factors - pili
2. Anti-phagocytic factors-
➔ Periplasmic flagella - AXIAL capsule, some components
FILAMENTS/ENDOFLAGELL of the cell wall
A (SPIROCHETES) 3. Enzymes
➔ Motility determination: a. Coagulase
b. Fibrinolysin (kinase)

PHA6114 LEC 2APH
c. Hyaluronidase
Effect Specific toxins Disruption of
(spreading factor, target specific clotting,
Duran-Reynal) host cells; the causing clots
4. Toxins - exotoxin & endotoxin type of toxin to form
varies with the throughout the
Comparis Exotoxin Endotoxin
bacterial species body [DIC]
on
a. Cytotoxin – Fever
Source Most commonly General toxin
kills host cells
associated with common to
Hypotension,
gram-positive almost all
b. Enterotoxin – shock, and
bacteria gram-negative
damages cells death
bacteria
of GIT
Release Produced and Released when
released by gram-negative c. Neurotoxin –
living bacteria; bacterial cell is interferes with
do not require destroyed nerve impulse
bacterial death transmission
for release
Toxicity HIGHLY TOXIC MODERATELY
Composit Polypeptides Composed of FATAL TO TOXIC FATAL
ion lipopolysaccha ANIMALS IN FOR ANIMALS
ride portion of MICROGRAM IN TENS TO
cell envelope QUANTITIES OR HUNDREDS
LESS OF
Heat HEAT-LABILE HEAT-STABLE MICROGRAM
stability EXCEPT FOR Withstand S
STAPHYLOCOC heating at
CAL temperatures Diseases TETANUS, UTI, TYPHOID
above 60°C for LOCK JAW (C.
ENTEROTOXIN hours without TETANI):
loss of toxicity TETANOSPASMI
Destroyed N
rapidly by NEUROTOXIN
heating at SPASMODIC
temperatures CONTRACTION
above 60°C S
Immunol HIGHLY WEAKLY BOTULISM (C.

ogic ANTIGENIC IMMUNOGENI BOTULINUM)
CONVERTED C NOT BOTULINUM
TO TOXOIDS CONVERTED TOXIN MOST
TO TOXOIDS POTENT
EXOTOXIN

PHA6114 LEC 2APH
NEUROTOXIN a. Tyndallization – flowing steam

FLACCID 100 oC for 30 minutes for 3
PARALYSIS successive days
** flaccid paralysis - usually people who
b. Inspissation – 75 to 80 oC for
have undergone botox procedure have
2 hours for 3 successive days
same facial expressions - face does not
move
Dry heat Flame
STERILIZATION -kills by - to sterilize inoculating
● Sterilization - process that kills all oxidation loops and needles
forms of microbial life, including Oven
bacterial spores - 160 to 180 oC for 1.5 to
Physical methods 3 hours
1. Heat - For glass wares, certain
metals, oils, petrolatum,
Moist heat Autoclave or powders
-coagulates - Most effective method of - Quality control/biologic
protein sterilization indicator:: spores of
- fastest and - 121 oC at 15 pounds Bacillus subtilis
simplest pressure per square inch
physical (psi) for 15 minutes INCINERATION
method of - Infectious medical waste - Most common method
sterilization is often sterilized at of treating infectious
132°C (270°F) for 30 to waste
60 minutes to allow - Hazardous material is
penetration of the steam literally burned to ashes
throughout the waste at temperatures of 870°
and the displacement of to 980°C
air trapped inside the - SAFEST METHOD TO
autoclave bag ENSURE THAT NO
- Quality control/ biologic INFECTIVE MATERIALS
indicator: spores of REMAIN IN SAMPLES
Bacillus OR CONTAINERS
stearothermophilus WHEN DISPOSE
- Prions, infective
Fractional proteins, are not
- alternate heating (kills eliminated using
vegetative cells), conventional methods;
incubation (spores therefore incineration is
germinate), heating (kills recommended
remaining vegetative
cells and spores) 2. Filtration

PHA6114 LEC 2APH
- Method of choice for antibiotic

containers
solutions, toxic chemicals,
radioisotopes, vaccines, and 2. Formald Sterilize HEPA filters in
carbohydrates, which are all ehyde BSCs
heat-sensitive vapor
and
- Filtration of liquids is accomplished
vapor
by pulling the solution through a phase
cellulose acetate or cellulose nitrate hydroge
membrane with a vacuum. n
- Filtration of air is accomplished peroxide
using high-efficiency particulate air
3. Glutarald Sporicidal (kills spores)
(HEPA) filters designed to remove
ehyde in 3 to 10 hours, is
organisms larger than 0.3 µm from used for medical
isolation rooms, operating rooms, equipment such as
and biologic safety cabinets (BSCs) bronchoscopes,
3. Ionizing radiation because it does not
- Plastic syringes, catheter or gloves corrode lenses, metal,
or rubber
- Short wavelength, high energy
gamma rays Cold sterilization
- Uses cathode rays (e.g. cobalt 60)
4. Peraceti Effective in the
Chemical methods c acid presence of organic
material, has also been
used for the surface
1. Ethylene Most common sterilization of surgical
oxide chemical sterilant instruments
Cold sterilization Cold sterilization
Used for materials

DISINFECTION
which cannot be
autoclaved ● Disinfection - process that destroys
pathogenic organisms, but not
Strict requirements of necessarily all microorganisms or
concentration, spores.
humidity Physical methods
Quality control: spores

of Bacillus subtilis Boiling 100°C for 15 minutes,
which kills vegetative
Used to sterilize plastic bacteria

PHA6114 LEC 2APH
Pasteurizatio Used mostly in the food agents used on Tincture

n industry, eliminates living tissue (alcohoholic) iodine,
food-borne pathogens iodophor
and organisms
responsible for food
spoilage Alcohol Ethyl or isopropyl
alcohol is
a. Batch method: 63°C for - Ethanol, nonsporicidal (does
30 minutes isopropanol, not kill spores) and
b. Flash method: 72°C for benzyl evaporates quickly
15 seconds alcohol
Denature 70% ethyl alcohol is
Disinfects and kills proteins; more effective as a
milk-borne pathogens and make lipids disinfectant than 95%
vegetable forms; soluble Skin ethyl alcohol because
endospores survive antiseptics the increased water
(H2O) hydrolyzing
Non-ionizing Long wavelength and low bonds in protein
radiation energy. molecules make the
killing of
Do not penetrate well, and microorganisms more
organisms must have effective
direct surface exposure,
such as the working Aldehydes (in Disinfectants; kill
surface of a BSC, for this solution) endospores
form of disinfection to
work - Formaldehy Toxic to humans
de,glutarald
ehyde
● Vinegar - natural disinfectant
Halogen Halogens, especially
Chemical methods iodine and chlorine, are
- Tincture frequently used as
(alcoholic) disinfectant
Disinfectants Phenol, phnenolics, iodine Iodine is prepared
- Destroys QUATS, chlorine, Iodophor = either as a tincture with
vegetative iodine + alcohol or as an
forms on Glutaraldehyde detergent iodophor coupled to a
inanimate neutral polymer (e.g.,
objects Chlorine povidone-iodine).
(microbicidal)
Chlorine is most often
Antiseptic Alcohol,
used in the form of
- Microbiostatic
sodium hypochlorite

PHA6114 LEC 2APH
**Benzalkonium chloride (Zephiran) -

(NaOCl), the
compound known as cationic; incompatible with anionic like soap
household bleach. (will produce neutral/0 net charge)
Heavy metals Because mercury is Biological safety cabinets

toxic to the
environment, heavy
metals containing
mercury are no longer
recommended
Eye drop solution

containing 1% silver
nitrate is still placed in
the eyes of newborns
to prevent infections
with N. gonorrhoeae
Quaternary Benzalkonium chloride

ammonium (Zephiran)
compounds
(QUATS) Used to disinfect
bench tops or other
surfaces in the
laboratory.
** Class II - common
Rapidly inactivated by ** Class III - hospitals and research centers
organic matter
Disadvantages: Transmission-based precautions
nonsporicidal, ● Airborne (Measles, varicella,
nontuberculoidal tuberculosis,smallpox)
- Place patient in private room
Phenolics Denature proteins;
disrupt cell that has monitored negative
membranes air pressure, 6-12 air
changes per hour, and
Disinfectants at high appropriate discharge of air
concentrations; used in outdoors or monitored HEPA
soaps at low
filtration of room air before
concentrations
air is circulated to other
areas of the hospital or
** Chlorine - in the form of chlorox cohorting of patients—that
is, placing patients with the

PHA6114 LEC 2APH
same infection in the same - Adenovirus, influenza virus,

room, if private rooms are mumps, Parvovirus B19,
not available rubella
- Health care workers (HCWs) - Place patient in private room
to wear respiratory without special air handling
protection when entering or ventilation or cohort
room of patient with known patients
or suspected tuberculosis - HCWs should wear mask
and, if not immune, for when working within 3 feet of
patients with measles or patient
varicella as well - Transfer patients out of their
- Transport patients out of room only after placement of
their room only after a surgical mask
placement of a surgical mask ● Contact
➔ On most N95 models, the nosepiece - Gastrointestinal, respiratory,
is meant to be molded to the user’s skin, or wound infections, or
facial structure using his/her index colonization with
fingers of both hands to press gently multidrugresistant bacteria
against the metal strip until it molds - Clostridium difficile
to a snug fit. - For diapered or incontinent
● Droplet patients: Escherichia coli
- Invasive Haemophilus O157:H7, Shigella, hepatitis
influenzae type b infection, A virus, or rotavirus
including meningitis, - Respiratory syncytial virus,
pneumonia, epiglottitis, and parainfluenza virus, and
sepsis enterovirus infections in
- Invasive Neisseria infants and young children
meningitidis infection, - Skin infections such as
including meningitis, diphtheria (cutaneous),
pneumonia, and sepsis herpes simplex virus
- Diphtheria (pharyngeal), (neonatal or
Mycoplasma pneumoniae mucocutaneous), impetigo,
- Pertussis, pneumonic plague major abscesses, cellulitis,
- Streptococcal pharyngitis, or decubiti, pediculosis (lice
pneumonia, or scarlet fever infestation), scabies (mite
in infants and young children infestation), staphylococci
furunculosis (boils) in infants
and young children, zoster

PHA6114 LEC 2APH
(disseminated or in the a. Obligate/strict aerobe

immunocompromised host) - require oxygen for growth
- Viral hemorrhagic infections - With catalase and
(Ebola, Lassa, or Marburg) superoxide dismutase (SOD)
enzymes that convert toxic
Bacterial growth factors product of oxygen to
Nutrients nontoxic substances
a. Carbon - synthesis of cellular b. Obligate/strict anaerobe
compounds - Cannot grow in presence of
➔ Lithotroph/autotroph - able oxygen
to grow by using CO2 s the Without catalase and
sole source of carbon superoxide dismutase (SOD)
➔ Heterotroph/organotroph - c. Facultative anaerobe
require an organic source of - Can grow wither with/without
carbon such as glucose oxygen
b. Nitrogen - synthesis of proteins, - Staphylococcus - catalase +
DNA, and RNA - Streptococcus - catalase -
c. Minerals - examples are sulfur and d. Aerotolerant anaerobe
magnesium - Can survive in the presence
d. Salt of oxygen but do not use
➔ Staphyloccocus oxygen in metabolism
➔ Vibrio sp. except V. cholerae e. Microaerophilic
and V. mimicus - Require reduced level of
e. Others oxygen to grow
➔ Increased protein (whole - Campylobacter - 5%
eggs) - Mycobacterium oxygen, 10% carbon
➔ X and V factors - Hemophilus dioxide, 85% nitrogen
● Hemophilus sp. require X and V f. Capnophilic
factors for growth: - Require an atmosphere
1. X factor/ hemin enriched with extra carbon
- From the degradation dioxide (5-10% CO2)
of hemoglobin
- Heat-stable
2. V factor/NAD/coenzyme 1
Aerobes Grow in AMBIENT
- Produced by some AIR, which contains
bacteria and yeasts 21% O2 and a small
- Heat-labile amount (0.03%) of
Oxygen and carbon dioxide availability CO2

PHA6114 LEC 2APH
● Capnophiles, such as Hemophilus

Anaerobes Cannot grow in the
presence of O2 and influenzae and Neisseria
the atmosphere in gonorrhoeae, require:
anaerobe jars, bags a. Increased concentrations of
or chambers is CO2 (5-10%)
composed of 5% to b. Approx. 15% O2
10% H2, 5% to 10%
➔ This atmosphere can be achieved
CO2, 80% to 90% N2
and 0% O2 by:
i. Candle jar (3% CO2)
Microaerophiles Grow under reduced ii. CO2 incubator, jar, or bag
O2 (5% to 10%) and
increased CO2 (8%
Temperature
to 10%); this
a. Psychrophilic/cryophilic -
environment can also
be obtained in cold-loving, optimal growth at
specially designed 10-200C
jars or bags b. Mesophilic - grow optimally at
moderate temperature, optimal
Capnophiles Requires increased 0
growth at 20-40 C; most
concentrations of
CO2 (5% to 10%) pathogenic; incubator temp: 35 +/-
and approximately 20C (33-37 0C)
15% O2; atmosphere c. Thermophilic - grow best at high
can be achieved by a temperatures, optimal growth at
candle jar or CO2 50-600C
incubator
pH
● most clinically relevant bacteria
prefer a neutral pH range, from
6.5-7.5
a. Acid-loving
- Lactobacillus (pH 3)
- Tomato juice agar
b. Alkali-loving
- Vibrio (pH 8-10)
- Alkaline peptone water
(APW)

PHA6114 LEC 2APH
Growth cycle: - Ex. media that contain

1. Lag phase blood/serum,
- little/ no multiplication plant/animal/yeast extract
- Period of adjustment c. Tissue - for organisms which
2. Log or exponential phase cannot grow on cell-free or artificial
- Organisms grow at media (Chilamydia/rickettsia/
maximum rate (exponential viruses)
rate)
- Organisms are most Function and use
sensitive to antimicrobials a. Simple/basal/supportive/general
3. Stationary or plateau phase isolation/general purpose media
- Growth ceases because - Support the growth of most
nutrients are exhausted or non fastidious bacteria
toxic metabolic products - Nutrient agar/broth
have accumulated - Trypticase soy agar/broth
4. Decline phase ● Nutritive/supportive media
- Viable count decreases - Tryptic soy agar, or nutrient
agar plates for bacteria
Culture media - Sabouraud’s dextrose agar
According to physical state/ consistency for fungi
a. Liquid - no solidifying agent; b. Enriched media
broth/infusion/milk - Contain nutrient supplement
b. Semisolid - contains 0.5 to 1% for fastidious bacteria
agar; transport media/motility media - Blood agar plate
c. Solid - Chocolate agar plate
- Liquefiable - contains
2-3% agar
- Non-liquefiable -
chopped meat
(anaerobes), rice
grains (fungi)
Composition
a. Synthetic/chemically-defined -
exact composition is known c. Enrichment media
b. Complex/non-synthetic - contains - Enhance the growth of an
at least one component which is not organism
chemically defined (not simple, no - Selenite broth - Salmonella,
chemical formula) Shigella

PHA6114 LEC 2APH
- Tetrathionate broth - ● Examples of selective culture

Salmonella, Shigella media
- Alkaline peptone water - 1. For mycobacterium
Vibrio tuberculosis (MTB)
● Enrichment media - Medium:
- Buffered charcoal-yeast Lowenstein-Jensen
extract agar, for medium
L.pneumophila - Inhibitor for other
● Enrichment broths organisms: malachite
- Thioglycollate for the green
isolation of anaerobes - Petragnani - for
- LIM (Todd-Hewitt with heavily contaminated
colistin and nalidixic acid) specimens, high
broth for selective conc. of malachite
enrichment of group B green
streptococci 2. For corynebacterium
- Gram-negative (GN) broth for diphtheria
the selective enrichment of - Medium: Tellurite
enteric gram-negative - Inhibitor: potassium
organisms tellurite
d. Selective media 3. For gram positive bacteria
- Select for growth of a - PEA (phenylethyl
particular organism, contains alcohol) inhibits gram
inhibitors negative bacteria
- Inhibitory agents used for 4. For neisseria gonorrhoeae
this purpose include dyes, - Contains antibiotics
bile salts, alcohols, acids, as inhibitors
and antibiotics ● Selective culture media for
● Inhibitors for gram positive neisseria gonorrhoeae
bacteria
1. Dyes: gentian violet, crystal
violet
THAYER Vancomycin – inhibits growth
2. Bile salt: sodium -MARTIN of gram positive bacteria
desoxycholate
● Inhibitors for gram negative Colistin – inhibits growth of
bacteria gram negative bacteria
1. Potassium tellurite except N. gonorrhoeae
2. Sodium azide
Nystatin – inhibits growth of

PHA6114 LEC 2APH
● Selective differential for

fungi
Staphylococcus sp.
MODIFIED Vancomycin – inhibits growth
THAYER of gram positive bacteria
-MARTIN Medium Mannitol salt agar
Colistin – inhibits growth of (MSA)
gram negative bacteria
CHO Mannitol
except N. gonorrhoeae
inhibitor 7.5 to 10% NaCl
Nystatin – inhibits growth of
fungi pH indicator Phenol red
Trimethoprim lactate – Mannitol fermenters Yellow colonies

prevents swarming of Proteus
sp. Non-mannitol Pink colonies
fermenters
MARTIN Vancomycin – inhibits growth
-LEWIS of gram positive bacteria
● Selective differential for Vibrio sp.
Colistin – inhibits growth of
gram negative bacteria Medium THIOSULFATE
except N. gonorrhoeae CITRATE BILE SALT
SUCROSE (TCBS)
Anisomycin – inhibits growth AGAR
of fungi
CHO Sucrose
Trimethoprim lactate –
prevents swarming of Proteus inhibitor Bile salts that inhibit
sp. growth of
gram-positive
NEW YORK Vancomycin – inhibits growth organisms
CITY AGAR of gram positive bacteria
pH indicator Bromthymol blue
Colistin – inhibits growth of
gram negative bacteria Mannitol Yellow colonies
except N. gonorrhoeae fermenters
Non-mannitol Green colonies

Amphotericin B – inhibits
fermenters
growth of fungi
Trimethoprim lactate – d. Differential media - provide distinct

prevents swarming of Proteus colonial appearances of microorganisms to
sp. aid in their identification

PHA6114 LEC 2APH
RAPID LATE NON-LACTO capsule)

LACTOSE LACTOSE SE
FERMENTE FERMENTE FERMENTE Enterobacter – pink
RS RS RS colonies with dark
center (fish eye)
With lactose With Without
permease beta-galacto lactose NON-LACTOSE Colorless colonies
and sidase only permease FERMENTERS
beta-galacto and
sidase beta-galactos
idase
Escherichia Salmonella Salmonella

arizonae
Klebsiella Shigella
Shigella
Enterobacte sonnei Proteus
r
● Maacconkey (MAC) agar
Citrobacter Providencia
Inhibitors Crystal violet, bile salt
Serratia Morganella
COH Lactose
Hafnia Edwardsiella
pH indicator Neutral red
Yersinia
Lactose Pink colonies
fermenters
Non-lactose Colorless colonies

fermenters
● Eosin-methylene blue (EMB) agar ● Family Micrococcaceae

- Genera: Staphylococcus,
CHO LACTOSE
Micrococcus, Planococcus,
LACTOSE PINK TO PURPLE and Stomatococcus
FERMENTERS COLONIES ➔ Laboratory diagnosis:
1. Gram staining:
E. coli – pink to purple - Staphylococci appear
colonies with green as gram-positive
metallic sheen
cocci, usually in
Klebsiella – pink clusters
mucoid colonies (large

PHA6114 LEC 2APH
- Micrococci typically isolate staphylococci

appear as from clinical materials
gram-positive cocci ➔ Phenylethyl
tetrads, rather large alcohol (PEA)
clusters ➔ Columbia
Colistin-Nalidi
xic Acid (CNA)
Agar
➔ Mannitol Salt
Agar (MSA)
Growth on Staphylococcus: white

BAP PINHEAD colonies with
characteristic hemolytic
pattern
Alpha - Partial lysis of red cells

(incomplet around colony
e) - Greenish discoloration
Hemolysis of area around colony
Beta - Complete lysis of red

2. Cultivation:
(complete) cells around colony
- Organisms will grow
Hemolysis - Clear area around
on 5% sheep blood
colony
and chocolate agars
- Grow well in Gamma - No lysis of red cells
broth-blood culture (nonhemol around colony
systems and ytic) - No change in agar
common nutrient
broths, such as Alpha-Prim - Small zone of alpha
thioglycollate, e surrounded by a zone
dextrose broth, and of beta hemolysis after
brain-heart infusion refrigeration
- Selective media can
Growth on Enhances pigmentation of
also be used to
Leoffler’s Staphylococcus spp.

PHA6114 LEC 2APH
● Staphylococc
Serum - S. aureus: golden
us: positive
Slant (LSS) yellow colonies
● Streptococcus
- S. citreus: lemon
: negative
yellow colonies
- S. albus: porcelain
4. Tests to differentiate
white colonies
Staphylococcus from
Growth on Selective and differential Micrococcus
Mannitol medium for
Salt Agar Staphylococcus spp. Tests Staphyloco Micrococcu
(MSA) Inhibitor: 7.5 to 10% NaCl ccus s
CHO: Mannitol
pH indicator: phenol red Aerobic Growth Growth
growth
- S. aureus: yellow
colonies (mannitol Anaerobic Growth No growth
fermenter) growth
- S. epidermidis:
Lysostaphin Susceptible Resistant
pink colonies
(nonfermenter of Furazolidone Susceptible Resistant
mannitol)
- S. saprophyticus: Bacitracin Resistant Susceptible
variable
Modified Negative Positive
oxidase
3. Catalase test
Glucose Fermenter Oxidizer
- Differentiates utilization
catalase-positive
micrococcal and
5. Coagulase
staphylococcal
- Most important
species from
pathogenic
catalase-negative
determinant of S.
streptococcal species
aureus
- Reagent: 3%
- Differentiates
hydrogen peroxide
Staphylococcus
(H2O2)
aureus (positive) from
- Positive result:
coagulase-negative
effervescence,
staphylococci
bubbles
(negative)

PHA6114 LEC 2APH
A. Slide coagulase - 5 μg novobiocin disk

Detects: cell-bound is gently pressed to
coagulase or the surface of the
clumping factor inoculated plate, and
Reagent: rabbit’s the plate is incubated
plasma overnight at 35 to
Positive: clumping 37oC
B. Tube coagulase - Zone of no growth
Detects: free that is 16 mm or
coagulase more in diameter
Reagent: rabbit’s indicates that the
plasma isolate is susceptible
Positive: gel-like to novobiocin
fibrin clot
S. Associated SENSITIVE
6. DNA Hydrolysis (DNASE epidermidis with TO
Test Agar) bacterial NOVOBIOCI
Determines the ability of an endocarditis N
organism to hydrolyze DNA following
- Medium is pale green the insertion
of artificial
because of the
heart
DNA-methyl green
complex S. Important RESISTANT
- If the organism saprophytic cause of TO
growing on the us UTI in NOVOBIOCI
young N
medium hydrolyses
women
DNA, the green color
fades and the colony
is surrounded by a PATHOGENIC DETERMINANTS OF
colorless zone STAPHYLOCOCCUS AUREUS
Medium: DNA medium 1. Protein A - inhibits phagocytosis
Positive result: clearing of ● Coagglutination: uses
the medium bacteria as inert particles to
which antibodies are
7. Novobiocin resistance test attached
Differentiate S. saprophyticus 2. Coagulase - conversion of
from other Staphylococcus fibrinogen to fibrin; may coat
spp. neutrophils with fibrin formed to

PHA6114 LEC 2APH
protect the organism from - Streptococcus spp. and

phagocytosis Enterococcus spp. Alloiococcus,
3. Staphylokinase (fibrinolysin) - which is catalase negative only
dissolves fibrin clots and may when tested on media devoid of
enable the infection to spread once whole blood (e.g. chocolate agar), is
clot is dissolved included because it morphologically
4. Lipase - hydrolyzes lipids in plasma resembles the viridans streptococci
and skin; associated with initiation
of skin infections such as boils LANCEFIELD CLASSIFICATION
5. Hyaluronidase - hydrolyzes - By antigenic character of a
hyaluronic acid in connective tissue, group-specific cell wall
spreading factor polysaccharide
6. DNase - degrades DNA - Almost 20 Lancefield groups are
7. Exfoliatins - hydrolyze tissue identified
through cleavage of stratum
granulosum; associated with Lancefield group antigen: A
staphylococcal scalded skin Species: S. pyogenes
syndrome Hemolysis: Beta
8. Leukocidins - lysis of neutrophils Disease associations:
and macrophages; inhibit - Infections include pharyngitis,
phagocytosis tonsillitis, or “strep throat” scarlet
9. Hemolysins - lyse erythrocytes fever, impetigo, cellulitis, wound
10. Enterotoxins - food poisoning and infections erysipelas
toxic shock syndrome - Necrotizing fasciitis (flesh-eating
disease)
● The “gold standard” for MRSA detection is - May be associated with acute
the detection of the mecA gene by using rheumatic fever (ARF)
molecular nucleic acid probes or PCR - Acute glomerular nephritis (AGN)
amplification may be seen in patient following
● VANCOMYCIN is the drug of choice and streptococcal pharyngitis or skin
sometimes the only drug available for infections
serious staphylococcal infections
Lancefield group antigen: B
FAMILY STREPTOCOCCACEAE Species: S. agalactiae
Genera Hemolysis: Beta
- Catalase-negative gram-positive Disease associations:
cocci

PHA6114 LEC 2APH
- Neonatal infections, including

ALPHA - Streptococcus
pneumonia, meningitis and pneumoniae, viridans
bacteremia streptococci
- Incomplete hemolysis
Lancefield group antigen: C - Green or brown zone
Species: S. dysgalactiae around the colonies
S. equi BETA - Group A, B, C, and some
Hemolysis: Beta group D
Disease associations: - Complete hemolysis
- Causes severe pharyngitis followed - Clear zone around the
by bacteremia colonies
- Opportunistic pathogen that may
GAMMA - Most group D
also be associated with pneumonia, - Non-hemolytic
cellulitis, and abscess
FAMILY STREPTOCOCCACEAE
Lancefield group antigen: D
1. Gram Staining
Species: E. faecalis
- Gram-positive cocci in pairs or in
E. faecium
chains
Common terms: Enterococcus
- S. pneumoniae is typically
Hemolysis: Alpha
lancet-shaped and occurs singly, in
Beta
pairs, or in short chains
Gamma
Disease associations:
2. Cultivation
- UTI and wound infections
- Grow on gram-positive selective
media such as CNA (Columbia agar
Lancefield group antigen: D
with colistin and nalidixic acid) and
Species: S. bovis group
PEA (phenylethyl alcohol agar)
Common terms: Nonenterococcus
- Except for Abiotrophia and
Hemolysis: Alpha
Granulicatella, the organisms will
Gamma
grow on standard laboratory media
Disease associations:
such as 5% sheep blood and
- Endocarditis, UTIs, pyogenic
chocolate agars
infections
- To detect genital carriage of group B
streptococci during pregnancy,
BROWN’S CLASSIFICATION
TODD-HEWITT BROTH
- Classified according to the pattern
of hemolysis
3. Catalase

PHA6114 LEC 2APH
- Differentiates catalase-positive presence of the enzyme

micrococcal and staphylococcal L-pyrrolidonyl arylamidase
species from catalase-negative - L-pyrrolidonyl- β-naphthylamide
streptococcal species substrate to produce a
Reagent: 3% hydrogen peroxide β-naphthylamine; β-naphthylamine
(H2O2) can be detected in the presence of
Positive result: effervescence, N,N-methylaminocinnamaldehyde
bubbles reagent by the production of a
Staphylococcus: positive bright red precipitate
Streptococcus: negative - Positive: bright red color within 5
minutes
4. Bile Esculin Hydrolysis - Positive for S. pyogenes and Group
- Presumptive identification of D Enterococcus
enterococci and organisms in the
Streptococcus bovis group 6. CAMP Test
- Test differentiates enterococci and - Christie, Atkins, and
group D streptococci from Munch-Peterson (CAMP) test
non-group D viridans streptococci - CAMP factor acts synergistically
- Determines ability to grow in 40% with the beta-lysin of
bile and esculin hydrolysis Staphylococcus aureus to cause
- Positive: growth and blackening of enhanced lysis of red blood cells
the agar slant - Positive: Enhanced hemolysis is
indicated by an arrowhead-shaped
zone of beta-hemolysis at the
Bile Growt PYR
Esculi h in juncture of the two organisms
n 6.5% - Positive for S. agalactiae
Hydrol NaCl - Negative: No enhancement of
ysis hemolysis
D + Growt +
Enterococcus h 7. Hippurate Hydrolysis
- End products of hydrolysis of
D + No - hippuric acid by hippuricase include
Non-enteroco growth glycine and benzoic acid
ccus
- Glycine is deaminated by the
oxidizing agent ninhydrin, which is
5. L-Pyrrolidonyl Arylamidase (PYR) Test reduced during the process
- Presumptive identification of group - Positive: deep purple color
A streptococci (Streptococcus - Positive for S. agalactiae
pyogenes) and enterococci by the
PHA6114 LEC 2APH
FAMILY STREPTOCOCCACEAE
BACITRACIN AND
Streptococc Not Lancefield grouped,
SULFAMETHOXAZOLE-TRIMETHOPRIM
us alpha hemolytic on BAP
SUSCEPTIBILITY
pneumoniae
Bacit SXT Notes Viridans Not Lancefield grouped,

racin streptococci alpha hemolytic on BAP
Taxo - S. mutans (associated
A with dental caries)
- S. uberis
Group A S R - Beta - S. mitis
(S. - PYR + - S. sanguis
pyogene - S. salivarius
s) - S. constellatus
- S. intermedius
Group B R R - Beta
(S. - CAMP +
agalactia TESTS TO DIFFERENTIATE S.
e) PNEUMONIAE FROM VIRIDANS
STREPTOCOCCI
Group C R* S - Beta S. VIRIDANS
PNEUMONI
Group D R R - Alpha,
AE
Enteroc beta,
occus gamma Mouse + -
- Bile esculin virulence
+
- Growth in Inulin + -
6.5% NaCl fermentatio
- PYRase + n
Group D R R - Alpha, Bile + -

Non-ent beta, solubility
erococc gamma
us - Bile esculin Optochin S R
+
- No growth Neufeld-Qu + -
in 6.5% ellung
NaCl
- PYRase (-)
S = susceptible
Bile - Differentiates
R = resistant Solubility Streptococcus
pneumoniae

PHA6114 LEC 2APH
- Two pathogenic species of

(positive-soluble) from
alpha-hemolytic Neisseria, N. gonorrhoeae and N.
streptococci meningitidis, are transmitted person
(negative-insoluble) to person. N. gonorrhoeae is
- Bile or a solution of a bile sexually transmitted, and N.
salt (e.g., sodium meningitidis is spread via
desoxycholate) rapidly
contaminated respiratory droplets
lyses pneumococcal
colonies; lysis depends
on the presence of an ● Gram-negative cocci except N. elongata
intracellular autolytic (rod)
enzyme, AMIDASE ● Obligate aerobes
- Positive: Colony ● Prefer increased carbon dioxide
disintegrates; an imprint ● Nonmotile
of the lysed colony may
● Catalase positive except N. elongata
remain in the zone
- Positive for ● Catalase positive
streptococcus
pneumoniae Oxidase Test
- Negative: Intact colonies 1. 1% tetramethyl-p-phenylenediamine
dihydrochloride, prepared fresh daily
Optochin - Determines the effect of
2. Remove colony with platinum loop or
(P Disk) Optochin
Suscepti (ETHYLHYDROCUPREI wooden applicator
bility Test NE HYDROCHLORIDE) a. Iron in the nichrome loop can
on an organism give a false positive reaction
- Optochin lyses b. Rub colony on a paper strip
pneumococci (positive and add a drop of reagent;
test), but or
alpha-streptococci are
c. Rub the colony on a piece of
resistant (negative test)
- Positive: Zone of paper containing the
inhibition ≥ 14 mm reagent; or
- Positive for d. Put a drop of reagent on the
Streptococcus colony
pneumoniae 3. Positive - turns DARK PURPLE in 10
seconds
FAMILY NEISSERIACEAE
Genera FAMILY NEISSERIACEAE
- Neisseria and Moraxella
N. - A leading cause of
- Oxidase-positive, gram-negative gonorrhoeae STD
diplococci - Other localized

PHA6114 LEC 2APH
cannot be used to diagnose gonorrhea in

infections include
pharyngitis, anorectal female patients.
infections and
conjunctivitis [e.g.
ophthalmia
neonatorium of
newborns (1%
AgNO3) acquired
during birth from the
infected mother]
- Disseminated
infections result when
the organism spreads
from a local infection
Selective Culture Media for Neisseria
Gonorrhoeae
N. - Life-threatening,
meningitidis acute, purulent Thayer- - Vancomycin: inhibits
meningitis. Meningitis Martin growth of gram positive
may be accompanied bacteria
by appearance of - Colistin: inhibits growth
petechiae (rash) that of gram negative bacteria
is associated with except N. gonorrhoeae
meningococcal - Nystatin: inhibits growth
bacteremia of fungi
(meningococcemia)
- Bacteremia leads to Modified - Vancomycin: inhibits
thrombocytopenia, Thayer- growth of gram positive
disseminated Martin bacteria
intravascular - Colistin: inhibits growth
coagulation, and of gram negative bacteria
shock except N. gonorrhoeae
- WATERHOUSE-FRID - Nystatin: inhibits growth
ERICHSEN of fungi
SYNDROME - Trimethoprim lactate -
(hemorrhage in prevents swarming of
adrenal gland) Proteus sp.
Martin-L - Vancomycin: inhibits

Gram-stained urethral discharge specimen ewis growth of gram positive
is useful for diagnosis of gonorrhea in male bacteria
patients but, because of the possible - Colistin: inhibits growth
presence of “look alike” bacteria, a of gram negative bacteria
gram-stained vaginal or cervical swab
- Anisomycin: inhibits

PHA6114 LEC 2APH
growth of fungi
- Trimethoprim lactate:
prevents swarming of Moraxella - Gram-negative
Proteus sp. diplococci
- Nonmotile
New - Vancomycin: inhibits
- Catalase positive
York City growth of gram positive
- Oxidase positive
Agar bacteria
- DNase positive
- Colistin: inhibits growth
- Grow on blood agar
of gram negative bacteria
- Negative for
carbohydrate utilization
- Amphotericin B: inhibits
MORAXELLA
growth of fungi
CATARRHALIS
- Trimethoprim lactate:
- 48-hr colony may
prevents swarming of
have elevated center
Proteus sp.
and thinner, wavelike
periphery (“wagon
wheel”)
- Colony can be swept
Gluco Malto Sucro Lacto across plate intact
se se se se (“hockey puck”)
Morax - - - -
ella FAMILY ENTEROBACTERIACEAE
Catarr - Gram-negative bacilli, non-spore
halis forming
Neiss + - - - - Facultative anaerobes
eria - Motile with PERITRICHOUS
Gonor FLAGELLA except Klebsiella and
rhoea Shigella (nonmotile)
e - Catalase positive except Shigella
N. + + - - dysenteriae
menin - OXIDASE NEGATIVE (except for
gitidis Plesiomonas sp.)
- Reduce nitrate to nitrite except for
N. + + +/- -
Erwinia and Pantoea agglomerans
subfla
va - Capable of fermenting glucose
- Grow well on MacConkey agar
N. + + - + (MAC), some as lactose fermenters
lacta (producing pink colonies on MAC)
mica
and some as lactose nonfermenters

PHA6114 LEC 2APH
(producing colorless colonies on Triple Sugar Iron Agar

MAC) - Used primarily to differentiate
- Aerogenic except for Shigella members of the Enterobacteriaceae
- H2S+ Salmonella, Proteus, Arizona, family from other gram-negative rods
Citrobacter, and Edwardsiella - Determines whether a gram-negative
rod ferments glucose and lactose or
Antigenic Structure sucrose and forms hydrogen sulfide
(H2S)
K - Capsular antigen,
antigen heat-labile Protein Beet extract, peptone,
- Some salmonellae have sources yeast extract, proteose
capsular (K) antigens, peptone
referred to as Vi
Carbohydra 10 parts lactose:10 parts
O - Somatic antigen, tes sucrose: 1 part glucose
antigen heat-stable
- Antibodies to O antigens pH Phenol red
are predominantly IgM indicator
H - Located in the flagella Sulfur Sodium thiosulfate

antigen - Denatured or removed by source
heat or alcohol
- H antigens agglutinate with H2S Ferrous sulfate
anti-H antibodies, mainly Indicator
IgG
Lysine Iron Agar
FAMILY ENTEROBACTERIACEAE - Can be used to determine the ability of
Culture the organism to deaminate lysine,
- Eosin/Methylene Blue Agar (EMB) decarboxylate lysine and produce H2S
- MacConkey Agar (MAC) Carbohyd Glucose
- MacConkey Sorbitol (MAC-Sor) rate
- Hektoen Enteric Agar (HEK)
- Xylose-Lysine Deoxycholate Agar (XLD) Amino Lysine
Acid
- Salmonella-Shigella Agar (SSA)
- Gram Negative Broth (GN) pH Bromocresol purple
- Cefsulodin Irgasan Novobiocin Agar Indicator
(CIN)
H2S Ferric ammonium citrate
Indicator

PHA6114 LEC 2APH
Indole Test - Slow urease producers: Citrobacter,

Detects: tryptophanase Klebsiella, Enterobacter, Yersinia, and
Medium: tryptone broth, SIM Serratia
Indicator: Kovac’s or Ehrlich’s reagent
(PDAB) ESCHERICHIA COLI
Positive result: red ring - A/A reaction on TSI
- IMViC results: ++--
MRVP Test - MUG positive (beta-D-glucuronidase,
Medium: MRVP broth or peptone-glucose electric blue fluorescence)
broth
METHYL RED - UTI - 90%
- Organism that produce enough acid - Sepsis
will overcome the neutralizing effect - Meningitis - (also Group B
of the buffer streptococci) causes meningitis in
- Positive result: red infants
VOGES PROSKAUER - Diarrheal diseases
- Detects: acetoin
(acetylmethylcarbinol)
Enterotoxigenic E. - Produces
- Reagents: KOH and alpha-naphthol
coli (ETEC) heat-labile (LT)
- Positive result: red
and/or heat
MOST ENTEROBACTERIACEAE GIVE stable (ST)
OPPOSITE MR AND VP REACTIONS enterotoxins;
genes of both
Citrate Utilization Test toxin reside on
- Determine of an organism can utilize a plasmid
citrate as sole source of carbon - LTs are closely
related in
- Medium: Simmons citrate agar (SCA)
structure and
- pH Indicator: bromthymol blue function to
- Positive result: growth and intense cholera toxin
blue color - “Montezuma;s
revenge” or
Urease Test “turista”,
- Medium: Christensen’s urea agar or traveler’s and
Stuart’s urea broth childhood
diarrhea
- pH Indicator: phenol red
characterized
- Positive result: red by profuse
- Rapid urease producers: Proteus, watery stool
Providencia (P. rettgeri) and Morganella
Enteroinvasive E. - Dysentery (i.e.,

PHA6114 LEC 2APH
coli (EIEC) necrosis, ground beef or

ulceration, and raw milk
inflammation of
the large
MacConkey Sorbitol (MAC-Sor)
bowel); usually
seen in young - Same as regular MacConkey except
children living in D-sorbitol is substituted for lactose
areas of poor - Sorbitol-negative organisms are
sanitation clear and may indicate E. coli
O157:H7
Enteropathogenic - Diarrhea in
E. coli (EPEC) infants in
developing, Enteroaggregative - Watery diarrhea
low-income E. coli (EAEC) that in some
nations; can cases can be
cause a chronic prolonged
diarrhea - Mode of
transmission is
Enterohemorrhagic - Toxin similar to
not well
E. coli (EHEC, Shiga toxin
understood
VTEC, or STEC) produced by S.
dysenteriae
- Most frequently KLEBSIELLA
associated with - Encapsulated strains produce mucoid
serotypes such colonies
as E. coli
- A/AG reaction on TSI
O157:H7
- Hemorrhagic - IMViC results: --++
colitis and the
sequelae - K pneumoniae- FRIEDLANDER’S
following BACILLUS, encapsulated and appears
infection of as mucoid colonies that tend to string
HEMOLYTIC-U - K. ozaenae - purulent sinus infection
REMIC
- K. rhinoscleromatis - granuloma of the
SYNDROME
(HUS) and nose and oropharynx
THROMBOTIC
THROMBOCYT ENTEROBACTER
OPENIA - A/A reaction on TSI
PURPURA - IMViC results: --++
(TTP)
- Transmitted by
- Opportunistic infections: UTI,
ingestion of
undercooked respiratory tract and wound infections

PHA6114 LEC 2APH
- Most predominant isolate is E. cloacae SHIGELLA

- Natural habitat of shigellae is limited to
CRONOBACTER SAKAZAKII (previously the intestinal tracts of humans and other
ENTEROBACTER SAKAZAKII) primates, where they produce
- Produces a yellow pigment that is BACILLARY DYSENTERY
enhanced by incubation at 25oC
Present Group type
SERRATIA designation
- Unique in their ability to produce the
enzymes DNase, lipase and gelatinase S. dysenteriae A
- S. marcescens and S. rubidaea produce
S. flexneri B
a red pigment PRODIGIOSIN
- S. odorifera produces a rancid S. boydii C
potato-like odor
S. sonnei D
- Common opportunistic pathogen in
hospitalized patients
CITROBACTER
SALMONELLA - Causes UTI and sepsis
- Sources of infection: water, milk and - Organisms resemble Salmonella but are
dairy products, shellfish (from ONPG positive and LDC negative
contaminated water), dried or frozen
eggs, meat and meat products and YERSINIA
household pets - Nonmotile at 37oC but motile at 22oC
- Cause enteritis, systemic infection and (except Y. pestis)
ENTERIC FEVER (TYPHOID FEVER) - Y. pestis has a characteristic BIPOLAR
- Cause ENTERIC FEVER: (SAFETY PIN) APPEARANCE in clinical
➔ S. paratyphi A (serogroup A) material, when stained with Wayson
➔ S. paratyphi B (serogroup B) stain
➔ S. choleraesuis (serogroup C) - Agent of plague (once referred to as the
➔ S. typhi (serogroup D) BLACK DEATH); a zoonosis transmitted
➔ S. typhi (most important by rat fleas. There are three types of
cause) plague:
- Bacteremia - S. choleraesuis 1. Pneumonic plague, which is
- Enterocolitis/gastroenteritis - most highly contagious and
common manifestation of salmonella transmitted from person to
infection person
2. Bubonic plague, in which
buboes (swollen lymph
nodes) are produced

PHA6114 LEC 2APH
3. Septicemic plague, which

the only
can lead to septic shock and urease-positive
death Providencia
YERSINIA ENTEROCOLITICA
EDWARDSIELLA
- Enterocolitis characterized by fever,
- Isolated from the environment and
diarrhea, and abdominal pain
many cold-blooded and warm-blooded
- Acute mesenteric lymphadenitis
animals including reptiles, freshwater
which may present clinically as
and aquarium fish, frogs and turtles
appendicitis (pseudoappendicular
- E. tarda - most human species
syndrome)
- To differentiate from E. coli: E. tarda is
nonlactose fermenter and H2S positive
Proteus - Produce infections in EXTENDED-SPECTRUM β-LACTAMASE

humans only when (ESBLs)
they leave the - Some gram-negative bacilli (e.g., some
intestinal tract
isolates of Klebsiella pneumoniae, K.
- UTI, bacteremia,
oxytoca, E. coli, Enterobacter spp.,
pneumonia,,
nosocomial infections Citrobacter spp., and Proteus mirabilis)
- Urease activity of can produce enzymes known as
lead to struvite EXTENDED-SPECTRUM
kidney stones β-LACTAMASES, ESBL-PRODUCING
(calculi) STRAINS may be resistant to certain
- P. mirabilis and many
drugs (e.g., penicillins, cephalosporins,
strains of P. vulgaris
exhibit swarming and aztreonam) to which most strains
motility of these species are susceptible
- Certain strains share
specific OTHER GRAM-NEGATIVE BACILLI
polysaccharides with Pseudomonas Aeruginosa
some rickettsia and - Obligate aerobe
are agglutinated by - Motile and rod shaped
sera from patients with
- Sweet or grape-like or corn-taco like
rickettsial diseases
odor
Providencia - Normal intestinal flora - Grows well at 42°C, and its growth
- UTI and occasionally at 42°C will differentiate it from other
other infections and Pseudomonas sp.
are often resistant to
antimicrobial therapy
- Providencia rettgeri is

PHA6114 LEC 2APH
- Grape like or “corn tortilla-like” odor Common Infections Attributed to P.

resulting from production of Aeruginosa
2-aminoacetophone ● Severe wound infections in burn
- Does not ferment carbohydrate patients, giving rise to blue-green
- Identification scheme for P. pus
Aeruginosa involves the following ● Urinary tract infections
conventional tests and ● Nosocomial pneumonia
characteristics: ● Septicemia in immunosuppressed
➢ Oxidase-positive patients and infants
➢ TSI with an alkaline/no ● Septic arthritis in intravenous drug
change (K/NC) reaction abusers
➢ Production of bright blush ● Swimmer’s ears
(procyanin) green ● Destructive eye infections (keratitis,
(pyoverdine), red (pyorubin), corneal ulcers) in contact lens
or brown (pyomelanin) wearers
diffusible pigment on ● Chronic lung infections in patients
Mueller-Hinton agar or with cystic fibrosis
trypticase soy agar
Burkholderia Causative agent of
Colonial Spreading and flat, Mallei GLANDERS in
appearance serrated edges; horses, mules, and
confluent growth; often donkeys; not part
shows metallic sheen; of human flora
blush gree, red, or
brown pigmentation; Burkholderia Causative agent of
colonies often Pseudomallei MELIOIDOSIS
beta-hemolytic;
grapelike or corn It has several
tortilla-like odor; forms, including the
mucoid colonies formation of skin
commonly seen in abscesses, sepsis
patients with cystic and septic shock,
fibrosis abscess formation
in several internal
Cetrimide Primarily used to isolate organs, and acute
and purify pulmonary disease.
Pseudomonas
aeruginosa from Burkholderia Environmental (soil,
contaminated Cepacia water, plants);
specimens survives well in
hospital

PHA6114 LEC 2APH
environment; not added to the

part of normal medium
human flora; may
colonize respiratory High pH of the
tract of patients medium (8.6)
with CYSTIC inhibits the growth
FIBROSIS of other intestinal
flora
Vibrio
- Facultative anaerobe Vibrio Cholerae
- Monotrichous - Divided into three major subgroups:
- Oxidase + V. cholerae O1, V. cholerae O129,
- Halophilic except V. cholerae and V. and V. cholerae non-O1
mimicus - Somatic antigens O1 and O139
associated with the V. cholerae cell
envelope are positive markers for
APW (Alkaline Alkaline peptone
Peptone Water) water (pH 8.4) may strains capable of epidemic and
be used as an pandemic spread of the disease
enrichment broth - Cholera toxin causes mucosal cells
for obtaining to hypersecrete water and
growth of vibrios electrolytes into the lumen of the
from stool
gastrointestinal tract
TCBS (Thiosulfate Selective medium - RICE WATER STOOLS composed
citrate bile salts) thiosulfate citrate of fluids and mucous flecks, are the
bile salts sucrose hallmark of cholera toxin activity
sugar - Fecal-oral route, by ingestion of
contaminated washing, swimming,
Contains 1%
cooking, or drinking water; also by
sodium chloride,
bile salts that ingestion of contaminated shellfish
inhibit the growth or other seafood
of gram-positive - Serogroups:
organisms, and ● Inaba
sucrose for the ● Ogawa
differentiation of
● Hikojima
the various Vibrio
spp. - Biotypes (Biovars)
Classical El Tor
Bromothymol blue
and thymol blue pH Red Negative Positive
indicators are Hemolysis

PHA6114 LEC 2APH
VP Negative Positive US wound infections

involving marine
Polymyxin Susceptible Resistant environment
B ● Halophilic
Agglutinati Negative Positive

on With Aeromonas
Chicken - Gram-negative straight rods with
RBCs rounded ends or coccobacillary
- Facultative anaerobes
- Typically oxidase and catalase
positive
V. ● Clinical significance:
Chromobacterium
ALGINOLY wound and ear
- Facultative anaerobic, motile,
TICUS infections
associated with gram-negative rod or cocci.
marine environment - Colonies are round, smooth,
● Halophilic convex, some strains are
● Sucrose beta-hemolytic; most colonies
fermentation appear black or very dark purple
positive
- Cultures smell of ammonium
V. ● Clinical significance: cyanide (almond-like)
PARAHEM gastroenteritis,
OLYTICUS usually associated Hemophilus
with contaminated - Non-motile, non-spore forming
seafood - Facultative anaerobes
● Halophilic - Most are oxidase and catalase
● Sucrose
positive
fermentation
negative - Preferred incubation: 35 to 37 oC
- Requires X and V factors
V. ● Clinical significance: ● X Factor/hemin
MIMICUS gastroenteritis and - From degradation of
ear infections
hemoglobin
associated with
marine environment - Heat-stable
● Nonhalophilic ● V Factor/NAD/Coenzyme I
● Sucrose - Produced by some
fermentation bacteria and yeasts
negative - Heat-labile
V. ● Clinical significance:
VULNIFIC septicemia and SPECIES REQUIRE BETA D-ALA

PHA6114 LEC 2APH
MENT HEMO
FOR LYSIS H. Aegypticus
- Formerly known as Koch-Week’s
X V bacillus
FAC FA - Closely resembles H. influenzae
TO CT
biotype III
R OR
- Causes pink eye conjunctivitis
H. + + - -
INFLUENZAE H. Ducreyi
- Formerly known as:
H. - + - +
PARAINFLUEN - Closely resembles H. influenzae
ZAE biotype III
- Causes chancroid or soft chancre
H. + + + -
HEMOLYTICUS
Campylobacter and Helicobacter
H. _ + + + - Small Curved Motile Gram-negative
PARAHEMOLY Bacilli
TICUS
Campylobacter
H. + + - -
- C. jejuni, C. coli (associated with
AEGYPTICUS
gastritis and diarrhea)
H. - - - + - C. jejuni have been recognized and
APHROPHILU include reactive arthritis and
S GUILLAIN-BARRÉ SYNDROME, an
acute demyelination (removal of the
H. - + - +
PARAPHROPH myelin sheath from a nerve) of the
ILUS peripheral nerves
- Microaerophilic and capnophilic,
H. DUCREYI + - - - motile, gram negative rods that are
H. Influenzae associated with gastritis and
- Formerly known as Pfeiffer’s bacillus diarrhea
- Six serotypes (a, b, c, d, e and f) - Optimum temperature for growth at
- Most frequently encountered 42 to 43 oC
serotype in infection: b_ - Motility: darting § Culture medium:
- Encapsulated strains are pathogenic Campy’s BAP, Skirrow’s
- Main cause of meningitis in children - Positive HIPPURATE HYDROLYSIS
<5 years old is an important characteristic for the
- Associated with respiratory identification of C. jejuni
conditions including EPIGLOTTITIS
PHA6114 LEC 2APH
Helicobacter Colonies: mercury

- Associated with peptic ulcer § droplets colonies
- The natural habitat is the human
stomach, where the organism is B. parapertussis
found in the mucus secreting cells - pertussis-like syndrome
- Current evidence may suggest a
role between H. pylori and peptic B. bronchiseptica
and duodenal ulcers - inhabits respiratory tract of canines
- Histology staining and culture of (kennel’s cough), infrequently
biopsies obtained from the stomach responsible for chronic respiratory
or duodenum are recommended for tract infections in humans
the identification
Brucella
Bordetella - Agents of Brucellosis, undulant
fever, Malta fever
- Small, nonmotile, aerobic,
gram-negative coccobacilli or short
rods
- Humans acquire infections through
B. pertussis the ingestion of contaminated
- agent of WHOOPING COUGH animal products, including meats
- Three stages of pertussis and milk; farmers can directly
(whooping cough) acquire infections through direct
1. CATARRHAL: general animal contact
flulike symptoms - Specimen: blood, bone marrow
2. PAROXYSMAL: - Culture medium for Brucella:
repetitive coughing Castañeda bottle (biphasic medium)
episodes
3. CONVALESCENT: Legionella
recovery phase - Naturally found in both natural and
- Ideal specimen: artificial water sources; the bacteria
nasopharyngeal swab have been found in ponds, creeks
Culture media: and streams, wet soil, water-cooling
1. Bordet-Gengou towers for air-conditioning and
medium heating systems, shower heads, and
(potato-blood-glycero plumbing systems
l) - Not usually detectable directly in
2. Regan-Lowe clinical material by Gram stain; the
(charcoal-cephalexin) use of 0.1% FUCHSIN substituted

PHA6114 LEC 2APH
for safranin in the Gram-stain through direct contact with blood or

procedure may enhance the visibility through animal bite or scratch
of the organisms - Bacterium can also be inhaled or
- L. pneumophila (Legionnaire’s acquired indirectly through insect
disease, Pontiac fever) vectors primarily ticks
- L. micdadei (Pittsburgh pneumonia) - Culture medium:
- L. bozemanni (Wiga’s agent of blood-glucose-cystine medium
pneumonia)
- Culture medium: buffered charcoal Gardnerella
yeast extract (BCYE) medium G. vaginalis
- Associated with bacterial vaginosis
Pasteurella - Gram-variable to gram-negative
- The organism is carried in the oral bacillus, previously known as
cavity and respiratory and Haemophilus vaginalis and
gastrointestinal tracts of cats and Corynebacterium vaginalis
dogs - Bacterial vaginosis (BV) is
- Humans may acquire the infection characterized by foul-smelling,
following contact with domestic grayish, vaginal discharge
animals that harbor the bacterium; - Cytology/Pap’s: clue cells
most frequently the route of - Whiff or Sniff test: 10% KOH
infection is from the bite or scratch - Organism can be isolated on:
of an infected animal, usually a cat human blood bilayer Tween medium
- Wound infections are the most
common type of Pasteurella Streptobacillus Moniliformis
infection - Gram-negative bacillus that requires
- Pasteurella multocida (agent of media containing blood, serum, or
pasteurellosis) ascites fluid as well as incubation
- Agent of shipping fever in cattles, a under carbon dioxide (CO2) for
hemorrhagic septicemia isolation from clinical specimens
1. RAT BITE, or possibly
Francisella through dire
- Faintly staining, gram negative 2. HAVERHILL FEVER from
coccobacilli that are non-motile and ingestion of contaminated
obligately aerobic food, such as unpasteurized
- Tularemia is a disease of the milk or milk products and,
rodents, primarily the rabbits less frequently, water
- Individuals who handle the infected
animal, acquire this zoonosis

PHA6114 LEC 2APH
Capnocytophaga Elizabethkingia Meningoseptica

- Gram-negative, fusiform-shaped - Previous names: Chryseobacterium
bacilli with one rounded end and meningosepticum, Flavobacterium
one tapered end and occasional meningosepticum
filamentous forms - Exposure of patients to
- Capnophilic, fusiform or filamentous contaminated medical devices or
bacilli solutions, but source is not always
- Characteristic motility: gliding known
- Occasionally may be transmitted
Calymmatobacterium from birth canal to neonate
- Agent of DONOVANOSIS,
GRANULOMA INGUINALE Actinobacillus Spp.
- Encapsulated, pleomorphic - Associated with granulomatous
gram-negative bacillus usually disease in animals and have been
observed in vacuoles in large associated with soft tissue infection
mononuclear cells in humans following animal bites
- Groups of organisms are seen within - Staining morphology gives the
mononuclear cells; this overall appearance of the dots and
pathognomonic entity is known as dashes of Morse code
Donovan body
- Closely related to Klebsiella GRAM-POSITIVE BACILLI
- Based on DNA-DNA hybridization, it Bacillus
has been recently proposed that the - Gram positive bacilli that form
name of this organism be changed spores aerobically
to Klebsiella granulomatis - Catalase positive
Cardiobacterium Hominis B. Anthracis

- Normally found in the upper - Most virulent, is the agent of anthrax
respiratory tract and is isolated as a 1. Cutaneous anthrax
rare cause of endocarditis 2. Woolsorter’s disease or
Chromobacterium Violaceum pulmonary anthrax
- Facultative anaerobic, motile, 3. Gastrointestinal anthrax
gram-negative rod or cocci - Medusa head colonies
- Most colonies appear black or very - From patient: encapsulated bacilli
dark purple - From culture: bamboo pole
- Cultures smell of ammonium arrangement
cyanide (almond-like) - String of pearls (MHA with penicillin)

PHA6114 LEC 2APH
B. Cereus C. Perfringens
- Food poisoning isolated from - Cause of myonecrosis (gas
several foods: rice, other cereals, gangrene) and food poisoning
vegetables and milk - In solid media and thioglycollate
- Most commonly encountered broth, spores are usually absent
species of Bacillus in opportunistic - Double zone of hemolysis
infections including - Lecithinase positive
POSTTRAUMATIC EYE - Reverse CAMP
INFECTIONS, endocarditis, and - Nagler reaction
bacteremia; infections of other sites
are rare and usually involve C. Botulinum
intravenous drug abusers or - Agent of food and wound botulism,
immunocompromised patients as well as infant botulism
- Botulism results from the liberation
of botulism toxin, a powerful
TESTS B. B. CEREUS
ANTHRACIS neurotoxin
- Spores: oval and subterminal
CATALASE + + - Lipase positive
LECITHINASE + +
C. Tetani
MOTILITY Non-motile Motile - Etiologic agent of tetanus, which
results from entry of the organism or
HEMOLYSIS Gamma Beta
spores into a puncture wound
ON BAP
- Produces tetanospasmin
(neurotoxin), associated with spastic
Clostridium contractions/lock jaw
- Gram positive bacilli that form
- Spores: round and terminal
spores anaerobically
- Catalase negative
C. Difficile
- ETHANOL SHOCK SPORE
- Important cause of antibiotic –
ISOLATION
associated pseudomembranous
➢ Clostridium species can be
colitis
recovered from mixed
- Spores: oval and terminal
populations of organisms
- Cultured on
and identified using the
cycloserine-cefoxitin-fructose agar
ethanol shock spore
(CCFA)
technique.

PHA6114 LEC 2APH
- Catalase positive
MOTI LECI LIPA LACT GLU
LITY THIN SE OSE COS - Glucose maltose fermented
ASE E - Sucrose not fermented
- Culture
C. - + - + + 1. LSS
Perfring
2. Pai’s coagulated egg
ens
3. Modified Tinsdale’s agar
C. + - + - + 4. Cystine tellurite blood agar
Botulinu (CTBA)
m
C. Tetani + - - - - Two media ● Both media

commonly used for contain a
C. + - - - + this purpose high
Difficile Cystine-tellurite concentratio
blood agar and n of
Corynebacterium Diphtheriae modified Tinsdale potassium
- Gram-positive bacilli, often in V, agar (TIN) § Both tellurite that
media contain a is inhibitory
picket fence, or Chinese letter
high concentration to normal
arrangements of potassium flora
- Toxigenic strains cause diphtheria tellurite that is ● C.
inhibitory to normal diphtheriae
flora § C. also
diphtheriae also produces a
produces a halo on halo on
both media § both media
● Brown halo
surrounding
the colony,
owing to
- KLEBS-LOEFFLER BACILLUS cystinase
- Produces a powerful exotoxin that activity, is a
first attacks the mucous membranes useful
differentiatin
of the respiratory tract; this results
g feature
from inflammation and because
PSEUDOMEMBRANE FORMATION only C.
OF THE OROPHARYNX leading to diphtheriae,
respiratory obstructions C. ulcerans,
- Non-motile and C.
pseudotube
- Nitrate reduction positive
rculosis
- Urease negative
PHA6114 LEC 2APH
- Immunocompromised: endocarditis,
produce a
brown halo septicemia, pneumonia and
● C. neonatal sepsis
diphtheriae
is Corynebacterium Minutissimum
distinguishe - Superficial, pruritic skin infections
d from the
known as erythrasma
other two
species by
its lack of Listeria Monocytogenes
urease - Major source of infection is
production contaminated food (cabbage, fruit,
dairy products)
Toxigenicity Test - Meningitis, pneumonia, abortion,
1. In vivo: animal inoculation test stillbirth, endocarditis, conjunctivitis
2. In vitro: Elek’s test and urethritis
3. SCHICK’ TEST: susceptibility test - Perinatal human listeriosis
(granulomatosis infanseptica)
Diphtheroids
C. Jeikeium Motility
- Formerly Group JK - Hanging drop: tumbling
- Resistant to a number of antibiotics - Semisolid medium: umbrella-like or
- Associated with endocarditis, inverted Christmas tree-like growth
pneumonia and peritonitis - Cultured on McBride’s medium
- Virulence test: Anton’s test –
C. Pseudodiphthericum organism is inoculated to the
- Normal conjunctival sac of rabbit
- Seen in throat
To distinguish LISTERIA from
C. Xerosis CORYNEBACTERIUM:
- Conjunctiva 1. Listeria: motile, salicin (+)
2. Corynebacterium: nonmotile, salicin
C. Acnes (P. Acnes) (-)
- Skin
Erysipelothrix Rhusiopathiae
Corynebacterium Amycolatum - Causative agent of ERYSIPELOID,
- Normal flora: Human conjunctiva, cutaneous inflammation of hand or
skin, nasopharynx fingers (seal finger or whale finger)

PHA6114 LEC 2APH
- Veterinary infection and - Easily killed by moist heat; boiling

occupational hazard for those for 10 minutes, pasteurization or
handling meat, poultry and fish steam under pressure (autoclave)
- Gelatin stab: test tube brush-like or - Tuberculosis usually begins in the
inverted x-mas tree like growth middle or lower lung, other areas for
dissemination include the spine,
Lactobacillus Acidophilus long bones, heart, meninges and
- Normal flora of the mouth, genitourinary system
gastrointestinal tract and vaginal - Direct sputum smear microscopy
canal: DODERLEIN BACILLI (DSSM) is fundamental to the
- Nonpathogenic and has little clinical detection of infectious cases and is
significance recommended for case finding
- Cultured on TOMATO JUICE AGAR among adults and children who can
expectorate. It is the PRIMARY
Kurthia Bessonnii DIAGNOSTIC METHOD adopted by
- Found in soil the National TB Control Program
- Opportunistic pathogen (NTP) among such individuals
because:
Rothia 1. It provides a definitive
- Normal flora of human mouth
diagnosis of active TB.
- Rare cause of abscess and
2. The procedure is simple.
endocarditis
3. It is economical.
4. A microscopy center could
Mycobacterium
be put up even in remote
- Aerobic (although some may grow in
areas
reduced oxygen concentrations)
- Non–spore forming (except for M.
Bacteriologically Confirmed TB
marinum)
1. Smear Positive
- Nonmotile, very thin, slightly curved
- A patient with at least one (1)
or straight rods
sputum specimen positive
- Cell wall contains
for AFB, with or without
N-glycolylmuramic acid instead of
radiographic abnormalities
N-acetylmuramic acid, and it has a
consistent with active TB.
very high lipid content
2. Culture Positive
- A patient with positive
Mycobacterium Tuberculosis
sputum culture for MTB
- Koch’s bacillus
complex, with or without

PHA6114 LEC 2APH
radiographic abnormalities ➢ ≥ 15 mm - Positive test in

consistent with active TB. individuals with no risk
3. Rapid Diagnostic Test Positive factors
- A patient with sputum ➢ ≥ 10 mm - Positive in recent
positive for MTB complex immigrants of high
using rapid diagnostic prevalence countries, IV drug
modalities such as Xpert users, employees of
MTB/RIF, with or without health-care and other
radiographic abnormalities high-risk facilities, persons
consistent. with certain clinical
conditions, and children
younger than 5 years of age
➢ ≥ 5 mm - Positive in persons
who have HIV infection or
other forms of
immunosuppression,
features on a chest xray
consistent with TB, or recent
contact with TB patients
Skin Test For TB
Antituberculosis Agents Commonly
Tested Against M. Tuberculosis
When taking anti tuberculosis drugs, you
have to monitor your liver enzymes
● Primary drugs: : Streptomycin,
Isoniazid, Rifampin, Ethambutol,
Pyrazinamide
○ If you are taking ethambutol
PPD (Purified Protein Derivative) you should see an eye
- Heat killed ammonium sulfate doctor because you cannot
- Precipitated organism is injected distinguish the color red and
intradermally (+ redness after 48 green
hours) ○ If you are taking rifampin,
- A positive PPD test indicates that every bodily discharge
the individual has previously been (sweat, urine, etc..) is colored
exposed to MTB or related orange or red. Thus, you
organism, but it does not cannot wear contact lenses
necessarily mean that he or she has
an active TB infection

PHA6114 LEC 2APH
because they will also be - Acid fast rod in nasal mucosa of

colored red or orange. patients with nodular variety of
○ Isoniazid is incorporated with Hansen’s disease
Vitamin B6 to prevent - Transmission occurs person to
peripheral neuritis (nerve person through inhalation or contact
ends become too sensitive) with infected skin
○ Primary drugs have less side - Armadillo is susceptible to Hansen’s
effects disease and has been used
● Secondary Drugs: : Ethionamide, experimentally
Capreomycin, Ciprofloxacin, - Smears of tissue juice, AF stain for
Ofloxacin, Kanamycin, Cycloserine, lepra cells: macrophages with AFB
Rifabutin - Cultured on foot pads of mice
- Treatment: sulfone dapsone
Media
1. Lowenstein-Jensen M. bovis
2. Petragnani - TB in cattles
3. American Thoracic Society - Bacillus of Calmette and Guerin
4. Middlebrook 7H10 and 7H11c (BCG) vaccine for MTB
- Clear agar: examination of
colonies M. avium
- Culture maintained for: 2 - TB in birds, chickens
months (60 days, 8 weeks)
- Colonies: tan to buff M. avium-intracellulare complex (MAI)
(nonpigmented), cauliflower - Battey bacillus
colonies - Common in AIDS patients
Stains M. kansasii
1. Ziehl-Neelsen - Yellow bacillus
2. Kinyoun
3. Fite-Faraco’s M. gordonae
4. Auramine-rhodamine stain (Truant’s) - Tap water bacillus
- Fluorescent organisms on
black background M. marinum
- Swimming pool granuloma
Mycobacterium Leprae
- Hansen’s disease or leprosy M. terrae
- Radish bacillus

PHA6114 LEC 2APH
M. ulcerans Nocardia
- Inert bacillus - Gram positive, PARTIALLY ACID
- Buruli ulcers FAST, aerobic
M. gastri - N. asteroides is the most clinically
- J bacillus relevant species; other species
include N. brasiliensis and N.
M. fortuitum-chelonae otitidiscaviarum
- Grows in MacConkey agar without - Primary pulmonary infection
crystal violet resembling tuberculosis
M. avium subs. paratuberculosis SPIRALS

- Known to cause an inflammatory Spirillum Minus
bowel disease (known as Johne’s - Gram-negative, helical, strictly
disease) in cattle, sheep, and goats aerobic organism
- Isolated from the bowel mucosa of - Causes rat-bite fever in humans and
patients with Crohn’s disease, a is referred to as SODOKU
chronic inflammatory bowel disease - Clinical signs and symptoms are
of humans similar to those caused by S.
moniliformis except that arthritis is
FUNGUS-LIKE BACTERIA rarely seen in patients with Sodoku
Actinomyces and swollen lymph nodes are
prominent; febrile episodes are also
more predictable in Sodoku
Borrelia
- Helically coiled bacteria transmitted
through arthropod vectors including
lice and ticks
- Gram positive but neither acid fast
nor stained with fungus stain, Borrelia recurrentis
anaerobic - Agent of louse-borne relapsing fever
- Causes chronic suppurative - High fever, muscle and bone pain,
granulomatous disease and confusion
- Agent of LUMPY JAW
- Colonies: MOLAR TOOTH Borrelia burgdorferi
COLONIES - Agent of Lyme Disease
- Transmitted by tick vectors
- Three stages:

PHA6114 LEC 2APH
1. Stage 1: appearance of 3. Latent syphilis: absence of

lesion: ERYTHEMA clinical symptoms
CHRONICUM MIGRANS 4. Tertiary syphilis: gummas,
2. Stage 2: dissemination neurosyphilis
through blood, affected
T. pallidum subsp. pertenue
areas may include the bones,
- Yaws (chronic nonvenereal disease
CNS, heart and liver
of skin and bones)
3. Stage 3: neurological
- Transmission: traumatized skin
abnormalities, arthritis and comes in contact with an infected
skin lesions lesion
Columbian Theory T. pallidum subsp. endemicum

- Christopher Columbus Crew - Bejel (Lesions in oral cavity, oral
- States that syphilis was endemic in mucosa, skin, bones and
Haiti and was subsequently nasopharynx)
contracted and carried to Europe by - Transmission: mouth to mouth by
Columbus crew. utensils
T. carateum
Treponema
- Pinta (ulcerative skin disease)
T. pallidum subsp. pallidum
- Transmission: traumatized skin
- Venereal syphilis
comes in contact with an infected
- Transmitted by sexual contact, direct
lesion
blood transmission or transplacental
route
Leptospira
- Patients with syphilis can be difficult
Leptospira biflexa
to diagnose because their clinical
- Nonpathogenic, found in water and
presentations can vary widely.
soil
Because the symptoms of syphilis
can mimic those of many other
Leptospira interrogans
diseases or conditions, the disease
- Cause of human and animal
has often been referred to as “THE
leptospirosis, a zoonosis
GREAT IMITATOR.”
- Parasitic on vertebrates other than
- Stages:
humans, including rodents, cattle,
1. Primary syphilis: hard
dogs, cats, raccoons and bats
chancre (painless and firm)
- Shed in the urine of these animals
2. Secondary syphilis:
and human acquire the infection
condylomata lata – wart-like
through direct contact with urine of
lesions in moist areas of the
animals who carry the organism
body

PHA6114 LEC 2APH
- Involves the kidney, liver and central C. pneumoniae (Chlamydophila

nervous system pneumoniae)
- Severe form of leptospirosis: Weil’s - Associated with mild respiratory tract
disease infections
- TWAR strain
Principal Leptospirosal Diseases
L. interrogans serovar
SUBTYPES CLINICAL
- icterohemorrhagiae: WEIL’S SYNDROME
DISEASE
- canicola: INFECTIOUS JAUNDICE A, B, Ba, C Endemic trachoma
- autumnalis: FORT BRAGG or (multiple or
PRETIBIAL FEVER persistent infections
that ultimately lead
- grippotyphosa: MARSCH FEVER
to blindness)
- hebdomadis: SEVEN DAY FEVER
- mitis/pomona: SWINEHERD’S D-K Urethritis, cervicitis,
DISEASE pelvic inflammatory
disease,
Culture epididymitis, infant
pneumonia and
- Blood is the most sensitive conjunctivitis
specimen during early infection;
urine should be cultured after L1, L2, L3 Lymphogranuloma
second week venereum
- Media include Ellinghausen,
McCullough, Johnson, Harris and Mycoplasma
Fletcher’s - Smallest free-living organisms,
found in several animals and plants
Chlamydia - Lack cell wall, pleomorphic in
- Obligate intracellular bacteria appearance
- Infectious particle: ELEMENTARY - Originally known as
BODIES pleuropneumonia-like organisms
- Metabolically active particle: (PPLOs), since they were first
RETICULATE BODIES discovered causing
pleuropneumonia in cattles
C. psittaci (Chlamydophila psittaci)
- Agent of psittacosis/ornithosis, a Mycoplasma pneumoniae
disease of birds, parrots, parakeets, - Eaton’s agent, frequent cause of
and cockatoos community-acquired pneumonia and
- Human may acquire the infection by tracheobronchitis in children and
inhalation of contaminated aerosols young adults
or fomites or through - Primary atypical pneumonia/Walking
person-to-person transmission pneumonia

PHA6114 LEC 2APH
Genital Mycoplasmas: M. hominis and U. - Other manifestation of infection,

urealyticum include conjunctivitis, pharyngitis,
- Can colonize adults and mild respiratory distress
asymptomatically and are also
cause of nongonococcal urethritis in
males GROUP SPECIE INFECTI TRANS
- M. hominis has also been identified S ON MISSIO
as an agent of salpingitis and N
postpartal fever in females
Spotted Rickettsi Rocky Ticks
Fever a Mountai
rickettsii n
Spotted
Fever
R. akari Rickettsi Mites

al pox
R. Australia Ticks
australis n/
Queensl
and tick
- Colonies of some species of typhus
Mycoplasma (e.g., M. hominis) are
R. Boutonn Ticks
referred to as fried-egg colonies, conorii euse
because they resemble fever
sunny-side-up fried eggs Mediterr
anean
Rickettsia and
- Include the genera Rickettsia, Israeli
Ehrlichia, Coxiella and Rochalimea spotted
fevers;
- Gram-negative, obligately
Indian
intracellular bacteria tick
- Infections are spread through insect typhus,
vectors such as lice, fleas and ticks Kenya
- All rickettsia, except Coxiella, cannot tick
survive outside the animal host or typhus
insect vector Typhus R. Epidemi Lice
- Signs of infection include fever, prowaze c typhus
headache, characteristic rash that kii
first appears on the wrists and Sporadic Flying
ankles typhus squirrels
Brill-Zins Reactiva
ser tion of

PHA6114 LEC 2APH
disease latent
infection
R. typhi Murine Fleas

typhus
Scrub R. Scrub Mites,

Typhus tsutsuga typhus chiggers
mushi
O.
tsutsuga
mushi
Q Fever Coxiella Q fever Ticks,

burnetti aerosol
Ehrlichio E. Human Ticks

sis chaffeen monocyt
sis e
ehrlichio
sis
E.. Human Ticks

phagocy granuloc
tophila yte
ehrlichio
E. sis
ewingii
Neoricke Sennets Ticks

ttsia u fever
sennets
u
(former
E.
sennets
u)
Rochali Rochali Trench Lice

mea mea fever
quintana
----------------------------------------------------------

PHA6114 LEC 2APH
MYCOLOGY FUNGI
- Study of fungi - Classified as thallophytes, organism
- Vernacular name of fungi is possess true nuclei and are
halamang singaw heterotrophic members of the plant
- Tinea versicolor (An-an) family that lack stems and roots
- Despite their great variation in
morphologic features, most fungi
share the following characteristics:
1. Chitin in the cell wall
2. Ergosterol in the cell
membrane
3. Reproduction by means of
spores, produced asexually
- Tinea ringworm (Buni) or sexually
4. Lack of chlorophyll
5. Lack of susceptibility to
antibacterial antibiotics
6. Saprophytic nature (derive
nutrition from organic
materials)
- Capable of 2 phases:
- Tinea cruris (Jock itch/ hadhad)
1. Multicellular MOLD PHASE
which consists of a cottony,
mycelial mass
2. Unicellular YEAST PHASE,
which is creamy, resembling
bacterial colony
Dimorphic When dimorphism is
- Tinea pedis (Athlete’s foot/alipunga) fungi temperature dependent, the
fungi are designated as
(temperature- thermally dimorphic. In general,
dependent) these fungi produce:
1. MOLD FORM AT 25° TO

30°C
2. YEAST FORM AT 35° TO
37°C
[D.N., S.L., & J.S.] | #2APHRPhCutie 1

PHA6114 LEC 2APH
SPORES INVOLVED IN SEXUAL

Medically important dimorphic
fungi are: REPRODUCTION
1. Histoplasma capsulatum
ASCOSPORES Contained in a saclike
2. Blastomyces dermatitidis
ascus
3. Coccidioides immitis (not
thermally dimorphic) ZYGOSPORES Involve the fusion of two
4. Paracoccidioides brasiliensis identical cells arising
5. Sporothrix schenckii from the same hypha
6. Penicillium marneffei
OOSPORES Involve the fusion of
cells from two separate,
MYCELIUM
non-identical hyphae
- An intertwining structure composed
of tubular filaments known as BASIDIOSPORES Contained in a
hyphae, the microscopic unit of club-shaped basidium
fungi - A perfect fungi exhibit sexual phase.
- consists of vegetative portion or
thallus, which grows in or on a 1. Conidia
substrate and absorbs nutrients, - Spores produced singly or
and a reproductive, or aerial, part, multiply in long chains or
which contains the fruiting bodies clusters by specialized
that produce the conidia or spores vegetative hyphae known as
conidiophores
ASEPTATE OR COENOCYTIC HYPHAE ● Macroconidia - large,
- No crosswall or division multicellular
- Zygomycetes: Rhizopus, Absidia ● Microconidia - small,
and Mucor unicellular
SEPTATE HYPHAE
- With crosswalls or divisions
- All fungi except Zygomycetes
2. Blastoconidia (blastospores)
- Develops as daughter cell
buds off the mother cell and
is pinched off

PHA6114 LEC 2APH
5. Sporangiospores
- Spores contained in a sporangia or
sacs that are produced terminally on
sporangophore or aseptate hyphae
- Unique to Zygomycetes
3. Chlamydoconidia
(chlamydospores)
- Thick walled, resistant, resting
spores produced by rounding up
and enlargement of terminal hyphal
cells ZYGOMYCETES
- spores germinate when favorable Aseptate (Nonseptate/coenocytic)
environmental conditions occur hyphae
a. Terminal - end of hypha 1. Rhizopus
b. Intercalary - within hypha - Large, broad, nonseptate
c. Sessile - side of hypha hyphae that produce
horizontal runners, or
stolons, which attach at
rhizoids
- Sporangiophores arise in
clusters at rhizoids and
terminate sporangia
4. Arthroconidia (arthrospores)
- Involve the simple fragmentation of
the mycelium
- Useful identification feature of C.
immitis and G. candidum
2. Absidia
- Similar to Rhizopus;
however, sporangiophores
arise between nodes from
which rhizoids are formed

PHA6114 LEC 2APH
● Irritated, oily skin

● Not shampooing enough
● A yeastlike fungus
(Malassezia) that feeds on
oils on the scalps of most
adults
● Dry skin
● Sensitivity to hair care
products (contact dermatitis)
3. Mucor ● Other skin conditions, such
- No rhizoids as psoriasis and eczema
➔ Irritation on forehead/ pimples -
probably because you are not
“hiyang” when it comes to your
shampoo
➔ Anti-dandruff shampoo - must be
used only for 2 weeks; common
side/adverse effects: hair
loss/excessive hair fall
➔ Every 3 months, change your
shampoo
SUPERFICIAL MYCOSES
- No cellular response by host
Ptyriasis (Tinea) versicolor

- (skin of chest, abdomen, back) –
Malassezia furfur
Figure: Malassezia spp. - Direct
Microscopic Exam resembling “Spaghetti
and meatballs” appearance
OLIVE-OIL LOVING FUNGI

- Malassezia furfur
- Cultivation requires an agar medium
overlaid with a long-chain fatty acid
(olive oil)
Figure: An-an
- Dandruff may cause several causes,
including:

PHA6114 LEC 2APH
Black piedra ➢ Trichophyton – Skin, Hair

And Nails
1. Microsporum
- invades the skin and hair
- M. canis (zoophilic)
- M. gypseum (geophilic)
- Piedraia hortai - M. audouinii (anthrophilic)
- Brown-black crust outside hairshaft host preference is MAN
White piedra
M. canis Fluoresce on Growth in
Wood’s lamp rice medium
M. audouinii Fluoresce on No growth

Wood’s lamp in rice
medium
- Trichosporon beigelii
- Light brown nodules on beard M. canis (zoophilic)
●
- Large, multicelled,
CUTANEOUS MYCOSES spindle-shaped,
- Dermatomycoses rough macroconidia
- Keratinized tissue - skin, hair, nails - Terminal ends
- Causes TINEA OR RINGWORM sometimes curved
● Tinea capitis - Ringworm of - Microcondia few or
the scalp absent
● Tinea barbae - Ringworm of - Green-yellow
the beard fluorescence of
● Tinea corporis - Ringworm ectothrix hairs
of the body ● M. audouinii (anthrophilic)
● Tinea cruris - Ringworm of - Conidia absent or
the groin (jock itch) bizarre if present
● Tinea unguium - Ringworm - Atypical vegetative
of the nails hyphae with terminal
● Tinea pedis - Athlete’s foot chlamydospores
- Caused by DERMATOPHYTES - Apple-green
➢ Microsporum – Skin, Hair fluorescence of
➢ Epidermophyton – Skin, ectothrix hairs
Nails 2. Epidermophyton
- Invades the skin and nails
PHA6114 LEC 2APH
- No microconidia - Negative: Trichophyton rubrum
●E. floccosum SUBCUTANEOUS MYCOSES

- Large, multi celled,
club-shaped, 1. Chromoblastomycosis
macroconidia, single - DEMATIACEOUS fungi (dark,
or in clusters of 2-3 slow-growing fungi)
- Microconidia not - Micro. of tissue: SCLEROTIC
formed BODIES
3. Trichophyton
● T. tonsurans
- ‘balloon forms’ aged
microconidia
● T. shoenleinii
- favic chandelier,
favus type of tinea
capitis
● Fonsecaea
- mixed sporulation
T. rubrum Abundant Negative - Acrotheca – conidia
wine-red hair baiting
in side
pigment test
- Cladosporium –
T. Scant red Positive hair conidia in chains
mentagroph pigment baiting test - Phialophora –
ytes conidia in clusters b.
Hair Baiting Test ● Phialophora

- only phialophora
sporulation
● Cladosporium
- only cladosporium
sporulation
2. Mycetoma
- Granulomatous tumor of
- Positive result: V-shaped
subcutaneous tissue a.
penetration of hair shaft
- Positive: Trichophyton
● Eumycotic (True Fungi)
mentagrophytes
- Exophiala

PHA6114 LEC 2APH
- Pseudoallescheria 4. Sporotrichosis
boydii
- most common
cause of
mycetoma
● Actinomycotic (Fngus-like
Bacteria)
- Do not stain with - Rose Gardener’s Disease
fungal stains - Sporothrix schenckii
- Actinomyces - Dimorphic fungus
- Nocardia
ORGANISM MYCELIAL YEAST

Actinomyce Gram Sulfur PHASE PHASE
s positive, granules (ROOM (37°C/TISS
anaerobic TEMP.) UES)
Nocardia Gram Partially Sporothrix Flowerette Cigar-shape
positive, acid-fast schenckii conidia d yeast cells
aerobic
ASTEROID Older cultures
3. Phaeohyphomycosis BODY - in – conidia on
tissue; side of hyphae
- Rare infection caused by
central halo (sleeve)
dematiaceous saprobes around
which invade organs (skin, degeneratin
lungs, brain) of g yeast;
immunosuppressed hosts concentric
- Exophiala (former radiating
Phialophora) jeanselmei eosinophilic
material
- Phialophora
(Ag-Ab
- Wangiella (former reaction)
Fonsecaea) dermatitidis
- Cladosporium trichoides

PHA6114 LEC 2APH
Systemic Mycoses
later, karge, stain
round,
thick-walled
Organism Mycelial Yeast Phase
knobby
Phase (37oC/tissues
tuberculated
(room )
macroconidi
temp.)
a forms
North Delicate, Thick-walled.
San Joaquin Coarse, Large, round,
American septate Large yeast
Valley Fever septate, thick-walled
Blastomycosi hyphae with cells with
branched spherules
s Gilchrist’s round or single bud on
Coccidioides hyphae that with
Disease pyriform a broad base;
immitis produce endospores
conidia broad
thick-walled observed in
Blastomyces borne singly isthmus at
barrel-shape tissue and
dermatitidis on constriction
d, direct
conidiophor
rectangular examination;
es or directly
arthroconidi not a true
on hyphae,
a that yeast
resembling
alternate
“lollipops”
with empty
South Small, Large. Round disjunctor
American septate, to oval, cells
Blastomycosi branched thick-walled
s hyphae with yeast cells Opportunistic Fungal Infections
intercalary with multiple
Paracoccidioi and terminal cuds, which 1. Candida spp.
des chlamydoco attach to ● Candida spp. Are
brasiliensis nidia; few mother cell responsible for the most
pyriform by narrow frequently encountered
microconidia constrictions; opportunistic fungal
resembles a infections
ship wheel
● C. albicans is the most
Darling’s Septate Small, commonly isolated yeast,
Disease hyphae with budding, but other emerging species
round to round to oval include Candida glabrata,
Histoplasma pyriform yeast cells; Candida parapsilosis,
capsulatum microconidia intracellular to
Candida tropicalis, Candida
on short mononuclear
branches or cells possible dubliniensis, and Candida
directly on with Giemsa krusei
hyphal stalk; or Wright’s

PHA6114 LEC 2APH
Candida albicans ● Demonstration of the

● Can be saprophytic in oral capsule by India ink stain
cavities, GI or vaginal tract ● Gram stain for the classic
● Potential pathogen for starburst pattern produced
immunosuppressed patients by Cryptococcus
● Positive for germ tube ● Urease positive, inositol
formation positive and nitrate negative
Identification of Unknown Yeast Isolate Cultured on:

1. SDA medium w/o
cycloheximide
First step Germ tube
formation 2. Birdseed/nigerseed/Staib’s
(assimilates creatinine)
Second step Morphology on
Most common cornmeal agar Latex agglutination test for
conventional carbohydrate
cryptococcal antigen in CSF
definitive utilization patterns
identification
Microscopic Laboratory Diagnosis
Candida albicans Identified by the
production of germ 1. Saline mount
tubes or
2. 10% KOH mount - dissolves keratin;
chlamydoconidia
gentle heat increases the rate of
Other Candida Most commonly clearing for better visualization of fungal
identified by the elements
utilization of 3. Lactophenol cotton blue (Aman
specific substrates medium)
and the
4. India ink or nigrosin
fermentation or
assimilation of 5. Calcoflour white stain (fluorescence
particular microscopy)
carbohydrates 6. Periodic acid Schiff (PAS) -
carbohydrate in fungus will stain
Germ tube Candida albicans, purplish red; H&E slides may be
formation Candida
restained by this
dubliniensis
7. Gram stain (Hucker modification) - all
fungi are gram positive
2. Cryptococcus neoformans 8. Giemsa, Gridley, PAS, GMS (Gomoro
● Encapsulated yeast cell in methenamine silver) used to locate H.
bird and bat droppings capsulatum in RE cells

PHA6114 LEC 2APH
9. Acridine orange, green fluorescence fungi, such as C. neoformans complex,

fungal elements, orange epithelial cells Candida krusei, and other Candida spp.,
Trichosporon spp., P. boydii, and
Aspergillus spp., are partially or
completely inhibited by cycloheximide.
Hair, Nails, or Skin Scrapings (for fungal
culture)
3. Dermatophyte Test Medium (DTM)
Container Clean, screw-top tube ● Indicator is phenol red
● Most dermatophytes
Patient Nails or skin: wipe with
preparation 70% alcohol produce red color
Special Hair: collect hair with intact 4. Cornmeal Agar

instruction shaft ● Stimulates chlamydospore
Nails: send clippings of
production of C. albicans
affected area
Skin: scrape skin at ● Plus 1% glucose to
leading edge of lesion differentiate T. rubrum from
T. mentagrophytes by
Transport to Within 24 hours/room amount of pigment
laboratory temperature
production
● Stimulate fungal sporulation
- use for slide culture
Laboratory Diagnosis
5. Czapek’s Agar
o
● Incubated at 30 C ● Species identification of
● Culture held for 21 days aspergilli
1. Sabouraud Dextrose Agar (SDA) 6. Birdseed/Nigerseed/Staib’s

● Primary isolation medium Medium
● Inhibitor for bacteria: acid pH ● C. neoformans produces
brown pigment
2. Mycosel or Mycobiotic
Contains: 7. Cottonseed Medium
● Chloramphenicol inhibits bacterial ● For Blastomyces
growth
● Cycloheximide inhibits saprophytic 8. Rice Medium
fungi
M. canis Fluoresce on Growth in
Wood’s lamp rice
medium

PHA6114 LEC 2APH
● Microscopic immunodiffusion test

M. Fluoresce on No growth
audouinii Wood’s lamp in rice with antisera
medium
Specific A band: Blastomyces
H and M bands: Histoplasma
Other Tests Laboratory Diagnosis
1. Germ tube
● C. albicans and C. dubliniensis
produce germ tube when incubated
with a sterile serum
2. L-Dopa Ferric Citrate Test

● test for phenol oxidase, (+) black, C.
neoformans
3. Urease Test _______________________________________

● Positive control C. neoformans;
negative control C. albicans
4. Hair Baiting Test

● V shaped penetration of hair shaft
● T. mentagrophytes (+)
● T. rubrum (-)
T. Abunda Negative Urease

rubrum nt hair negative
win-red baiting
pigment test
T. Scant Positive Urease

mentagr red hair positive
ophytes pigment baiting
test
5. Exoantigen Test
● Serologic confirmation for systemic
fungi

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PHA6114 LEC 2APH

PHARMACEUTICAL MICROBIOLOGY AND PARASITOLOGY

[D.N. & S.L.] | #2APHRPhCutie 1

[D.N. & S.L.] | #2APHRPhCutie 2

ORIGIN OF GRAM STAIN

[D.N. & S.L.] | #2APHRPhCutie 3

[D.N. & S.L.] | #2APHRPhCutie 4

[D.N. & S.L.] | #2APHRPhCutie 5

[D.N. & S.L.] | #2APHRPhCutie 6

[D.N. & S.L.] | #2APHRPhCutie 7

[D.N. & S.L.] | #2APHRPhCutie 8

THE MOSQUITO - A PERSISTENT AND

[D.N. & S.L.] | #2APHRPhCutie 9

THE DISCOVERY OF THE HEREDITARY

[D.N. & S.L.] | #2APHRPhCutie

[D.N. & S.L.] | #2APHRPhCutie 11

[D.N. & S.L.] | #2APHRPhCutie 1

[D.N. & S.L.] | #2APHRPhCutie 2

Nucleolus Present Absent Bacteria

[D.N. & S.L.] | #2APHRPhCutie 3

[D.N. & S.L.] | #2APHRPhCutie 4

[D.N. & S.L.] | #2APHRPhCutie 5

In contrast to medium ingredients designed

[D.N. & S.L.] | #2APHRPhCutie 6

When growing on solid media in petri

At concentrations below about 107, cells

[D.N. & S.L.] | #2APHRPhCutie 7

[D.N. & S.L.] | #2APHRPhCutie 8

[D.N. & S.L.] | #2APHRPhCutie 9

Most probable number (MPN) counts

[D.N. & S.L.] | #2APHRPhCutie

[D.N. & S.L.] | #2APHRPhCutie 11

[D.N. & S.L.] | #2APHRPhCutie 12

[D.N. & S.L.] | #2APHRPhCutie 13

[D.N. & S.L.] | #2APHRPhCutie

● Pharmacopoeias and regulatory

[D.N. & S.L.] | #2APHRPhCutie 15

WEEK 3 Bacterial cell components

Chapter 2: Bacteriology Cell wall

● defines the shape of bacteria

Cytoplasmi Phospholipid Phospholipid

Cell Absent Present

Site of Cytoplasmic Mitochondria

[D.N., S.L., & J.S] | #2APHRPhCutie 1

2. Core looking for WBCs, epithelial cells,

[D.N., S.L., & J.S] | #2APHRPhCutie 2

[D.N., S.L., & J.S] | #2APHRPhCutie 3

[D.N., S.L., & J.S] | #2APHRPhCutie 4

➔ Clostridium tetani spores

PARTS EXTERNAL TO CELL WALL

[D.N., S.L., & J.S] | #2APHRPhCutie 5

a. Tumbling motility: Listeria

➔ Peritrichous - flagella all

[D.N., S.L., & J.S] | #2APHRPhCutie 6

Immunol HIGHLY WEAKLY BOTULISM (C.

[D.N., S.L., & J.S] | #2APHRPhCutie 7

NEUROTOXIN a. Tyndallization – flowing steam

[D.N., S.L., & J.S] | #2APHRPhCutie 8

- Method of choice for antibiotic

Cold sterilization Cold sterilization

Used for materials

Quality control: spores