CLBACT1 : BSMLS

CLINICAL BACTERIOLOGY FOR MED LAB SCIENCE : LECTURE

MICROBIOLOGY BIOGENESIS
- Study of organisms and agents too small to be seen by Biogenesis is any process by which lifeforms produce other
the unaided eye lifeforms. For example, a spider lays eggs that become other
- Study of microorganisms spiders. A demonstrative experiment, which showed biogenesis
BRANCHES OF MICROBIOLOGY right down to the bacterial level, was devised by Louis Pasteur
Bacteriology in 1859.
- study of bacteria
Mycology Theodore Schwann (1810 – 1882)
- study of fungus/fungi - Allowed air (that passed thru a red-hot tube) to enter a
Virology sterile medium
- study of viruses - He observed that no growth occurred in a flask
Phycology containing nutrient solution
- study of algae Georg Friedrich Schroder and Theodore van Dusch
Protozoology - They observed that no growth occurred after allowing
- study of protozoans air to pass through sterile cotton wool placed in a flask
THE DISCOVERY OF MICROORGANISMS of the heat-sterilized medium
Lucretius (98 – 55 BC) Felix Pouchet
Girolamo Fracastoro (1478 – 1553) - In 1859, He was able to grow microorganisms without
- Diseases were caused by invisible living creatures air contamination
Francesco Stelluti (1625 and 1630) Rudolf Virchow (1885)
- He made the earliest microscopic observations on - He challenged spontaneous generation with the
bees and weevils using a microscope probably concept of biogenesis
supplied by Galileo - “Cells arise from pre-existing cells”
Anton Van Leeuwenhoek (1632 – 1723) Louis Pasteur (1822 – 1895)
- The first true microbiologist - Placed sterile broths in flasks and drew the necks in a
- Constructed a simple microscope using double convex variety of curves
lens - Bacteria got trapped in necks
- He described the microorganisms from the pond water - No growth in broths
as animalcules - He resolved the issue of spontaneous generation in
- He used his self-made single lens microscope with 50 1861
– 300x magnification - Other contributions
o Developed anthrax vaccine
SPONTANEOUS GENERATION o Rabies vaccine
- This was the idea that living organism can give rise o Fermentation (Pasteurization)
or develop to non-living organisms. John Tyndall (1820 – 1893)
Recipe for Mice - He showed that dust carry germs which contaminates
- Dirty Shirt + Wheat + 21 Days (Time) sterile broth
- He made a specially designed box, after allowing the
Aristotle (384 – 322 BC) dust particles to settle, he carefully placed tubes of
- Simpler invertebrates could arise from spontaneous sterile infusions in the box, as long as the dust is not
generation distributed, the infusion was sterile
Francesco Redi (1626 – 1697) - The pathway of light is seen through air because it
- He demonstrated that maggots do not arise refracted by dust particles (Tyndall Effect)
spontaneously from decaying meat but came from flies - Other contributions:
- Place meat in 3 containers o Tyndallization – form of sterilization for three
o One uncovered = w/ maggots consecutive days
o One covered with paper = no maggots o Endospores – spores found within bacteria
o One covered with gauze = maggots on gauze o Fractional Distillation
John Needham (1713-1781) THE RECOGNITION OF THE MICROBIAL ROLE IN
- He observed that boiled mutton broth (tightly sealed) DISEASE
eventually became cloudy with microorganisms Then, diseases were caused by:
- Organic matter possessed a vital force that confers - Invisible organisms
life to non-living matter - Supernatural forces
Lazzaro Spallanzani (1729 – 1799) - Miasma
- He improved the previous experiments of Needham o Poisonous vapor
- Heated sealed flasks containing water and seeds then - Imbalances in the 4 humors
placed in boiling water for ¾ of an hour o Blood
- No growth as long as seal stays in place o Phlegm
o Air carried germs to the medium o Yellow Bile
o Black Bile

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FERMENTATION AND PASTEURIZATION KOCH’S POSTULATE
- Theodore Schwann stated that yeast cells were responsible ✓ A microorganism is present in all cases of diseases and
for the conversion of sugars to alcohol, however he said that absent in healthy individuals
fermentation was not due to microorganisms but to a ✓ The microorganism that is suspected case can be isolated
chemical instability that converted sugars to alcohols and grown in a pure culture
- Pasteur described that certain microorganism knows as ✓ The isolated organism can cause disease in an
yeast converts sugar to alcohol in the absence of air experimental susceptible animal
(fermentation) ✓ The organism can be re-isolated from the experimentally
- Souring and spoilage are caused by different susceptible animal
microorganisms called bacteria Fannie Eilshemius Hesse
- In the presence of air, bacteria change the alcohol in the - Suggested the use of agar as a solidifying agent
beverage into vinegar (acetic acid) - Heating the bear and Richard Petri
wine just enough to kill most of the bacteria (pasteurization). - Developed the petri dish (plate)
Charles Chamberland
PASTEUR’S CONTRIBUTION TO SCIENCE - Created a porcelain bacterial filter (1884) and developed
✓ He disproved the theory of spontaneous generation anthrax vaccine together with Pasteur
✓ He developed vaccines against anthrax (1861) and Martinus Beijerinck and Sergie Winogradsky
rabies (1885) - Developed the enrichment-culture technique and the use
✓ He improved the wine industry (theory of fermentation) of selective media
IMMUNOLOGICAL STUDIES – VACCINATION
Agostino Bassi Edward Jenner (1749 – 1823)
- 1835 – silkworm diseases were caused by a fungus - found a way to protect people from small pox
- First showed a microorganism can cause disease Pasteur used the term
M.J Berkeley - “Vaccine” (Latin “vacca” -cow)
- the great potato blight in Ireland is caused by a fungus Emil von Behring (1854-1917) & Shibasaburo Kitasato
Joseph Lister (1827 – 1912) (1852-1931)
- Father of Antisepsis - Prepared antitoxins for diphtheria and tetanus
- He developed the antiseptic system of surgery MODERN THERAPY” “MAGIC BULLET”
- He demonstrated the use of phenol for treating surgical CHEMOTHERAPY
wound and also sprayed phenol over the surgical area - The treatment of disease using chemical substances
Ignaz Philipp Semmelweis (1840s) - It also refers to chemical treatment of non-infectious
- He demonstrated the transmission of childbed fever diseases, such as cancer
- He noticed that the doctors who had not washed their o Synthetic drugs – prepared from chemical in the
hands had much higher mortality rates laboratory
- He concluded that the puerperal fever was septic and o Antibiotics – produced naturally by bacteria and
contagious fungi to act against microorganisms
- He ordered the students to wash their hands with Ellie Metchnikoff (1845-1961)
Chlorinated Lime then reduces mortality rate from 12.24 - Phagocytosis
to 1.27% in 2 years Landsteiner
Robert Koch (1843 – 1910) - 1902, ABO Blood Group
- German physician known as the “Father if Schaudinn & Hoffman
Bacteriologic Techniques) - 1906, Treponema Pallidum causes Syphilis
- He established the first proof that bacteria indeed cause Wasserman
diseases - 1906, developed the complement fixation test for
- Formulated Germ Theory syphilis
o States that for every disease, a living organism is Paul Ehrlich
responsible - Discovered salvarsan (arsphenamine) for treatment of
- Also known as the “Cause and Effect” syphilis
- Other contributions” - Chemotherapeutic agent
o Bacillus Anthracis – causes Anthrax D’ Herelle & Twort
o Cholera - Discovered bacterial viruses (bacteriophage)
o Mycobacterium tuberculosis Waksman
o Pure culture using agar (with Fannie Eilshemus - Discovers Streptomycin in 1944
Hesse) Alexander Fleming
o He developed culture media for observing growth of - Discovered penicillin (Penicillium notatum)
bacteria isolated from the human body. Howard Florey and Ernst Chain
- Made the purification process for penicillin

OTHER SCIENTIST
Gaffky – Salmonella
Loeffler – Corynebacterium
Kitasato – Clostridium
Ricketts – Typhus
Jenner – Small Pox Vaccine

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 SCOPES OF MICROBIOLOGY - Creates a magnified image by using a series of glass
MEDICAL MICROBIOLOGY lenses that focuses a beam of light onto or through an
object.
- identify the agent causing an infectious disease and plan
- Convex objective lenses are used to enlarge the image
measures to eliminate it
formed.
- involved in tracking down new, unidentified pathogens
PUBLIC HEALTH MICROBIOLOGY TYPES
• Bright field microscope
- tries to control the spread of communicable diseases • Dark field microscope
- monitor community food establishments and water
• Phase contrast microscope
supplies
• Fluorescence microscope
IMMUNOLOGY
BRIGHT FIELD MICROSCOPE
- concerned with how the immune system protects the
- Ordinary type of microscope
body from pathogens and the response of infectious
- Dark image, bright background
agents
PARFOCAL
- deals with nature and treatment of allergies and
- image should remain in focus when objectives are
autoimmune diseases
changed.
AGRICULTURAL MICROBIOLOGY VIRTUAL IMAGE
- combat plant diseases that attack important food crops - enlarged specimen image.
- work on methods to increases soil fertility DARK FIELD MICROSCOPE
MICROBIAL ECOLOGY - Field surrounding specimen appears black, specimen is
- study relationships between microorganisms and their brightly illuminated.
habitats - Used for:
- use of microorganisms in bioremediation to reduce o Living unstained cells and organisms
pollution o Internal structures
FOOD AND DAIRY MICROBIOLOGY o Treponema pallidum (spirochetes).
- prevent microbial spoilage of food and transmission of - Uses dark field condenser that blocks light that enters the
food bone diseases objective directly.
INDUSTRIAL MICROBIOLOGY - Directs light to the specimen at an oblique angle.
- microorganisms are used to make products such as
PHASE CONTRAST MICROSCOPE
antibiotics, vaccines, steroids, alcohols and other - Background is light, unstained object appears dark and is
solvents, vitamins, amino acid and enzymes well defined.
MICROBIAL PHYSIOLOGY AND BIOCHEMISTRY - Converts slight differences in refractive index and cell
density into easily detected variations in light intensity.
- study of the synthesis of antibodies and toxins, microbial
CONDENSER: Annular stop
energy production
- opaque disk with a thin transparent ring that produces a
MICROBIAL GENETICS AND MOLECULAR BIOLOGY hollow cone of light.
- focus on nature of genetic information and how it - Used for:
regulates the development and function of cells and o Unpigmented living cells
organisms o Detection of bacterial components and inclusion
- produce new microbial stains that re more efficient in bodies
synthesizing useful products o Eukaryotic cells.
MICROSCOPY FLUORESCENCE MICROSCOPE
Refraction - Molecules absorb energy, becomes excited, and
- occurs when a ray of light passes from one medium to releases trapped energy as light.
another and the ray is bent at the interphase - Specimen is exposed to UV light, violet or blue light and
Refractive Index forms an image of the object with the resulting
- the measure of how greatly a substance slows the fluorescence light
velocity of light FLUOROCHROMES – stains used.
o the direction and magnitude of bending is • Fluoresce brightly upon exposure to light of a
determined by the refractive indices of the 2 media specific wavelength.
forming the interface • Microscope forms an image of fluorochrome-
Focal Point labeled organisms.
- point at which rays of parallel light meets as focused by a
ELECTRON MICROSCOPE
convex lens
Focal Length - One of the most advanced and important types of
- the distance between the center of the lens and the focal microscopes.
point - Has the highest magnifying capacity.
NOTE: - Electrons are used to illuminate the tiniest particles.
Focal Strength is related to Focal Length - More powerful than light microscope.
- the shorter the focal length, the higher its magnification - Energy source: beam of electrons
ELECTRONS
• Short wavelength
TYPES OF MICROSCOPES • Strikes most objects in its path
LIGHT MICROSCOPE • Travels in a vacuum to avoid contact with
- Uses visible light to detect objects. deflecting air molecules.
- Used for visualizing fine details of an object.

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 • Magnets focuses beam on object. RESOLUTION
• Image is created in a monitor, viewed by the - It is the ability of the lens to separate or distinguish
technologist. between small objects that are close together.
• Increases resolution of the microscope ERNST ABBE
significantly. - Formulated the Abbe Sine equation which shows the
TRANSMISSION ELECTRON MICROSCOPE maximum resolution for a microscope.
- Traditional form of EM microscope
- Ultrathin slices of microorganisms or viruses are placed
on a wire grind.
- STAIN: GOLD OR PALLIDUM
- Densely coated parts of the specimen deflect electron
As d becomes smaller, the resolution increases.
beam and both dark and light areas show on the image.
WAVELENGTH
SCANNING ELECTRON MICROSCOPE - Important factor
- Contemporary form of EM microscope - Must be shorter than the distance between two objects.
- Gives lower magnification than TEM. - Shorter wavelength, higher resolution.
- Three-dimensional views of microorganisms and other - Uses the blue end of visible spectrum (450-500nm).
objects. NUMERICAL APERTURE (N SIN θ)
- STAIN: GOLD OR PALLIDUM - θ – ½ angle of cone light entering an objective.
- Whole objects are used. - N – refractive index
SCANNING TUNNELING MICROSCOPE - The N of air is 1.00
- Structure of a surface is studied using a stylus that scans - No lens working in air can have NA of greater than 1.
surface at a fixed distance. - To increase refractive index with the use of oil immersion
- Electrons tunnel between the surface and stylus, (oil which has the same refractive lens as glass)
producing an electrical signal. - Air is replaced by oil immersion.
- Shows a three-dimensional image of a sample. TYPICAL N.A VALUES
- Stylus tip is formed by one single atom. 4x 0.1
- Scans across surface at a distance of only an atom’s 10x 0.25
diameter. 40x 0,65
- Vertical movement of stylus is recorded. 100x 1,25
PARTS OF THE BRIGHT FIELD MICROSCOPE - The maximum theoretical resolving power of a
OCULAR LENS microscope with oil immersion objective (NA 1.25) and
- Remagnifies image formed by the objective lens. blue green light is approximately 0.2 UM
- *Lens should be small to achieve magnification with good - A bright field microscope can distinguish between 2 dots
results around 0.2 UM apart.
WORKING DISTANCE
BODY TUBE
- The distance between objective and glass slide.
- Transmits image from the objective lens to the ocular. - Distance between 2 front surfaces of the lens and surface
ARM of the cover glass (if one is used) or the specimen when
- Used for holding the microscope. it is sharp focus
NOSEPIECE - Objectives with large numerical apertures and great
- Holds the objectives. resolving powers have short working distances
OBJECTIVE LENSES
- Primary lenses that magnify the specimen.
- Most important part of the microscope.
MECHANICAL STAGE
- Holds the slide in place.
CONDENSER
- Focuses light through the specimen.
DIAPHRAGM
- Controls the amount of light entering the condenser.
COARSE ADJUSTMENT KNOB
- Focuses the image under low power and moves the
stage.
FINE ADJUSTMENT KNOB
- Sharpens the image under all types of objectives.
ILLUMINATOR
- Light source.
BASE
- Supports the microscope.

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 TAXONOMY 4-KINGDOM CLASSIFICATION BY HERBERT COPELAND (1938)
- Bacterial taxonomy is concerned with the naming of - Plants
bacterial organisms and with organizing these names o Includes Fungi
according to various criteria. - Animals
- The basic taxon is the species. - Protista
- Greek o Amoebas, Diatoms, Some Unicellular and
o Taxis – arrangement or order Multicellular Eukaryotes
o Nomos – law - Monera/Prokaryotes
o Nemein – to distribute or govern o Bacteria
- Taxonomy is the science of biological classification 5-KINGDOM CLASSIFICATION BY ROBERT WHITTAKER (1957)
3 PARTS OF TAXONOMY - Plants
CLASSIFICATION - Animals
- Arrangement of organisms into groups or taxa (sing. - Protista
taxum) o Algae and Protozoa
- Based on mutual similarity or relatedness - Monera/Prokaryotae
NOMENCLATURE o Bacteria and Archaeans
- Fungi
- Branch of taxonomy concerned with assignment of TAXONOMIC RANKS (DESCENDING)
names to taxonomic groups in agreement with
- Kingdom
published rules
- Division
IDENTIFICATION - Phyla
- Process of determining that a particular isolate belong - Classes
to a re-organized taxon - Orders
SYSTEMATICS - Families
- The scientific study of organisms with the ultimate - Genera
object of characterizing them in an orderly manner - Species
- Encompasses LINNAEAN SCHEME OF CLASSIFICATION
o Morphology - Bacteria may be classified as to:
o Ecology o Kingdom
o Epidemiology o Class (-aceae)
o Biochemistry o Order (-ales)
o Molecular biology o Family (-aceae)
o Physiology o Tribe (-ieae)
BERGEY’S MANUAL OF SYSTEMATIC BINOMIAL SYSTEM
BACTERIOLOGY – By Carl Von Linne/ Carolus Linnaeus
- This is the most widely followed classification and - Latinized, italicized name
nomenclature system in the US and was first published in GENERIC NAME
1974. - First part
- The American Society for Microbiology (originally the - Capitalized
Society of American Bacteriologists) has for decades - May be changed
published a compilation of known bacteria, first as Bergey's SPECIFIC EPITHET
Manual of Determinative Bacteriology and more recently
as Bergey's Manual of Systematic Bacteriology. - Second part
- Multiple editions of Bergey's Manual of Determinative - Stable
Bacteriology, published between 1923 and 1994, organized - The oldest epithet for a particular organism takes
bacteria in groups by phenotypic characteristics, with no precedence and must be used
attempt to sort out higher phylogenetic relationships. - Names may be shortened by abbreviating the genus name with
- They were very useful for identifying unknown bacterial a single capital letter. e.g. E. coli
cultures The first edition of Bergey's Manual of Systematic TERMINOLOGIES
Bacteriology, which came out in four volumes from 1984 Species
through 1989, attempted to organize bacterial species - The basic taxonomic group in microbial taxonomy
according to known phylogenetic relationships Bacterial Species
BACTERIAL CLASSIFICATION - Collection of strains that share many stable properties
KINGDOMS and differ significantly from other groups of strains
2-KINGDOM CLASSIFICATION BY ARISTOTLE (384 – 322 BC) Strain
- Population of organisms that descends from a single
- Plants
organism or pure culture isolates
- Animals
Type strain
3-KINGDOM CLASSIFICATION BY ERNST HAECKEL (1735)
- One strain of species that is more fully characterized
- Plants than other strains
- Animals Biovars
- Protista - Variant bacterial strains
o Includes Singles Celled Organisms, Bacteria, - Characterized by biochemical physiological
Protozoa, Fungi and Algae differences
Morphovars
- Differ morphologically

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Serovars o Requirement for temperature, pH, oxygen, and
- Distinctive antigenic property osmotic concentration
Genus GENETIC ANALYSIS
- Well-defined group of one or more species that is - Chromosomal gene exchange thru
clearly separate from other genera o Transformation
CHARACTERISTICS USED IN TAXONOMY ▪ Can occur between different bacterial
- Morphological characteristics species but only rarely between genera
- Physiological and metabolic characteristics o Conjugation
- Ecological characteristics o Plasmids
- Genetic analysis MOLECULAR CHARACTERISTICS
- Molecular characteristics COMPARISON OF PROTEINS
o Comparison of proteins - Amino acid sequences are direct reflections of mRNA
o Nucleic acid base composition sequences
o Nucleic acid hybridization - Closely related to the structure of the genus coding for
o Nucleic acid sequencing their synthesis
MORPHOLOGICAL CHARACTERISTICS DIRECT APPROACH
REASONS - Determines amino acid sequence of proteins with the
a. Easy to study and analyze same function
b. Structural features depend on the expression of many - Slow and expensive
genes INDIRECT APPROACH
c. Usually genetically stable
Electrophoretic mobility
d. Normally do not vary greatly with environmental
- Relationships at species and subspecies level
changes
Antibodies
e. Morphological similarity is a good indication of
- Can discriminate between very similar proteins
phylogenetic relatedness
Immunologic
MORPHOLOGICAL FEATURES - Compare proteins from different microorganisms
- Cell shape Enzymes
- Cell size - Physical, kinetic and regulatory properties
- Colonial morphology - Enzyme behavior reflects amino acid sequence
- Ultrastructural characteristics
NUCLEIC ACID BASE COMPOSITION
- Staining behavior
- Cilia and flagella
G + C CONTENT
- Mechanism of motility - Reflects the base sequence
- Endospore shape and location - Determined from melting temperature (Tm) of DNA
- Cellular inclusions - DNA with a greater G + C content will have a higher
- color melting point
PHYSIOLOGICAL AND METABOLIC - Use spectrophotometer – absorbance of 260 nm UV
CHARACTERISTICS light by DNA‘s increase during DNA separation
- Directly related to the nature and activity of microbial IMPORTANCE
enzymes and transport proteins - Can confirm a taxonomic scheme developed using
- Provides indirect comparison of microbial genomes other data
- Some important physiological and metabolic - Useful in characterizing bacterial genera since the
characteristics used for classification and variation within a genus is usually less than 10%
identification NUCLEIC ACID HYBRIDIZATION
o Carbon and Nitrogen content - Compare similarity between genomes
o Cell wall constituents - If a mixture of single stranded DNA formed by heating
o Energy sources dsDNA is cooled and held at a temperature about 25C
o Fermentation products below the Tm, strands with complementary base
o General nutritional status sequences will reassociate to form stable dsDNA,
o Growth temperature optimum and range while non-complementary strands will remain single
o Luminescence - Incubation of the mixture at 30 – 50C below the Tm will
o Mechanisms of energy conversion allow hybrids of more diverse ssDNA‘s to form
o Motility NUCLEIC ACID SEQUENCING
o Osmotic tolerance - Genome structure can only be directly compared by
o Oxygen relationships sequencing DNA and RNA
o pH optimum and growth range - rRNA‘s are most ideal for studies of microbial evolution
o Photosynthetic pigments and relatedness since they are essential to a critical
o Salt requirements and tolerance organelle found in all microorganisms
o Secondary metabolites formed NUMERICAL TAXONOMY
o Sensitivity to metabolic inhibitors and antibiotics
- Quantitative approach
o Storage inclusions
- Grouping by numerical methods of taxonomic units into
ECOLOGICAL CHARACTERISTICS taxa on the basis of their character states
- Life cycle patterns - Based on general similarity as judged by comparison
- Nature of symbiotic relationships of many characteristics, each given equal weigh
- Ability to cause disease in a particular host
- Habitat preferences

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 BACTERIAL GENETICS GENETIC DIVERSITY
GENETICS - Ways in which bacteria acquire new genetic
- Study that defines and analyzes heredity and genetic information.
variations. GENETIC MUTATIONS
GENE - Mutation is a change in the DNA base sequence that
- Basic unit of heredity. alters the structure and function of the protein in the
- A segment of DNA that carries information for a specific cell.
biochemical or physiologic property. Change is transmissible to offspring:
- MUTAGEN
GENOME
o Substance that increases the rate of mutation.
- All genes taken together within an organism. - MUTANT
- Set of chromosomes. o Organism containing the mutation.
CHROMOSOME
- Rod-shaped structure carrying genes. CATEGORIES OF MUTATION
- Contains nucleoproteins (nucleic acid and protein). BENEFICIAL MUTATION
NUCLEOSIDE - Mutation that is beneficial to the organism.
- Purine or pyrimidine base linked to a sugar molecule. HARMFUL MUTATION
- Ex. Adenosine, guanosine - Mutation which leads to the production of a non-
NUCLEOTIDE functional gene.
- Basic unit of a nucleic acid composed of sugar, - Lethal Mutation
phosphate and a base. o leads to the death of the organism.
- Phosphate esters. SILENT MUTATION
- Ex. Adenosine monophosphate, adenosine - Mutation that has no effect on the cell.
triphosphate. - Mutation that causes no change in function.
NUCLEIC ACID TYPES OF MUTATION
- Polynucleotide SUBSTITUTION
DEOXYRIBONUCLEIC ACID (DNA) - A mutation that replaces one base in a DNA with a
- Most common macromolecule that encodes genetic different base.
information. - The change in the codon may cause a different amino
- Twisted ladder in appearance. acid to be inserted at that point in the polypeptide.
RIBONUCLEIC ACID (RNA) SUBSTANCE NORMAL EFFECT OF
- Also encodes genetic information for viruses. SEQUENCE MUTATION
- TYPES: Possible change in base
o mRNA – messenger RNA; responsible for DNA ACA-CCC-AGG- ACA-CAC-AGG-TTT
communicating DNA gene sequences. TTT
o rRNA – ribosomal RNA; structural and change in codon
functional components of ribosomes. mRNA UGU-GGG-UCC- UGU-GUG-UCC-AAA
o tRNA – transfer RNA; attaches amino acids AAA
to protein chains being made at ribosomes. Change in amino acid order
DNA RNA Amino Acid Cys-Gly-Ser-Lys Cys-Val-Ser-Lys
NITROGENOUS Adenine Adenine Sequence
BASES Guanine Guanine
Cytosine Cytosine FRAME SHIFT MUTATION
Thymine Uracil
- A mutation that inserts or deletes a base in a DNA
5-SUGAR Deoxyribose Ribose sequence.
CONTENT
FACTORS THAT CAUSE MUTATION
ARRANGEMENT Double stranded Single stranded
a. Viruses
b. Chemicals (industrial chemicals, pesticides, food
REPLICATION OF GENETIC INFORMATION additives, hair dyes, and cosmetics).
- Involves a complicated process mediated by the c. Ultraviolet light (overexposure to sum)
enzyme d. X-rays
DNA polymerase.
UNWINDING MUTAGEN
- For enzymes and cofactors to act on the DNA - The agents that cause mutation which include
molecule. chemical agents or various types of radiation.
UNZIPPING - Ames Test
- DNA polymerase opens the replication fork. o developed by Bruce Ames; standard test for
SYNTHESIS OF THE NEW DNA mutagenicity.
- Each parent strand serves as a template from which a
complementary strand is produced.
TERMINATION OF REPLICATION
- Occurs when two replication forks meet.

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RECOMBINATION
- Process in which a new recombinant chromosome,
one with a genotype different from either parent, is
formed by combining genetic material from two
organisms.
TYPES OF RECOMBINATION
GENERAL RECOMBINATION
- Most common form
- Involves a reciprocal exchange between a pair of
homologous DNA sequences.
SPECIFIC RECOMBINATION
- The genetic material is not homologous with the
chromosome it joins.
- Enzymes responsible for this event are specific for the
particular virus and its host.
REPLICATIVE RECOMBINATION
- Accompanies the replication of genetic material and
does not depend on sequence homology.
- Used by genetic elements that move about the
chromosome.
PLASMIDS
- Small, circular DNA molecules that can exist
independently of host chromosomes
- Replicon
o a DNA molecule or sequence that has a
replication origin and is capable of being
replicated
CLASSIFICATION OF PLASMIDS
EPISOME
- A plasmid that can exist either with or without being
integrated into the host’s chromosome.
CONJUGATIVE PLASMIDS
- Have genes for pili and can transfer copies of
themselves to other bacteria during conjugation.
FERTILITY FACTOR / F FACTOR
- Plays a major role in conjugation.
- Bears genes responsible for cell attachment and
plasmid transfer between specific bacterial stains
during conjugation.
RESISTANCE FACTORS / R FACTOR
- Have genes for enzymes capable of destroying or
modifying antibiotics.
- Genes coding for resistance to antibiotics (ampicillin,
chloramphenicol and kanamycin have been found in
plasmids). - Conjugative plasmids; they can spread
throughout a population (not as rapidly as the F factor).
- Readily transferred between species, further promoting
the spread of resistance.
COL PLASMIDS
- Plasmids with genes that give them competitive
advantage in the microbial world.
- Code bacteriocins;
o Colicins – bacteriocins produced by E. coli
that destroy other bacteria.
VIRULENCE PLASMIDS
- Makes the host more pathogenic because the
bacterium is better able to resist host defense of to
produce toxins.
METABOLIC PLASMIDS
- Carry genes that degrade substances such as
aromatic compounds (toluene), pesticides (2,4-
dichlorophenocyacetic acid), and sugars (lactose).

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 BACTERIAL CONTROL LYOPHILIZATION
- Inhibiting the growth of microorganisms - Combines dehydration and freezing (freeze-drying).
- “In Vitro” - Frozen in a vacuum.
DEFINITION OF TERMS - To preserve foods, antibiotics, antisera,
STERILIZATION microorganisms, and other biological materials.
- Complete destruction of all living organisms including - Doesn’t kill bacteria BUT prevents them from
cells, spores, and viruses. reproducing.

STERILE SEPSIS
– devoid of microbial life. - Presence of pathogens in blood or tissues.

DISINFECTION ASEPSIS
- Destruction or removal of pathogens from non-living - Absence of pathogens.
objects by physical or chemical methods.
ASEPTIC TECHNIQUE
PASTEURIZATION - Techniques employed to eliminate and exclude
- For disinfecting liquids. pathogens.
- Originally used by Pasteur for wine disinfection. - Involves handwashing, use of sterile gloves, masks,
- Now used for milk. gowns, sterilization of materials, use of disinfectants,
and antiseptics.
DISINFECTANTS
- Chemicals used to disinfect inanimate objects.
ANTISEPSIS
- Strong chemical substances that cannot be used on - Prevention of infection
living tissues. - Developed by Joseph Lister
o Refers to the use of antiseptics.
ANTISEPTICS
ANTISEPTIC TECHNIQUE
- Used to disinfect skin and other living tissues.
- Type of aseptic technique.
SANITATION - PHENOL: used by Lister to cleanse surgical wounds.
- Reduction of microbial populations to levels considered
as safe by public health standards.
STERILE TECHNIQUE
- Practiced when it is necessary to exclude all
MICROBICIDAL AGENTS microorganisms from a particular area.
- Disinfectants that kill microbes
PHYSICAL METHODS TO INHIBIT MICROBIAL
o Germicidal agents (germicides)
o Biocidal agents (biocides) GROWTH
o Microbicidal agents (microbicides) • Heat
• Radiation
BACTERICIDAL AGENTS • Pressure
• Sonic disruption - Desiccation
- Disinfectants that specifically kill bacteria but not
necessarily the bacterial endospores. • Filtration
HEAT
SPORICIDAL AGENT - Most practical, efficient, and inexpensive method for
inanimate objects and materials that can withstand
- Used to kill bacterial endospores. high temperature.
- Kills by coagulating proteins.
FUNGICIDAL AGENTS (FUNGICIDE) - Factors: Heat and time
- Kill fungi and fungal spores. *The higher the temperature, the shorter the time required to kill
the organisms.
ALGICIDAL AGENTS (ALGICIDES) THERMAL DEATH POINT (TDP)
- Kills algae in swimming pools and hot tubs. - Lowest temperature that will kill all organisms in a standardized
pure culture within a specified period.
VIRICUDAL/VIRUCIDAL AGENTS
- Destroys viruses. THERMAL DEATH TIME (TDT)
- Length of time necessary to sterilize a pure culture at a
MICROBISTATIC AGENTS specified temperature.
- Drug or chemical that inhibits growth and reproduction
of microorganisms. DRY HEAT
- Kills by oxidation
BACTERIOSTATIC AGENTS Hot air oven.
– 160C to 165C for 2 hours
- Specifically inhibits the metabolism and reproduction of – 170C to 180C for 1 hour
bacteria.

JASCHA KEAN ROSALES-LBH 9

 – For metals, glassware, some powders, oils, and - May be placed in rooms, cabinets containing
waxes. instruments, cloth equipment, liquid and other
Incineration inanimate objects
- Burning - Reduces number of microorganisms in the air
- For contaminated disposable materials. - Disadvantages:
o DNA replication is inhibited
- FLAMING o forms thymine dimers
o for wire loops, wire needles, forceps, mouth of o can damage the cornea and skin
tubed media, and rim of petri dishes. o Do not penetrate cloth, glass and metals
MOIST HEAT X-RAYS
- Coagulation accompanied by hydrolysis. - With higher energy and higher penetrating power than
Autoclave UV
- Uses steam under pressure. - Produces hydroxyl radicals by the hydrolysis of water
- 1210C at 15 psi for 15-30 minutes. - breaks covalent bonds on the DNA
- Pressure-sensitive autoclave tapes and solutions - Spores are resistant due to its low water content
containing bacterial spores used for quality control. BETA RAYS
Boiling
- 1000C, 15-30 minutes
- Kills vegetative forms but not spores and viruses. GAMMA RAYS
Fractional sterilization - From cobalt 60
- Tyndallization - Can be used to process chicken and red meat
- Steam for 30 minutes for 3 consecutive days - For food processing
- Arnold sterilizer - May kill Salmonella and Campylobacter in chickens
Inspissation o Chicken that is irradiated is marked with the
- 75-800C for 2 hours, 3 successive days. green international symbol for radiation
Pasteurization MECHANICAL METHODS
- For milk and wine - disintegrate bacteria
Sonic vibration
- Uses sound waves
- For delicate equipment
Trituration
- grinding
Agitation
- shaking
FILTRATION
- Used to separate cells, larger viruses, bacteria and
certain other microorganisms from the liquid or gases
in which they are suspended
COLD - Sintered glass, plastic films, unglazed porcelain,
- Doesn’t kill microorganisms. asbestos, diatomaceous earth, cellulose membrane
- Slows down their metabolism. filters
Refrigeration - HEPA – high efficiency particulate air
- slows down growth. o To protect workers
Slow freezing
OSMOTIC PRESSURE
- Not used as ice crystals are formed.
Rapid freezing - by plasmolysis
- Uses nitrogen - Example:
- For preservation o immersion of meat in salt solution
- Places bacteria under suspended animation o Fruits and vegetables in sugar solution
DESSICATION VARIABLES OF DISINFECTION
1. Concentration
- Foods may be preserved by drying
2. Time
- However, some microbes remain viable even after
3. Temperature
drying
4. pH
- Example:
N = 1/CT
o N. gonorrheae
Where:
o Mycobacterium tuberculosis
N = number of surviving bacteria
RADIATION C = concentration
- For prevention of food spoilage, sterilization of heat T = time
sensitive equipment, preparation of vaccine - increased concentration and increased time =
- Sunlight decreased survivors
o Includes IFR, visible light, UVR
o Kills only those exposed to direct sunlight
UVR
- Uses UV lamp or germicidal lamp

JASCHA KEAN ROSALES-LBH 10

 CHEMICAL METHODS TO INHIBIT MICROBIAL GLUTARALDEHYDE
GROWTH - a high-level disinfectant and chemical sterilant
ALCOHOL - Acidic glutaraldehyde is not sporicidal
- "alcohol" refers to two water-soluble chemical - Should be made alkaline to become sporicidal
compounds—ethyl alcohol and isopropyl o Should be used within 14 days because
- alcohol— polymerization blocks aldehyde functional
- bactericidal, tuberculocidal, fungicidal, and virucidal group which is responsible for its biociddal
- do not destroy bacterial spores. activity
- cidal activity drops when diluted below 50% - Glutaraldehyde is used most commonly as a high-level
concentration, disinfectant for medical equipment
- optimum bactericidal concentration is 60%–90% - Mode of action
solutions in water (volume/volume) o results from its alkylation of sulfhydryl,
- mode of action: hydroxyl, carboxyl, and amino groups of
o denaturation of proteins. microorganisms, which alters RNA, DNA, and
o absolute ethyl alcohol, a dehydrating agent, is protein synthesis
less bactericidal than mixtures of alcohol and HYDROGEN PEROXIDE
water because proteins are denatured more - bactericidal, virucidal, sporicidal, and fungicidal
quickly in the presence of water. properties
CHLORINE AND CHLORINE COMPOUNDS - Mode of action
HYPOCHLORITES o produce destructive hydroxyl free radicals
- available as liquid (e.g., sodium hypochlorite) or solid that can attack membrane lipids, DNA, and
(e.g., calcium hypochlorite) other essential cell components.
- They have a broad spectrum of antimicrobial activity o Catalase, produced by aerobic organisms
- do not leave toxic residues and facultative anaerobes, is overwhelmed by
- unaffected by water hardness o the concentrations used for disinfection
- inexpensive IODOPHORES
- fast acting - Iodophors can be used both as antiseptics and
- remove dried or fixed organisms and biofilms from disinfectants
surfaces - a combination of iodine and a solubilizing agent or
- low incidence of serious toxicity carrier
- mode of action: - the resulting complex provides a sustained-release
o oxidation of sulfhydryl enzymes and amino reservoir of iodine and releases small amounts of free
acids iodine in aqueous solution.
o ring chlorination of amino acids - Example:
o loss of intracellular contents o povidone-iodine, a compound of
o decreased uptake of nutrients polyvinylpyrrolidone with iodine.
o inhibition of protein synthesis - retain the germicidal efficacy of iodine but are
o decreased oxygen uptake generally are non-staining and relatively free of toxicity
o oxidation of respiratory components and irritancy
o decreased adenosine triphosphate - Mode of action
production; o Iodine can penetrate the cell wall of
o breaks in DNA; and depressed DNA microorganisms quickly, and the lethal effects
synthesis are believed to result from disruption of
FORMALDEHYDE protein and nucleic acid structure and
- used as a disinfectant and sterilant in both its liquid and synthesis.
gaseous states. ORTHO-PHTHALALDEHYDE (OPA)
- water-based solution called formalin, which is 37% - Ortho-phthalaldehyde is a high-level disinfectant
formaldehyde by weight. - It contains 0.55% 1,2-benzenedicarboxaldehyde
- bactericide, tuberculocide, fungicide, virucide and (OPA).
sporicide - OPA solution is a clear, pale-blue liquid with a pH of
- Disadvantages 7.5
o Prolonged exposure can lead to contact - Advantages
dermatitis o excellent stability over a wide pH range (pH
o Fumes are irritating to the eye and respiratory 3–9)
system o not a known irritant to the eyes and nasal
- Mode of action passages
o By alkylating the amino and sulfhydryl groups o does not require exposure monitoring,
of proteins and ring nitrogen atoms of purine o has a barely perceptible odor, and requires no
bases activation
o Carcinogen - Disadvantages
o limit exposures o it stains proteins gray (including unprotected
skin) and thus must be handled with caution

JASCHA KEAN ROSALES-LBH 11

 - Mode of action - copper-8-quinolinolate
o interact with amino acids, proteins, and o fungicide against Aspergillus, copper-silver
microorganisms ionization for Legionella disinfection
o However, OPA is a less potent cross-linking - organic mercurials
agent than glutaraldehyde. o as an antiseptic (e.g., mercurochrome) and
o This is compensated for by the lipophilic preservative/disinfectant (e.g., thimerosal)
aromatic nature of OPA DETERGENTS/SOAPS
o appears to kill spores by blocking the spore - surfactants interact with the lipid in the cell membrane
germination process and with the surrounding water
PERACETIC ACID - increases the surface tension
- AKA peroxyacetic acid - Example: Quaternary ammonium (Quats or
- Has rapid action against all microorganisms. Zephiran)
- Advantages ETHYLENE OXIDE
o harmful decomposition products - for sterilization of heat-sensitive equipment
o enhances removal of organic material - Most effective cold sterilization technique
o leaves no residue.
CRYSTAL VIOLET (GENTIAN VIOLET)
o remains effective in the presence of organic
matter - skin antiseptic
o sporicidal even at low temperatures - binding of + charged dye molecule to the – charged
- Disadvantages PO4 groups of nucleic acid
o can corrode copper, brass, bronze, plain MALACHITE GREEN
steel, and galvanized iron - In LJ medium, it kills other bacteria except M.
o can be reduced by additives and pH tuberculosis
modifications.
o It is considered unstable, particularly when MEDIUM USE COMMENTS
diluted Blood agar For most fastidious Tryptic soy agar with 5%
o for example, a 1% solution loses half its bacteria sheep blood added
strength through hydrolysis in 6 days, use. Type O Allows differentiation of
whereas 40% peracetic acid loses 1%–2% of hemolysis/hemolytic
its active ingredients per month pattern
- Mode of action Chocolate Enriched medium for Supplies X and V
o it denatures proteins, disrupts the cell wall agar Haemophilus and factors.
permeability, and oxidizes sulfhydryl and Neisseria Incubate in increased
sulfur bonds in proteins, enzymes, and other CO2
metabolites
- Uses NOTE:
o To chemically sterilized medical, surgical and Preferred blood for the preparation of BAP
dental equipments 1st – sheep
PHENOLICS 2nd – Horse
- Fist germicide used by Lister 3rd – Rabbit
- Derivatives of phenol
- Phenolics are absorbed by porous materials, and the Human blood is not preferred because it contains nonspecific
residual disinfectant can irritate tissue. inhibitors
- depigmentation of the skin was reported to be caused - Citrate: inhibit grown of β-hemolytic streptococcus
by phenolic germicidal detergents - Dextrose: alter type of hemolysis
- Mode of action Blood is added at 40 – 50 C
o In high concentrations,
▪ acts as a gross protoplasmic poison, c. Enrichment culture medium
▪ penetrates and disrupts the cell wall - Increase the number of pathogens that are
▪ precipitates cell proteins. outnumbered by non-pathogens.
o Low concentrations, - Extends the lag phase of non-pathogens while
▪ inactivation of essential enzyme decreasing the lag phase of pathogens
systems and leakage of essential - Used to propagate the growth of certain group of
metabolites from the cell wall organisms
METALS AS MICROBICIDES - They contain specific nutrients
- silver - Examples:
o used for prophylaxis of conjunctivitis of the o Gram negative broth (GN)
newborn, topical therapy for burn wounds, o Selenite broth
and bonding to indwelling catheters o Tetrathionate broth
- Zeolite ceramic coatings with Zn and Ag o Alkaline peptone water
o Inactivation of bacteria on stainless steel o Buffered charcoal yeast extract agar
- silver, iron, and copper o Thioglycolate
o used for environmental control, disinfection of 1. Selective culture medium
water, or reusable medical devices Selects the growth of a particular organism at the same time
inhibiting the growth of other organisms
- Example:

JASCHA KEAN ROSALES-LBH 12

 o MSA 1. Ideally, specimens should be transported to the laboratory
o SSA within 30 minutes of collection
2. Differential culture medium a. For anaerobic bacteria, transport should not take more than
- Distinguishes group of organisms based on cultural 10 minutes
characteristics b. For CSF samples, it should be transported within 15 minutes
- Example: 2. All specimen containers should be leak-proof
o Emb 3. All specimens should be transported within sealable, leak-
o HEA proof, plastic bag; specimen
3. Transport Medium bags should be marked with biohazard label
- Used when there is an anticipated delay in bringing the 4. Use of appropriate special preservatives or holding media for
specimen into the laboratory transport of specimen
- it can hold the specimen within 30m minutes delayed for more than 30 minutes is important in ensuring
o Pike Media – S. pyogenes organism viability
o JEMBEC – Neisseria a. Ex. Changes in temperature – Nesseria meningitidis
o Alkaline Salt Transport Medium – V. cholera Changes in pH – Shigella spp
o Glycerol Saline Transport Media – Salmonella
typhi Basis for specimen rejection
o Mishulow‘s Medium – Bordetella - Unlabeling or improperly labeled specimen
4. Culture Medium for Sensitivity or Susceptibility testing - Improper collection site
- Prolonged transit (over 2 hours without preservation)
- Used to demonstrate the antibiotic resistance or - Improper
sensitivity or an organism to different antibiotics - Specimen received in formalin
o Mueller Hinton Agar (MHA) – for fastidious - Saliva instead of sputum
organisms - Insuffiecient quantity
▪ pH – 7.2 – 7.4 Fragile organism
▪ depth – at least 4 mm - Salmonella, shigella
o Middlebrook 7H 10, 7H 11 – for - Streptococcus pneumoniae
Mycobacterium - Anaerobes
o Wilkin-Chalgren Agar – for anaerobic - Haemophilus influenzae
bacteria - Chlamydia
BIOCHEMICAL MEDIUM - Neisseria meningitis
- Used to demonstrate biochemical activities of bacteria - Neisseria gonorrhea
that is useful in their identification SPECIMEN COLLECTION, TRANSPORT AND
- Example: PROCESSING
o TSIA Objectives
o Citrate • To isolate and identify microorganisms from clinical
o IMViC (opposite reaction) specimens rapidly
o LIA • To provide the physician with information concerning
SPECIMEN COLLECTION AND HANDLING the presence or absence of microorganisms that may
Specimen collection and transportation are critical be involved in the infectious disease process
considerations, because any results • To determine the susceptibility of microorganisms to
the laboratory generates is limited by the quality of the specimen antimicrobial agents
and its condition on arrival in UNIVERSAL PRECAUTIONS FOR HEALTHCARE
the laboratory PROFESSIONALS
Careful skin preparation before procedures such as blood - Use appropriate barrier protection to prevent skin and
cultures and spinal taps mucous membrane exposure
decreases the chance that organisms normally present on the o Gloves
skin will contaminate the ▪ For touching blood and body fluids,
specimen mucous membranes or non-intact skin
General Considerations ▪ For handling items soiled with blood
1. Specimens should be collected during the acute (early) ▪ When performing venipuncture and other
phase of an illness (or within 2-3 days for viral infection) vascular access procedures
2. If possible, specimens should be collected before ▪ Should be changed after each contact with
antibiotics are administered patient
3. Swabs are generally poor specimens (except nares and o Masks, eyeware/faceshields
throat specimens) if tissue and needle aspirates can be ▪ Worn during procedures that are likely to
obtained generate droplets of blood
o For anaerobes, aspirates are preferred to o Gowns/aprons
swabs ▪ Worn during procedures that are likely to
4. The specimen collected should be a representation of the general splashes of blood and other body
diseased area fluids
5. The quantity of the specimen should be sufficient enough - Frequent hand washing
for diagnostic testing - Take precautions to prevent injuries caused by
needles, scalpels and other sharp instruments or
Specimen Transport devices

JASCHA KEAN ROSALES-LBH 13

 o To prevent needlestick injuries, needles should
not.
▪ be recapped
▪ purposely bent or broken by hand
▪ Removed from disposable syringes
o Sharps should be placed in puncture-resistant
containers for disposal
- Minimize mouth to mouth resuscitation
- Healthcare workers with exudative lesions or weeping
dermatitis should refrain from all direct patient care and
from handling patient care equipment
- To clean up spills:
o Put on PPE
o Wipe up excess material with disposable towels
and place in a container for sterilization
o Disinfect the area with either a commercial EPA-
approved germicide or household bleach
(sodium hypochlorite)
▪ Dilution: 1:100 (smooth surfaces) 1:10
(porous or dirty surfaces), and should not
be more than 24 hours old
o If spills have broken glasses
▪ Cover spill with disposable toweling
▪ Saturate with commercial germicide or
1:10 household bleach solution
▪ Stand for 10 minutes then cleans as above
SPECIMEN
- represents a portion of human material that is to be
studied
o It should represent the infectious disease
o Quantity should be adequate
o avoid contamination
o Forwarded promptly to the clinical lab
o obtain specimen before administration of
antimicrobial agents
SPECIMEN COLLECTION
Aseptic technique
- procedures to prevent unwanted microorganisms from
contaminating the clinical specimen
Swab
- Sterile rayon or dacron tipped polystyrene applicator
Needle aspiration
- For blood and CSF
- A sample fluid is drawn into a sterile tube that has been
treated with heparin or potassium oxalate
Intubation
- Inserting of a tube into a body canal or hollow organ
- To collect specimen from the stomach
- Levin tube
Catheter
- Tubular instrument used for withdrawing or introducing
fluids from or into a body cavity
- Urine specimen:
o Hard catheter – if urethra is narrow or has
strictures
o French catheter – soft tube for single
specimen sample
o Foley catheter – for multiple samples over a
prolonged period
- Clean catch method if without a catheter
Sputum
- Collect using sputum cups
SPECIMEN HANDING
Specimen should be labelled properly
- Name
JASCHA KEAN ROSALES-LBH
- Registration number
- Location of patient
- Type and source of specimen
- Tests to be performed
TRANSPORT
- Speed is important
- Use appropriate transport media if needed
- Special precautions for the type of microorganism to be
isolated
- Example
o aerobes and anaerobes
BACTERIAL CULTURES
- transport at room temp unless otherwise specified
ABSCESS
• Tissue or aspirates are always superior to swab
specimens.
- Remove surface exudate by wiping with sterile saline
or 70% alcohol.
- Aspirate with needle and syringe. Cleanse rubber
stopper of anaerobic transport vial with alcohol; allow
to dry 1 min before inoculating
- Push needle through septum and inject all abscess
material on top of agar.
- If a swab must be used, pass the swab deep into the
base of the lesion to firmly sample the fresh border.
Transport time < 2 hours.
ANAEROBIC CULTURES
• Tissue or aspirates are preferred rather than swabs.
Fluid collections should be aspirated through
disinfected tissue or skin. For superficial ulcers, collect
material from below the surface (after surface
debridement or use a needle and syringe). Submit
specimens using anaerobic transport media:
- Anaerobic vial
o Cleanse rubber stopper with alcohol; allow to
dry 1 min before inoculation; push needle
through septum and inject specimen on top of
agar
o Anaerobic jar
- Sterile container
o may be used for tissue if transported to the
microbiology lab immediately (add drops of
sterile saline to keep small pieces of tissue
moist).
- Anaerobic swab
o swab specimens are suboptimal, aspirate
preferred.
o Deliver all specimens to the laboratory
immediately after collection.
- Anaerobic flora is prevalent on mucosal surfaces of
the oral cavity, upper respiratory, gastrointestinal, and
genital tracts; specimens collected from these sites
should not ordinarily be cultured for anaerobic bacteria.
The following is a list of specimens that are likely to be
contaminated with anaerobic normal flora and are NOT
routinely accepted for anaerobic culture.
o Throat or nasopharyngeal swabs
o Gingival or other intraoral surface swabs
o Expectorated sputum
o Sputum obtained by nasotracheal or
endotracheal suction
o Bronchial washings
o Voided or catheterized urine
o Vaginal or cervical swabs
14
 o Gastric and small bowel contents (except for
"blind loop" or bacterial overgrowth
syndrome)
o Feces (except for specific etiologic agents
such as C. difficile and C. botulinum)
o Rectal swabs - Surface swabs from ulcers
and wounds (collect material from below the
surface)
o Material adjacent to a mucous membrane that
has not been adequately decontaminated
BLOOD
a. Adult – Cleanse skin
- Use a side-to-side motion to scrub the site for a full 30
sec; allow site to dry completely (at least 30 sec) before
venipuncture. Do not touch site after prep.
- Draw 20 mL of blood and inoculate each bottle with 10
mL of blood. Do not vent or overfill bottles. Adding low
(<8 mL) or high (>10 mL) volumes may adversely affect
the recovery of organisms.
- Transport time <2 h.
- For adults with a suspected bloodstream infection
(BSI), collect two initial sets of blood cultures
sequentially from separate phlebotomy procedures
followed by a third and a fourth set at 4–6-hour intervals
(will detect >99% of BSIs).
o Three sets of blood cultures collected within a
24-hour period will detect 96.9 - 98.3% of
BSIs.
o A single set of blood cultures to detect BSIs in
adults is inadequate (only 73% sensitivity)
o two sets of blood cultures will allow detection
of 87.7-89.7% of BSI episodes.
• If patient is allergic to chlorhexidine, prep site with a
povidone iodine swab stick applied in concentric circles
(start at center).
o Allow to dry at least 1 min before
venipuncture.
o If patient is allergic to iodine, cleanse site with
70% alcohol for 60 sec.
b. Pediatric – Prepare skin and bottles as for adult.
BONE MARROW ASPIRATE
- Prepare puncture site as for surgical incision. Inoculate
blood culture or Isolator (lysis-centrifugation) tube.
o Transport time <2 hours.
o Routine bacterial culture of bone marrow is
rarely useful.
BURN
- Clean and debride burn.
o Place tissue in sterile screw-cap container
o Transfer aspirates to a sterile container.
o These are processed for aerobic culture only.
▪ Quantitative culture may or may not
be valuable.
o A 3 to 4 mm punch biopsy specimen is
optimum when quantitative cultures are
ordered.
▪ Cultures of surface samples can be
misleading.
CATHETER TIPS
- Only intravascular catheter tips from pediatric patients
and peritoneal dialysis catheters are routinely accepted
for culture.
o Send 5 cm of distal tip in sterile screw–cap
container Transport time <15 min.
JASCHA KEAN ROSALES-LBH
o Foley catheters are not accepted for culture
since growth represents distal urethral flora.
CEREBROSPINAL FLUID (CSF)
- Aseptically collect CSF from a lumbar puncture into
sterile tubes
o Send second tube (>3 mL) to the Microbiology
Laboratory.
o Transport time <15 min.
o Cerebrospinal fluid for bacterial culture should
never be refrigerated.
DECUBITUS ULCER
- A swab is not the specimen of choice. Cleanse surface
with sterile saline. Submit tissue or aspirate
inflammatory material from the base of the ulcer in a
sterile tube or anaerobic system.
o Transport time <2 hours.
EAR
a. Inner ear
- Tympanocentesis should be reserved for complicated,
recurrent, or chronic persistent otitis media.
- For intact eardrum, clean ear canal with soap solution
and collect fluid via syringe aspiration. Submit in sterile
container.
- For ruptured eardrum, collect fluid on flexible shaft
swab via an auditory speculum. Transport time <2
hours.
b. Outer ear
- Use moistened swab to remove any debris or crust
from ear canal.
- Obtain sample by firmly rotating swab in outer canal.
- For otitis externa, vigorous swabbing is required
o surface swabbing may miss streptococcal
cellulitis.
EYE
a. Conjunctiva
- Sample each eye with separate swabs (premoistened
with sterile saline) by rolling over conjunctiva. When
only one eye is infected, sampling both can help
distinguish indigenous microflora from true pathogens.
b. Corneal scrapings
- Collected by ophthalmologist. Using sterile spatula,
scrape ulcers and lesions; inoculate scraping directly
onto media (BHI with 10% sheep blood, chocolate, and
inhibitory mold agar). Prepare 2 smears by rubbing
material onto 1-2 cm area of slide. Transport time <15
min.
c. Vitreous fluid
- Prepare eye for needle aspiration of fluid. Transfer fluid
to sterile tube. Transport time <15 min
GENITAL
- Cultures for Neisseria gonorrhoeae should be collected
with a sterile swab and inoculated directly to a Jembec
plate
o place white tablet in hole of Jembec plate to
provide CO2 enriched atmosphere, close top
of the plate tightly and place in ziploc bag
provided,
o keep at room temperature,
o deliver to lab as soon as possible).
o If a Jembec plate is unavailable, an aerobic
culturette swab may be used if transported to
laboratory immediately.
15
a. Endocervical
- Remove cervical mucus with swab and discard.
- Insert a second swab into endocervical canal and
rotate against walls.
- Allow time for organisms to absorb onto the swab
surface.
b. Urethral
- Collect urethral specimens at least 1 h after patient has
urinated.
- Insert small swab 2-4 cm into urethral lumen, rotate,
leave for 2s to facilitate absorption.
RESPIRATORY, LOWER
- Transport time <2 hours.
a. Bronchoalveolar lavage or brush, endotracheal aspirate
- Collect fluid in a sputum trap
- transfer to leak-proof container for transport in
pneumatic tube system, place brush in sterile container
with 1 mL sterile saline.
b. Sputum, expectorated
- Patient should rinse mouth and gargle with sterile
water prior to collection
- instruct patient to cough deeply. Collect specimen in
sterile transport containers
c. Sputum, induced
- Have patient brush gums and teeth
- then rinse mouth thoroughly with sterile water. Using a
nebulizer, have the patient inhale 20-30 mL of 3 to 10%
sterile saline. Collect sputum in sterile container.
RESPIRATORY, UPPER
- Transport time ≤2 hours.
a. Oral
- remove oral secretions and debris from surface of
lesion with a swab.
- Use a second swab to vigorously sample lesion,
avoiding normal tissue.
- Superficial swab specimens should not be submitted.
- Tissue or needle aspirates are preferred.
b. Nasal swabs
- Insert a sterile swab into the nose until resistance is
met at the level of the turbinates (approximately 1-2 cm
into one nostril).
- Rotate the swab against the nasal mucosa for 3 sec.
- Apply slight pressure with a finger on the outside of the
nose to ensure good contact between swab and inside
of nose.
- Using the same swab, repeat for the other nostril
c. Sinus aspirates
- Aspirate with needle and syringe.
- Cleanse rubber stopper of anaerobic transport
transport vial with alcohol
- push needle through septum and inject specimen on
top of agar.
d. Throat
- Routine throat cultures will be processed only for
growth of ß-hemolytic Streptococcus species
- arrange for provision of special media if culture for
other organisms (Corynebacterium diphtheria,
Neisseria gonorrhoeae) is desired.
- Do not obtain throat samples if epiglottis is inflamed, as
sampling may cause serious respiratory obstruction.
- Sample the posterior pharynx, tonsils, and inflamed
areas with a sterile swab.
STERILE BODY FLUIDS (OTHER THAN CSF)
a. Transport fluid to laboratory in sterile, leak-proof
container (BD Vacutainer, no additive, yellow top) or
anaerobic transport vial.
JASCHA KEAN ROSALES-LBH
b. Cleanse rubber septum of container with 70% alcohol.
Allow septum to dry for 1 min before inoculating.
c. Disinfect overlying skin with iodine or chlorhexidine
preparation. Obtain specimen with needle and syringe.
Push needle through septum of transport container and
inject fluid.
d. Amniotic and culdocentesis fluids should always be
transported in an anaerobic system. Agar in anaerobic
vial should be clear before inoculation; inject fluid on
top of agar.
e. Submit as much fluid as possible. NEVER submit a
swab dipped in fluid. NEVER inject fluid into swab
container.
f. One aerobic blood culture bottle inoculated at bedside
(up to 10 mL) is highly recommended provided
adequate sample is available.
- If blood culture bottle is inoculated, submit separate
aliquot in anaerobic vial or sterile container for
preparation of cytocentrifuged Gram stain and
inoculation of solid media (allows quantitation, aids in
culture interpretation).
g. Transport time ≤15 min, room temperature
STOOL
- Submit 10-20 g in sterile container.
- Transport time ≤1 hour. Refrigerate if transport is
delayed.
- Stools are cultured to isolate bacterial causative agents
of diarrheal illness; Salmonella, Shigella,
Campylobacter, and Shiga toxin producing E. coli.
- Routine stool culture includes EIA for Shiga toxin from
E. coli.
- Cultures for Yersinia are performed by special request.
- Stools for C. difficile toxin detection must be
transported to the laboratory immediately or
refrigerated if transport is delayed.
- Surveillance cultures may be ordered on Bone Marrow
transplant and other immunocompromised patients to
detect overgrowth of normal flora by Staph aureus,
yeast or a gram-negative bacillus.
TISSUE
- Submit in anaerobic collection jar or sterile screw-cap
container
- add drops of sterile saline to keep small pieces of
tissue moist.
- Transport time <15 min.
URINE
- Collect 1-10 mL of urine in a sterile specimen container
- Transport urine specimens to the microbiology
laboratory or refrigerate within 30 minutes.
- Refrigerated specimens should be delivered to the lab
as soon as possible, and may be rejected if not
received within 24 hours of collection.
a. Midstream clean catch method:
- Patients should be instructed to wash hands prior to
collection and offered exam gloves.
o Female patients should be instructed to sit on
toilet with legs apart and spread labia with one
hand. First void in toilet and then, continuing
to void, hold specimen container in
"midstream" to collect sample.
o Male patients should be instructed to retract
foreskin if uncircumcised. First void in toilet
and then, continuing to void, hold specimen
container in "midstream" to collect sample.
16
Straight catheter - Lactose – lactose fermenters will produce colonies
- Thoroughly cleanse the urethral opening with soap and with various shades of red, non-lactose fermenters
water. Rinse area with wet gauze pads. Aseptically produce colorless colonies
insert catheter into the bladder. After discarding initial HEKTOEN ENTERIC AGAR
15 to 30 mL of urine, collect urine for submission in a - To increase yield of Salmonella and Shigella Species
sterile container. relative to other microbiota
Indwelling catheter - High bile salt – inhibits G(+) bacteria and retards the
- Clamp catheter below port and allow urine to collect in growth of many coliform strains
tubing.
ENRICHMENT MEDIA
- Disinfect the catheter collection port with 70% alcohol.
• Addition of blood, serum or extracts to tryptic Soy Agar
- Use needle and syringe to aseptically collect 5-10 mL
• For fastidious bacteria
freshly voided urine though catheter port. Transfer to
• To isolate bacteria from CSF, pleural fluid, sputum and
sterile container. Do not collect urine from collection
wound abscess
bag.
Ileal conduit
BLOOD AGAR
- Remove the external device and discard urine within - - + citrated blood for variable hemolysis
device. 3 HEMOLYTIC PATTERNS
- Gently cleanse the stoma with 70% alcohol followed by α hemolysis
povidone-iodine swab stick - greenish to brownish halo around the colonies
- Using sterile technique, insert a double catheter into (Streptococcus gordonii and S. pneumoniae
the cleansed stoma, to a depth beyond the fascial β hemolysis
level, and collect the urine into a sterile container. - complete lysis; clearing effect around colonies (S.
- Use of a double catheter helps to minimize aureus, S. pyogenes)
contamination of the specimen with skin flora. γ hemolysis
ISOLATION OF PURE BACTERIAL CULTURES FROM - no change in medium (S. epidermidis, S.
SPECIMEN saprophyticus)
SELECTIVE MEDIA CHOCOLATE AGAR
• Prepared by the addition of specific substances to a - Made from heated blood
culture medium - Provides growth factors
• Permit growth of one group of bacteria while inhibiting - Haemophilus influenzae, Neisseria gonorrhea
others CHARACTERISTIC MEDIA
SALMONELLA-SHIGELLA AGAR (SS) • Used to test bacteria for particular metabolic activities,
- used to isolate Salmonella and Shigella species products or requirements
- Its bile salt mixture inhibits many groups of coliforms UREA BROTH
- Both Salmonella and Shigella produces colourless - urease
colonies because they are unable to ferment lactose - Some enteric bacteria are able to break down urea to
- Lactose fermenters produce pink colonies CO2 and NH3 in the presence of urease
MANNITOL SALT AGAR (MS)
- Used for the isolation of staphylococci TRIPLE SUGAR IRON SUGAR AGAR (TSIA)
- 7.5% salt - inhibits growth of many groups of bacteria - Contains glucose, lactose and sucrose, ferrous
- Mannitol – for differentiating pathogenic from non- ammonium sulfate and sodium thiosulfate
pathogenic staphylococci since pathogenic staph - For the ID of enteric organisms by their ability to attack
ferments mannitol the sugars and liberate sulfides
BISMUTH SULFITE AGAR CITRATE AGAR
- Used for isolation of Salmonella typhi - sodium citrate
- For food specimens and stool o serves as sole source of carbon
- S. typhi reduces sulfite to sulfide - Ammonium phosphate
- (+) black colonies with metallic sheen o sole source of nitrogen
DIFFERENTIAL MEDIA - To differentiate enteric bacteria on the basis of citrate
• The incorporation of certain chemicals may result in utilization
diagnostically useful growth or visible change in the LYSINE IRON AGAR (LIA)
medium after incubation - To differentiate bacteria that can either deaminate or
EOSIN METHYLENE BLUE AGAR (EMB) decarboxylate the amino acid lysine
- Differentiate between lactose and non-lactose - Lysine
fermenters (E. coli – lactose fermenter, produces dark o permits enzyme detection
colony with metallic sheen; S. typhi – appear colorless - Ferric ammonium citrate
- Also contain 2 dyes – eosin and methylene blue o for detection of H2S detection
MACCONKEY AGAR - Sodium thiosulfate
- For the selection and recovery of Enterobacteriaceae SULFIDE INDOLE MOTILITY AGAR (SIM)
and related gram-negative rods - 3 tests
- Bile salt and crystal violet – inhibits G (+) and some o Production of sulfides
fastidious G (-) o Formation of indole (metabolic product from
tryptophan utilization)
o Motility

JASCHA KEAN ROSALES-LBH 17

 - Used for the differentiation of enteric organisms • for Mycobacterium species
Escherichia coli
- Negative for H2S, Positive for Indole, Undetermined PRINCIPLE
motility - designed for bacteria whose cell wall contains long
Staphylococcus aureus chain fatty acids (mycolic acid)
- Negative for H2S, Negative for Indole, Undetermined o mycolic acid renders the cells resistant to
motility decolorization
Salmonella arizonae o Acid fast organisms may be Gram (+)
- Positive for H2S, Negative for Indole, Positive for
REAGENTS
motility
Enterobacter aerogenes Carbol Fuchsin
- Negative for H2S, Negative for Indole, Positive for - initial stain
motility Acid Alcohol
Proteus vulgaris - decolorizer
- Positive for H2S, Positive for Indole, Positive for - HCl + ethyl alcohol
motility Methylene Blue
STAINING TECHNIQUES FOR LIGHT MICROSCOPY - counter stain
- or Malachite Green
GRAM’S STAINING
- Hot Method – Ziehl - Neelsen
• a principal stain
o uses steam as a mordant
• most clinically significant bacteria are detected except:
- Cold Method – Kinyoun Modification
o intracellular bacteria
o uses phenol or tergitol as mordant
o bacteria that lack cell wall
NOTE:
o bacteria with insufficient dimensions to be
resolved by light (i.e. spirochetes) Acid Fast Bacilli
• provides preliminary diagnosis for initial treatment - stains RED or PINK
Non-Acid Fast Bacilli
PRINCIPLE
- stains BLUE or GREEN
- Gram (+) bacteria have thicker peptidoglycan layer (40)
with numerous teichoic acid cross-linkages than that of
G (-) (1or 2)
o teichoic acid prevents decolorization
o G (+) bacteria may lose CW integrity by:
▪ antibiotic treatment
▪ old cells
▪ use of autolytic enzymes
RULE
1. All cocci are Gram (+) except:
- Neisseria
- Branhamella
- Veilonella
2. All bacilli are Gram (-) except:
- Bacillus
- Clostridium
- Corynebacterium
- Erysipelothrix
- Lactobacillus
- Listeria
- Mycobacterium
REAGENTS
Crystal Violet
- initial stain
Gram’s Iodine
- mordant
95% Alcohol
- decolorizer
Safranin
- counter stain
NOTE:
Gram (+)
- stains BLUE / VIOLET
Gram (-)
- stains RED / PINK

ACID FAST STAINING

• other commonly used stain for light microscopy

JASCHA KEAN ROSALES-LBH 18

 ANTIMICROBIAL CHEMOTHERAPY - PBP‘s (penicillin binding proteins)
- Treatment of diseases through chemical compounds o site in the cell wall of bacteria where the beta-
- 17th century lactam ring binds to
• Drugs have been used for the treatment of infectious - Examples:
diseases o Penicillins – penicillin, ampicillin, piperacillin
• Example: o Cephalosporins – cefalozin, cefuroxime,
o Quinine for malaria ceftriaxone
o Emetine for amoebiasis o Monobactams – aztreonams
PAUL EHRLICH o Carbapenems – imipenem, meropenem
GLYCOPEPTIDES
- The science of chemotherapy began with Paul Ehrlich
- He is the father of chemotherapy - Vancomycin
- Discovered salvarsan or arsphenamine or 606 o binds to precursors of cell wall synthesis
- Contributions: o Usually ineffective against G- bacteria
o He formulated the principles of selective - Bacitracin
toxicity o inhibits the recycling of certain metabolites
o Recognized the specific relationships required for maintaining the peptidoglycan
between microbial pathogens and drugs layer
o Development of drug and drug resistance o It is toxic and thus for topical use only
o The role of combined therapy INHIBITORS OF CELL MEMBRANE FUNCTION
ANTIBIOTICS POLYMYXIN B AND COLISTIN
- Chemotherapeutics of microbial origin - Disrupt cell membrane
- Sources of antibiotics - Leads to leakage of macromolecules and ions
o From fungi or bacteria essential for cell survival
o Chemically prepared - More active against G- bacteria
CHARACTERISTICS OF ANTIBIOTICS INHIBITORS OF PROTEIN SYNTHESIS
- Selective toxicity AMINOGLYCOSIDES
o Implies that a drug is harmful to a parasite - Binds to protein receptors on the organisms 30S
without being harmful to the host ribosomal unit
- Kill or inhibit the growth of pathogens - Ex. Gentamycin, tobramycin, amikacin, netilmicin,
- Cause no allergic reaction in the host streptomycin and kanamycin
- Be stable when stored in solid or liquid form - For G+ and G- bacteria
- Remain in specific issues in the body long enough to - Not for anaerobes
be effective MACROLIDE-LINCOSAMIDE-STREPTOGAMIN (MLS)
- Kill the pathogen before they mutate and become
GROUP
resistant to it
CLASSIFICATION OF ANTIBACTERIAL AGENTS - Binds to receptors on the bacterial 50S ribosomal
subunit
BROAD V. NARROW SPECTRUM ANTIBIOTICS
- Ex. Erythromycin, azithromycin, clarithromycin,
- Narrow spectrum antibiotics clindamycin
o Kills only a limited group of organisms OXAZOLIDINONES
o G+ or G-
o Ex. Vancomycin, nalidixic acid, colistin - Synthetic drug
- Broad Spectrum antibiotics - Not expected to be affected by drug resistance
o For both G+ and G- bacteria mechanisms
o Ex. Ampicillin, chloramphenicol and - Ex. Linezolid
tetracycline CHLORAMPHENICOL
COMPETITIVE INHIBITORS - Inhibits addition of new amino acids by binding to the
- Inhibit growth of microorganism by competing with an 50S ribosomal subunit
enzyme needed to produce an essential metabolite - For G- and G+ bacteria
- Bacteriostatic TETRACYCLINS
o inhibit growth - Binds to the 30S ribosomal subunit
- Examples: sulfa drugs - Inhibits tRNA –amino acid complex from binding to the
MECHANISM OF ACTION ribosome
1. Inhibition of cell wall synthesis - For G- and G+ bacteria
2. Inhibition of cell membrane function INHIBITORS OF DNA AND RNA SYNTHESIS
3. Inhibition of protein synthesis (inhibition of translation FLUOROQUINOLONES
and transcription of genetic material) - AKA quinolones
4. Inhibition of nucleic acid synthesis (either DNA or - Derived from nalidixic acid
RNA synthesis) - Binds and interferes with DNA gyrase for bacterial DNA
5. Inhibition of enzyme activity supercoiling
INHIBITION OF CELL WALL SYNTHESIS - Ex. Ciprofloxacin and ofloxacin
BETA-LACTAM ANTIMICROBIAL AGENTS - For G- and G+ bacteria
- Antibiotics with the beta-lactam ring
- Binds enzymes involved in cell wall synthesis

JASCHA KEAN ROSALES-LBH 19

METRONIDAZOLE MECHANISMS BY WHICH BACTERIA BECOME
- Direct interaction between the activated drug and DNA RESISTANT TO ANTIMICROBIAL AGENTS
that results to the breakage of the DNA strand
- Activation happens at anaerobic conditions
- For anaerobic G- bacteria
RIFAMPIN
- Binds to the enzyme DNA –dependent RNA
polymerase and inhibits synthesis of RNA
- For G+ bacteria
INHIBITORS OF OTHER METABOLIC PROCESSES
SULFONAMIDES
- Binds to dihydropteroate synthase to disrupt the folic
acid pathway
- Folic acid PW is important for the precursors needed
for DNA synthesis
- For G+ and G- bacteria
TRIMETOPRIM
- Targets dihydrofolate reductase in the folic acid PW
- For G+ and G- bacteria
NITROFURANTOIN
- May have several targets involved in bacterial protein
and enzyme synthesis
- May directly damage DNA
- For G+ and G- bacteria causing UTI
DRUG RESISTANCE
- “Superbugs”
o Bacteria that become resistant to one or more
antimicrobial agents
- Multi-drug-resistant
o pathogens that are resistant to several
different antimicrobial agents
SUPERBUGS
MRSA (Methicillin-Resistant Staphylococcus Aureus)
MRSE (Methicillin-Resistant S. Epidermidis)
- Resistant to all anti-staphylococcal drugs except
vancomycin, synercid and zyvox
VISA (Vancomycin-Intermediate S. Aureus)
- Organism has developed resistance to the usual
dosage of vancomycin, thus higher dosages are
needed.
VRE (Vancomycin-Resistant Enterococcus Spp)
- Resistant to most anti-enterococcal drugs including
vancomycin
- Enterococcus causes nosocomial (hospital acquired)
UTI
MDRTB (Multi-Drug-Resistant Mycobacterium Tuberculosis)
- Resistant to all antitubercular drugs and combinations
of these drugs
MULTI-DRUG-RESISTANT strains of:
- pseudomonas spp.
- Salmonella spp.
- Shigella spp.
- Neisseria Gonorrhoeae
BETA-LACTAMASE PRODUCING strains of:
- Streptococcus pneumoniae
- Haemophilus influenzae

JASCHA KEAN ROSALES-LBH 20

 ANTIMICROBIAL SUSCEPTIBILTY TESTING
EMERGENCE OF ANTIMICROBIAL RESISTANCE
- Susceptibility testing of individual isolates is important
with species that may possess acquired resistance
mechanism
- Examples:
o Members of Enterobacteriaceae
o Pseudomonas species
o Staphylococcus species
o Enterococcus species
o Streptococcus pneumoniae
RATIONALE FOR PERFORMING SUSCEPTIBILITY
TESTING
- To confirm susceptibility to chosen empirical (simplest)
antimicrobial agents
- To detect resistance in individual bacterial isolates
NOTE:
Group A Antimicrobial
- First generation
- Always start with group a
COMMONLY USED SUSCEPTIBILITY TESTING
METHOD
BROTH DILUTION TESTS
- Also known as macrobroth or tube dilution method
- One of the earliest antimicrobial susceptibility
testing methods
- The antibiotic-containing tubes are inoculated with a
standardized bacterial suspension of 1-5x105CFU/mL
- Overnight incubation at 35 C or 37 C
- Tubes are examined for visible bacterial growth as
evidenced by turbidity
- The precision of this method is considered to be plus
or minus 1 two-fold concentration
o Due to manually preparing serial dilutions of
the antibiotics
- Advantages:
o Generate quantitative result (MIC)
- Disadvantages:
o Tedious- manual task of preparing the
antibiotics solutions of each test
o The possibility of errors in preparation of the
antibiotic solutions
o The relatively large amount of reagents and
space required for each test
NOTE:
MIC
- Minimum Inhibitory Concentration
MLC
- Minimum Lethal Concentration
- It can be adapted to miniaturization and
mechanization of the test
o Use microdilution trays – small, disposable
tray
o Standard trays contain 96 wells
o Each containing a volume of 0.1 mL that
allows approximately 12 antibiotics to be
tested in a range of 8 two-fold dilutions in a
single tray
- Inoculation of panels with the standard 5x105CFU/mL
is accompanied using a disposable device that
transfers 0.01 – 0.05 mL of standardized bacterial
suspension into each well of the microdilution tray or
by use if a mechanized dispenser
JASCHA KEAN ROSALES-LBH
- Following incubation, MICs are determined using
manual or automated viewing device for inspection of
each of the panel walls for growth
- The advantage of the microdilution procedure
includes:
o The generation of MICs
o The reproducibility and convenience of having
prepared panels
o The economy of reagents and space that
occurs due to the miniaturization of the test
o There is also assistance in generating
computerized reports if an automated panel
reader is used
- The main disadvantage:
o Some inflexibility of drug selections available
in standard commercial panels
ANTIMICROBIAL GRADIENT METHOD
- the antimicrobial gradient diffusion method uses the
principle of establishment of an antimicrobial
concentration gradient in an agar medium as a means
of determining susceptibility
- The Etest (bioMérieux AB BIODISK)
o Is a commercial version
o It employs thin plastic test strips that are
impregnated on the underside with a dried
antibiotic concentration gradient
o marked on the upper surface with a
concentration scale
- 5 or 6 strips may be placed in a radial fashion on the
surface of an appropriate 150-mm agar plate that has
been inoculated with a standardized organism
- After overnight incubation, the tests are read by
viewing the strips from the top of the plate.
- The MIC is determined by the intersection of the lower
part of the ellipse shaped growth inhibition area with
the test strip.
- The gradient diffusion method has intrinsic flexibility
by being able to test the drugs the laboratory chooses.
- Etest strips cost approximately $2-$3 each and can
represent an expensive approach if more than a few
drugs are tested
- This method is best suited to situations in which an MIC
for only 1 or 2 drugs is needed or when a fastidious
organism requiring enriched medium or special
incubation atmosphere is to be tested (eg, penicillin
and ceftriaxone with pneumococci)
- Generally, Etest results have correlated well with MICs
generated by broth or agar dilution methods.
- However, there are some systematic biases toward
higher or lower MICs determined by the Etest when
testing certain organism-antimicrobial agent
combinations.
- This can represent a potential shortcoming when
standard MIC interpretive criteria derived from broth
dilution testing are applied to Etest MICs that may not
be identical
DISK DIFFUSION METHOD (KIRBY-BAUER TEST)
- The disk diffusion susceptibility method is simple and
practical and has been well standardized.
- The test is performed by applying a bacterial inoculum
of approximately 1-2×108CFU/mL to the surface of a
large (150 mm diameter) Mueller-Hinton agar plate
- Up to 12 commercially-prepared, fixed concentration,
paper antibiotic disks are placed on the inoculated agar
surface.
21
 - Plates are incubated for 16–24hrs at 35°C prior to - Exceptions can occur if the antibiotic is highly
determination of results. concentrated in a body fluid such as urine, or if higher
- The zones of growth inhibition around each of the than normal dosages of the antibiotic can be safely
antibiotic disks are measured to the nearest administered (ex. Penicillins and Cephalosporins)
millimeter. - At times, the “intermediate” can also mean that certain
- The diameter of the zone is related to the susceptibility variables in the susceptibility test may not have been
of the isolate and to the diffusion rate of the drug properly controlled, and that the values have fallen into
through the agar medium. a “buffer zone” separating susceptible from resistant
- The zone diameters of each drug are interpreted using strains
the criteria published by the Clinical and Laboratory - It is important that the tables used for susceptibility test
Standards Institute (CLSI, formerly the National interpretations represent the most current criteria
Committee for Clinical Laboratory Standards or - The CLSI documents are reviewed and updated
NCCLS) or those included in the US Food and Drug frequently, usually once per year
Administration (FDA)-approved product inserts for - Used of old or outdated information from the original
the disks. editions of FDA-approved drugs labels or older CLSI
- The results of the disk diffusion test are “qualitative” in tables could represent a serious shortcoming in the
that a category of susceptibility (ie, susceptible, reporting of patients results.
intermediate, or resistant) is derived from the test
rather than an MIC.
- Advantages
o test simplicity
o does not require any special equipment,
o the provision of categorical results easily
interpreted by all clinicians,
o flexibility in selection of disks for testing.
o It is the least costly of all susceptibility
methods ($2.50-$5 per test for material)
- Disadvantages
o lack of mechanization or automation of the
test.
o not all fastidious or slow growing bacteria can
be accurately tested by this method
o the disk test has been standardized for testing
streptococci, Haemophilus influenzae, and
N. meningitidis through use of specialized
media, incubation conditions, and specific
zone size interpretive criteria
INTERPRETATION OF SUSCEPTIBILITY TEST
RESULTS
- both MIC values and disk diffusion zone diameters
must be interpreted using a table of values that relate
to proven clinical efficiency of each antibiotic and for
various bacterial species
KIRBY-BAUER TEST
- pharmacokinetic and pharmacodynamic data
- clinical studies results (including comparisons of MIC
and zone diameter with microbiological eradication and
clinical efficiency) obtained during studies prior to FDA
approval and marketing of antibiotic
SUSCEPTIBLE RESULT
- Indicates that the patient organism should respond to
therapy with that antibiotic using the dosage
recommended normally for that type of infection and
species
- An organism with a MIC or zone size interpreted a
“resistant” should not be inhibited by the
concentrations of antibiotic achieved with the dosages
normally used with that drug
INTERMEDIATE RESULT
- Indicates that a microorganism falls into a range of
susceptibility in which the MIC approaches or exceeds
the level of antibiotic that can ordinarily be achieved
and for which clinical response is likely to be less than
with susceptible strain

JASCHA KEAN ROSALES-LBH 22

 MICROBIAL ECOLOGY o Endoparasite
Ecology ▪ A parasite established within the
- The systematic study of the interrelationships that exist body of its host and produces an
between organisms and their environment infection
Microbial Ecology TYPE OF HOST
- The study of the numerous interrelationships between INTERMEDIATE HOST
microorganisms and the world around them - Harbors the asexual or immature stage of the parasite
- Involves: - First Intermediate host
o How microbes interact with other microbes o Harbor the larval stage
o How microbes interact with other organisms - Second Intermediate host
other than microbes o Harbors the infective stage
o How microbes interact with the non-living
DEFINITIVE HOST
world around them
SYMBIOTIC RELATIONSHIPS - Harbors the sexually mature form of the parasite
SYMBIOSIS RESERVOIR HOST
- Defined as the living together or close association of - Alternate host
two dissimilar organisms PARATENIC HOST
- Usually, two different species - Transport host
SYMBIONTS/SYMBIOTES OTHER RELATIONSHIPS
- Any microorganism that spends a portion or all of its NEUTRALISM
life associated with another organism of a different - A symbiotic relationship in which neither the symbiont
species is affected by the relationship
CATEGORIES OF SYMBIOSIS - A situation in which different microorganisms occupy
ECTOSYMBIOSIS the same ecological niche but have absolutely no effect
- One organism remains outside the other on each other
ENDOSYMBIOSIS SYNERGIST (SYNERGISTIC INFECTIONS)
- One organism is present within the other - Two or more microorganisms ―team up‖ to produce a
disease that neither could cause by itself
THREE TYPES OF SYMBIOTIC RELATIONSHIP
- ANUG
COMMENSALISM o Acute Necrotizing Ulcerative Gingivitis or
- Relationship in which one organism, the commensal, Vincent‘s disease or trench mouth
benefits while the other, the host is neither harmed nor o Caused by Fusobacterium, Actinomyces,
helped Prevotella spp
- Both the host and the commensal ―eat at the same - Bacterial vaginosis
table o Mobiluncus and Gardnerella spp
- E.g. Entamoeba coli and man INDIGENOUS MICROFLORA OF HUMANS
MUTUALISM - Also referred to as normal flora
- A symbiotic relationship that is beneficial to both - Includes all microbes that reside on or within that
symbionts person
- The mutualist and the host are metabolically - A fetus has no indigenous microflora, during and after
dependent on each other delivery, a newborn is exposed to many
- E.g. Lichens microorganisms from its mother food, air and
o association between ascomycetes (fungus) virtually everything that touches the infant
and certain genera of either green algae or CATEGORIES OF MICROORGANISMS
cyanobacteria RESIDENT
PARASITISM - Normally grow on or in the skin
- Branch of biology which deals the study of the TRANSIENT
phenomena of dependence of one living organism on - Those organisms that are temporarily present
another temporarily or permanently for the purpose of
FACTORS THAT AFFECT THE MICROFLORA OF
procuring food and shelter
- Parasite
THE SKIN
o the organism that lives in the body of another THE SKIN IS SUBJECTED TO PERIODIC DRYING
organism for survival - Lack of moisture drives many resident microbiotas into
- Host a dormant state
o an organism that harbors a parasite and - Scalp, ears, axillary areas, genitourinary and anal
provides physical protection and nourishment regions, perineum, palms have moisture that is
to the specific parasite sufficient to support a resident microbiota
- Categories of Parasites: THE SKIN HAS A SLIGHTLY ACIDIC PH (4-6)
o Ectoparasite - Due to the organic acids produced by normal
▪ Attached to the skin or temporarily staphylococci and secretions from skin oil and sweat
invades the superficial tissues of the glands
host‘s body - The acidic pH discourages colonization by many
microorganisms - Sweat contains a high concentration
of NaCl

JASCHA KEAN ROSALES-LBH 23

 - It makes the skin hyperosmotic and osmotically - The phagocytic action of the alveolar
stresses most microorganisms macrophages
THE SKIN CAN SECRETE CERTAIN INHIBITORY ORAL CAVITY
SUBSTANCES MOUTH
• It may be bactericidal and/or bacteriostatic - Organisms should resist the mechanical removal by
• Sweat glands adhering to surfaces like the gums and teeth
o release lysozymes (muramidase) - Mouth is colonized within hours after a human is born
o an enzyme that lyses S. epidermidis and Initial microbiota
other gram + bacteria by hydrolyzing the B – • Streptococcus
1,4 glycosidic bonds in the bacterial cell wall • Neisseria
peptidoglycan • Actinomyces
• Oil glands • Veillonella
o secrete complex lipids that may be partially • Lactobacillus
degraded by enzymes from As teeth grows
Propionibacterium acnes that can change it - Streptococcus parasanguis
to oleic acid that have strong antimicrobial - S. mutans
activity against gram negative bacteria and - S. salivarius – attaches to the buccal and gingival
some fungi epithelial surfaces and colonizes the saliva
MICROBES ON THE SUPERFICIAL SKIN EYE
• Staphylococcus epidermidis - Commensals of the Conjunctiva
• Aerobic corynebacteria o S. epidermidis
ORGANISMS THAT NORMALLY OCCUR IN THE o S. aureus
SCALP o Aerobic corynebacteria (diphtheroids)
• Pitysporum ovale o S. pneumoniae
• Pitysporum orbicularis - Cultures from the eyelids
MICROBES FOUND IN SKIN GLANDS - Branhamella catarrhalis
• Propionibacterium acnes - Escherichia
o normally harmless but is associated with acne - Klebsiella
vulgaris and comedones - Proteus
TRANSIENT FLORA FOUND AROUND ORIFICES - Enterobacter
- Neisseria
• Staphylococcus aureus
- Bacillus
o nostrils and perianal region
• Clostridium perfringens EXTERNAL EAR
o colonizes the perineum and thighs especially - Coagulase negative Staphylococci
those patients who suffer from diabetes - Corynebacterium
NOSE AND NASOPHARYNX - Less frequently seen
NOSE o Bacillus
o Microccocus
- S. aureus o Neisseria
- S. epidermidis - Occasionally seen
- Diphtheroids o Proteus
NASOPHARYNX o Escherichia
- Lies above the soft palate o Pseudomonas
- Streptococcus pneumoniae – non-encapsulated o Fungi
- Neisseria meningitidis – non-encapsulated o Aspergillus
- Haemophilus influenzae o Alternaria
OROPHARYNX o Penicillum
- Lies between the soft palate and the upper edge of the o Candida
epiglottis o Saccahromyces
o S. aureus STOMACH
o S. epidermidis - Most bacteria are killed due to the very acidic pH (2 –
o Alpha hemolytic streptococci (S. oralis, S. 3) of the gastric contents
gordinii, S. salivarius) - Stomach contains less than 10 viable bacteria per mL
o Diphtheroids of gastric fluid
o Branhamella catarrhalis - Streptococcus, Staphylococcus, Lactobacillus,
PALATINE AND PHARYNGEAL TONSILS Peptostreptococcus and Candida spp.
- Micrococcus SMALL INTESTINES
- Porphyromonas, Prevotella, and Fusobacterium - Duodenum
RESPIRATORY TRACT o Gram + cocci and rods
- The upper and the lower respiratory tract (trachea, o Contains few microorganisms because of the
bronchii, bronchioles, alveoli) do not have a normal combined influence of the stomach‘s acidic
microbiota juices and the inhibitory action of bile and
- Reasons: pancreatic secretions
- The continous stream of mucus generated by the
ciliated epithelial cells

JASCHA KEAN ROSALES-LBH 24

 - Jejunum MICROBIAL MECHANISMS OF PATHOGENICITY
o Enterococcus faecalis Pathogen
o Lactobacilli - A microorganism capable of causing a disease
o Diphtheroids - Able to overcome the host‘s defenses
o Candida albicans - Could be due to toxins, enzymes, capsules, etc…
- Ileum Pathology
o Anaerobic gram-negative bacteria - Scientific study of diseases (pathos – suffering; logy –
o Family Enterobacteriaceae study)
o The microbiota takes on the characteristics of ASPECTS OF PATHOLOGY
the colon microbiota
- Etiology
LARGE INTESTINES o study of the cause of disease
- Has the largest microbial population of the body - - Pathogenesis
Microbiota consists primarily of anaerobic, gram -, non- o development of the disease
sporing bacteria and gram +, spore forming and non- - Pathology
sporing rods o concerned with the structural and functional
- The vast majority of microorganisms are anaerobic changes brought about by diseases and its
FUNGI final effect
- Candida albicans DISEASE
PROTOZOANS - Occurs when an infection results in any change from
- Trichomonas hominis the state of health
- Entamoeba humanni - An abnormal state in which part or all of the body is not
- Endolimax nana properly adjusted or is not capable of carrying its
- Iodamoeba butschlii normal function
- Entamoeba coli - An infection may exist in the absence of any detectable
INITIAL RESIDENTS disease - Signs and symptoms of a disease
o Dolor
- Bifidobacterium genus o Calor
o found in breastfed infants because milk o Rubor
contains a disaccharide amino sugar that they o Tumor
require as growth factor o Functio laesa
- Lactobacillus
SYMPTOM
o found in formula-fed infants because the
formula lacks the required growth factor for - Some evidence of a disease that is experienced or
bifidobacterium percieved by the patient
GENITOURINARY TRACT - Subjective
- Ex: ache or pain. Tinnitus, blurred vision, nausea,
UPPER GENITOURINARY TRACT
dizziness, itching and chills
- Includes the kidneys, ureters and urinary bladder - Symptomatic disease (clinical dse)
- Usually free of microorganisms o patient experiences symptoms
LOWER GUT - Asymptomatic disease (subclinical dse)
- Distal urethra and external opening of the urethra o patient is unaware because he does not
o Neisseria spp. experience symptoms
o Staphylococci SIGN OF A DISEASE
o Streprococci - Objective evidence of a disease
o Enterococci - Ex: abnormal heart or breath sounds, blood pressure,
o Diphtheroids pulse rate, abnormal lab results, radiographs,
o Mycobacteria ultrasounds, CT scans
o Mycoplasma COMMUNICABLE DISEASE
o Enteric Gram – rods - An infectious disease that is transmissible from one
GENITALIA person to another
VAGINA - spreads from one host to another, either directly or
- Microflora varies with the stage of sexual development indirectly
- Puberty and following menopause - illness due to the transmission of the products of an
o Alkaline secretions: diphtheroids, etiologic agent or reservoir to a susceptible host
streptococci, staphylococci, and coliforms directly or indirectly
- Childbearing years - Ex: gonorrhea, chicken pox, measles, genital herpes,
o Acidic secretions (pH 4 – 5): lactobacilli, α – typhoid fever
hemolytic CONTAGIOUS DISEASE
o streptococci, staphylococci and yeasts - A communicable disease that is easily transmitted
- easily spread from one person to another
o illness due to direct transmission of etiologic
agent from reservoir to susceptible host
- Ex: influenza

JASCHA KEAN ROSALES-LBH 25

 INFECTION Latent infection
- Colonization by a pathogen - it may go from being symptomatic to asymptomatic and
- A person can be infected by a certain pathogen but not then sometime later go back to being symptomatic
have the infectious disease - Ex: Cold sores, genital herpes, shingles, syphilis
- caused by that pathogen due to the following reasons: ACCORDING TO EXTENT OF HOST INVOLVEMENT
o The microbe may land on an anatomical site Localized infection
where it is unable to multiply - Remain in one site or may spread
o Many pathogens must attach to specific - Ex: pimples, boils, abscesses
receptor sites before they are able to multiply Systemic infection
and cause damage - AKA generalized infection
o Antibacterial factors may be present at the - When the infection has spread throughout the body
site where the pathogen lands (lysozymes in - Happens when pathogens are not contained at the
saliva, sweat and tears) original site of infection
o The indigenous microflora may inhibit the - Ways in which pathogens are spread:
growth of the foreign microbe o Lymph
o The indigenous microflora may produce o Blood
antibacterial factors o Phagocytes
o The individual‘s nutritional and overall health Focal
status often influences the outcome of the - can arise from infections in the teeth, tonsils or
pathogen/host encounter sinuses
o The person may be immune to that particular - Bacteremia
pathogen o bacteria in the blood
o Phagocytes my engulf and destroy the - Septicemia
pathogen o bacteria in the blood actually multiplies
CLASSIFICATION OF INFECTIOUS DISEASES - Toxemia
ACCORDING TO HOW THEY BEHAVE IN THE HOST: o toxins in the blood
- Communicable disease - Viremia
- Contagious disease o viruses in the blood
- Non-communicable ACCORDING TO EXTENT OF INFECTION
o is not spread from one host to another Primary infection
ACCORDING TO FREQUENCY OCCURRENCE - first infection that often allows another organism to
Incidence appear on the scene
- frequency/ number of new occurrences of disease, Viral respiratory infection
injury or death, i.e. number of transitions from well to - destroys cilia of the respiratory tract
ill, from uninjured to injured, or from alive to dead in the Secondary infection
study population DURING THE TIME PERIOD BEING - caused by organisms following an initial or primary
EXAMINED infection
Prevalence o Bacterial pneumonia
- number of persons in a defined population who have a THE NATURAL HISTORY OF DISEASE
specified disease or condition AT A POINT IN TIME The Etiologic Agent
(usually the time a survey is done) - The causative agent of a disease
- Sporadic - Genetic/Intrinsic
o occasionally - Acquired/Extrinsic:
- Endemic - Physical
o a disease that is constantly present in a o radiation, increase or decrease in
population temperature
- Epidemic - Chemical
o if many people in a given area acquire a o lead, alcohol, mercury, chloroform
certain disease in a relatively short period of - Nutritional
time o malnourishment
- Pandemic - Infectious
o occurs worldwide o bacterial, viral, fungal, parasitic
ACCORDING TO SEVERITY OR DURATION OF A ENVIRONMENT
DISEASE - Involves the mode of transmission
Acute infections CONTACT TRANSMISSION
- has rapid onset, followed by a relatively rapid recovery Direct contact transmission
- Ex: measles, mumps, influenza - through physical contact between its source and a
Chronic infection susceptible host. No intermediate object is involved
- diseases that have an insidious (slow) onset and lasts (touching, kissing, sexual intercourse)
a long time. Indirect contact transmission
- Ex: tuberculosis, leprosy (Hansen‘s disease), syphilis - transmitted by means of non-living objects (fomites)
Subacute infections Droplet transmission
- disease having a sudden onset and then develop into - microbes are spread in droplet nuclei that travel only
a long-lasting disease short distances
- Ex: bacterial endocarditis (infection on heart)

JASCHA KEAN ROSALES-LBH 26

VEHICULAR TRANSMISSION Casual, acute or transient carriers
Waterborne transmission - harbor the pathogen for only a brief period of time
- spread by water contaminated with untreated or poorly (hours, days or weeks)
treated sewage Chronic carriers
Foodborne transmission - harbor the pathogen for long periods (months, years or
- transmitted by food that re incompletely cooked, poorly life)
refrigerated, or prepared under unsatisfactory PORTAL OF ENTRY
conditions MUCOUS MEMBRANES
Airborne transmission - respiratory, GIT and GUT, conjunctiva
- spread of agents of infection by droplet nuclei in dust SKIN
that travel more than one meter from the reservoir to
- cuts, abrasions, hair follicles, sweat gland ducts
the host
PARENTERAL
VECTORS
- microorganisms are deposited directly into the tissues
- (living things that carry pathogens from one host to
beneath the skin or into the mucous membranes when
another)
these barriers are penetrated or injured. Punctures,
Mechanical transmission
injections, bites, cuts, wounds, surgery, splitting due to
- passive transport of the pathogens on the insect‘s feet
swelling or drying
or other body parts
Biological transmission
SUSCEPTIBILITY OF THE HOST
- active transmission wherein the pathogen multiplies Depends on:
inside the arthropod • Pathogenicity of the organism
HOST • Non-specific and specific defense mechanism of the
- A susceptible host host
- responsible for the degree to which the individual can PORTAL OF EXIT
adapt to the stressors produced by the agent ACTIVE ESCAPE
- influenced by: - takes place when a pathogen actively moves to a portal
- genotype of exit and leaves the host
o nutritional status PASSIVE ESCAPE
o immune system - occurs when a pathogen or its progeny leaves the host
o social behavior in feces, urine, droplets, saliva or desquamated cells
THE PATHOGEN FACTORS
- the agent causes the disease PORTAL OF ENTRY
o Viruses
• Salmonella species – typhoid – swallowed
o Bacteria
o Protozoa • Staphylococcus aureus – pneumonia – respiratory
o Parasite route
o Protein o Furuncles – skin penetration
o Food poisoning – gastrointestinal
RESERVOIR
- site of natural environmental location in which the VIRULENCE
pathogen is normally found living the from which - intensity of the pathogenicity
infection of the host can occur o Ability to multiply in vitro
SOURCE o Example: Neisseria vs MTB
- the location from which the pathogen is immediately NUMBER OF PATHOGENS
transmitted to the host directly or indirectly - in general, more pathogens would mean more chances
- animate sources – humans and animals of infection
- inanimate sources – water, soil or food RESISTANCE OF THE HOST
- period of infectivity – the time during which the source STAGES OF A DISEASE
is infectious or disseminating the pathogen PERIOD OF INCUBATION
CARRIER
- The time interval between the actual infection and the
- an infected individual who is a potential source of first appearance of any signs and symptoms
infection for others - It is dependent on the virulence of the microorganism
4 TYPES OF CARRIERS and the resistance of the host
Active carrier • Factors that affect the length of incubation
- an individual who has an overt clinical case of a o Overall health and nutritional status of the
disease host
Convalescent carrier o The immune status of the host
- an individual who has recovered from the infectious (immunocompetent or immunocompromised)
disease but continues to harbor large numbers of o The virulence of the pathogen
pathogen o The number of pathogens that enter the body
Healthy carrier PRODROMAL PERIOD
- an individual who harbors the pathogen but is not ill
An incubatory carrier - A relatively short period that follows the period of
- an individual who is incubating the pathogen in large incubation
numbers but is not yet ill - Characterized by early, mild symptoms of disease,
such as general aches and malaise

JASCHA KEAN ROSALES-LBH 27

 - Patient feels “out of sorts” ACTIVE ENTRY
PERIOD OF ILLNESS/CLINICAL PERIOD/FASTIGIUM/ACME
- Attach to the epithelial surface
- During this period, the disease is most active - Thru the production of lytic substances that alter the
- Host exhibits overt signs and symptoms such as fever, host tissue by
chills, sore throat, muscle pain, photophobia etc o Attacking the ground substances and
- communicable diseases are most easily transmitted at basement membranes of integuments and
this time intestinal linings
PERIOD OF DECLINE (DEFERVESCENCE) o Degrading carbohydrates-protein complexes
- Signs and symptoms of the disease subsides between cells or on the cell surface
- The fever decreases, and the feeling of malaise (glycocalyx)
diminish o Disrupting the cell surface
- Patient is vulnerable to secondary infections PASSIVE ENTRY
PERIOD OF CONVALESCENCE - Not related to the pathogen itself
- The person regains strength and the body returns to its o Small breaks, lesions or ulcers in a mucus
pre-disease state membrane that permit initial entry
- Recovery has occurred o Wounds, abrasions or burns
- Longer for viral respiratory diseases o Arthropods vectors that create small wounds
VIRULENCE while feeding
- The degree or intensity of pathogenicity o Tissue damage caused by other organisms
3 CHARACTERISTICS THAT DETERMINES o Existing eukaryotic internalization pathways
VIRULENCE ATTACHMENT
INVASIVENESS - Pathogen attaches to some tissues within the body
- Adherence
- The ability of the organism to spread to adjacent or
o Due to adhesins
other tissues
INFECTIVITY MULTIPLICATION
- The pathogen may multiply in one location of the body,
- The ability of the orgnaims to establish a focal point of
resulting in a localized infection
infection
- The pathogen may multiply throughout the body
PATHOGENIC POTENTIAL
INVASION/SPREAD
- The degree that the pathogen causes morbid
- Term commonly used to describe the entry of bacterial
symptoms
into host cells
- Toxigenicity
o Ability to produce toxins, chemical EVASION OF HOST DEFENSES
substances that will damage the host and
produce disease DAMAGE TO HOST TISSUES
LETHAL DOSE (LD50), INFECTIVE DOSE (ID50) - Damage maybe so extensive as to cause the death of
- Used to measure virulence the patient
- Refers to the dose that will either kill or infect 50% of VIRULENCE FACTORS
an experimental group of hosts within a specific period - Attribute that enables pathogens to attach, escape
Some organisms are better able to cause disease destruction and cause disease
- 10 shigella cells can cause shigellosis - They are phenotypic characteristics
- 100-1,000 cells of salmonella are needed to cause o Dictated by organism genotype
salmonellosis
- S. pyogenes ADHERENCE DESCRIPTION
o Some can cause scarlet fever, while some FACTORS
produce flesh eating enzymes Filamentous Causes adherence to erythrocytes
DETERMINANTS OF INFECTIOUS DISEASE Hemagglutinin
To induce an infectious disease, a pathogen must be able to: Fimbriae Filamentous structures that help attach
- Initially be transported to the host bacteria to solid surface
- Adhere to, colonize, or invade the host Glycocalyx A layer of exopolysaccharide fibers with a
- Multiply (grow) or complete its life cycle or in the host (Capsule) distinct outer margin that surrounds many
- Initially evade host defense mechanism cells
- Possess the mechanical, chemical or molecular ability It inhibits phagocytosis and aid in adherence
to damage the host
STEPS IN THE PATHOGENESIS OF INFECTIOUS Lectin Any carbohydrate-binding protein or
DISEASES glycoprotein of non-immune origin
ENTRY Ligand A low molecular weight molecule that exhibits
specific binding to a complementary site on a
- Penetration of the skin and mucus membranes
high molecular weight molecule such as
- Inoculation of the pathogen into bodily tissues by an
proteins
arthropod
- Inhalation Mucus Gel The glycoprotein layer or mucopolysaccharide
- Ingestion layer of glycosaminoglycan covering animal
- Introduction of the pathogen into the GUT cells mucosal surfaces
- Introduction directly into the blood

JASCHA KEAN ROSALES-LBH 28

Pili Filamentous structures that bind prokaryotes Immunoglob Streptococcus cleaves IgA into Fab
together for the transfer of genetic material ulin A pneumoniae and Fc fragments
protease
Receptors Complementary binding sites that bind Lecithinase Clostridium spp. destroys the
specific ligands or adhesins lecithin(phosphatidylch
S Layer The outermost regularly structured layer of oline) component of
cell envelope of some archaeobacteria and plasma membranes,
eubacteria that may promote adherence to allowing the pathogen
surfaces to spread
Leukocidins Staphylococci, Cause degranulation
Slime A tenacious bacterial film that is less compact Pneumococci, of lysosomes within
than a capsule Streptococci leukocytes, which
Teichoic Acid Cell wall components in gram positive decreases host
& Lipoteichoic bacteria that aid in adhesion resistance
Acid Also kills leukocytes
Porins Salmonella inhibits leukocyte
MICROBIAL PRODUCTS INVOLVED IN PATHOGEN typhimurium phagocytosis bt
DISSEMINATION IN A MAMMALIAN HOST activating the
adenylate cyclase
PRODUCT ORGANISM MECHANISM OF system
INVOLVED ACTION Protein A Staphylococcus located on cell wall.
Coagulase Staphylococcus Coagulates the aureus IgG binds to protein A
aureus fibrinogen in plasma. by its Fc end, thereby
The clot protects the preventing
pathogen from complement from
phagocytosis and interacting with bound
isolates it from other IgG
host defenses Streptokinas Group A, C & G A protein that binds to
Collagenase Clostridium spp. breaks down collagen e streptococci, plasminogen and
that forms the (Fibrinolysis, Staphylococci activates the
framework of Staphylokina production of plasmin,
connective tissues se) thus digesting fibrin
allows the pathogen to clots, this allows the
spread pathogen to move from
Deoxyribonu Group A Streptococci, lowers viscosity of the clotted area
clease (with Staphylococci, exudates, giving the
Ca and Mg) Clostridium perfingens pathogens more
mobility
Elastase and Pseudomonas Cleaves laminin
Alkaline aeruginosa associated with
protease basement membranes
Exotoxin B Group A Streptococci, degrades proteins
(cysteine Streptococcus
Protease) pyogenes
Hemolysins Staphylococci, lyse eryhtocytes,
Streptococci, E. coli, causing anmenia and
C. perfingens weakened host
defenses, make iron
available for microbial
growth
Hyaluronida Group A, C & G hydolyzes hyaluronic
se streptococci, acid, a constituent of
Staphylococci, intercellular groumd
Clostridia substance that
cements cells together
and renders
intercellular spaces
amenable to passage
by a pathogen
H2O2 and Mycoplasma spp. produce as metabolic
NH3 Ureaplasma spp. waste.
they are toxic and
famage epithelia in
respiratory and
urogenital systems

JASCHA KEAN ROSALES-LBH 29

 IMMUNOLOGY LACRIMAL APPARATUS
- The branch of microbiology that deals with the study of - Protects the eyes
body defenses against invading microorganisms - A group of structures that manufactures and drains
IMMUNE RESPONSE tears
- Involve complex interactions among many different - Contains enzymes (lysozymes)
types of body cells and cellular secretions - Flushing action of tears
- Types: SALIVARY GLANDS
o Specific - Produce saliva that help to dilute the number of
o Non-specific microorganisms and wash them from the surface of the
SUSCEPTIBILITY teeth and the mucous membrane of the mouth to
- Vulnerability or lack of resistance prevent colonization by microbes
RESISTANCE CILIARY ESCALATOR
- The ability to ward off disease thru the body defenses - Covers the cell of the mucous membrane or the lower
2 TYPES OF RESISTANCE respiratory tract
NON-SPECIFIC RESISTANCE - It propels inhaled dust and microorganism, that have
become trapped in mucus, upward toward the throat
- refers to all body defenses that protect the body from
EPIGLOTTIS
any kind of pathogens
o resistance that is not acquired through - Covers the larynx during swallowing and prevent
contact with an antigen microorganisms from entering the lower respiratory
o innate tract
SPECIFIC RESISTANCE (IMMUNITY) URETHRA
- refers to defenses (antibodies) against specific - Cleansed by the flow of urine to prevent microbial
microorganisms Susceptibility colonization in the urinary tract
LINES OF BODY DEFENSES VAGINAL SECRETIONS
FUNCTION - Move microorganisms out of the female body
1. Some defenses are designed to keep out some CHEMICAL FACTORS
microorganisms SEBUM
2. Other defenses remove the microorganism if they do - Composed of fatty acids which inhibit the growth of
get in certain pathogenic bacteria and fungi
3. Other combats them if they remain inside LYSOSYME/MURAMIDASE
NON-SPECIFIC RESISTANCE - Present in perspiration which helps maintain body
1. First line of body defense: temperature
o Skin - Eliminates certain waste and flush microorganisms
o Mucous membranes from the surface of the skin
2. Second line of body defense GASTRIC JUICE
o Phagocytosis
- Mixture of HCl, enzymes and mucus that produce a
o Complement system
very high acidity of gastric juice
FIRST LINE OF BODY DEFENSE - The acidity destroys bacteria and most bacterial toxins
MECHANICAL FACTORS/PHYSICAL BARRIERS TRANSFERRINS
SKIN
- Iron-binding protein in blood that inhibits bacterial
- Closely packed cells, continuous layering and growth by reducing the amount of available iron
presence of keratin, provides a formidable barrier to SECOND LINE OF DEFENSE
the entrance of microorganisms
RETICULOENDOTHELIAL SYSTEM
- few microbes are capable of penetrating the skin
- involves mononuclear phagocytic cells
- due to the presence of dry, acidic pH and fatty acids,
- flushing action of lymph fluid
alcohols
- sloughing removes attached bacteria PHAGOCYTOSIS
- SALT - Is the ingestion of a microorganism or nay particulate
- cool temperature matter by a cell (phagocyte)
MUCOUS MEMBRANE MECHANISM OF PHAGOCYTOSIS
CHEMOTAXIS
- Line the gastrointestinal, urinary and reproductive
tracts - The chemical attraction of phagocytes to
- Inhibit the entrance of many microorganisms but they microorganisms
offer less protection than the skin ADHERENCE
- in the respiratory tract and GIT - Is the attachment of the phagocytes plasma
- mucus traps microbes membranes to the surface of the microorganisms of
- microorganisms are constantly driven upwards by cilia their foreign materials
- rapid shedding of cells - It is mediated by opsonins through the process of
- MALT opsonization
- enzymes are also secreted in the GIT - Opsonization
o coating of a microorganism with certain
plasma proteins (opsonin) that promote the

JASCHA KEAN ROSALES-LBH 30

 attachment of the microorganisms to the - IL-1 induces the hypothalamus to release
phagocytes prostaglandin that sets the hypothalamus‘ thermostat
INGESTION at a higher temperature, thereby causing fever
- Occurs when the plasma membrane of the phagocytes - During fever, blood vessels constrict, rate of
extends projections called pseudopods that engulf that metabolism increases and shivering occurs, all of
microorganism which raise the body temperature
DIGESTION SIGNIFICANCE OF FEVER
- IL-1 steps up the production of T-lymphocytes
- Takes place when phagosomes (membrane containing - High temperature intensifies the effect of interferon
the ingestion antigen) fuses with the lysosome of the - Inhibits growth of some microorganisms by decreasing
phagocytes with subsequent release of the proteolytic the amount of iron available to them
enzymes with digest the antigen - Helps body tissues to repair quickly since the increase
WHITE BLOOD CELLS OR DERIVATIVES OF WBC in body
WHITE BLOOD CELLS - temperature speeds up the body‘s reaction
- Increase in number during bacterial infections THE COMPLEMENT SYSTEM
(leukocytosis) - A defensive system of serum proteins that participate
Neutrophils in lysis of foreign cells, inflammation, and phagocytosis
- Highly phagocytic and motile TWO PATHWAYS OF ACTIVATION
- Active in the initial stage of an infection Classical pathway
Basophil - activated by an immune complex
- Release substance known as histamine Alternative pathway
- Histamine - activated through direct interaction with a bacterium
o important in inflammation and allergic
CONSEQUENCES OF COMPLEMENT ACTIVATION
responses
Eosinophil Opsonization
- Produce toxic proteins against certain parasites such - Coating of antigen to facilitate phagocytosis
as helminthes Inflammation
Monocytes - Complement casues basophils and mast cells to
- Not actively phagocytic in blood, but when they enter release
the body tissues and mature, they become - histamine and other substances that stimulates the
macrophage inflammatory reaction
INFLAMMATION Cytolysis
- A defensive response against microbial infection, - Due to the destruction of plasma membrane causing
physical and/or chemical agents the cellular contents to break out
- Release of monokines, IL – 1 and TNF - The activated components of the complement proteins,
- Results to vasodilation causing edema at the with C9 proteins possibley playing a key role, attacks
infection site with the accumulation of plasma the invading cell‘s membrane and produce circular
o Fibrin limits the spread of microbes lesion called transmembrane channels, this leads to
- Integrins and selectins causes leukocytes to attach to loss of ions and eventual cytolysis
blood vessels and promote their movement across the INTERFERONS
blood vessels going to the irritant (chemotaxis) - Classes of similar antiviral proteins produced by certain
- Chemokines also induce chemotaxis animal cells after viral stimulation
5 SIGNS OF INFLAMMATION o Produced by virus infected cells
• Rubor – redness o It interfered with viral multiplication
o Produced by fibroblasts in connective tissues,
• Calor – heat
by lymphocytes and by other leukocytes
• Dolor – pain
o Interferons diffuse to uninfected neighboring
• Tumor – swelling cells to manufacture mRNA for the synthesis
• Functio laesa – loss of function of antiviral proteins (AVP)
FUNCTIONS OF INFLAMMATION o AVP are proteins which functions as enzymes
- To destroy infectious agents and remove its by- that disrups various stages of viral
products from the body multiplication
- To limit the effects of the injurious agent and its by- THIRD LINE OF DEFENSE
products by confining or walling it off
IMMUNE RESPONSE
- To repair or replace tissue damaged by the injurious
agent or its by products - Specific host defense mechanism that represents the
STAGES OF INFLAMMATION third line of defense against pathogens
- Vasodilation and increased membrane permeability IMMUNITY
- Phagocyte migration - Involves a specific defensive response when the host
o Margination is invaded by foreign organism or other foreign
o Diapedesis substances
- Repair - Results from the production of specialized lymphocytes
FEVER and antibodies
- A systemic response of the body to microbial invasion, LYMPHOCYTES
which is due to the release of IL-1 (endogenous - Not phagocytic but play a key role in specific immunity
pyrogen)

JASCHA KEAN ROSALES-LBH 31

 ANTIGENS milk), hout
- Organisms or substances that provide a response from Blood the
the immune system Lymp body,
ANTIBODIES blood
Placental yes No No No No
- Special glycoproteins produced by the lymphocytes to transfer
recognize, bind with, inactivate and destroy specific
Compleme yes yes No No No
microorganisms
nt
ACQUIRED IMMUNITY activation
- Refers to the protection and organism develops Function Enhance Especially Localiz Not Allergic
against certain types of microbes of foreign substances phagocyt effective ed know reactio
Types: osis, against protect n but ns;
- Acquired active immunity neutraliz microoga ion in prese possibl
o When a person is exposed to ed toxins nisms and mucos nce y
microorganisms/foreign substances and the and agglutinati al on c expulsi
immune system responds by producing viruses, ng surfac cells on or
specialized lymphocytes and specialized and antigens; es may lysis of
proteins called antibodies protects first indica protoz
- Acquired passive immunity fetus and antibody te oan
o Antibodies are transferred from one organism newborn produced functi parasit
to another. The recipient does not actively in on in es
synthesize the antibody but has them response initiati
provided by an outside source to initial on of
NATURALLY ACQUIRED IMMUNITY infection immu
- Obtained when a person is exposed to ann antigen in ne
the course of daily life and the immune system respo
responds by producing antibodies and specialized nse
lymphocytes
Types: IMMUNE SYSTEM
- Naturally acquired Active Immunity ORGANS OF THE IMMUNE SYSTEM
o Obtained when a person is exposed to 1. Lymph nodes
antigens in the course of daily life 2. Bone marrow
- Naturally acquired passive immunity 3. Spleen
o Involves natural transfer of antibodies 4. Liver
from the mother to infant 2 MAIN COMPONENTS OF THE IMMUNE SYSTEM
ARTIFICIALLY ACQUIRED IMMUNITY 1. HUMORAL IMMUNE RESPONSE
- Involves transfer of performed antibodies - Antibodies
Types o proteins secreted by plasma cells
- Artificially acquired active immunity (differentiated B-cells) as a reaction to
o Results from vaccination or immunization antigen stimulation
o Vaccines – specially prepared antigens o The system involves antibodies
▪ Live produced by B-cells in response to a
▪ Attenuated specific antigen
▪ Toxoid o Antibodies primarily defend against
▪ Recombinant bacteria, viruses and toxins in blood
- Artificially acquired passive immunity plasma and lymph
o Involves introduction of antibodies into the - Antigens
body o foreign substances that provide antibody
o Antibodies come from an animal or person formation
who is already immune to the disease 2. CELLULAR IMMUNE RESPONSE
SERUM - The system depends on T cells and does not involve
- Where antibodies can be found in an immune antibody production
animal or person - Cellular immunity is primarily a response to intracellular
ANTISERUM parasites, transplanted tissues and cancer cells
- Generic term from blood-derived fluids containing o T cells contain antigen receptors attached to
antibodies their surfaces which allow T cells to recognize
and react to an antigen
Characteri IgG IgM IgA IgD IgE
o Most effective against bacteria and antigen
stics
located within phagocytes or infected host
Location Blood, Blood, Secreti B-cell Bound
cells and against fungi, protozoan and
lymph, lymph, B ons surfac to mast
helminthes
intestine cell (tears, e, cells
o It also responds to transplanted tissue by
surface saliva, blood, and
mounting an immune response to reject it
mucus, lymph basoph
intestin ils
e, throug
TYPES OF T CELLS

JASCHA KEAN ROSALES-LBH 32

 - T helper cells (T4) cells
o necessary for B cell activation and T cell
dependent antigen
- Delayed hypersensitivity T cells (TD cells)
o provide protection against infectious agents and
cause inflammation associated with tissue
transplant rejection
- T suppressor cells (Ts)
o regulate immune response and help maintain
tolerance
- T cytotoxic cells (Tc)
o destroys target cells on contact
OTHER CELLS INVOLVED IN CELL - MEDIATED
IMMUNITY
- Killer cells
o attack antibody-coated target cells
- Natural killer cells
o attack and destroy target cells
MEDIATED IMMUNITY
- conferring immunity to microorganisms that are located
intracellular and therefore protetedted

JASCHA KEAN ROSALES-LBH 33

 MICROCOCCACEAE FAMILY - Uses Phenol Red as Indicator
Staphylococcus family o S. aureus
- mostly associated with human infection ▪ Yellow color
Micrococcus VJ (VOGEL JOHNSON)
- normal flora - Contains Mannitol and Tellurite
Planococcus o Staphylococcus aureus
Stomatococcus ▪ Black with Yellow Halo
DIFFERENTIAL PARAMETERS FOR o Staphylococcus epidermis
STAYPHYLOCOCCUS FROM MICROCOCCUS ▪ Black without Yellow Halo
STAPHYLOCOCCUS MICROCOCCUS BAIRD PARKER
Catalase + + - LiCl, Egg Yolk Emulsion & Tellurite
Aerobic Growth + + o Staphylococcus aureus
Anaerobic Growth + - ▪ Black with Clear Zone (lipolysis of egg)
OF Test Fermentative Oxidative o Staphylococcus epidermis
Modified Oxidase - + ▪ Black without clear zone
Benzidine - + CHAPMAN STONE
Lysostaphin S R
Sensitivity - Enhances pigmentation
Bacitracin R S - Contains mannitol and gelatin
Sensitivity o Staphylococcus aureus
▪ Yellow halo
STAPHYLOCOCCUS o Staphylococcus epidermis
- Carbohydrate fermenters ▪ No Yellow Halo
- Produces pigments from white to deep yellow on DNAse AGAR
culture media o Clearing around colony
- Some are non-fermenters
TYPES OF HEMOLYTIC PATTERNS
CAUSES: BETA HEMOLYSIS
o Suppuration and other pyogenic infections
- The complete lysis of red blood cells and hemoglobin
o Septicemia
- This results in complete clearing of the blood around
o food poisoning
the colonies
- With available hemolytic reactions
- With more than 30 species ALPHA HEMOLYSIS
IMPORTANT: - Partial lysis of red blood cell as and hemoglobin
- This result in a greenish-grey discoloration of the blood
- Staphylococcus aureus
around the colonies
- Staphylococcus epidermidis
- Staphylococcus saprophyticus GAMMA HEMOLYSIS
Rosenbach - No hemolysis
- Identified 2 species according to pigment produced - Results in no change in the medium
o Staphylococcus aureus (YELLOW) THE STAPHYLOCOCCACAE
o Staphylococcus albus (WHITE) STAPHYLOCOCCUS AUREUS
▪ Later renamed epidermidis - The most pathogenic due to its invasiveness or the
MORPHOLOGY: virulence factors (Pathogenic Determinants)
- Spherical about 1um arranged in clusters VIRULENCE FACTORS OF STAPHYLOCOCCUS
- Gram positive AUREUS
- Non-motile and non-sporeformers SURFACE COMPONENTS
CULTURE: CAPSULE
- Aerobic and microaerophilic - Antiphagocytic
- 37C, room temp. (pigment production) PROTEIN A
o S. aureus
▪ Gray to deep golden yellow - The most important virulence factor
o S. epidermidis - It inhibits complementary fixation and ADCC by binding
▪ Gray to white Fc portion of the IgG
- Requires 9% NaCl - Disrupts opsonization and phagocytosis
- Resistant to drying LIPOTEICHOIC ACID & TEICHOIC ACID
CULTURE MEDIA - Promote adherence to mucosal surfaces and
BAM (BLOOD AGAR MEDIA) persistence in tissues by binding to fibronectic
- Teichoic Acid
- S. aureus
o reaction with AB is used in the diagnosis of
o B- Hemolysis
endocarditis
- S. epidermidis
o Y- Hemolysis ENZYMES
MSA (MANNITOL SALT AGAR CATALASE
- 7.5% salt - Reduces phagocytic killing by converting H2O2 to H2O
- Fermentation - Differentiates staphylococcus from streptococcus
o Acid production

JASCHA KEAN ROSALES-LBH 34

COAGULASE - Characterized by rapid onset of fever, diarrhea,
- Helps encase infection by forming fibrin layer around desquamating rash, multisystem organ involvement,
abscess kidney failure, shock with generalized flushing of the
- Clots oxalated or citrated blood skin and mucus membrane
- Positive in staphylococcus aureus only - It could develop into shock and multiorgan failure
Degradative enzyme SUPPURATIVE INFECTIONS
- SUPERFICIAL ABSCESSES
Hyaluronidase - Involving sweat or sebaceus glands or hair follicles
- Duran Reynal Factor - Pimple/acne
- Hydrolyzes the hyaluronic acid present in intracellular o Infection of the sebaceous gland
ground substances of connective tissues - Folliculitis
- Facilitates spread of infection o Most superficial
Lipase o Infection of the hair follicle
- Lipid Hydrolyzing Enzyme o Stye
- Essential in the invasion of healthy cutaneous and ▪ Folliculitis of the hair follicle
subcutaneous tissues FURUNCLES OR BOILS
Protease
- Subcutaneous abscesses
- Protein Hydrolyzing Enzyme
- Result in the formation of a focal suppurative lesion
Staphylokinase
- Proteolytic Enzyme
CARBUNCLES
- Has fibrinolytic activity - Larger infections that can lead to bacteremia
- Antigenically and enzymatically different from - Similar to furuncles but has multiple foci and extend to
streptokinase the deeper layer of fibrous tissue
PENICILLINASE (B-LACTAMASE) - Limited to the neck and upper back where the skin is
thick and elastic
- Confers antibiotic resistance by clearing B-Lactam ring
of penicillins
DIFFUSE SKIN INFECTION
- It catalyzes the hydrolysis of the beta-lactam ring of - Impetigo: superficial, spreading, crusty skin lesion
some penicilins and cephalosporins generally seen in children
- Produces penicilloic acid - When crust are removed, a red weeping denuded
- Renders the antibiotic ineffective surface is exposed
- Also called Cephalosporinase Penicillinase DEEP LOCALIZED INFECTIONS
TOXINS Osteomyelitis
LEUKOCIDINS (CYTOLYTIC) - Acute and chronic infection of the bone marrow
- The Panton Valentin toxin Septic joint
- Damage and lyse leukocytes to limit host response - Arthritis resulting from acute infection of the joint space
- Release tissue damaging substances OTHER INFECTIONS
ENTEROTOXINS (A-E) - Acute endocarditis
- Acts as suprantigens - Septicemia and severe necrotizing pneumonia
- Responsible for gastrointestinal food poisoning - Meningitis
- Heat stable - Endocarditis
EXFOLIATIVE TOXINS (A,B) - Post operative infections
NOTE:
- Cause splitting of cell junctions (desmosomes) in
S. aureus is one of the most common causes of nosocomial
epidermis
infections.
- Responsible for scalded skin syndrome
Progression to septicemia is often a terminal event
TOXIC SHOCK SYNDROME
DISEASE CAUSED BY S. EPIDERMIDIS & S.
- Acts as superantigen SAPROPHYTICUS
- Promotes massive cytokine release - These bacteria normally benign colonizers of the skin
- Causes toxic shock syndrome and are less virulent than S. aureus, although their
DISEASES CAUSED BY S. AUREUS virulence factors are similar (except for their lack of
TOXIN MEDIATED DISEASES coagulase)
FOOD POISONING DUE TO ENTEROTOXIN (A-E) - S. epidermidis
- Characterized by nausea, vomiting and diarrhea o can adhere to artificial heart valves, vascular
developing 4 hours after eating contaminated food catheters, shunts, and prosthetic joints,
SCALDED SKIN SYNDROME colonizing the implant area and causing tissue
destruction mediated by degradative enzymes
- Due to exfoliative toxin
- S. saprophyticus
- Characterized by blister-like lesions widely
o Is a frequent cause of urinary tract infections
disseminated over the body
(UTI’s) in sexually active young women
- Large areas of desquamated epithelium but no
scarring
TOXIC SHOCK SYNDROME
- Dues to TSST-1(superantigen)
- Common among young women using tampons

JASCHA KEAN ROSALES-LBH 35

 DIAGNOSIS HOT-COLD HEMOLYSIS TEST
Clinical: - Staphylococcus aureus is the only staphylococcus
- exudates from s lesion species that has B-hemolysin and thus can damage
Laboratory: RBC’s at a cold temp. (4C)
- gram + cocci in clusters
TEST THAT DIFFERENTIATES THE COAGULASE
Specimen:
NEGATIVE STAPHYLOCOCCUS
- Surface swab, pus, blood, tracheal aspirates
NOVOBIOCIN SENSIVITY TEST
LAB IDENTIFICATION
TEST THAT DIFFERENTIATED STAPHY FROM - Resistant: S. saprophyticus
- Sensitive: S. epidermidis
STREPTO
HOW IS METHICILLIN RESISTANT S. AUREUS
CATALASE TEST
(MRSA) IDENTIFIED?
- Separates Staphy and Strepto being the former is
SCREENING TEST
catalase positive and negative for the latter
- The test makes use of 3% Hydrogen Peroxide - Salt agar plate
- Bubbling indicates a positive result GOLD STANDARD
- Considerations in catalase test - Detection of the mecA gene by PCR
o Use TSA (trypticase Soy Agar) DETECTION OF MRSA
o Use of blood agar is discovered because it has PHENOTYPIC DETECTION SYSTEMS
catalase that produces a false positive result
Deisc diffusion test
DIFFERENTIAL TEST FOR THE THREE SPECIES OF - Colony suspension prepared form 5 colonies and
STAPHY plated on Muller Hinton agar containing 2-4% NaCl at
COAGULASE TEST neutral pH
- The test is positive for S. aureus - Oxacillin disc (1ug) placed and incubated at 35C for 24
- Negative to S. epidermidis & S. saprophyticus hours
Method Slide Screening Test Tube Confirmatory Test - < 10mm is considered resistant
Reagent Human plasma Rabbit plasma - > 13mm is considered sensitive
Detects “Bound” coagulase “free” coagulase - For isolated with intermediates results
Result Clumping Clot formation o Test for mec A, PBP2a
(+): S. Aureus (+): s. Aureus o Cefoxitin disc test
(-): S. Saprophyticus & (-): s. Saprophyticus & s. o Oxacillin MIC test
s. Epidermidis Epidermidis o Oxacillin salt agar screen test may be performed
Considerations If negative confirm by False positive test caused - Any growth within the zone of inhibition indicates
more sensitive tube by citrate-positive oxacillin resistance
method organism that makes use HOW IS VANCOMYCIN RESISTANT S. AUREUS
of citrate and release IDENTIFIED?
calcium, so a clot occurs - Use Vancomycin Agar Plate
in the absence of - Macrolide resistance
coagulase o D-Zone Test with the use of clindamycin and
Citrate positive organism: erythromycin
Serratia, pseudomonas, HOST DEFENSES
enterococcus - It is a normal flora of the skin
- Antibodies may be produced but protein A prevents
OTHER TESTS EMPLOYED TO DIFFERENTIATE opsonization and the action of complement
COAGULASE POSITIVE STAPHYLO FROM EPIDEMIOLOGY
COGUALSE NEGATIVE STAPHYLO - Hospital strains have become penicillin resistant
DNASE TEST (MRSA, VRSA)
Reagent - Ubiquitous
- Nutrient agar base medium with Toluidine Blue CONTROL
o Result: Clearing of the dye Sanitary
- Nutrient agar based medium flooded with Dilute - Proper food handling
Hydrochloric Acid - Cleaning of hospitals
o Result: change in color from Blue Green to Pink Prevention
GROWTH ON MEDIA WITH HIGH SALT CONC. (7.5%) - Staphvax (experimental)
Mannitol Salt Agar Chemotherapeutic
- With phenol red as an indicator - Penicillin, Methicillin, Oxaxillin, Cephalosporin,
- S-100 Agar Vancomycin
Phenyl Ethyl Alcohol (PEA)
- For “contaminated” specimens such as stool
Chrom Agar
- Media containing chromogenic substrates that can be
formulated to provide specific colors to develop in
colonies of a particular genus or species
Chrom Agar MRSA

JASCHA KEAN ROSALES-LBH 36

 THE STREPTOCOCCACAE Streptolysin 0 and S
B. STREPTOCOCCUS - lyse blood cells and platelets; stimulates release of
lysosomal enzymes
- FORMS PAIRS OR CHAINS
- MOST GROUP A, B AND C ARE ENCAPSULATED TOXINS
- GRAM + AND NON-MOTILE COCCI Pyrogenic or Erythrogenic Exotoxins
- WITH DIFFERENT REACTIONS TO HEMOLYSIS - act as superantigens
CLASSIFICATION DISEASES CAUSED BY STREPTOCOCCUS
HEMOLYTIC PATTERN DY SHITH AND BROWN PYOGENES
TYPES OF DESCRIPTION EXAMPLE OF SUPPURATIVE INFECTIONS
HEMOLYSIS STREPTOCOCCUS Erysipelas
Alpha incomplete or partial Viridans streptococci - characterized by fever, headache, swollen lymph
hemolysis. S. pneumonia nodes and well demarcated swollen, erythematous
Green or Brown color area on the face
currounding the colony - Butterfly rash
Beta Complete hemolysis S. pyogenes - aka St. Anthony’s fire
clearing or colorless S. agalactiae Impetigo
zone surrounding the - Honey-crusted skin lesions over the face or trunk and
colony itching
Gamma Lack of hemolysis. Enterococcus - S. Pyogenes is the second most common cause of
No apparent change in infection
color or area Pharyngitis
surrounding the colony - Fever, inflamed throat with possible exudates
PHYSIOLOGIC PROPERTY BY BERGEY - Adherence of microbe to the pharyngeal epithelium
due to lipoteichoic acid covering the pili
Streptococcus Growth In Pyoderma
10C 37C 45C 6.5% Skimmed milk
NacL w/ Methylene - Multiple, 1- or 2- mm abscesses over the body
Blue - Commonly occurs during warm, humid weather and
Pyogenic - + - - - among children in close contact with each other
Enterococcus + + + + + Necrotizing fasciitis
Viridans - + + - - - Rapidly expanding area of erythema, bulla formation,
Lactic + + - - + and anesthesia around a wound which leads to tissue
ANTIGENIC PROPERTY BY REBECCA LANCEFIELD destruction
- This is accompanied by fever and other evidence of
- Characteristics of the c carbohydrate found on the cell systemic toxicity and possible shock and multisystem
wall organ failure
- Streptococcus is classified from A to S Cellulitis
- Precipitin reactions with specific antisera determines - A common, potentially serious bacterial skin infection.
arrangement - The affected skin appears swollen and red and is
- A-H and K-U typically painful and warm to the touch.
- A-D, F and G are pathogenic to man - Affects the skin on the lower legs, but it can occur in
GROUP A: STREPTOCOCCUS PYOGENES the face, arms and other areas.
VIRULENCE FACTORS OF S. PYOGENES - It occurs when a crack or break in your skin allows
SURFACE COMPONENTS bacteria to enter.
Capsule - Left untreated, the infection can spread to the lymph
- Anti-phagocytic nodes and bloodstream and become life-threatening.
M Protein - It is not usually spread from person to person
- Inhibits opsonization: immunogenic TOXIN-MEDIATED DISEASES
- main virulence factor Scarlet Fever
- appears as hair-like structures on the cell wall - Strawberry tongue and diffuse sandpaper-like
- Resists phagocytosis erythematous rash over the body within 24 to 48 hours
C. F. protein after the onset of streptococcal pharyngitis
- lipoteichoic acid: mediate adherence to - Rash fades in 5 - 7 days with desquamation
mucoepithelium by binding to fibronectin Streptococcal Toxic Shock Syndrome
ENZYMES - Cellulitis, shock, multisystem organ failure and
C5a peptidase generalized skin flushing
- reduces Inflammatory responses mediated by C5a AUTOIMMUNE SEQUELAE
DNase Acute Glomerulonephritis
- aids in bacteria spread by reducing viscosity of - Hematuria, red blood cell casts, proteinuria,
abscess material hypertension and periorbital edema with history of
Hyaluronidase recently treated skin infection
- promotes tissue destruction and bacterial spread Rheumatic Fever
Streptokinase - Fever, heart failure, migrating inflamed joints that are
- promotes bacterial spread into tissue by breaking down not symmetrically involved and a history of recent sore
blood cells throat

JASCHA KEAN ROSALES-LBH 37

LABORATORY IDENTIFICATION from diseases is less clear
Specimen than with exotoxin.
- Throat swabs, pus and lesion samples, sputum, blood Converted to antigenic. Not converted to toxoids
or spinal fluid Non-toxic toxoids by
Culture formalin, acid, heat, etc.
- On sheep blood agar, it appears as small, opalescent Toxoids are used to
colonies surrounded by a large zone of b -hemolysis immunize (tetanus toxoid)
Presumptive identification Highly toxic. moderately toxic.
- Bacitracin susceptibility test (taxo a) Fatal to animals in Fatal for animals in 10-100
- Makes use of a differential disk with 0.02 - 0.04 units. microgram quantities or less od microorganism
Of bacitracin Usually bind to specific Specific receptors not found
- The test is considered 100% accurate receptors on cells on cells
- Any zone of inhibition is reported as "presumptive beta Usually do not produce fever Usually produces fever in the
hemolytic streptococcus a by bacitracin” in the host host by release of
BIOCHEMICAL TEST FOR CONFIRMATION interleukin-1 and other
mediators
Pyr (pyrolidonase) hydrolysis test
- Which is based on presence of an aminopeptidase Frequently controlled by Synthesis is directed by
enzyme that degrades the substrate PRY (L- extrachromosomal genes extrachromosomal genes
pyrrolidonyl-b-naphthylamide) (Plasmids)
- The positive result is the production of red color is
indicated by p-dimethyl amino cinhamaldehyde EXOTOXINS
- Aside from grp a streptococci (b-hemolytic) CATEGORIES:
aerococcus, enterococcus and gernella (alpha or non- NEUROTOXINS
hemolytic) will also give a positive result to this test - Clostridium botulinum
- Clostridium tetani
TOXIGENICITY - Staphylococcus aureus
INFECTION - Vibrio cholerae
- Result from the pathogen’s growth and reproduction ENTEROTOXINS:
that often produce tissue alterations - V. cholera
INTOXICATION - E. coli
- Disease that results from the entrance of toxins into the CYTOTOXINS
body of the host - Shigella spp
TOXINS - Vibrio parahaemoliticus
- A specific substance, often a metabolic product of the - S. aureus
organism, that damages the host - C. difficile
- Can induce disease even in the absence of the Effects
pathogen that produced it - Inhibition of protein synthesis
TOXEMIA - Inhibition of nerve synapse functions
- Disruption of membrane transport
- The condition caused by toxins that have entered the
blood of the host
EXOTOXIN VS ENDOTOXIN DIPHTHERIAE TOXIN
EXOTOXIN ENDOTOXIN - Produced by Corynebacterium diphtheriae
Excreted by living cells. Integral part of the cell wall of - Action:
High conc. In liquid medium G- bacteria. o Causes ADP ribosylation of EF2 (elongation
Released on the bacterial factor needed for protein synthesis)
death and in part during - EF2 + NAD -> EF2-ADP-ribose + Nicotinamide + H+
growth. o EF2-ADP-Ribose – inactive substance that
May not need to be released stops protein synthesis
to have biologic activity. o
Produced by both G- & G+ Found only in G- bacteria - Result:
bacteria o Causes cell death and the respiratory tract
Polypeptides with a MW of Lipopolysaccharide o Lethal dose is 40 ng
10K – 900K complexes. o Pseudomembranous
Lipid A portion probably ▪ Composed of dead cells and debris
responsible for toxicity TETANUS TOXIN (TETANOSPASMIN
Relatively unstable, toxicity Relatively stable, with stand - By Clostridium tetani
often destroyed by heating heating at temp. above 60C - A neurotoxin
and temp. above 60C for hours without loss of - Small amount can be lethal
toxicity - Action:
Highly antigenic. Weakyly immunogenic. o Inhibits action of the neurotransmitter glycine
stimulates formation of high Antibodies are antitoxic and - Effect:
titer antitoxin. (Antitoxin protective. o Spasms occur because toxins inhibit the
neutralizes toxins) Relationships between inhibitory transmitter glycine
antibody titers and protection

JASCHA KEAN ROSALES-LBH 38

BOTULINUM TOXIN - Activation of clotting, forming microthrombi even in the
- By Clostridium botulinum absence of wound
- The most potent toxin known Result:
- Action: - Consumptive coagulopathy because all factors will be
o Inhibits of the action of the faciliatory used up
transmitter acetylcholine at the synapse - Bleeding
- Effect: - Platelets are important for vascular integrity
o Patient experiences paralysis like difficulty in FEVER
swallowing and breathing - Due to endogenous pyrogen
ENTEROTOXIN o Acts on the hypothalamus (thermoregulatory)
Heat labile o An IL-1
- By Escherichia coli causing diarrhea to the host HYPOTENSION
- Action: - Result to shock
o Toxin stimulates adenylate cyclase which - Bradykinin causes vasodilation
increases the amount of cyclic-AMP Erythrogenic toxin/Pyrogenic toxin
- Effect: - In Streptococcus pyogenes
o Hypersecretion of chloride ions - Similar to TSS toxin
Heat stable
- In bacillus cereus
- Guanylate cyclase is activated
PERTUSSIS TOXIN
- In Bordetella pertussis
- Causes whooping cough
- Promotes ribosylation of ADP and promotes
lymphocytosis
ANTHRAX TOXIN
- Bacillus anthracis
- Can exist in spores
- 3 factors
o Edema factor
▪ Swelling on the skin
o Protective antigen
o Lethal factor
▪ Causes cell death
TOXIC SHOCK TOXIN (TSS)
- In staphylococcus aureus and streptococcus
- Seen in woman using tampons
- Actions:
o Stimulates class II MHC protein which
activates Helper T cell which secretes IL-1 an
IL-2
- Effect:
o IL-1 is vasodilator. Blood pressure falls as a
result in the dilation of blood vessels resulting
to shock
ALPHA TOXIN
- In Clostridium perfringens
- A phospholipase/ lecithinase
- Also secretes collagenase, hyaluronidase, protease
and DNAse
- Action:
o Phospholipase destroys the cell membrane
- Effect:
o Cell death
EFFECTS OF ENDOTOXINS
- Complement cascade
o Activates the alternate pathway
- Macrophages activation
o For antibody production
DISSEMINATED INTRAVASCULAR COAGULATION
(DIC)
Action:
- Activates the Hageman Factor (F12)

JASCHA KEAN ROSALES-LBH 39