OVERVIEW: THE SCIENCE OF MICROBIOLOGY, ★ Viruses are not living organisms because

CELL STRUCTURE & CLASSIFICATION OF they do not follow the cell theory (they are not
BACTERIA cellular). REMEMBER: cells are the basic
structural unit of all organisms; all cells
Microorganisms come from pre-existing cells
● Refer to organisms that exist as single cells or cell
clusters
● It also includes viruses, which are MICROSCOPIC
but not cellular
● Also known as MICROBES

Microbiology
● mikros – “small” + bios – “life” + logia – “study of”
● Refers to the scientific study of microbes

FOUNDATION OF MICROBIOLOGY

Microbes – What comes to mind?

● Diseases
● Infections
● Epidemics VIRUS
● Food Spoilage
● Lack many of the attributes of the cells (i.e. ability to
★ Only 1% of all known bacteria cause human replicate)
diseases ● Vary in diameter from 15 to 300 nm
★ 95% of known bacteria are non-pathogenic ● Reproduction can be acquired only after infecting
another cell
Benefits to humans: ● Host-virus interactions
● Bacteria are primary decomposers. ● Differ from other microbes in at least two ways:
● They recycle nutrients back into the environment A. Simple and acellular (obligate intracellular
(sewage treatment plants) parasites)
● Microbes produce various food products B. Absence of both DNA and RNA in the same
(fermentation): cheese, pickles, sauerkraut, green virion
olives, yogurt, soy sauce, vinegar, bread beer, wine,
alcohol ● Virophages
- Viruses that infect other viruses
★ Microbes are used to produce antibiotics - Highly specific
● Ilosome (Erythromycin)
● Generally small and consist of:
★ Bacteria synthesize chemicals that our body - Nucleic acid molecule ─ Either DNA or RNA
needs but cannot synthesize. - Capsid
● Vitamin B (for neural development) ○ Protein coat that encloses the nucleic acid
● Vitamin K molecule
○ Sometimes enveloped by lipids, proteins
★ Recombinant DNA Technology and carbohydrates
● Gene Therapy ○ Proteins (frequently glycoproteins)
● Genetic Engineering determine specificity of host-virus
interaction
★ Bacteria can be manipulated to produce ○ Protects the nucleic acid
enzymes and proteins they normally would not ○ Facilitates attachment and penetration by
produce the virus
● Insulin – genetically derived
● Human Growth Hormone
● Interferon-antiviral proteins given to
patients with chronic hepatitis

★ Can carry out photosynthesis more than plants

can perform

___________________________________________

Cell Theory
1. All living organisms are composed of one or more
cells.
2. The cell is the basic unit of structure and
organization in organisms.
3. Cells arise from pre-existing cells.
 DOMAIN EXAMPLES

Domain Eukarya ● Animals

(Eukaryotic) ● Fungi
● Protozoans
● Helminths

Domain Bacteria ● Bacteria

(Prokaryotic)

Domain Archaea ● Contains organisms that can

live in extreme environments;
Not medically important

TERM DEFINITION

Prokaryotes ● pro – “before” + karyote –

“nucleus”
CLASSIFICATION OF MICROBES ● Domain Bacteria, Archaea

Hierarchical Levels of Taxonomy Eukaryotes ● eu – “true” + karyote – “nucleus”

● Genus - consists of species that differ from each ● Domain Eukarya
other in certain ways but are related by descent
● Species - group of closely related organisms that
breed among themselves
★ Note: of these ranks, the family, genus, and
species are most useful.
● Binomial nomenclature - uses the genus and
species names to identify each creature.

Three Domain of Life

● Bacteria
● Archaea
● Eukarya
 PROKARYOTE VS EUKARYOTE CELLULAR MICROBES
PROKARYOTE EUKARYOTE
● With true nucleus
Nuclear Absent Present ● Contain membrane-bound organelles, microtubules,
Membrane (nucleoid) (nucleus) and microfilaments
● Taxonomic groupings based on shared morphologic
Nucleoli Absent Present properties

❖ Algae
Site of ribosome Cytosol Nucleoli - all organisms that produce oxygen as a
production product of photosynthesis
- Many are unicellular while some may form
large multicellular structures
Presence of Absent Present
membrane–bound
organelles ❖ Protozoans
- unicellular, non-photosynthetic, with three
forms:
Number of 1 >1 ● Flagellates (flagella) – resemble algae
chromosomes and May not have chloroplasts
● Amoeboids (pseudopodia)
Site of ATP Cellular Mitochondria / ● Ciliated (cilia)
production membrane Chloroplast ● Sporozoa (non-motile)

❖ Fungi
Presence of Present Absent
● non-photosynthetic, grow as a mass of
peptidoglycan
layer branching, interlacing filaments (“hyphae”)
○ mycelium
● Evolutionary offshoot of protozoa
● The mycelial forms are called molds, while the
non-mycelial / unicellular forms are called
OVERVIEW OF THE CELLULAR MICROBES yeasts.
DOMAIN DOMAIN DOMAIN
EUKARYA BACTERIA ARCHAEA
CLASSIFICATION OF BACTERIA
Fungi Bacteria “Living on the
Multicellular, Unicellular, edge” organisms Criterion for Classification of Bacteria
eukaryotes prokaryotes residence in 1. Culture Media
extreme A. Nonselective Media
Helminths environments ● support the growth of many different
Multicellular, bacteria to cultivate as many species as
eukaryotes Not medically possible
important ● Give rise to numerous bacteria colonies
Protozoans ● Ex. Blood agar and chocolate agar
Unicellular,
eukaryotes

CLASSIFICATION REMARKS

Bacteria ● Unicellular
● Has cell wall formed by
peptidoglycan layers –
except Mycoplasma
● Has both DNA & RNA

Fungi ● Multicellular
● Has cell wall formed by chitin
● Has both DNA & RNA

Protozoan ● Unicellular
● Absence of cell wall
● Has both DNA & RNA
Helminth ● Multicellular
● Absence of cell wall
● Has both DNA & RNA
● From Kingdom Animalia
Arthropod ● Multicellular
● Has both DNA & RNA
● From Kingdom Animalia
B. Selective Media
● used to eliminate large numbers of
irrelevant bacteria in specimens
● Incorporation of an inhibitory agent that
specifically selects against the growth of
irrelevant bacteria
Examples:
➔ MacConkey agar - contains bile that
secretes for the Enterobacteriaceae
 ➔ CNA blood agar - selects
Staphylococci and Streptococci;
contains Colistin and Nalidixic Acid,
gram-positive bacteria

STEP PROCEDURES REAGENT

1 Primary Stain Crystal Violet

2 Mordant Iodine

3 Decolorizing agent Alcohol/acetone

4 Counterstain Safranin

C. Differential media
● some bacteria produce characteristic
pigments upon culture while bacteria can
be differentiated on the basis of their
complement of extracellular enzymes
● Enzyme activity detected as zones of
clearing surrounding colonies grown in
the presence of insoluble substrates
● Formulated to display a color change
when the growing of bacteria metabolizes
an ingredient
● Ex. Agar in a medium with RBC

CLASSIFICATION OF BACTERIA
GRAM (+) VS GRAM (-)
2. Bacterial Microscopy
● divides bacteria on the basis of fundamental
differences in the structure of their cell walls COMPONENT GRAM (+) GRAM (-)
CELLS CELLS

Peptidoglycan Thicker; Thinner

multilayer

Teichoic acids Yes No

Lipopolysaccharides No Yes

Periplasmic space No or small Yes

❖ Gram Staining
● also called Gram's method, is a method of
staining used to distinguish and classify
bacterial species into two large groups:
gram-positive bacteria and gram-negative
bacteria.
 Disinfection Eliminates many Application of
or all pathogenic rubbing
microorganisms, alcohol – to
except bacterial inanimate objects
spores,
on inanimate
objects

Antisepsis Refers to the Preoperative skin

application of prep
chemicals on the
surface of the Hand rubbing
HIGHLIGHTS IN THE HISTORY OF MICROBIOLOGY skin and mucous
membranes
HISTORICAL CONTRIBUTION
FIGURE
Sanitization Reducing Heating cutlery
microbial (e.g. at KFC!)
Elie ● Discovered the process of
contamination to
Metchnikoff phagocytosis
an acceptable
● Nobel Prize in Physiology or
“safe” level
Medicine 1908
(terminology used
in food safety)
Robert Koch ● Established experimental steps
for directly linking a specific
Degerming Physical removal Handwashing with
microbe to a specific disease
or Cleaning of soap and running
microorganisms by water
Louis Pasteur ● Disproved the theory of
using such things
spontaneous generation or
as soaps or
abiogenesis – the
detergents
hypothetical process by
which living organisms
develop from nonliving STERILIZATION TECHNIQUES
matter
● Developed the first vaccine
against rabies Autoclaving ● Exposure to steam (moist
● Developed pasteurization heat) at 121 Celsius under a
pressure of 15 lb/in2 for
Joseph Lister ● Performed surgery using 15-20 minutes
aseptic conditions using phenol
Ethylene oxide ● Gas used in hospitals for the
Anton Van ● Discovered animalcules under gas sterilization of heat-sensitive
Leeuwenhoek the light microscope materials (e.g. surgical
● Credited as the first person instruments and plastics)
to see microbes
Glutaraldehyde ● Chemical used to sterilize
respiratory therapy equipment,
Koch’s Postulates endoscopes, and hemodialysis
1. The microbe must be found in diseased but not equipment
healthy individuals
2. The microbe must be cultured from the diseased Filtration ● Use of filter composed of
individual nitrocellulose with pore size
3. The cultured microbe should cause disease of 0.22 mm
when introduced into a healthy organism ● Preferred method of sterilizing
4. The microbe must be reisolated from the solutions (with heat-sensitive
inoculated, diseased experimental host and components)
identified as being identical to the original
specific causative agent Sterilization by ● Uses dry heat at 180 Celsius
dry heat for 2 hours
● Primarily used for glassware
METHODS OF MICROBIAL CONTROL
PHYSICAL AGENTS USED IN DISINFECTION
METHOD DEFINITION EXAMPLE
Flash pasteurization ● Heating milk at 72 Celsius
Sterilization Destroys or Autoclaving for 15 seconds → rapid
eliminating ALL cooling
forms of microbial Exposure to ● Sufficient to kill the
life, including ethylene vegetative cells of
bacterial spores oxide gas (for milk-borne pathogens (e.g.
surgical Mycobacterium Bovis,
instruments) Salmonella, Streptococcus,
Listeria, Brucella), but not
Filtration (for most to sterilize milk
IV solutions)
 Arrangement of Spirochetes:
UV light ● Greatest antimicrobial
activity is achieved at 250
❖ Vibrio
to 260 nm ❖ Spirillum
● For disinfection of air in the ❖ Spirochete
OR
SHAPES OF BACTERIA
X Rays ● More powerful than UV
light
● Cannot sterilize due to the
lower amount of water in
spores

CHEMICAL AGENTS USED IN DISINFECTION

Chlorhexidine ● To disinfect the

surgeon’s hand prior to
surgery.

Iodophor ● To disinfect surgical site

prior to surgery

70% ethanol ● To disinfect skin prior to

venipuncture or
immunization
● To disinfect stethoscope

Thimerosal ● Used as a preservative

in vaccines

Hydrogen peroxide ● To cleanse wounds

Hypochlorite (bleach) ● To cleanup blood spill

from a patient with
hepatitis B, hepatitis C or
HIV

Benzalkonium ● To disinfect floor of OR

chloride Bacterial Cell Wall
● All bacteria have a cell wall composed of
peptidoglycan except Mycoplasma
BACTERIAL STRUCTURE ● Peptidoglycan = sugar backbone (glycan) +
peptide side chains (peptido) cross-linked by
Bacterial Shape and Size transpeptidase

Three shapes:
1. cocci (spheres)
Arrangement of Cocci:
● pairs (diplococci)
● chains (streptococci)
● clusters (staphylococci)
● sarcina (eight-membered cocci
● formation

BACTERIAL STAINING

NAME REMARKS

Giemsa stain ● Rickettsia, Chlamydia,

Trypanosomes, Plasmodium,
Borrelia, Helicobacter pylori
2. bacilli (rods)
Periodic ● Stains glycogen,
3. spirochetes (spirals) acid-Schiff mucopolysaccharides
● Leptospira (PAS) stain ● Used to diagnose Whipple
● Treponema disease (Tropheryma whipplei)
● Borreliella (the new name of Borrelia since ● PAS the sugar (glycogen)
2019)
 ESSENTIAL COMPONENTS OF BACTERIA
India Ink stain ● Uses negative staining
technique
● Cryptococcus neoformans COMPONENTS DESCRIPTION FUNCTION

Mucicarmine ● It directly stains the thick Cell Wall As described previously

stain polysaccharide capsule of
Cryptococcus neoformans with Cytoplasmic Lipoprotein Site of oxidative and
a red membrane bilayer ETC enzymes

Silver stain ● Coccidioides, Pneumocystis Ribosome RNA and protein Protein synthesis
jirovecii, Legionella,
Helicobacter pylori Nucleoid DNA Genetic material

Mesosome Invagination of Participates in cell

Fluorescent ● Used to identify bacteria, viruses, plasma division and
antibody stain P. jirovecii, Giardia lamblia, membrane secretion
Cryptosporidium
● FTA-ABS for syphilis Periplasm Space between Contains many
the plasma hydrolytic
membrane and enzymes, including
outer beta-lactamase
Bacteria NOT seen in Gram Stain
membrane

NAME REASON ALTERNATIVE

APPROACH

Mycobacteriae Too much mycolic ACID-FAST

acid (lipid) in cell STAIN
wall so dye cannot
penetrate Positive - red
Negative - blue

Spirochetes Too thin to see DARK FIELD

MICROSCOPY

Mycoplasma No cell wall NONE

Very small (SEROLOGIES)
NON-ESSENTIAL COMPONENTS OF BACTERIA
Chlamydiae Intracellular GIEMSA STAIN
→ INCLUSION
Lacks classic BODIES COMPONENT FUNCTION
peptidoglycan
because of ↓ Capsule Protects against phagocytosis
muramic acid
Pilus or Attachment (ordinary pili)
Legionella Poor uptake of red SILVER STAIN fimbria
counterstain Conjugation (sex pili)

Intracellular Glycocalyx Mediates adherence to surfaces

→ biofilms

Flagellum Motility
COMPONENTS OF BACTERIA
Spore Resistance to heat, and chemicals
Essential
● Refers to structures the bacterium cannot live Plasmid Genes for antibiotic resistance and
without. toxins
● It is inherited from the parents via the chromosome
itself by binary fission.
● Vertical transfer Capsules
● All bacterial capsules are composed of
Non-essential polysaccharide EXCEPT Bacillus anthracis
● Acquired from genetic mobile elements via special
genetic transfers (e.g. conjugation, transduction, ❖ Spore
transformation) ● formed by gram-positive rods, especially
● Horizontal transfer Bacillus and Clostridium spp
● Makes the bacteria more virulent
Plasmids
● Extrachromosomal, double-stranded, circular DNA
is capable of replicating independently of the
bacterial chromosome.
● Can sometimes be integrated into the bacterial
chromosome → called episomes

Significance of Plasmids
● Antibiotic resistance
● Resistance to heavy metals
● Resistance to UV light
● Exotoxins and several enterotoxins.
● Bacteriocins – toxic proteins produced by
certain bacteria that are lethal for other
bacteria.
BACTERIAL GROWTH
● Bacterial growth is a coordinated process of
increase in individual cell mass and size and
duplication of the chromosome, followed by cell
division
● Bacterial reproduction occurs via binary fission

BACTERIAL GROWTH CYCLE

PHASE REMARKS

PHASE I: ● Phase during which vigorous

LAG PHASE metabolic activity occurs, but
cells do not divide
● Can last for minutes up to hours
● Zero growth rate

PHASE II: ● When rapid cell division

LOG OR occurs
EXPONENTIAL ● b-lactam antibiotics are most
PHASE efficacious during this phase

PHASE III: ● Occurs when nutrition depletion

STATIONARY or toxic products cause growth
PHASE to slow until the number of new
cells produced balances the
number of cells that die
● When bacterial spores are
formed
● Zero growth rate

PHASE IV: ● Marked by a decline in the

DEATH PHASE number of viable bacteria
● Negative growth rate
 BACTERIAL OXYGEN METABOLISM

Aerobic and Anaerobic Growth

● oxygen metabolism generates toxic products
such as superoxide and hydrogen peroxide
● superoxide dismutase, peroxidase, and
catalase are needed to survive in aerobic
environments

CULTIVATION OF MICROORGANISMS

Bacterial Nutrition
● Water constitutes 80% of the total weight of
bacterial cells.
Aerobic Metabolism ● Proteins, polysaccharides, lipids, nucleic acids,
● Obligate Aerobes muropeptides, and low molecular weight
○ completely dependent on oxygen for compounds make up the remaining 20%
ATP-generation ● For growth and multiplication, the minimum
nutritional requirements are:
● Microaerophiles ○ water
○ use fermentation but can tolerate low amounts ○ source of carbon
of oxygen because they have Superoxide ○ a source of nitrogen
dismutase (SOD) ○ some inorganic salts
● Desiccation is important to eliminate bacteria
Anaerobic Metabolism
● Facultative Aerobes TERMS:
○ utilize oxygen if it is present, but can use
fermentation in its absence ❖ Culture Medium – nutrient material prepared for
the growth of microorganisms in a laboratory
● Aerotolerant Anaerobes
○ exclusively anaerobic but insensitive to the ❖ Inoculum – microbes that are introduced into a
presence of oxygen culture medium to initiate growth

● Obligate Anaerobes ❖ Culture – the microbes that grow and multiply in or

○ cannot grow in the presence of oxygen on a culture medium
because they lack SOD, peroxidase, and
catalase. ❖ Batch culture – if many of them are inoculated at
the same time

★ The medium must be sterile – must initially contain

no living microorganism
❖ Nutrient agar – default medium of choice BACTERIAL GENETICS
❖ Peptone and Beef extract – primary sources of
protein ➔ DNA transfer between bacterial cells:
❖ NaCl – provide ions
❖ Agar – complex upon which all is suspended, upon
solidification, then add water

Types of Plating
● For urine – single streaking all
the time
● For blood and other body
fluids – quadrant streaking A bacteriophage is a type of virus that infects
● Aims to isolate bacterial cells bacteria. In fact, the word "bacteriophage" literally
that can be characterized means "bacteria eater," because bacteriophages
destroy their host cells.

Culture in tubes – agar slant or agar broth

● Strictly aerobes – streak on surface
● Anaerobes – stab

Plating Method
● Pour Plate Method
● Spread Plate Method

❖ Pour Plate Method

● Start inoculum, place agar, swirl, and allow the
agar to harden
● Inoculum dies if the agar is too hot. If too cold,
impossible to pour
● Advantages: gives more even lawn
● Disadvantages: requires universal bottles of
agar at just the right temperature
❖ Spread Plate Method
● Use L-rod after inoculum introduction, spread
evenly and place agar
VIRAL REPLICATION
2 cycles:
❖ Lytic Cycle
➢ The lytic cycle involves the reproduction of
viruses using a host cell to manufacture more
viruses; the viruses then burst out of the cell.
❖ Lysogenic Cycle
➢ The lysogenic cycle involves the incorporation
of the viral genome into the host cell genome,
infecting it from within.

● Advantage: easier to do
● Disadvantage: may give an uneven lawn

NORMAL FLORA

● Refer to microbes that are permanent residents of

the body
● Normal flora are low-virulence organisms in their
usual anatomic site

❖ Resident microbiota
➢ Microbes regularly found in a given area of the
body at a given time

❖ Transient microbiota
➢ are nonpathogenic or potentially pathogenic
microbes that inhabit the skin or mucous
membranes for hours, days, or weeks
❖ Colonization resistance
➢ occurs when normal flora occupy receptor
sites preventing pathogens from binding

Skin Staphylococcus epidermidis

Nose Staphylococcus aureus

Mouth Viridans Streptococci

Intestinal bacteria play a role in
Dental plaque Streptococcus mutans ● Vitamin K synthesis
● Conversion of bile pigments and bile acids
● Absorption of nutrients and breakdown products
Colon Bacteroides, Escherichia coli
(not always protective, ie. ammonia)
Bifidobacteria – dominant intestinal
● Antagonism of microbial pathogen
microbiota in among breastfed children

Vagina Lactobacillus vaginalis

Escherichia coli
Streptococcus agalactiae

★ NSVD infants harbor bacterial communities (in all

body habitats) that are most similar in composition
to the vaginal community of their mothers.

★ CS-delivered infants lack bacteria from the vaginal

community (eg. Lactobacillus, Prevotella,
Atopobium, Sneathia spp) instead, harbor bacterial
communities most similar to the skin communities
of the mother.
NORMALLY STERILE AREAS
★ Viridans streptococci become established as the ❖ Cerebrospinal fluid
most prominent resident flora within 4-12 h after ❖ Blood
birth and remain so for life. ❖ Middle ear (small bones — the hammer (malleus),
anvil (incus) and stirrup (stapes))
How are Dental Caries produced? ❖ Paranasal sinuses
- Involves genetic, hormonal, and nutritional Influences ❖ Tissues
❖ Bladder
1. Plaque formation – an essential first step in caries ❖ Uterus
production ❖ Fallopian tubes
2. Acid formation – in large amounts and processed
by Streptococci and Lactobacilli from carbohydrates
● S. mutans – considered to be the dominant
organism for the initiation of caries

In all, there are thought to be 300–400 bacterial species

present in mature dental plaque.

How are caries controlled?

➢ Physical removal of the plaque
➢ Limit sucrose intake
➢ Good nutrition (adequate protein intake)
➢ Reduce acid in the mouth (limit CHO, cleansing)
➢ Fluoride – enhances acid resistance of the enamel

★ Control of periodontal disease: remove calcified

deposits and good mouth hygiene.

Stomach
● Acidic pH
○ Low bacterial content
○ Only H. pylori present – located on the
epithelial side of gastric mucosa
○ Protects against infection of some enteric
pathogens (Vibrio cholera)
● Pyloric obstruction favor growth of gram-positive
cocci and bacilli
● Increase in pH (towards alkalinity) cause a great
increase in microbial flora (including fecal microbes)