MICROBIOLOGY

THE SCIENCE OF MICROBIOLOGY and CELL STRUCTURE

Carmela Reyes, MD
1st Shift | 14 August 2018

TOPIC OUTLINE o PrPc is expressed on the surface of neurons

I. Introduction o Conformational change occurs in prior protein
II. General Classification of Microbes ▪ PrPc (normal or cellular form) to PrPSc (disease-
A. Viruses causing conformation)
B. Prions ▪ PrPSc , when present in an individual, is capable of
C. Prokaryotes: Bacteria and Archaebacteria recruiting PrPC and converting it to the disease form.
D. Protists: Algae, Protozoa, Fungi, Slime Molds ▪ Thus, prions replicate using the PrPc substrate that is
III. General Mechanisms How Microbes Cause Illnesses in Man present in the host
IV. Cell Structure - Prion diseases of importance
A. Types of Microscopes o PrPSc prevents normal
i. Light Microscope transmission of
ii. Electron Microscope neurotransmitters causing
B. Eukaryotic Cell (Mitotic Spindle) dementia, Alzheimer’s
C. Prokaryotic Cell (Binary Fission) disease, Kuru and Mad Cow
V. Gram Staining Bacteria Disease
A. Gram Positive vs. Gram Negative Cell Wall o Scrapie, Kuru
VI. Protein Secretion System o Creutzfeldt-Jakob disease
VII. Sporulation and Germination (CJD)
VIII.Stains o Gerstmann-Straussler-
IX. Cell Division and Cell Groupings Scheinker disease
o Fatal familial insomnia
I. Introduction o Bovine-spongiform Prion: Abnormal isoform of PrPc
Microbiology encephalopathy
- The study of microorganisms
o A large and diverse group of microscopic organisms that exist VIRUSES VIROIDS PRIONS
as single cells or cell clusters Obligate Intracellular Obligate Abnormal form of
o Also includes viruses: microscopic but not cellular. Agents intracellular agents cellular protein
Consist of either DNA Consist only of Consist only of
II. General Classification of Microbes or RNA surrounded RNA; no protein protein; no DNA or
Viruses by a protein coat coat RNA
- Lack many attributes of cells: Does not replicate
o Only replicates when it infects a host or a living cell Prokaryotes
- Known to infect all cells, including microbial cells - Relatively small size; absence of a nuclear membrane
o Virophages – virus that infects other viruses - Most have a single chromosome
o Bacteriophage – virus infecting bacteria - Nucleoid: Cell containing DNA
o Viroids – small single stranded, covalently closed circular - Two Major Subdivisions: Bacteria and Archaebacteria
RNA molecules causing a number of transmissible plant - Bacteria
diseases o Peptidoglycan cell wall
- Viral Particles o Phospholipid cell membrane
o Generally small o Ribosomes
o Nucleic acid molecule ▪ 70s sedimentation rate: 30s & 50s subunit
▪ Either DNA or RNA (but never both) o DNA contains exons only
▪ Enclosed in a protein coat (capsid) - Archaebacteria
▪ Always have an intermediate nucleic acid in the form of o Some species are killed by contact with oxygen
Mrna o Others grow at temperature exceeding that of boiling water
- DNA viruses replicate by going inside the cell  nucleus  DNA o Unique lipid cell membrane (isoprenoids) allowing them to
of the host  DNA of the virus makes mRNA  ribosomes  withstand extreme conditions
produces bacterial proteins  assembled to become viral o Presence of introns within genes
particles ▪ Segments of DNA that interrupts informational DNA
within genes
Prions ▪ An intriguing trait shared by archaebacteria and
- Proteinaceous and infectious in nature eukaryotes
- No nucleic acids: No ability to replicate in man ▪ Main function is not established
- Cellular form of the prion protein (PrPc) is encoded by the host’s
chromosomal DNA.

Leandro L. Espino [MG, SG] 1 of 6

 MICROBIOLOGY – SCIENCE OF MICROBIOLOGY and CELL STRUCTURE

Protists
- Microbial eukaryotes
- Has “true nucleus”
- Membrane-bound organelles, microtubules, and microfilaments of
eukaryotes form a complex intracellular structure
- Protists are members of four following major groups: algae,
protozoa, fungi, and slime molds.

Algae Life cycle of an Fruiting body of a

- Used to denote all organisms that produce O2 as a product of
photosynthesis
- One of the major subgroups: Cyanobacteria (blue-green bacteria)
are prokaryotic and no longer termed as algae
- Photosynthetic eukaryotic organisms
o Contains chlorophyll
o Many algal species are unicellular, and some may form large
multicellular structures
o Some produce toxins
▪ Dinoflagellates – unicellular; causes algal blooms, or red
tides.
▪ Produces neurotoxins such as saxitoxin and
gonyautoxin
Alga dinoflagellate (Red Tide)
Protozoa
- Unicellular nonphotosynthetic protists
- Heterotrophs – nutritional requirements through organic
compounds
- Major groups: Flagellates, Amoebae, Ciliates, Sporozoa
Fungi
- Non-photosynthetic protists
- Coenocyte (a multinucleated mass of continuous cytoplasm)
confined in a series of branching tubes
- Molds: mycelial forms
- Yeasts: do not form a mycelium; grow as single cells that bud;
presence of transitional forms
Slime Molds
- Diverse forms
- Characterized by the presence of Plasmodium, an amoeboid
multinucleate mass of cytoplasm; analogous to mycelium
- Growth depends on nutrients provided by bacterial or, in some
cases, plant cells; growth is in response to increasing levels of 3,5-
cyclic AMP
acellular slime mold cellular slime mold
III. General Mechanisms How Microbes Causes Illnesses in Man
- Virus: Nucleic acid integrated to host (provirus)
- Prions: folding of normal prion materials
- Prokaryotes and Protists: Pathogenic structures
o Gram (+): Exotoxin (released by bacteria)
o Gram (-): Endotoxin (attached to the cell wall of bacteria)
IV. Cell Structure
Types of Microscopes
Light Microscope
- Bright-Field Microscope
o Most commonly used
o Consists of two series of lenses: Objective (100x) and ocular
(10x) lens = 1000x magnification
o Specimens are rendered visible due to differences in
contrast between them and the surrounding medium.
o Dyes (stains) can be used to stain cells or their organelles
and increase their contrast
- Phase Contrast Microscope
o Developed to improve contrast difference between cells and
the surrounding medium, making it possible to see living
cells without staining.
o Dark image on a light background
- Dark-Field Microscope
o A light microscope in which lighting system has been
modified to reach the specimen from the sides only
o Uses a special condenser that both blocks direct light rays
and deflects light off a mirror on the side of the condenser at
an oblique angle
o This creates a “dark field” that contrasts against the
highlighted edge of the specimens and results when the
oblique rays are reflected from the edge of the specimen
upward into the objective of the microscope.
o Useful for Treponema pallidum
- Fluorescence Microscope
o Used to visualize specimens that fluoresce, the ability to
absorb short wavelengths of light (ultraviolet) and give off
light at a longer wavelength (visible)
o Fluorochromes – group of fluorescent dyes used to stain
organisms or cells that do not naturally fluoresce
▪ Auramine O: glows yellow when exposed to UV light;
strongly absorbed by M. tuberculosis
o Fluorescent-antibody (FA) technique or
Immunofluorescence – principal use of fluorescence
microscope
▪ Specific antibodies are chemically labeled with a
fluorochrome, fluorescein isothiocyanate (FITC).
▪ Antibodies to Legionella pneumophila

Leandro L. Espino [MG, SG] 2 of 6

 MICROBIOLOGY – SCIENCE OF MICROBIOLOGY and CELL STRUCTURE

- Differential Interference Contrast (DIC) Microscope - Cell wall: outer wall composed of cellulose, chitin, or silica
o Employ a polarizer to produce polarized light - Mitochondria able to self-replicate because it has its own DNA
o The polarized light beam passes through a prism that - Mitotic spindle is the best differentiating criteria for eukaryotes
generates two distinct beams; these beams pass through the - E.R. and Lysosomes are exclusive to eukaryotes
specimen and enter the objective lens, where they are
recombined into a single beam. Prokaryotic Cell (Binary Fission)
o Slight differences in refractive index creates an interference STRUCTURE CHARACTERISTICS FUNCTION
effect Nucleoid No nuclear membrane Genetic Material
o Spores, vacuoles, and granules appear three-dimensional ATP production;
o Useful for unstained cells Cell Membrane Phospholipids
protein secretion
Peptidoglycan, murein,
Electron Microscope Cell Wall Antigenic; rigidity
or mucopeptide
- The superior resolution of the electron microscope is because of AFB Wall Mycolic Acids Caseous necrosis
electrons that have a much shorter wavelength than the photons of Archaebacteria Pseudomurein Rigidity
white light Remarks: cause of
- Transmission Electron Microscope (TEM) L Forms Absent cell wall
chronic inflammation
o Shows internal structures of the cell Remarks: Resistant
- Scanning Electron Microscope (SEM) Mycoplasma Absent cell wall
to PCN group
o Has lower resolving power than TEM Capsule/
o Useful for providing three-dimensional images on the Glycocalyx/ Carbohydrates Antigenic; adherence
surface of microscopic objects Slime Layer
- Confocal Scanning Laser Microscope (CSLM)
Monotrichous;
o Couples a laser light source to a light microscope
Lophotrichous;
o A laser beam is bounced off a mirror that directs the beam Flagella Locomotion
Peritrichous
through a scanning device then the laser beam is directed
(Flagellin)
through a pinhole that precisely adjusts the plane of focus of
Virulence factor;
the beam to a given vertical layer within the specimen Pili L hairs (Pilin)
adherence
o Equipped with computer software to assemble digital images
Virulence factor;
for subsequent image processing Fimbriae L fringes (Pilin)
adherence
- Scanning Probe Microscope
o Measures surface features by moving a sharp probe over the Virulence factor;
Adhesin Pilin
object’s surface adherence
o Enables scientists to view atoms or molecules on the surface Transfers F+ (Fertility
Sex Pili Pilin
of a specimen factor in plasmid)
e.g. Scanning tunneling microscope and atomic force Extra-chromosomal
Plasmid Resistance Factor
microscope DNA

Eukaryotic Cell (Mitotic Spindle) - Capsule: For vaccine production; makes use of carbohydrates of
STRUCTURE CHARACTERISTICS FUNCTION the capsule; not all bacteria have capsule
Nucleus Bounded Genetic Material - Flagella: Three types of arrangement are known: monotrichous
Ribosomal RNA (single polar flagellum), lophotrichous (multiple polar flagella),
Nucleolus Within Nucleus and peritrichous (flagella distributed over the entire cell).
synthesis
o Flagellin: protein subunit
RER: Protein
- Plasmids: Extra-chromosomal DNA; used for cloning
Synthesis
Endoplasmic Membrane bound - Those with sex pili are males; females do not have sex pili
Reticulum channels - Virulence factor, resistance factor, and fertility factors in plasmids
SER: Lipid and CHO
may be exchanged between bacteria through the process of
synthesis
conjugation, contributing to resistance
Golgi Apparatus Stack of membranes Packaging
Mitochondria 70S; self-replicating ATP production
V. Gram Staining Bacteria
Membrane enclosed - Most bacteria are classified as gram positive or gram negative
Lysosomes Digestion
sacs according to their response to the Gram-staining procedure
Gliding; contraction; - Developed by Hans Christian Gram
Cytoskeleton Filaments
cytokinesis - Gram Positive: retains the crystal violet (a purple dye)-iodine
Flagella/Cilia Tubulin Locomotion complex after a brief wash with alcohol or acetone. Thus, gram-
Plasmid Extrachromosomal positive bacteria will look purple under the microscope.
Resistance Factor
(Absent in Man) DNA - Gram Negative: does not retain the dye-iodine complex and
become translucent, but they can then be counterstained with
- Cell membrane: phospholipid bilayer safranin (a red dye). Thus, gram-negative bacteria will look red
o Asymmetric membrane; no peptidoglycan layer under the microscope.

Leandro L. Espino [MG, SG] 3 of 6

 MICROBIOLOGY – SCIENCE OF MICROBIOLOGY and CELL STRUCTURE
Gram Positive versus Gram Negative Cell Wall
- Peptidoglycan Layer
o A complex polymer consisting of alternating N-
acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)
connected by  1→4 linkages
o Gram Positive: Thick peptidoglycan layer
o Gram Negative: Thin peptidoglycan layer
- Special Components of Gram-Positive Cell walls
o Teichoic Acid: encompasses all wall, membrane, or
capsular polymers containing glycerophosphate or ribitol
phosphate residues.
▪ Wall teichoic acid (WTA): covalently linked to
peptidoglycan
▪ Lipoteichoic acid (LTA): covalently linked to
membrane glycolipid
▪ Together with Peptidoglycan, WTA and LTA make up a
polyanionic network or matrix, that provides functions
relating to elasticity, porosity, tensile strength, and
electrostatic properties of the envelope.
o Teichuronic Acid: similar to teichoic acids but the repeat
units include sugar acids instead of phosphoric acids. They
are synthesized in place of teichoic acids when phosphate is
limiting.
o Polysaccharides
▪ Neutral sugars: mannose, arabinose, rhamnose,
glucosamine
▪ Acidic sugars: glucuronic acid and mannuronic acid
o Periplasmic space: B-lactamase; (extended B-lactamase in
Gram Negative)
o Principle why it is blue-stained
▪ No lipoprotein present
 soluble)  upon application of alcohol it is washed off
▪ The alcohol will trap the crystal violet, then it will bind
(plus the iodine) with teichoic acid and to the
peptidoglycan
A: Teichoic acid structure. The segment of a teichoic acid made of
phosphate, glycerol, and a side chain, R. R may represent D-alanine,
glucose, or other molecules. B: Teichoic and lipoteichoic acids of the
gram-positive envelope.
- Special Components of Gram-Negative Cell walls
o Outer Membrane: Asymmetrically bilayered structure
▪ Present ONLY in gram-negative bacteria
▪ Inner leaflet: resembles composition of cell membrane
▪ Outer leaflet: contains distinctive component,
Lipopolysaccharide (LPS)
▪ Has the ability to exclude hydrophobic molecules:
protect cell from deleterious substances such as bile
salts
▪ Lipid in nature – low molecular weight hydrophilic
structures enter the cell via passive diffusion through
porin channels: sugar, amino acids, and certain ions
▪ Large antibiotic molecules penetrate the outer
membrane relatively slowly, which accounts for the
relatively high antibiotic resistance of gram-negative
bacteria
o Lipopolysaccharide (LPS)
▪ Has been called the endotoxin of gram-negative
bacteria, which is extremely toxic to animals, because it
is firmly bound to the cell surface and is released only
when cells are lysed.
▪ Consists of complex glycolipid called “Lipid A” attached
to a polysaccharide made up of a core and terminal
series of repeat units
▪ Lipid A: causes fever; toxic moiety of LPS (endotoxin)
which is release upon destruction of gram-negative
bacteria upon giving antibiotics
▪ The negatively charged LPS molecules are
noncovalently cross-bridged by divalent cations (ie,
Ca2+ and Mg2+); this stabilizes the membrane and
provides a barrier to hydrophobic molecules.
o Lipoprotein: Functions to stabilize the outer membrane and
anchor it to the peptidoglycan layer
o Periplasmic space: Space between the inner and outer
membranes; contains the peptidoglycan layer and a gel-like
solution of proteins
o Principle why it is pink-stained
▪ Most of the peptidoglycan has been washed away by
the process of gram stain
▪ Crystal violet  iodine  alcohol (decolorization)
▪ Through the structure lipoprotein (which is alcohol
(Remember: lipoprotein attaches the peptidoglycan
layer to the outer membrane)
Molecular representation of the envelope of a gram-negative bacterium
Leandro L. Espino [MG, SG] 4 of 6
 MICROBIOLOGY – SCIENCE OF MICROBIOLOGY and CELL STRUCTURE
Comparison of Features of Gram- Positive and Gram-Negative
Bacteria
Taken from: Jawetz, Melnick and Adelberg’s Medical Microbiology
(27th edition)
VI. Protein Secretion System
- Excretion of hydrolytic exoenzymes and pathogenicity factors
o Hydrolytic exoenzymes are secreted to degrade organic
polymers to small subunits small enough to penetrate the
cell membrane as a source of nutrients
o Gram-positive bacteria: secrete directly into the external
environment
o Gram-negative bacteria: secrete into the periplasmic space
Six pathways of protein secretion
- Protein Secretion Systems
o Exotoxins are released by bacteria via protein secretion
system
o Six pathways of protein secretion have been described in
bacteria: Type I, Type II, Type III, Type IV, Type V and
Type VI secretion systems
▪ Type I and IV: described in both Gram (+) and Gram (-
)
▪ Type II, III, V, and VI: found only in Gram (-)
▪ Type I and III: traverse both inner membrane (IM) and
outer membrane (OM) in one step; proteins secreted
do not have a leader sequence and are exported intact
▪ Type II and V: cross the IM and OM in several steps;
proteins are synthesized as pre-proteins containing an
extra leader or signal sequence of 15 – 40 amino acids
(most commonly about 30 amino acids) at the amino
terminal and require the sec system for transport
across the IM.
o The Sec-dependent and Tat pathways deliver proteins
from the cytoplasm to the periplasmic space
▪ Type II, V and sometime type IV: complete the
secretion process begun by the Sec-dependent
pathway.
▪ Type II: The Tat system appears to deliver proteins
only to this pathway.
▪ Type I and III: bypass the Sec-dependent and Tat
pathways, moving proteins directly from the cytoplasm,
through outer membrane, to the extracellular space
(Sec-independent)
▪ Type IV system can work either with the Sec-
dependent pathway or can work alone to transport
proteins to the extracellular space.
▪ Proteins translocated by the Sec-dependent pathway
and the type III pathway are delivered to those
systems by chaperone proteins.
VII. Sporulation and Germination
- Members of several bacterial genera are capable of forming
endospores.
- The two most common are gram-positive rods: the obligately
aerobic genus Bacillus and the obligately anaerobic genus
Clostridium.
- Endospore:
o Core = chromosomes
o Spore wall = peptidoglycan
o Cortex = thickest layer; sensitive to lysozyme
o Coat = keratin like; resistant to anti-microbials
o Exosporium = protein, lipids, carbohydrates
Sporulating cells of bacillus species. A: Unidentified bacillus from soil.
B: Bacillus cereus. C: Bacillus megaterium.
Leandro L. Espino [MG, SG] 5 of 6
 MICROBIOLOGY – SCIENCE OF MICROBIOLOGY and CELL STRUCTURE

Self-Assessment
• Heat resistance of spores is due to: Calcium dipicolinate
• Type of Electron Microscope showing internal structures of the
cell: Transmission Electron Microscope
• Thicker peptidoglycan is seen in: Gram-positive bacteria
• The type of cell wall with the outer membrane: Gram-negative cell
wall
• The toxic moiety of Lipopolysaccharide: Lipid A
• Microbes without nucleic acids: Prions
• End color of gram-positive wall: Purple
• Give the microbial groupings: Viruses, prokaryotes, prions,
Protista
• The unbound nucleus is found in: Prokaryotes
• ATP production in bacteria occurs in: Cell/cytoplasmic
membrane
• Mitochondria are believed to be part of: Prokaryotic cell
• The structure containing the F+ factor: Plasmids
• Bacteria with F+ are also known as: male

References
- Powerpoint Presentation of Dr. Reyes
The stages of endospore formation.
- UST FMS Section D 2020 Trans: The Science of Microbiology
- Instalinotes Microbiology
VIII. Stains
- Carroll, K.C, Morse, S.A., Mietzner, T., and Miller, S. (2016).
- Stains combine chemically with the bacterial protoplasm; if the cell
Jawetz, Melnick, & Adelberg’s Medical Microbiology (27e). New
is not already dead, the staining process itself will kill it. The
York: Lange.
process is thus a drastic one and may produce artifacts.
- Gram stain: affinity for peptidoglycan
- Acid Fast stain: mycolic acids; retain carbolfuchsin when
decolorized with hydrochloric acid
- Negative stain: staining the background with an acidic dye;
o Nigrosin is commonly used.
- Flagella stain: Tannic acid to form precipitate then basic stain
- Capsule stain: Background; negative staining procedure.
- Nucleoid stain: Feulgen stain specific for DNA
- Spore stain: Malachite green and Carbolfuchsin

IX. Cell Division and Cell Groupings

- Cell Division
o Most bacteria divide by binary fission into two equal
progeny cells.
o The chromosomes, which have doubled in number
preceding division, are distributed equally to the two
daughter cells
- Cell Groupings
o Certain characteristic groupings results, if the cells remain
temporarily attached after division.
o Coccal forms: chains (streptococci), pairs (diplococci),
cubical bundles (sarcinae), or flat plates
o Rods may form pairs or chains
o Characteristic postdivision movements:
▪ “Whipping” motion: bring cells into parallel positions
▪ Repeated whipping result in the “palisade”
arrangement characteristic of diphtheria bacilli

Leandro L. Espino [MG, SG] 6 of 6