THE CELL

MICROSCOPY

 Zaccharias Janssen
 Inventor of compound microscope (poor quality)
 Joseph Jackson Lister
 Invented better compound microscope
Three important parameters:

 MAGNIFICATION
 The ratio of an object’s image size to its real size
 TOTAL MAGNIFICATION: objective lens x ocular lens

Objective Ocular lens Total magnification

lens
Scanner 4x 10x 40x
Low power objective 10x 10x 100x
High Power objective 40x 10x 400x
Oil immersion objective 100x 10x 1000x

 RESOLUTION / RESOLVING POWER

 Measure of the clarity of the image
 It is the minimum distance two points can be separated and still be distinguished as separate points.
 It is the ability of the lenses to distinguish fine detail and structure.
 CONTRAST
 The difference in brightness between the light and dark areas of an image.
 Better contrast when changing the refractive index
 Refractive - It is the measure of the light bending ability of the medium.
o STAINING TECHNIQUE - to change the refractive index.

PARTS OF THE MICROSCOPE

1. LENS SYSTEM
 Objective lens – initial magnification
 Ocular lens – further magnification
 Coarse adjustment knob – moves the mechanical stage noticeably
 Fine adjustment knob – it sharpens the image
2. ILLUMINATION SYSTEM
 Light source
 RHEOSTAT - It regulates the intensity of the light
 Condenser – it focuses light on the specimen and controls the light for uniform illumination
 Iris diaphragm
 Field diaphragm
3. BODY
 Base
 Body tube
 Nosepiece
TYPES OF MICROSCOPES

Microscope Type Distinguishing Features Principal Uses Additional notes

Brightfield Uses visible light as a source of To observe various  Spx appears against a
illumination; cannot resolve stained specimen and to bright background
structure small than about 0.2 count microbes; does  Cannot resolve
μm; specimen appears against a not resolve very small structures smaller than
bright background. Inexpensive specimens, such as about 0.2 μm
and easy to use. viruses.  Leeuwenhoek’s: cannot
resolve structure smaller
than 1 μm
Darkfield Uses a special condenser with an To examine living  Very important in
opaque disk that blocks light microorganisms that are examination of
from entering the objective lens invisible in brightfield Spirochetes (a
directly; light reflected by microscopy, do not prokaryotic bacteria)
specimen enters the object lens, stain easily or are  Ex. of Spirochetes:
and the specimen appears light distorted by staining; Treponema pallidum-
against a black background. frequently used to causes syphilis
detect Treponema
pallidum.
Phase-contrast Uses a special condenser To facilitate detailed  Direct and reflected or
containing an annular (ring- examination of the diffracted light rays are
shaped) diaphragm. The internal structures of brought together to
diaphragm allows direct light to living specimens. produce the image
pass through the condenser,  No staining required.
focusing light on the specimen  Halo around the image
and a diffraction plate in the  Useful in examining
objective lens. Direct and living unpigmented
reflected or diffracted light rays cells.
are brought together to produce
the image. No saining required
Differential Like phase-contrast, uses To provide three-  Can give almost 3D
Interference differences in refractive indexes dimensional images.  Uses 2 beams of light
Contrast (DIC) to produce images. Uses two separated by prisms
beams of light separated by  Has 2 types:
prisms; the specimen appears o Modulation
colored as a result of the prism Contrast- aka
effect. No staining required. Hoffman
o Interference
Contrast- aka
Nomarski
Fluorescence Uses an ultraviolet or near- For Fluorescent-  Principle: Fluorescent-
ultraviolet source of illumination antibody techniques antibody technique /
that causes fluorescent (immunofluorescence) Immunofluorescence
compounds (green-colored) in a to rapidly detect and  Examples of
specimen to emit light. identify microbes in Fluorochromes:
tissues or clinical • Auramine O -
specimens. used for
Mycobacterium
tuberculosis
(color yellow)
• FITC
(Fluorescein
isothiocyanate)-
Bacillus
anthracis (color
apple green)
Confocal Uses a single photon to To obtain two- and  Gives 2D and 3D image
 illuminate one plane ofa three- dimensional of cells for biomedical
specimen at a time. images of cells for applications
biomedical
applications.
Electron Uses a beam of electrons instead To examine viruses or  Uses beam of electrons
Transmission of light; electrons pass through the internal instead of light
the specimen; because of the ultrastructure in thin  Electrons pass through
shorter wavelength of electrons, section of cell (usually the spx
structures small than 0.2 μm can magni  Because of the shorter
be resolved. The image wavelength of electrons,
produced is two-dimesional. structures smaller than
0.2 μm can be resolved.
 Image produced is 2D
Scanning Uses a beam of electrons instead To study the surface  Uses electron beams
of light; electrons are reflected features of cells and instead of light;
from the specimen; because of viruses (usually electrons are reflected
the shorter wavelength of magnified 1000- from the spx
electrons, structures smaller than 10,000X)  Because of the shorter
0.2 can be resolved. The image wavelength of electrons,
produced appears three structures smaller than
dimensional. 0.2 μm can be resolved.
 Image produced is 3D

CELL FRACTIONATION

 Useful in studying cell and structure

 Principle: used to isolate/fractionate cell components based on size and density
 Cells are homogenized in a blender to break them up. The resulting mixture is centrifuged. The supernatant
(liquid) is poured into another tube and centrifuged at a higher speed for a longer period. This process is
repeated several times. This “differential centrifugation” results in a series of pellets, each containing diff. cell
components.
 Supernatant - upper liquid portion of pellets
 Lower speed - larger components are left behind (nuclei & mitochondria)
 Higher speed - smaller components are left (ribosomes)

 Results: In early experiments, researchers used microscopy to identify the organelles in each pellet and
biochemical methods to determine their metabolic functions. These identifications established a baseline for this
method, enabling today’s researchers to know which cell fraction they should collect to isolate and study
particular organelles.
THREE MAJOR DOMAINS

 Domain - highest in the hierarchy (taxonomy)

MNEMONICS: King Phillip Came Over For Great Spaghetti
Kingdom – Phyla – Class – Order – Family – Genus – Species
1. BACTERIA

 Lack a membrane-bounded nucleus and mitochondria

 Surrounded by a cell wall
 Divide by binary fission
2. ARCHAEA

 Cell walls lack peptidoglycan

• Can be stained by Gram (+) or Gram (-)
 Share common characteristics with Bacteria except peptidoglycan
 Structure of cell envelope from archaea helps them to survive toxic environment, particularly enzymes present
help the organism survive from stressful and toxic conditions.
3. EUKARYA

 Cells contain an elaborate network of internal membranes, a membrane-bounded nucleus, and mitochondria.
 DNA is organized into true chromosomes, and cell division takes place by means of mitosis.
MICROORGANISMS

ACELLULAR CELLULAR
Viruses Prokaryotes
 Eubacteria
- Gram Positive & Gram Negative
 Cyanobacteria
- Blue Green Algae
 Archaebacteria
Eukaryotes
• Parasites
• Fungi

PROKARYOTES
DNA is not enclosed in a membrane, and is usually a
singular circularly arranged chromosome
 Vibrio cholerae - an exemption because it has 2
chromosomes
DNA is not associated with histones
Lack membrane-enclosed organelles
Cell wall always contain the complex polysaccharide
peptidoglycan aka Murein Layer
EUKARYOTES
DNA is found in the cell’s nucleus, which is
separated from the cytoplasm by a nuclear membrane,
and DNA is found in multiple chromosomes.
DNA is consistently associated with chromosomal
proteins (histones) and non-histones
• Histones- highly basic proteins found in eukaryotic
cell
nuclei that pack and
order the DNA into
structural unit
Have a number of membrane-bound organelles
including mitochondria, Golgi apparatus, ER,
Lysosomes, and sometimes, chloroplasts
Cell walls (when present) are chemically simple
  Thick if Gram +
 Thin if Gram -
Usually divide by binary fission (during this process,
DNA makes a copy of itself then the cell splits into 2
copies
Ureaplasma & Mycoplasma- have no cell wall
Cell division usually involves mitosis (chromosomes
replicate and an identical set is distributed into each of 2
nuclei)
Animals & protozoans- no cell wall

PROKARYOTE EUKARYOTE
Nuclear Body No nuclear membrane Classic membrane-bound
Cell Division Binary fission Mitosis
Cell Wall Eubacteria- Peptidoglycan Animals & Protozoan – no CW

Archaebacteria- Resembles Fungi & Plants- with CW

peptidoglycan
Cytoplasmic Membrane Phospholipid bilayer (without CHO Phospholipid bilayer (with CHO &
& sterol component; except sterol component)
Mycoplasma)
Organelles Present, no membrane- enclosed Present w/ membrane- enclosed
Site of Energy Production Cytoplasmic membrane Mitochondria
Site of Protein Synthesis Free ribosomes Rough ER
Bacteria - single-cell prokaryotic organisms
Fungi - single cell or multicellular eukaryotic microorganisms
Yeast - unicellular, eukaryotic microorganisms
Parasites - single cell or multicellular eukaryotic organisms
Viruses - dependent on host cells for survival and therefore are not considered cellular organisms but rather infectious
agents.
PROKARYOTES
BACTERIA

 Unicellular organisms that lack a nuclear membrane and true nucleus

 Classified as prokaryotes (Greek: before kernel, ‘nucleus’), having no mitochondria, ER, or Golgi bodies
BACTERIAL MORPHOLOGY

 Vary in size, morphology, and cell-to-cell arrangements and in the chemical composition and structure of the
cell wall
 Bacterial cell wall differences provide basis for the gram stain (most fundamental test used in bacterial
identification)
BACTERIAL SIZE

 Most clinically relevant bacterial species range in size from 0.25 to 1 μm in width and 1 to 3 μm in length
 Bacterium is some hundred-fold larger than a virus, and ten-fold smaller than eukaryotic cell
BACTERIAL SHAPE
a) Common bacterial cellular morphologies include:

I. Cocci- circular
II. Coccobacilli- ovoid
III.Bacillus- rod shaped
IV. Fusiform- tapered, pointed ends
V. Curved
VI. Spiral- helical, like corkscrew
 Spirochetes vary in length and the number of helical turns
 All Spirochetes are spiral, but not all helical bacteria are Spirochetes
VII. Pleomorphic- no defined shape
o Rhizobium & Corynebacterium- example of pleomorphic
o Most are called Monomorphic (one/single shaped)
o Adenoma- many shape

BACTERIAL ARRANGEMENT
I. Pairs- Diplo
II. Chains- Strepto
III. Grape-like clusters- Staphylo
IV. Group of 4- Tetrad
V. Packets of 8- Sarcinae
VI. Palisades- side by side
VII. Chinese characters

BACTERIAL CELL STRUCTURE

1. CELL ENVELOPE
b) Outermost structure; comprises:
A. Outer membrane
 in gram negative bacteria only
 Functions as the cell’s initial barrier to the environment
 Serve as primary permeability barriers to hydrophilic and hydrophobic compounds and contain
essential enzymes and other proteins located in the periplasmic space.
 Bilayer structure composed of Lipopolysaccharide
 Lipopolysaccharide- gives the surface of gram-negative bacteria a net negative charge. Its strong
negative charge is important for evading phagocytosis and actions of complement (host’s defense
against bacteria)

Porins
 protein structures scattered throughout lipopolysaccharide macromolecules
 Water-filled structures that control the passage of molecules/nutrients (nucleotides, disaccharide,
peptides, amino acids, Vit. B12, Iron) and other solutes, including antibiotics, through the outer
membrane
 Number and types of porins may vary with bacterial species
 Influence the extent to which various substances pass through the outer membranes of different
bacteria

Murein Lipoproteins
 facilitate the attachment of the outer membrane to the next internal layer in the cell envelope, the cell
wall

B. Cell Wall
 aka Murein Layer or Peptidoglycan
 Composed of the peptidoglycan macromolecule
 Gives the bacteria cell shape and strength to withstand changes in environmental osmotic pressures
that would otherwise result in cell lysis
 Composition:
- A backbone composed of alternating sugar components
- N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) connected by B 1-4 linkage
 - NAG & NAM molecules are linked in rows of 10 to 65 sugars that makes up carbohydrate
backbone
- NAG & NAM linkage is linked by polypeptide (may sugar or glycan + peptide or peptido =
peptidoglycan)
 Diaminopimelic acid (DAP)- unique element of bacterial cell wall
 Penicillin - important antibiotic that interferes with the linkage of peptidoglycan
 Some components responsible for pathogenicity
 M-proteine (present in Streptococcus pyogenes)
 Mycoloc acid (Mycobacterium tuberculosis)
 Mycobacterium spp. Have an unusual cell wall structure:
 Cell wall contains N-glycolilmuramic acid instead of N-acetylmuramic acid
 Has a very high lipid content which creates hydrophobic permeability barrier
 Different types of cell wall structure traditionally have been categorized according to their staining
characteristics.

 Major types of cell walls: GRAM-POSITIVE AND GRAM-NEGATIVE TYPES

 Mycobacteria – stain gram-positive, have a modified cell wall called an acid-fast cell
wall
 Mycoplasmas and Ureaplasma – microorganisms that have no cell wall

C. Periplasm or Periplasmic Space

a) in gram negative bacteria only

D. Cytoplasmic or Cell membrane

b) encloses the cytoplasm

 If gram positive bacteria, B and D only are present. If gram negative, all are present
 Outer membrane and Periplasm are unique in gram negative bacteria

a) GRAM – POSITIVE CELL WALL

 Composed of a very thick protective peptidoglycan (murein) layer
 Consists of glycan (polysaccharide) chains of alternating N-acetyl-d-glucosamin (NAG) and N-acetyl-
d-muramic acid (NAM)
 Many antibiotics effective against gram-positive organisms (e.g., penicillin) act by preventing
synthesis of peptidoglycan
  Other component of the gram-positive cell wall that penetrate to the exterior of the cell are:

TEICHOIC ACID and TEICHURONIC ACID

 Accounts for up to 50% of the dry weight of the wall and 10% of the dry weight of the total cell
TEICHOIC ACID
 Anchored to the peptidoglycan (N-acetylmuramic acid)
 Glycerol or ribitol phosphate polymers combined with various sugars, amino acids, and amino sugars
(composition)
 Has 2 types:
 Wall teichoic acid – covalently link to the peptidoglycan
 Membrane teichoic acid – linked to membrane glycolipid (associated with lipids)
 LIPOTEICHOIC ACID
 Anchored to the plasma membrane
 Linked to the next underlying layer, plasma membrane or cellular membrane
DIFFERENT BACTERIAS THAT ADDS VIRULENCE
Streptococcus pneumoniae
 Teichoic acid pairs the antigenic determinants (Forssman antigen)
Streptococcus pyogenes
 Its lipoteichoic acid is associated with M protein
 M protein + LTA = facilitates attachment of S. pyogenes to cell
TEICHURONIC ACIDS
 Similar polymers, but the repeat units include sugar acids (eg, N-acetylmannosuronic or d-
glucosuronic acid) instead of phosphoric acids
 Synthesized in place of teichoic acids when phosphate is limiting
POLYSACCHARIDES
 Subunits of polysaccharides in the cell wall of gram positive:
 Mannose, arabinose, glucosamine and acidic sugars
b.) GRAM – NEGATIVE CELL WALL
 The cell wall and outer membrane is part of the cell envelope of gram-negative bacteria
 Composed of two layers:
 INNER PEPTIDOGLYCAN LAYER
- Much thinner that in gram-positive cell walls
 OUTER MEMBRANE
- Outside the peptidoglycan layer is an additional outer membrane
- Contains proteins, phospholipids, and lipopolysaccharide (LPS)
 LIPOPOLYSACCHARIDE (LPS)
 Three regions of Lipopolysaccharide (LPS)
1. ANTIGENIC O – SPECIFIC POLYSACCARIDE
 Helps in distinguishing species of gram negative
2. CORE POLYSACCARIDE
 Composed of ketodeoxyoctanoic acid (KDO) and heptose
3. INNER LIPID A
 Liquid portion of the LPS
  Also called endotoxin
 Responsible for producing fever and shock conditions in patients infected with gram
negative bacteria
 Beta – hydroxy myristic acid
- Unique to Lipid A
-
 LPS Functions
 Vital in evading the host defenses
 Contribute to the negative charge of the bacterial surface, which stabilizes the membrane
structure
 Considered as an endotoxin
c.) ACID-FAST CELL WALL
 Have a gram-positive cell wall structure
 Contain a waxy layer of glycolipids and fatty acid (mycolic acid) bound to the exterior of the cell wall
 More than 60% of the cell wall is lipd
MYCOLIC ACID
 Prevents uptake of the dye
 Major lipid component
 Strong “hydrophobic” molecule that forms a lipid shell around the organism and affects its permeability
 Makes mycobacterium spp. Difficult to stain with the Gram stain
Mycobacterium and Nocardia
 Stain a faint blue (gram-positive) color
 Best stained with an acid-fast stain
d.) ABSENCE OF CELL WALL
 Lack a cell wall and contain sterols in their cell membranes
 Lack the rigidity of the cell wall
 Seen in various shapes microscopically (ex. Mycoplasma and ureaplasma)

PERPLASMIC SPACE
 Typically found only in gram-negative bacteria
 Bounded by the internal surface of the outer membrane and the external surface of the cellular membrane
encompassing the thin peptidoglycan layer
 Contains the murein layer, consists gilllike matrix containing nutrient-binding proteins that assist in the
capture of nutrients from the environment
 Contains several enzymes involved in the degradation of macromolecules and detoxification of environmental
solutes, including antibiotics that enter through the outer membrane
 Hydrolytic enzymes – alkaline phosphatase and 5’ nucleosidase (inactivates antibiotics)
 Detoxifying - beta lactamase, amino glycoside phosphorylase (inactivates antibiotics)
 Periplasmic space is absent in gram-positive bacteria
CYTOPLASMIC (INNER MEMBRANE)
 Present in both gram-positive and gram-negative bacteria and I the deepest layer of the cell envelope
 Consist of phospholipid bilayer, various proteins (70%), including a number of enzymes vital to cellular
metabolism
 Serves as an additional osmotic barrier absence of sterols

Exceptions:
 Incorporate sterols (e.g., cholesterol), into their membranes when growing in sterol-containing media