Bio 120 lecture 3 2011 2012

MICROBIAL CELL
BIOLOGY
Biology 120 Lecture 3

Reference: Chapter 3 and 4 TORTORA

Parungao-Balolong 2011
Tuesday, July 5, 2011

LECTURE OUTLINE
• Observation of
Microorganisms

• Size, Shape and
arrangement of Cells

• Functional Anatomy
of Prokaryotes

of Eukaryotes Parungao-Balolong 2011

OBSERVATION OF
MICROORGANISMS
• Units of • 1000 nm = 1
Measure µm

• 1 µm = 10–6 m • 0.001 µm = 1
= 10 –3 mm nm

• 1 nm = 10 –9 m
= 10–6 mm


OBSERVATION OF
MICROORGANISMS
• Simple Microscope (1 • Compound Microscope
lens) ( more than 1 lens)

• Magniﬁcation differs

OBSERVATION OF
MICROORGANISMS

• RECALL: Microscopy!
• Resolution
• Refractive Index
• Immersion Oil

TYPES OF
MICROSCOPES

• Brightﬁeld
versus Darkﬁeld


TYPES OF MICROSCOPES

• Phase Contrast
• accentuates diffraction of
light that passes through
the specimen

• Differential Interference
Contrast

• uses two beams of light


• Fluorescence
• ﬂuorescent substance absorbs
UV light and emit visible light

• Confocal
• laser illuminates each plane in
a specimen (3D image)

• *** ﬂuorochromes


• Measures sound waves
that are reﬂected back
from an object

• For microbial cells on
surface


ELECTRON MICROSCOPY

• Transmission Electron Microscope (Res 2.5nm)
• Scanning Electron Microscope (Res 20nm)

PREPARING SPECIMENS: WET
MOUNT

• Wet Mount
• viable
• heat ﬁxing not
done


WET MOUNT


STAINING SPECIMENS
•Stained
•dead
•ﬁxation (heat or ethanol)
•Smear
• Simple
• Differential
• Special

SMEAR
PREPARATION
FROM
COLONIES OR
BROTH
CULTURE


FIXATION

• Process by which the internal and external structures
of cells and microorganisms are preserved and ﬁxed
in position

• Inactivates enzymes that might disrupt cell
morphology

• Toughens cell structures to prevent changes during
staining and observation

• Usually microbes are killed when ﬁxed


TYPES OF FIXATION
•Heat-fixation •Chemical fixation
• By flaming air-dried • Used to protect fine
smear gently cellular substructure;
morphology of larger,
• Adequately preserves more delicate microbes
overall morphology but
not structures within • Penetrates cells and
cells react with cellular
components (proteins
and lipids)

• Example: alcohol,
formaldehyde, acetic

STAINING BACTERIA
Acid-Fast staining

Simple staining

Capsule staining

Flagella staining
Gram staining

SIZE, SHAPE &
ARRANGEMENT OF CELLS
• Average Size: • Environmental
0.2um-1.0um x Factors
2-8um
• pleiomorphism
• Shape Based on:
• devoid of cell wall
• Genetics
• monomorphism
• uniqueness for
each bacteria Parungao-Balolong 2011

SIZE, SHAPE &


SIZE, SHAPE &

Stella sp. Haloarcula sp.


FUNCTIONAL ANATOMY
OF PROKARYOTES
• Basics of Prokaryotes
• One circular chromosome not enclosed in a
membrane

• no histones
• no organelles
• Cell walls with peptidoglycans
• Divides via Binary Fission Parungao-Balolong 2011

FUNCTIONAL ANATOMY
OF PROKARYOTES


FUNCTIONAL ANATOMY
OF PROKARYOTES
Plasma Selectively permeable barrier, mechanical
Membrane boundary of cell, nutrient and waste
transport, location of many metabolic
processes (respiration, photosynthesis),
detection of environmental cues for
chemotaxis

Gas Vacuole Buoyancy for ﬂoating in aquatic
environments
Ribosomes Protein synthesis

Inclusion Bodies Storage of carbon, phosphate and other
substances


PLASMA MEMBRANE

 Phospholipid
bilayer
 Peripheral
proteins
 Integral proteins
 Transmembrane
proteins


PLASMA MEMBRANE
 Membrane is as
viscous as olive
oil.
 Proteins move to
function.
 Phospholipids
rotate
and move laterally.

PLASMA MEMBRANE
 Selective permeability allows passage of some molecules
 Enzymes for ATP production
 Photosynthetic pigments on foldings called chromatophores or
thylakoids; chlorosomes; photosynthetic lamellae

green sulfur bacteria

purple sulfur bacteria

purple non-sulfur bacteria Parungao-Balolong 2011

PLASMA MEMBRANE
 Damage to the membrane by alcohols,
quaternary ammonium (detergents),
and polymyxin antibiotics causes
leakage of cell contents.


PLASMA MEMBRANE
 EUBACTERIA
 Ester linkage
 Weaker linkage
 ARCHAEBACTERIA
 Ether linkage
 Stronger linkage
 NOTE: POINT OF LINKAGE = POINT
OF BREAKDOWN

MEMBRANE
TRANSPORT: RECALL
 Simple diffusion:
Movement of a solute
from an area of high
concentration to an
area of low
concentration.
 Facilitative diffusion:
Solute combines with
a transporter protein
in the membrane.


MEMBRANE
TRANSPORT: RECALL


MEMBRANE
TRANSPORT: OSMOSIS

 

 Osmosis: The movement of water

 Osmotic pressure: The pressure needed to stop
the movement of water across the membrane.

INCLUSION BODIES
Metachromatic granules Phosphate reserves
(volutin)
Corynebacterium diptheria

Polysaccharide granules Energy reserves
Example: Glycogen

Lipid inclusions (PHBs) Energy reserves
Mycobacterium, Bacillus, Azotobacter

Sulfur granules Energy reserves
Corynebacterium diptheria

Carboxysomes
Ribulose 1,5-diphosphate carboxylase for CO2 ﬁxation
Nitrifying Bacteria

Gas vacuoles/Gas vesicles Protein covered cylinders

Magnetosomes Iron oxide
(destroys H2O2)
Aquaspirillum magnetotacticum


INCLUSION BODIES


RIBOSOMES

16S ribosomal DNA =
prokaryotes

18S ribosomal DNA =
eukaryotes Parungao-Balolong 2011

FUNCTIONAL ANATOMY
OF PROKARYOTES
Nucleoid Localization of Genetic Material
(DNA)

Periplasmic Contains hydrolytic enzymes and
Space binding proteins for nutrient
processing and uptake
Cell Wall Gives bacteria shape and
protection from lysis in dilute
solutions


PERIPLASMIC SPACE/
PERIPLASM
• Space between the
plasma membrane
and the outer
membrane

• 40% in G- minimal
in G+

• Loose PG (murein)
and some proteins

• For Gram +
counterpart are
exoenzymes


CELL WALL
 Prevents osmotic lysis
 Made of peptidoglycan (in bacteria)


PEPTIDOGLYCAN (PG)
 Polymer of disaccharide
N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)
 Linked by polypeptides


PEPTIDOGLYCAN (PG)


PEPTIDOGLYCAN (PG)

 Thick peptidoglycan
 Teichoic acids
 In acid-fast cells, contains mycolic acid

PEPTIDOGLYCAN (PG)

 Thin peptidoglycan
 No teichoic acids
 Outer membrane

TEICHOIC ACIDS : G+
 Lipoteichoic acid links
to plasma membrane
 Wall teichoic acid links
to peptidoglycan
 May regulate
movement of cations
 Polysaccharides
provide antigenic
variation

OUTER MEMBRANE : G-

 Lipopolysaccharides, lipoproteins, phospholipids
 Forms the periplasm between the outer membrane and the plasma
membrane.
 Protection from phagocytes, complement, and antibiotics
 O polysaccharide antigen, e.g., E. coli O157:H7
 Lipid A is an endotoxin
 Porins (proteins) form channels through membrane.

CELL WALLS & GRAM STAIN
 Crystal violet-iodine crystals form in cell.
 Gram-positive
Alcohol dehydrates peptidoglycan
CV-I crystals do not leave
 Gram-negative
Alcohol dissolves outer membrane and leaves
holes in peptidoglycan.
CV-I washes out


CELL WALLS & GRAM STAIN


GRAM + VS GRAM -


WHEN GRAM STAIN NOT APPLICABLE
 Mycoplasmas
 Lack cell walls
 Sterols in plasma
membrane
 Archaea
 Wall-less or
 Walls of
pseudomurein (lack
NAM and D amino

TARGETING THE CELL WALL
 Lysozyme digests disaccharide in peptidoglycan.
 Penicillin inhibits peptide bridges in peptidoglycan.
 Protoplast (complete) is a wall-less cell.
 Spheroplast (partial) is a wall-less Gram-positive
cell.
 L forms (reversible) are wall-less cells that swell
into irregular shapes.
 Protoplasts and spheroplasts are susceptible to
osmotic lysis.

PROTOPLASTS


FUNCTIONAL ANATOMY
OF PROKARYOTES
Capsules and Slime Layers Resistance to phagocytosis,
adherence to surfaces

Fimbriae and Pili Attachment to surfaces,
bacterial mating

Flagella Movement

Endospore Survival under harsh
environmental conditions


FIMBRIAE & PILI

 Fimbriae allow
attachment

 Pili are used to
transfer DNA from
one cell to another


FIMBRIAE & PILI


THE F PLASMID


ENDOSPORE
 Resting cells
 Resistant to desiccation,
heat, chemicals
 Bacillus, Clostridium
 Sporulation: Endospore
formation
 Germination: Return to
vegetative state


ENDOSPORE RESISTANCE
 15% of spore dry weight consists of dipicolinic acid complexed
with calcium ions
 DPA directly involved in spore heat resistance
 GENERA:
• Clostridium
• Bacillus
• Sporolactobacillus
• Sporosarcina


OUTSIDE THE WALL:
GLYCOCALYX
 Outside cell wall
 Usually sticky
 A capsule is neatly organized
 A slime layer is unorganized and
loose
 Extracellular polysaccharide allows
cell to attach
 Capsules prevent phagocytosis

OUTSIDE THE WALL:
GLYCOCALYX

Capsule Staining


OUTSIDE THE CELL
WALL : FLAGELLA
 Outside cell wall
 Made of chains of
flagellin
 Attached to a protein
hook
 Anchored to the wall
and membrane by the
basal body

OUTSIDE THE CELL
WALL : FLAGELLA


MOTILITY
 Rotate flagella to run or tumble
 Move toward or away from stimuli (taxis)
 Flagella proteins are H antigens
(e.g., E. coli O157:H7)


MOTILITY


CYTOPLASM
 Cytoplasm is the substance inside the
plasma membrane.

 Nuclear area (nucleoid)


FUNCTIONAL ANATOMY
OF EUKARYOTES
• Observation of
Microorganisms

• Size, Shape and
arrangement of Cells

of Prokaryotes

of Eukaryotes Parungao-Balolong 2011

FUNCTIONAL ANATOMY OF
EUKARYOTES


PROKARYOTES VERSUS
EUKARYOTES


Bio 120 lecture 3 2011 2012

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