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MICROBIAL CELL BIOLOGYBiology 120 Lecture 3
Reference: Chapter 3 and 4 TORTORA
Parungao-Balolong 2011Tuesday, July 5, 2011
LECTURE OUTLINE
•Observation of Microorganisms
•Size, Shape and arrangement of Cells
•Functional Anatomy of Prokaryotes
•Functional Anatomy of Eukaryotes
Parungao-Balolong 2011Tuesday, July 5, 2011
OBSERVATION OF MICROORGANISMS•Units of
Measure
• 1 µm = 10–6 m = 10–3 mm
• 1 nm = 10–9 m = 10–6 mm
• 1000 nm = 1 µm
• 0.001 µm = 1 nm
Parungao-Balolong 2011Tuesday, July 5, 2011
OBSERVATION OF MICROORGANISMS
• Simple Microscope (1 lens)
• Compound Microscope ( more than 1 lens)
Parungao-Balolong 2011• Magnification differsTuesday, July 5, 2011
OBSERVATION OF MICROORGANISMS
•RECALL: Microscopy!
•Resolution
•Refractive Index
•Immersion Oil
Parungao-Balolong 2011Tuesday, July 5, 2011
TYPES OF MICROSCOPES
•Brightfield versus Darkfield
Parungao-Balolong 2011Tuesday, July 5, 2011
TYPES OF MICROSCOPES
•Phase Contrast
•accentuates diffraction of light that passes through the specimen
•Differential Interference Contrast
•uses two beams of light
Parungao-Balolong 2011Tuesday, July 5, 2011
TYPES OF MICROSCOPES
• Fluorescence
•fluorescent substance absorbs UV light and emit visible light
•Confocal
• laser illuminates each plane in a specimen (3D image)
• *** fluorochromes
Parungao-Balolong 2011Tuesday, July 5, 2011
TYPES OF MICROSCOPES
•Measures sound waves that are reflected back from an object
• For microbial cells on surface
Parungao-Balolong 2011Tuesday, July 5, 2011
ELECTRON MICROSCOPY
•Transmission Electron Microscope (Res 2.5nm)
• Scanning Electron Microscope (Res 20nm)
Parungao-Balolong 2011Tuesday, July 5, 2011
PREPARING SPECIMENS: WET MOUNT
•Wet Mount
• viable
• heat fixing not done
Parungao-Balolong 2011Tuesday, July 5, 2011
WET MOUNT
Parungao-Balolong 2011Tuesday, July 5, 2011
STAINING SPECIMENS•Stained
•dead
•fixation (heat or ethanol)
•Smear
• Simple
•Differential
• Special
Parungao-Balolong 2011Tuesday, July 5, 2011
SMEAR PREPARATION
FROM COLONIES OR
BROTH CULTURE
Parungao-Balolong 2011Tuesday, July 5, 2011
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
Parungao-Balolong 2011Tuesday, July 5, 2011
TYPES OF FIXATION
•Heat-fixation
• By flaming air-dried smear gently
• Adequately preserves overall morphology but not structures within cells
•Chemical fixation
• Used to protect fine cellular substructure; morphology of larger, more delicate microbes
• Penetrates cells and react with cellular components (proteins and lipids)
• Example: alcohol, formaldehyde, acetic
Parungao-Balolong 2011Tuesday, July 5, 2011
STAINING BACTERIA
Parungao-Balolong 2011
Simple staining
Acid-Fast staining
Gram staining
Capsule staining
Flagella staining
Tuesday, July 5, 2011
SIZE, SHAPE & ARRANGEMENT OF CELLS
•Average Size: 0.2um-1.0um x 2-8um
•Shape Based on:
•Genetics
•monomorphism
•uniqueness for each bacteria
•Environmental Factors
•pleiomorphism
•devoid of cell wall
Parungao-Balolong 2011Tuesday, July 5, 2011
SIZE, SHAPE & ARRANGEMENT OF CELLS
Parungao-Balolong 2011Tuesday, July 5, 2011
SIZE, SHAPE & ARRANGEMENT OF CELLS
Parungao-Balolong 2011Tuesday, July 5, 2011
SIZE, SHAPE & ARRANGEMENT OF CELLS
Parungao-Balolong 2011Tuesday, July 5, 2011
SIZE, SHAPE & ARRANGEMENT OF CELLS
Parungao-Balolong 2011Tuesday, July 5, 2011
SIZE, SHAPE & ARRANGEMENT OF CELLS
Parungao-Balolong 2011Tuesday, July 5, 2011
SIZE, SHAPE & ARRANGEMENT OF CELLS
Parungao-Balolong 2011
Stella sp. Haloarcula sp.
Tuesday, July 5, 2011
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 FissionParungao-Balolong 2011
Tuesday, July 5, 2011
FUNCTIONAL ANATOMY OF PROKARYOTES
Parungao-Balolong 2011Tuesday, July 5, 2011
FUNCTIONAL ANATOMY OF PROKARYOTES
Parungao-Balolong 2011
Plasma Membrane
Selectively permeable barrier, mechanical 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
Tuesday, July 5, 2011
PLASMA MEMBRANE
Parungao-Balolong 2011
Phospholipid
bilayer
Peripheral
proteins
Integral proteins
Transmembrane
proteins
Tuesday, July 5, 2011
PLASMA MEMBRANE
Parungao-Balolong 2011
Membrane is as
viscous as olive
oil.
Proteins move to
function.
Phospholipids
rotate
and move laterally.Tuesday, July 5, 2011
PLASMA MEMBRANE
Parungao-Balolong 2011
Selective permeability allows passage of some molecules
Enzymes for ATP production
Photosynthetic pigments on foldings called chromatophores or
thylakoids; chlorosomes; photosynthetic lamellae
purple non-sulfur bacteria
purple sulfur bacteria
green sulfur bacteria
Tuesday, July 5, 2011
PLASMA MEMBRANE
Parungao-Balolong 2011
Damage to the membrane by alcohols,
quaternary ammonium (detergents),
and polymyxin antibiotics causes
leakage of cell contents.
Tuesday, July 5, 2011
PLASMA MEMBRANE
Parungao-Balolong 2011
EUBACTERIA
Ester linkage
Weaker linkage
ARCHAEBACTERIA
Ether linkage
Stronger linkage
NOTE: POINT OF LINKAGE = POINT OF BREAKDOWN
Tuesday, July 5, 2011
MEMBRANE TRANSPORT: RECALL
Parungao-Balolong 2011
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.
Tuesday, July 5, 2011
MEMBRANE TRANSPORT: RECALL
Parungao-Balolong 2011Tuesday, July 5, 2011
MEMBRANE TRANSPORT: OSMOSIS
Parungao-Balolong 2011
Osmosis: The movement of water
Osmotic pressure: The pressure needed to stop the movement of water across the membrane.
Tuesday, July 5, 2011
MEMBRANE TRANSPORT: RECALL
Parungao-Balolong 2011Tuesday, July 5, 2011
INCLUSION BODIES
Parungao-Balolong 2011
Metachromatic granules (volutin)
Phosphate reservesCorynebacterium diptheria
Polysaccharide granulesExample: Glycogen
Energy reserves
Lipid inclusions (PHBs) Energy reservesMycobacterium, Bacillus, Azotobacter
Sulfur granules Energy reservesCorynebacterium diptheria
Carboxysomes Ribulose 1,5-diphosphate carboxylase for CO2 fixationNitrifying Bacteria
Gas vacuoles/Gas vesicles Protein covered cylinders
Magnetosomes Iron oxide (destroys H2O2) Aquaspirillum magnetotacticum
Tuesday, July 5, 2011
INCLUSION BODIES
Parungao-Balolong 2011Tuesday, July 5, 2011
RIBOSOMES
Parungao-Balolong 2011
16S ribosomal DNA = prokaryotes
18S ribosomal DNA = eukaryotes
Tuesday, July 5, 2011
FUNCTIONAL ANATOMY OF PROKARYOTES
Parungao-Balolong 2011
Nucleoid Localization of Genetic Material (DNA)
Periplasmic Space
Contains hydrolytic enzymes and binding proteins for nutrient processing and uptake
Cell Wall Gives bacteria shape and protection from lysis in dilute solutions
Tuesday, July 5, 2011
PERIPLASMIC SPACE/PERIPLASM
Parungao-Balolong 2011
• 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
Tuesday, July 5, 2011
CELL WALL
Parungao-Balolong 2011
Prevents osmotic lysis
Made of peptidoglycan (in bacteria)
Tuesday, July 5, 2011
PEPTIDOGLYCAN (PG)
Parungao-Balolong 2011
Polymer of disaccharide
N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)
Linked by polypeptides
Tuesday, July 5, 2011
PEPTIDOGLYCAN (PG)
Parungao-Balolong 2011Tuesday, July 5, 2011
PEPTIDOGLYCAN (PG)
Parungao-Balolong 2011
Thick peptidoglycan
Teichoic acids In acid-fast cells, contains mycolic acid
Tuesday, July 5, 2011
PEPTIDOGLYCAN (PG)
Parungao-Balolong 2011
Thin peptidoglycan
No teichoic acids
Outer membraneTuesday, July 5, 2011
TEICHOIC ACIDS : G+
Parungao-Balolong 2011
Lipoteichoic acid links
to plasma membrane
Wall teichoic acid links
to peptidoglycan
May regulate
movement of cations
Polysaccharides
provide antigenic
variation
Tuesday, July 5, 2011
OUTER MEMBRANE : G-
Parungao-Balolong 2011
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.
Tuesday, July 5, 2011
CELL WALLS & GRAM STAIN
Parungao-Balolong 2011
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
Tuesday, July 5, 2011
CELL WALLS & GRAM STAIN
Parungao-Balolong 2011Tuesday, July 5, 2011
GRAM + VS GRAM -
Parungao-Balolong 2011Tuesday, July 5, 2011
GRAM + VS GRAM -
Parungao-Balolong 2011Tuesday, July 5, 2011
WHEN GRAM STAIN NOT APPLICABLE
Parungao-Balolong 2011
Mycoplasmas
Lack cell walls
Sterols in plasma
membrane
Archaea
Wall-less or
Walls of
pseudomurein (lack
NAM and D amino
Tuesday, July 5, 2011
TARGETING THE CELL WALL
Parungao-Balolong 2011
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.
Tuesday, July 5, 2011
PROTOPLASTS
Parungao-Balolong 2011Tuesday, July 5, 2011
FUNCTIONAL ANATOMY OF PROKARYOTES
Parungao-Balolong 2011
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
Tuesday, July 5, 2011
FIMBRIAE & PILI
Parungao-Balolong 2011
Fimbriae allow
attachment
Pili are used to
transfer DNA from
one cell to another
Tuesday, July 5, 2011
FIMBRIAE & PILI
Parungao-Balolong 2011Tuesday, July 5, 2011
THE F PLASMID
Parungao-Balolong 2011Tuesday, July 5, 2011
ENDOSPORE
Parungao-Balolong 2011
Resting cells
Resistant to desiccation,
heat, chemicals
Bacillus, Clostridium
Sporulation: Endospore
formation
Germination: Return to
vegetative state
Tuesday, July 5, 2011
Parungao-Balolong 2011Tuesday, July 5, 2011
Parungao-Balolong 2011Tuesday, July 5, 2011
ENDOSPORE RESISTANCE
Parungao-Balolong 2011
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
Tuesday, July 5, 2011
OUTSIDE THE WALL: GLYCOCALYX
Parungao-Balolong 2011
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
Tuesday, July 5, 2011
OUTSIDE THE WALL: GLYCOCALYX
Parungao-Balolong 2011
Capsule Staining
Tuesday, July 5, 2011
OUTSIDE THE CELL WALL : FLAGELLA
Parungao-Balolong 2011
Outside cell wall
Made of chains of
flagellin
Attached to a protein
hook
Anchored to the wall
and membrane by the
basal bodyTuesday, July 5, 2011
OUTSIDE THE CELL WALL : FLAGELLA
Parungao-Balolong 2011Tuesday, July 5, 2011
OUTSIDE THE CELL WALL : FLAGELLA
Parungao-Balolong 2011Tuesday, July 5, 2011
MOTILITY
Parungao-Balolong 2011
Rotate flagella to run or tumble
Move toward or away from stimuli (taxis)
Flagella proteins are H antigens
(e.g., E. coli O157:H7)
Tuesday, July 5, 2011
MOTILITY
Parungao-Balolong 2011Tuesday, July 5, 2011
MOTILITY
Parungao-Balolong 2011Tuesday, July 5, 2011
CYTOPLASM
Parungao-Balolong 2011
Cytoplasm is the substance inside the
plasma membrane.
Nuclear area (nucleoid)
Tuesday, July 5, 2011
FUNCTIONAL ANATOMY OF EUKARYOTES
•Observation of Microorganisms
•Size, Shape and arrangement of Cells
•Functional Anatomy of Prokaryotes
•Functional Anatomy of Eukaryotes
Parungao-Balolong 2011Tuesday, July 5, 2011
FUNCTIONAL ANATOMY OF EUKARYOTES
Parungao-Balolong 2011Tuesday, July 5, 2011
PROKARYOTES VERSUS EUKARYOTES
Parungao-Balolong 2011Tuesday, July 5, 2011
PROKARYOTES VERSUS EUKARYOTES
Parungao-Balolong 2011Tuesday, July 5, 2011