Bio 120 lecture 3 2011 2012

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

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

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

TYPES OF MICROSCOPES

•Brightfield versus Darkfield

•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

• 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

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

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 FissionParungao-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

PLASMA MEMBRANE

Phospholipid

bilayer

Peripheral

proteins

Integral proteins

Transmembrane

proteins

PLASMA MEMBRANE

Membrane is as

viscous as olive

Proteins move to

function.

Phospholipids

rotate

and move laterally.Tuesday, July 5, 2011

PLASMA MEMBRANE

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

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: OSMOSIS

Osmosis: The movement of water

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

INCLUSION BODIES

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

INCLUSION BODIES

RIBOSOMES

16S ribosomal DNA = prokaryotes

18S ribosomal DNA = eukaryotes

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

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)

Thick peptidoglycan

Teichoic acids In acid-fast cells, contains mycolic acid

PEPTIDOGLYCAN (PG)

Thin peptidoglycan

No teichoic acids

Outer membraneTuesday, July 5, 2011

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

L forms (reversible) are wall-less cells that swell

into irregular shapes.

Protoplasts and spheroplasts are susceptible to

osmotic lysis.

PROTOPLASTS

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

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

Anchored to the wall

and membrane by the

basal bodyTuesday, July 5, 2011

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

•Functional Anatomy of Prokaryotes

•Functional Anatomy of Eukaryotes

FUNCTIONAL ANATOMY OF EUKARYOTES

PROKARYOTES VERSUS EUKARYOTES

Bio 120 lecture 3 2011 2012

prokaryotes

cells functional

cell walls

functional

plasma membrane

gram

motility parungao

fimbriae amp

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