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

oil.

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)


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 -


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




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 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


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


PROKARYOTES VERSUS EUKARYOTES


Technology

Bio 120 lecture 3 2011 2012