MICROBIOLOGY Chapter 3

Microscopy

Why do we use stains?

Bacteria are
colorless very small
just barely visible under the 1000X

Observe size and shape more easily

Simple Stains
One dye ---> one color Determine morphology and arrangement

BASIC dye which has a POSITIVE charge that is attracted to the slightly negative charge of the cell wall
Acidic dyes have a NEGATIVE charge and are repelled by the negative charge of the cell wall. Bacteria appear white/clear in a colored background

Differential Stain
Involves at least 2 Different Colored Dyes Used to distinguish between cell structures Cell wall content
Gram-positive vs. gram negative cells Acid fast vs. non acid fast cells

Interior cell structures

endospores

3 Most Commonly Used Differential Stains

Gram Stain
a thick peptidoglycan cell wall (Gram +) (Gram -) a cell wall that has both an inner and outer membrane, but less peptidoglycan.

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Acid Fast Stain

Cell walls that contain mycolic acid (+) tuberculosis, Mycobacterium leprae Mycobacterium

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Endospore Stain
Heat is used to force stain into the endospore a survival structure for some bacteria genera Bacillus (Bacillus anthracis) and Clostridium (Clostridium tetanii, C. botulinum)

GRAM STAIN

Acid Fast Bacilli Mycobacterium tuberculosis

MICROBIOLOGY Chapter 4
Cell Structure and Function

Prokaryotes
Bacteria no membrane-bound nucleus ONE circular chromosome Cell walls contain peptidoglycan Divide by BINARY FISSION Lack membrane-enclosed organelles DNA is not associated with histones

Size, Shape and Arrangement of Bacterial Cells

Prokaryotic Cell Structures

Structures External to the Cell Wall

Glycocalyx protects cell from dehydration Polysaccharides and/or polypeptides Sticky Capsule - well organized - contributes to VIRULENCE
*Protect bacteria from the host immune system stealth bomber

Slime layer - loosely attached, unorganized EPS - extracellular polysaccharide(sugar) allows bacteria to attach to environmental surfaces

Flagella
Virulence factor 3 parts Filament, hook, basal body
flagellin protein H antigens in typing bacteria, E. coli O157:H7

Function Motility Taxis movement toward(+) or away from (-) a stimulus Chemotaxis Phototaxis

Flagella

Fimbriae
Attachment to other cells and surfaces VIRULENCE factor Neisseria gonorrheae

CELL WALL

Gram-negative cell wall

Lipopolysaccharide (LPS) component
Lipid A
ENDOTOXIN (VIRULENCE FACTOR)
FEVER SHOCK

Polysaccharide portion - O antigen

Used to type bacteria, e.g. E. coli O157:H7

Homework
List at least 5 differences that you can use to distinguish gram-positive cell walls from gram-negative cell walls. See Table 4.1

STRUCTURES INTERNAL TO THE CELL WALL

Plasma Membrane (inner membrane) Fluid Mosaic Model composed of Phospholipids and Proteins Phospholipid bilayer
Water soluble head end (phosphate) Hydrophobic hydrocarbon tails

Proteins
peripheral integral

Cell Membrane Functions

Selective Barrier
Selectively Permeable to: Small molecules Lipid soluble substances Large molecules, e.g. proteins cannot pass through Transporter molecules have a role

Enzymes attached to the cell membrane breakdown of nutrients Energy production

Simple Diffusion

Facilitated Diffusion uses transporter proteins (permeases)

OSMOSIS

Passive Processes
Do not require Energy
Diffusion Facilitated Diffusion Osmosis

Active Processes
Active Transport
Requires the expenditure of ATP Movement of substances from low concentration to high concentration

Group Translocation - only in prokaryotes

Requires the use of PEP (high E compound) Changes the chemistry of a substance as it is brought into the cell.

Cytoplasm
Thick, semitransparent, aqueous (80% water) gel
Substances dissolved in the cytoplasm include proteins, carbohydrates, lipids, inorganic ions. Found within the confines of the cell membrane Contains: Nucleoid Ribosomes Inclusions

Nucleoid
Bacteria have a single, circular chromosome that contains all the info required for the structure and function of that cell ******************************************************** Plasmids small, circular, extrachromasomal piece of DNA 5-100 genes May code for toxins (diphtheria) or antibiotic resistance

Ribosomes
Site of protein synthesis 70 S differ from eukaryotic ribosomes (80S) Target for antibiotics
Streptomycin and gentamicin attach to the 30S subunit Erythromycin and chloramphenicol attach to the 50S subunit

Inclusions
Reserve deposits of substances stored in the cytoplasm Storage of nutrients
polysaccharide granules lipids sulfur

Gas vacuoles - buoyancy

Endospores
Sporulation/sporogenesis
Germination Clostridium and Bacillus genera

Prokaryotic Cell vs. Eukaryotic Cell

Study Table 4.2

List at least 5 properties you could use to distinguish a prokaryotic cell from a eukaryotic cell.

Eukaryotic Cell

Flagella and Cilia

Function is motility
9 pairs of microtubules in a ring + one central paid of microtubules
Microtubules are composed of tubulin

Flagella are long and few in number Cilia are short and numerous

Cell Wall and Glycocalyx

Cell Wall
Plants and some fungi - cellulose Most fungi - chitin Yeast cell - glucan and mannan Animal cells have a glycocalyx provides support means of attachment to other cells LACK A CELL WALL

Plasma Membrane
Phospholipid bilayer containing proteins
Carbohydrates are attached to the proteins of the cell membrane Contains sterols

Movement across the membrane is by osmosis, diffusion, facilitated diffusion, active transport, pinocytosis and phagocytosis

Cytoplasm
Gel between the cell membrane and the nuclear membrane
The following organelles are found within the cytoplasm: nucleus, endoplasmic reticulum, ribosomes, Golgi complex, lysosomes, vacuoles, mitochondria, chloroplasts, peroxisomes, centrosomes

Nucleus
Spherical to ovoid Contains the cells DNA combined with histone proteins Surrounded by the nuclear envelope Nucleoli are located within the nucleus and are sites for ribosomal RNA synthesis Cell division in eukaryotes involves mitosis and meiosis

Endoplasmic Reticulum
Network of tubules throughout the cytoplasm of a cell. Rough ER has ribosomes attached to its outer membrane surface
synthesizes secretory proteins and membrane molecules continuous with the nuclear membrane

Smooth ER lacks ribosomes

synthesizes fats, steroids and phospholipids

Ribosomes
May be found free in the cytoplasm or attached to the outer surface of the rough ER
Site of protein synthesis in the cell Consists of a 60S and a 40S subunit

Golgi Complex
Composed of flattened sacs, cisterns Functions
Membrane formation and Protein secretion

Golgi complex continued

Lysosomes
Formed from Golgi complexes Contain digestive enzymes

Vacuoles
Membrane enclosed cavities Derived from Golgi complexes or endocytosis Function is often storage
In plants vacuoles are often very large and may contain nutrients, such as starch

Mitochondria
Site of ATP production
cristae Matrix

Contain 70S ribosomes and DNA Multiply by binary fission inherited only from the cytoplasm of the mothers ova maternal lines can be studied based on mitochondrial DNA

Chloroplast

in algae and green plants

contains chlorophyll and the enzymes necessary for photosynthesis Contain 70S RNA and their own DNA Replicate by binary fission independent of the nucleus

Endosymbiotic Theory
Evidence that some organelles arose from engulfed prokaryotes
Chloroplasts and mitochondria contain 70S ribosomes Chloroplasts and mitochondria contain their own DNA Chloroplasts and mitochondria replicate by binary fission and their replication is independent of the nucleus