Chap. 3- Microscopy

what is a light microscope?

• any kind of microscope that uses visible light to observe a specimen

• compound light microscope- uses 2 lenses to magnify the image

what is the ocular lens?

• the eyepiece

  • magnifies by 10x

the objective lens?

• lens closest to the specimen

  • magnifies b/w 10x-100x

• total magnification = ocular lens x objective lens

specimen stage

• stand, holds the ‘glass card’

condenser lens

• focuses the light

iris diaphragm lever

• controls the amount of light that enters the objective lens 

rheostat

controls the brightness of the light

resolution

• ability to distinguish fine detail and structure

• distinguish 2 points a certain distance apart

  • resolving power of 4nm

  • 2 points can be distinguished if they are at least 4 nm apart

• light has to pass b/w 2 objects for them to be seen as 2 separate things

• need light of a short-enough wavelength to fit b/w them, or will appear as 1 object

• general principle: the shorter the wavelength, the better the resolution

electron microscope

• uses electrons instead of light

  • electrons travel in much shorter waves than light

  • resolving power is greater

  • greater magnification (up to 500 000x)

  • view viruses and internal cell structures

what are 2 types of electron microscope?

• transmission (TEM)- internal structures

  • very thin slices can be cut from sample

  • samples generally stained with a metal (osmium, uranium) to make structures opaque to electrons

• scanning (SEM)- surfaces, less powerful

atomic force (AFM)

• to see molecules

• uses thin metal probe to scan a specimen, revealing bumps and depression

viewing objects- humans vs microscopes

• human eye can see an object about 0.2nm

• compound light microscope → 0.2um

• electron microscope → 2nm

why do we stain microorganisms?

• microorganisms are colourless when seen through a microscope, we use stains to prepare for viewing

• stains are composed to positively and negatively charged ions, one of which is coloured- chromophore

• simple stain- only 1 dye used to highlight the entire microorganism

steps to adding stain on microorganism

1. smear sample on slide

2. fix with heat

3. add stain

4. wash,dry and view

how do the stains work?

• bacteria have a net neg. charge on their outer surface

  • charge attracts stains w/ positively charged chromophores and repels stains w negatively charged chromophores

• positive stains

  • will bind to the bacterium

  • bacterium appears coloured

  • background appears clear

    • ex.) crystal violet

• negative stains

  • will NOT bind to the bacterium

  • bacterium appears clear

  • background is coloured

    • ex.) nigrosin, india ink

• different stains, react differently with different bacteria, thus can be used to distinguish b/w them

  • ex.) gram stain

gram stain

• differentiates bacteria based on structure of the cell wall

• bacteria w a thick cell wall retain the primary stain crystal violet and appear purple- gram positive

  • ex.) Streptococcus pyogenes

• bacteria w a thin cell wall lose crystal violet during destaining, take on the colour of the counterstain safranin and appear pink- gram negative

  • ex.) E.coli

• spore stain and flagella stain

spore stain

• stains an internal structure of some bacteria

• primary stain colours endospores green

• counterstain (safranin) colours thr rest of cell red (pink)

  • ex.) Bacillus anthracis

flagella stain

• stains an external structure

• mordant thickens the flagella so they can be observed under light microscope

acid-fast stain

• detects the presence of a waxy compound in cell wall

• used to identify the genus Mycobacterium

  • ex.) Mycobacterium tuberculosis, Mycobacterium leprae

• Mycobacterium cell wall retains the dye carbol fuchsin

• counterstain with methylene blue stains non acid-fast bacteria and tissues blue

capsule stain

• detects a thick layer of polysaccharide outside the cell- capsule

  • negative stain colours the background

  • positive stain colours the cell

  • the capsule does not take up most dyes and remains colourless

    • ex.) Streptococcus pneumoniae

prokaryote

• DNA is not enclosed within a nucleus

• usually DNA is arranged as one circular chromosome

• they lack membrane-enclosed organelles

• single celled organisms: Bacteria, Archaea

eukaryotes

• DNA is found in nucleus- surrounded by nuclear membrane

• DNA arranged as multiple chromosomes

• the have membrane-enclosed organelles

• single celled or multicellular organisms

  • ex.) Algae, Protozoa. Fungi. Plants. Animals

types of bacteria

morphology (shape)

• Coccus/ Cocci - spherical

• Bacillus/ Bacilli - rod shaped

• vibrio - curved

• Spirillum/ Spirilla - spiral shaped

• Spirochete - corkscrew shaped

capsule and slime layers

• sticky, gelatinous layer external to the cell

• composed of polysaccharide, protein or both

• capsule- layer is organized and firmly attached to the cell wall

  • protection against phagocytosis

    • ex.) Streptococcus pneumoniae

      • with a capsule: causes disease

      • w/o capsule: no disease

• slime layer - layer is unorganized and loosely attached to the cell wall

slime layers often allow bacteria to attach to surfaces

• medical implants, water pipes, teeth

  • ex.) Streptococcus mutans

    • makes polysaccharide slime from sucrose

    • attaches to teeth, can lead to cavities

what is a flagella?

• long protein appendages

• used in motility,

• is semi-rigid, helical, turns like a propeller

bacteria cells have 4 typical arrangements of flagella

• Monotrichous- a single polar flagellum

• Lophitrichous- two or more flagella orginating from one pole

• Amphitrichous- tufts of flagella orginating from one pole

• Peritrichous- flagella distributed all over the cell

flagellar motility

• flagella turn causing call to move in one direction- “run”

• reverse direction causing a random change in direction- “tumble”

in what way does flagella allow chemotaxis?

• movement toward or away from a stimulant

• toward a nutrient (attractant)

• away from a toxin (repellent)

  • ex.) E.coli will move toward glucose

what are fimbriae and pili?

• short, hair-like appendages

• hollow

fimbriae

• allow the cell to adhere to surfaces

• contribute to pathogenicity

  • ex.) some strains of E.coli have fimbriae that allow them to attach to the intestinal wall

pili

• allows attachment of two bacteria to each other

• involved in transfer of genetic material b/w bacteria

  • ex.) E.coli’s sex pilus

bacterial cell wall

• semi-rigid structure giving shape to the cell

• major function is to prevent rupture of the cell- protects against environmental changes

• useful in the identification of bacteria- ie. the gram stain

• composed of the complex macromolecule: PeptidoGlycan

peptidoglycan

• mesh-like structure composed of amino acids and polysaccharide

• polysaccharide portion is composed of two alternating monosaccharides

  • N-acetyl glucosamine (NAG)

  • N-acetyl muramic acid (NAM)

• peptide portion composed of short chains of amino acids

a generalized view of peptidoglycan

• polysaccharide chains run parallel

• peptide chains link polysaccharides together

• forms a mesh-like net surrounding the cell

the gram positive cell wall

• made of thick layers of peptidoglycan outside of plasma membrane

  • also contains teichoic acids

    • wall teichoic acids: attached to the peptidoglycan

    • lipoteichoic acids: attached to plasma membrane and extend through the peptidoglycan

• gram positive bacteria have only one membrane → cytoplasmic membrane

the gram negative cell wall

• thin peptidoglycan layer that is sandwiched b/w two membranes

• outer membrane made of lipids (phospholipids), proteins and lipopolysaccharides (LPS)

• polysaccharide portion of LPS is composed of O-sugars

  • useful for distinguishing gram negative bacteria

  • ex.) E.coli O157:H7

• lipid portion of LPS is toxic

• referred to as endotoxin

how does the gram stain works?

gram positive cells

• thick peptidoglycan traps the crystal violet- stain purple

gram negative cells

• thin peptidoglycan does not trap crystal violet, and the outer membrane gets disrupted by alcohol

• crystal violet is washed away

• safranin counterstain stains the cells pink

what is so special about peptidoglycan?

• completely different from anything found in animal cells

• many antibiotics have been discovered that act against peptidoglycan

  • ex.) penicillin- inhibits production of peptidoglycan

  • also degraded by one of our own natural defenses: lysozyme

    • found in tears, saliva, mucous

the cytoplasmic membrane

• composed of a phospholipid bilayer

• separates the interior (cytoplasm) from the outside environment

  • serves as a semi-permeable barrier

  • selectively allows inflow and outflow of materials

• exists in a semi-fluid state

antimicrobial agents

• alcohols disrupt the membrane

• can be used as a disinfectant

internal components cytoplasm

• the substance inside the plasma membrane

  • about 80% water

  • contains most of the ‘stuff’ needed for life

    • sugars, amino acids, nucleotides, etc

    • enzymes

    • some functional structures

the nucleoid

• contains the bacterial chromosome (DNA)

  • all genetic info required for cell’s structures and functions

• not surrounded by a nuclear membrane

• may also contain plasmids:

  • smaller double stranded DNA molecules

  • contain non-essential genes

    • ex.) genes for antibiotic resistance

ribosomes

• site of protein synthesis (translation)

• made of protein and ribosomal RNA (rRNA)

• ribosomes of bacteria differ from eukaryotic ribosome

  • several antibiotics target bacterial ribosomes

    • ex.) Streptomycin, Erythromycin

    • prevent bacteria from making new proteins

what is the size of bacteria ribosome?

two parts:

• 30S subunit

• 50S subunit

• together form the complete 70S ribosome

what is the size of eukaryotic ribosome?

80S ribosome

what are some antibiotics that target bacterial ribosomes?

streptomycin and erythromycin- prevent the bacteria from making new proteins

storage granules (inclusion bodies) + examples

• usually deposits or granules of nutrients, stored for later use

• ex.)

  • sulfur granules

  • polysaccharides (glycogen)

  • lipid inclusion

  • enzymes

  • magnetite

• variety of inclusion bodies occur in different bacterial species- can serve as a basis for identification

what are endospores?

• formed mostly by some gram-positive bacteria

• special resting structure- allows bacteria to enter dormant state

• remains dormant until good growth conditions occur

  • can form new population

in what way are endospores durable?

• resists heat, desiccation, chemicals, radiation

• some endospores can survive in boiling water for hours

sporulation (steps)

1. cell replicates its DNA

2. septum forms, dividing the cell into unequal compartments

3. larger compartment engulfs the smaller

4. peptidoglycan and other protective material forms around the forespore

• spore coat

1. finished spore is freed from the mother cell as the mother cell dies

microorganisms in eukaryotic cell

• algae

• fungi

• protozoa

structure of eukaryotic cell

• higher organisms: plants and animals

  • larger and more complex than prokaryotes

  • genetic material is housed in a nucleus

  • membrane bound organelles

cytoplasmic membrane

• same basic structure as in prokaryotic cells

• contains phospholipids, proteins and

  • sterols make membrane relatively rigid compared to bacteria

what is cell wall made of?

• cellulose (algae, plants)

• chitin (fungi, insects)

cell wall

• not all eukaryotes have a cell wall

  • allows endocytosis

• simple structure compared to bacteria

what is the cytoplasm?

• substance inside the plasma membrane, but outside the nuclear membrane

• cytoplasm has complex internal structure- cytoskeleton

  • proteins filaments on the inside of the plasma membrane

  • provides support and shape

  • transports substance through the cell

membrane bound organelles

• structures with specialized function

• not present in bacteria

what are examples of membrane bound organelles?

• nucleus- compartment holding genetic material

• mitochondria- site of energy production

• chloroplasts- site of photosynthesis in algae and plant cells

flagellum and cilia

• long flexible projections that contain protein and cytoplasm

• move in a whip-like fashion

what can flagellum and cilia be used for?

• motility 

• sweeping material past stationary cells

prokaryotic flagella

• made of protein subunits

• protrude through cell wall and cell membrane

• stiff, twirl like a propeller

eukaryotic flagella

• a bundle (9+2) of microtubules- made of protein

• covered by cell membrane

• whipping action