Chapter 3 - Microscopy

What is a light microscope?

• Any microscope using visible light to observe a specimen

What is a compound light microscope?

• Microscope using two lenses to magnify the image

What are the parts of the light microscope? (6)

• Ocular Lens

• Objective Lens

• Specimen Stage

• Condenser Lens

• Iris Diaphragm Lever

• Light Source Rheostat

What is the ocular lens + function? (1.1)

• The eyepiece

  • magnifies by 10x

What is the objective lens?

• Lens closest to the specimen

  • Magnifies between 10x-100x

How do you calculate total magnification?

• Multiply ocular lens magnification by objective lens magnification

What is resolution and the minimum distance? (1.2)

• The ability to distinguish 2 points a certain distance apart

  • ex. fine detail + structure

  • has to be at least 4 nm apart

What is the resolution general principle?

• The shorter the wavelength, the better the resolution

What is an electron microscope?

• Uses electrons to observe a speciman instead of light

What is the benefit of using electrons to observe a specimen? (1.1)

• Electrons travel in much shorter wavelengths = greater magnification

  • up to 500,000x 

What is the function of an electron microscope?

• Allows us to view viruses + internal cell structures

What are the two types of electron microscopes?

• Transmission

• Scanning

What is the function of a transmission electron microscope?

• Used to see internal structures

What is the function of a scanning electron microscope?

• Used to see surfaces → less powerful microscope

What is the function of atomic force + how does it work?

• Used to see molecules

  • Uses a thin metal probe to scan a specimen → reveals bumps + depressions

What is the difference between the human eye vs compound light microscope + electron microscope? (3)

• Human eye sees objects about 0.2 mm

• Compound light microscope → 0.2 μm

• Electron microscope → 2 nm

What is the function of a stain? (1.1)

• Prepares the microorganism for view when using a microscope

  • microorganisms are colourless = need for stain

What are stains composed of? (2)

• Positively charged ions

• Negatively charged ions

What is a chromophore?

• The part of the molecule responsible for its colour

What is a simple stain?

• One dye used to highlight the entire organism

What are the steps to using simple stains? (4)

• Smear sample on slide

• Fix with heat

• Add stain

• Wash, dry, view

How do stains work? (1.1)

• Bacterias have a net negative charge on outer surface

  • Charge attracts stains w/ positively charged chromophores + repels stain w/ negatively charged chromophores

How do positive stains work? (1.2)

• Stain binds to bacterium

  • Bacterium = coloured

  • Background = clear

What is an example of a positive stain?

• Crystal violet

How do negative stains work? (1.2)

• Doesn’t bind to bacterium

  • bacterium = clear

  • background = coloured

What are examples of negative stains? (2)

• Nigrosin

• India ink

What is a differential stain?

• A stain that reacts differently w/ different bacteria = used to distinguish between them

What is a gram stain?

• A stain that differentiates bacteria based on the structure of the cell wall

What does it mean to be gram positive + a bacteria example?

• Bacteria with a thick cell wall retains primary stain crystal violet → appears purple

  • ex. streptococcus pyogenes

What does it mean to be gram negative + a bacteria example? (1.2)

• Bacteria with a thin cell wall loses crystal violet during destaining 

  • takes on the colour of the counterstain safranin = pink

  • ex. e. coli

What is a spore stain?

• A stain that stains an internal structure of bacteria

What are the parts of a spore stain? (2)

• Primary stain - colours endospores green

• Counterstain (safranin) - colours rest of cell pink

What is a flagella stain?

• Stains an external structure

How does a flagella stain work?

• Mordant thickens flagella → can be observed under light microscope

What is the function of an acid-fast stain + example? (1.1)

• Detects the presence of a waxy compound in cell wall

  • used to identify the genus mycobacterium

How do acid-fast stains work? (2)

• Mycobacterium cell wall retains the dye, carbol fuchsin

• Counterstain w/ methylene blue stains non acid-fast bacteria + tissues blue

What is the function of a capsule stain?

• Detects a thick layer of polysaccharide outside the cell (capsule)

How does a capsule stain work? (3)

• Negative stain colours background

• Positive stain colours cell

• Capsule doesn’t take up most cells = remains colourless

What are the characteristics of a prokaryote? (4)

• DNA not enclosed within a nucleus

• DNA → one circular chromosome

• Lacks membrane-enclosed organelles

• Single celled organisms: bacteria, archaea

What are the characteristics of a eukaryote? (4)

• DNA found in nucleus, surrounded by a nuclear membrane

• DNA arranged as multiple chromosomes

• Membrane-enclosed organelles

• Single celled or multicellular organisms

What are the shapes of bacteria? (5)

• Coccus - spherical

• Bacillus - rod shaped

• Vibrio - c shaped

• Spirillum - spiral shaped

• Spirochete - corkscrew shaped

What are capsules + slime layers (generally)?

• A sticky, gelatinous layer external to the cell

  • composed of polysaccharides, proteins, or both

What is a capsule?

• A organized and firmly attached layer → cell wall

What is a slime layer?

• An organized layer loosely attached to the cell wall

What is the relation between capsules + virulence? (1.1.2)

• Protection against phagocytosis

  • ex. streptococcus pneumoniae

    • capule: causes disease

    • no capsule: no disease

What is a function of the slime layer + example? (1.1)

• Allows bacteria to attach to surfaces

  • ex. streptococcus mutans

What is flagella + its usage? (1.1.1)

• Long protein appendages

  • Semi-rigid, helical, turns like a propeller

    • Used in motility

What are the typical arrangements of flagella? (4)

• Monotrichous

• Lophotrichous

• Amphitrichous

• Peritrichous

What is the monotrichous arrangement of flagella?

• A single polar flagellum

What is the lophotrichous arrangement of flagella?

• Two or more flagella originating → one pole

What is the amphitrichous arrangement of flagella?

• Tufts of flagella originating from opposite poles

What is the peritrichous arrangement of flagella?

• Flagella distributed all over the cell

What is flagellar motility? (2)

• Running - flagella turning = cell moves in one direction

• Tumbling - periodically flagella reverse direction = random change in direction

What is chemotaxis? (1.1)

• Movement away or towards a stimulant

  • allowed by flagella

What can flagellar protein be used for in relation to stains?

• Can be used to distinguish among strains of a species

What are fimbriae + pili (general)? (1.1)

• Short, hairlike appendages

  • hollow

What is the function of fimbriae? (1.1)

• Allows the cell ro adhere to surfaces

  • contributes to pathogenicity

What is the function of pili? (1.1)

• Allows attachment of two bacteria to each other 

  • involved in transfer of genetic material → bacteria

What is the bacterial cell wall?

• Semi-rigid structure giving shape → cell

What is the function of the bacterial cell wall? (1.2)

• Prevent rupture of the cell

  • protects against environmental changes

  • useful in the identification of bacteria

What is the bacterial cell wall composed of?

• Complex macromolecule - PeptidoGlycan

What is peptidoglycan? (1.2.2)

• Mesh-like structure made of amino acids + polysaccharide

  • Peptide portion - short chains of amino acids

  • Polysaccharide portion - two alternating monosaccharides

    • N-acyetyl glucosamine (NAG)

    • N-acetyl muramic acid (NAM)

What is the structure of peptidoglycan?

• Polysaccharide chains run parallel 

• Peptide chains link polysaccharides together

  • = a mesh-like net surrounding the cell

What is the gram positive cell wall composed of?

• Thick layers of peptidoglycan outside of plasma membrane

  • teichoic acids:

    • wall teichoic acids - attached to peptidoglycan

    • lipoteichoic acids - attached to plasma membrane + extend through peptidoglycan

What is the gram negative cell wall composed of? (1.2.3)

• A thin peptidoglycan layer sandwiched between two membranes

  • outer membrane:

    • phospholipids,

    • proteins,

    • lipopolysaccharides (LPS)

  • polysaccharide portion: O-sugars

How does the gram stain work on gram positive cells?

• Thick peptidoglycan traps crystal violet → stains purple

How does the gram stain work on gram negative cells? (1.3)

• Thin peptidoglycan doesn’t trap crystal violet

  • outer membrane gets disrupted by alcohol

  • crystal violet gets washed away

  • safranin counterstain → stains pink

What is special about peptidoglycan? (2)

• Completely different from anything in animal cells

• Many antibiotics have been discovered to act against peptidoglycan

What makes the cytoplasmic membrane?

• composed of a phospholipid bilayer

What is the function of the cytoplasmic membrane? (1.2)

• Separates interior cytoplasm from external environment

  • Serves as a semi-permeable membrane

  • Allows inflow + outflow of material

What is the function of antimicrobial agents? (1.1)

• Disrupts the cytoplasmic membrane

  • can be used as a disinfectant

What is cytoplasm?

• The substance inside the plasma membrane

What is the cytoplasm composed of? (6)

• 80% water

• sugars

• amino acids

• nucleotides

• enzymes

• functional structures

What is the nucleoid + characteristics? (1.2)

• Contains bacterial chromosome

  • not surrounded by a nuclear membrane

  • may contain plasmids

What is the bacterial chromosome?

• All the genetic information required for a cell’s structure + function

What are plasmids? (1.1)

• Smaller double stranded DNA molecules

  • contains non-essential genes

What are ribosomes?

• The site of protein synthesis

What are ribosomes made of?

• Protein + ribosomal RNA

What are the parts of the ribosome? (2.1)

• 30S subunit

• 50S subunit

  • together forms the complete 70S ribosome

What is the difference between bacterial ribosomes + eukaryotic ribosomes? (1.1.1 + 1)

• Bacterial ribosomes - 70S 

  • several antibiotics target bacterial ribosomes

    • prevents bacteria from making new proteins

• Eukaryote - 80S

What are storage granules (inclusion bodies)?

• Deposits or granules of nutrients, stored for later use

What are examples of storage granules? (5.1)

• Sulfur granules

• Polysaccharides

• Lipid inclusions

• Enzymes

• Magnetite

  • Variety of inclusion bodies → serve as a basis for identification

What are endospores?

• A special resting structure → allows bacteria to enter dormant state

  • formed by gram-positive bacteria

What is a dormant state?

• An inactive state where growth + development stop and metabolism is slowed

Why are endospores beneficial?

• They’re extremely durable

  • resists heat, desiccation, chemicals, radiation

What is the process of sporulation? (5)

1. Cell replicates DNA

2. Septum forms → divides cell into unequal compartments

3. Larger compartment engulfs the smaller

4. Peptidoglycan + other protective material forms around forespore (spore coat)

5. Finished spore is freed from mother cell + mother cell dies

What kingdoms are eukaryotes?

• Microorganisms: algae, fungi, protozoa

• Higher organisms: plants + animals

What is the cell wall composed of? (2)

• Cellulose in plants + algae

• Chitin in fungi + insects

What is the cytoskeleton + its function? (2.2)

• Internal structure in the cytoplasm

• Protein filaments on inside of plasma membrane

  • provides support + structure

  • Transports substances through cell

What are eukaryotic flagellum and cilia? (2.2)

• Long flexible projections that contain protein + cytoplasm

• Moves in a whiplike fashion

• Function:

  • Motility

  • Sweeping material past stationary cells