Module 2: Chapter 2 - Basic components of living systems

what does cell theory state?

• both plant and animal tissue is composed of cells

• cells are the basic unit of all life

• cells only develop from existing cells

how does a compound light microscope work?

• has two lenses: the objective lens (placed near to the specimen), and an eyepiece lens (viewing the specimen)

• objective lens produces a magnified image, which is magnified again by the eyepiece lens

• this configuration allows for much higher magnification and reduced chromatic aberration than that in a simple light microscope

• illumination is usually provided by a light underneath the sample

• opaque specimens can be illuminated from above with some microscopes

what are the main 4 ways to prepare a microscope sample?

• dry mount

• wet mount

• squash slides

• smear slides

how does dry mounting work?

• solid specimens viewed whole or cut into thin slices with a sharp blade (sectioning)

• specimen placed on centre of slide and a cover slip is placed over sample

examples of specimens that can be viewed with dry mounting

• hair

• pollen

• dust

• muscle tissues or plants can be sectioned and viewed this way

how does wet mounting work?

• specimens suspended in a liquid such as water or an immersion oil

• cover slip placed on from an angle

give 2 examples of liquids that specimens can be suspended in for wet mounting

• water

• an immersion oil

examples of specimens that can be viewed with wet mounting

aquatic samples and other living organisms

how do squash slides work?

• a wet mount is first prepared

• a lens tissue is used to gently press down the cover slip

• depending on the material, potential damage to a cover slip can be avoided by squashing the sample between 2 microscopic slides

• good technique for soft samples

• care needs to be taken that the cover slip is not broken when being pressed

what types of samples, and an example, can squash slides be used to view?

• soft samples

• root tip squashes are used to look at cell division

how do smear slides work?

• the edge of a slide is used to smear the sample, creating a thin, even coating on another slide

• cover slip placed over the sample

example of a specimen viewed using smear slides

• a sample of blood - good way to view the cells in the blood

what is a photomicrograph?

a photograph of the image seen using an optical (light) microscope

what is resolution?

the ability to distinguish between 2 points - the shortest distance between 2 objects that are still seen as separate objects

what is magnification?

the number of times greater an image is than the object (the real thing)

what is the energy source used for an optical microscope?

visible light

what is the energy source used for a scanning electron microscope?

a beam of high energy electrons

what is the energy source used for a transmission electron microscope?

a beam of high energy electrons

is a vacuum necessary in an optical microscope?

no

is a vacuum necessary in a scanning electron microscope?

yes

is a vacuum necessary in a transmission electron microscope?

yes

why are vacuums necessary in electron microscopes?

Air molecules (like oxygen and nitrogen) will scatter, deflect, or block the electrons if they are not removed, destroying image resolution

how can a specimen be made more visible in an optical microscope?

• adjust light levels

• staining

how can a specimen be made more visible in a scanning electron microscope?

improve electrical conductivity

• i.e. coat with a metal like gold - prevents build up of static charge

how can a specimen be made more visible in a transmission electron microscope?

• ultra-thin sectioning

• staining to increase image contrast

can living material be viewed in an optical microscope?

yes

can living material be viewed in a scanning electron microscope?

no

can living material be viewed in a transmission electron microscope?

no

why can’t living material be viewed in electron microscopes?

• living specimens cannot survive in a vacuum

• complex preparation process (dehydration, fixation with chemicals, heavy metal staining, thin sectioning) that kills the cells

• high-energy beam of electrons destroys/damages living cells

how does an optical (light) microscope work?

visible light passes from a bulb through a specimen and glass lenses, which refract light to focus the image

what are the advantages of an optical microscope?

• can view living specimens

• cheap and portable

• simple/fast sample preparation

• preserves natural colour or uses standard stains

what is an optical microscope used to view?

• whole cells

• living & non-living tissues

• larger cell structures/organelles (e.g., cell walls, vacuoles, nuclei, chloroplasts at low detail)

what are the disadvantages of an optical microscope?

• low resolution and low maximum magnification

• cannot view small organelles or internal sub-cellular ultrastructure

can optical microscopes produce colour images?

yes - displays natural specimen colours or colours from stains

what is the maximum magnification of an optical microscope?

x1,500 (up to ~x2,000)

what is the resolution of an optical microscope?

(0.2 µm)

how does a scanning electron microscope (SEM) work?

• beam of electrons scanned back and forth across the surface of a metal-coated specimen

• scattered/reflected electrons are detected to form an image

what are the advantages of a scanning electron microscope (SEM)?

• high resolution & magnification compared to light microscopes

• provides detailed 3D surface views

• specimens do not need to be ultra-thin

what is a scanning electron microscope (SEM) used to view?

• 3D surface structures of cells, tissues, organisms, and organelles

• non-living material

• e.g. pollen grains, cell surface membranes, cilia, insects

what are the disadvantages of a scanning electron microscope (SEM)?

• specimen must be dead (vacuum required)

• expensive and non-portable

• complex preparation (can cause artifacts)

• lower resolution than TEM

• 3D surface only

can a scanning electron microscope (SEM) produce colour images?

no - naturally produces black and white/grayscale images

what is the maximum magnification of a scanning electron microscope (SEM)?

x500,000

what is the resolution of a scanning electron microscope (SEM)?

3 – 10 nm

how does a transmission electron microscope (TEM) work?

• use electromagnets to transmit a beam of electrons through an ultra-thin, metal-stained specimen

• denser areas absorb more electrons and appear darker

what are the advantages of a transmission electron microscope (TEM)?

• highest resolution and magnification available

• reveals internal sub-cellular ultrastructure and small organelles in high detail

what is a transmission electron microscope (TEM) used to view?

• internal ultrastructure of organelles

• non-living material

• e.g. mitochondrial cristae, chloroplast grana, ribosomes, nuclear pores, lipid bilayers

what are the disadvantages of a transmission electron microscope (TEM)?

• specimen must be dead (vacuum required)

• complex, skilled prep (ultra-thin sectioning etc. - artifacts common)

• expensive and large

• produces flat 2D images only

can a transmission electron microscope (TEM) produce colour images?

no - produces 2D black and white/grayscale images

what is the maximum magnification of a transmission electron microscope (TEM)?

> x500,000 (up to ~x2,000,000)

what is the resolution of a transmission electron microscope (TEM)?

0.5 nm

what is the comparative order of resolution for optical, SEM, and TEM?

TEM (0.5 nm) > SEM (3–10 nm) > optical microscope (200nm)

why do electron microscopes have a much higher resolution than optical microscopes?

• electron beams have a shorter wavelength than visible light waves (which has ~400–700 nm)

• structures smaller than half the wavelength of light cannot be resolved by light

why does a TEM have a higher resolution than an SEM?

• in TEM, electrons pass directly through an ultra-thin section, minimizing electron scattering

• SEM relies on electrons bouncing off a metallic surface coating, which limits maximum resolving power

what are artefacts?

objects or structures seen through a microscope created during the processing of the specimen

name 2 positively charged dyes

• crystal violet

• methylene blue

name a negatively charged material that positively charged dyes would be attracted to

cytoplasm

name 2 negatively charged dyes

• nigrosin

• Congo red

what repels negatively charged dyes such as nigrosin or Congo red?

the negatively charged cytosol

where do dyes like nigrosin or Congo red (negatively charged) go?

stay outside cells, leaving the cells unstained, which then stand out against the stained background (a negative stain technique)

what is differential staining?

multiple different stains or dyes are used to bind selectively to different cellular structures or types of organisms

what can differential staining differentiate between?

• 2 types of organisms that would otherwise be hard to identify

• different organelles of a single organism within a tissue sample

what is a counterstain?

a second, contrasting dye applied to color parts of a specimen that did not retain the primary stain, making background structures, different cell types, or specific organelles clearly visible

what is the Gram stain technique used for?

• used to separate bacteria into 2 groups: Gram-positive bacteria and Gram-negative bacteria

how does the Gram stain technique work?

• Crystal violet is applied to a bacterial specimen on a slide

• Iodine is added, which fixes the dye

• Slide is washed with alcohol

• Gram-positive bacteria retain the crystal violet stain and will appear blue or purple under a microscope

• Gram-negative bacteria have thinner cell walls and therefore lose the stain

• They are then stained with safranin dye, which is called a counterstain

• These bacteria will then appear red/pink

• Gram-positive bacteria are susceptible to the antibiotic penicillin, which inhibits the formation of cell walls

• Gram-negative bacteria have much thinner cell walls that are not susceptible to penicillin

how is a sample prepared for staining?

• first it is placed on a slide and allowed to air dry

• then it is heat-fixed by passing through a flame

• the specimen will adhere to the microscope slide and will take up stains

what is the Acid-fast technique used for?

to differentiate species of Mycobacterium from other bacteria

what is the Acid-fast technique?

• a lipid solvent is used to carry carbolfuchsin dye into the cells being studied

• the cells are then washed with a dilute acid-alcohol solution

• Mycobacterium are not affected by the acid-alcohol and retain the carbolfuchsin stain, which is bright red

• other bacteria lose the stain and are exposed to a methylene blue stain (which is blue)

what are the staged of slide production/preparation?

• fixing

• sectioning

• staining

• mounting

what does fixing entail in the preparation of a slide?

chemicals like formaldehyde are used to preserve specimens in as near-natural a state as possible

what does (paraffin) sectioning entail in the preparation of a slide?

• specimens are dehydrated with alcohols and then placed in a mould with wax or resin to form a hard block

• this can then be sliced thinly with a knife called a microtome

what does mounting entail in the preparation of a slide?

the specimens are secured to a microscope slide and a cover slip placed on top

what is the magnification formula?

magnification = image size / actual size (“I AM” in a triangle)

in terms of dividing by 1000, what is the order of units from metre to nanometre?

metre, millimetre, micrometre, nanometre

what is an electron micrograph?

a photograph or digital image taken through an electron microscope or similar device to show a magnified image of an item

what is a prokaryotic cell (by definition)?

a single celled organism that contains no nucleus or other membrane bound organelles

what are the organelles common to all eukaryotic cells?

• cell surface membrane

• ribosome

• cytosol

• centriole

• rough ER

• smooth ER

• cytoskeleton

• secretory vesicle

• golgi apparatus

• nucleus

• nucleolus

• vesicle

• microtubule network

what is the function of the nucleus?

• contains coded genetic information as DNA molecules

• directs proteinsynthesis, controlling cellular metabolic activities

how does the nucleus control cellular metabolism?

directs protein synthesis, which produces the enzymes required for metabolic reactions to occur

what is the nuclear envelope?

a double membrane that surrounds the nucleus to protect it from damage

what is the function of nuclear pores?

allow molecules to move into and out of the nucleus

what is the function of the nucleolus?

• to produce ribosomes

• RNA is used to make ribosomal RNA, which then combines with proteins to form ribosomes

how is DNA removed from the nucleus?

transcribed to smaller RNA molecules and leaves through the nuclear pores

why must DNA be transcribed into RNA to leave the nucleus?

DNA itself is too large to leave the nucleus through nuclear pores to reach ribosomes in the cytoplasm

what protein does DNA associate itself with?

histones

what is the name of the complex form when DNA associates with histones?

chromatin

how are chromosomes formed?

chromatin coils and condenses to form chromosomes - these only become visible when cells are preparing to divide

what is the main function of mitochondria?

site of the final stages of aerobic cellular respiration, converting energy stored in organic molecules into ATP

what is the number of mitochondria in a cell generally a reflection of?

the amount of energy it uses, so very active cells usually have a lot of mitochondria

what type of membrane does mitochondria of?

a double membrane

what are cristae?

folds of the inner membrane of mitochondria

what is the function of the cristae?

provides a large surface area containing enzymes needed for aerobic respiration

what is the matrix?

the fluid interior of mitochondria

what does the membrane forming the cristae contain?

the enzymes used in aerobic respiration

why do mitochondria contain a small amount of DNA (mitochondrial (mt) DNA)?

so they can produce their own enzymes and reproduce themselves

what is the structure of a vesicle?

a membranous sac, with a single membrane, containing fluid inside

what is the function of vesicles?

to store and transport materials inside the cell

what is the function of a secretory vesicle?

carries modified proteins to the cell surface membrane and fuses with it to release contents outside the cell via exocytosis

what is a lysosome?

specialised forms of vesicles that contain hydrolytic enzymes

what is the function of a lysosome?

• breaking down waste material in cells, including old organelles

• breaking down pathogens ingested by phagocytic cells

• programmed cell death or apoptosis

where is the cytoskeleton present?

throughout the cytoplasm of all eukaryotic cells