Chapter 2.1 Microscopy Flashcards
Cell theory, development of the microscope
Definition of microscope
Instrument which enables you to magnify an object hundreds, thousands or even hundreds of thousands of times.
Function of microscopes
They make the individual cells of multicellular organisms visible, allowing us to discover details of the structures and how these details relate to their functions.
1st type of microscope
Light microscope. Developed in 16th to 17th century
When could individual cells be seen
Mid 19th century
3 parts of cell theory
- both plant and animal tissue is composed of cells
- cells are the basic unit of all life
- cells only develop from existing cells
Benefits of light microscopy
Easily available
Relatively cheap
Can be used out in the field
Observe living and dead prepared specimens
History of light microscope
Romans experimented with glass
Objects looked bigger when viewed through glass that’s thicker in the middle than at the edges
13th century - invention of spectacles
Dutch spectacle makers invented telescope
Galileo Galilei developed compound microscope
Galileo instrument first to be named a microscope
Stage 1 of development of cell theory
1665 - Robert hooke observed the structure of thinly sliced cork
Used early light microscope
Described compartments as cells
Was observing dead plant tissue so he could only see cell walls
Stage 2 of development of cell theory
1674-1683
Leeuwenhoek developed technique for creating powerful glass lenses
Used handcrafted microscopes to examine samples of pond water
First to observe bacteria and protoctista ‘little animals’
Now called microorganisms
Observed red blood cells, sperm cells and muscle fibres
Stage 3 of development of cell theory
1832 - Dumortier observed cell division in plants
Disproved theory that New cells arise from within old cells
Disproved that cells formed spontaneously from non-cellular material
Virchow published these findings in 1855
Stage 4 of development of cell theory
1833 - Brown described plant cell nucleus
Stage 5 of development of cell theory
1837 - 1838
Schleiden- all plant tissues are composed of cells
Purkyně - used microtone to make ultra-thin slices of tissue for microscopic examination
Animals are composed of cells
Basic cellular tissue of animals is clearly analogous to that of plants
Schwann - all living things are composed of cells and cell products
Stage 6 of development of cell theory
1844 (1855) - Remak observed cell division within animal cells
Disproved that new cells originated from within old cells
Virchow plublis
Stage 7 of development of cell theory
1860 - Pasteur disproved spontaneous generation of cells.
Demonstrated that bacteria would only grow in sterile nutrient broth after being exposed to air
How does a compound light microscope work?
2 lenses - configuration allows for higher magnification, reduced chromatic aberration
objective lens (placed near specimen) produces magnified image,
eyepiece lens (where specimen is viewed) magnifies again
Illumination usually provided by light underneath sample
Opaque specimens an be illuminated from above
Types of sample preparation
Dry mount
Wet mount
Squash slides
Smear slides
Steps for a dry mount
Solid specimen can be viewd whole Can be sectioned (cut into thin slices using a blade) Specimen placed on centre of slide Cover slip placed over the top ( used on hair, pollen)
Steps for a wet mount
Specimen suspended in liquid (water or immersion oil)
Cover slip placed on from an angle
(aquatic samples)
Steps for squash slides
Wet mount prepared
Lens tissue gently pressed down on cover slip
Depending on material, potential damage can be avoided by squashing the sample between 2 microscope slides
(good for soft samples)
(root tip squashes used to look at cell division)
Steps for smear slides
Edge of a slide smears sample
Creates thin and even coating
Cover slip placed over sample
(used to view cells in blood)
Types of basic light microscopy
Brightfield microscopy - Sample is illuminated from below with white light Observed from above Wide-field microscopy - Whole same illuminated at once
Use of stains
Images have low contrast as most cells don’t absorb much light
Cytosol and other cell structures are often transparent
Different components in cells take up stains to different degrees
Stains increase contrast
Increased contrast allows components to become visible so they can be identified
Prepare sample for staining
Place on slide
Air dry
Heat-fix by passing thrugh a flame
Specimen will adhere to micrscope slide and will take up stains
What is differential staining?
Using multiple stains. It can distinguish between two types of organisms that would’ve been hard to identify. Can also differentiate between different organelles of a single organism within a tissue sample.
How do crystal violet and methylene blue work as stains?
Positively charged. Attracted to negatively charged materials in cytoplasm. Results in staining of cell components.
Negative stain technique
When you stain the background.
Can use nigrosin or congo red
Negatively charged dye is repelled by negatively charged cytosol.
Dyes stays outside of cell, cell remains unstained
Cell stands out against stained background
Gram stain technique
Separates bacteria into gram-positive and gram-negative
Apply crystal violet to bacterial specimen (on a slide)
Apply iodine to fix the dye
Wash slide with alcohol
Gram+ retains crystal violet stain - appears blue/purple under microscope
Gram- has thinner cell walls so loses stain.
Stain gram- with safranin dye (counterstain) bacteria appears red.
Difference between gram+ and gram-
Gram+ is susceptible to antibiotic penicillin (inhibits formation of cell walls) whilst gram- has thinner cell walls that are not suscetible to penicillin.
What is acid-fast technique used for
Differentiate species of mycobacterium from other bacteria
Steps for acid-fast technique
Lipid solvent carries carbolfuschsin dye into cells being studied
Cells washed with dilute acid-alcohol solution
Mycobacterium are not affected by acid-alcohol solution
They retain the carbolfuchsin stain
Appear bright red
Other bacteria lose stain and are exposed to methylene blue stain (appear blue)
Stages for preparing slides
Fixing - chemicals (formaldehyde) used to preserve specimens in near-neutral state
Sectioning - specimen dehydrated with alcohols. placed in mould with wax/resin to form hard block. Sliced thinly with microtone
Staining - specimen treated with multiple stains to show different structures
Mounting - specimen secured to microscope slide and cover slip placed on top
Risk management
Stains can be toxic or irritants
Identify any procedures that may cause harm
CLEAPSS provides support for practical work in schools
Advice provided to schools and employees
Many slides are preprepared due to toxic stains and long complex process of preparing
Rules for scientific drawings
Title State magnification Sharp pencil White, unlined paper Use majority of paper Smooth continuous lines No shading Clearly defined structures Proportions correct Label lines don't cross or have arrowheads Label libes parallel to top of the page and drawn with ruler
