Unit 2 - Basic Components of Living Things Flashcards
When were light microscopes developed?
In the 16th and 17th centuries.
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
What are the two lenses on a light microscope called and what do they do?
Objective lens - produces a magnified image of the specimen
Eyepiece lens - magnifies the image produced by the objective lens
Give two advantages of light microscopes.
Any two of:
- Easily available
- Relatively cheap
- Easy to use
- Can be used in the field
- Can be used to observe living organisms as well as dead organisms
Give two disadvantages of light microscopes.
Any two of:
- Low magnification
- Low resolution
- Specimen has to be thin and transparent
- Can’t give detailed information about internal cell structure
How is a slide prepared using the dry mount method?
Solid specimens are viewed whole or cut into thin slices. The specimen is placed on the centre of a slide and covered with a cover slip.
Which specimens are viewed with the dry mount method?
Hair, pollen, dust, insect parts and muscle/plant tissue.
How do you prepare a slide using the wet mount method?
The specimen is suspended in liquid with the cover slip placed on at an angle.
Which specimens are viewed with the wet mount method?
Aquatic samples and other living organisms.
How do you prepare squash slides?
A wet mount is prepared, then a lens tissue is used to press down the cover slip.
Which specimens are viewed as squash slides?
Soft samples such as root tip squashes.
How do you prepare smear slides?
The edge of a slide is used to smear the sample, creating a thin, even coating on another slide. A cover slip is then placed over the sample.
Which specimens are viewed as smear slides?
Blood
Why do specimens have to be thin in slide preparation for light microscopes?
So enough light can pass through the specimen to produce a clear image.
Why, when preparing a wet mount, should the refractive index of the medium be roughly the same as that of glass?
So the light passes through without bending too much and the image isn’t distorted.
Why must the cover slip on a wet mount be placed at an angle?
To avoid air bubbles forming between the cover slip and the specimen.
Why are stains used in slide preparation?
To increase the contrast of structures in the cell. The different components stain to different degrees so they are more visible and it is easier to identify them.
How are slides prepared before being stained?
The specimen is placed on a slide and allowed to air dry, then passed through a flame. This fixes the specimen to the slide, allowing it to take up stains.
What are two positively charged stains used?
Crystal violet and methylene blue. Because these stains are positively charged, they are attracted to the negatively charged material in the cytoplasm, leading to the staining of cell components.
What are two negatively charged stains used?
Nigrosin and Congo red. These stains are repelled by the negatively charged material in the cytoplasm, so leave cells unstained. This means the cells stand out against an unstained background, and is called a negative stain technique.
What is differential staining?
Staining used to distinguish between two types of organisms that would otherwise be difficult to identify.
How are Gram positive bacteria and Gram negative bacteria different in structure?
Both have a layer of peptoglycan (mesh-like layer of sugars and amino acids) on the cell wall but Gram positive bacteria has a thicker layer than Gram negative bacteria. Gram negative bacteria also has an extra layer of lipopolysaccharide.
Give two examples of Gram positive bacteria.
Staphylococcus spp. and streptococcus spp.
Give two examples of Gram negative bacteria.
E. coli and salmonella.
What are the four steps of the Gram stain technique?
- Staining with crystal violet which will leave both variants blue/purple.
- Specimen is treated with a mordant (solution of iodine and potassium iodide) which fixes the dye.
- Decolourisation with a mixture of alcohol and acetone.
Gram positive bacteria keeps the crystal violet stain while Gram negative bacteria is decolourised because it has thinner cell walls. - Counterstaining with either safranin or fuchsin dye.
Gram positive bacteria is blue/purple from the crystal violet while Gram negative bacteria will be red/pink
What is the acid-fast technique used for?
To differentiate species of acid-fast bacteria from other bacteria.
Give an example of acid-fast bacteria.
Mycobacterium
What is the structure of acid-fast organisms?
Acid-fast organisms have high levels of mycolic acid, fatty acids, waxes and complex lipids in their cell walls, meaning the cell walls are wax-like and virtually impermeable.
What are the four stages of the acid-fast technique?
- A lipid solvent is used to carry carbolfuchsin dye into the cells of the specimen. Both variants will be coloured red.
- The red stain is fixed by heating the slide.
- Cells are washed with a dilute acid and alcohol solution.
The acid-fast bacteria retains the stain, but other bacteria will lose the stain - Counterstaining with methylene blue.
The acid-fast bacteria will be red and other bacteria will be blue.
How is carbolfuchsin dye able to permeate the cell walls of acid-fast bacteria?
Carbolfuchsin contains phenol, which helps dissolve the waxy cell wall.
Why can acid-fast bacteria retain a carbolfuchsin stain after applying a decolouriser?
The dilute acid and alcohol solution cannot permeate the acid-fast bacteria’s cell walls, due to the high level of mycolic acid.
What are the stages of producing pre-prepared slides?
Fixing - preserving specimens in as close to a natural state as possible
Sectioning - specimens are dehydrated with alcohol and placed in a mould with wax/resin to form a hard block, then sliced thinly with a microtome
Staining - often multiple stains are used
Mounting - specimens are secured to a microscope slide and a cover slip is placed on top
Define magnification.
Magnification describes how many times larger the image is than the actual object.
Define resolution.
Resolution is the ability to distinguish between two separate objects which are close together.
What is the equation for magnification?
Magnification = Image size / Actual size
How are microscopes calibrated using an eyepiece graticule and stage micrometer?
The eyepiece graticule (inside the eyepiece lens) is a glass disk with a 1-100 scale (no units). The size of the divisions between the scale numbers differs between the different objective lenses (which all give a different magnification). The stage micrometer, a slide with an accurate measuring scale, is used to determine the size of the divisions at each magnification.
When were electron microscopes developed?
In the 20th century.
How do electron microscopes work?
A beam of electrons with a wavelength under 1nm is used to illuminate the specimen.
Give two advantages of electron microscopes.
- High magnification (could be up to x500,000)
- High resolution
Give two disadvantages of electron microscopes.
- Expensive
- It’s easier to damage the specimen
- They require more preparation
What is the average resolution of a light microscope?
200nm.
What is the average resolution of an electron microscope (transmission and scanning)?
Transmission: 0.5nm
Scanning: 3-10nm
TEM have a higher resolution.