2.1- 2.3 Microscopy Flashcards
What is a microscope?
an instrument which enabes you to magnify an object hundreds, thousands and even hundreds of thousands of times
The first type of microscopes to be developed were?
light microscopes
When was light microscopes developed?
16th to 17th century
What does cell theory state?
1) both plant and animal tissue is composed of cells
2) cells are the basic unit of all life
3) cells only develop from existing cells
Advantages of light microscopes?
- easily available, relatively cheap
- used out in the field
- observe living organisms as well as dead, prepared speciments
a compound light microscope has two lenses. What are they
objective lens, placed near to specimen and eyepiece lens, through which the specimen is viewed
what do the objective and eyepiece lens do?
objective lens- produces a magnified image
eyepiece lens- magnifies it again by eyepiece lens
what does this objective/ eyepiece lens configuration allow?
allows for much higher magnification and reduced chromatic aberration than that in a simple light microscope
Illumination is provided where?
light underneath the sample
How may opaque specimens be illuminated
can be illuminated from above with some microscopes
What is the dry mount method of sample preparation
- sectioning (solid specimens are viewed whole/ cut into very think slices with a sharp blade)
- speciment placed on the centre of the slide and a cover slip is placed over sample
e. g. hair, polen and insect parts. Muscle tissue or plants can be sectioned and viewed in this way.
what is the squash slides method of sample preparation
-good technique for soft samples
-wet mount first prepared
-lens tissue is used to gently press down cover slip
-depends on material, potential damage to a cover strip can be avoided by squashing the sample between two microscope slides
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what is the smear slides method of sample preparation
-edge of a slide is used to smear the sample, creating a thin, even coating on another slide.
what is the wet mount sample preparation
- specimens suspended in a liquid such as water or an immersion oil
- cover slip placed on from an angle
what needs to be cared for in squash slides
cover slip is not broken when being pressed
what is an example of a smear slide?
sample of blood, good way to view the cells in the blood
wet mount method can be used for what method?
aquatic samples and other living organisms
what is necessary in scientific drawings
- a title
- magnification
- sharp pencil for drawings and labels
- white unlined paper
- use as much of paper as possible
- smooth continuous lines
- do not shade
- draw clearly defined structures
- ensure proportions are correct
- label lines should not cross and no arrow heads
- label lines should be parallel to the top of page and drawn with a ruler
What can differential staining do?
- distinguish between two types of organisms that would otherwise be hard to identify
- can also differentiate between different organelles of a single organism with a tissue sample
What is the Gram stain technique used for?
To separate bacteria into two groups, Gram-positive bacteria and Gram-negative bacteria
How is the gram stain method carried out?
Crystal violet is first applied to a bacterial specimen on a slide, then iodine, which fixes the dye. Slide is washed with alcohol
How do we tell which one in gram-negative bacteria and one is gram-positive bacteria?
Gram Stain technique- Gram positive bacteria retain the CRYSTAL VIOLET STAIN and will appear blue or purple under a microscope
Gram negative bacteria- thinner walls, and therefore LOSE THE STAIN
What happes after the gram-negative bacteria is identified by the gram stain technique?
They are then stained with safranin dye, which is called a counterstain. Bacteria will appear red
Gram-positive bacteria are susceptible to what?
antibiotic penicillion, which inhibits the formation of cell walls
Why is gram-negative bacteria have not susceptible to penicillin?
- has much thinner cell walls
- penicillin inhibits the formation of cell walls
What is the acid-fast technique used for?
to differenciate species of Mycobacterium from other bacteria
How is the acid fast technique carried out?
a lipid solvent is used to carry carbolfuchsin dye into the cells being studied
-cells are then washed with a dilute acid- alcohol solution
How can we tell which one is a mycrobacterium or other bacteria
mycrobacterium- not affected by the acid-alcohol and retain the carbolfuchsin, which is bright red
other bacteria- lose the stain, and are exposed to a methylene blue stain, which is blue
What do stains do?
Increase contrast as different components within a cell take upp stains to different degrees
The increase in contrast allows?
components to become visible so they can be identified
How do we prepare a sample for staining?
it is first placed on a slide and allowed to air dry
- then heat-fixed by passing through a flame
- specimen will adhere to the microscope side and will then take up stains
Images of light microscope tends to have low contrast why?
-most cells do not absorb a lot of light -resolution is limited by the wavelength of light and diffraction of light as it passes through the sample
The cytosol (aqueous interior) of cells and other cell structures are often transparent
Stains increase contrast
What are the stages involved in the production of tgese slides that have been pre-prepared
- Fixing
- Sectioning
- Staining
- Mounting
Describe the FIXING in the production of these slides
chemicals like formaldehyde are used to PRESERVE SPECIMENS in as near -natural a state as possible
Describe the SECTIONING in the production of these slides
specimens are dehydrated with alcohols and then placed in mould with wax or resin to form a hard block
-this can then be sliced thinly with a knife called a microtome
Describe the STAINING in the production of these slides
specimens are often treated with multiple stains to show different structures
Describe the MOUNTING in the production of these slides
specimens are then secured to a microscope slide and a cover slip placed on top
Many of the stains used in the preparation of slides are toxic or irritants, then what must be carried out?
a risk assesment before any practical, identifies any procedures involved that may result in harm
What does CLEAPSS do?
provide student safety sheets that identify specific risks, advice on the measures to be taken to reduce these risks and the action to be taken in emergency
in schools, many of the microscopy slides that are used are bought in ready prepared and pre-stained. Why?
Not only because of the harmful nature of the stains but also because of the long complex process needed to produce high quality sections
