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Biology: Module 2: Foundations in Biology > Micrsocopy & Staining > Flashcards

Micrsocopy & Staining Flashcards

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1
Q

Use of microscopy

A

To observe and investigate different types of cell and cell structure in a range of eukaryotic organisms

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2
Q

Types of microscopy

A
  • Light microscope
  • transmission electron (TEM)
  • scanning electron (SEM)
  • laser scanning confocal
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3
Q

Types of mount

A
  • Wet
  • Dry
  • Squash
  • Smear
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4
Q

How to do a wet mount

A
  1. Suspend the specimen in liquid
  2. Place cover slip on from an angle
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5
Q

Examples of liquids used in wet mounts

A
  • Water
  • Immersion oil
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6
Q

Organisms that can be viewed in wet mounts

A

Aquatic organisms

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7
Q

How to do a dry mount

A
  1. Section the sample if it is too large
  2. Place specimen on centre of the slide
  3. Put cover slip on top
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8
Q

Things that can be viewed in dry mounts

A
  • Hair
  • Pollen
  • Dust
  • Insect parts
  • Parts of muscle tissue
  • Parts of plant
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9
Q

How to do a smear slide

A
  1. Use the edge of a slide to smear out the sample on another slide
  2. Put cover slip over the sample
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10
Q

How to do a squash slide

A
  1. Prepare a wet mount
  2. Use a lens tissue or two microscope slides to press down on the cover slip
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11
Q

What can you view with a squash slide?

A

Root tips during cell division

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12
Q

What can you view with a smear slide?

A

Blood

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13
Q

Why must a sample be thin for light microscopy?

A

So the light can shine through it and details can be seen.

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14
Q

How a light microscope works

A
  1. Objective lens produces a magnified image
  2. Image magnified again by the eyepiece lens
  3. Illumination provided by a light underneath the sample
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15
Q

How to calibrate a microscope

A
  1. Stage micrometer on the stage and the eyepiece graticule in the eyepiece
  2. Get in focus and align micrometer with the eyepiece graticule
  3. Take readings from both the micrometer and the graticule
  4. Use ratios to find how much one graticule division is worth
  5. Find the magnification factor and measure
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16
Q

Why must the liquid medium used in wet mounts have a similar refractive index to glass?

A

To prevent diffraction between the liquid and the glass and thus preventing image distortion.

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17
Q

Why are cover slips placed on wet mounts at an angle?

A

To prevent the trapping of air bubbles.

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18
Q

Purpose of differential staining

A

To identify different cellular components and cell types

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19
Q

Examples of differential staining

A
  • Gram stain technique
  • Acid-fast technique
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20
Q

Purpose of the Gram stain technique

A

To differentiate between Gram positive and Gram negative bacteria.

21
Q

Purpose of the Acid Fast technique

A

To differentiate between species of Mycobacterium and other bacteria

22
Q

Stages in pre-preparation of slides

A
  • Fixing
  • Sectioning
  • Staining
  • Mounting
23
Q

Fixing

A

Using chemicals like formaldehyde to preserve specimens in a near-natural state.

24
Q

Sectioning

A

Dehydrating a sample with alcohol and then placing it in a mould of resin or wax to form a hard block before slicing it with a microtome into thin slices.

25
Q

Staining

A

Treating specimens with multiple stains to show different structures.

26
Q

Mounting

A

Securing a specimen onto a microscope slide under a cover slip.

27
Q

Advantages of staining

A
  • See more detail
  • Increases contrast
  • Allows you to identify different cells and cellular components like organelles
28
Q

Magnification Formula

A

Magnification = Image size / Object Size

29
Q

Difference between magnification and resolution

A
  • Magnification is how many times larger the image is than the actual size of the object
  • Resolution is how detailed the image is and the ability to distinguish between two points that are close together.
30
Q

Resolution of light microscopes

A

200nm

31
Q

Magnification of light microscopes

A

1500x

32
Q

Resolution of a transmission electron microscope

A

0.5nm

33
Q

Magnification of a transmission electron microscope

A

500,000x

34
Q

Resolution of a scanning electron microscope

A

3-10nm

35
Q

Magnification of a scanning electron microscope

A

100,000x

36
Q

How to tell the difference between an SEM image and a TEM image

A
  • Both images are black and white but TEM images can add false color using computer system.
  • SEM = 3D
  • TEM = 2D
  • SEM has higher resolution
  • SEM image only allows to see outside of cell rather than inside cell deatil
37
Q

Comparison points for Light Microscopes

A
  • Cheap to purchase and operate * small and portable
  • Simple and easy sample preparation
  • Vacuum not required
  • Natural colour of sample is maintained
  • Magnifies only up to 2000 times
  • Specimens can be living or dead
  • Stains are often needed to make the cells visible
38
Q

Comparison points for Electron Microscopes

A
  • Expensive to buy and produce electron beam
  • Large and requires special room
  • Lengthy and complex smaple prep
  • Vacuum is required
  • All images are black and white
  • Magnification is x500000
  • Specimens are dead
  • Electron beam can damage specimens as they must be stained with electron-dense chemical
39
Q

Why might organelles appear to be different sizes down a microscope?

A
  • Cut in a transverse or longitudinal planes
  • Natural variation in shape
  • Some may have just divided or be growing
  • An artefact may have deformed it
40
Q

Advantages of filming when doing microscopy

A
  • Can use time lapse
  • Continuous record
  • You don’t need to be constantly looking
  • Not dependent on drawing or describing ability
  • Easy to see detail
41
Q

Positively charged dyes

A
  • Crystal violet
  • Methylene blue
42
Q

Negatively charged dyes

A
  • Nigrosin
  • Congo red
43
Q

Why do we use staining?

A
  • To increase contrast because cells don’t absorb much light
  • The crystal of cells and other cell structures are often transparent.
44
Q

What is methylene blue?

A

It is an all-purpose stain

45
Q

What is differential staining?

A

Some stains bind to specific cell structures staining each structure differently so the structures can be easily identified.

46
Q

Acetin Orcein binds to….

A

DNA + stains chromosomes dark red.

47
Q

Eosin stains…..

A

Cytoplasm.

48
Q

Sudan red stains….

A

Lipids.

49
Q

Iodine in KI solution stains….

A

The cellulose in plant cell walls yellow & Starch granules blue/black might look violet under MS

Biology: Module 2: Foundations in Biology (10 decks)

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