2.1 - Microscopy (2.1.1)

Question 1

What 3 things does ‘cell theory’ state?

Answer 1

• Both plant and animal tissue is composed of cells
• Cells are the basic unit of all life
• Cells only develop from existing cells

Question 2

When was the first cell observed?

Answer 2

1665

Question 3

Which English scientist observed the first cells?

Answer 3

Robert Hooke

Question 4

Which Dutch biologist observed the first living cells?

Answer 4

Anton van Leeuwenhoek

Question 5

When did the evidence for the origin of new plant cells arise?

Answer 5

1832

Question 6

When was the first nucleus observed?

Answer 6

1833

Question 7

Who observed the first nucleus?

Answer 7

Robert Brown

Question 8

When did the birth of a universal cell theory arise?

Answer 8

1837 - 1838

Question 9

Which German scientist proposed that all plant tissues are composed of cells?

Answer 9

Matthias Schleiden

Question 10

Which Czech scientist was the first to use a microtome to make ultra thin slices of tissue for microscopic examination?

Answer 10

Jan Purkyně

Question 11

Based on his observations, what did Jan Purkyně propose?

Answer 11

That not only are animals composed of cells but also that the “basic cellular tissue is clearly analogous to that of plants”

Question 12

Which German physiologist is credited with the ‘birth’ of cell theory?

Answer 12

Theodor Schwann

Question 13

What did Theodor Schwann, a German physiologist, declare?

Answer 13

“all living things are composed of cells and cell products”

Question 14

Which Polish/German biologist was the first to observe cell division in animal cells, disproving the existing theory that new cells originate from within old cells?

Answer 14

Robert Remak

Question 15

When did the evidence for the origin of new animal cells arise?

Answer 15

1844 (1855)

Question 16

Which German biologist published Robert Remak’s findings as his own in 1855?

Answer 16

Rudolf Virchow

Question 17

Who disproved the theory of spontaneous generation of cells?

Answer 17

Louis Pasteur

Question 18

When did Louis Pasteur disprove the theory of spontaneous generation of cells?

Answer 18

1860

Question 19

How did Louise Pasteur disprove the theory of spontaneous generation of cells?

Answer 19

By demonstrating that bacteria would only grow in a sterile nutrient broth after it had been exposed to the air

Question 20

What are the two lenses of a compound light microscope?

Answer 20

The objective lens and an eyepiece lens

Question 21

In a compound light microscope, what does the objective/eyepiece lens configuration allow for?

Answer 21

Much higher magnification and reduced chromatic aberration than that in a simple light microscope

Question 22

What is chromatic aberration?

Answer 22

Chromatic aberration, also known as color fringing, is a color distortion that creates an outline of unwanted color along the edges of objects in a photograph

Question 23

Describe how a dry mount is prepared

Answer 23

Solid specimens are viewed whole or cut into very thin slices with a sharp blade, this is called sectioning. The specimen is placed on the centre of the slide and a cover slip is placed over the sample.

Question 24

Describe how a wet mount is prepared

Answer 24

Specimens are suspended in a liquid such as water or an immersion oil. A cover slip is placed on from an angle, as shown.

Question 25

What are some examples of things that can be prepared as a dry mount? (3)

Answer 25

• Hair
• Pollen
• Dust

Question 26

What are some examples of things that can be prepared as a wet mount? (2)

Answer 26

• Aquatic samples
• Other living organisms

Question 27

Describe how a squash slide is prepared

Answer 27

A wet mount is first prepared, then 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 a sample between two microscope slides.

Question 28

What is using squash slides a good technique for?

Answer 28

Soft samples

Question 29

What is an example of something that can be prepared using a squash slide?

Answer 29

Root tip squashes - they’re used to look at cell division

Question 30

Describe how a smear slide is prepared

Answer 30

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.

Question 31

What is an example of a smear slide?

Answer 31

A sample of blood

Question 32

In basic light microscopy the sample is illuminated from below with white light and observed from above. What type of microscopy is this?

Answer 32

Brightfield microscopy

Question 33

In basic microscopy the sample is illuminated from below with white light and observed from above. The whole sample is illuminated. What type of microscopy is this?

Answer 33

Wide-field microscopy

Question 34

What is resolution limited by?

Answer 34

The wavelength of light and diffraction of light as it passes through the sample

Question 35

What is diffraction?

Answer 35

Diffraction is the bending of light as it passes close to the edge of an object

Question 36

What is the cytosol of cells?

Answer 36

The aqueous interior

Question 37

How do stains on cells increase contrast?

Answer 37

Different components within a cell take up stains to different degrees. The increase in contrast allows components to become visible so they can be identified.

Question 38

How do you prepare a sample for staining?

Answer 38

It is first placed on a slide and allowed to air dry. This is then heat-fixed by passing through a flame. The specimen will adhere to the microscope slide and will then take up stains.

Question 39

What stain is used to stain the nuclei of white blood cells to the colour purple?

Answer 39

Wright’s stain (a mixture of eosin red and methylene blue dyes)

Question 40

How do dyes like crystal violet or methylene blue lead to the staining of cell components?

Answer 40

Crystal violet or methylene blue are positively charged dyes, which are attracted to negatively charged materials in cytoplasm leading to staining of cell components.

Question 41

How are dyes like nigrosin or Congo red used as a negative stain technique?

Answer 41

Dyes such as nigrosin or Congo red are negatively charged and are repelled by the negatively charged cytosol. These dyes stay outside cells, leaving the cells unstained, which then stand out against the stained background. This is a negative stain technique.

Question 42

What is the purpose of differential staining?

Answer 42

Differential staining can distinguish between two types of organisms that would otherwise be hard to identify. It can also differentiate between different organelles of a single organism within a tissue sample.

Question 43

Describe the gram stain technique in full detail

Answer 43

Gram stain technique is used to separate bacteria into two groups, Gram - positive bacteria and Gram - negative bacteria. Crystal violet is first applied to a bacterial specimen on a slide, then iodine, which fixes the dye. The slide is then washed with alcohol. The 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. 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.

Question 44

Describe the Acid-fast technique in detail

Answer 44

The Acid-fast technique is used to differentiate species of Mycobacterium from other bacteria. 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.

Question 45

In the acid-fast technique, what dye is carried into the cells being studied?

Answer 45

Carbolfuchsin dye

Question 46

You will often look at slides that have been pre-prepared. Describe the 4 stages that are in this preparation process.

Answer 46

• Fixing: chemicals like formaldehyde are used to preserve specimens in as near-natural a state as possible.
• Sectioning: 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.
• Staining: specimens are often treated with multiple stains to show different structures.
• Mounting: the specimens are then secured to a microscope slide and a cover slip placed on top.

Question 47

Many of the stains used in the preparation of slides are toxic or irritants. What does this mean for the user?

Answer 47

A risk assessment must be carried out before any practical is started to identify any procedures involved that may result in harm

Question 48

Describe all the safety measures that are put in place for the use of stains (that are toxic or irritants) (4)

Answer 48

• A risk assessment must be carried out before any practical is started to identify any procedures involved that may result in harm.
• CLEAPSS (Consortium of Local Education Authorities for the Provision of Science Services) is the organisation that provides support for the practical work carried out in schools. One of the main areas covered is health and safety, including risk assessment. Advice and support is provided to all types of educational establishments and their employees about all aspects of practical work such as the use of chemicals or living organisms, laboratory design, and even where to obtain the right equipment.
• CLEAPSS provide student safety sheets that identify specific risks, advise on the measures to be taken to reduce these risks and the action to be taken in any emergency.
• In fact, in schools many of the microscopy slides that are used are bought ready-prepared and pre-stained, not only because of the harmful nature of the stains but also because of the long complex process needed to produce high quality sections

Question 49

What are the 11 rules to follow when producing good scientific drawings?

Answer 49

• Include a title
• State magnification
• Use a sharp pencil for drawings and labels
• Use white, unlined paper
• Use as much of the paper as possible for the drawing
• Draw smooth, continuous lines
• Do not shade
• Draw clearly defined structures
• Ensure proportions are correct
• Label lines should not cross and should not have arrow heads
• Label lines should be parallel to the top of the page and drawn with a ruler