Using a microscope

Question 1

what is the field of view

Answer 1

This is the lit circle when looking through the ocular lens. it refers to how much of the sample you can see when magnified

Question 2

what is the smallest field of view

Answer 2

The smallest field of view is when using the highest magnification objective lens.

Question 3

what is the largest field of view

Answer 3

The largest field of view is when using the lowest magnification objective lens

Question 4

what can an optical microscope be used to view

Answer 4

Living organisms such as Paramecium and Amoeba
Smear preparations of human blood and cheek cells
Thin section of animal, plant and fungal tissue, such as bone, muscle, leaf, root or fungal hyphae.

Question 5

what happens if specimens are viewed with a high magnification

Answer 5

not everything is in sharp focus, this shows the specimen has depth

Question 6

describe the art of focusing

Answer 6

Step 1 – Obtain a slide and place it on the stage over the central hole
Step 2 – Rotate the nosepiece so that low power objective is in line with the body tube
Step 3 – Raise the body tube using the coarse focussing knob
Step 4 – To begin to focus on low power, use the coarse focussing knob. When the specimen comes into view, then use the fine focussing knob to bring the specimen into sharp focus.
Step 5 – Adjust the diaphragm (amount of light) to get the most detailed view.
Step 6 – Increase to a higher magnification by rotating the nosepiece until the objective click into place
Step 7 – use only the fine adjustment knob to focus images on medium and high power.

Question 7

why does the orientation of a specimen appear reversed/upside down

Answer 7

The lens system, particularly the objective lenses and ocular lens, flips the image both horizontally and vertically. This means that the image you see through the microscope is a mirror image of the actual specimen.

Question 8

what happens when you move the slide or adjust the focus

Answer 8

the direction of movement is opposite to how it appears through the eyepiece

Question 9

how do we prepare a dry mount sample (for e.g. hair)

Answer 9

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 10

how do we prepare a wet mount sample (for e.g. aquatic samples)

Answer 10

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

Question 11

how do we prepare a squash slide (for e.g root top squashes)

Answer 11

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 the sample between two microscope slides. Using squash slides is a good technique for soft samples. Care needs to be taken that the cover slip is not broken when being pressed.

Question 12

how do we prepare a smear slide (for e.g. blood)

Answer 12

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 13

what is the advantage of making a temporary mount of cells

Answer 13

they are quick to prepare and can be observed while they are alive

Question 14

why should the specimens used be thin

Answer 14

to allow light to pass through them, which is necessary for viewing the details of the specimen under a microscope.

Question 15

why should the cover slip be placed on a wet mount at an angle

Answer 15

to reduce or prevent air bubbles from being trapped under the cover slip, which can interfere with the clarity of the image.

Question 16

why are biological specimens often difficult to distinguish

Answer 16

they are often colourless

Question 17

what is a stain

Answer 17

coloured chemicals that bind to specific molecules in or on the specimen enhancing visibility. some stains bind to specific cell structures, allowing different structures to be distinguised within a single preparation.

Question 18

how does staining the specimen help

Answer 18

Staining enhances the visibility of these structures by binding coloured chemicals to specific molecules, making it easier to observe and differentiate various components.

Question 19

give three examples of how stains can be used

Answer 19

• increase contrast so different parts of a cell can be distinguised.
• to observe the location of certain chemicals in a cell
• differentiate between organisms that can be difficult to tell apart

Question 20

what is differential staining

Answer 20

using two different stains. this helps distinguish between different types of organisms or organelles within a sample

Question 21

name two types of stains

Answer 21

methylene blue and iodine

Question 22

how to prepare a cheek slide

Answer 22

1) Add a very small drop of sodium chloride solution to the microscope slide. This is to ensure the sample remains hydrated.
2) Use a sterile spatula or toothpick to obtain a sample. Rub it gently inside your cheek to collect cheek cells.
3) Place the specimen into the drop of sodium chloride solution on the slide by swirling the end of the spatula or toothpick in the solution. Dispose of the used spatula or toothpick properly as it is a biohazard.
4) Add a drop of methylene blue stain to the sample. This stain is used because cheek cells and epithelial cells are very transparent and need to be coloured to be visible under the microscope.
5) Carefully place the cover slip on top of the stained sample. Hold it at a right angle to the slide and gently lower it to avoid air bubbles.
6) If there is too much liquid and it seeps out from the sides of the cover slip, use a paper towel to blot away the excess. Treat the paper towel as a biohazard and dispose of it properly.

Question 23

how to prepare an onion slide

Answer 23

1) Peel off a thin, transparent layer of onion cells from the onion. This layer is the epidermal cells.
2) Use a pair of tweezers to transfer the onion cell layer onto the microscope slide. 3) Spread the layer out gently to prevent it from folding or curling. Alternatively, you can peel the layer directly onto the slide.
4) Apply a few drops of iodine stain to the specimen. This will wet the mount and stain the cells, making them more visible under the microscope.
5) Carefully place the cover slip on top of the stained sample. Hold the cover slip at a right angle from one side and gently lower it to avoid trapping air bubbles.

Question 24

why did the onion cell need to be stained

Answer 24

To observe the cells. nuclei and cell walls absorb the stain more strongly, so that they can be distinguished.

Question 25

discuss the criteria for a good slide

Answer 25

A clean slide - no smudges or fingerprints
Complete water or stain coverage - under the coverslip not flooded – blot excess with paper towel
No visible air bubbles
Specimen is centred on the slide
Specimen is in focus

Question 26

describe the process of drawing slides

Answer 26

1) Use a prepared slide such as a transverse section through a dicot leaf. Set it up on the microscope. Focus the specimen under low power.
2) Use a sharp HB pencil.
3) Use a title that explains exactly what the drawing is and the magnification used.
4) Indicate the scale - i.e. how much bigger your drawing is than the size of the image.
5) Make a low-power plan of the specimen to show where the different tissue areas are, and do not draw any individual cells.
6) Use clear unbroken lines and do not shade any areas.
7) Label the areas shown on the low-power plan.
8) Indicate on the plan a portion of the tissues that you will include in a high-power drawing.
9) Make sure that this area of the specimen on the slide is directly over the hole in the microscope stage.`
10 ) Turn the nosepiece and bring the bigger objective lens into place over it. Make sure that it fully clicks into place.
11) Use the fine-focus knob to bring the specimen into sharp focus.
12) Make a separate drawing of two or three cells from each region that you highlighted in step 5. Draw clear unbroken lines and do not shade.
13) Label as many structures as you can see and identify. Use a ruler to draw the label lines and make sure that each label points exactly to the structure identified.

Question 27

rules for scientific drawing

Answer 27

• Use a sharp pencil on plain white unlined paper
• Drawings and must be done in pencil
• Place your drawing in a circle which represents the field of view
• Draw smooth continuous lines (no sketchy lines)
• Do no shade
• Diagram accurately represents what was being observed
• Your drawing must be proportionally correct
• Accuracy – pay attention to shape, size and thickness.
• Label lines should be draw with a ruler, should be horizontal and not cross or have arrowheads
• Write label at the end of the line – keep writing horizontal
• Include annotations to describe details such as colour, shape and thickness
• Write the title of what is being drawn
• State the magnification

Question 28

what is an eyepiece graticule

Answer 28

a measuring device. it is placed in the eyepiece of a microscope and acts as a ruler when you view an object under the microscope

Question 29

what is a stage graticule

Answer 29

a precise measuring device. it is a small scale that is placed on a microscope stage and used to calibrate the value of eyepiece divisions at different magnifications.

Question 30

give another name for a stage graticule

Answer 30

stage micrometer

Question 31

how are graticules used`

Answer 31

a microscope eyepiece can be fitted with a graticule. this graticule is transparent with a small ruler etched on it. as the specimen is viewed the eyepiece graticule scale is super imposed on it and the dimensions of the specimen can be measured

Question 32

what is the scale of the eyepiece graticule

Answer 32

arbitrary so it represents different lengths at different magnifications. the image of the specimen looks bigger at higher magnification but the actual specimen has not increased in size

Question 33

what needs to happen to the eyepiece scale for each objective lens

Answer 33

the eyepiece has to be calibrated for each different objective lens

Question 34

what is a stage graticule used to

Answer 34

to calibrate the eyepiece graticule

Question 35

how is a stage graticule used to calibrate the eyepiece graticule

Answer 35

a microscopic ruler on a special slide is called a stage graticule. this is placed on the microscopic stage. the ruler is 1mm long and divided into 100 divisions. each division is 0.01mm or 10μm.

Question 36

how to calibrate the eyepiece graticule

Answer 36

1) insert an eyepiece graticule into the x10 eyepiece of your microscope. this ruler has a total of 100 divisions.
2) place a stage graticule on the microscope stage and bring it into focus using the lower power objective lens (x4). total magnification - x40
3) align the eyepiece graticule and the stage graticule. check the value of one eyepiece division at this magnification of your microscope.
4) if the stage graticule (1mm or 1000μm) corresponds to 40 eyepiece divisions.
5) each eyepiece division = 1000/40μm= 25μm
6) now use the x10 objective lens on your microscope (total magnification is x100) and focus on the stage graticule
7) align them both
8) 100 eyepiece divisions now correspond with 1mm or 1000μm
9) therefore one eyepiece division = 1000/100μm = 10μm

Question 37

what should you do when you are finished with the microscope

Answer 37

• If viewing another slide, start on low power and repeat the focussing process.
• If finished for the day, remove the slide from the stage and return to origin.
• Clean any slides made by you
• Rotate the eyepiece to the lowest objective
• Turn the light off.
• Place the cover on the microscope.