Unit 2

Unit 2: Microscope Use and Cell Length CalculationsBy Patricia G. Wilber, Heather Fitzgerald and Karen Bentz. Copyright Central New Mexico Community College, 2015.

IntroductionIn microbiology laboratory, you will observe cells using the microscope to determine cell length, shapes and arrangements. You will use the 100X lens to magnify your specimens to a total magnification of 1000X.

I. Parts of the Microscope.Procedure

1. Obtain a microscope from the cabinet and put it on your desk. Carry the scope using the handle on the back and one hand under the scope for support.

2. Put your name next to the number of the scope you have chosen on the sign out sheet provided by your instructor. You will use the SAME SCOPE every class.

3. Please locate the following parts of the microscope on the image provided and the scope you have obtained. Your scope maybe slightly different from the image. Know what these parts do. If you have problems locating a part or understanding a function, be sure to ask for help.

Eyepiece (ocular) lens Objective lenses: 4X, 10X, 40X and 100X (oil immersion lens) Condenser aperture lever or Iris lever Light source Slide holder Slide adjustment knobs Coarse focus/adjustment knob Fine focus/adjustment knob

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Light Source

Slide Control Knobs

Fine Focus Knob

Coarse Focus Knob

Light Control Knob

Power On/Off Switch

Condenser Lens

Slide Holder

Ocular Lenses

Objective Lens, 4X

Stage

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Figure 2-1. Compound microscope.

Figure created by Karen Bentz, 2016

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II. Using the Microscope.

Materials Microscope Kimwipes Gram+ and Gram – prepared slides Immersion oil

ProcedureA. General Procedures for Use of the Scope

1. You will have an assigned microscope in this lab. You will use the same microscope for each lab, and you are responsible for its upkeep.

2. Go to the microscope cabinet and pick up your microscope. Always use two hands to carry the microscope.

3. Use Kimwipes and lens cleaner to clean all the lenses.4. Make sure the lowest objective (4X) lens is in position. (The lowest lens should already

be in place, but if it is not, place your index finger and thumb on the revolving nosepiece and move it into position.)

5. Place your slide in the slide holder on the stage with the beam of light passing through your specimen. (Hint: when choosing a specimen, choose a slide where you can see the color of the stain on the slide easily right when you pick it up.)

6. Move the stage up to its highest position using the coarse focus knob.7. Turn the light switch on and turn up the light. Adjust the light so it is comfortable for

your eyes.Double check: Is your specimen centered over the condenser lens with light shining through?

8. To focus in on your specimen, use the coarse focus knob and lower the stage until your specimen appears. Then sharpen the focus. Re-center.

9. Adjust the amount of light with the iris lever (See the diagram! Ask for help if you need it!) and light intensity knob.

10. WITHOUT LOWERING THE STAGE, use the revolving nosepiece to click the next objective lens (10X) in position. Focus with the fine focus knob until your image appears very sharp.

11. Re-center! Adjust the light! 12. WITHOUT LOWERING THE STAGE, gently click the next objective lens (40X) into place. It

might seem like there is not sufficient space between the stage and this lens, but if you had a sharp focus with the previous lens, there should be enough room.

13. Focus with the fine focus knob only. Re-center. Increase the amount of light!14. WITHOUT LOWERING THE STAGE, move the 40X lens out of the way. Do not put the

100X lens in place yet.15. Put a drop of oil directly on top of the coverslip.16. Rotate the 100X lens in place. DO NOT move the stage! Use the fine focus and bring the

object into view.

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17. You may need to turn the fine focus knob a few turns away from you in order to find the object. Adjust the amount of light!

NOTE: If you can’t find anything, you must go back to the 10X lens without getting oil on the 40X lens!

Before Putting the Scope Away1. Lower the stage with the coarse focus knob, and remove the slide.2. Dim the light all the way and turn off the light.3. With Kimwipes and lens cleaner, clean the slide, the ocular lenses and all the objective

lenses, especially the 40X and 100X. Check the body of the scope and clean that as well. 4. Wrap the cord.5. Have your scope inspected by your lab instructor.6. Return your scope to its spot in the cabinet, with the lenses facing IN.

Important Microscope Tips If you skip or disregard the steps, using the scope can be frustrating and you may even

damage the microscope and/ or the slide. Always start with the 4X objective lens! If you don’t, you may accidentally damage the

scope or slide. More than likely you will not be able to focus on your specimen. Most microscopes are parfocal, which is the ability to maintain focus when switching

between lenses. However, most parfocal microscopes still need some fine-focusing and re-centering, especially when switching to the higher objective lenses.

Use only the fine adjustment knob with the 40X and 100X lenses; do not use the coarse adjustment knob because there is very little room for movement of the 40X objective lens, and if the lens moves very much it may hit the slide and crack it.

The final (and longest) objective lens is called the oil immersion lens (100X) and can be used only with the application of immersion oil onto the slide. You will always need to go to this lens when observing bacteria.

DO NOT GET OIL ON THE 40X LENS!! Let your instructor know if there are any problems with your microscope.

B. Using the Microscope to Observe Bacteria.If you have carefully followed the steps of microscopy listed above, you should now have something in view! You can now use your specimen to observe cell shapes and arrangements, and determine cell size.

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Cell shape and arrangement.

Figure 2-2. Three basic cell shapes.

Figure created by Patricia. G. Wilber

Figure 2-3. Cell arrangements

Single (mono) Diplo Staphylo StreptoFigure created by Patricia. G. Wilber

Figure 2-4. Cell shape and arrangementA. Bacillus anthracis, shape: bacillus; arrangement: strepto-

Accessed 4/23/15 from http://commons.wikimedia.org/wiki/File:Anthrax_cells.jpg . The image is in the public domain.

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Bacilli Cocci SpirilliBacillus (singular) Coccus (singular) Spirillum (singular)

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B. Staphylococcus epidermidis, shape: coccus: arrangement: staphylo-

Accessed: 4/23/15. http://commons.wikimedia.org/wiki/File:Staphylococcus_epidermids.jpg#filelinks

Four basic cell arrangements . Dr. Sahay Creative Commons Attribution-Share Alike 3.0 Unported license.

C. Shape: sprillum; arrangement: mono-

Accessed 4/23/15. https://www.flickr.com/photos/occbio/6414380027/sizes/m/ Mark Perkins (All sizes of this photo are available for download under a Creative Commons Attribution-non-commercial 2.0 generic license)

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Video Links:

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Calculating Total Magnification, Field Diameter and Cell Size.

Calculating Field Diameter and Cell Size: https://mediasite.cnm.edu/Mediasite/Play/b363fd8840eb4100a2d1ed4914d5f46f1d

Video by Corrie Andries

You will need to be able to calculate cell size. To do that, you first need to calculate total magnification and field diameter.

Total Magnification1. Formula:

Total Magnification (TM) = Ocular Lens Value x Objective Lens Value2. Use your scope to determine the ocular lens and the objective lens value. 3. Fill the values into the table and calculate total magnification.

Table 2-1. Total Magnification for Your MicroscopeOcular Lens Value x Objective Lens Value = Total Magnification10X x 4X =

x =

x =

x =

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Field Diameter1. Formula:

(Objective Lens for the Known Field Diameter) X (Known Field Diameter)= (New Objective Lens) X (Unknown Field Diameter of the New Objective Lens)

We will solve for the Unknown Field Diameter, so we can rearrange the formula to make our lives a little easier:Unknown Field Diameter of the New Objective Lens =(Objective Lens Value for the Known Field Diameter ) X (Known Field Diameter)

(New Objective Lens Value)

2. Here is a table with values for a hypothetical microscope. The values are different than your actual scope. Figure out the Unknown Field Diameter for the hypothetical example for the 6X and 60X objective lenses.

3. Table 2-2. Practice Calculating Field Diameter for a Hypothetical Scope.Objective Lens Field Diameter3X 3 mm

6X

60X

Answers: Unknown Field Diameter for the 6X objective lens: 3X x 3 mm = 1.5 mm 6X

Unknown Field Diameter for the 60X lens: 3X x 3 mm = 0.15 mm OR 6X x 1.5 mm = 0.15 mm 60X 60X

4. Now, figure out the Field Diameters for your actual scope. First, look at your scope, then put the objective lens values in the appropriate column in Table 2-3.

Table 2-3. Calculating Field Diameter for Your Microscope Objective Lens Field Diameter in

mmField Diameter in μm

4X 5mm

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5. You may assume that the field diameter for the 4X objective lens is 5mm (that value has been put into Table 2-3 for you). Since you know both the objective lens value (4X) and the field diameter, you can derive the field diameter of any (all!) of the other objective lenses with the same formula (with different numbers!) you used to calculate the Field Diameter for the hypothetical scope.

6. On your mark! Get set! Go! Calculate the field diameter for the 10X objective lens on your microscope and record your answer in Table 2.3.

7. Calculate the field diameter for the 40X and 100X objective lenses and record youranswers in Table 2.3.

8. Convert your field diameters in Table 2.3 into micrometers (μm).

NOTE: Since total magnification is directly proportional to objective lens magnification,you can also use total magnification (TM) instead of objective lens magnification tocalculate field size. You will end up with the same results.Cell Length Calculation.

1. Formula: Field Diameter = Cell Length

# of cells that fit across field diameter

2. To estimate the length of a cell, use the following steps:Step 1. Estimate how many cells could be lined up end to end across the field diameter.Step 2. Divide the Field Diameter by the number of cells that could fit across the field diameter.

For example, to estimate the length of the cells in Figure 2-5:

Figure 2.5. Bacillus-shaped cells (mono- arrangement in case you were wondering) in a hypothetical field of view.

Field diameter (the line across the field of view) is 120um

Figure created by Patricia G. Wilber

Since the field diameter of Figure 2.5 is 120 μm. If you lined the cells up across the field diameter, even though many cells are “seen”, about eight cells could fit across the diameter. So:

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120μm/8 cells = 15 μm/cell The length of one cell is 15 μm.

If 75 Escherichia coli (more commonly known as E. coli) bacilli shaped bacterial cells could be lined up across a field diameter of 180 μm, what is the approximate length of one E. coli cell?

Answer: 180 μm/75 cells = 2.4 μm per cell

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Observing Bacterial Cells1. Obtain one prepared slide.2. Using proper technique, focus at TM 1000X.3. Draw your specimen and fill in the information in Figure 2-6.4. Get another slide or switch with your lab partner and fill in Figure 2-7

Figure 2-6.Name of specimen: _______________

TM used:________________________

Field diam: ______________________

Number of cells that fit across: ______

Cell size in mm:___________________

Cell size in μm:____________________

Cell shape: _______________________

Cell arrangement:__________________

Figure 2-7Name of specimen: _______________

TM used:________________________

Field diam: ______________________

Number of cells that fit across: ______

Cell size in mm:___________________

Cell size in μm:____________________

Cell shape: _______________________

Cell arrangement:__________________

The authors of this lab unit would like to thank Andrea Peterson and Deyanna Decatur for testing new media and organisms, our associate dean Linda Martin for many kinds of aid, Michael Jillson and Alex Silage for IT support, and our dean John Cornish .

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