Microbiology and Microscopy Experiment 4

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Microbiology and Microscopy Experiment 4
Cappuccino and Sherman

• Professor Sidelsky
• psidelsky_at_gmail.com

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Introducing your microscope

• Always clean lenses before starting
• Always with both hands supporting base
• Store with arm facing the door. Cover
• Focus with scanning and low before proceeding to
  high and oil
• Learn the most effective ways to regulate and
  manage light

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Microscope Parts
LENSES
ADJUSTMENTS

• Eyepiece( Ocular)
• Binocular two eyepieces magnification 10x
• Objective lenses 4( see chart)
• Condenser lens collects and directs light
  through the lenses

• Condenser adjustment raises and lowers the
  condenser lens for better focus
• Mechanical stage adjustment Knobs that move the
  mechanical stage
• Coarse adjustment used with scanning and low
  power
• Fine adjustment used with high and oil

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Binocular Microscopes
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Checklist

• Find the following parts on your microscope,and
  be sure to know the functions of each    Coarse
  adjustment knob    Fine adjustment knob   
  Eyepieces    Objective lenses- 4x, 10x, 40x,
  100x oil    Light source (lamp)     On-off knob
  for light    Condenser adjustment knob   
  Diaphragm lever 

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Microscope Anatomy Relate to function
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Olympus web site on the anatomy of a microscope

• http//www.olympusmicro.com/primer/anatomy/anatomy
  .html

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Eyepiece

• Adjustment of the Interpupillary Distance The
  interpupillary distance is the distance between
  the centers of your two pupils. The distance
  between the two eyepieces of the binocular
  microscope must correspond to your interpupillary
  distance.
• Each person has his or her own interpupillary
  distance and the microscope must be adjusted for
  your specific distance.

• This is true of any binocular microscope. During
  your first session with a new microscope you must
  determine the correct interpupillary distance and
  set the microscope for that distance. If you look
  through the eyepieces and see two images, the
  interpupillary distance is not correct. To
  correct it, slide the eyepieces closer together
  or farther apart until the two fields merge to
  form a single circle of light. The interpupillary
  distance is now correct for you.

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Mechanical Stage and Adjustments

• Holds slide in position
• Movement of slide on X and Y axis with adjustment
  knobs
• Permits student to locate specific site on slide

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Condenser lens and iris diaphragm
Condenser lens
Iris diaphragm
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Microscope lenses
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Lenses
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Objective Lenses
Objective Lens Mag Total Mag Color of Lens Use
Scanning 5 50 Red Observe the entire slide. Focus with coarse adjustment
Low 10 100 Yellow Tweak focus with coarse. Make sure the image is clear and centered
High 40 400 Blue Focus only with fine adjustment
Oil 100 1000 White Apply oil and then turn high power objective lens and focus with fine adj
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Coarse and fine adjustment

• Use the coarse adjustment to assist you in
  focusing on scanning and low Do not use the
  coarse adjustment on high and oil

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Microscope terms

• Microscope terms
• Magnification
• Total magnification
• Resolution
• Depth of field
• Size of field
• Working distance
• Parfocal
• Parcentric

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Total Magnification

• The magnification of the objective lens
• X
• the magnification of the ocular( 10)

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Resolution

• Resolution is the ability to see two points as
  separate entities
• Resolution is determined by the magnification and
  the numerical aperture The numerical aperture
  is the opening at the bottom of the lens

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Numerical aperture

• A measure of the lenses ability to gather light
  and focus an image at a precise focal lengthh

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Terms

• Size of field the diameter of the field of view
  at different magnifications
• Depth of field the depth of the image through
  which it is possible to focus
• Working distance - the distance between the
  stage and the bottom of the lens
• Ease of focus ability to bring image into focus

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More Terms

• Parfocal The quality of a microscope that
  enables one change from a lower power to a higher
  power and still retain the focus
• Parcentric If the image is basically in the
  center of the field of view it will remain in
  the center even if the magnification is changed.

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Practice Slide for technique

• Use a prepared slide to view bacterial cells.
  Place it on the Stage
• Observe the slide on scanning, low, and high
  power.
• Adjust the light and draw on high power.

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High Power

• Always begin focus on scanning or low. Look over
  the slide to choose and optimal section for
  viewing.
• If your focus is sharp and clear, turn the
  revolving nosepiece to high (blue)
• Use the fine adjustment, focus carefully you
  are very close to the slide.

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Light Adjustment( Three ways to adjust the light)

• Iris Diaphragm lever under the stage. This
  works like the iris of the eyes
• Condenser lens collects and directs the light.
  Use the condenser adjustment to move the lens
  closer to and further from the stage.
• Light intensity adjustment( diopter) can also be
  checked.

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Oil immersion lens

• This lens provides the best resolution and the
  highest magnification ( 1000x)
• Focus first on high power. Tweak your focus
  until it is perfect with the fine adjustment.
• Turn the revolving nosepiece until it is between
  the high and oil immersion lens, use oil dropper
  to place a drop over the center of the slide.
  Slowly turn lens into the oil. Then again use
  the fine adjustment to make the image clear.

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Oil immersion lens
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Pond Water

• Practice using microscope with the slides of pond
  water.
• Draw your slides on scanning, low, high, and oil
• Review your procedures for using coarse and fine
  adjustment, the condenser knobs, and mechanical
  stage

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Stagnant Water Slide
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Cleaning Up

•  1.  Put low power objective lens in
  place. 2.  Lower stage all the way down. 3. 
  Clean oil off immersion lens. 4.  Remove slide
  and clean off oil. 5.  Turn light off. 6. 
  Unplug and cover.

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