Microbiology and MicroscopyMicrobiology and Microscopy Experiment 4 – Cappuccino and ShermanExperiment 4 – Cappuccino and Sherman

Professor SidelskyProfessor Sidelsky

psidelsky@gmail.compsidelsky@gmail.com

Introducing your Introducing your microscopemicroscope

Always clean lenses before startingAlways clean lenses before starting Always with both hands supporting Always with both hands supporting

basebase Store with arm facing the door. CoverStore with arm facing the door. Cover Focus with scanning and low before Focus with scanning and low before

proceeding to high and oilproceeding to high and oil Learn the most effective ways to Learn the most effective ways to

regulate and manage lightregulate and manage light

Microscope PartsMicroscope Parts

Eyepiece( Ocular)Eyepiece( Ocular)

Binocular –two eyepieces – Binocular –two eyepieces – magnification 10xmagnification 10x

Objective lenses – 4( see Objective lenses – 4( see chart)chart)

Condenser lens – collects Condenser lens – collects and directs light through and directs light through the lensesthe lenses

Condenser adjustment – Condenser adjustment – raises and lowers the raises and lowers the condenser lens for better condenser lens for better focusfocus

Mechanical stage Mechanical stage adjustment – Knobs that adjustment – Knobs that move the mechanical move the mechanical stagestage

Coarse adjustment – used Coarse adjustment – used with scanning and low with scanning and low powerpower

Fine adjustment – used Fine adjustment – used with high and oilwith high and oil

LENSES ADJUSTMENTS

Binocular MicroscopesBinocular Microscopes

ChecklistChecklist

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

Microscope Anatomy – Relate to function

Olympus web site on the Olympus web site on the anatomy of a microscopeanatomy of a microscope

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

EyepieceEyepiece Adjustment of the Adjustment of the

Interpupillary Distance The Interpupillary Distance The interpupillary distance is interpupillary distance is the distance between the the distance between the centers of your two pupils. centers of your two pupils. The distance between the The distance between the two eyepieces of the two eyepieces of the binocular microscope must binocular microscope must correspond to your correspond to your interpupillary distance.interpupillary distance.

Each person has his or her Each person has his or her own interpupillary distance own interpupillary distance and the microscope must and the microscope must be adjusted for your be adjusted for your specific distance. specific distance.

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

Mechanical Stage and Mechanical Stage and AdjustmentsAdjustments

Holds slide in Holds slide in positionposition

Movement of slide Movement of slide on X and Y axis on X and Y axis with adjustment with adjustment knobsknobs

Permits student to Permits student to locate specific site locate specific site on slideon slide

Condenser lens and iris Condenser lens and iris diaphragmdiaphragm

Condenser lens

Iris diaphragm

Microscope lensesMicroscope lenses

LensesLenses

Objective LensesObjective LensesObjective LensObjective Lens MagMag Total MagTotal Mag Color of Color of

LensLensUseUse

ScanningScanning 55 5050 RedRed Observe the entire Observe the entire slide. Focus with slide. Focus with coarse adjustmentcoarse adjustment

LowLow 1010 100100 YellowYellow Tweak focus with Tweak focus with coarse. Make sure coarse. Make sure the image is clear the image is clear and centeredand centered

High High 4040 400400 BlueBlue Focus only with Focus only with fine adjustmentfine adjustment

OilOil 100100 10001000 WhiteWhite Apply oil and then Apply oil and then turn high power turn high power objective lens and objective lens and focus with fine adjfocus with fine adj

Coarse and fine Coarse and fine adjustmentadjustment

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

Microscope termsMicroscope terms

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

Total MagnificationTotal Magnification

The magnification of the objective lens The magnification of the objective lens

X X

the magnification of the ocular( 10)the magnification of the ocular( 10)

ResolutionResolution

Resolution is the ability to see two points Resolution is the ability to see two points as separate entitiesas separate entities

Resolution is determined by the Resolution is determined by the magnification and the numerical aperture magnification and the numerical aperture – The numerical aperture is the opening – The numerical aperture is the opening at the bottom of the lensat the bottom of the lens

Numerical apertureNumerical aperture

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

TermsTerms

Size of field – the diameter of the field of Size of field – the diameter of the field of view at different magnificationsview at different magnifications

Depth of field – the depth of the image Depth of field – the depth of the image through which it is possible to focusthrough which it is possible to focus

Working distance - the distance between Working distance - the distance between the stage and the bottom of the lensthe stage and the bottom of the lens

Ease of focus – ability to bring image into Ease of focus – ability to bring image into focusfocus

More TermsMore Terms

Parfocal – The quality of a microscope Parfocal – The quality of a microscope that enables one change from a lower that enables one change from a lower power to a higher power and still retain power to a higher power and still retain the focusthe focus

Parcentric – If the image is basically in Parcentric – If the image is basically in the center of the field of view – it will the center of the field of view – it will remain in the center even if the remain in the center even if the magnification is changed.magnification is changed.

Practice Slide for techniquePractice Slide for technique

Use a prepared slide to view Use a prepared slide to view bacterial cells. Place it on the Stagebacterial cells. Place it on the Stage

Observe the slide on scanning, low, Observe the slide on scanning, low, and high power.and high power.

Adjust the light and draw on high Adjust the light and draw on high power.power.

High PowerHigh Power

Always begin focus on scanning or Always begin focus on scanning or low. Look over the slide to choose low. Look over the slide to choose and optimal section for viewing.and optimal section for viewing.

If your focus is sharp and clear, turn If your focus is sharp and clear, turn the revolving nosepiece to high (blue)the revolving nosepiece to high (blue)

Use the fine adjustment, focus Use the fine adjustment, focus carefully – you are very close to the carefully – you are very close to the slide.slide.

Light Adjustment( Three Light Adjustment( Three ways to adjust the light)ways to adjust the light)

Iris Diaphragm – lever under the stage. Iris Diaphragm – lever under the stage. This works like the iris of the eyes This works like the iris of the eyes

Condenser lens – collects and directs Condenser lens – collects and directs the light. Use the condenser the light. Use the condenser adjustment to move the lens closer to adjustment to move the lens closer to and further from the stage.and further from the stage.

Light intensity adjustment( diopter) Light intensity adjustment( diopter) can also be checked.can also be checked.

Oil immersion lensOil immersion lens This lens provides the best resolution and This lens provides the best resolution and

the highest magnification ( 1000x)the highest magnification ( 1000x) Focus first on high power. Tweak your Focus first on high power. Tweak your

focus until it is perfect with the fine focus until it is perfect with the fine adjustment.adjustment.

Turn the revolving nosepiece until it is Turn the revolving nosepiece until it is between the high and oil immersion lens, between the high and oil immersion lens, use oil dropper to place a drop over the use oil dropper to place a drop over the center of the slide. Slowly turn lens into center of the slide. Slowly turn lens into the oil. Then again use the fine the oil. Then again use the fine adjustment to make the image clear.adjustment to make the image clear.

Oil immersion lensOil immersion lens

Pond WaterPond Water

Practice using microscope with the Practice using microscope with the slides of pond water.slides of pond water.

Draw your slides on scanning, low, Draw your slides on scanning, low, high, and oilhigh, and oil

Review your procedures for using Review your procedures for using coarse and fine adjustment, the coarse and fine adjustment, the condenser knobs, and mechanical condenser knobs, and mechanical stagestage

Stagnant Water SlideStagnant Water Slide

Cleaning UpCleaning Up

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