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USE OF THE MICROSCOPE

The microscope is absolutely essential to the microbiology lab: most microorganisms cannotbe seen without the aid of a microscope, save some fungi. And, of course, there are somemicrobes which cannot be seen even with a microscope, unless it is an electron microscope,such as the viruses.

You will be using an assigned light microscope for a variety of lab exercises through thesemester, everything from viewing pond water to identification of your unknown bacterium.Therefore, it is extremely important that you understand how to use the microscopeeffectively and how to use different types of microscopy----brightfield, phase-contrast, anddarkfield. These Nikon microscopes have a revolving condenser, with specific settings forthe 3 kinds of microscopy: the settings are labeled with white letters that can be seen in thefront. Bright field is “0”, darkfield is “DF”, and phase-contrast is “PH.” There are 3 settingsof phase-contrast, one for each of the lenses—PH1 for 10X, PH3 for 40X, PH4 for 100X. Ifyou forget, look at the markings in red on the objective lenses.

HANDLING THE MICROSCOPE:

Carry it with 2 hands---one on the arm and the other under the base. Clean ALL objective lenses and the ocular with lens paper BEFORE you even place a

slide on the stage, and it is a good idea to wipe the condenser lens also. The lastperson using the microscope may have left it dirty: it is imperative to begin with cleanlenses.

Use lens paper (ONLY) to remove any oil from the 100X lens. Once oil has been added to the slide, do not move back to the 40X lens to focus: oil

should never get on this lens. If this happens, it will be very difficult to get all of the oiloff, and you will have to clean the lens thoroughly.

Place the microscope back into the correct spot in cabinet, with the arm toward you. Loosely wind the electrical cord around the cord holder on the back of the arm. Do not

bend the cord at the microscope socket.

Determination of total magnification = ocular lens (10X) X objective lens (10X, 40X, 1 00X)

OBJECTIVES:

Identify the parts of the microscope and their functions.Become familiar with the 3 variations of light microscopy.Learn how to use the microscope effectively, and particularly the oil immersion lens.Determine total magnification of the specimen, using various objective lenses.Be able to switch objective lenses, while focused on the specimen, without moving the stage.Handle the microscope safely and clean it.Explain principles and terms used in microscopy and focusing.

Fall 2011 – Jackie Reynolds, Richland College

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MATERIALS NEEDED:

microscopelens paperbibulous paperoil dropperprepared wet mounts or stained smears

THE PROCEDURES:

You will use a variety of specimens with your microscope----a wet mount, a boughtprepared smear, and a stained smear that you have made. Refer to the lab exercisePREPARATION OF SPECIMENS.

BEFORE PUTTING A SLIDE ON THE MICROSCOPE STAGE:

1. Find all of the structures on the microscope (diagrams below) being sure that youknow their functions. Rotate the condenser so that you see all of the settings (whiteletters are engraved into the front of the condenser dial). Also, move the irisdiaphragm left and right so you can see the effect on the amount of light.

2. The NIKON microscopes have 3 types ofcondenser lenses for 3 types of l ightmicroscopy:

brightfield (O on condenser) darkfield (DF on condenser) phase-contrast (condenser settings: PH

1 for 10X, PH3 for 40X, PH4 for 1 00X)3. Start with brightfield microscopy ALWAYS.

The brightfield condenser has a 0 etched inwhite.

4. Raise the condenser stage ALL THEWAY UP. There is a special knob for thecondenser stage under the mechanicalstage . The condenser gathers all available light from the lamp and directs it up to thestage. We always have the condenser stage closest to the mechanical stage whenviewing microorganisms. When viewing largest objects, like worms or insects, youcan move the condenser down to improve light density hitting the specimen, but not formicroorganisms.

5. Turn the brightness control knob ALL the way up, and then back off 1/4 of a turn.This is where the control knob will stay: do not touch it again. Your light amountcoming up through the condenser is controlled by the iris diaphragm.

6. Rotate the revolving nosepiece until the low power 10Xobjective lens snaps intoplace.

7. Bring the stage all the way up, using the coarse adjustment knob. Keep an eye onthe distance between the slide and the lens to MAKE SURE that you do not crash thelens into the stage.

9. Clean all lenses (oculars, objective lenses, and lens on condenser) with LENS PAPER.10. Set the ocular lenses to the correct distance for your face (the oculars can be moved

apart or closer together for your own needs). These ocular eyepiece lenses are both

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10X magnification.11.You will want to reduce the light coming through the condenser, so close your iris

diaphragm so that you get better contrast of the specimen.

TO VIEW A SPECIMEN:

1. Place the wet mount or prepared smear on the stage, and secure it inside of the stageholder.

2. Try to guesstimate where the specimen is located on the slide, and place it in thecenter of the hole allowing light through the stage.

3. While looking through the ocular eyepiece, lower the stage SLOWLY using the coarseadjustment knob. Be sure that you are looking through the binocular head of themicroscope with BOTH eyes.

4. As soon as you see the specimen, STOP using the coarse adjustment, and switchover to the fine adjustment knob. After focusing at the beginning with the coarseadjustment knob, it is NOT TOUCHED AGAIN. All focusing will now be done with thefine adjustment knob.

5. CHANGING OBJECTIVES:

The Nikon lenses are PARFOCAL---the objectives arealigned so that rotation to another lens can be donewithout major focusing. Rotate the 40X lens in place,making sure that it snaps into place. Your specimenshould still be seen in the field of vision, but 4 timeslarger now. Use your fine adjustment knob to clarify theobjects.IF YOUR FIELD OF VISION IS FUZZY, AND NOAMOUNT OF FOCUSING BRINGS THE OBJECT INTOVIEW, YOU PROBABLY HAVE OIL RESIDUE ON THE40x OBJECTIVE. IT HAS TO BE CLEANED WELLWITH LENS PAPER.

TO MOVE INTO OIL IMMERSION, 100XMAGNIFICATION:

1. Do NOT MOVE the focus knobs or the stage knobs. Swing the 40X objective (highdry) out of the way. Place a single drop of immersion oil on the slide right over wherethe light is coming through the stage, and rotate the 100X objective (oil immersion)into place. The lens will actually GO INTO THE OIL DROP.

2. Now look through the oculars, increasing your light with your iris diaphragm lever.Your object should still be in the field of vision, probably out of focus. Use the fineadjustment knob to focus clearly.

3. Once you have gone into oil immersion, do NOT GO BACK TO THE 40XOBJECTIVE. The objective will get oil on it, and you will have to really clean it to getthe oil off. The 1 0X can be returned to, since the lens should not touch the slideanyway.

4. Once through with the microscope, use the lens paper to wipe the oil from the100X objective lens.

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USING DARKFIELD AND PHASE-CONTRAST:

Once you are looking at your object using brightfield microscopy, you can easily switch toanother type of microscopy: Just rotate the condenser knob. Darkfield and phase-contrastmicroscopies have particular condenser lenses required for proper visualization.HOWEVER:

Darkfield is used for wet mounts, using 10X and 40X (1 00X will not show well).Be sure that your iris diaphragm is OPEN all the way.

Phase-contrast is used for wet mounts also, although SOMETIMES it is helpfulfor delineating subtle shapes and colors that cannot be readily seen usingbrightfield. Be sure that you are using the correct condenser setting for thatparticular objective lens. Depending on whether you want to use the 10X, 40X,or 1 00X objective lens, you will have to change the phase-contrast condenserlens to the appropriate setting.

PLACING MICROSCOPES BACK INTO THE CABINETS: YOU are responsible for your assigned microscope! There is only 1 person in each

lab who is assigned that particular microscope, so if someone else complains about itbeing left with oil or a slide on the stage, you or another person who is assigned thatparticular scope will be reprimanded.

Make sure that the 10X low power lens is in place, pointing towards the stage---not the1 00X oil immersion lens. The lens could hit against the stage and get scratched.

Turn the coarse adjustment knob so that the stage is far from the lens. Do NOT drag it across the table, making annoying grating noises. Wind the electrical cord around the cord holder properly. Remove any slide left on the stage. PLACE YOUR MICROSCOPE BACK IN ITS NUMBERED POSITION IN THE

CORRECT CABINET. THE MICROSCOPE GOES INTO THE CABINET WITH ARM OUT.

TROUBLESHOOTING:Focus fuzzy?Probably oil on the lens. Clean with lens paper thoroughly. If that does not help, use lenscleaner with lens paper. If that doesn’t work, ask instructor for some help. Xylene may beused, but SPARINGLY since it can destroy the glue seating the lens.

Light off center?Do you have the lens correctly in place? As you rotate the nosepiece, each lens should“click” into place, and you will know that it is in the correct position.

Found the specimen on low power, but lose it when moving into a high power?

Not focusing on the specimen, but rather dirt on slide.

Specimen off-center on slide: when moving to a higher lens magnification, the specimen isoutside the field of vision Slide has been turned upside down, with specimen facing down towards stage.

PART OF MICROSCOPE FUNCTION

1. coarse adjustment knob general focus, particularly for 10X lens2. fine adjustment knob fine focus, particularly for 1 00X lens3. arm infrastructure of the microscope4. power switch/brightness control on/off switch for light, changes intensity5. base infrastructure of the microscope6. condenser knob condenser movement7. iris diaphragm lever control of cone of light coming through

condenser8. objective lens magnification of 10X, 40X, or 100X9. revolving nosepiece attachment of objective lenses10.ocular eyepiece lens magnification of 1 0X11.stage holder hold slide in place12.stage holder knobs movement of stage, 2 directions13. stage placement of slide

LABORATORY REPORT SHEET

QUESTIONS:

1. Which objective lens is also called the oil-immersion lens?

2. How do the functions of the substage condenser and the iris diaphragm within thecondenser differ?

3. What term is used to describe the feature of the microscope that makes it possible to moveamong the objective lenses with just MINOR focusing?

4. What condenser setting value do you want when you are using the 1 00X objective lens?

5. What would be the total magnification of a specimen using the 40X objective lens?

6. Why move the 10X objective lens into place when putting the microscope back into thecabinet?

7. What is parfocal?

8. Why reduce your light when using the 10X objective lens?