1

Microscope operation

The practical is divided into a number of sections.

You will need your Practical Manual.

2

Familiarisation of the parts and use of the light microscope

The structure and use of the light microscope.

Parts of the microscope:1. Refer to the Practical Manual Introduction Section C.2. Using this as a guide, get out the microscope and

place it on the desk in front of you. Identify the parts of the instrument, noting the functions of each. When you feel conversant with this proceed to the next step.

3. Use the following presentation for microscopes with an inbuilt light source.

3

Getting a microscope The microscopes

are stored in boxes on trolleys

Each box has a number, as does the microscope

The numbers on the microscope, the box and its marked position on the trolley MUST all agree

Key and lock

Box and microscope number

4

Opening the box Please remember

the position and number of the microscope so that you can ensure that it is returned to the correct box and position on the trolley

Open the box with the key attached to the box

5

Picking up the microscope You are to ALWAYS

use two hands when carrying a microscope

Place one hand around the PILLAR of the microscope and the other under the BASE

Do not carry the box to your desk as there is insufficient room

Base

Pillar

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Turning the eyepiece Place the

microscope squarely on the table in front you, with the pillar away from you

Undo EYE PIECE TURRET lock and then rotate the turret towards you

Lock into position, finger tight

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Eyepiece to back

Eyepiece

turret

Eyepiece

Locking

knob

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Eyepiece in correct position

Eyepiece

Locking knob

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Eyepiece and eyepiece tube

Please be careful carrying microscopes as the eyepiece is easily removed from the eyepiece tube

This is a 10x eyepiece

10

Positioning the microscope Move the

microscope until it is in a comfortable position for you

The front side of the microscope should be facing you

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Turning on the lamp The microscope has a built in lamp, so

locate the on/off switch on the left-hand side and turn it on

Locate the brightness adjustment on the left-hand side High Low

On/Off switch Brightness adjustment

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Comparison of light intensities

Low intensity brightness of lamp

High intensity brightness of lamp

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Right sideEyepiece

Eyepiece tube

Rotating turretObjectives

Mechanical stageCondenser

Slide holder

Light

Base

Stage movement knobs

Coarse focusing knobFine focusing knob

Cord

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Left side

On/Off switchBrightness adjustment

Condenser knob

Coarse focusing knob

Fine focusing knob

Eyepiece

Light

Eyepiece tube

Rotating turret for objectivesObjectives

Eyepiece turret

Slide holder

Mechanical stage

4x

10x

40x

20x

Iris diaphragm

Base

Pillar

StopCondenser

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Front

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The iris diaphragm Locate the iris

diaphragm which is in the lower section of the condenser

Locate the flat silver lever on the right hand side of condenser which is used to open and close the iris

Do not touch.This is the centring

lock for the condenser

Iris diaphrag

m

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The iris diaphragm

Stage

Condenser

Iris diaphragm

Filter slot

Condenser knobLight

Stage knobs

Slide holder

Iris lever

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The iris diaphragm

Position backwards towards pillar = fully closed

Lever in back positio

nIris

almost closed

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The iris diaphragm

Position towards you = fully open

Open the iris diaphragm of the condenser fully

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Adjusting the condenser Locate the

condenser and its adjustment knob on the left-hand side

Set the condenser, using its focusing adjustment knob, so that the top of the lens of the condenser is about 2 mm below the upper surface of the stage

Condenser

Adjustment knob

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Position of condenser

Top of the lens of the

condenser

Stage openin

g

About 2 mm

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Focus adjustment knobs Locate the fine and

coarse focus adjustment knobs on both sides

Lower the microscope STAGE with the coarse focus adjustment knob so that the objectives on the rotating nosepiece will NOT hit the stage when rotated into position

Fine Coarse

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Of interest NOTE that the

stage and condenser move together

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Focusing using low power

For most microscopes this isOBJECTIVE 4xEYEPIECE 10x

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Rotating objective lenses Swing the low

power (4x) objective into position above the stage

Practice clicking the objectives into place, noting that the objectives SHOULD be changed in one direction only from LOW 4x to 10x to HIGH 40x

4x

10x

40x

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N.B. Always focus the

microscope with the low power (4x) objective first

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Objective characteristics

4x = low power objective Red ring, shortest in length widest field of view, brightest image

10x = Mid range objective Yellow ring, mid length Reduced field of view, image duller

40x = High power objective End sprung loaded, blue ring, longest Very limited field of view, image very

dull

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10x objective

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40x objective

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Slide holder on movable stage

Make sure that slide holder is positioned to back of the stage and that the two knurled knobs are finger tightSlide holder

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Slide into slide holder To place the slide in

position on stage

press lever at A and slide holder arm will swing to side

A

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Slide to stage (do not remove)

Place the slide against the left side and top of the slide holder

Press on A Lever swings

outwards Insert slide

Slowly release the movable section to hold the slide

Spring-loaded lever

A

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Movable stage The stage is moved

by two knobs on the right-hand side

The larger top knob moves the stage towards and away from you

The smaller lower knob moves the stage from left to right

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Horizontal and vertical stage scales

2 scales on the stage One at the back

which shows the amount that the microscope is moved from side to side

One on right-hand side for forward and backward movement Coordinates can also

be recorded of particular points of interest on slide

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Positioning the slide Use these knobs to

position the slide so that the OBJECT you wish to view is placed in the centre of the condenser lens

N.B. Scales for both movements (one on right for away and towards, and one at back for right to left) measure how much the slide is moved

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To start focusing Looking from the

side, using the coarse focus knob, lower the low power objective until it is about 6 mm above the glass cover strip of the slide

6 mm

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N.B.

These microscopes have STOPS on the left side of the stage mechanism to prevent damage to slides so DO NOT FORCE the KNOB past these

When you feel resistance to the free turning of the knob STOP because this point should allow all the objectives to be swung into place without them touching the slide

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Microscope stop – left side

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Field of view illumination The field of view of

the microscope should now be fairly evenly illuminated. Close the iris diaphragm until the light is about half its maximum intensity.

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N.B. N.B. A common cause of poor

definition of a specimen is over-illumination. Best definition is often obtained by cutting down light, not by increasing it.

Also remember that the light intensity can be reduced:

by changing the light intensity slide on the sideOR by reducing the diameter of the iris diaphragmOR by lowering the condenser

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Focusing on low power Keeping both eyes

open, look through the eyepiece and LOWER the STAGE by means of the coarse focus adjustment until the specimen comes into focus. Use the fine focus adjustment knob for critical focus

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N.B.

Never focus by moving the stage with the slide towards the objective lens. When you are looking down the eye piece you are unaware of where the objective lens is in relation to the slide, and it is easy to crush the cover slip and possibly the slide itself.

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Focusing using high power

These microscopes have two higher power objectives:

10x and 40x (100x oil immersion)

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Centring to point of interest

After finding the object with the 4x, move the slide until the object of interest lies in the exact centre of the field of view using the stage moving knobs

At this point you will find out whether your microscope is parafocal

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Parafocal

Parafocal means that once the microscope has been focused in one power, it is approximately in focus in the other magnifications

These microscopes should be parafocal

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Using the 10x objective Swing in the 10x

objective, watching to see that it does not touch the slide. The lower end of the objective will come close to it

A partial revolution of the fine adjustment focusing knob should bring the object into focus, if it is in the field of view

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Using the 40x objective Swing in the 40x

objective, watching to see that it does not touch the slide

Open the iris diaphragm slightly

Using the fine focus adjustment ONLY, bring the object into focus

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Problems If the object is not

in the field, or the slide has moved accidentally, swing in the 4x objective, then centre the object and proceed as before

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N.B.

Always return to low power before removing the slide from the stage.

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General precautions (1) Always be sure that the objectives will

not contact the stage or slide when moving them into a new position

ALWAYS FOCUS STAGE DOWN, never up, while looking through the microscope. This will avoid breaking the cover slip, crushing your specimen, damaging a prepared slide or even damaging the objective lens or condenser lens

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General precautions (2)

KEEP BOTH EYES OPEN ALL OF THE TIME, and alternate the viewing eye, to avoid eye strain and fatigue. This will seem very difficult at first, but practice will overcome the difficulty

Cover slips MUST always cover objects under examination, to protect the lens and the specimen

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General precautions (3) NEVER TOUCH ANY OF THE GLASS

PARTS OF THE MICROSCOPE WITH YOUR FINGERS

If a lens system is dirty or you have any trouble with the microscope, report it to your teacher. DO NOT ATTEMPT TO CLEAN a lens system with a handkerchief, tie, or anything else. Certain cloths will scratch the soft glass of the lens system

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Putting the microscope away

ENSURE that the following steps are carried out:

Swing the 4x objective into operational position

Unlock the turret and rotate to back, and lock here. Finger tight ONLY

Remove the slide from the stage. Allow stage clip to return to position on the stage

Carefully return the microscope to the trolley and place in the appropriate box.