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

6 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

7 Eyepiece to back Eyepiece turret Eyepiece Locking knob

8 Eyepiece in correct position Eyepiece Locking knob

9 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

11 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

12 Comparison of light intensities Low intensity brightness of lamp High intensity brightness of lamp

13 Right side Eyepiece Eyepiece tube Rotating turret Objectives Mechanical stage Condenser Slide holder Light Base Stage movement knobs Coarse focusing knob Fine focusing knob Cord

14 Left side On/Off switch Brightness adjustment Condenser knob Coarse focusing knob Fine focusing knob Eyepiece Light Eyepiece tube Rotating turret for objectives Objectives Eyepiece turret Slide holder Mechanical stage 4x 10x 40x 20x Iris diaphragm Base Pillar Stop Condenser

15 Front

16 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 diaphragm

17 The iris diaphragm Stage Condenser Iris diaphragm Filter slot Condenser knob Light Stage knobs Slide holder Iris lever

18 The iris diaphragm Position backwards towards pillar = fully closed Lever in back position Iris almost closed

19 The iris diaphragm Position towards you = fully open Open the iris diaphragm of the condenser fully

20 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

21 Position of condenser Top of the lens of the condenser Stage opening About 2 mm

22 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 FineCoarse

23 Of interest NOTE that the stage and condenser move together

24 Focusing using low power For most microscopes this is OBJECTIVE 4x EYEPIECE 10x

25 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

26 N.B. Always focus the microscope with the low power (4x) objective first

27 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

28 10x objective

29 40x objective

30 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 tight Slide holder

31 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

32 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

33 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

34 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

35 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

36 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

37 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

38 Microscope stop – left side

39 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.

40 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 side OR by reducing the diameter of the iris diaphragm OR by lowering the condenser

41 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

42 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.

43 Focusing using high power These microscopes have two higher power objectives: 10x and 40x (100x oil immersion)

44 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

45 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

46 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

47 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

48 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

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

50 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

51 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

52 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

53 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.