MICROSCOPE

AMAILA QAISAR
M.PHIL MLS (UHS, LAHORE)
COURSE TITLE: GENERAL MICROBIOLOGY
COURSE CODE MLMB-513
 MICROSCOPE

• THE LIGHT MICROSCOPE IS ONE OF THE MOST BASIC AND ESSENTIAL EQUIPMENT USED IN ANY
LABORATORY.
• IT IS USED FOR VISUALISING VERY SMALL OBJECTS LIKE CELLS, BACTERIA, PARASITES, THEIR OVA/
CYSTS AND CRYSTALS ETC., THAT ARE OTHERWISE NOT VISIBLE TO THE
NAKED EYE.
• IT COMPRISES A SERIES OF LENSES, WHICH MAGNIFY AN ILLUMINATED SMALL OBJECT SEVERAL TIMES
TO MAKE IT RECOGNISABLE WITH THE NAKED EYE AND TO STUDY ITS DETAILS.
• SUCH A MICROSCOPE IS CALLED COMPOUND LIGHT MICROSCOPE.
Typical Compound Microscope
 WHAT IS THE PURPOSE OF A
MICROSCOPE?
• TO MAGNIFY OR ENLARGE THE IMAGE OF TINY OBJECTS.
• THE MICROSCOPES WE WILL USE THIS YEAR IN SCIENCE CAN
MAGNIFY OBJECTS 40 TIMES, 100 TIMES, AND 400 TIMES THE SIZE
OF THE OBJECT.
WHAT TYPE OF MICROSCOPE WILL WE
BE USING IN LABORATORIES?
• IN LABORATORIES, WE USE A COMPOUND LIGHT MICROSCOPE
FOR MOST OF OUR LAB ACTIVITIES.
• A LIGHT MICROSCOPE USES LIGHT AS A SOURCE OF ENERGY FOR
VIEWING OBJECTS.
• A COMPOUND MICROSCOPE CONTAINS TWO LENSES FOR
MAGNIFYING A SPECIMEN AT THE SAME TIME, THE EYEPIECE
(OCULAR) AND ONE OF THE OBJECTIVE LENSES.
• WHETHER A LIGHT MICROSCOPE IS MONOCULAR (HAVING ONE EYEPIECE)
OR BINOCULAR (HAVING TWO EYEPIECES) OR MULTIHEAD (USED BY MORE THAN ONE
OBSERVERS SIMULTANEOUSLY) THE BASIC COMPONENTS REMAIN
THE SAME.
• IT HAS THREE BASIC COMPONENTS:
• FOOT PIECE
• BODY
• EYE PIECE
 FOOT PIECE/ BASE
• SUPPORTS THE WHOLE MICROSCOPE
• USED TO CARRY THE MICROSCOPE
• WHEN CARRYING A MICROSCOPE, ALWAYS HAVE ONE HAND ON
THE ARM AND ONE HAND ON THE BASE. USE TWO HANDS!!
 FOOT PIECE

IT FORMS THE BASE OF THE MICROSCOPE AND PROVIDES STABILITY TO THE BODY AND EYEPIECES.
• THE LIGHT SOURCE, WITH OR WITHOUT ITS CONTROLS, IS USUALLY INCORPORATED IN THE BASE.
• IN SOME OLD OR FIELD MICROSCOPES A MIRROR IS PROVIDED IN PLACE OF A LIGHT SOURCE.
• THIS ALLOWS THE USE OF NATURAL OR EXTERNAL SOURCE OF LIGHT TO ILLUMINATE THE OBJECT.
• ONE SIDE OF THE MIRROR IS CONCAVE AND IS USED WHEN MORE INTENSE LIGHT IS REQUIRED TO
ILLUMINATE A SMALL FIELD.
• THE OTHER SIDE OF THE MIRROR IS CONVEX AND IS USED WHEN LESS INTENSE (DIFFUSE)
LIGHT IS REQUIRED TO ILLUMINATE A BROAD FIELD.
Eyepiece
 EYEPIECE
• ALSO KNOWN AS THE OCULAR
• CONTAINS THE FIRST LENS YOU LOOK THROUGH - USUALLY A
MAGNIFICATION OF 10X
• LOCATED ON THE TOP OF THE BODY TUBE
• THE OBSERVER, TO LOOK AT THE OBJECT UNDER EXAMINATION USES THIS PART OF THE
MICROSCOPE.
• THE MICROSCOPE HAVING ONE EYEPIECE IS CALLED MONOCULAR
• WHEREAS THE MICROSCOPE WITH TWO EYEPIECES IS CALLED BINOCULAR MICROSCOPE.
• THE EYEPIECE CONSISTS OF A SYSTEM OF LENSES THAT FURTHER MAGNIFY THE IMAGE
PRODUCED BY THE OBJECTIVE.
• THE MAGNIFICATION POWER OF THE EYEPIECE IS INSCRIBED ON IT E.G., X10. IN BINOCULAR
MICROSCOPE TWO EYEPIECES ARE INSTALLED IN A TUBE PROVIDED WITH A PRISM TO DIVERT
THE INCIDENT LIGHT TO BOTH EYEPIECES EQUALLY.
THE OBSERVER CAN ADJUST THE DISTANCE BETWEEN TWO EYEPIECES (INTER-PUPILLARY
DISTANCE) TO HIS CONVENIENCE.
MOVEMENT OF THE EYEPIECE IN THE HOLDING TUBE ALLOWS DIOPTER SETTING FOR AN
INDIVIDUAL OBSERVER.
 BODY

• THE BODY OF THE MICROSCOPE IS MOUNTED ON THE FOOT PIECE.

• IT HOLDS A SUB-STAGE CONDENSER, A STAGE AND A NOSEPIECE.
• SUB-STAGE CONDENSER IS COMPOSED OF A SYSTEM OF LENSES AND DIAPHRAGM.
• THE INTENSITY OF LIGHT AND THE SIZE OF FIELD ILLUMINATED BY IT ARE CONTROLLED BY
MOVING THE
Nosepiece
 NOSEPIECE
• HOLDS THE OBJECTIVE LENSES
• ROTATES TO ENABLE MAGNIFICATION
• LOCATED AT THE BOTTOM OF THE BODY TUBE
• IN MODERN MICROSCOPES IT COMPRISES A REVOLVING DEVICE TO HOLD 4-5 OBJECTIVES
OF DIFFERENT MAGNIFICATION.
• THE DEVICE HELPS IN BRINGING THE REQUIRED OBJECTIVE OVER THE OBJECT FOR
EXAMINATION.
 OBJECTIVE LENSES
• USED IN COMBINATION WITH THE EYEPIECE TO PROVIDE A RANGE
OF MAGNIFICATION
• MAGNIFICATION RANGES FROM 40X TO 400X
• LOCATED ON THE NOSEPIECE AT THE BOTTOM OF THE BODY TUBE
• AN OBJECTIVE COMPRISES A SYSTEM OF LENSES, WHICH MAGNIFY THE IMAGE SEVERAL TIMES.
• EACH OBJECTIVE IS MARKED WITH A COLOURED LINE, WHICH INDICATES
ITS MAGNIFICATION.
• THE MAGNIFICATION IS ALSO ENGRAVED ON THE OBJECTIVE IN NUMERIC ALONG WITH OTHER
INFORMATION.
• FOR EXAMPLE A DRY HIGH POWER OBJECTIVE HAS A BLUE LINE AND IS ENGRAVED WITH
FOLLOWING:
PLAN 40/0.65
160/0.17
THIS MEANS THAT THIS PARTICULAR OBJECTIVE HAS A MAGNIFICATION OF X40 AND HAS A
NUMERICAL
APERTURE 0.65 AT A TUBE LENGTH OF 160 MM WHEN A COVER GLASS OF 0.17 MM
THICKNESS IS USED.
• WORD PLAN DENOTES THE TYPE OF OBJECTIVE.
• FOLLOWING ARE THE COMMON OBJECTIVES INSTALLED IN
AN ORDINARY LIGHT MICROSCOPE:
• SCANNER: RED LINE, X4 MAGNIFICATION
• LOW POWER: YELLOW LINE, X10 MAGNIFICATION
• DRY HIGH POWER: BLUE LINE, X 40 MAGNIFICATION
• OIL IMMERSION: WHITE LINE, X100 MAGNIFICATION
Arm
 ARM
• SUPPORTS THE UPPER PARTS OF THE MICROSCOPE
• USED TO CARRY THE MICROSCOPE
• WHEN CARRYING A MICROSCOPE, ALWAYS HAVE ONE HAND ON
THE ARM AND ONE HAND ON THE BASE. USE TWO HANDS!!
 STAGE
• SUPPORTS THE SLIDE
• THE SLIDE CONTAINS THE SPECIMEN OR OBJECT THAT YOU ARE
VIEWING WITH THE MICROSCOPE.
• THE STAGE IS A DEVICE FOR HOLDING THE OBJECTS FOR EXAMINATION.
• IT HAS A HOLE IN THE MIDDLE OVER WHICH THE OBJECT IS PLACED.
• EXACTLY UNDERNEATH THE HOLE IS THE SUBSTAGE CONDENSER.
• THE STAGE MAY BE A FIXED STAGE WITH CLIPS TO HOLD THE OBJECT IN PLACE.
• BUT IN MOST MICROSCOPES IT IS PROVIDED WITH A MECHANICAL DEVICE TO MOVE THE OBJECT IN BOTH
PLANES (MECHANICAL STAGE).
• THE DEVICE IS MARKED ON BOTH AXES FOR NOTING THE GRID REFERENCE OF THE FIELD EXAMINED.
• THIS HELPS IN LOCALISING THE FIELD IN FUTURE EXAMINATION OF THE SAME OBJECT.
Stage Clip
 STAGE CLIP
• HELPS TO HOLD THE SLIDE IN PLACE
• USUALLY ONE ON EACH SIDE OF THE HOLE (STAGE OPENING) = 2
STAGE CLIPS
• THE STAGE OPENING ALLOWS LIGHT TO PASS FROM THE LIGHT
SOURCE TO THE LENSES.
Light Source
 LIGHT SOURCE
• PROVIDES LIGHT NECESSARY FOR VIEWING THE SPECIMEN
• USUALLY EITHER A MIRROR OR ILLUMINATOR
• SENDS LIGHT THROUGH THE STAGE OPENING TO THE DIAPHRAGM
Diaphragm
 DIAPHRAGM
• WHEEL OR LEVER LOCATED BELOW THE STAGE OPENING
• REGULATES THE AMOUNT OF LIGHT THAT CAN ENTER THE LENSES
• MAY NEED TO BE ADJUSTED BASED ON THE THICKNESS OF THE
SPECIMEN BEING STUDIED
Coarse Adjustment Knob
 COARSE ADJUSTMENT KNOB
• RAISES AND LOWERS THE STAGE OR OBJECTIVE LENSES
• USED ONLY WHEN FOCUSING THE LOW POWER (4X) OBJECTIVE
LENS
Fine Adjustment Knob
 FINE ADJUSTMENT KNOB
• RAISES AND LOWERS THE STAGE OR OBJECTIVE LENSES A SMALL
DISTANCE FOR EXACT FOCUSING
• USED WHEN FOCUSING THE MEDIUM POWER (10X) AND HIGH
POWER (40X) OBJECTIVE LENSES
• THE LIGHT CONSTITUTES THE RAW MATERIAL OF THE LIGHT MICROSCOPY. THE LIGHT IS A
FORM OF ENERGY THAT TRAVELS IN WAVES.
• WAVELENGTH IS THE DISTANCE BETWEEN TWO CORRESPONDING POINTS ON ADJACENT
WAVES AND DETERMINES THE COLOUR OF LIGHT.
• THE VISIBLE LIGHT IS A MIXTURE OF SEVEN DIFFERENT COLOURS WITH WAVELENGTH (Λ) IN
THE RANGE OF 400- 750 NM
• THE FREQUENCY (F), I.E. THE NUMBER OF VARIATIONS PER SECOND, OF THESE WAVES IS
RESPONSIBLE FOR DIFFERENCES IN COLOUR.
• WHEREAS THE AMPLITUDE, I.E., VERTICAL DISPLACEMENT OF THE WAVE FROM THE OPTICAL
AXIS DETERMINES THE INTENSITY OR BRIGHTNESS.
• THE LIGHT RAYS WHEN PASS FROM AIR TO A DENSE MEDIUM E.G., THE LENS OF THE
MICROSCOPE, CHANGE THEIR DIRECTION AND SPEED. THIS IS CALLED REFRACTION.
• THE REFRACTIVE INDEX OF AIR IS 1.0 WHEREAS THAT OF GLASS AND CEDAR WOOD
OIL IS 1.5.
• IF THE REFRACTIVE INDEX OF ALL THE MEDIA IS SAME IT RESULTS IN BETTER MAGNIFICATION.
• SIMILARLY LIGHT RAYS, WHILE PASSING THROUGH AN OBJECT, LOOSE SOME OF THEIR
INTENSITY. THIS IS CALLED ABSORPTION.
• NOT ALL THE LIGHT RAYS SUCCEED IN ENTERING FROM ONE MEDIUM TO OTHER. SOME OF
THESE CHANGE THEIR DIRECTION. THIS IS CALLED DIFFRACTION.
 LENSES

A LENS IS AN OPTICAL ELEMENT COMPOSED OF GLASS OR OTHER TRANSPARENT MATERIAL.

• THERE ARE TWO BASIC TYPES OF LENSES.
• FIRST ARE POSITIVE, CONVEX LENSES, WHICH CAUSE LIGHT RAYS PASSING THROUGH THEM
TO CONVERGE TO FORM AN IMAGE.
• SECOND ARE NEGATIVE, CONCAVE LENSES, WHICH CAUSE LIGHT RAYS PASSING THROUGH
THEM TO DIVERGE TO FORM AN IMAGE.
• EACH TYPE OF LENS HAS SPECIFIC ABILITY TO DELINEATE DETAILS OF AN OBJECT UNDER
EXAMINATION. THIS IS CALLED RESOLUTION.
• IT IS THE SMALLEST DISTANCE (IN ΜM) BETWEEN TWO STRUCTURAL ELEMENTS THAT CAN
STILL BE VISUALLY DISTINGUISHED FROM EACH OTHER.
• THE RESOLUTION (R) OF THE LENS IS DETERMINED BY ITS NUMERICAL APERTURE (NA) AND
THE WAVELENGTH (Λ) OF THE ILLUMINATING LIGHT.
• SHORTER THE WAVELENGTH BETTER IS THE RESOLUTION
• R ΜM = 1.2Λ ΜM/ 2NA
THE NUMERICAL APERTURE (NA) IS THE RATIO OF THE DIAMETER OF THE LENS TO ITS FOCAL
LENGTH.
• IT CAN BE CALCULATED BY THE FORMULA:
NA = N SIN U
WHERE N IS THE REFRACTIVE INDEX AND U IS THE ANGLE OF APERTURE.
• FOCAL LENGTH IS THE DISTANCE BETWEEN THE LENS AND THE OBJECT FROM WHICH ALL
RAYS OF LIGHT ARE BROUGHT TO A POINT OR FOCUS.
 ABERRATIONS

• ALL LENSES HAVE CERTAIN INHERENT DEFECTS (ABERRATIONS).

• THESE ARE OF SIX TYPES BUT TWO ARE IMPORTANT.
• CHROMATIC ABERRATIONS ARE RESPONSIBLE FOR COLOUR FRINGES ON THE MARGINS OF IMAGE.
• SPHERICAL ABERRATIONS ARE RESPONSIBLE FOR POOR IMAGE DEFINITION AND CONTRAST.
SPHERICAL ABERRATIONS CREATE CURVED IMAGES OF FLAT OBJECTS.
• THESE ARE CORRECTED BY USING A COMBINATION OF LENSES OF VARIOUS SHAPES AND TYPES IN
AN OBJECTIVE.
• WORKING DISTANCE IS THE DEPTH OF SPACE IN MM BETWEEN THE TOP SURFACE OF THE OBJECT
AND THE FRONT SURFACE OF THE OBJECTIVE.
• IT REDUCES WITH INCREASE IN POWER OF THE LENS. FOR THIS REASON HIGH POWER LENSES ARE
PROVIDED WITH SPRING LOADED FRONT PART TO AVOID DAMAGE TO THE LENS OR OBJECT.
• DEPTH OF FOCUS IS THE DISTANCE THROUGH WHICH ALL PARTS OF THE OBJECT IMAGE ARE CLEARLY
IN FOCUS SIMULTANEOUSLY.
• FIELD OF VIEW IS THE AREA OF AN OBJECT THAT CAN BE SEEN.
• MAGNIFICATION IS THE DEGREE OF ENLARGEMENT OF THE VISUAL IMAGE OF AN OBJECT PRODUCED BY THE OPTICAL
SYSTEM OF THE MICROSCOPE.
• THERE ARE TWO MAGNIFYING OPTICAL SYSTEMS IN THE MICROSCOPE, OBJECTIVE AND THE EYEPIECE.
• FINAL MAGNIFICATION OF THE IMAGE IS THE PRODUCT OF MAGNIFICATION OF
OBJECTIVE AND THE EYEPIECE.
• FOR EXAMPLE WHEN USING AN OBJECTIVE OF X40 AND AN EYEPIECE OF X10 MAGNIFICATION, THE FINAL
MAGNIFICATION OF THE OBJECT WILL BE 40X10=X400.
• INCREASING MAGNIFICATION REDUCES THE DEPTH OF FOCUS AS WELL AS FIELD OF VIEW.
• ALSO WITH INCREASING MAGNIFICATION GREATER AMOUNT OF LIGHT IS REQUIRED
TO ILLUMINATE THE FIELD.
 HOW TO OPERATE A COMPOUND LIGHT
MICROSCOPE

• THE MICROSCOPE SHOULD BE PLACED ON A LEVEL BENCH, WHICH SHOULD BE FREE OF VIBRATIONS.

• 2. THE POWER SOCKET, TO WHICH THE MICROSCOPE IS PLUGGED, SHOULD NOT BE LOOSE AND SPARKING.
•
3. THE HEIGHT OF THE MICROSCOPE OR CHAIR SHOULD BE ADJUSTED IN SUCH A WAY THAT THE EYES OF THE
USER ARE RIGHT ON THE EYEPIECES WHILE MAINTAINING THE NORMAL CURVATURES OF THE BACKBONE.
• 4. THE MICROSCOPE SHOULD THEN BE ADJUSTED FOR THE OPTIMUM RESOLUTION AND
CONTRAST TO ENSURE MAXIMUM DEFINITION OF SPECIMEN
DETAILS.
 KOEHLER TECHNIQUE

• IT CAN BE DONE BY USING KÖEHLER TECHNIQUE AS UNDER

• TURN ON THE MICROSCOPE AT VERY LOW ILLUMINATION AND GIVE 1-2 MIN TO THE FILAMENT OF THE BULB TO WARM.
• THEN ADJUST THE LIGHT INTENSITY.
• PLACE THE SPECIMEN ON THE STAGE, SWITCH TO X10 OBJECTIVE AND FOCUS.
• CLOSE THE IRIS DIAPHRAGM OF THE SUBSTAGE CONDENSER AND RAISE THE SUBSTAGE CONDENSER TO THE TOP
“STOP”.
• CLOSE THE FIELD IRIS DIAPHRAGM OF THE LIGHT ASSEMBLY IN THE BODY.
• MOVE THE SUB-STAGE CONDENSER DOWN UNTIL THE IMAGE OF THE FIELD IRIS DIAPHRAGM IS IN SHARP FOCUS.
• NOW RE-FOCUS THE SPECIMEN.
 • CENTRE FIELD DIAPHRAGM IMAGE BY USING ADJUSTMENT SCREWS IN THE CONDENSER.
• ENLARGE FIELD DIAPHRAGM IMAGE UNTIL IT IS JUST OUT OF THE FIELD OF VIEW AND THE ENTIRE AREA UNDER
OBSERVATION IS ILLUMINATED.
• REMOVE ONE EYEPIECE AND LOOK DOWN THE TUBE.
• ADJUST THE APERTURE OF DIAPHRAGM WHILE OBSERVING THE CIRCULAR BEAM OF THE LIGHT SO THE LIGHT
BEAM FILLS 75% OF THE FIELD.
• REPLACE THE EYEPIECE. ADJUST THE DIOPTRE SETTING AND INTER-PUPILLARY DISTANCE.
• PLACE YOUR FOREARMS FLAT ON THE SURFACE OF THE TABLE WHILE USING MICROSCOPE. PERIODICALLY
LOOK AWAY, PREFERABLY OUT OF WINDOW OR TO A PICTURE OR ANY PLEASANT OBJECT.
 OIL IMMERSION MICROSCOPY

• OIL IMMERSION MICROSCOPY IS EXTENSIVELY USED TO IDENTIFY VERY SMALL OBJECTS AND TO
STUDY FINER DETAILS OF THE CELLS.
• IT REQUIRES THE USE OF SPECIALLY CONSTRUCTED OBJECTIVES WITH A SMALL
WORKING DISTANCE.
• AIR (REFRACTIVE INDEX 1.0) IN THE LIGHT PATH OF THE OBJECT SPACE IS REPLACED WITH OIL
(REFRACTIVE INDEX 1.5-1.6).
• THIS IMPROVES RESOLUTION. OIL IMMERSION OBJECTIVES OF VARIOUS MAGNIFICATIONS ARE
AVAILABLE BUT THE MOST COMMONLY USED HAS MAGNIFICATION OF X100.
• FOLLOWING PROCEDURE SHOULD BE ADOPTED FOR OIL IMMERSION MICROSCOPY:
1. ADJUST THE MICROSCOPE.
2. PLACE THE OBJECT ON THE STAGE AND FOCUS WITH X10 OBJECTIVE.
3. SELECT THE VIEWING AREA.
4. ROTATE THE OBJECTIVE OUT OF LIGHT PATH.
5. PLACE A DROP OF OIL OVER THE OBJECT, IN THE CENTRE OF LIGHT BEAM.
6. WATCHING FROM THE SIDE, CAREFULLY SWING IN THE OIL IMMERSION OBJECTIVE.
7. FOCUS CAREFULLY USING FINE ADJUSTMENT KNOB.
8. AFTER EXAMINATION WIPE OFF THE OIL AND CLEAN THE OBJECTIVE AS WELL AS THE OBJECT WITH
A PIECE OF SOFT TISSUE PAPER
 CARE OF MICROSCOPE

• MICROSCOPE IS VERY DELICATE EQUIPMENT. PROPER CARE NOT ONLY ENHANCES PRECISION
BUT ALSO INCREASES ITS LIFE.

• FOLLOWING POINTS ARE HELPFUL IN THE CARE OF MICROSCOPE:

1. PROTECT FROM HEAT.
2. CLEAN IT DAILY. WHEN NOT IN USE, KEEP IT COVERED WITH A PLASTIC COVER OR A PIECE
OF CLOTH BUT NOT WITH MESH GAUZE.
• 3. CLEAN THE OBJECTIVES WITH SOFT TISSUE PAPER SOAKED IN XYLOL AND THEN WITH LINT FREE
CLOTH. BE CAREFUL AS EXCESS OF XYLOL MAY DISSOLVE THE CEMENT WITH WHICH LENS IS FIXED IN
THE OBJECTIVE AND MAY TRICKLE INTO IT. DO NOT CLEAN
WITH ALCOHOL.
4. REMOVE THE DUST FROM THE EYEPIECES WITH THE HELP OF SOFT TISSUE PAPER.
5. ALWAYS USE SOFT TISSUE PAPER OR LINT FREE CLOTH FOR CLEANING LENSES AND NEVER RUB BUT
WIPE GENTLY. THIS PROTECTS LENSES FROM SCRATCHES.
6. SWITCH OFF THE POWER AT THE END OF
MICROSCOPY SESSION.
 TROUBLE SHOOTING AND REMEDIES

• 1. NO LIGHT: THIS MAY HAPPEN IF THE POWER CONNECTION IS LOOSE, THE BULB IS LOOSE OR
FUSED, BRIGHTNESS CONTROL DIAL IS AT LOWEST LEVEL, OBJECTIVE IS NOT CLICKED IN PLACE,
DIAPHRAGM IS COMPLETELY CLOSED OR NOT CENTRED OR FUSE IS BLOWN. THE CAUSE SHOULD BE
RECOGNISED AND REMOVED.
2. INSUFFICIENT LIGHT: THIS MAY RESULT FROM LOW SET BRIGHTNESS CONTROL DIAL, TOO LOW
CONDENSER AND CLOSED CONDENSER DIAPHRAGM. CHECK AND CORRECT ACCORDINGLY.
3. TOO BRIGHT LIGHT: BRIGHTNESS CONTROL SET TOO HIGH FOR THE OBJECTIVE BEING USED.
• 4. FLICKERING: FLICKERING RESULTS FROM LOOSE POWER CONNECTION, DEFECTIVE BULB
SOCKET, CORROSION OF BULB PINS AND IMPROPERLY
INSTALLED BULB.
5. DOES NOT FOCUS WITH HIGH OBJECTIVE: THE SPECIMEN SLIDE IS PLACED UP SIDE DOWN.
6. BUBBLES OR DARK WAVES ACROSS THE FIELD: CONTACT BETWEEN THE OIL AND OIL
IMMERSION OBJECTIVE IS BROKEN. CLEAN THE SLIDE AND ADD
MORE OIL.
 SPECIAL TYPES OF MICROSCOPES

• DARK GROUND MICROSCOPE

IT IS ALSO CALLED DARK FIELD ILLUMINATION MICROSCOPE.

• THERE ARE CERTAIN MICROORGANISMS, WHICH ARE VERY DIFFICULT TO STAIN E.G., SPIROCHETES.
TO VISUALISE THEM UNDER MICROSCOPE DARK FIELD ILLUMINATION IS USED.

• THE MICROORGANISMS APPEAR BRIGHT AGAINST A DARK BACKGROUND. IT IS SIMILAR TO DUST PARTICLES SEEN IN
A BEAM OF LIGHT FROM A VENTILATOR IN A DARK ROOM.

• IN THIS MICROSCOPE A SPECIAL CONDENSER WITH A CENTRAL BLACK AREA IS

PLACED JUST BEHIND THE OBJECTIVE.

• A DARK GROUND, PHASE CONTRAST MICROSCOPE CAN BE MADE FROM AN ORDINARY MICROSCOPE. FOR THIS, CUT
OUT A THICK TALC SHEET OF THE SIZE OF A FILTER.

• COLOUR THE CENTRAL TWO THIRD WITH BLACK INK. PLACE IT ALONG THE FILTER IN THE HOLDER BELOW THE
CONDENSER.
 FLUORESCENT MICROSCOPE

• CERTAIN DYES HAVE THE CHARACTERISTIC OF GLOWING WHEN EXPOSED TO ULTRAVIOLET LIGHT.
• IN FLUORESCENT MICROSCOPE THE OBJECT IS STAINED WITH THESE (FLUOROCHROME) DYES.
• THE LIGHT SOURCE OF MICROSCOPE IS REPLACED WITH A SOURCE THAT PROVIDES ONLY ULTRAVIOLET LIGHT.
• THE OBJECT APPEARS AS A GLOWING PARTICLE AGAINST A DARK BACKGROUND.
• RHODAMINE AND AURAMINE ARE COMMONLY USED FLOUROCHROME DYES.
• IF AN ANTIBODY IS ATTACHED TO THESE FLOUROCHROME DYES, THE PRESENCE OF A SPECIFIC ANTIGEN CAN
BE DETECTED. THIS IS CALLED IMMUNOFLUORESCENT
MICROSCOPY
 PHASE CONTRAST MICROSCOPE

• THIS MICROSCOPE IS USED FOR OBSERVING UNSTAINED LIVING ORGANISM WITH GOOD
CONTRAST AND HIGH RESOLUTION.
• IT IS USEFUL FOR THE STUDY OF STRUCTURE OF LARGE MICROORGANISMS,
TISSUES AND CELLS.
• UNSTAINED BACTERIA AND CELLS CONSIST OF ALTERNATE STRIPS OF MATERIEL OF DIFFERENT
REFRACTIVE INDICES THAT CAUSE THE LIGHT TO ACQUIRE SMALL PHASE DIFFERENCES.
• THESE DIFFERENCES ARE EXAGGERATED BY CAUSING THE DIRECT AND DIFFRACTED
RAYS TO PASS THROUGH DIFFERENT THICKNESS OF GLASS IN PHASE PLATE.
• DIRECT AND DIFFRACTED LIGHT BEAMS ARE THEN RECOMBINED TO PRODUCE AN IMAGE
• ELECTRON MICROSCOPE
THIS MICROSCOPE IS USED TO SEE VIRUSES OR PARTS OF CELLS SMALLER THAN THE LIMITS OF
RESOLUTION OF THE LIGHT MICROSCOPE.
• IT UTILISES A BEAM OF ELECTRONS INSTEAD OF VISIBLE LIGHT AND ELECTROMAGNETIC FIELDS IN PLACE
OF OPTICAL
LENSES.
• AN OBJECT FORMS AN IMAGE IN THE ELECTRON MICROSCOPE BECAUSE ITS SOLID CONTENT
SCATTERS THE ELECTRON BEAM AND SO CASTS A SHADOW IN THE ELECTRON BEAM.
• THE IMAGE CANNOT BE SEEN WITH EYE. INSTEAD, IT IS FOCUSED ON A SCREEN AND/OR IS
PHOTOGRAPHED.
• FURTHER MAGNIFICATION AND RESOLUTION CAN BE OBTAINED BY ENLARGING THE PHOTOGRAPHS.