Microscope PDF Lab Manual

MICROSCOPE   GOALS   The   idea   behind   conversion   is   a   simple   one.     If   the   The...

MICROSCOPE   GOALS   The   idea   behind   conversion   is   a   simple   one.     If   the   The  goals  of  this  exercise  are:   units   you   have   are   larger   than   the   units   you   want,   to  review  metric  system  measurements you   multiply   by   the   factor   difference   between   the   to  properly  use  and  care  for  a  sensitive  scientific units   (see   table   1).     For   example,   let’s   convert   200   instrument meters   into   centimeters.     You   have   just   memorized   to  teach  you  the  techniques  required  to  prepare that   1   m   =   100   cm.     Since   a   meter   is   larger   than   a   cells  for  viewing  with  a  microscope centimeter,  we  should  multiply.    By  how  much?  The   to  give  you  a  sense  of  the  size  of  cells difference   between   meters   and   centimeters   is   100,   so   multiply   the   number   of   meters   by   100.         For   to   survey   microscopic   organisms   from   a   few example,  200  m  x  100  =  20,000  cm.       kingdoms  of  life   TABLE  1.    Metric  Measurements   By  the  end  of  this  lab  you  should:   identify  the  parts  of  a  microscope  and  functions “Saying”   LENGTH   MULTIPLE   know   how   to   properly   carry,   use   and   store   a Kids   kilometer  (km)   1000.0   microscope   Have   hectometer   100.0   know  how  to  prepare  a  wet  mount  slide Dropped   decameter   10.0   view  and  focus  specimens  under  a  microscope Over   meter  (m)   1.0   compare  the  sizes  of  different  kinds  of  cells Dead   decimeter   0.1   estimate  the  size  of  a  cell  by  using  the  size  of  the Converting   centimeter  (cm)   0.01   field  of  view   Metric   millimeter  (mm)   0.001   0.0001   0.00001   A. METRIC  SYSTEM  REVIEW Micrometer  (µm)   0.000001   Scientists   (and   most   of   the   world)   use   the   metric   How   would   you   convert   2   millimeters   to   system   to   communicate   information   concerning   the   centimeters?     Since   a   millimeter   is   smaller   than   a   temperature,   length,   weight   and   volume   of   a   centimeter,   so   what   you   have   is   smaller   than   what   material.     When   using   the   metric   system,   the   you   want,   so   you   divide.     The   difference   between   measurements   of   temperature,   length,   weight,   millimeters   and   centimeters   is   10;   so   divide   the   volume  are  degrees  Celsius  (or  centigrade),  meters,   number  of  millimeters  by  10.       grams  and  liters.   2  millimeters/10  =  0.2  centimeters.   The   metric   system   is   a   convenient   one   to   use   Double-­‐check  that  you  are  moving  the  decimal  in  the   because  it  is  based  on  the  decimal  equivalents  of  the   correct  direction.    Your  conversion  equation  should   measurement   unit.     This   means   that   the   always  have  the  format  of  [big  number]  x  [small  unit]   measurement   is   multiplied   or   divided   by   ten.   The   =  [small  number]  x  [big  unit].     fractions   or   smaller   parts   of   a   measurement   are   named   using   the   prefixes   “centi-­‐”   (hundredth),   Conversions  to  Memorize:   “milli-­‐”   (thousandth)   and   “micro-­‐”   (millionth).     The   multiple   is   “kilo-­‐”   (thousand).     Table   1   lists   the   1  km  =  1,000  m   measurements   for   length   in   meters.     But   the   same   1      m  =  100  cm   principle   is   used   for   volume   (liters)   and   weight   1  cm  =  10  mm   (grams).      The  Greek  letter  “µ”    stands  for  “micro-­‐”.                      1  mm  =  1,000  µm   Conversions.     One   metric   unit   is   converted   to   another   for   reasons   of   comparison.     If   one   Ø PRE-LAB: Go  to  the  Data  Sheet  (page  5) measurement   is   in   centimeters,   and   another   is   in   and complete  the  problems  for  Metric millimeters,  it  is  easier  to  compare  the  lengths  if  the   System  Conversions.   units  used  are  the  same.       Bio 101 Microscope - 1 B. COMPOUND  LIGHT  MICROSCOPE The   compound   light   microscope   is   an   useful   tool   for   studying   biology,   to   view   small   specimens   that   are   not   visible   to   the   naked   eye.       The   microscope   uses   bright   light   to   illuminate   through   the   specimen  and  provides  an  inverted  image  at  high  magnification  and   resolution.     There   are   two   lenses   that   magnify   the   image   of   the   specimen  -­‐-­‐  the  objective  lens  on  the  nosepiece  and  the  ocular  lens   (or   eyepiece).     To   determine   the   total   magnification   of   the   specimen,   multiply   the   objective   lens   magnification   with   the   ocular   lens   magnification.   Ø Calculate  the  three  total  magnifications  possible  with your  microscope  and  fill  in  Table  1  on  the  Data  Sheet. As   the   compound   light   microscope   uses   expensive   glass   lenses   similar   to   professional   camera   lenses,   the   lenses   can   only   be   cleaned   with   special   lens   paper   or   cloth.     Any   other   type   of   paper   (tissue,   paper   towel,   etc)   can   scratch   the   lenses.     In   addition,   water   and   solvents  can  damage  the  various  metal  fittings.    So  be  sure  to  follow   these   important   rules   and   protocol   about   microscope   use.     Each   person   should   get   a   compound   light   microscope   from   the   storage   cabinet  at  the  back  of  the  room.   General  Rules:   Always  START  and  END  with  the  low  power  lens  when  putting  on  OR  taking  away  a  slide. Do  not  get  any  portion  of  the  microscope  wet  -­‐  especially  the  stage  and  objective  lenses. Use  only  lens  paper  to  clean  microscope  lenses. Carrying  the  Microscope:   1. Always  use  one  hand  around  the  microscope  arm  and  one  hand  under  the  microscope  base. 2. Carry  it  in  a  vertical  position  without  swinging,  tipping,  dropping  or  bumping  the  microscope. 3. Place  the  microscope  on  a  table  with  the  arm  toward  you. Cleaning  the  Microscope  –  only  use  LENS  paper   If   needed,   obtain   a   small   square   of   lens   paper   (and   ONLY   lens   paper)   and   gently   wipe   the   microscope  lenses  directly  across,  in  this  order:     1. the  lower  surface  of  all  the  objective  lenses 2. the  ocular  lens 3. the  condenser  lens  and  the  light  housing. Identifying  the  Microscope  Parts   £ Eyepiece  (ocular  lens)   £ Nosepiece  Ring  (turret)   £ Objective  Lenses  (low,  medium,  high  power)   £ Stage   £ Stage  Controls   £ Iris  Diaphragm   £ Condenser  Lens   £ Light  Source   £ Coarse  Focus  Adjustment  Knob   £ Fine  Focus  Adjustment  Knob   Bio 101 Microscope - 2 C. OBSERVING  SPECIMENS In  order  to  observe  specimens  with  the  compound  light  microscope,  always  start  with  the  low  power  objective   lens   and   the   nosepiece   raised   high   above   the   stage,   before   placing   the   slide   on   the   stage.     Ensure   that   your   specimen   is   in   the   circle   of   view   (over   the   light   source).     Focus   on   the   specimen   at   low   power   first,   BEFORE   attempting  to  look  at  specimen  at  higher  magnification.    As  your  microscope  is  PARFOCAL,  it  remains  generally   in  focus  at  the  higher  magnifications,  so  that  you  should  ONLY  adjust  with  the  Fine  focus  knob  when  using  the   medium  and  high  power  objective  lenses.   £ Obtain  a  slide  with  the  letter  “e”  from  the  demo  table.   £ Follow  the  steps  below  to  properly  use  the  microscope.   £ Focus,  observe  and  draw  the  letter  “e”  with  low  power  objective  lens.   Ø On   the   Data   Sheet,   draw   what   you   see   with   the   focused   microscope,   with   the   circle   on paper  representing  the  field  of  view  (circle  of  light).  Label  the  total  magnification. £ Repeat  with  medium  power  objective  lens  and  again  at  high  power  objective  lens.   Using  the  Microscope  (protocol  steps)   1. Before  starting  to  place  a  slide  onto  the  stage,  check  that  the  LOW  power  objective  lens  is  in place  (directly  above  the  light  source). 2. Rotate  the  large  nosepiece  disc  that  holds  the  objective  lenses.    Do  NOT  grab  an  objective  lens to  rotate  the  nosepiece!    You  may  hear  a  soft  click,  that  confirms  the  objective  lens  is  in  place. 3. Using  the  coarse  adjustment  knob,  raise  the  nosepiece  high. 4. Carefully,  gently  place  the  slide  between  the  stage  clips,  with  the  brand  name  on  the  left  side. 5. Center  the  “e”  over  the  light  with  the  stage  control  knobs. 6. Lower  the  nosepiece  all  the  way  down  to  the  stage. 7. Look   through   the   eyepiece   and   adjust   the   intensity   of   light   with   the   diaphragm   lever   as needed. 8. Adjust  with  the  fine  adjustment  knob  until  the  letter  “e”  is  in  focus. 9. Try  to  have  both  eyes  open  to  reduce  eyestrain. 10. Your  instructor  will  come  to  check  your  focus  and  help  as  needed. Focusing  with  Parfocal  Medium  and  Higher  Power  Objective  Lenses   11. While  low  power  objective  lens  is  in  place,  move  stage  control  knobs  so  that  the  “e”  is  in  the middle  of  the  field  of  view. 12. Rotate  the  nosepiece  disc  to  bring  the  medium  power  objective  lens  clicked  into  place. 13. You  must  NOT  touch  the  coarse  focus  knob  again. 14. Use  the  FINE  focus  knob  to  focus  the  specimen  at  this  magnification,  usually  need  less  than  a quarter  of  a  turn.    Draw  as  needed. 15. Again  place  the  specimen  in  middle  of  the  field  of  view. 16. Rotate  the  nosepiece  disc  to  click  the  high  power  objective  lens  in  place. 17. Use  the  FINE  focus  knob  to  focus  the  specimen,  usually  need  less  than  a  quarter  of  a  turn. 18. When  finished,  rotate  the  low  power  objective  lens  in  place.    Raise  the  nosepiece  away  from the  stage  with  coarse  focus  know.    Remove  the  slide  from  the  stage  clips. Bio 101 Microscope - 3  WORK  EFFICIENTLY  in  this  long  lab:    Work  with  your  lab  partners.    Each  partner  should  prepare  and  focus  a   different   specimen   slide   to   the   best   magnification   to   see   details   of   three   whole   cells.     Then   all   lab   partners   can   rotate   around   the   table   and   observe   and   draw   from   each   microscope.   You   must   make   your   own   drawings   and   measurements.    Copying  from  someone  else’s  drawing  is  considered  cheating.    You  will  not  be  graded  on  artistic   ability,  just  on  your  ability  to  see  the  details  of  each  specimen.    Your  instructor  will  also  explain  the  structures  of   each  specimen  on  the  video  microscopy  monitor!   LAB  GRADING:     For  each  specimen,  use  the  ONE  magnification  at  which  you  can  most  clearly  see  whole  cells   with   maximum   detail.     Draw   and   color   at   least   three   cells   from   each   specimen   as   accurately   as   possible.     Label   the   specimen  name,  total  magnification  used,  arrow,  and  identifiable  cell  structures.   DRY  PREPARED  SPECIMEN  SLIDES   WET  MOUNTS  THAT  YOU  PREPARE   Spirogyra:  cell  wall,  nucleus,  chloroplasts Elodea  leaf:  chloroplasts Paramecium:  cytoplasm,  vacuoles,  nucleus Human  cheek:  nucleus Human  blood:  nucleus,  white  &  red  blood Onion:  cell  wall,  nucleus Human  sickle  cell  anemia:  sickle  cells Pond  water Amphibian  blood:  white,  red  blood Preparing  Wet  Mounts   Onion  epidermis   Obtain  a  slide  from  the  container  of  alcohol.    Unless   {Note:   Iodine   can   stain   skin   &   clothes.}     Place   a   drop   otherwise  indicated,  use  a  flat  slide  that  contains  no   of   iodine   on   a   dry   microscope   slide.     Peel   the   thin,   depressions.     Alcohol   kills   cells!     DRY   it   off   with   a   white,   transparent   membrane   from   the  inner   concave   quarter   strip   of   paper   towel,   not   expensive   lens   side   of   an   onion   section   and   quickly   place   it   paper!     Place   it   on   the   lab   bench.     Place   a   small   drop   smoothly   into   the   iodine   drop.   Count   slowly   to   ten.     of   the   medium   (water   or   stain   usually)   on   the   slide   Gently   cover   with   a   dry   coverslip.     Place   the   slide   first!     Obtain   your   specimen   and   place   it   in   the   inside   a   folded   towel   and   gently   dab   out   excess   medium.    Obtain  a  coverslip  (very  thin,  small  square   iodine.     Look   under   medium   power   to   find   the   cell   glass)  from  the  container  of  alcohol.    Be  very  careful   wall,   cytoplasm  and  nucleus  of  the  rectangular  cells.     when   drying   -­‐   they   break   easily.   (Where   do   you   Human  Cheek  cells   dispose  of  broken  glass?)    ________   {Note:   Methylene   blue   stains   skin   &   clothes.}   Place   Hold   the   cover   glass   at   an   angle   on   the   slide   and   a   drop   of   methylene   blue   stain   on   a   slide.  Rub  a  clean   allow  it  to  fall  over  the  specimen.      This  gets  all  of  the   toothpick  on  the  inside  of  your  cheek  very   gently   only   air  out  from  under  the  cover  glass  and  minimizes  the   one   or   two   times.     DO   NOT   gouge   the   inside   of   your   number  of  air  bubbles  in  with  your  specimen!   cheek!     DO   NOT   draw   blood!   Rub   toothpick   onto   slide.     Cover   and   gently   dab   out   excess   stain   with   cover glass cover glass paper   towel  strip.     Throw   away   your   slimy   toothpick,   specimen specimen as   no   one  else  wants  to  clean  it  up  for  you!       slide Figure 1. Proper placement of a cover glass. slide Pond  water  or  culture   DO NOT GET OBJECTIVE LENSES WET!!! Wipe Go   to   the   "Single   Depression   Microscope   Slide"   jar   to   immediately with lens paper if needed. dry   off   a   thick   glass   depression   slide.     The   depression   slide   has   a   curved   indent   in   the   middle   of   the   Elodea  leaf   slide,   which   allows   the   living   creatures   to   move   Put   a   drop   of   water   onto   a   dry   slide.     With   around   and   not   get   squashed.   Put   a   drop   of   methyl   forceps,  place  a  very  small  leaf  (or  use  half  of  a   cellulose   and   a   drop   from   the   pond   water   or   cultures.   larger   leaf)   into   the   water   and   coverslip.   Carefully   put   on   a  cover   slip.     If   you   have   too   much   Examine   the   rectangular   cells   at   high   power   to   liquid   on   the   slide,  then  gently  use  a  corner  of  a  paper   find  the  cell  walls,  nucleus,  cytoplasm,  and  green   towel   to   absorb   the   excess   liquid.     Use   the   low   chloroplasts.    As  you  focus  through  the  leaf,  you   and   medium   power   objectives   only!       will   be   able   to   distinguish   different   cell   layers   These   creatures   are   very   difficult   to   find   and   move   very   through   the   leaf.     Cytoplasmic   streaming   occurs   quickly!   The   organisms   are   swimming   in   the   liquid,   often,   which   is   evident   by   chloroplasts   moving   so   just   take   a   drop   of   it   and   place   both   water   and   inside  the  cells.     organisms  on  a  slide.   Bio 101 Microscope - 4 Partners:   Name:   Bio  101  Lab:    MICROSCOPE  DATA  SHEET   PRE-­‐LAB:      METRIC  CONVERSIONS   1. 1  cm  =  _________  mm  =  _______________  µm 6. 0.025  m  =  __________  cm  =  ____________  mm 2. 1000  µm  =  _________  mm  =  ____________  cm 7. 45  mm  =  _________  cm  =  _______________  m 3. 100  mm  =  ________  cm  =  _______________  m 8. 185  µm  =  __________  mm  =  ____________  cm 4. 1  km  =  ___________  m  =  _______________  cm 9. 0.04  km  =  __________  m  =  ______________  cm 5. 0.123  mm  =  ________  cm  =  ____________  µm 10. 54,321  µm  =  __________  mm  =__________  cm TABLE  1.  TOTAL  MAGNIFICATION  CALCULATION   Low  Power   Medium  Power   High  Power   Ocular  Lens  Magnification   10  x   Objective  Lens  Magnification   Total  Specimen  Magnification   Drawings  of  the  Letter  “E”  Slide   Specimen  _____________   Specimen  _____________   Specimen  _____________   Total  Mag  ____________   Total  Mag  ____________   Total  Mag  ____________   Ø How  does  the  orientation  of  the  letter  “e”  when  observing  under  a  light  microscope  DIFFER  from  observing with  your  naked  eye?  Name  two  differences.  ____________________________________________________ Ø While  looking  at  the  stage,  move  the  slide  VERY  SLIGHTLY  to  the  right.    Which  way  did  the  letter  move  in  the field  of  view?  _______________________________ Ø While  looking  at  the  stage,  move  the  slide  VERY  SLIGHTLY  to  the  left,  which  way  did  the  letter  move  in    the  field of  view?  ________________________________ Bio 101 Microscope - 5 Microscope  Lab  Data  Sheet  (page  2)   Name:   Specimen  Observations  –  color  and  label   Specimen  _____________   Specimen  _____________   Specimen  _____________   Total  Mag  ____________   Total  Mag  ____________   Total  Mag  ____________   Specimen  _____________   Specimen  _____________   Specimen  _____________   Total  Mag  ____________   Total  Mag  ____________   Total  Mag  ____________   Specimen  _____________   Specimen  _____________   Specimen  _____________   Total  Mag  ____________   Total  Mag  ____________   Total  Mag  ____________   Bio 101 Microscope - 6  Microscope  Lab  Data  Sheet  (page  3)   Name:   LAB  CHECK-­‐OUT     Cleaning  Wet  Mounts:     To  put  the  microscope  away,  follow  these  directions:   q Rinse  slide  into  beaker  of  water,  place  cover  slip   and  slide  into  proper  jars   q low  power  objective  in  position   q Put  Prepared  Slides  back  onto  the  correct  tray  on   q raise  nosepiece  to  the  top  position   the  demo  table.   q remove  slide  from  stage,  turn  off  light   q Tighten  all  reagent  bottle  caps.    Clean  up  demo   table.   q be  sure  black  bar  from  stage  clips  does  not  stick  out   q Wipe  off  all  tables  with  wet  sponge.   q unplug,   wrap   cord   according   to   your   instructor’s   instructions   q carry   microscope   properly   to   cabinet   and   place   it   Instructor  signature_________________   on  the  correct  shelf  space   STUDY  QUESTIONS   1. Name  the  parts  of  the  microscope. 2. How  is  a  microscope  properly  carried? 3. How   is   the   microscope   properly   put   away? 4. What  is  the  magnification  of a) high power  objective  lens? b) medium power  objective  lens? c) low power  objective  lens? d) ocular  lens? 5. How   is   the   total   magnification   of   a   specimen determined? 6. When   the   magnification   increases,   how   does   the size  of  the  field  of  view  change? 7. Why  is  it  important  to  place  the  medium  on  a  slide before  selecting  the  specimen  to  be  mounted? 8. Name   one   way   to   be   sure   the   specimen   can   be found   in   the   field   of   view   when   you   change magnification. 9. What   are   the   distinguishing   characteristics   of   a plant  cell  versus  an  animal  cell? Bio 101 Microscope - 7