2

 

Acknowledgements  

 

 

I  would  like  to  thank  Tokyo  Metropolitan  University  for  

providing  me  with  lab  facilities  as  well  as  guidance  on  how  to  

use  the  equipment  present  in  their  labs.  

 

 

I  would  also  like  to  thank  Mr.  John  Fortin  for  the  supervision  and  

guidance  he  provided  during  the  process  of  my  research.  

 

 

And  lastly,  I  would  like  to  thank  my  parents  who  were  

supportive  and  had  encouraged  me  during  this  research.    

 

 

 

 

 

 

 

 

3

Introduction  

 

Drosophila  melanogaster  commonly  known  as  fruit  flies  is  well  studied  and  

experimented  organism;  it  is  often  seen  around  rotten  fruits  for  instance  rotten  

bananas.  Fruit  flies  have  three  major  body  components  thorax,  abdomen.  The  head  has  

two  prominent  eyes,  the  heart  and  part  of  digestive  system  attached  to  the  thorax  and  

the  abdomen  contains  the  remainder  of  the  digestive  system  and  reproductive  system.    

The  reason  for  D.  melanogaster’s  vast  and  continued  research  is  because  it  only  has  a  

generation  time  of  two  weeks  and  is  a  convenient  tool  for  genetic  research  because  

their  entire  genome  has  been  sequenced.  An  adult  fly  can  lay  hundreds  of  eggs  in  one  

day  and  therefore  many  generations  can  be  raised  in  relatively  short  period  of  time.      

Drosophila  was  mainly  an  organism  studied  for  genetics  however  recently  researchers  

and  scientists  have  started  to  look  closely  at  developmental  biology.    In  the  area  of  

developmental  biology  many  areas  have  and  are  being  researched,  one  of  them  is  

nutrition.  Some  studies  have  taken  place,  which  are  regarding  ideal  nutrition  for  

longevity  of  flies,  testing  flies’  senses  by  using  different  types  of  nutrient  conditions  and  

so  on.    

Studies  have  proposed  that  the  different  varieties  of  yeast  within  various  media  were  

influencing  factors  in  fruit  fly  nutrition  and  subsequent  development  (Baumberger,  

1917).  Another  research  showed  that  developmental  and  fecundity  rates  varied  when  

fruit  flies  were  reared  on  media  of  sugar,  tomatoes,  and  grapes  (Jaenike,  1986).  

 

Researchers  have  found  recently  that  fruit  flies  have  pancreatic  like  cell  in  their  brain  

known  as  corpora  cardiaca  (CC)  which  is  also  necessary  for  larval  glucose  homeostasis  as  

well  (Brownlee,  2004).  The  resemblance  between  mammalian  structure  and  fly  will  be  

helpful  to  make  connections  and  make  educated  predictions  about  subject  possibilities  

for  further  studies  in  biological  areas.    

The  experiment  will  examine  the  effect  of  different  levels  of  added  glucose  in  media  on  

the  eclosion  rate  of  D.  melanogaster.    

4

 

The  preliminary  research  conducted  showed  that  numerous  studies  have  been  

conducted  regarding  the  nutrient  media  of  fruit  flies  to  find  out  the  correlation  between  

certain  nutrients,  vitamins  and  carbohydrates  and  the  fecundity  rate,  eclosion  rate  or  

longevity  of  insects.  Therefore  I  formulated  an  objective  to  investigate  the  effect  of  

glucose  supplementation  in  nutrient  medium  on  Drosophila  melanogaster.    

The  Metropolitan  University  has  offered  to  supply  with  D.melanogaster  and  apparatus  

that  will  be  required  to  conduct  this  research.  The  flies  in  the  university  are  fed  with  

instant  medium  which  comes  in  powder  form  and  mixed  with  water  which  gives  it  a  

spongy  quality.  The  instant  medium’s  ingredients  and  name  are  unknown  because  of  

confidentiality  reasons.    The  independent  variable  in  this  experiment  will  be  glucose  

concentration  since  the  ingredients  of  the  media  are  unknown  it  must  be  taken  under  

consideration  that  their  may  or  may  not  be  glucose  in  the  media.    Commercially  

available  dry  mixtures  of  nutrient  medium  aim  to  standardize  the  ingredients  for  rearing  

the  fruit  fly  (Flagg,  2005).    Therefore  it  is  considerable  to  assume  that  there  should  be  

glucose  present  in  the  media  since  all  living  organisms  need  glucose  since  it  is  essential  

for  survival.    

 

 

 

 

 

 

 

 

 

 

 

 

5

Research  Question  

 

Investigating  the  effect  of  glucose  supplementation  in  nutrient  medium  on  Drosophila  melanogaster  eclosion  number  

 

Aim  

The  purpose  of  the  experiment  is  to  find  out  the  ideal  level  of  glucose  required  for  

D.melanogaster  to  eclose  in  the  ultimate  period  of  time.  Preliminary  research  has  

shown  that  many  studies  have  been  conducted  regarding  the  nutrient  media  of  insects  

to  find  out  their  ideal  nutritional  requirements,  longevity,  and  fecundity  rates.  This  study  

will  show  how  glucose  will  affect  the  rate  of  flies  hatching  from  larva  the  results  of  this  

experiment  may  benefit  in  further  experiments  where  there  is  need  of  controlling  the  

time  when  flies  hatch  for  example  to  evade  predation  or  to  study  predation  behavior.    

 

Hypothesis  

It  is  assumed  that  D.  melanogaster  will  eclose  at  expected  time  in  10%  concentrated  

glucose  media.  As  it  is  known  that  blood  glucose  level  is  maintained  by  homeostasis  in  

which  case  we  can  assume  if  the  glucose  level  is  high  (and  no  insulin  to  maintain)  then  

as  in  humans  diabetes  is  acquired  as  we  know  corpora  cardiaca  cells  are  similar  to  the  

mammalian  pancreatic  cells  -­‐  insects  may  not  be  able  to  develop  on  highly  concentrated  

glucose  nutrient  medium  because  of  dehydration  that  may  occur  to  their  cells.  

 

 

Apparatus  

∗ Pipette  (+/-­‐  0.5mL)  

∗ Graduated  cylinder  (+/-­‐  0.5mL)  

∗ Beaker  (+/-­‐  0.5mL)  

∗ Electrical  scale  (+/-­‐  o.oo5g)    

∗ Microwave  (used  during  the  making  of  medium)  

∗ 250mL  culture  vial   (quantity:  36vials   for  3   trials,  with   total  of  9  vials   for  all   the  

6

trials  for  all  concentrations)    

∗ Incubator  with  consistant  temperature  of  25  degree  centigrade  

∗ Trays  to  hold  the  culture  vials    

∗ Tweezers  used  to  count  the  number  of   fruit   flies  and  to  separate  eclosed-­‐dead  

flies  from  the  nutrient  medium  

∗ Anesthesia  (  Triethylamine)  to  immobilize  the  flies  during  data  collection  

 

Variables  

The  independent  variable  chosen  for  this  experiment  is  the  glucose  concentration.  

Additional  glucose  will  be  added  to  the  nutrient  medium,  measuring  up  to  10%,  20%  and  

30%.  The  result  will  be  recorded  starting  the  first  eclosion  (number  of  flies  hatched)  in  

any  of  the  cultural  vials.  

The  dependent  variable  in  this  experiment  is  the  number  of  flies  which  will  eclose  in  the  

given  time,  until  no  more  flies  eclose.    

The  controlled  variables  are  temperature,  for  which  an  incubator  set  on  constant  

temperature  of  25  degree  centigrade  which  is  an  ideal  environment  for  D.melanogaster  

growth  (Tatum,  1939).  Equal  amount  of  liquid  and  powder  of  the  nutrient  media  except  

the  glucose  concentration  which  will  be  different  as  it  is  the  independent  variable.  

Number  of  flies  added  in  each  cultural  vial,  five  male  and  females  which  will  be  added  

on  the  first  day  of  the  experiment  and  taken  out  24  hours  after  they  were  put  into  all  

cultural  vials  which  are  of  equal  sizes  and  shape.    To  minimize  the  error  that  may  be  

introduced  and  to  control  the  experiment  the  adult  flies  which  will  be  added  in  the  

cultural  vial  for  reproducing  will  be  all  from  the  same  generation.    

 

 

 

 

 

 

7

Method  

 

The  investigation  includes  three  trials  of  the  experimentation  for  accuracy.  Each  trial  

consisted  of  two  culture  vials  for  each  concentration  of  additional  glucose  (0%,  10%,  

20%  and  30%).  The  flies  were  reared  in  250mL  culture  vials  with  foam  plugs  to  secure  

the  top  opening.  

The  instant  medium  was  made  using  the  following  procedure:  

To  make  10%  concentrated  glucose  solution  for  the  instant  medium,  2g  of  glucose  was  

measured  on  electronic  weight  scale,  water  was  added  using  a  pipette  in  the  beaker  till  

it  covered  the  glucose  grains,  then  the  beaker  containing  the  glucose  and  water  were  

heated  in  microwave  oven  till  it  started  to  boil.  The  dissolved  glucose  was  mixed  till  the  

grains  had  melted  (this  was  essential  so  that  it  would  not  introduce  error  in  the  creation  

of  instant  media  so  that  if  any  glucose  grain  remaining  left  will  not  introduce  any  error)  

added  in  graduated  cylinder  and  water  was  added  till  the  solution  rose  till  20mL.  (Same  

steps  were  repeated  for  20%  with  4g  of  glucose  and  30%  with  6g  of  glucose).  

 

Five  female  and  male  adult  D.  melanogaster  were  released  in  each  culture  vial,  the  flies  

were  released  according  to  the  day  trial  had  started.  (Trial  1:  On  30th  July  flies  were  

released,  31st  eggs  were  laid  and  flies  were  taken  out  of  the  culture  vial.  All  this  was  

conducted  at  same  time  everyday  on  2:00pm  which  was  decided  arbitrarily,  to  keep  

consistency  and  avoid  any  error.    

All  the  flies  were  added  in  consistent  manner  in  the  culture  vial  before  all  three  trials  

and  all  nutrient  medium  was  made  at  same  time.  The  culture  vials  were  labeled  

according  to  their  concentrations  and  were  kept  in  the  incubator  with  25  degree  

centigrade  controlled  temperature  until  next  day.  The  adult  flies  were  taken  out  and  the  

eggs  were  kept  in  the  incubator  till  expected  day  of  eclosion  which  according  to  prior  

research  (from  secondary  source)  ranges  from  8  to  10  days.  Until  than  the  larva  was  left  

to  feed  on  the  instant  medium,  once  the  flies  had  started  to  eclose  the  culture  vials  

were  observed  daily  for  more  fly  eclosions.    

8

The  culture  vials  consisting  of  the  D.  melanogaster  eggs  were  observed  daily  from  the  

5th  day  from  when  the  trial  had  started  and  data  was  recorded  from  the  first  fly  which  

had  hatched.        

When  the  flies  had  started  to  eclose,  flies  were  transferred  into  separate  culture  vials  

and  were  sprayed  by  Trithylamine  (anesthesia)  until  they  were  immobilized  and  then  

were  dumped  on  white  paper  if  finding  any  difficulties  to  count,  flies  were  transferred  

under  the  dissecting  microscope  for  better  view.  This  way  data  collection  took  place  and  

afterwards  healthy  flies  were  returned  to  the  lab  and  flies  which  have  been  dead  were  

disposed  into  a  fly  morgue  (flask  containing  water  with  salt).    Exact  steps  were  repeated  

for  all  three  trials  of  the  experiment.  The  D.melanogster  were  counted  until  there  were  

no  further  eclosions.    

Results/Findings  

 

The  data  is  complied  using  Microsoft  Excel  spreadsheet  and  the  difference  between  

each  concentration  was  compared.  As  represented  in  Figure  B1  which  clearly  suggests  

that  there  is  a  peak  in  eclosion  rate  for  ideal  nutrient  medium  which  would  be  the  

medium  without  any  additional  glucose  added.  The  figure  also  suggests  similar  pattern  

of  eclosion  for  10%  added  glucose  however  the  number  of  flies  that  have  been  eclosed  

has  decreased.      20%  added  glucose  has  similar  results  as  well.  However  the  graphical  

representation  suggests  some  major  alteration  had  occurred  in  30%,  despite  the  

number  of  eclosed  flies  being  fewer,  there  were  some  flies  which  did  eclose  seemingly  

normal  and  healthy  however  in  delay.  In  the  30%  concentrated  glucose  culture  vial  most  

larvas  had  failed  to  eclose  therefore  suggesting  that  the  additional  glucose  of  30%  had  a  

detrimental  effect  on  the  flies  and  the  number  of  flies  eclosed.      

 

 

 

 

 

9

Rate  of  eclosion  when  additional  glucose  is  added  

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

8 10 12 14 16 18 20 22 24 26

days

eclos

ion ra

te (a

vera

ge n

umbe

r of f

lies)

0%

10%

20%

30%

 Figure  B1  

Chi  -­‐squared  test  was  conducted  to  analyze  the  data  with  respect  to  D.melanogaster  

selection  of  certain  glucose  concentration  (no  additional,  10%,  20%  &  30%)  and  also  to  

show  any  differences  between  the  trials  of  same  concentration.    

Two  sets  of  Chi-­‐squared  test  were  conducted  to  test  the  Null  hypothesis,  for  1  –  there  

are  no  preferences  for  specific  trial  within  the  three  trials  of  same  concentration  which  

was  proven  to  be  rejected.  And  2-­‐  there  are  no  preferences  for  one  specific  type  of  

concentration  out  of  the  4  tested  which  was  also  proven  to  be  rejected.  The  two  tests  

conclude  that  there  are  differences  within  the  each  trial  of  same  concentration  as  well  

as  difference  between  each  concentration  which  greatly  contributes  to  the  research  by  

showing  that  there  is  a  amount  which  his  ideal  and  popular  trend  amongst  

D.melanogaster.  

 

 

 

 

 

10

Discussion  

 

Current  research  has  show  that  D.  melanogaster  have  cells  which  sense  blood  sugar  and  

secrete  insulin  as  pancreatic  beta  cells  do  in  mammals  these  cells  present  in  the  fly  are  

corpora  cardiaca  cells  otherwise  known  as  CC  cells.  This  suggests  that  the  mechanism  of  

homeostasis  of  fruit  flies  is  related  the  homeostatic  functions  in  humans.  The  CC  cells  

are  essential  for  larval  glucose  homeostasis.  They  also  produce  adipokinetic  hormone  

which  is  a  polyperptide  with  similar  functions  to  glucagon.  The  glucose  regulation  by  CC  

cells  is  disabled  when  exposed  to  sulphonylureas  –  which  is  antibiotic  drug  used  in  the  

management  of  diabetes  mellitus  type  2,  they  act  by  increasing  insulin  release  from  

beta  cells  in  pancreas.  CC  cells  are  regulators  of  glucose  homeostasis  and  use  glucose  

sensing  and  response  mechanisms  similar  to  islet  cells.  It  is  also  known  that  deficiency  of  

CC  cells  does  not  result  in  growth  reduction  or  developmental  delay.  In  the  experiment  

conducted  there  was  growth  reduction  and  developmental  delay  from  the  statement  

above  it  is  suggested  that  CC  cells  may  or  may  not  be  in  deficiency  but  this  had  no  effect  

on  the  results.The  product  of  CC  cell  is  adipokinetic  hormone  referred  as  AKH  which  is  

similar  to  mammalian  glucagons  in  pancreatic  alpha  cells.  AKH  is  essential  regulator  of  

haemolymph  carbohydrate  concentrations  in  D.  melanogaster.  (Kim,  Rulifson,  2004)  

 

According  to  research  conducted  by  scientists  flies  during  starvation  had  increased  

severity  of  hypoglycemia  –  pathologic  state  produced  by  a  lower  than  normal  level  of  

glucose  in  blood,  in  insects  which  lacked  CC  cells.  This  also  showed  that  AKH  may  be  

required  for  compensation  to  maintain  the  glucose  circulation.  (Kim,  Rulifson,  2004)  

 

The  concentration  of  glucose  in  the  blood  is  relatively  constant.  Glucose  is  essential  for  

tissue  respiration  and  to  provide  energy  to  perform  vital  activities.  The  sugar  required  

immediately  for  energy  in  the  cells  is  changed  in  the  liver  to  glycogen.  The  glycogen  

molecules  are  built  up  by  combining  many  glucose  molecules.  Some  of  this  insoluble  

glucose  is  stored,  when  the  blood  sugar  falls  below  a  certain  level  the  glycogen  is  

11

changed  back  to  glucose  and  releases  it  into  the  circulation.  The  muscle  glycogen  is  not  

returned  to  the  circulation  but  is  used  by  muscle  cells  as  source  of  energy  during  

muscular  activity  such  as  moving  their  legs  and  wings.  Excess  glucose  not  stored  as  

glycogen  is  converted  into  fat.    

Generally  homeostatic  control  involves  negative  feedback  which  for  instance,  if  there  is  

too  much  glucose  in  blood  (stimulus)  it  stimulates  pancreas  (beta  cells)  to  secrete  more  

insulin  which  causes  the  liver  to  convert  glucose  to  glycogen  and  the  blood  glucose  level  

falls  (negative  feedback).    And  when  glucose  level  raises  pancreas  (alpha  cells)  secrete  

glucagons  which  causes  the  lives  to  convert  glycogen  to  glucose.  (Greenwood,  

Shepherd,  Allen,  Butler  47)  

 

Cells  are  adapted  to  keep  consistency  of  water  in  relation  to  salts  to  perform  its  

functions.  Water  is  the  solvent  of  the  solutes  in  the  cell.  Each  cell  has  been  adapted  to  a  

defined  quantity  of  water  in  relation  to  salts  in  it  to  perform  its  functions.  Homeostatic  

mechanisms  generally  maintain  this  concentration.  High  glucose  in  the  D.  

melanogaster’s  had  created  and  hypertonic  environment  (the  environment  was  more  

concentrated  externally  than  cell  concentration)  which  means  the  cells  had  been  

dehydrated  because  of  net  water  loss  and  high  concentration  of  glucose  in  relation  to  

water  potential.    

 

Generally  to  prevent  these  situations  cells  osmoregulate  to  keep  the  balance  but  as  the  

results  show  that  in  20%  and  30%  concentrated  glucose  most  flies  has  failed  to  survive  

because  of  net  water  loss.  The  results  suggest  that  the  glucose  level  in  the  medium  may  

have  exceeded  the  ideal  level.  (Greenwood,  Shepherd,  Allen,  Butler  47)  

 

In  humans  inability  to  control  blood  glucose  level  causes  diabetes,  where  the  hormones  

can  not  produce  sufficient  amount  of  insulin  to  convert  glucose  into  glycogen  for  

storage.  This  results’  in  high  blood  sugar  level  because  the  glucose  is  not  taken  by  body’s  

cells.    

12

 

Another  possible  conclusion  of  the  results  acquired  may  be  that  the  larva  may  have  not  

fed  on  the  supplementation  because  it  was  not  according  to  their  taste  and  starvation  

led  to  their  delay  in  development.  According  to  one  of  the  research  where  they  had  test  

the  same  independent  variable  had  results  which  show,  nymph  who  were  fed  diets  

supplemented  with  glucose  consumed  less,  gained  less  weight,  developed  slower  and  

had  lower  rate  of  survival  than  nymphs  who  were  fed  the  same  diet  without  added  

glucose  supplementation  (Silverman  ).  

 

Sources  of  error  

 

The  major  concerns  of  the  experiment  were  that  the  ingredients  for  the  nutrient  

medium  were  unknown  due  to  confidentiality  reasons.  Therefore  it  is  possible  that  

glucose  may  already  be  present  in  the  media,  the  experiment  was  conducted  keeping  in  

mind  that  their  may  or  may  not  be  glucose  already  included.  It  is  not  difficult  to  see  that  

there  may  already  be  glucose  added  since  the  nutrient  medium  used  in  labs  are  made  so  

they  are  suited  to  the  fly  with  all  the  ideal  level  for  ingredients  used.  The  experiment  

was  conducted  under  considering  that  the  (10%,  20,  30%)  were  added  glucose  and  that  

the  number  0%  referred  to  no  additional  glucose  added  for  the  experiment.  Although  

this  may  not  have  affected  the  results  greatly,  it  is  hard  to  conclude  regarding  the  ideal  

glucose  level  for  D.melanogaster  since  it  is  unknown  whether  glucose  is  originally  

present  or  not  in  the  instant  medium  used.    

Looking  at  results  given  in  appendix  A,  TableA3  we  can  see  that  there  was  only  one  fly  

eclosed  during  the  whole  trial  3  for  20%  concentration  which  may  suggest  that  there  

may  have  been  source  of  error  during  the  experiment  for  this  particular  trial,  since  there  

seemed  to  be  a  significant  difference  just  in  this  trial  we  can  assume  the  other  trials  

went  fairly  accurate.  The  possible  errors  may  be  that  there  was  a  human  error  of  

precision  while  making  the  instant  medium  out  of  the  instant  medium  powder.  The  

proportion  of  water  in  terms  of  the  powder  may  have  been  altered  (excess  of  water  or  

13

less  water  causing  the  media  to  become  highly  concentrated)  which  may  have  caused  

failure  in  flies  eclosing,  another  possibility  may  be  that  the  sponge  plugs  used  to  secure  

the  top  of  the  cultural  vial  may  be  kept  open  which  resulted  into  the  flies  that  were  

eclosed  to  escape.    

Another  factor  of  slight  error  that  should  be  taken  under  consideration  is  that  the  flies  

were  taken  out  of  the  incubator  every  day  after  the  first  7  days  to  observe  any  changes  

or  eclosion.  This  had  exposed  the  flies  to  the  room  temperature  which  would  have  

introduced  change  in  to  the  constant  temperature  of  25  degree  centigrade.  However  

this  should  not  have  introduced  any  major  source  of  error.  

The  instant  nutrient  medium  is  a  sticky  substance  which  may  have  also  altered  some  of  

the  results.  Some  of  the  larva  or  flies  eclosed  had  got  stuck  into  the  medium,  larva  

which  got  stuck  were  unable  to  hatch  and  died  and  flies  which  got  stuck  and  died  were  

difficult  to  count  since  there  may  be  a  possibility  of  damaging  or  slicing  the  body  into  

pieces  and  introducing  error  during  counting  even  though  the  flies  were  counted  under  

the  dissecting  scope  there  is  always  possibility  of  slight  error.      

 

             

                         

14

 Bibliography  

 

 

BAUMBERGER,  J.  (1917).  Solid  media  for  rearing  Drosophila  .  The  American  Naturalist,  51,  447-­‐448        Brownlee,  Christen.  "Flies  "R"  Us.."  Vol.  166  Issue  121  Science  News  8/09/2004  180  -­‐  181.  27/08/2007  <http://web.ebscohost.com/ehost/detail?vid=5&hid=17&sid=c6d7589e-­‐583b-­‐4eb6-­‐b64b-­‐53b3ce8926b1%40SRCSM1>.      FLAGG,  R.O.  (2005).  Carolina  Drosophila  Manual,  Carolina  Biological  Supply  Company      Greenwood,  Shepherd,  Allen,  Butler,  Tracey,  Lyn,  Richard,  Dan.  Biology  1.  2.  BIOZONE      JAENIKE,  J.  (1986).  Feeding  behavior  of  future  fecundity  in  Drosophila,  The  American  Naturalist,  127,  118-­‐123      Kim,  Seung  K.  and  Rulifson,  Eric  J..  "Conserved  mechanisms  of  glucose  sensing  and  regulation  by  Drosophila  corpora  cardiaca  cells.."  Nature  16/09/2004  316  -­‐  320.  19/08/2007  .      Silverman,  Jules  .  "Effects  of  glucose-­‐supplemented  diets  on  food  intake,  nymphal  development,  and  fecundity  of  glucose-­‐averse,  non-­‐glucose-­‐averse,  and  heterozygous  strains  of  the  German  cockroach,Blattella  germanica  ."  Entomologia  Experimentalis  et  Applicata  Volume  76,  Number  1  Jul,  1995  7-­‐14.  <http://www.springerlink.com/content/gl9162r57020m125/>.    TATUM,  E.  (1939).  Nutritional  requirement  of  Drosophila  melanogaster  Proceedings  of  the  National  Academy  of  science  of  the  united  stated  of  America,  25,  490-­‐497      

     

15

Appendix A:  Raw  Data    

Table A1 Table A2

Table A3

16

Appendix B:  Processed  Data  

 

Table B1

17

Table B2

18

Table B3

19

Table B4                                          

20

   

Rate  of  eclosion  when  additional  glucose  is  added  

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

8 10 12 14 16 18 20 22 24 26

days

eclo

sion

rat

e (a

vera

ge n

umbe

r of

flie

s)

0%

10%

20%

30%

 Figure  B1                                        

21

     

Appendix C:  Chi-­‐Squared  Test    

O  =  Observed    E  =  Expected  χ2  =  Chi-­‐square  

   Table  C1.1    

Category     O   E   O  -­‐  E   (O  -­‐  E)2   (O  -­‐  E)2  /  E  round  1  -­‐10%   202   154   48   2304   14.96  round  2  -­‐10%   98   154   -­‐56   3136   20.36  round  3  -­‐10%   162   154   8   64   0.42  

TOTAL   462            Σ   35.74      Table  C1.2    

Category     O   E   O  -­‐  E   (O  -­‐  E)2   (O  -­‐  E)2  /  E  round  1  -­‐20%   203   95.33   107.67   11592.11   121.60  round  2  -­‐20%   82   95.33   -­‐13.33   177.78   1.86  round  3  -­‐20%   1   95.33   -­‐94.33   8898.78   93.34  

TOTAL   286            Σ   216.80      Table  C1,3    

Category     O   E   O  -­‐  E   (O  -­‐  E)2   (O  -­‐  E)2  /  E  round  1  -­‐30%   41   60.33   -­‐19.33   373.78   6.20  round  2  -­‐30%   87   60.33   26.67   711.11   11.79  round  3  -­‐30%   53   60.33   -­‐7.33   53.78   0.89  

TOTAL   181            Σ   18.87              

22

     

   Table  C2.1                        

Category     O   E   O  -­‐  E   (O  -­‐  E)2   (O  -­‐  E)2  /  E  round  1  -­‐  0%   257   175.75   81.25   6601.56   37.56  round  1  -­‐  10%   202   175.75   26.25   689.06   3.92  round  1  -­‐  20%   203   175.75   27.25   742.56   4.23  round  1  -­‐  30%   41   175.75   -­‐134.75   18157.56   103.31  

    TOTAL  =   703          Σ   149.02      Table  C2.2                          Category     O   E   O  -­‐  E   (O  -­‐  E)2   (O  -­‐  E)2  /  E  round  2  -­‐  0%   125   98   27   729.00   7.44  round  2  -­‐  10%   98   98   0   0.00   0.00  round  2  -­‐  20%   82   98   -­‐16   256.00   2.61  round  2  -­‐  30%   87   98   -­‐11   121.00   1.23  

    TOTAL  =   392          Σ   11.29    Table  C2.3                

Category     O   E   O  -­‐  E   (O  -­‐  E)2   (O  -­‐  E)2  /  E  round  3  -­‐  0%   154   92.5   61.5   3782.25   40.89  round  3  -­‐  10%   162   92.5   69.5   4830.25   52.22  round  3  -­‐  20%   1   92.5   -­‐91.5   8372.25   90.51  round  3  -­‐  30%   53   92.5   -­‐39.5   1560.25   16.87  

    TOTAL  =   370          Σ   200.49