T6 Chen, Goyal, Puri, Shin

Medium Modification to Determine a Cost-Efficient Growth Environment for

Yeast (Saccharomyces cerevisiae)
BRIEF BACGR!"#D$
Many biotechnological processes used in industry bank on the reliable and cost-effect production
of Saccharomyces cerevisiae, commonly known as aker!s "east# "east can grow in the presence of $ust
sugars, which pro%ide a source of glucose# &owe%er, yeast grows much faster when pro%ided with
proteins as well as biotin, trace metals, and salts, which are needed for growth# Proteins, which ser%e to
pro%ide amino acids as well as perform en'ymatic acti%ities, can be supplied through peptone(partially
digested proteins# "east e)tract is obtained by the autolysis of yeast cells, and therefore, pro%ides growing
yeast with all the necessary components, including biotin, trace metals and salts, as well as proteins#
*lthough yeast has been pro%en to grow effecti%ely with $ust de)trose, pre%ious e)perimentation
has suggested that the yeast e)tract has the most significant effect on the reproduction of yeast# +The
addition of protein and yeast cell e)tract hydrolysates allow faster growth so that during e)ponential or
log-phase growth, the cells di%ide e%ery ,- minutes#.
/
Moreo%er, during the lag phase secondary
metabolites re0uired for growth are produced# Since the yeast e)tract contains ready-made secondary
metabolites it reduces the time re0uired for the lag phase to transition into the log phase#
Cost-efficiency of yeast growth is rele%ant in se%eral sectors of industry# "east is used as a fermenting
agent in bread-making, wine-making, the production of cheese and yogurt, and the production of %arious
other foods that re0uire fermentation# esides fermentation, yeast can be used to create yeast e)tract
spread for direct consumption# 1t is in the best interest of industrial manufacturers to determine the
specific combinations of yeast e)tract, peptone, and de)trose that produce the desired results according to
each manufacturer2s financial situation and3or demand for their product#
%Y&!'%E(I()!B*EC'I+E()AIM($
The preliminary aim of the e)periment was to determine the ratios of yeast e)tract, peptone and
de)trose, which would constitute the optimum growth medium for yeast# The primary aim of the
e)periment was to determine the most cost-effecti%e of these optimum growth mediums#
The established standard media ratio was /#4g 5"east e)tract67 8g 5Peptone67 8g 59e)trose6# 1t was
hypothesi'ed that the concentration of 8g 5"east e)tract67 8g 5Peptone67 8g 59e)trose6 will yield the
highest growth rate# The additional amount of yeast e)tract will pro%ide the necessary secondary
metabolites for the yeast resulting in a reduction of the lag phase# 1t is also hypothesi'ed that the
concentration of #:4g 5"east ;)tract67 8g 5Peptone67 8 g 59e)trose6 will be the most economical# *fter the
lag phase, peptone becomes the most crucial component for the growth phase# ;conomical effecti%eness
is determined by the ratio of 5amount of cells produced6 3 5doubling time < cost6# This combination will
gi%e the highest ratio due to the reduction of the most e)pensi%e component, yeast e)tract, as well as
maintaining the crucial component of peptone#
*s an e)tension of the e)periment, the relationship between the growth rate constant and the
%olume of media used was in%estigated#
GE#ERA, &R!'!C!,$
=ur protocol has been designed around the aforementioned hypotheses and ob$ecti%es#
-ee. /$
Goals7
To determine the optimum concentration of yeast using the standard media mi)ture to find greatest
growth rate constant#
/
Talron iotech >T9#, +"P9,. Mar# ?, @--A, Mar# @?, @--A Bhttp733www#talron#co#il3products3prod4/#htmC#
/
T6 Chen, Goyal, Puri, Shin
Plan7
/6 Test three different concentrations of yeast using two trials 56 total samples6 using standard
media mi)ture ratio 5"east ;)tract7 Peptone7 9e)trose6 5/#4g78g78g6 5A- m> used for each
sample6
/#- mg3ml3absorbance
-#4 mg3ml3absorbance
-#@4 mg3ml3absorbance
@6 9ecide which concentration of yeast is the most effecti%e using an *D=E* test 5nF86,
and if
needed, onferroni2s correction#
86 Make media mi)tures for week @#
-ee. 0$
The concentration of yeast determined in week / will be used for all further e)perimentation#
Goals7
To determine the optimum concentration of yeast e)tract in the media mi)ture that will yield
either the greatest growth rate constant#
Plan7
/6 Test three different ratios of media mi)ture modifying the concentration of yeast e)tract ratios
using two trials 56 total samples6 5"east ;)tract7 Peptone7 9e)trose6 5g3#/4- >6 5A- m> used for
each sample6
/#47 8#-7 8#- 5standard basis of comparison6
-#:47 8#-7 8#- 5decreasing concentration 4-G6
8#-7 8#-7 8#- 5increasing concentration /--G6
@6 9etermine length of lag and log phase
86 9ecide which is the most effecti%e concentration of yeast e)tract for the media mi)ture, using
an *D=E* test 5nF86 and onferroni2s correction if need be, which will be denoted as H#
A6 Make media mi)tures for week 8
-ee. 1$
Goals7
To determine the optimum concentration of peptone in the media mi)ture which will yield either
the greatest growth rate constant#
Plan7
/6 Test three different ratios of media mi)ture modifying concentration of peptone ratios
using two
trials 56 total samples6 5"east ;)tract7 Peptone7 9e)trose6 5:#4 g3#/4- >6 5A- m> used for
each
sample6
H7 8#-7 8#- 5standard basis of comparison6
H7 /#47 8#- 5decreasing concentration 4-G6
H7 6#-7 8#- 5increasing concentration /--G6
@6 9etermine length of lag and log phase#
86 9ecide which is the most effecti%e concentration of peptone, using an *D=E* test 5nF86 and if
needed, onferroni2s correction, for the media mi)ture#
9etermine the most cost-effecti%e combination between the 8 optimum combinations use *D=E* testing
5nF86 and if needed, onferroni2s correction, as well as the yield ratio#
(&ECIFIC ME'%!D($
@
T6 Chen, Goyal, Puri, Shin
Ior all e)perimentation7
Time constraints and efficiency allowed only for @ samples per each ratio#
=ne person pipetted the media into each 4-m> test tube#
=ne person inoculated each sample with a specified %olume of yeast using the same pipettes and
clean tips#
;%ery indi%idual sample was inoculated 4 minutes apart# *fter the final inoculation, absorbance
readings were taken e%ery 4 minutes using the spectrophotometer, with 8- minutes between readings
for each sample, for the duration of the lab 5generally A-4 hrs6, in order to maintain consistency and
conduct more accurate measurements# The spectrophotometer was 'eroed for e%ery sample using the
specific standard media solution#
;ach sample has its own dropper and its own tube for absorbance readings in order to a%oid
contamination#
-ee. /$
The standard media ratio of 5"east ;)tract7 Peptone7 9e)trose65/#4g78g78g6 was used with %arying
amounts of yeast#
@ samples of /#- M F @- m> of standard media J @#@@ m> yeast
@ samples of -#4 M F @- m> of standard media J /#-4 m> yeast
@ samples of #@4 M F @- m> of standard media J #4/8 m> yeast
ecause there was a limited amount of standard media and other groups had to use it for
e)perimentation, only @- m> of media could be used in order to maintain @ trials per sample#
Do lag phase was witnessed, and thus its length could not be measured# Similarly, the entire log phase
was not witnessed so its length could not be measured either#
The media was made for week @ using the combinations pre%iously stated in the general protocol# *ll
reagents were weighed using the Mettler electronic balance with precision of J #--/# ;ach media mi)ture
was made in /4- m> of water 5measured using /4- m> %olumetric flask6# *ll media mi)tures, which were
autocla%ed, were prepared in sterili'ed containers#
-ee. 0$
*ll samples were calculated to ha%e a #@4 M concentration#
9ifferent media mi)ture ratios 5"east ;)tract7 Peptone7 9e)trose6 were used with the same amount of
yeast#
@ samples of #@4 M F A- m> of media 5/#47 8#-7 8#-6 J /#-8 m> of yeast
@ samples of #@4 M F A- m> of media 5#:47 8#-7 8#-6 J /#-8 m> of yeast
@ samples of #@4 M F A- m> of media 58#-7 8#-7 8#-6 J /#-8 m> of yeast
Do lag phase was witnessed, and thus its length could not be measured# Similarly, the entire log phase
was not witnessed so its length could not be measured either#
The media was made for week 8 using the combinations pre%iously stated in the general protocol#
&owe%er, rather than increasing the peptone by /--G, it was inade%ertently decreased by :4G# *ll
reagents were weighed using the Mettler electronic balance with precision of J #--/# ;ach media mi)ture
was made in /4- m> of water 5measured using /4- m> %olumetric flask6# *ll media mi)tures, which were
autocla%ed, were prepared in sterili'ed containers#
-ee. 1$
*ll samples were calculated to ha%e a #@4 M concentration
9ifferent media mi)ture ratios 5"east ;)tract7 Peptone7 9e)trose6 were used with the same amount of
yeast#
@ samples of #@4 M F A- m> of media 5#:47 /#47 8#-6 J /#-8 m> of yeast
@ samples of #@4 M F A- m> of media 5#:47 #:47 8#-6 J /#-8 m> of yeast
/ sample of #@4 M F /?#4 m> of media 5#:47 8#-7 8#-6 J #A:A m> of yeast
/ sample of #@4 M F /? m> of media 5#:47 8#-7 8#-6 J #A6@ m> of yeast
8
T6 Chen, Goyal, Puri, Shin
Part of the lag phase was witnessed for all samples, and thus when determining the growth rate constant,
the appropriate absorbance readings were used#
Media mi)ture ratio 5#:478786 was spilled before inoculation, and in order to maintain a #@4 M
concentration as well as allow T@ to use the media for their e)perimentation, the amount of yeast was
ad$usted as such#
RE(",'($
"east was grown for a period of A#4 hours in standard solution, %arying initial yeast concentrations#
Two samples were tested for each concentration of /M, #4M, and #@4M#
Growth Rate Constants
Yeast Concentration Av2 (tdev
/#- M #---,4 :#/;-4
-#4 M #--@ -
#@4 M #--8 /#A;-A
'a34e /5 Avera2e 2rowth rate constants o3served for sam64es with varied 7east concentrations#
Fi2ure /5 Effects of initia4 ce44 concentration shown throu2h a 4ineari8ed 2ra6h of A3sor3ance vs5
'ime#
*bsorbance indicates concentration of yeast cells in solution# Slopes indicate the growth rate
constant K in the e0uation ln5*3*o6F Kt# Iigure / shows that the growth rate constant increased with a
decrease in initial concentration of yeast cells# Plots with the largest slope shown in brown and purple
were the #@4M samples#
Gi%en an optimum initial cell concentration of #@4M, yeast was grown for A#4 hours %arying yeast
e)tract concentration while peptone and de)trose le%els were held constant at 8 grams# "east e)tract
concentrations of #:4, /#4, and 8#- 5g3/4- ml6 were tested# There was a discrepancy in spectrophotometer
readings at /4- minutes and subse0uently only data obtained from /4--@:- minutes was used for
analysis#
Growth Rate Constants
Yeast E9tract (2) Av2 (tdev
-#:4 #--4? -
/#4 #--A6 #---@
8 #--8? -
'a34e 05 Avera2e 2rowth rate constants o3served for sam64es with amounts of 7east e9tract varied#
A
T6 Chen, Goyal, Puri, Shin
Fi2ure 05 Effects of 7east e9tract concentration shown throu2h a 4ineari8ed 2ra6h of A3sor3ance vs5
'ime5
*bsorbance indicates concentration of yeast cells in solution# Slopes indicate the growth rate
constant K in the e0uation ln5*3*o6F Kt# Iigure shows that the growth rate constant increased with a
decrease in yeast e)tract concentration although further statistical analysis showed no statistical difference
between the /#4 and 8#- g3/4- ml concentrations 5pC#-/6:6# Plots with the largest slope shown in light
blue and yellow were the #:47878 ratios#
Lsing #@4M and #:4 g3/4- ml as the optimum growing conditions of initial cell concentration and
yeast e)tract, peptone content was %aried at #:4, /#4, and 8#- g3/4- ml# //- ml of the #:47878 "P9 broth
was spilled during preparation# Samples for that ratio were prepared at /? and /?#4 ml rather than A- ml#
Fi2ure 15 Effects of 6e6tone concentration shown throu2h a 4ineari8ed 2ra6h of A3sor3ance vs5
'ime5
*bsorbance indicates concentration of yeast cells in solution# Slopes indicate the growth rate
constant K in the e0uation ln5*3*o6F Kt# This figure indicates that there was no statistical difference in
slope between yeast cells grown in the 8 %ariations of peptone concentration 5pC#-46#
4
T6 Chen, Goyal, Puri, Shin
Growth Rate Constants
&e6tone (2) Av2 (tdev
-#:4 #--844 :#-:;--4
/#4 #--884 :#-:;--4
8 #--8: #---/A/
'a34e 1# 'a34e of the 2rowth rate constants o3served for sam64es with varied 6e6tone
concentrations5
*lthough %alues indicate a possible trend of increasing growth rate with increasing peptone
concentration, statistical testing indicates that the %alues are not significantly different 5pC#-46#
The cheapest combination was determined to be #:47 #:47 8 while the fastest growing combination was
determined to be #:47 87 8# The e0uation "MF/---35cost<doubling time6 was used to determine a
0uantitati%e yield ratio#
Com6osition Cost (:) Dou34in2 'ime (min) Yie4d Ratio
/#47 87 8 -#?- @8/ 4#A/
#:47 87 8 -#6? /@- /@#@4
#:47 #:47 8 -#8? /,4 /8#4
'a34e ;5 'he cost< dou34in2 time< and 7ie4d ratios of the standard< fastest 2rowin2< and chea6est
com3inations of media#
The 8? cent combination had the highest yield ratioN it produced the most cells at the smallest
e)pense of time and cost# The double times of the ?- cent combination and the 8? cent combination were
found to be statistically similar 5pC #-/6:6# The 6? cent combination was about half the doubling time of
the standard media and close to half of the double time of the 8? cent combination because there were
found to be statistically similar#

Fi2ure ;5 Corre4ation coefficients of 4ineari8ed data for o6timum sam64es and the tota4 avera2e5
The a%erage correlation coefficient for all samples 5nF/?6 was #,,@?/:J #--68,6# The 8 optimum
mi)tures were analy'ed separately# Ohile the /#47 87 8 5#,,644 J #--/6@66 and #:47 #:47 8 5#,?4? J #
--6-?/6 combinations lay within the total a%erage standard de%iation, the #:47 87 8 5#,?/:4 J #--@6/66
combination does not# The initial cell concentration of the 8 optimum combinations was #@4M while the
total a%erage also included the combinations with #4M and /M#
*D=E* testing showed that there was a statistical difference between the #@4M , #4M, and /M
combinations of initial yeast concentration 5pB#-/6:6# Conse0uently, #@4M was determined to be the
optimum initial cell concentration since it yielded the lowest doubling time of @8/ minutes# The doubling
times for #4M and /M solutions were determined to be 8A: and ::- minutes, respecti%ely# *D=E* testing
also showed that there was a statistical difference between the three concentrations of yeast e)tracted
6
T6 Chen, Goyal, Puri, Shin
tested 5pB #-/6:6# Ohile the growth in the #:4 g3/4- ml was significantly different from growth in both
the /#4 and 8#- g3/4- ml solutions 5pB #-/6:6, the latter did not show a difference amongst each other
5pC #-/6:6# 9oubling times were as follows /@-, /4@, and /?@ minutes# >astly, it was determined through
*D=E* testing that there was no statistical difference in solutions with %aried peptone concentration
5pC#-46#
*D=E* testing showed that there was a statistical difference between #:47 #:47 8 combination and /#47
87 8 and :47 87 8 combinations respecti%ely 5pB #-/6:6# 1t also showed that there was no statistical
significance between /#47 87 8 and :47 87 8 combinations 5pC #-/6:6#
The most cost-efficient growth medium was determined to be a solution of ratio #:47 #:47 8# The
fastest growth actually occurred with the #:47 87 8 ratio but the doubling time was offset by its high price#
The preceding results are based on the assumption that there is no relationship between the growth
rate constant and the %olume of media used#
DI(C"((I!#$
1n week /, it was found that the samples with #@4M initial concentration of yeast yielded the shortest
doubling time# This can be attributed to the fact that the media mi)ture as a whole was a limiting reagent
at higher concentrations of yeast# *t that point in the e)perimentation, it was unknown which component
of the media mi)ture was the limiting reagent#
1n week @, results pro%ed to be counterintuiti%e to the hypotheses# The least amount of yeast e)tract
produced the highest rate of growth# 1t is uncertain as to why the results were as such, howe%er there are
some possible e)planations# "east e)tract is thought to reduce lag time because it pro%ides the secondary
metabolites, which would otherwise ha%e to be made during the lag phase# ecause, no lag time was
witnessed during the duration of the e)periment, it is unknown if the lag time was shortened, and if it was
how it affected growth# *nother possible solution is that the yeast may ha%e been the limiting reagent, and
thus larger amounts of yeast e)tract would be unnecessary# This e)planation is somewhat consistent with
the e)perimental findings# 1t was statistically pro%en that the media combinations with /#4 g and 8 g of
yeast e)tract were similar 5pC #-/6:6# &owe%er, these results were not consistent with the theory of
limiting reagents and that the samples with the e)traneous yeast e)tract should ideally yield statistically
similar growth rate constants as the #:4g combination# Iurthermore, it is possible that there were
competing organisms in the solution that could ha%e used the e)cess yeast e)tract and thereby multiplied
0uickly enough to consume other resources that the yeast might need as well# This e)planation is %alid in
that it is ine%itable that there is at least some type of contamination in the samples# 1n addition, it has been
found that certain types of bacteria such as ;# coli use yeast e)tract as part of their growth medium#
1n week 8, the results concluded that peptone did not ha%e a discernable effect on the growth of the
yeast cells assuming there was no relationship between the growth rate constant and the %olume of media
used# Though the amount of peptone was inad%ertently decreased by :4G rather than increased by /--G
for two trials, the results show that an increased amount of peptone would not likely ha%e affected the
results because the growth rate constants for %arying amounts of peptone pro%ed to be statistically similar#
* higher amount of peptone would most likely only raise the cost of the media mi)ture, rather than
increase the growth rate# &owe%er, only further e)perimentation can clarify the %alidity of this assertion#
These results, howe%er, are contradictory to those of week @# The #:47878 ratio in week @ yielded a
growth rate constant of #--4?J- and in week 8, the ratio yielded a growth rate constant of #--8:J #
---/A/# *lthough the growth rate constants were shown to be statistically similar in week 8, the 8#- g
combination of peptone 0ualitati%ely showed the highest growth rate constant 5pC#-46# This could
possibly be because this solution 5#:478786 was not tested under the same conditions as the other two
samples# Testing for this combination was done with /? ml and /?#4 ml of solution because the original
container was spilled during preparation rather than A- ml samples# 1t is probable that an element of
human error was present due to the time constraints of sampling 5e%ery 4 minutes6 and the haste at which
the problem of spilling the solution, in order to maintain an initial concentration of #@4 M of yeast, was
dealt with# Pualitati%ely, there was found to be a positi%e correlation between peptone and the growth rate
constant at a constant concentration of #@4M# 1f there is a direct relationship between the %olume of media
:
T6 Chen, Goyal, Puri, Shin
used and the growth rate constant 5see *PP;D91H6, it was concluded that 8 g of peptone yielded the
highest growth rate constant 5pB#-/6:6#
*nalysis of the correlation coefficients of all growth rate constants showed that there is limited
%ariance in the data, and thus there is a greater %alidity in the actual growth rate constants# The total
a%erage correlation coefficient for all samples 5nF/?6 was #,,@?/: J #--68,6# * %alue of #,,@?/: is
deemed as a %ery strong correlation and these e)perimental %alues ha%e a great degree of %alidity# Ohile
the /#47878 and #:47 #:47 8 combinations lay within the total a%erage standard de%iation, the #:47878
combination does not# The total a%erage correlation coefficient ranges as far down as #,?6A@/, whereas
the #:47 87 8 combination ranges as far high as #,?A866, a total difference of #--@-44# Dormally, there
would be cause for concern if the correlation coefficient did not fall within the standard de%iation of the
a%erage, but because the correlation coefficient for the #:47878 mi)ture is still relati%ely high 5#,?/:4 J #
--@6/66 the %alue of the growth rate constant was deemed %alid# * correlation %alue of #,?/:4 shows a
%ery strong correlation, and though error analysis may deem it as erroneous, the number in itself cannot
be disregarded#
The a%erage e)pense determined by the ratio used and the standard price 5Media *6 pro%ided was
Q#?- per /4- ml of media# The two cost-effecti%e prices deri%ed were Q#6? per /4- ml 5Media 6, with
half the doubling time of the yeast in the standard media and Q#8? 5Media C6 per /4- ml of media
pro%iding the same yeast production as the standard media# The Q#6? per /4- ml would be %ery cost
efficient if a company desired a greater yeast production# 1f howe%er, the company was e)periencing
monetary distress and desired to cut production costs but maintain the yeast yield, they could reduce the
costs by Q#A@ and still get the standard media yield by using Media C# 1f there is a direct relationship
between the %olume of media used and the growth rate constant, the cost-effecti%e combinations remain
the same as when there was no assumed relationship 5see *PP;D91H6#
1n e)trapolating this in%estigation, carrying out trials on the effect of %ariable de)trose concentration
on yeast growth would be useful such that e%en more cost-effecti%e media ratios can be sought# Gi%en
more time, the de)trose in%estigation would be a priority# *dditionally, gi%en time and the appropriate
e0uipment, 0uestioning the effects of temperature and p& on yeast production in the most effecti%e
growth media would be an interesting in0uiry# To obtain a more accurate cost-effecti%e media mi)ture,
better obser%ation of the entire lag and log phases is re0uired# Testing the effect of different conditions on
increasing3decreasing the duration of the lag phase is also an important aspect of future e)perimentation#

?
T6 Chen, Goyal, Puri, Shin
A&&E#DI=$
1n e)tension of the data presented, it was noted that the %olume of media used for each sample was
inconsistent due to the limited resources as well as accidents# 1t was thought that there might be a direct
relationship between the growth rate constant and the %olume of media used, though the sample as a
whole will ha%e the same concentration# There were A ratios 5? trials6 that had the same concentrations
and media mi)ture ratios, howe%er, the %olumes of media used %aried# Lsing the formula7
K/<E/ 5greater %olume of media used in m>6 F R<K@<E@ 5smaller %olume of media used in m>6
where K was the growth rate constant for the corresponding %olume, R was determined for each
combination# There were eight %iable combinations for which R were determined# The a%erage R was
determined to e0ual 8#@8?# Lsing the aforementioned formula, data was e)trapolated so that all samples
had growth rate constants corresponding to using A- m> of media# Ior those samples that used A- m> of
media, the %alues were kept the same#
Growth Rate Constants
Yeast Concentration Avg Stdev
1M 0.00155 7.10E-05
.5M 0.0032 0
.25M 0.00485 2.10E-04
'a34e /5 Avera2e 2rowth rate constants o3served for sam64es with varied 7east concentrations#
1t was found through *D=E* testing and onferroni2s correction, that all three growth rate constants
were statistically different 5p B#-/6:6# Therefore, the most optimum concentration to be used was #@4M#
Growth Rate Constants
Yeast E9tract (2) Av2 (tdev
-#:4 #--4? -
/#4 #--A6 #---@
8 #--8? -
'a34e 05 Avera2e 2rowth rate constants o3served for sam64es with amounts of 7east e9tract varied#
Ior further information, refer back to this M;SL>TS section because they are identical#
Growth Rate Constants
&e6tone (2) Av2 (tdev
-#:4 #--884 -
/#4 #--844 #---@
8 #--4A4 -
'a34e 15 Avera2e 2rowth rate constants o3served for sam64es with varied 6e6tone amounts5
Through *D=E* testing, it was found that there no statistical difference between #:4 g and /#4 g of
peptone 5pC#-/6:6# &owe%er, there was a statistical difference between 8 g and #:4g and 8 g and /#4 g
5pB#-/6:6# 1t was determined that the optimum amount of peptone to be used was 8 g#
Ohen performing an *D=E* test of the three weeks between optimum growth rate constants, where
the concentration stayed the same for weeks @ and 8, there was no statistical difference 5pC#-46# The
5#:478786 combination was determined to be more cost-effecti%e than the standard, because it yields the
same product at a cost of /@ cents less#
Ohen performing an *D=E* test between the standard, #:47878, and #:47#:478 combinations, it was
found that all growth rate constants were statistically different 5pB#-/6:6# The doubling times differed
from those found in the M;SL>TS section, but ultimately the most cost-effecti%e combinations remained
the same#
Com6osition Cost (:) Dou34in2 'ime (min) Yie4d Ratio
/#47 87 8 -#?- /A8 ?#:4
#:47 87 8 -#6? /@- /@#8/
:47 #:47 8 -#8? /,4 /8#A?
,