Lab 2 Bacterial Growth Curve PDF

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Lecture notes of prof nalina natraja...

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Lab$2:$Determination$of$Bacterial$Growth$Curve$ # Objectives:$ # 1.#To#understand#the#different#phases#of#a#standard#growth#curve.# 2.#To#understand#and#perform#direct#measurement#of#bacterial#growth#through#serial# dilutions#and#standard#plate#counts.# 3.#To#understand#and#perform#indirect#measurement#of#bacterial#growth#through# spectrophotometer#readings#and#optical#density#measurements.# # Introduction:$ # Bacterial#population#growth#studies#require#inoculation#of#viable#cells#into#a#sterile#broth# medium#and#incubation#of#the#culture#under#optimum#temperature,#pH,#and#aeration# conditions.#Under#these#conditions,#the#cells#will#reproduce#rapidly#by#binary#fission#and# the#dynamics#of#the#microbial#growth#can#be#charted#by#means#of#a#population#growth# curve,#which#is#constructed#by#plotting#the#increase#in#cell#numbers#versus#time#of# incubation.#This#chart#can#be#used#to#delineate#stages#of#the#growth#cycle.#It#also#facilitates# measurement#of#cell#numbers#and#the#rate#of#growth#of#a#particular#organism#under# standardized#conditions#as#expressed#by#its#generation#time.#Generation#time#is#defined#as# the#time#required#for#a#microbial#population#to#double.# # The#stages#of#a#typical#growth#curve#(figure#below)#are:# 1. Lag$phase:$#When#the#cells#are#adjusting#to#their#new#environment.#During#this# phase,#cellular#metabolism#is#accelerated,#resulting#in#rapid#biosynthesis#of#cellular# macromolecules,#primarily#enzymes,#in#preparation#for#the#next#phase#of#the#cycle.# Although#the#cells#are#increasing#in#size,#there#is#no#cell#division#and#therefore#no# increase#in#numbers.# # 2. Logarithmic$(log)/Exponential$phase:#Under#optimum#nutritional#and#physical# conditions,#the#physiologically#robust#cells#reproduce#at#a#uniform#and#rapid#rate#by#

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# binary#fission.#Thus#there#is#a#rapid#exponential#increase#in#population,#which# doubles#regularly#until#a#maximum#number#of#cells#is#reached.#The#length#of#the#log# phase#varies,#depending#on#the#organisms#and#the#composition#of#the#medium.# # 3. Stationary$phase:#During#this#stage,#the#number#of#cells#undergoing#division#is# equal#to#the#number#of#cells#that#are#dying.#There#is#no#further#increase#in#cell# number#and#the#population#is#maintained#at#its#maximum#level#for#a#period#of#time.# The#primary#factors#responsible#for#this#phase#are#the#depletion#of#some#essential# metabolites#and#the#accumulation#of#toxic#acidic#or#alkaline#end#products#in#the# medium.# # 4. Decline$or$death$phase:#Because#of#the#continuing#depletion#of#nutrients#and# buildup#of#metabolic#wastes,#the#microorganisms#die#at#a#rapid#and#uniform#rate.## #

# # Figure#1#–#Typical#bacterial#growth#curve# # If#this#cell#division#occurs#at#a#steady#rate#–#such#as#when#the#cells#have#adequate#nutrients# and#compatible#growing#conditions#–#we#can#plot#numbers#of#cells#vs.#time#(Figure#2).#By# converting#the#vertical#axis#values#to#a#logarithmic#scale#you#will#note#that#a#steady#rate#of# growth#is#reflected#as#a#straight#line#(Figure#2).#

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# Figure#2.##Plotting#the#numbers#of#cells#vs.#time#on#a#linear#scale#graph#(left).##Conversion#of# the#vertical#axis#values#to#logarithmic#scale#would#produce#a#straight#line#on#the#on#a#semiT log#scale#graph#(right)#(Figures#borrowed#from#Lindquist,#2006)# # Once#all#of#the#date#has#been#compiled,#both#colonyTforming#units#(CFU)#per#ml#and# absorbance#can#be#plot#on#the#same#graph#(Figure#3).##Note#that#the#absorbance#units# should#also#be#on#a#logarithmic#scale.#A#straight#line#of#best#fit#can#be#drawn#among#the# CFU/ml#plots#to#represent#the#phases#of#growth.#

# Figure#3.##Plot#of#log10CFU/ml#versus#time#on#a#semiTlog#scale.##Absorbance#values#are#plot# on#the#same#graph#in#logarithmic#scale#values#(Figure#borrowed#from#Lindquist,#2006).# 3

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# You#will#determine#generation#time#with#indirect#and#direct#methods#by#using#data#you# collect,#once#it#has#been#plotted#onto#a#graph#like#the#one#shown#below#(Figure#4).#Indirect# determination#is#made#by#simple#extrapolation#from#the#log#phase#as#illustrated#in#the# figure#below.#Select#two#points#on#the#optical#density#(OD)#scale,#such#as#0.2#and#0.4,#that# represent#a#doubling#of#turbidity.#Using#a#ruler,#extrapolate#by#drawing#a#line#between#each# of#the#selected#optical#densities#on#the#ordinate#and#the#plotted#line#of#the#growth#curve.# Then#draw#perpendicular#lines#from#these#end#points#on#the#plotted#line#of#the#growth# curve#to#their#respective#time#intervals#on#the#abscissa.#With#this#information,#determine# the#generation#time#as#follows:#

# Figure#4.##Plot#of#log10CFU/ml#versus#time#on#a#semiTlog#scale.##Absorbance#values#are#plot# on#the#same#graph#in#logarithmic#scale#values#(Figure#borrowed#from# http://faculty.sdmiramar.edu/dtrubovitz/micro/microhandouts/labs/growthcurve.pdf)# # 4

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# The#direct#method#uses#the#log#of#cell#number#scale#on#the#growth#curve#and#the#following# formula:# GT#=#t#log#2/[log#b#T#log#B]# # Where#GT#=#generation#time;#B#=#number#of#bacterial#CFUs#at#some#point#during#the#log# phase;#b#=#number#of#bacterial#cells#at#a#second#point#of#the#log#phase;#and#t#=#time#in#hours# or#minutes#between#B#and#b.# # Upon#completion#of#the#laboratory#exercise#and#laboratory#report#the#student#will#be# expected#to#be#able#to:# • Describe#the#objectives#of#the#growth#curve#laboratory.# • Explain#what#the#growth#curve#was#measuring.# • Define#generation#and#doubling#time.# • Calculate#generation#time#using#both#numerical#data#and#data#from#a#graph.# • Describe#the#limitations#of#the#testing#systems#used.# #

Prep$Room$Order$List$(4$students$per$group$=$6$groups$per$section)$ 1. Overnight#E.#coli#11303#culture#(40#ml#per#section)#grown#in#TSB#maintained#in#37°C# shaking#water#bath#(~#108#CFU/ml).# 2. Sterile#TSB#–#60#ml#in#a#sterile#bottle#with#stir#bar#(autoclaved)#–#1#per#group#–# warmed#in#37°C#incubator#or#water#bath# 3. Beaker#for#water#bath# 4. Hot#plate#–#1#per#group# 5. Thermometer#–#1#per#group# 6. Timer# 7. Vortex#mixers# 8. Sterile#Phosphate#buffer#in#tubes:#9.9#ml#–#12#per#group# 9. Sterile#Phosphate#buffer#in#tubes:#9.0#ml#–#14#per#group# 10. Ice#bucket#–#1#per#group# 11. Sterile#TSB#(for#OD#dilutions)#=#25#ml/#group# 12. Spec#20D#–#1#per#group# 13. Cuvette#–#2#per#group# 14. Rinse#water#bottles# 15. Beaker#to#collect#rinsing/#waste#–#1#per#bench# 16. Large#bottle#to#collect#waste#T#1#per#section#(to#autoclave#waste)# 17. Sterile#water#–#1L#(backup#dilutions)# 18. TSA#plates#–#24#per#group#+#2#per#class#for#controls# 19. Alcohol#&#Spreader# 20. Micropipettes#P1000,#P100# 21. Sterile#micropipette#tips#–#P100,#P1000#

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# Week$1$O$Procedure$ Work#in#groups$of$4$students#in#order#to#complete#spectrophotometer#readings#and# plating#in#the#allowed#time.#Suggested#distribution#of#responsibilities:# Member#1:#responsible#for#absorbance#measurements# Member#2:#responsible#for#dilution## Member#3:#responsible#for#plating# Member#4:#responsible#for#temperature#maintenance#(crucial#to#maintain#constant#37°C# water#bath#at#all#times),#timing#and#helping#out#with#team#work# 1. Turn#ON#and#set#your#spectrophotometer#to#600#nm.#Use#TSB#to#blank#the#machine.# 2. Setup#your#water#bath#at#your#bench#by#taking#~#200#ml#of#water#in#the#big#beaker# and#keep#it#on#the#hot#plate.#Keep#your#hotplate#at#the#lowest#setting#and#maintain# water#bath#at#37°C#(thermometer#always#in#the#waterbath).## 3. Collect#and#label$your$dilution$tubes#(12#x#9.9#ml#and#14#x#9.0#ml#per#group).#Find# out#whether#you#are#in#Group$A$or$B.# 4. Collect#and#label#your#TSA$plates$with$appropriate$dilutions,$time$point#–#24#per# group# 5. Each#group#will#receive#a#sterile#bottle#containing#60#ml#of#TSB#(preTwarmed#to# 37°C)#with#a#stir#bar.# 6. When#you#are#ready,#notify#your#instructor#and#your#lab#instructor#will#inoculate# your#flask#with#6#ml#of#E.#coli#that#is#in#“log#phase”#(exponential#growth#phase).## 7. Swirl#the#flask#to#make#an#even#suspension#of#bacteria.## a. Member$1:#pipette$5.0$ml#out$of$the$flask#into#a#cuvette#(this#will#be#your#0# time#point,#be#sure#to#record#the#time#(e.g.#t=0#at#8:55#am))#and#record#the# absorbance#at#600#nm#using#sterile#TSB#as#blank#(expected#Abs#~#0.1).## b. Member$2:$pipette$100$µ µ l$of$culture$from$the$flask$into$a$9.9$ml$tube$$ (10O2$dilution)#for#serial#dilution#and#plating#(t#=#0).#See#the#dilution#schedule# provided#in#Figure#5.# 8. Record#absorbance$readings$every$10$minutes#for#the#next#70#minutes#(i.e.#8$ readings).#

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# 9. At#any#time#point,#if#the#absorbance#reading#is#above#0.8,#perform#1:1#dilution#and# measure#the#absorbance.#You$need$to$multiply$the$absorbance$by$dilution$factor$ during$analysis$of$the$results.# 10. Perform#dilution#and#plating#every#10#minutes#for#the#next#50T60#minutes#(i.e.#6#time# points:#follow#Table#2).#For#the#first$2$time$points,#plate#3#dilutions#(from$10O4,$10O5,$ 10O6$tubes)#in#duplicates;#for#the#next$2$time$points,#plate#(from$10O4,$10O5,$10O6$ tubes)#in#single#plates;#for#the#last$2$time$points,#plate#3#dilutions#(from$10O5,$10O6,$ 10O7$tubes)#in#single#plates.# 11. Always#keep#the#inoculated#bottle/flask#covered#and#always#in#the#water#bath# (beaker)#at#37°C.# 12. If#it#is#necessary#to#hold$the$samples#(if#you’re#running#late#on#plating),#place#your# 10T2#dilution#tube#(first#dilution#tube)#on#ice.# 13. At#the#end#of#the#lab,#turn#the#petri#dishes#upside#down#to#prevent#condensation# from#falling#on#the#agar.#Stack#them#(6#per#stack),#tape#them,#label#them#and#place# plates#on#a#labelled#tray#to#be#stored#in#37°C#incubator.# 14. Monitors#will#measure#the#concentration$of$the$original$culture$by$McFarland$ Standard#as#well#as#by#absorbance$at$600$nm.# 15. Monitors#will#prepare#controls#and#incubate#them#at#37°C:# a. positive:#streak#E.coli#culture## b. negative:#sterile#TSB#plates# c. negative:#sterile#TSB#broth#in#a#tube# # #

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Dilution

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# Table$1:#Absorbance#readings#of#E.coli#grown#on#TSB#at#37°C#measured#at#600#nm# (submit$at$the$end$of$the$lab$–$1$copy$per$group)# $ Group$A$time$points$(min):#0,#10,#20,#30,#40,#50,#60,#70# Group$B$time$points$(min):#0,#5,#15,#25,#35,#45,#55,#65# Section:## Group:##

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# # Table$2:#Time#course#colony$counts#of#E.coli#grown#on#TSB#at#37°C,#plated#on#TSA#and# incubated#at#37°C#for#48#hours#(submit$at$the$end$of$the$lab$–$1$copy$per$group)# Group$A$time$points$(min):#0,#10,#20,#30,#40,#50# Group$B$time$points$(min):#0,#5,#15,#25,#35,#45# Section:## Group#(circle#one):## #

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WEEK$2$–$FollowOup$ # 1. Count#all#colonies#on#dilution#plates# 2. Calculate#the#CFU/ml#for#each#time#point.##Record#data#on#Group#Data#Sheet# 3. Construct#plots#of#CFU/ml#vs.#time#and#OD600#vs.#time#on#linear#graph# 4. Construct#plots#of#CFU/ml#vs.#time#on#semiOlog#graph#(on#Excel#as#well#as#by$hand)# 5. Calculate#the#Generation#time#(Gt)#of#your#culture#based#on#both#direct#(CFU/ml)# and#indirect#measurements#(Absorbance)# 6. Compare#all#the#graphs#and#understand#the#relationship#between#viable#count#and# optical#density#at#600nm.### # References:# 1. Blaber,#Michael.##1998.##Lecture#12#–#Growth#in#Bacterial#Populations.##BCH5425# Molecular#Biology#and#Biotechnology#Florida#State#University.# http://www.mikeblaber.org/oldwine/bch5425/lect12/lect12.htm# 2. Lindquist,#John#L.##2006.##Bacteriology#102#–#Exp.#5.4:#The#Growth#Curve#Experiment.# Department#of#Bacteriology#University#of#Wisconsin.## http://www.splammo.net/bact102/102gcurve.html# 3. Todar,#Kenneth.#2008.##The#Growth#of#Bacterial#Populations#(Chapter#4).##Todar’s# Online#Textbook#of#Bacteriology.##www.textbookofbacteriology.net/growth.htm# 4. Bowdoin#College,#New#England:# http://www.bowdoin.edu/biology/grants/spec/pdf/bacterialTgrowth.pdf# # # ## # #

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# Appendix$ # Growth$Rate$and$Generation$Time$ $ The#rate#of#exponential#growth#of#a#bacterial#culture,#under#standard#nutritional#conditions# (culture#medium,#temperature,#pH,#etc.),#is#expressed#as#generation#time#or#doubling#time#of# the#bacterial#population.##Generation$time#(Gt)#is#defined#as#the#time#interval#required# (time#(t))#for#the#cells#(or#population)#to#divide#(n=number#of#generations).### # Generation#times#for#bacteria#vary#from#about#12#min#to#24h#or#more#(Todar,#2008).##The# generation#time#for#E.coli#under#laboratory#conditions#is#15T20#min,#but#in#the# gastrointestinal#tract,#the#generation#time#is#estimated#to#be#12T24h#(Why#do#you#think#this# is?).##For#most#bacteria#that#can#be#cultured#in#a#laboratory,#generation#times#range#from# about#15#min#to#1h.##The#mean#generation#time#(or#doubling#time)#(Gt)#can#be#calculated# from#deriving#the#following#equation)1:## # Gt=Δt/n# # Where:# t=time#interval#in#hours#or#minutes# n#=#number#of#generations#(number#of#times#the#cell#population#doubles#during#the#time# interval)# # Bacteria#divide#by#binary#fission,#which#is#an#exponential#growth.##Therefore,#an#expression# of#growth#by#binary#fission#can#be#expressed#as#equation)2:# N=No2n## # The#number#of#generations#(n)#can#be#calculated#if#we#know#the#initial#population#(No)#and# the#population#(Nt)#after#time#(t),#from#equation)3.#Solving#for#n$(number$of$generations):# # log10N=log10NO#+#nlog102# n=logNTlogNo/log2# n=logNOlogNo/0.301# # Where:# No#=#the#number#of#cells#at#time#zero#(beginning#of#the#time#interval)# N=#the#number#of#cells#at#the#end#of#the#time#interval# # During#exponential#growth#phase,#the#rate#of#an#increase#of#cells#is#proportional#to#the# number#of#bacteria#present#at#that#time.##This#constant#of#proportionality#is#an#index#of# growth#rate#and#is#called#the#growth#rate#constant.##The#growth$rate$constant$(μ)#can#be# determined#from#the#following#equation,#equation)4:# # lnN#T#lnNO#=#μ(tTtO)# log10NTlog10NO#=#(μ/2.303)(tTtO)# μ$=$(log10NOlog10NO)$2.303/(tOtO)#

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# # The#exponential#growth$rate$(k)#for#a#specific#strain#under#specific#conditions#can#be# determined#using#equation)5:# logN=#logNo#+#ktlog2# k=logNOlogNo/Δtlog102# # You#can#obtain#the#number#of#generations/hour#by#solving#the#growth#rate#(k).##In#essence,# the#exponential#growth#rate#IS#the#number#of#generations#per#unit#time.##Therefore,#the# growth#rate#can#also#be#written#as#the#reciprocal#to#generation#time#(how#long#it#takes#for# each#generation):#Gt=1/k.#c.f.#equation#6#and#equation#1.# # To#calculate#growth$rate$(k)#from#a#graph,#you#can#measure#the#slope#of#the#exponential# phase#between#any#two#points#(log#Cfu/ml)#(Figure#3).##Two#points#on#the#straight#line# drawn#through#the#exponential#phase#(Cfu/ml)#must#be#chosen,#while#also#making#note#of# the#time#interval#between#them.#Keep#in#mind#that#the#values#you#are#to#use#must#be# converted#to#logarithms#for#the#formula.### # From#Figure#3#our#two#points#are:#Nt=1X1010#(at#5.75#hours)#and#NO=1X108#(at#2.75#hours).## The#time#interval#(in#hours)#between#the#2#points#=#t#=#3.##Using#equation#5,#we#can#calculate# the#growth#rate#(k),#which#is#the#number#of#generations#(doublings)#per#hour:# # k=log10NTlog10No/tlog102# k=10T8/(0.301)(3)=#2.21#gen/hr# # Generation#time,#which#is#the#time#it#takes#for#the#population#to#double,#can#then#be# obtained:# # Gt=1/k#=#1/2.21#=#0.45#hour/gen#=#27#min/gen# # When#we#graph#the#CFU/ml#and#absorbance#on#the#same#graph,#we#would#hope#to#see#an# upward#trend#for#both.##

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LAB$2$O$Write$up$and$Marking$Scheme$(attach$to$your$report)$ Total:$/30$ $ Name:_________________________________$$ #

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1. Present#your#data#in#properly$labeled$Tables# • Calculate#CFU/mL#at#each#dilution#and#present#that#in#your#Table#1–# show#a#sample#calculation# • Group#data#absorbance#(600nm)#over#time#–#Table1# • Group#data#of#colony#counts#over#time#(incl.#CFU/ml)#–#Table#2# # # 2. Plot#the#following#graphs:# a. CFU/ml$vs.$time$on$linear#graph#(in#Excel)# b. OD600$vs.$time#on#linear#graph#(in#Excel)# c. CFU/ml$vs.$time$on$semiOlog$graph#(in#Excel)# d. CFU/ml$vs.$time$on$semiOlog$graph#by$hand### • Include#Figure###and#Descriptive#title#at#the#bottom#of#the#figure# • Interpret$each$graph$in$2O3$sentence$below$the$graph$ # #

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# 3. Calculate#the#generation#time#(Gt)#of#your#cul...