creditspage videopage helppage "info" "scrollbarinfo" "hotwordfound" "menubarinfo" enterpage leavepage enterpage - 2 - - 1 - leavepage scrollbarinfo hotwordfound menubarinfo "info" buttonup buttonup - 2 - - 1 - How to use this program "info" buttonup buttonup - 3 - - 2 - Buttons and scroll bars "info" buttonup buttonup - 4 - - 3 - Push button "info" buttonup buttonup - 6 - - 5 - Checkbox "info" buttonup buttonup - 5 - - 4 - Rounded button "info" buttonup buttonup - 7 - - 6 - Radio button "info" buttonup buttonup - 9 - - 8 - Special buttons at the foot of the page backPage "info" buttonup buttonup - 12 - - 11 - NextPage "info" buttonup buttonup - 13 - - 12 - ExitProgram "info" buttonup buttonup - 10 - - 9 - FirstPage "info" buttonup buttonup - 11 - - 10 - Other featuress at the foot of the page Hotword Hotword "info" buttonup buttonup - 14 - - 13 - "info" buttonup buttonup - 15 - - 14 - Menubarem "info" buttonup buttonup - END - - 15 - Windows "info" buttonup buttonup - 8 - - 7 - Scroll bars - - - - - - - 1 - - - - - - - HOW TO USE THIS PROGRAM This page contains a number of terms and items that you may not be familiar with if you have not used a similar program before. Just click on one of the objects in the blue boxes on the right for further information... - - - - - - - 2 - - - - - - - BUTTONS AND SCROLL BARS Buttons, as their name suggests, are items that you can "push" by clicking on them using the left button on the mouse. Scroll bars help you move around text. - - - - - - - 3 - - - - - - - PUSH BUTTONS Push buttons, as their name suggests, can be "pushed" by clicking on the button using the left button on the mouse. The word on the push button will indicate what will happen when you press it. - - - - - - - 4 - - - - - - - ROUNDED BUTTONS Rounded buttons are identical in use to push buttons. To "push" the button, simply click on it. The word on the button will indicate what will happen when you press it. - - - - - - - 5 - - - - - - - CHECKBOXES Checkboxes are a special type of button. A cross in the box indicates that you have selected the option on the button label. Test this by clicking on the button a few times... - - - - - - - 6 - - - - - - - RADIO BUTTONS Radio buttons are a special type of button. A dot in the circle indicates that you have selected the option on the button label. Test this by clicking on the button a few times... - - - - - - - 7 - - - - - - - SCROLL BARS Scroll bars are found at the right hand side of some boxes containing text. Click on to move upwards and to move downwards through the text. To move more quickly, keep the mouse button pressed down. - - - - - - - 8 - - - - - - - SPECIAL BUTTONS AT THE FOOT OF THE PAGE These buttons are at the foot of every page in this program. They are used to navigate your way through the program or to exit it. Click on one of the buttons in the Special Buttons box for more information... - - - - - - - 9 - - - - - - - THE EXIT BUTTON This button is on the far left at the foot of each page. Click on it when you wish to end the program. As a precaution, you will be asked whether you really want to quit before the tutorial ends. - - - - - - 10 - - - - - - BUTTON This button is on the inside left at the foot of every page. Click on it to return to the Main menu. - - - - - - 11 - - - - - - BUTTON This button is on the inside right at the foot of every page. Click on it to move back to the previous page. - - - - - - 12 - - - - - - BUTTON This button is on the far right at the foot of every page. Click on it to move on to the next page. - - - - - - 13 - - - - - - HOTWORDS Hotwords are found buried in normal text and are either in a bold typeface or are highlighted by a box around the word. When you click on the hotword, you will get more information on that word. - - - - - - 14 - - - - - - THE MENUBAR The menubar is the bar along the top of the page just below the title. If you click on any of the words there, a drop-down menu will appear. Click a word or phrase on the drop-down part to choose an option e.g. to print your data or to get more help. - - - - - - 15 - - - - - - WINDOWS Windows is the system that this computer uses to run programs. Each program runs in a separate "window" with a border. - - - - - END - - - - -dows and using this computer, then select Help on the menubar. - - - - - END - - - - -ND - - - - --------- - - - - - - - - - - - - - - - - - - - ---- - - --- - - ---- - --- - ---- ----------- - - ---------- ------------------- --- ----- ------ ----- - - - - - ----------- - - - - - - - - - "scrollbarinfo" buttonup buttonup scrollbarinfo "hotwordfound" buttonup buttonup hotwordfound "menubarinfo" buttonup buttonup menubarinfo menubarinfo The menu bar is this bar along the top of the page. Click on OK to hide this box.. "menubarinfo" buttonup buttonup menubarinfo scrollbarinfo Click on this arrow to move upwards through the text. Keep the mouse button pressed down to move more quickly. Hold the mouse button down and drag the small box up and down the scroll bar to move up and down through the text. Click on this arrow to move downwards through the text. Keep the mouse pressed down to move more quickly. Click on OK to hide this box. scrollbarinfo buttonup buttonup scrollbarinfo hotwordfound Yes! You've found a hotword. Click on OK to hide this box. s box. "hotwordfound" buttonup buttonup hotwordfound aimspage You have three aims in this tutorial: l To gain an understanding of the different methods that are used to monitor the growth of bacteria. l To use a computer simulation of these methods to study the growth of a bacterium in batch culture. l To use the data obtained from these experiments to determine the effect of temperature on the growth rate and cell yield for this bacterium. The tutorial ends with six questions that you should be able to answer if you have completed the rest of the tutorial successfully. Have fun!!fun! successfully. Have fun! "info" buttonup buttonup - 2 - - 1 - Aims of this tutoriallm endbackground buttonup buttonup BackPage buttonUp buttonUp ExitProgram "This will the tutorial. Are you sure want quit?" f"Yes" "&No" SysSuspendMessages buttonUp buttonUp This will end the tutorial. Are you sure you want to quit? MainMenu "MainMenu" buttonUp buttonUp MainMenu Click on a grey button to choose an option...ft here to leave this page.... Bacterial growth NextPage buttonUp buttonUp creditspage CreditssaataauormationG BACTERIAL GROWTH: A Computer-based Simulation Exercise M. I. Tait, P. G. Newman, J. A. Hamnett, A. M. Rajnicek & J. I. Prosser Department of Molecular and Cell Biology, University of Aberdeen, Aberdeen AB9 1AS Mike Tait, Department of Molecular and Cell Biology, University of AberdeenOriginal concept: Jim Prosser Mike Tait, Department of Molecular and Cell Biology, University of Aberdeennn Programming: Mike Tait Time-lapse video: Ann Rajnicek Video capture: Greg Newman Original concept: Jim Prosser Version: 1.02 Date: 5th March 1993 analysepage8 Effect of temperatureeon curveon one of the icons in the blue Data box at the left side of the screen to select the data set you wish to view. Click on the Graph or Table icons in the blue View box on the right side of the screen to show the data in graphic or tabular form. Note that if you have not run the relevant experiments, no data will be available. Click on the Question icon to view the questions that are posed for each data set. There are six in all. Click on the Information icon to see this screen again.n. screen again.k on the Information icon to see this screen again... "runpage2" buttonup buttonup runpage2 runpage2 "screenA" "analysepage8" "screenK" "screenU" "screen2" "screen3" "screen4" "screen5" "screen6" "biomassscreen" buttonup buttonup screenA analysepage8 screenK screenU screen2 screen3 screen4 screen5 screen6 biomassscreen "screenA" "analysepage8" "screenK" "screenU" "screen2" "screen3" "screen4" "screen5" "screen6" "biomassscreen" buttonup buttonup screenA analysepage8 screenK screenU screen2 screen3 screen4 screen5 screen6 biomassscreen Time ln biomass [ln(mg/ml)] at temperature: (hours) 25 C 35 C 45 0 -3.00 -3.00 -3.00 1 -2.66 -3.00 -3.00 2 -2.04 -2.30 -3.00 3 -1.35 -1.11 -3.00 4 -0.62 0.05 -2.41 5 0.07 0.84 -1.43 6 0.76 0.85 -0.40 7 0.96 0.85 0.55 8 0.96 0.85 0.59 10 0.96 0.86 0.60 12 0.97 0.86 0.600000 screenA screenK screenU screen2 screen3 screen4 screen6 screen5 biomassscreen Cannot calculate biomass values until nephalometer calibration curve is available analysepage9 Effect of temperatureeon curveon one of the icons in the blue Data box at the left side of the screen to select the data set you wish to view. Click on the Graph or Table icons in the blue View box on the right side of the screen to show the data in graphic or tabular form. Note that if you have not run the relevant experiments, no data will be available. Click on the Question icon to view the questions that are posed for each data set. There are six in all. Click on the Information icon to see this screen again.n. screen again.k on the Information icon to see this screen again... "runpage2" buttonup buttonup runpage2 runpage2 "screenA" "analysepage9" "screenK" "screenU" "screen2" "screen3" "screen4" "screen5" "screen6" "biomassscreen" buttonup buttonup screenA analysepage9 screenK screenU screen2 screen3 screen4 screen5 screen6 biomassscreen "screenA" "analysepage9" "screenK" "screenU" "screen2" "screen3" "screen4" "screen5" "screen6" "biomassscreen" buttonup buttonup screenA analysepage9 screenK screenU screen2 screen3 screen4 screen5 screen6 biomassscreen screenK screenA screenU screen2 screen3 screen4 screen5 screen6 biomassscreen biomassscreen biomassscreen Cannot calculate biomass values until nephalometer calibration curve is availableeeeeee 0 2 4 6 8 10 12555444 biomass Time (hours))l))gding Cle cell numberse cell numberserssss EnterBook Reader c"File" c"Edit" c"Text" c"Page" c"Help" c"&Go e"&Title c"Go e"&Main c"Go c"Go e"&Aims" c"Go e"&Background information" c"Go e"&Calibrate nephelometer" c"Go e"&Run experiment" c"Go e"&Data analysis" c"Go e"Cr&edits" c"Go starting values 4entrance TitlePage "startpage" MainMenu "mainmenu" "helppage" "aimspage" BackgroundInformation "infopage" CalibrateNephelometer 4seencalibinfo "calibratepage" RunExperiment 4runpage " & DataAnalysis 4analysepage Credits "creditspage" LeaveBook FALSE "tbkdlg.dll" "tbkwin. "kernel" "user" BackgroundInformation EnterBook CalibrateNephelometer MainMenu RunExperiment DataAnalysis Credits TitlePage LeaveBook EnterBook sizetopage &Go to &Title page Go to &Main menu Go to &Help Go to &Aims Go to &Background information Go to &Calibrate nephelometer Go to &Run experiment Go to &Data analysis Go to Cr&edits Go to entrance TitlePage startpage MainMenu mainmenu helppage aimspage BackgroundInformation infopage CalibrateNephelometer calibratepage calibratepage seencalibinfo RunExperiment runpage runpage DataAnalysis analysepage analysepage Credits creditspage LeaveBook tbkdlg.dll tbkwin.dll kernel 4duration,runstage,runpage,temperature, Zn,m,culturecolor "runpage4" "You need prepare sterilise your medium f"OK" ?have just inoculated ehear a whimper turn dlaboratory supervisor behind k&"He says nothing." f"Whistle nonchalantly" "timeflies" /20) ,n,100 (88-n)*20 "timer" "Your cultures turned a strange colour."&CRLF&" realise that they contaminated because you forgot flasks."& mutters something about giving up research ever wanders f"Blush" "datacollected" "screen" & (n+48) "analysepage5" "analysepage6" "analysepage8" "analysepage9" "screenK" buttonup buttonup runpage4 runpage4 You need to prepare and sterilise your medium first. You have just inoculated an old culture. You hear a whimper and turn to see your laboratory supervisor behind you. He says nothing. Whistle nonchalantly inoculated timeflies culture timer timeflies Your cultures have turned a strange colour. You realise that they are all contaminated because you forgot to sterilise your flasks. Your laboratory supervisor mutters something about giving up research for ever and wanders out of the laboratory. Blush datacollected screen analysepage5 analysepage6 analysepage8 analysepage9 screen analysepage8 analysepage9 screenK analysepage5 screenK analysepage6 n:by1 n:to1 culturecolor runstage runpage temperature duration analysepage4 calibratepage calibratepage nephdatacollected Nephelometer data collected tube11 flask1 E E c bung1 reading 4seencalibinfo buttonup buttonup seencalibinfo biomass Biomass 26 mg/ml dilution Dilution 0.2 dilutionsbiomass 5.2 4.7 4.2 3.6 3.1 2.6 2.1 1.6 1.0 0.5 0 Biomass in each test tube (mg/ml) tube9 tube2 tube1 tube8 tube7 tube6 tube4 tube3 tube5 reset 4calibstage,biomassstage "calibratepage" "dilution" "dilutionsbiomass" "datacollected" "nephdatacollected" reading buttonup buttonup biomass calibratepage dilution dilutionsbiomass dilutions datacollected nephdatacollected reading calibstage biomassstage reset dilutions tube0 4biomassstage,calibstage "dilutionsbiomass" "datacollected" --reveal on analysepages "biomassscreen" "analysepage2" "analysepage3" "analysepage5" "analysepage6" "analysepage8" "analysepage9" buttonup buttonup biomass dilutionsbiomass datacollected biomassscreen analysepage2 analysepage3 analysepage5 analysepage6 analysepage8 analysepage9 biomassstage calibstage Determine biomass 4calibstage "dilution" buttonup buttonup dilution calibstage Dilute cell suspension Take readings Make sub-dilutions datacollected All data collecteddto Anal tube: tubescreen timeflies Time flies... "reading" buttonup buttonup reading Calibrating the nephelometer When you use a nephelometer to monitor the growth of a culture, it will give you only relative values. If you want to convert these values into, for example, biomass values, then you must first calibrate your nephelometer using a suspension of cells containing a known biomass. By diluting the original cell suspension appropriately and then making a series of sub-dilutions from this, you can determine the nephelometer readings at different biomass concentrations. This is simulated for you on this page. Click on the information symbol to see this box again. Click on OK to hide this box. "info" buttonup buttonup analysepage4 1. What was the highest nephelometer reading that could be used to estimate the biomass concentration in the tube inserted into the nephelometer? 2. How could you adapt the method used so that you could determine the biomass values in cultures with high cell concentrations? determine the biomass values in cultures with high cell concentrations???4. What is the mass of a single E. coli cell? Click on each question for a hint.a hint.... Questions on calibration dataeon one of the icons in the blue Data box at the left side of the screen to select the data set you wish to view. Click on the Graph or Table icons in the blue View box on the right side of the screen to show the data in graphic or tabular form. Note that if you have not run the relevant experiments, no data will be available. Click on the Question icon to view the questions that are posed for each data set. There are six in all. Click on the Information icon to see this screen again.n. screen again.k on the Information icon to see this screen again... analysepage7 3. How and why would the values for the total and viable cell numbers change if you incubated the cultures longer? 4. For what practical reasons do you think that more values have been obtained for biomass than for viable counts?an for viable counts?ore values have been obtained for biomass than for viable counts?n for biomass/nephalometer readings?t.int. Click on each question for a hint.l? Click on each question for a hint.a hint.... Questions on growth curve dataon one of the icons in the blue Data box at the left side of the screen to select the data set you wish to view. Click on the Graph or Table icons in the blue View box on the right side of the screen to show the data in graphic or tabular form. Note that if you have not run the relevant experiments, no data will be available. Click on the Question icon to view the questions that are posed for each data set. There are six in all. Click on the Information icon to see this screen again.n. screen again.k on the Information icon to see this screen again... analysepage: 5. At which temperature was the cell yield (Y) greatest? (Assume that all of the glucose in the medium was utilised by the culture at each temperature). 6. At which temperature was the specific growth rate (m) greatest??? occur?tilised at each temperature, what can you say about the growth yield (cell biomass produced/amount of substrate consumed) 4. What is the mass of a single E. coli cell? Click on each question for a hint.a hint.... Questions on temperature dataeon one of the icons in the blue Data box at the left side of the screen to select the data set you wish to view. Click on the Graph or Table icons in the blue View box on the right side of the screen to show the data in graphic or tabular form. Note that if you have not run the relevant experiments, no data will be available. Click on the Question icon to view the questions that are posed for each data set. There are six in all. Click on the Information icon to see this screen again.n. screen again.k on the Information icon to see this screen again... "runpage2" buttonup buttonup runpage2 runpage2 4calibstage,biomassstage "You will need dilute the original cell suspension 4you asub-dilutions."& k&"If x, your ed suspensions ebe so dense that fnephelometer readings be offscale." f"Cancel" "dilutionsbiomass" prepare laboratory supervisor points out have fact already done k&"He does politely, but sense be watching carefully future." overhear asome sarcastic comment technician about planet k&"With embarassment, realise taken necessary gs." f"Beam me up, Scotty" buttonup buttonup You will need to dilute the original cell suspension before you make sub-dilutions. If not, your sub-diluted suspensions will be so dense that the nephelometer readings will be offscale. Cancel dilutions dilutionsbiomass As you start to prepare your dilutions, your laboratory supervisor points out that you have in fact already done this. He does this politely, but you sense that he will be watching you carefully in future. Cancel You overhear your laboratory supervisor make some sarcastic comment to the lab technician about your planet of origin. With embarassment, you realise that you have already taken the necessary nephelometer readings. Beam me up, Scotty calibstage biomassstage MainPoints nephelometry runpage3 Setting the duration The length of time you run an experiment when growing a bacterium in batch culture depends on what questions you wish to answer. If you want to know the cell yield, then the culture must be allowed to reach stationary phase. If you want to know how the total and viable cell counts differ in death phase, then the culture must obviously be allowed to reach this phase. Click on your required duration in the box.or click on OK to hide this box......................de this box.box.re-run it for a longer period. Use the up and down arrows to set the duration you require. 4duration selectedtextlines *120)+120 buttonup buttonup duration duration 4 hours 6 hours 8 hours 10 hours 12 hours 14 hours "durationinfo" buttonup buttonup durationinfo analysepage5 Time Nephelometer ln biomass ln total cells ln viable cells (hours) readings ln(mg/ml) ln(nos.x 10 ) ln(nos.x 10 ) 0 1.5 -3.00 - -1.83 1 1.5 -3.00 - - 2 3.0 -2.30 -0.22 -1.20 3 10.2 -1.11 0.47 -0.60 4 32.3 0.05 2.23 1.10 5 71.7 0.84 2.81 1.74 6 72.1 0.85 3.14 2.08 7 72.2 0.85 - - 8 72.3 0.85 3.06 2.07 10 73.1 0.86 3.23 1.97 12 73.2 0.86 3.17 2.08 Growth curve data at 35 Ccurveon one of the icons in the blue Data box at the left side of the screen to select the data set you wish to view. Click on the Graph or Table icons in the blue View box on the right side of the screen to show the data in graphic or tabular form. Note that if you have not run the relevant experiments, no data will be available. Click on the Question icon to view the questions that are posed for each data set. There are six in all. Click on the Information icon to see this screen again.n. screen again.k on the Information icon to see this screen again... "biomassscreen" "analysepage5" "screen2" "screen3" "screen4" "screen5" "screen6" "screenK" buttonup buttonup biomassscreen analysepage5 screen2 screen3 screen4 screen5 screen6 screenK "biomassscreen" "analysepage5" "screen2" "screen3" "screen4" "screen5" "screen6" "screenK" buttonup buttonup biomassscreen analysepage5 screen2 screen3 screen4 screen5 screen6 screenK screen2 screen3 screen4 screen5 screen6 screenK biomassscreen Cannot calculate biomass values until nephalo-meter calibration curve is prepared infobackground buttonup buttonup TOPICS Click on one of the phrases below for further information on that topic. Bacterial cell replication The batch growth cycle Measurement of growth 1. Viable cell counts 2. Total cell counts 3. Biomass determination 4. Nephelometry Bacterial growth Background informationnnrmationinformation BackPage buttonUp buttonUp NextPage buttonUp buttonUp ExitProgram "This will the tutorial. Are you sure want quit?" f"Yes" "&No" SysSuspendMessages buttonUp buttonUp This will end the tutorial. Are you sure you want to quit? MainMenu "MainMenu" buttonUp buttonUp MainMenu Click on one of the buttons on the right or left here to leave this page... selectedtextlines 4videoin "infopage" "growthcycle" "measurement" "viablecell" "totalcell" "biomass" "nephelometry" buttonup buttonup infopage growthcycle measurement viablecell totalcell biomass nephelometry videoin Bacterial cell replication The batch growth cycle Measurement of growth 1. Viable cell counts 2. Total cell counts 3. Biomass determination 4. Nephelometry infopage "video" 5800,4050 4videoin "videoinfo" enterpage leavepage enterpage video video videoin leavepage videoinfo Bacteria grow by increasing in length. When a certain cell size is reached, DNA replication begins. Once this is complete, a septum is formed, splitting the cell into two equal halves...al halves.qual halves.er to see a time-lapse video of bacteria growing....ing......rder to see a time-lapse video of bacteria growing... cover 4videoin Zvertpos 4050 3550 5800, "searching" "videopage" buttonup buttonup video video video searching videopage vertpos:by vertpos:to vertpos videoin 4videoin Zvertpos 4050 3550 5800, "searching" "videopage" buttonup buttonup video video video searching videopage vertpos:by vertpos:to vertpos videoin replication Bacterial cell replication Click on one of the buttons on the video recorder to see a time-lapse video of bacteria growing... "videoinfo" buttonup buttonup videoinfo searching Searching tape... videoinfo The tape shows Enterobacter cloaceae cells growing on a surface. In real time, it lasts 2 hours 15 minutes.In real time, it lasts 2 hours 15 minutes "videoinfo" buttonup buttonup videoinfo video Zvertpos "video" 4050 3550 5800, videoin buttonUp buttonUp video video video videoin vertpos:by vertpos:to vertpos runpage3 analysepage1 analysepage10 biomass replication Biomass determination The biomass of a culture can be determined by removing the cells from the medium (e.g. by centrifugation) and then determining the dry weight. This method is relatively insensitive for small volumes of culture as it is difficult to weigh such low biomasses accurately..... Centrifuge culturespension pellet hhe Weigh pellety and reweigh nephelometry "lid" "tube:" 3862,2500 leavepage leavepage tube: replication Nephelometry cycle tube: reading "lid" "tube:" 3862,2500 "reading" buttonup buttonup tube: reading Light source Cross-section of test tube containing cell suspension Detectorreee "lid" "reading" "tube:" 3862,2500 2500 3200 3862,n buttonUp buttonUp reading tube: tube: reading Take reading In the nephelometer, a detector reads the amount of light scattered by a cell suspension. If the culture is very dense, this must first be diluted to a concentration that is on-scale..eading on the nephelometer scale. Reader & Author MainMenu Page Design growthcycle analysepage6 biomassdata biomassscreen Growth curve at 35 Ction curveon one of the icons in the blue Data box at the left side of the screen to select the data set you wish to view. Click on the Graph or Table icons in the blue View box on the right side of the screen to show the data in graphic or tabular form. Note that if you have not run the relevant experiments, no data will be available. Click on the Question icon to view the questions that are posed for each data set. There are six in all. Click on the Information icon to see this screen again.n. screen again.k on the Information icon to see this screen again... cellnosdata screen2 screen3 screen4 screen5 screen6 screenK 0 2 4 6 8 10 12555444 biomass Time (hours))l))gding nos..rs)))) x 10 ml) Biomass Total cell numbers Viable cell numberssss "screen2" "analysepage6" "screen3" "screen4" "screen5" "screen6" "biomassscreen" "screenK" buttonup buttonup screen2 analysepage6 screen3 screen4 screen5 screen6 biomassscreen screenK "screen2" "analysepage6" "screen3" "screen4" "screen5" "screen6" "biomassscreen" "screenK" buttonup buttonup screen2 analysepage6 screen3 screen4 screen5 screen6 biomassscreen screenK analysebackground measurement analysepage2 analysepage6 analysepage: infopage viablecell analysepage7 analysepage9 calibrationpage biomass videopage "searching" "infopage" 4videoin frame " & n n > 1 a" & n-1 "videoinfo" "video14" enterpage leavepage enterpage searching infopage video video frame videoin leavepage videoinfo video14 videoin I:N:NT replication Bacterial cell replication cover Click on one of the buttons on the video recorder to see a time-lapse video of bacteria growing... "videoinfo" ouseenter mouseleave mouseenter buttonup mouseenter mouseleave buttonup videoinfo BackPage "aimspage" buttonUp buttonUp aimspage NextPage "growthcycle" buttonUp buttonUp growthcycle videoinfo The tape shows Enterobacter cloaceae cells growing on a surface. In real time, it lasts 2 hours 15 minutes.In real time, it lasts 2 hours 15 minutes "videoinfo" buttonup buttonup videoinfo frame "video" & n n > 1 buttonup buttonup video video frame frame "video" & n n > 1 buttonup buttonup video video frame Bacteria grow by increasing in length. When a certain cell size is reached, DNA replication begins. Once this is complete, a septum is formed, splitting the cell into two equal halves...al halves.qual halves.er to see a time-lapse video of bacteria growing....ing......rder to see a time-lapse video of bacteria growing... video1 :PHYSSIZE video2 :PHYSSIZE video3 :PHYSSIZE video4 h%:PHYSSIZE video5 ):PHYSSIZE video6 x.:PHYSSIZE video7 3:PHYSSIZE video8 7:PHYSSIZE video9 <:PHYSSIZE video10 @:PHYSSIZE video11 E:PHYSSIZE video12 I:PHYSSIZE video13 0N:PHYSSIZE video14 R:PHYSSIZE 4calibstage,biomassstage "You will need prepare dilutions the cell suspension 4you can take readings" f"Cancel" "Your laboratory supervisor points out tolerantly that ahave only one test tube containing diluted culture really give a calibration regret having those extra pints Jnight." f"Consider going teetotal" ahead "timeflies" "tubescreen" (2447+n*453),1065 "tube11" (11-n)*16 --reveal data on analysepages "biomassscreen" "analysepage2" "analysepage3" "analysepage5" "analysepage6" "analysepage8" "analysepage9" "datacollected" "nephdatacollected" approach nephelometer, realise already taken necessary k&"Besides, someone ] now."& k&"This person 6 foot tall wearing a karate suit." f"Sidle off" buttonup buttonup You will need to prepare dilutions from the cell suspension before you can take any readings Cancel Your laboratory supervisor points out tolerantly that you have only one test tube containing diluted culture and that you really need to prepare sub-dilutions to give you a calibration curve. You regret having those extra pints last night. Consider going teetotal timeflies tubescreen tubescreen tube11 reading reading reading reading reading tube11 tubescreen biomassscreen analysepage2 analysepage3 analysepage5 analysepage6 analysepage8 analysepage9 datacollected nephdatacollected timeflies As you approach the nephelometer, you realise that you have already taken the necessary readings. Besides, someone else is using the nephelometer now. This person is 6 foot tall and wearing a karate suit. Sidle off n:to1 calibstage biomassstage calibratebackground endbackground runpage1 "iconinfo" leavepage leavepage iconinfo Running your experiment Click on one of the icons in the blue Settings box at the left side of the screen to set the temperature and/or duration of each experiment. When you are ready to run the experiment, click on the three icons in the blue Run box on the right side of the screen to prepare and sterilise your growth medium and then to inoculate each flask. To give you reliable results, each experiment will be performed in triplicate. To read this information again, click on the icon in the Info box on the left hand side of the screen.n. "iconinfo" buttonup buttonup iconinfo iconinfo ICONS Icons are these symbols in the blue boxes at either side of the screen. Click on OK to hide this box.uration of each experiment. When you are ready to run the experiment, click on the three icons in the blue box on the right side of the screen to prepare and sterilise your growth medium and then to inoculate each flask. To give you reliable results, each experiment will be preformed in triplicate. Click on OK to hide this box..to hide this box... "iconinfo" buttonup buttonup iconinfo runbackground growthcycle 4phase, newphase U"lagline" 120,50,100 m"acccurve" 120,50,100 U"logline" 120,50,100 m"deccurve" 120,50,100 U"statline" 120,50,100 m"deathcurve" 120,50,100 "gcinfo" & enterpage leavepage enterpage phase newphase leavepage lagline acccurve logline deccurve statline deathcurve gcinfo phase gcinfo0 When growth medium is inoculated with bacteria, the cell population normally grows in a manner similar to that shown in this graph. Click at different points on the graph for more information... gcinfo3 3. LOG (EXPONENTIAL) PHASE The cells grow at their maximum growth rate. The gradient of this line is the specific growth rate. This varies with cell type and growth medium. gcinfo2 2. ACCELERATION PHASE Each cell, once adjusted to the growth medium, starts to grow. As more and more cells start dividing, the growth rate of the culture increases. gcinfo1 1. LAG PHASE The cells become adjusted to their new environment. If the inoculum contains cells growing rapidly under the same conditions, there may be no lag phase. om a simil gcinfo5 5. STATIONARY PHASE The cells cease growing when their environment changes, usually when a nutrient is exhausted. They are, however, still metabolically active. lag phase. graphpaper :*a"* 4phase, newphase U"lagline" 120,50,100 m"acccurve" 120,50,100 U"logline" 120,50,100 m"deccurve" 120,50,100 U"statline" 120,50,100 m"deathcurve" 120,50,100 "gcinfo" & buttonup buttonup lagline acccurve logline deccurve statline deathcurve gcinfo gcinfo phase newphase replication 4newphase "graphpaper" buttonup buttonup buttonup graphpaper newphase The batch growth cycle logline 4newphase 0,50,100 "graphpaper" buttonup buttonup buttonup graphpaper newphase statline 4newphase 0,50,100 "graphpaper" buttonup buttonup buttonup graphpaper newphase 4newphase "graphpaper" buttonup buttonup buttonup graphpaper newphase ln cell numbersss 4newphase "graphpaper" buttonup buttonup buttonup graphpaper newphase acccurve 4newphase 0,50,100 "graphpaper" buttonup buttonup buttonup graphpaper newphase deathcurve 4newphase 0,50,100 "graphpaper" buttonup buttonup buttonup graphpaper newphase deccurve 4newphase 0,50,100 "graphpaper" buttonup buttonup buttonup graphpaper newphase lagline 4newphase 0,50,100 "graphpaper" buttonup buttonup buttonup graphpaper newphase gcinfo4 4. DECELERATION PHASE Increasing numbers of cells stop growing. Synthesis of secondary metabolites such as antibiotics normally starts at this time. his time. time. hase. gcinfo6 6. DEATH PHASE The number of viable cells decreases and some cells lyse. Others can utilise the nutrients released by lysed cells in a process called cryptic growth. led cryptic growth. gcinfo7 SEMILOGARITHMIC PLOT Bacterial growth data are usually plotted using a semilogarithmic plot i.e. a plot of the natural log (ln) of cell numbers (or biomass) against time. info8 THE GROWTH YIELD The growth yield (Y) is the biomass of cells formed divided by the amount of substrate (e.g. glucose) utilised during growth. biomass) against time. 4newphase "graphpaper" buttonup buttonup buttonup graphpaper newphase 4newphase "graphpaper" buttonup buttonup buttonup graphpaper newphase gcinfo8 THE SPECIFIC GROWTH RATE The specific growth rate (m) of a bacterial culture is the gradient of the line during log phase. It varies with cell type and growth conditions. gcinfo9 aimspage titlebackground starting values 4entrance 4temperature,duration,runstage,runpage,analysepage 4data,view,seencalibinfo,biomassstage,calibstage /"runbackground" "6 hours" ackground enterbackground buttonup enterbackground runbackground temperature duration 6 hours seencalibinfo biomassstage calibstage temperature duration runstage runpage analysepage entrance buttonup runpage2 startpage Zhorizpos -2100 5400 "runner" .,2340 5400 8400 j,2340 enterpage leavepage enterpage runner horizpos:by horizpos:to horizpos leavepage runner horizpos:by horizpos:to horizpos MCBcoursewaree courseware MCBBBBBBeware buttonUp buttonUp Start tutorial Department of Molecular and Cell Biology University of Aberdeennnnnnnn $ k+ runner '$ k+ 3 Y$ "3 Y$ Zhorizpos 5400 8400 "runner" -,2340 -2100 5400 [,2340 buttonUp buttonUp runner runner horizpos:by horizpos:to horizpos swim! Bacterial growth Use the mouse to move the arrow and then click on the grey button to start the tutorial... startpage runpage2 settemperature Setting the temperature The aim of the series of experiments you have been asked to run is to determine the effect of growth temperature on the specific growth rate and cell yield of E. coli cells. Your laboratory supervisor has asked that you investigate the following three temperatures. Click on the required temperature in the box. ture. Click on OK to hide this box.he temperatureicon. Click on OK to hide this box.ter. member that your aim is to determine the effect of temperature on the specific growth rate and the cell yield......... "temperatureinfo" buttonup buttonup temperatureinfo 4temperature selectedtextlines *10)+15 buttonup buttonup temperature temperature 10 hours 12 hoursrsrsrs 14 hours analysepage3 calibrate runpage4 Time drifts sleepily by... shaker flask1 E E c flask2 E E c R * O flask3 E E c culture bungs bung1 timer "timer" "000 buttonup buttonup timer 000 min timer 000 min99999996592 Smiffs timeflies Time flies... datacollected Data collected. measurement "viableinfo" leavepage leavepage viableinfo Growth of bacterial cultures can be measured in four main ways: 1. By counting the number of viable cells present. 2. By counting the total number of cells present. 3. By measuring the biomass. 4. By measuring the cell density using a nephelometer or a spectrophotometer. The last method is the one that is most frequently used as all of the other methods are time-consuming..g. "viableinfo" buttonup buttonup viableinfo replication Measurement of growth viableinfo Viable cells Viable cells, by definition, are cells that are capable of growing when transferred to a fresh growth medium. Although a viable cell is clearly a living cell, a non-viable cell is not necessarily dead. This is because a cell that cannot grow on the growth medium used for the viablity test may still be capable of growing on another medium. the one used for the viability test. "viableinfo" buttonup buttonup viableinfo runpage4 analysepage3 nephgraph Line fitted by regression of first six data points. biomassscreen Biomass (mg/ml))gding Nephelo-meter Reading 0 1 2 3 4 5444 "biomassscreen" "analysepage3" buttonup buttonup biomassscreen analysepage3 "biomassscreen" "analysepage3" buttonup buttonup biomassscreen analysepage3 Nephelometer calibration curveon one of the icons in the blue Data box at the left side of the screen to select the data set you wish to view. Click on the Graph or Table icons in the blue View box on the right side of the screen to show the data in graphic or tabular form. Note that if you have not run the relevant experiments, no data will be available. Click on the Question icon to view the questions that are posed for each data set. There are six in all. Click on the Information icon to see this screen again.n. screen again.k on the Information icon to see this screen again... System Sans Serif Arial se Study One rminal urier New System Arial Arial Arial Arial Arial Arial Arial Symbol System Times New Roman Wingdings Times New Roman ngdings nAJpp sans serif sans serif Arial c"File" c"Help" Me" Main Menu" 2 Me" HelpMainMenu X"user" --bringWindowToTop a Windows function that puts the + whose passed front %other windows. toolbook MfAppName bringwindowtotop( doesn't even un-iconize SizeToPage" app isn't running, so we'll "casehelp.tbk" LeaveBook terBook HelpMainMenu EnterBook LeaveBook EnterBook sizetopage Help Me Help Main Menu Help Me HelpMainMenu bringWindowToTop sysWindowHandle bringwindowtotop send SizeToPage casehelp.tbk fAppName toolbook LeaveBook mes New Roman Arial MCB Bacterial growth Arial System > ngdings Times New Roman Times New Roman ciences Symbol System ngdings all Fonts ngdings mes New Roman System Wingdings Wingdings :PRINTLAYOUT gD|D|} s Rmn gD|D|} Arial Arial mes New Roman mes New Roman Times New Roman Wingdings ngdings Arial + whose passed front %other windows. toolbook MfAppName bringwindowtotop( doesn't even un-iconize SizeToPage" app isn't running, so we'll \work\utl\bmshelp.tbk" LeaveBook FALSE "tbkdlg.dll" "tbkwin. "kernel" terBook HelpMainMenu EnterBook LeaveBook EnterBook sizetopage &Help Me Help Main Menu Help Me HelpMainMenu bringWindowToTop sysWindowHandle bringwindowtotop send SizeToPage \toolbook\work\utl\bmshelp.tbk fAppName toolbook LeaveBook tbkdlg.dll tbkwin.dll kernel System Arial mes New Roman Arial Arial Wingdings HelpMainMenu X"user" --bringWindowToTop a Windows function that puts the + whose passed front %other windows. toolbook MfAppName bringwindowtotop( doesn't even un-iconize SizeToPage" app isn't running, so we'll \work\utl\bmshelp.tbk" LeaveBook FALSE "tbkdlg.dll" "tbkwin. "kernel" terBook HelpMainMenu EnterBook LeaveBook EnterBook sizetopage &Help Me Help Main Menu Help Me HelpMainMenu bringWindowToTop sysWindowHandle bringwindowtotop send SizeToPage \toolbook\work\utl\bmshelp.tbk fAppName toolbook LeaveBook tbkdlg.dll tbkwin.dll kernel Arial EnterBook Reader c"File" c"Edit" c"Text" c"Page" c"Help" starting values 4entrance LeaveBook FALSE "tbkdlg.dll" "tbkwin. "kernel" "user" EnterBook LeaveBook EnterBook sizetopage entrance LeaveBook tbkdlg.dll tbkwin.dll kernel Times New Roman Times New Roman Wingdings EnterBook Reader c"File" c"Edit" c"Text" c"Page" c"Help" c"&Go e"&Title c"Go e"&Main c"Go c"Go e"&Aims" c"Go e"&Background information" c"Go e"&Calibrate nephalometer" c"Go e"&Run experiment" c"Go e"&Data analysis" c"Go e"C&redits" c"Go starting values 4entrance TitlePage "startpage" LeaveBook FALSE "tbkdlg.dll" "tbkwin. "kernel" "user" terBook TitlePage EnterBook LeaveBook EnterBook sizetopage &Go to &Title page Go to &Main menu Go to &Help Go to &Aims Go to &Background information Go to &Calibrate nephalometer Go to &Run experiment Go to &Data analysis Go to C&redits Go to entrance TitlePage startpage LeaveBook tbkdlg.dll tbkwin.dll kernel mainbackground buttonup buttonup BackPage buttonUp buttonUp NextPage buttonUp buttonUp ExitProgram "This will the tutorial. Are you sure want quit?" f"Yes" "&No" SysSuspendMessages buttonUp buttonUp This will end the tutorial. Are you sure you want to quit? MainMenu "MainMenu" buttonUp buttonUp MainMenu Click on a grey button to choose an option...ft here to leave this page.... Bacterial growth runpage1 analysepage5 infobackground titlebackground analysebackground buttonup buttonup BackPage buttonUp buttonUp NextPage buttonUp buttonUp ExitProgram "This will the tutorial. Are you sure want quit?" f"Yes" "&No" SysSuspendMessages buttonUp buttonUp This will end the tutorial. Are you sure you want to quit? MainMenu "MainMenu" buttonUp buttonUp MainMenu Click on a grey button to choose an option...ft here to leave this page.... Bacterial growth Analyse dataauormation Calibration Growth curve Temperature Dataasss Graph Table Question Informationnn Table Question Information Informationnn Question Information x"analysepage1" buttonup buttonup analysepage1 analysepage1 analysepage showdata 4data, view, analysepage buttonup buttonup analysepage analysepage 4data "showdata" buttonup buttonup buttonup showdata 4data "showdata" buttonup buttonup buttonup showdata 4data "showdata" buttonup buttonup buttonup showdata 4view,analysepage,data "showdata" buttonup buttonup buttonup showdata analysepage 4view,analysepage,data "showdata" buttonup buttonup buttonup showdata analysepage 4view,analysepage,data "showdata" buttonup buttonup buttonup showdata analysepage loc, isshift, isctrl rightbuttondoubleclick rightbuttondoubleclick isctrl isshift runbackground buttonup buttonup BackPage buttonUp buttonUp NextPage buttonUp buttonUp ExitProgram "This will the tutorial. Are you sure want quit?" f"Yes" "&No" SysSuspendMessages buttonUp buttonUp This will end the tutorial. Are you sure you want to quit? MainMenu "MainMenu" buttonUp buttonUp MainMenu Click on a grey button to choose an option...ft here to leave this page.... Bacterial growth Temperature Duration Informationn Settings temperature duration 6 hourssurs "runpage2" buttonup buttonup runpage2 runpage2 runpage "runpage3" buttonup buttonup runpage3 runpage3 runpage "runpage4" buttonup buttonup runpage4 runpage4 runpage Prepare Sterilise Inoculateeatelate "runpage4" "prepared" "sterilised" "inoculated" "datacollected" timer "000 "bungs" "culture" 60,95,100 4runstage buttonup buttonup runpage4 runpage4 prepared sterilised inoculated datacollected 000 min bungs culture culture culture prepared runstage timer runpage "runpage4" 4runstage "You have just sterilised old culture."& k&"Are you sure 5gained anything k&"Choose one the following options." f"No" ymedium that already sterile."& k&"Your laboratory supervisor frowns but says nothing." f"Cancel" "bungs" 60,90,100 must prepare your f"OK" buttonup buttonup runpage4 runpage4 You have just sterilised an old culture. Are you sure you have gained anything from this? Choose one of the following options. You have just sterilised medium that is already sterile. Your laboratory supervisor frowns but says nothing. Cancel bungs culture sterilised You must prepare your medium first. runstage runpage prepared inoculated sterilised Run experimenttumationQ Information x"runpage1" buttonup buttonup runpage1 runpage1 runpage MainBackground MenuBackground analysepage1 Click on one of the icons in the blue Data box at the left side of the screen to select the data set you wish to view. Click on the Table or Graph icons in the blue View box on the right side of the screen to show the data in tabular or graphic form. Note that if you have not run the relevant experiments, no data will be available. Click on the Question icon to view the questions that are posed for each data set. There are six in all. Click on the Information icon to see this screen again... this screen again. screen again.k on the Information icon to see this screen again... "iconinfo" buttonup buttonup iconinfo iconinfo ICONS Icons are these symbols in the blue boxes at either side of the screen. Click on OK to hide this box.uration of each experiment. When you are ready to run the experiment, click on the three icons in the blue box on the right side of the screen to prepare and sterilise your growth medium and then to inoculate each flask. To give you reliable results, each experiment will be preformed in triplicate. Click on OK to hide this box..to hide this box... "iconinfo" buttonup buttonup iconinfo Analysing your dataation dataeon one of the icons in the blue Data box at the left side of the screen to select the data set you wish to view. Click on the Graph or Table icons in the blue View box on the right side of the screen to show the data in graphic or tabular form. Note that if you have not run the relevant experiments, no data will be available. Click on the Question icon to view the questions that are posed for each data set. There are six in all. Click on the Information icon to see this screen again.n. screen again.k on the Information icon to see this screen again... analysepage2 Nephelometer calibration dataeon one of the icons in the blue Data box at the left side of the screen to select the data set you wish to view. Click on the Graph or Table icons in the blue View box on the right side of the screen to show the data in graphic or tabular form. Note that if you have not run the relevant experiments, no data will be available. Click on the Question icon to view the questions that are posed for each data set. There are six in all. Click on the Information icon to see this screen again.n. screen again.k on the Information icon to see this screen again... Biomass concentration in Nephelometer nephelometer (mg/ml) reading 0 0 0.52 16 1.04 32 1.56 48 2.08 64 2.60 80 3.12 89 3.64 97 4.16 offscale 5.2 offscale "biomassscreen" "analysepage2" buttonup buttonup biomassscreen analysepage2 "biomassscreen" "analysepage2" buttonup buttonup biomassscreen analysepage2 biomassscreen totalcell helppage analysepage8 calibratebackground buttonup buttonup BackPage buttonUp buttonUp NextPage buttonUp buttonUp ExitProgram "This will the tutorial. Are you sure want quit?" f"Yes" "&No" SysSuspendMessages buttonUp buttonUp This will end the tutorial. Are you sure you want to quit? MainMenu "MainMenu" buttonUp buttonUp MainMenu Bacterial growth loc, isshift, isctrl B"reset" rightbuttondoubleclick rightbuttondoubleclick reset reset isctrl isshift Click on a grey button to choose an option...ft here to leave this page.... "planinfo" buttonup buttonup planinfo Calibrate nephelometerrt MainMenu Analyse data Run experiment Calibrate nephelometerrt 4analysepage buttonup buttonup analysepage analysepage Analyse 4runpage buttonup buttonup runpage runpage 4seencalibinfo "calibratepage" buttonup buttonup calibratepage calibratepage seencalibinfo Calibrate Background information How to use this programme buttonup buttonup Aims of the tutorialll "aimspage" buttonup buttonup aimspage Button Main menuuninformationY "infopage" buttonup buttonup infopage viablecell "platingout" "counting" "calculation" "serialdilutions" leavepage leavepage platingout counting calculation serialdilutions There are three steps in determining the viable count: Click on one of these steps for further information. 2. Samples from the lower dilutions are plated out. 3. After incubating these plates, the number of colony- forming units are counted. Click on one of these stages for further information. replication Viable cell counts serialdilutions tube: flask1 E E c bung1 tube: tube: tube: tube: tube: Dilution: 10 10 10 10 10 10 The culture is diluted in a series of ten-fold dilutions -1 -2 -3 -4 -5 -6777 arrow2 arrow1 arrow2 arrow2 arrow2 arrow2 platingout tube: tube: tube: 10 10 10 10 10 10 10 10 The culture is diluted in a series of ten-fold dilutions -4 -5 -6 -4 -5 -6777 10 10 10 10 10 10 10 10 The culture is diluted in a series of ten-fold dilutions -4 -5 -6 -4 -5 -6777 counting Z#2#W# 30-300 colonies OK for countingm# >300 colonies Too many to count r$*$o$ <30 colonies Not statistically valid selectedtextlines "platingout" "counting" "calculation" "serialdilutions" buttonup buttonup platingout counting calculation serialdilutions counting serialdilutions calculation platingout platingout serialdilutions calculation counting platingout serialdilutions counting calculation 1. The culture is diluted in a series of ten-fold dilutions. 2. The lower dilutions are plated out and incubated. 3. The number of colonies on each plate are counted. 4. The cell count in the original culture is calculated. forming units are counted. calculation 103 colonies on a plate from the 10 dilution corresponds to 1.03 x 10 cells/ml in the original undiluted cell suspensionnn plated out Z*P*W* totalcell replication Total cell counts Coverslip Drop of cultureepp Microscope slideeee To be statistically valid, around 100 cells are counted and the average number of cells per small square calculated. Since the volume of liquid above each square is known, the concentration of cells can then be calculated........................using sing Grid etched on surface of slide. This is visible under the microscope Cells can be counted using the microscope quare can be counted l\VN\r xtrffnz |`fnx tHXR\ d@JVdzxdr .NJR\V\lxnrz :D<4BR^fdrlr &&.,&2@DBVfl`jt ,(&""".6:@@NV\RXn x2&"2. ,,.6:@:NH<@Vlz .JD:66:24@6@