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...
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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.
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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.
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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.
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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...
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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...
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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.
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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...
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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.
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BUTTON
This button is on the inside left at the foot of every page.
Click on it to return to the Main menu.
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BUTTON
This button is on the inside right at the foot of every page.
Click on it to move back to the previous page.
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BUTTON
This button is on the far right at the foot of every page.
Click on it to move on to the next page.
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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.
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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.
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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.
The menu bar is this bar along the top of the page.
Click on OK to hide this box..
"menubarinfo"
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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.
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hotwordfound
Yes! You've found a hotword.
Click on OK to hide this box.
s box.
"hotwordfound"
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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"
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- 2 -
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Aims of this tutoriallm
endbackground
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BackPage
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ExitProgram
"This will
the tutorial.
Are you sure
want
quit?"
f"Yes"
"&No"
SysSuspendMessages
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This will end the tutorial. Are you sure you want to quit?
MainMenu
"MainMenu"
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MainMenu
Click on a grey button to choose an option...ft here to leave this page....
Bacterial growth
NextPage
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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...
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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
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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"
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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
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c"File"
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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"
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"tbkdlg.dll"
"tbkwin.
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BackgroundInformation
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CalibrateNephelometer
MainMenu
RunExperiment
DataAnalysis
Credits
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TitlePage
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MainMenu
mainmenu
helppage
aimspage
BackgroundInformation
infopage
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calibratepage
calibratepage
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RunExperiment
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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"
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"analysepage9"
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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
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temperature
duration
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nephdatacollected
Nephelometer data collected
tube11
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E E c
bung1
reading
4seencalibinfo
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biomass
Biomass
26 mg/ml
dilution
Dilution
0.2
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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)
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on analysepages
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biomassstage
calibstage
Determine biomass
4calibstage
"dilution"
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dilution
calibstage
Dilute cell suspension
Take readings
Make sub-dilutions
datacollected
All data collecteddto Anal
tube:
tubescreen
timeflies
Time flies...
"reading"
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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"
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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"
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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
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duration
duration
4 hours
6 hours
8 hours
10 hours
12 hours
14 hours
"durationinfo"
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durationinfo
analysepage5
Time Nephelometer ln biomass ln total cells ln viable cells
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"
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"screen4"
"screen5"
"screen6"
"screenK"
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Cannot calculate biomass values until nephalo-meter calibration curve is prepared
infobackground
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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
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NextPage
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ExitProgram
"This will
the tutorial.
Are you sure
want
quit?"
f"Yes"
"&No"
SysSuspendMessages
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This will end the tutorial. Are you sure you want to quit?
MainMenu
"MainMenu"
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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"
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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
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enterpage
video
video
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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
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4050
3550
5800,
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replication
Bacterial cell replication
Click on one of the buttons on the video recorder to see a time-lapse video of bacteria growing...
"videoinfo"
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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"
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5800,
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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
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tube:
replication
Nephelometry
cycle
tube:
reading
"lid"
"tube:"
3862,2500
"reading"
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tube:
reading
Light source
Cross-section of test tube containing cell suspension
Detectorreee
"lid"
"reading"
"tube:"
3862,2500
2500
3200
3862,n
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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
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0 2 4 6 8 10 12555444
biomass
Time (hours))l))gding
nos..rs))))
x 10 ml)
Biomass
Total cell numbers
Viable cell numberssss
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analysebackground
measurement
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analysepage:
infopage
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calibrationpage
biomass
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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
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mouseenter
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videoinfo
BackPage
"aimspage"
buttonUp
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aimspage
NextPage
"growthcycle"
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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
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frame
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video
video
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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
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video11
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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"
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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
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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" &
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lagline
acccurve
logline
deccurve
statline
deathcurve
gcinfo
gcinfo
phase
newphase
replication
4newphase
"graphpaper"
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graphpaper
newphase
The batch growth cycle
logline
4newphase
0,50,100
"graphpaper"
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statline
4newphase
0,50,100
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4newphase
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newphase
ln cell
numbersss
4newphase
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4newphase
0,50,100
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deathcurve
4newphase
0,50,100
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4newphase
0,50,100
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lagline
4newphase
0,50,100
"graphpaper"
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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"
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graphpaper
newphase
4newphase
"graphpaper"
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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.
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
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temperatureinfo
4temperature
selectedtextlines
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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
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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
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c"Go
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c"Go
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BackPage
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ExitProgram
"This will
the tutorial.
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f"Yes"
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MainMenu
Click on a grey button to choose an option...ft here to leave this page....
Bacterial growth
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analysepage5
infobackground
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analysebackground
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NextPage
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ExitProgram
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want
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f"Yes"
"&No"
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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"
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analysepage1
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analysepage
showdata
4data, view, analysepage
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Bacterial growth
Temperature
Duration
Informationn
Settings
temperature
duration
6 hourssurs
"runpage2"
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runpage2
runpage2
runpage
"runpage3"
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runpage3
runpage3
runpage
"runpage4"
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runpage4
runpage4
runpage
Prepare
Sterilise
Inoculateeatelate
"runpage4"
"prepared"
"sterilised"
"inoculated"
"datacollected"
timer
"000
"bungs"
"culture"
60,95,100
4runstage
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runpage4
runpage4
prepared
sterilised
inoculated
datacollected
000 min
bungs
culture
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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"
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MainBackground
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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"
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biomassscreen
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"biomassscreen"
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helppage
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MainMenu
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Bacterial growth
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"planinfo"
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planinfo
Calibrate nephelometerrt
MainMenu
Analyse data
Run experiment
Calibrate nephelometerrt
4analysepage
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analysepage
Analyse
4runpage
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4seencalibinfo
"calibratepage"
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seencalibinfo
Calibrate
Background information
How to use this programme
buttonup
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Aims of the tutorialll
"aimspage"
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aimspage
Button
Main menuuninformationY
"infopage"
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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
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