MMBB255 Week 5 Background
1
Growth and enumeration of viable cells and unknown two
I. Objective
The goal of this experiment is examine the kinetics of bacterial growth and to learn how to determine the
number of cells in a broth culture using a spectrophotometer and to correlate this value with the number of
colony forming units that result from viable cells. You will start unknown number two and follow up on
several of the previous experiments.
II. Background
The growth rate of microbial cells is highly variable
doubling time: the time it takes for a
and depends on factors that include the type of organism,
population of cells to double in number.
the pH of the medium, the temperature, and nutritional
factors. For example, Escherichia coli, an organism found
in the intestinal tract, grows with a doubling time of about 30 min at 37°C in rich medium at pH 7 containing
a carbon source such as glucose. If incubated at 30°C, the same organism in the same medium would grow
with a doubling time >40 min. In contrast, a microbe isolated from the soil might grow best at 30°C and have
a doubling time of 4-8 hours.
In order to determine the doubling time of an organism growth curves are required. A growth curve is
generated when you monitor the cell number over time. Cell numbers are generally on the y-axis and are in
base10 log while time is on the x-axis in normal form (this is called semilog graphing; one axis is in log
while the other is arithmetic). A typical growth curve will have four phases: the lag phase (where the cells
are adapting to the growing conditions), the exponential phase (where the cells have unrestricted growth),
the stationary phase (where the cells have run out of food or toxic by-products have accumulated), and the
death phase (death and cell lysis). The doubling time is determined from the exponential phase of growth.
In your professional capacity, whether it be medicine, environmental science, food microbiology, or
research, it will be important that you know how to quantify microbes in a sample. For example, if your job
involves advising the public of water quality standards, you must be able to report the numbers of
microorganisms in a water sample and determine if the numbers are within a safe limit for human
consumption. The following topics summarize the most common quantification procedures:
A. Quantifying total cell number using a spectrophotometer
We will use a
The detector in the
spectrophotometer this week to
spectrophotometer receives all of
estimate cell numbers based on the
the light because water does not
principle that cells absorb light. The
absorb.
sample to be measured is placed
%T = 100 or A = 0.0
between a light source and a
Water
Light Source
detector. The detector measures
The detector receives less light
light. The spectrophotometers that
because the microbial cells
you will use have two scales: one
absorb some of the light. The
scale indicates the amount of light
amount of light absorbed
absorbed by the sample called
depends on the cell density.
absorbance and the other is used to
Cells
measure the amount of light
transmitted (passes through) by a sample called percent transmittance. A sample, such as air or water,
which does not absorb light will have an absorbance = 0. (Absorbance = Abs or Awavelength and in older
literature is called optical density or OD). Because air and water do not absorb light, all of the light will
pass through to the detector. Hence, the % transmittance (%T) of air is 100%.
We will measure absorbance of cells at 600 nm. We use this wavelength because absorbance of light
by other molecules in the cell, such as flavins and carotenoids, is minimal at
this position.
Equations that relate OD to %T are provided in the box; Beer’s law gives a
direct linear relationship of Abs to concentration and the abbreviation
definitions are below:
I0 = light before sample
I = light that passes through the sample
a = molar absorptivity in M-1cm-1
b = light pathlength of the sample in cm
c = concentration in M
MMBB255 Week 5 Background
2
This method can be used to estimate the total number of cells in a sample, which includes living cells,
dead cells, and noncellular material that absorbs/scatters the light. To correlate the Abs (absorbance) with
numbers of viable cells (living cells capable of cell division) we will be doing plate counts. This
correlation is be done by removing an aliquot of cells from the growing culture, taking a spctrophotmetric
reading, then diluting the cells, and then plating the dilutions (either by spread plate or pour plate,
methods). Each viable cell will be able to grow in the agar medium and will produce a colony. These
colonies, now called a colony forming unit or CFU, are counted and then calculated to get the cell
concentrations.
B. Quantification of cells via direct and electronic methods
There are several methods for direct counting of total cell number. One of the most common is to
count them by eye under the microscope using a Petroff-Hauser chamber. A Petroff-Hauser chamber has
an exact grid etched into a glass slide. Cells are placed on the grid, covered with an absolutely flat cover
slip, and counted within the gridded spaces. The grid with the cover slip gives a known volume which
when combined with the number of cells counted in the grid will give you the concentration (cells/ml) of
total cell number. Again, this is a total cell count that includes both viable (live) and non-viable (dead)
cells.
An electronic method uses the fact that cells have a different conductivity than an aqueous solution.
Two chambers are connected by a small hole just big enough to let a cell through. A known volume is
pumped through the hole and when a cell enters the hole the conductance changes and is counted.
C. Quantification of cells by viable plating techniques
The most accurate method for determining the number of
viable (living cells capable of replication) cells is by plating
aliquots of cell dilutions. There are various ways of plating the
aliquots; the spread plate method, the pour plate method, and
calibrated loop method are some examples. After a period of
time (usually 24-48 hrs), the number of colonies (called colony
forming units or cfu) on each plate can be counted. Only
statistically valid plates, those that have between 25 and 250
colonies per plate, are counted (it used to be 30-300). This method is based on the assumption that each
colony arose from a single cell. Hence, by counting the number of colonies and factoring in the various
dilutions that were performed, you can estimate (extrapolate) the number of viable cells in the starting
material (see the equations to the right). Dilutions and extrapolations generated from dilution series are
particularly important in medicine, immunology, food microbiology, and ecology
Here is an example of a dilution series and the pour plate method of plating to determine the number of
viable cells in a sample:
1. A water sample (tube A) from a contaminated aquifer has an Abs600= 0.17 (this will look a little
cloudy or turbid). You do a dilution series:
You add 1 ml of the sample to 99 ml of diluen
You add 1 ml of dilution 1 to 99 ml of diluent
You add 1 ml of dilution 2 to 9 ml of diluent
You add 1 ml of dilution 3 to 9 ml of diluent
dilution 1 (tube B) a 1:100 dilution for a 10-2 total dilution
dilution 2 (tube C) a 1:100 dilution for a 10-4 total dilution
dilution 3 (tube D) a 1:10 dilution for a 10-5 total dilution
dilution 4 (tube E) a 1:10 dilution for a 10-6 total dilution
2. 1 ml of dilutions 2, 3, and 4 were added aseptically to empty, sterile petri plates and tryptic soy agar
(TSA), at 55°C, was immediately aseptically poured into each petri dish, mixed in a figure eight, and
allowed to solidify. The plated sample was inverted and incubated at 37°C. The next day, colonies
were counted.
Your counts were:
plate 1 (from tube C)
plate 2 (from tube D)
plate 3 (from tube E)
= >>1000 (too numerous to count TNTC)
= 450 (TNTC-not statistically valid)
= 56
These are good results because plate 3 contains a statistically valid
number of colonies to count. Statistically valid counts are between 25
and 250 colonies per plate.
3. Extrapolating, calculating, the original sample's viable cell number is
next. Using the number of colonies from plate 3, you plug it into the
equation here along with the corresponding total dilution and volume of
sample added to the plate (see the work to the right).
MMBB255 Week 5 Background
3
Tuesday’s Procedures:
A. Practice streak: examining – Finished.
1. Remove your streak plate from the bucket. You should see three different colony morphologies.
2. Examine the colony morphology. Record your colony observations (week 1 has the criteria). Decide
if you need to improve your streaking technique and how you would.
Colony type 1
Colony type 2
Colony type 3
Colony description:
morphology, color,
texture, size etc.
B. Technique: Dilutions and the spread plate method – Work in Pairs.
Background: Another way to obtain isolated colonies is to spread a small amount of liquid containing cells
on a plate. This is the spread-plate technique. Before spreading, you will need to make a few serial
dilutions of your cell culture as it contains millions of bacteria per ml. You must understand how to
perform a serial dilution and how to quantify using this technique. There are practice problems that are
highly recommended for you to work
out.
1. Make serial dilutions of the cell
mixtures. Label the dilution tubes
you prepared last Tuesday with
numbers 1 through 8. Although you
added 10 ml peptone water (the
diluent) to each tube, some liquid is
lost during the autoclaving process.
As a result, each tube contains
about 9 ml of peptone water, which
is ideal for the dilutions we want to
make. The diagram to the right is an
example to help you visualize the
procedure.
a. Mix on the vortexer and remove
1 ml of sample that you used to
practice streak last week (a
mixture of three organisms).
Add it to the 9 ml of peptone
water in tube #1. Use aseptic
technique. Your lab instructor
will demonstrate the transfer
technique. Remember to dispose
the used pipette tips into the
small biohazard bags.
b. Mix tube 1 on the vortexer (this
is crucial). This is a 1:10 dilution (also written as 1/10 or 10-1) for a total dilution of 10-1
c. Take a new sterile tip and remove 1 ml from tube 1 and add it to 9 ml (1:10) of peptone water in tube
#2. This is a 1:10 dilution for a total dilution of 10-2 (relative to the starting material).
MMBB255 Week 5 Background
4
d. Continue as follows. Using a new tip for each tube and mixing each tube before aseptically
transferring; until the sample has been transferred to the last tube (as below):
Add 1 ml from tube 2 to the 9 ml of peptone water in tube #3.
Add 1 ml from tube 3 to the 9 ml of peptone water in tube #4.
Add 1 ml from tube 4 to the 9 ml of peptone water in tube #5.
Add 1 ml from tube 5 to the 9 ml of peptone water in tube #6.
Add 1 ml from tube 6 to the 9 ml of peptone water in tube #7.
Add 1 ml from tube 7 to the 9 ml of peptone water in tube #8.
1:10 dilution for a 10-3 total dilution
1:10 dilution for a 10-4 total dilution
1:10 dilution for a 10-5 total dilution
1:10 dilution for a 10-6 total dilution
1:10 dilution for a 10-7 total dilution
1:10 dilution for a 10-8 total dilution
2. Estimate the concentration of cells in each dilution. Before you can spread a sample on the plate, you
need to decide which dilutions are most likely to have a reasonable number of cells so that you will
have the appropriate number of colonies. The most statistically valid spread plates should have
between 25 and 250 colonies per plate (a colony forming unit—cfu). We will assume that each cell
will give rise to one colony. Further, we will estimate that a turbid sample (+++) will contain about 8 x
109 cells per ml, a (++) 8 x 108 cells per ml, and a (+) 8 x 107 cells per ml.
Some simple calculations are in order. We will use a turbid sample of (+++) as an example.
• If you remove 1 ml of the sample and dilute it in 9 ml diluent (see tube #1), you now have 8 x 109
cells put into a total volume of 10 ml or 8 x 108 cells/ml.
• If you remove 0.1 ml from this tube and spread it onto the agar surface, there will be 8 x 107 cells
on the plate and each can give rise to one colony. Eighty million colonies on one plate would
produce a ‘lawn’ of growth, so this is not a good dilution tube to use.
Now do the same type of calculations for the remaining dilutions in the table below and choose three
dilutions that you think will contain a reasonable number of cells in a 0.1 and 1 ml volume. Our mixture
of three organisms is a (+++) turbidity.
Tube #
1
2
3
4
5
6
7
8
Concentration of the
inoculum (the
starting culture)
8 x 109
8 x 108
Cells/ml after
dilution
Cells in 0.1 ml
8 x 108
8 x 107
Based on our original estimate of cells per ml, which three tubes are most likely to yield 25-250
colonies on the spread plates (you want to pick one above and below your ideal tube)?
Tubes:_____________________
The reason it is important to sample from three different dilutions is because our calculations are based
on the fact that the starting culture contains about 8 x 109 cell per ml. By sampling several dilutions, we
should obtain at least one plate with a reasonable number of colonies in case our original estimate of
cells in our culture is incorrect.
3. Spread the cells on the agar. When all your dilutions are done, you are ready to spread. Aseptically
remove 1.0 ml (the table above is using 0.1 ml) from one of the dilution tubes that your calculations
show should give a reasonable number of colonies and dispense the liquid onto the surface of one of
your TY agar plates made previously and stored in your cabinet. Make sure your lab partner gets a
chance to do this spread plate technique. Dispose the pipette tips correctly.
a. Take your metal rod that looks like a metal triangle, immerse the triangle end into the ethanol. Place
the ethanol coated end into the flame of your Bunsen burner in order to ignite the ethanol (this is
fun). Let the ethanol burn off away from the flame (in order to prevent it from getting too hot). Be
careful not to let the ethanol run down the handle of the metal triangle or you might burn your
fingers.
MMBB255 Week 5 Background
5
b. After the ethanol is burned off, allow the rod to cool briefly, place the sterile metal triangle end on
the dilution sample you placed on the plate, and spread the liquid all over the surface of the plate.
Rub smoothly and evenly over the surface. Place the contaminated metal triangle back into the
ethanol for the next plate and if done spreading, flame the stick one last time to decontaminate it and
put the stick back were it came from.
c. Repeat steps a and b with the other two plates, using a different dilution tube for each plate. Be sure
to label each plate with the volume and dilution number of the inoculum.
d. Invert the plate and incubate at 30°C until Thursday.
C. Fermentations: Wine making and beer continued.
1. The wine is still in the primary
fermentation stage. The beer will
be bottled Thursday.
2. Check the trap to make sure the seal
is intact and fermentation is
occurring. You will need to keep
doing this until the fermentation is
finished.
3. Once no more bubbles are rising in
the flask, we will take the final
potential alcohol reading and bottle
the wine if worthy.
Normally the wine is transferred to a new container for another 2 to 4
weeks to finish fermentation and improve flavor (this leaves behind the
'sludge'). We will skip this due to time constraints. This is called the
secondary fermentation.
To age the wine, it is transferred to a flavoring container (oak barrels are
particularly good) and then bottled. Aging for an additional 2 weeks to
several years will add complexity and improve the flavor. Most wines
today are clarified and sulfites are added to help preserve the wine. We
will simply transfer the primary fermentation, trying not to disturb the
bottom sludge, to a sterile/clean bottle. Before bottling, the finished
product will be analyzed.
D. Fermentations: cheese tasting – Finished.
At this stage, the cheese should be firm and ready to eat. Homemade cheese is a good substitute for
cream cheese in baking. Bacteria, (primarily Lactococcus lactis, Lactococcus cremoris, Streptococcus
thermophilus and Lactobacillus bulgaricus) are involved in the early stages of fermentation for most types of
cheeses – unripened soft (cottage, cream and mozzarella), ripened (brie), semisoft (blue/Roquefort), and hard
ripened cheeses (cheddar, swiss, and parmesan). Following the initial processing (as you just completed)
addition of Penicillium sps, Brevibacterium sps, Lactobacillus sps, and Propionibacterium yields ripened
cheeses. Taste your cheese. Enjoy!
E. Fermentations: sampling the soda – Finished.
The soda should be complete by this time. Because
the culture has not been allowed to advance to an
anaerobic fermentation stage (we stopped it by putting it
into the 4°C), no alcohol is produced. Be careful not to
disturb the yeast in the sediment when pouring the soda.
Although ingestion of the yeast will not harm you, it will
spoil the sweet taste of the soda pop.
1. Microscopic examination of the soda pop.
Remove a small amount of the sediment and
prepare a wet mount. Record your observations at
1000X.
wet mount
MMBB255 Week 5 Background
6
F. Unknown #2: Isolation & biochemical characterization – Work individually. 50 pts.
Due date:_________________
Name: ___________________
Section: __________________
Unknown Number: ________
1. You will be given a mixture of two organisms in broth (everyone has one red and one non-red one).
Record your unknown number above.
2. Streak for isolation. Mix the broth before you streak and label your plate with your name, section, and
unknown number. Invert your plate in a bucket that will be incubated at 30°C. After 24-48 hrs, we will
grade your technique. Your grade will be based on good isolation of the two different colony types
without contamination and good technique. The more isolated colonies you get and use of the whole
plate will yield more points.
Your streaking grade (15 pts.): _____________________
3. Identify the two colony morphologies and prepare a Gram stain from each type. Remember to put
in all the prerequisite observations for both colony and cellular observations (at 1000 X) to get all
points. (18 pts.)
Colony type #1
Colony type #2 (Red)
Colony characteristics
(size, color, texture,
smell, etc.)
Gram reaction
Cell Observations
4. Get a pure culture of your unknown. Use an isolated colony from your streak plate to obtain a pure
culture of the non-pigmented (not red) unknown on a second plate. Incubate at 30°C and check your
growth after 24-30 hrs.
5. Biochemical tests. Use your pure culture of your unknown to perform the following tests during the
biochemical experiment (Week 6). Record your results below. (14 pts.)
Sugar fermentation
Glucose
positive or negative
Sucrose
Lactose
Enzyme production
Catalase
Oxidase
Urease
Motility
(motile or non-motile)
positive or negative
6. Based on the results above and the organisms we have seen so far this semester, I suspect this
organism is (write and spell it correctly): (3 pts.)
MMBB255 Week 5 Background
7
Thursday’s Procedures
A. Fermentations: bottling of the beer.
1. You will analyze the results of the primary fermentation for it’s potential alcohol (using a
hydrometer), pH (using pH meter), and any contamination (doing a Gram stain).
pH: __________
%Potential Alcohol: ________ Final %Potential Alcohol: ___________
Any contamination (Gram Stain)?
2. It is time to bottle the beer when bubbling ceases. Transfer the primary fermentation to a clean,
sterile carboy with a valve. This is what you will analyze in step 1. Add about 3/4 cup (or 70-80 g) of
corn sugar dissolved in a small amount of sterile water. This will be the final bit of substrate for the
yeast that are still alive and present in the mixture. This final addition of sugar is just enough to
generate new growth so that more CO2 will be produced in the bottles - providing the carbonation
that is associated with beer.
3. Mix. Using the valve on the carboy transfer the mixture (this is called racking) into sterile bottles to
within about 1 inch from the top.
4. Cap the bottles and leave at room temperature for about 1 week. Transfer into refrigeration
temperatures for at least a day before testing the final product.
B. Technique: the spread plate method for isolation and quantification - continued.
Your spread plate should give you two types of information – 1) information about the different types of
microbes in your culture and 2) an estimate of the total number of viable cells in the original culture.
1. Identification of colony types. You should see the same three different colony morphologies that you
obtained from your practice streak plate that we examined on Tuesday. Compare the two methods.
Which method gives the best results?
2. Quantification. Count the number of colonies on the plates (colony forming units or cfu) that fall
within 25-250 CFU. Of that total include numbers for each organism (i.e. each colony type). Record
this in the table below and then calculate the concentration of viable cells of each organism that were
in the original sample by extrapolation. Some terms: a dilution is 1x10-4 or 1:10000; a dilution factor
is the inverse of the dilution (i.e. 1/dilution) so 1x104 and 10000. The table below is in the form of
this equation: cells of sample/ml = (cfu) x (1/dilution of sample)÷(volume plated in ml) (A
hypothetical example is given first).
Dilution tube used
for spread plate
Actual # of
colonies
x dilution factor
÷volume
plated
Estimate of cells in the
original sample
#5
256
1x105
0.1 ml
2.56 x 108
C. Unknown continued.
1. Make sure you do a pure culture of your unknown (see the unknown instructions in Tuesday’s
procedures). If you do not have your non-red organism isolated then you must talk with your TA.
The Gram stains can be done when you have time as long as you refrigerate your plates.
MMBB255 Week 5 Background
D. Comparing growth of a bacterium on different substrates – 4 Teams.
You will monitor growth of E. coli in two different media and at two different temperatures. You will form 4
teams. During the lab period each team will take 3 time points. At each time point you will take turns within
your team (so everyone gets a chance to do all the techniques) to:
1. measure the Abs (OD) at 600 nm with a spectrophotometer.
2. make serial dilutions and pour plates for viable cell counts.
The media (medium is singular, media is the plural form) was inoculated in the morning (before lab) with an
overnight culture of Escherichia coli by your lab instructor. Please record the zero time. We will be
monitoring throughout the day so be careful not to contaminate your medium; use aseptic techniques.
•Team A will monitor growth in Luria-Bertani (LB) broth + glucose at room temp (≈22°C).
•Team B will monitor growth in Luria-Bertani (LB) broth + glucose at 37°C.
•Team C will monitor growth in Luria-Bertani (LB) broth + lactose at room temp.
•Team D will monitor growth in Luria-Bertani (LB) broth + lactose at 37°C.
Do the following three steps every 30 min (i.e., for the 9:30 lab, you will take a sample at 10:00, 10:30 and
11:00). Each team will take their measurements at each time point in the following order:
1. Take the flask out of the shaker and aseptically remove 5 ml of culture into a sterile test tube and then
put the flask back onto the shaker ASAP.
2. One person will aseptically remove 1 ml from the 5 ml sample and make the first serial dilution and
pass the remaining 4 ml to their partner who will follow the instruction in step 3. Follow the
instructions a – e below to finish the dilutions and plate counts.
a. Aseptically remove 1 ml from your 5 ml sample tube and transfer it to a dilution bottle containing
99 ml of sterile diluent. Give the remaining 4 ml to your partner so that s/he can take a
spectrophotometer reading. Mix by shaking the bottle in an arc with a paper towel covering the lid
(the standard is 25 arcs). This is dilution #1, 1:100 (1/100) or 10-2 total dilution.
b. Aseptically transfer 1 ml from dilution #1 to another dilution bottle containing 99 ml of sterile
diluent. Mix by shaking the bottle in an arc with a paper towel covering the lid (the standard is 25
arcs). This is dilution #2, 1:100 (1/100) or 10-4 total dilution.
c. Aseptically transfer 1 ml from dilution #2 to another dilution bottle containing 99 ml of sterile
diluent. Mix by shaking the bottle in an arc with a paper towel covering the lid (the standard is 25
arcs). This is dilution #3, 1:100 (1/100) or 10-6 total dilution.
d. Aseptically transfer 1 ml from dilution #3 to a test tube containing 9 ml sterile diluent. Mix with
the vortexers or by hand. This is dilution #4, 1:10 (1/10) or 10-7 total dilution.
e. Pour plating your cells from dilutions. Before plating your dilution, label the bottom of the plates
with the dilution (i.e. 10-7), time point, group, and section. Do the following:
• Aseptically place 1 ml from dilution #4 in the center of a sterile petri dish.
• Aseptically place 1 ml from dilution #3 in the center of a sterile petri dish.
• Aseptically place 1 ml from dilution #2 in the center of a sterile petri dish.
Remove a flask of tryptic soy agar (TSA) tempered in the 55°C waterbath. Aseptically pour the
TSA to just cover the bottom of one plate and mix the agar with the 1 ml of diluted cells by
moving the petri dish gently over the bench forming an figure 8 pattern. Repeat with the other two
plates. Allow the plates to solidify on your bench. When the plates have solidified invert and
incubate at 37°C for 24 hrs.
3. To measure Absorbance you need to transfer your sample into a spectrophotometer (spec) tube; an
optically clear tube that fits in the sample holder – part B in the picture on the next page. Use the
same machine for all the time points. The spec tubes we are using need a minimum of 3.5 ml of
sample; luckily we have 4 ml left of sample. You will transfer the entire leftover sample into the spec
tube; there is no need to use aseptic technique here, just pour the contents in. You will measure the
absorbance at 600 nanometers (nm) and record your readings in the table on the next page and in the
computer. Follow the following procedures to take your measurements:
8
MMBB255 Week 5 Background
9
Important Notes:
1. Zeroing a machine means to make it read 0. Since a spectrophotometer reads light (detects light)
that means you need a 0 %Transmittance (T) or infinity Absorbance (A). Your light path must be
blocked for this to occur which means there must be no tubes in the machine. This will correct for
any machine error (i.e. leaking light).
2. Blanking a machine means to obtain 100% of the signal (light in our case) which is 100 %T or 0
A. This always should be done with everything that is in the sample except for the thing you are
trying to measure (cells in this week’s experiment thus your blank will be LB broth). This will
correct for any incident absorbance in the sample that we do not want to measure.
3. These machines have a linear response, i.e. the readings will be directly proportional to amount of
analyte (cells), between 0.1 and 1.0 A. If you go over you should dilute your sample (usually a
1:10 will work fine).
Procedures for using the Spectronic 20D+ spectrophotometer:
a. Turn the machine on with knob A. Let it warm up for 15 min.
b. Set the desired wavelength (λ) to desired wavelength using knob C.
c. Select the proper filter by flipping
the E switch to be in correct
wavelength range.
d. Zero (see the notes above) the
machine by turning knob A until
the %T is 0. The reading will
flash 1.99 if you try to read
absorbance. Use the mode button
to see the %T scale for reading.
e. Set the mode to absorbance (also
called OD for optical density).
f. Blank (see the notes above) the
machine. Wipe the outside of the
blank tube with a Kimwipe and
insert it into the sample holder
(B). Set the Absorbance to 0 (or
100 %T) using knob D.
g. Wipe the outside of the sample tube and insert it into the sample holder (B). Read the absorbance
of the sample off the digital readout.
h. Record the reading
Absorbance readings for Team ________
Time- record real time
t= 0 min (we will give you this
reading)
t=30 min
t=60 min
t=90 min
Absorbance600nm
MMBB255 Week 5 Background
Study Questions
1. What are the four phases of a growth curve?
2. Can you operate a spectrophotometer? What is the difference between blanking and zeroing the machine?
What does this machine detect and which scale is a reading of this detection and which scale is derived
from this?
3. Does a spectrophotometer and direct counts measure viable cells? Why?
4. What are some direct cell counting methods?
5. How would you do a viable cell count? What two plating methods could you use to do this?
6. Can you do a dilution problem? Try to answer the dilution problem sets.
7. What is the statistically correct number of colonies for plate counts?
8. Why did we add corn sugar (glucose) to the beer before we bottled it?
9. Can you manually plot growth curve data on a piece of semi-log graph paper and determine the doubling
time from the plot? What are the four phases of growth?
10. Define doubling time.
10