5-6 Generating a growth curve In this experiment, the classic bacterial growth

curve will be demonstrated. A culture of Escherichia coli will be sampled at hourly or half-
hourly intervals from the time of inoculation of the culture (0-time) through a 7 to 9-hour
incubation period. The periodic samplings will be plated to determine viable counts (as colony-
forming units per ml of culture) over the incubation period such that a growth curve may be
plotted. From the graph, we may note the stages in the growth of the culture as it grows into the
stationary phase. Additionally we will be able to determine the growth rate and generation
time of E. coli under our experimental conditions from two points in the exponential phase of the
graph.

Figure 5-16 Bacterial Growth

Graphing of bacterial growth on a linear scale. By definition, bacterial growth is cell

replication - i.e., growth of the culture. Most species of bacteria replicate by binary fission,
where one cell divides into 2 cells, the 2 cells into 4, the 4 into 8, etc. If this cell division occurs
at a steady rate - such as when the cells have adequate nutrients and compatible growing
conditions - we can plot numbers of cells vs. time such as on the graph at right. Before too long,
we will need to extend the paper vertically as the population continues to double. For a culture
where cells divide every 20 minutes, one cell can result in 16,777,216 (i.e., 2 24) cells after just 8
hours - barring nutrient depletion or other growth-altering conditions.Figure 5-17 Bacterial
Growth

Graphing of bacterial growth with cell number on a log scale.

If we were to convert our vertical axis to a logarithmic scale - as on the graph at right - we will
not need as many sheets of graph paper, and we will find that a steady rate of growth is reflected
as a straight line. (On the vertical axis, the same distance on the paper is covered with each
doubling.) This type of graph paper is called semilogarithmic graph paper on which we will be
plotting our class results. The numbers we plot will fall on the graph at the same place the
logarithms of these numbers would fall when plotted on conventional graph paper.

The example below shows the type of graph we may obtain from our class data. We can plot
both colony-forming units (CFUs) per ml and absorbance on the same graph, remembering that
the absorbance units should also be on a logarithmic scale. Rather than "connecting the dots," we
draw the best straight line among our CFU/ml plots to represent the phases of growth - lag,
exponential, and the start of the maximum stationary phase.

Figure 5-18 Two measurements of growth

Example data showing a plot of cell number by VPC and by turbidity.

For the growth rate formula we are about to use, we need to choose two points on the straight
line drawn through the exponential phase, also making note of the time interval between them.
As we will be converting our numbers to logarithms for the formula, why not choose two points
for which the logs are easy to obtain? (For example, the log of 1X10 10 is simply 10.)

 Higher CFU/ml = Xt = 1X1010 (at 5.75 hours)

 Lower CFU/ml = X0 = 1X108 (at 2.75 hours)
 Time interval (in hours) between the 2 points = t = 3

Using the first formula, we find the growth rate which is the number of generations (doublings)
per hour:
Figure 5-19 Calculating the growth rate

Use this formula to determine the growth rate k

With the second formula, we find the generation time which is the time it takes for the
population to double:

Figure 5-20 Generation time

The generation time is the reciprocal of the growth rate.

When we graph the CFUs/ml and absorbance on the same graph, we would hope to see an
upward trend for both. Sometimes the absorbance continues to rise after the CFUs/ml level off
into the maximum stationary phase. What would be the cause of that?

With a clear graph, one should be able to determine the generation time without the use of
formulas. Just look for a doubling of the population and the time it takes for that to happen. For
example - in the above graph - the time it takes to go from 3 X 109 to 6 X 109 appears to be
approximately 30 minutes, which is close to the generation time determined above.

In preparation for this exercise, be sure to read the relevant material in your textbook, and look
over the procedure below.

Precautions regarding observance of aseptic technique:

1. Remember the proper procedure for holding the tubes during inoculations. Do not allow
the plugs or caps to contact the desk top or anything else.
2. When using the pipettor, hold one tube at a time.
3. Do not pipette from tube to tube with the tubes sitting open and vertical in the test tube
rack!
4. Also, one person should not be holding the tubes while another makes the transfers.
5. Remember to use a new tip each time you begin to use a more dilute concentration of
cells

Period 1

Materials
Samples (5-6 ml) which were taken at hourly or half-hourly intervals from a culture of E. coli
growing in Nutrient Broth+0.2% yeast extract, incubated at 37°C on a shaker. These samples
have been kept on ice for use in this experiment, and each pair will use one sample.

The following are provided for each pair of students:

7-9 nine ml dilution blanks

8 tubes of melted Plate Count Agar (PCA) in test tubes (15-20 ml/tube) - in 50°C water bath

8 empty, sterile petri dishes

Pipettors (P1000) and sterile tips

Spectrophotometer tube and spectrophotometer

1. Each pair will pick up one culture from the ice-water bath on the front table. Record the
number on the tube. It represents the age of the culture at which time the sample was
taken.
2. With the P1000 (blue) pipettor set at 1.0 ml, transfer 1 ml of the culture to the first nine
ml dilution blank (for the first 1/10 dilution to work with in Step 5).
3. Aseptically dump the remainder of the culture into the small spectrophotometer tube (to
work with in step 4).
4. With the culture in the spectrophotometer tube, one person in the pair will obtain and
record the absorbance reading of the culture while the other begins the next step.
5. With additional dilution blanks, make dilutions as specified below. Inoculate 1 ml from
each of the four specified dilutions into each of two petri plates; plate inoculations can be
made concurrently with preparation of the dilutions.

The dilutions to be plated are as follows:

For 0-hour through 2-hour sampling times: 10-4, 10-5, 10-6, 10-7
For 2.5-hour through 4.5-hour sampling times: 10-5, 10-6, 10-7, 10-8
For 5-hour through 9-hour sampling times: 10-6, 10-7, 10-8, 10-9

1. For each plate, obtain a tube of melted PCA from the water bath and pour the contents
into the plate. Mix the medium and inoculum by carefully swirling and allow the plates to
solidify.
2. Incubate the plates inverted at 30°C until the next period.
Period 2

1. Each pair will determine the total plate count (no. of colony-forming units/ml of culture).
Be sure you are counting colonies of all sizes. (E. coli typically produces small, lens-
shaped colonies when growing below the surface.) Turn your result in to the instructor
along with the absorbance reading. Be sure you have indicated the sampling time! Results
will be compiled and presented next period.

For Your Assignment:

1. Plot the plate count data on semi-logarithmic graph paper. Rather than generating a
growth curve by connecting the dots, draw the best straight lines through the lag and
exponential phases. (Transitions between the growth phases can be rounded out.)
2. Determine the growth rate and generation time for the particular strain and cultural
conditions in our experiment. Remember that the points you need to calculate these
values are to be taken from the best straight line drawn through the exponential phase. Do
not use individual data points from the class data. Also, be sure to indicate the proper
units (gen/hr or hr/gen). Show your calculations!
3. Plot the absorbance readings on semi-logarithmic graph paper. Note any similarities in
the graph generated and that for the plate count data. Both the absorbance and plate count
plots can be made on the same graph. Do not use the absorbance readings for any
calculations of growth rate or generation time.
 Historical Background
1815 Kirchoff first indicated the presence of enzymes in living systems
1833 A. Payen & Persoz reported a re-usable factor from malt extract and called it
DIASTASE
1837 Berzilius recognised the catalytic nature of biological diastase
1860 L. Pasteur reported fermentation of food stuffs by living cells
1878 Kühne - term 'enzyme': Greek "in yeast"
1894 Emil Fisher studied enzyme specificity for substrate and proposed Lock &
Key theory

1897 Hans & Eduard Buchner – filtrates of yeast extracts could catalyse
fermentation! No need to living cells
Also prepared pure extracts of “Zymase” from yeast

1903 Henri – first successful mathematical model

1913 Michaelis and Menten – Kinetic theory of enz action.
1958 Koshland – “Induced fit” model
1926 James B. Sumner isolated as well as crystallised
“Urease” from jack beans. He was the first one
to postulate the proteinaceous nature of enz
thou the idea remained unnoticed. Was
awarded NOBLE PRIZE
1930 John Northrop and Stanley prepared pure crystals
of PEPSIN & TRYPSIN
1930s JBS Haldane wrote the treaty “ ENZYMES” and
suggested weak interactions b/w E and S
1953 Sanger determined aa seq of INSULIN
1960s AA seq of RNAse
1965 3-dim str of LYSOZYME by X-ray Crystallo.
1965 Monod, Wyman and Changeux – allosteric
regulation
1969 Ist enz chemically synthesised “RIBONUCLEASE”
 How to define enzyme activity?

Physical properties of an enzyme most often is measured by relative rate by

which the substrate is converted to ---> product

• 1 unit ACTIVITY= International unit (IU)

amount enzyme which converts 1 μmole substrate to product per min at
25oC
– e.g. IU= 10 μmole/min

• 1 unit SPECIFIC ACTIVITY

# IU of enzymatic activity per mg of total protein present
– e.g. 10 μmole/min/mg protein or 10 IU/mg protein
 Enzyme assays
• Enzyme assays are laboratory methods for measuring enzymatic
activity. They are vital for the study of enzyme kinetics and
enzyme inhibition.
• The assay is the act of measuring how fast a given (unknown)
amount of enzyme will convert substrate to product (the act of
measuring a velocity).
• Enzyme assays measure either the disappearance of substrate over
time or the appearance of product over time.
• An assay requires to determine the concentration of a product or
substrate at a given time after starting the reaction.
 Measuring enzyme activity
• Enzymes are usually present in very small quantities in
biological fluids
• Therefore, Enzymes are not directly measured
• They are commonly measured in terms of their catalytic activity
• We don’t measure the molecule …
• But we measure how much “work” it performs (catalytic
activity)
• That means the rate at which it catalyzes the conversion of
substrate to product
• The enzymatic activity is a reflection of its concentration
• Activity is proportional to concentration
 Types of Enzyme assay
Enzyme assays can be split into two groups
according to their sampling method:
• Continuous assays, where the assay gives a
continuous reading of activity,
– multiple measurements, usually of absorbance change,
are made during the reaction,
– either at specific time intervals (usually every 30 or 60
seconds)
– or continuously by a continuous-recording
spectrophotometer.
– These assays are advantageous over fixed-time methods
because the linearity of the reaction may be more
adequately verified.
Discontinuous assays, where samples are taken, the
reaction stopped and then the concentration of
substrates/products determined.

– the reaction proceeds for a designated time,

– the reaction is stopped (usually by inactivating the enzyme
with a weak acid),
– a measurement is made of the amount of reaction that has
occurred.
 Features of a good E.A.

• 1. Simple and Specific

• 2. Rapid ( one doesn’t need to wait for hrs or
weeks for the results to appear)
• 3. Sensitive ( v little sample)
• 4. Easy to use
• 5. Economical
 Continuous assays
1. Spectrophotometric:
• The spectrophotometric assay is the most common method of
detection in enzyme assays.
• It uses a spectrophotometer, a machine used to measure the amount
of light a substance's absorbs, to combine kinetic measurements and
Beer's law by calculating the appearance of product or disappearance
of substrate concentrations
• If this light is in the visible region we can actually see a change in the
color of the assay, these are called colorimetric assays.
• UV light is often used, since the common coenzymes NADH and
NADPH absorb UV light in their reduced forms, but do not in their
oxidized forms.
Decrease in absorption at 300nm indicates that the rxn is moving in forward direction in
Ist case and increase in abs at 290nm in the IInd case
 COUPLED REACTIONS:
• In many reactions, changes in substrates or products are not
observable by spectrophotometric methods because they do
not absorb light.

• Even when such enzyme reaction does not result in a change in

the absorbance of light, it can still be possible to use a
spectrophotometric assay for the enzyme by using a coupled
assay.

• These reactions can be measured by coupling them to enzymes

that can be detected via a spectrophotometer.

• Here, the product of one reaction is used as the substrate of

another, easily-detectable reaction.
• This help to follow the first enzymatic reaction.
The assay mixture wud contain– Citrate, aconitase, isocitrate DH and NAD+ with Mn2+
2. Flurescence method/Fluorimetric:
 Fluorescence is when a molecule emits light of one

wavelength after absorbing light of a different wavelength.

 Uses a Fluorometer

 Fluorometric assays use a difference in the fluorescence of

substrate from product to measure the enzyme reaction.

 FLAVIN COMPOUNDS: fluorescence in reduced form and

loose their flouescence in oxidised form

 An example of these assays is again the use of the nucleotide coenzymes
NADH and NADPH.

 Here, the reduced forms are fluorescent and the oxidised forms non-
fluorescent.

 Oxidation reactions can therefore be followed by a decrease in fluorescence

and reduction reactions by an increase.

 More sensitive than spectrophotometric assays, but can suffer from

interference caused by impurities and the instability of many fluorescent
compounds when exposed to light.

 Detection in small quantities

 Non dangerous
3. Calorimetric: is the measurement of the
heat released or absorbed by chemical
reactions.
• These assays are very general, since many
reactions involve some change in heat and
with use of a micro-calorimeter, not much
enzyme or substrate is required.
• These assays can be used to measure
reactions that are impossible to assay in
any other way.
4. Chemiluminescent: is the emission of light
by a chemical reaction.
• Some enzyme reactions produce light and
this can be measured to detect product
formation.
• These types of assay can be extremely
sensitive, since the light produced can be
captured by photographic film over days or
weeks,
• but can be hard to quantify, because not all
the light released by a reaction will be
detected.
 5. MANOMETRIC METHOD

Emission/ absorption of gas is measured by WARBURG Manometer

6. ELECTRODE METHOD
7. POLARIMETRIC METHOD
 Discontinuous assays
Discontinuous assays are when samples are taken from an enzyme
reaction at intervals and the amount of product production or substrate
consumption is measured in these samples by different chemical
methods.
Radiometric: Radiometric assays measure the incorporation of
radioactivity into substrates or its release from substrates.
• The radioactive isotopes most frequently used in these assays are
14C, 32P, 35S and 125I.
• Since radioactive isotopes can allow the specific labelling of a single
atom of a substrate, these assays are both extremely sensitive and
specific.
• They are frequently used in biochemistry and are often the only way
of measuring a specific reaction in crude extracts (the complex
mixtures of enzymes produced when you lyse cells).
• Radioactivity is usually measured in these procedures using a
scintillation counter., which measures the ionizing radiation.
• V sensitive but hazardous
Chromatographic: Chromatographic assays measure
product formation by separating the reaction mixture
into its components by chromatography.
• This is usually done by high-performance liquid
chromatography (HPLC), but can also use the
simpler technique of thin layer chromatography.
• Although this approach can need a lot of material,
its sensitivity can be increased by labelling the
substrates/products with a radioactive or
fluorescent tag.