Generation Time Of Bacteria
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Contents
1
Introduction
2
Generation Time
3
Methods Of Cell Mass Measurement
4
Methods Of Cell Number Measurement
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Contents
5
The Bacterial Growth Curve
6
Turbidity
7
The Plate Count
8
Direct Microscopic Method
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Measurement of Bacterial Growth
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Growth is an orderly increase in the quantity of
cellular constituents. It depends upon the ability
of the cell to form new protoplasm from nutrients
available in the environment. In most bacteria,
growth involves increase in cell mass and
number of ribosome, duplication of the bacterial
chromosome, synthesis of new cell wall and
plasma membrane, partitioning of the two
chromosomes, septum formation, and cell
division. This asexual process of reproduction is
called binary fission.
Measurement of Bacterial Growth
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Generation time
Time taken for a cell population to double in
numbers and thus equivalent to the average
length of the cell cycle
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Table 2. Generation times for some common bacteria under
optimal conditions of growth.
Medium
Generation Time
(minutes)
Glucose-salts
17
Bacillus megat
Sucrose-salts
erium
25
Bacterium
Escherichia
coli
Streptococcus
lactis
Streptococcus
lactis
Staphylococcus
aureus
Milk
26
Lactose broth
48
Heart infusion
broth
27-30
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Table 2. Generation times for some common bacteria under
optimal conditions of growth.
Lactobacillus
acidophilus
Milk
66-87
Rhizobium
japonicum
Mannitol-saltsyeast extract
344-461
Mycobacteriu
m
Synthetic
tuberculosis
792-932
Treponema
pallidum
1980
Rabbit testes
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Methods for Measurement of Cell Mass
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Methods for measurement of the cell mass involve
both direct and indirect techniques.
1. Direct physical measurement of dry weight,
wet weight, or volume of cells after centrifugation. .
Wet and Dry Weights: A known volume of a
microbial sample is centrifuged so that the cells
form a pellet and are separated from the medium.
The supernatant medium is discarded and the cell
pellet can be weighed and the mg cells/ml of
culture can be determined (wet weight).
Methods for Measurement of Cell Mass
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- The cell pellet can be dried before weighing to get
mg cell/ml (dry weight).
Filtration ( preparation after staining with acridine orange SEM)
Methods for Measurement of Cell Mass
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2. Direct chemical measurement of some
chemical component of the cells such as total N,
total protein, or total DNA content.
3. Indirect measurement of chemical activity
such as rate of O2 production or consumption,
CO2 production or consumption, etc.
4. Turbidity measurements employ a variety of
instruments to determine the amount of light
scattered by a suspension of cells. Particulate
objects such as bacteria scatter light in proportion
to their numbers.
Methods for Measurement of Cell Numbers
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Measuring techniques involve direct counts,
visually or instrumentally, and indirect viable cell
counts.
1. Direct microscopic counts are possible using
special slides known as counting chambers. Dead
cells cannot be distinguished from living
ones. Only dense suspensions can be counted
(>107 cells per ml), but samples can be
concentrated by centrifugation or filtration to
increase sensitivity.
Methods for Measurement of Cell Numbers
2. Electronic counting chambers count
numbers and measure size distribution of cells.
Such electronic devices are more often used to
count eukaryotic cells such as blood cells.
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Methods for Measurement of Cell Numbers
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3. Indirect viable cell counts, also called plate
counts, involve plating out (spreading) a sample
of a culture on a nutrient agar surface. The
sample or cell suspension can be diluted in a
nontoxic diluent (e.g. water or saline) before
plating. If plated on a suitable medium, each
viable unit grows and forms a colony. Each
colony that can be counted is called a colony
forming unit (cfu) and the number of cfu's is
related to the viable number of bacteria in the
sample.
Table 1. Some Methods used to measure bacterial growth
Method
Application
Comments
Direct microscopic
count
Enumeration of
bacteria in milk
or cellular
vaccines
Cannot distinguish living
from nonliving cells
Viable cell count
(colony counts)
Enumeration of
bacteria in milk,
Very sensitive if plating
foods, soil, water,
conditions are optimal
laboratory
cultures, etc.
Turbidity
measurement
Estimations of large
numbers of
bacteria in clear
liquid media and
broths
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Fast and nondestructive, but
cannot detect cell
densities less than 107
cells per ml
Table 1. Some Methods used to measure bacterial growth
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Measurement of total
cell yield from
very dense
cultures
only practical application
is in the research
laboratory
Measurement of
Biochemical
activity e.g. O2
uptake CO2
production, ATP
production, etc.
Microbiological
assays
Requires a fixed standard
to relate chemical
activity to cell mass
and/or cell numbers
Measurement of dry
weight or wet
weight of cells or
volume of cells
after centrifugation
Measurement of total
cell yield in
cultures
probably more sensitive
than total N or total
protein measurements
Measurement of total
N or protein
The Bacterial Growth Curve
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The Bacterial Growth Curve
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TURBIDITY
When you mix the bacteria growing in a liquid
medium, the culture appears turbid. This is
because a bacterial culture acts as a colloidal
suspension that blocks and reflects light
passing through the culture. Within limits, the
light absorbed by the bacterial suspension will
be directly proportional to the concentration of
cells in the culture. By measuring the amount
of light absorbed by a bacterial suspension,
one can estimate and compare the number of
bacteria present.
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TURBIDITY
The instrument used to measure turbidity is a
spectrophotometer (see Fig. 1). It consists of
a light source, a filter which allows only a
single wavelength of light to pass through, the
sample tube containing the bacterial
suspension, and a photocell that compares the
amount of light coming through the tube with
the total light entering the tube.
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Figure 1
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TURBIDITY
The ability of the culture to block the light can
be expressed as either percent of light
transmitted through the tube or the amount of
light absorbed in the tube (see Fig. 2). The
percent of light transmitted is inversely
proportional to the bacterial concentration.
(The greater the percent transmittance, the
lower the number of bacteria.) The
absorbance (or optical density) is directly
proportional to the cell concentration. (The
greater the absorbance, the greater the
number of bacteria.)
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Figure 2
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TURBIDITY
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Turbidimetric measurement is often correlated
with some other method of cell count, such as
the direct microscopic method or the plate
count. In this way, turbidity can be used as an
indirect measurement of the cell count. For
example:
1. Several dilutions can be made of a bacterial
stock.
2. A Petroff-Hausser counter can then be used to
perform a direct microscopic count on each dilution.
3. Then a spectrophotometer can be used to
measure the absorbance of each dilution tube.
TURBIDITY
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4. A standard curve comparing absorbance to the
number of bacteria can be made by plotting
absorbance versus the number of bacteria per cc
(see Fig. 3).
5. Once the standard curve is completed, any
dilution tube of that organism can be placed in a
spectrophotometer and its absorbance read. Once
the absorbance is determined, the standard curve
can be used to determine the corresponding
number of bacteria per cc (see Fig. 4).
McFarland 0.5 Standard
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Figure 3
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Figure 4
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TURBIDITY
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MATERIALS
Spectrophotometer, sample test, broth tubes , pipette
PROCEDURE
1. Let the spectrophotometer (opposite) "warm up" for at
least 15 minutes so you get the correct reading.
2. Adjust the wavelength to 600nm (green light). Place a
cuvette containing a blank of medium in the machine and
adjust so the reading is zero.
3. Place a cuvette containing your sample in the machine
and read the optical density (O.D.).
N.B. You can only accurately read OD up to a value of about
2.0. Above this level readings are not accurate. If the
reading from your sample is higher than 2.0, make a 10fold dilution and record the OD of this. (Don't forget to
multiply the reading by 10 to take account of the dilution).
TURBIDITY
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TURBIDITY
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THE PLATE COUNT (VIABLE COUNT)
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The number of bacteria in a given sample is
usually too great to be counted directly. However,
if the sample is serially diluted (see Fig. 5) and
then plated out on an agar surface in such a
manner that single isolated bacteria form
visible isolated colonies (see Fig. 6), the
number of colonies can be used as a measure of
the number of viable (living) cells in that known
dilution.
THE PLATE COUNT (VIABLE COUNT)
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However, keep in mind that if the organism
normally forms multiple cell arrangements, such
as chains, the colony-forming unit may consist of
a chain of bacteria rather than a single bacterium.
In addition, some of the bacteria may be clumped
together. Therefore, when doing the plate count
technique, we generally say we are determining
the number of Colony-Forming Units (CFUs) in
that known dilution. By extrapolation, this number
can in turn be used to calculate the number of
CFUs in the original sample.
THE PLATE COUNT (VIABLE COUNT)
Normally, the bacterial sample is diluted by
factors of 10 and plated on agar. After
incubation, the number of colonies on a dilution
plate showing between 30 and 300 colonies
(see Fig. 7) is determined. A plate having 30300 colonies is chosen because this range is
considered statistically significant. If there are
less than 30 colonies on the plate, small errors
in dilution technique or the presence of a few
contaminants will have a drastic effect on the
final count. Likewise, if there are more than 300
colonies on the plate, there will be poor
isolation and colonies will have grown together.
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THE PLATE COUNT (VIABLE COUNT)
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Generally, one wants to determine the number of
CFUs per milliliter (ml) of sample. To find this,
the number of colonies (on a plate having 30-300
colonies) is multiplied by the number of times the
original ml of bacteria was diluted (the dilution
factor of the plate counted). For example, if a
plate containing a 1/1,000,000 dilution of the
original ml of sample shows 150 colonies, then
150 represents 1/1,000,000 the number of CFUs
present in the original ml. Therefore the number of
CFUs per ml in the original sample is found by
multiplying 150 x 1,000,000 as shown in the
formula below:
THE PLATE COUNT (VIABLE COUNT)
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The number of CFUs per ml of sample =
The number of colonies (30-300 plate) X
The dilution factor of the plate counted
In the case of the example above, 150 x
1,000,000 = 150,000,000 CFUs per ml.
For a more accurate count it is advisable to plate
each dilution in duplicate or triplicate and then
find an average count.
Figure 5
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Figure 6 & 7
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THE PLATE COUNT (VIABLE COUNT)
MATERIALS
6 tubes each containing 9.0 ml sterile saline, 3
plates of Trypticase Soy agar, 2 sterile 1.0 ml
pipettes, pipette filler, turntable, bent glass rod,
dish of alcohol
ORGANISM
Trypticase Soy broth culture of Escherichia coli
PROCEDURE
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1.0 Milliliter (ml) Pipette
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Using a Pipette to Remove Bacteria from a Tube
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Using a Vortex Mixer to Mix Bacteria Throughout a Tube
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Using a Pipette to Transfer Bacteria to an Agar Plate
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Using a Bent Glass Rod and a Turntable to Spread a
Bacterial Sample
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Dilution of Bacterial Sample, Step 1
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Dilution of Bacterial Sample, Step 2
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Dilution of Bacterial Sample, Step 3
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Dilution of Bacterial Sample, Step 4
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Dilution of Bacterial Sample, Step 5
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Dilution of Bacterial Sample, Step 6
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Dilution of Bacterial Sample, Step 8
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Dilution of Bacterial Sample, Step 9
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Result
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1. Choose a plate that appears to have between
30 and 300 colonies.
Sample 1/100,000 dilution plate (Figure a).
Sample 1/1,000,000 dilution plate (Figure b).
Sample 1/10,000,000 dilution plate (Figure c).
2. Count the exact number of colonies on that
plate using the colony counter
3. Calculate the number of CFUs per ml of
original sample
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Result
Choice
a
b
c
Result Of Viable Count And O.D.
Time (h):
8
O.D600 :
3.8
Viable Count:
10-1 dilution:
10-2 dilution:
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Result Of Viable Count And O.D.
10-3 dilution:
10-4 dilution:
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Result Of Viable Count And O.D.
10-5 dilution:
10-6 dilution:
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Result Of Viable Count And O.D.
Time (h):
Viable cells/ml:
O.D. 600:
0.5
3.1x101
0.64
1.0
3.2x101
0.65
1.5
8.4x101
0.85
2.0
2.58x102
1.10
2.5
8.57x102
1.36
3.0
2.11x103
1.55
3.5
9.80x103
1.89
4.0
2.04x104
2.05
4.5
5.92x104
2.28
5.0
1.90x105
2.53
5.5
3.15x105
2.64
6.0
1.71x106
3.01
6.5
4.33x106
3.21
7.0
1.42x107
3.47
7.5
3.11x107
3.64
8.0
8.70x107
3.77
8.5
3.39x108
4.16
9.0
1.04x109
4.40
9.5
1.04x109
4.40
10.0
9.68x108
4.39
10.5
1.04x109
4.40
11.0
1.03x109
4.40
11.5
1.04x109
4.40
12.0
4.45x108
4.22
18.0
3.49x102
1.16
24.0
3
0.13
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Calculation of Generation Time
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Because of the very large differences in the number of
cells present at the peak and at the start/end of the
experiment, it's hard to see what's going on from this
graph.
It's much easier to see the whole experiment if you plot the
number of viable cells on a logarithmic scale (or more
simply, plot the log of cell number).
Calculation of Generation Time
the log plot
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Calculation of Generation Time
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As you can see, the indirect method of counting (optical
density) closely parallels the direct method (viable count).
(At later time points, you can see that the number of
viable cells declines faster than the optical density of the
culture.
It will be even easier to see the results if we concentrate
on the first 12 hours of the experiment.
Calculation of Generation Time
The graph of the results reveals FOUR distinct phases
which occur during the growth of a bacterial culture.
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Calculation of Generation Time
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When growing exponentially by binary fission, the increase
in a bacterial population is by geometric progression. If we
start with one cell, when it divides, there are 2 cells in the
first generation, 4 cells in the second generation, 8 cells in
the third generation, and so on. The generation time is
the time interval required for the cells (or population) to
divide.
B = number of bacteria at the beginning of a time interval
b = number of bacteria at the end of the time interval
G=
t
3.3 log b/B
Calculation of Generation Time
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Example: What is the generation time of a bacterial population that
increases from 10,000 cells to 10,000,000 cells in four hours of growth?
G=
t_____
3.3 log b/B
G = 240 minutes
3.3 log 107/104
G = 240 minutes
3.3 x 3
G = 24 minutes
DIRECT MICROSCOPIC METHOD (TOTAL CELL COUNT)
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In the direct microscopic count, a counting
chamber consisting of a ruled slide and a coverslip
is employed.. It is constructed in such a manner
that the coverslip, slide, and ruled lines delimit a
known volume. The number of bacteria in a
small known volume is directly counted
microscopically and the number of bacteria in
the larger original sample is determined by
extrapolation
DIRECT MICROSCOPIC METHOD (TOTAL CELL COUNT)
The Petroff-Hausser counting
chamber for example, has small
etched squares 1/20 of a millimeter
(mm) by 1/20 of a mm and is 1/50
of a mm deep.
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DIRECT MICROSCOPIC METHOD (TOTAL CELL COUNT)
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If the bacteria are diluted, such as by mixing the bacteria with
dye before being placed in the counting chamber, then this
dilution must also be considered in the final calculations.
The formula used for the direct microscopic count is:
The number of bacteria per cc =
The average number of bacteria per large double-lined
square X
The dilution factor of the large square (1,250,000) X
The dilution factor of any dilutions made prior to placing
the sample
in the counting chamber, e.g., mixing the bacteria with
dye
DIRECT MICROSCOPIC METHOD (TOTAL CELL COUNT)
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DIRECT MICROSCOPIC METHOD (TOTAL CELL COUNT)
A variation of the direct microscopic count has
been used to observe and measure growth of
bacteria in natural environments. In order to
detect and prove that thermopiles bacteria were
growing in boiling hot springs, T.D. Brock
immersed microscope slides in the springs and
withdrew them periodically for microscopic
observation. The bacteria in the boiling water
attached to the glass slides naturally and grew
as microcolonies on the surface.
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Methods for Measurement of Cell Numbers
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The MPN Method
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The MPN Method
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The MPN Method
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