MICROBIAL BIOCHEMISTRY
LESSON 11:
GENERATION TIME , ISOLATION AND PRESERVATION OF BACTERIAL CULTURES
This lesson will cover the following topics in detail:
1. Growth Rate and Generation Time
Relationship between the number of bacteria in a
population at a given time and the number of divisions.
Calculation of Generation Time
2. Isolation of pure cultures and preserving bacterial
cultures
The streak plate method
The Pour plate and Spread plate Technique
Micromanipulator Techniques.
3. Preserving bacterial cultures
Periodic Transfer to Fresh Media
Preservation by Overlaying Cultures with Mineral Oil
Preservation by Lyophilization (Freeze-Drying)
Storage at Low Temperatures
Introduction
An individual bacterial cell will divide and eventually become a
visible mass of cells known as a colony
If instead of a single cell, the solid media is initially populated
with a large number of cells, confluent growth or a lawn of bacteria
will be visible
Growth Rate and Generation Time
In the laboratory, under favorable conditions, a growing
bacterial population doubles at regular intervals. Growth is by
geometric progression: 1, 2, 4, 8, etc. or 20, 21, 22, 23.........2n
(where n = the number of generations). This is called expo-
nential growth. In reality, exponential growth is only part of
the bacterial life cycle, and not representative of the normal
pattern of growth of bacteria in Nature.
Generation time is the time it takes for a population of
bacteria to double in number. For many common bacteria,
the generation time is quite short, 20-60 minutes under
Copy Right: Rai University
2.722
optimum conditions. For most common pathogens in the
body, the generation time is probably closer to 5-10 hours.
Because bacteria grow by geometric progression and most have
a short generation time, they can astronomically increase their
number in a short period of time.
a. Relationship between the number of bacteria in a population
at a given time and the number of divisions
The relationship between the number of bacteria in a popula-
tion at a given time (N), the original number of bacterial cells in
the population (No), and the number of divisions those bacteria
have undergone during that time (n) can be expressed by the
following equation:
Each succeeding generation, assuming no cell death, doubles
the population .The total population N at the end of a given
period would be expressed
N= 1 X 2n (1)
However, under practical conditions the number of bacteria No
inoculated at time zero is not 1 but more likely several thousand
so the formulas
N= No X 2n (2)
n= 3.3(log10 N- log10N0) (3)
Thus the number of generations that have taken place can be
calculated by above equation 3,provided we know the initial
population(No) and population after growth has occurred(N)
For example, Escheichia coli, under optimum conditions, has a
generation time of 20 minutes. If one started with only 10 E.
coli (No = 10) and allowed them to grow for 12 hours (n = 36;
with a generation time of 20 minutes they would divide 3 times
in one hour and 36 times in 12 hours), then plugging the
numbers in the formula, the number of bacteria after 12 hours
(N) would be
10 X 236 = N= 687,194,767,360 E. coli
As mentioned above, bacterial growth rates during the phase of
exponential growth, under standard nutritional conditions
(culture medium, temperature, pH, etc.), define the bacteriums
generation time. Generation times for bacteria vary from about
12 minutes to 24 hours or more. The generation time for E. coli
in the laboratory is 15-20 minutes, but in the intestinal tract, the
coliforms generation time is estimated to be 12-24 hours. For
most known bacteria that can be cultured, generation times
range from about 15 minutes to 1 hour. Symbionts such as
Rhizobium tend to have longer generation times. Many
lithotrophs, such as the nitrifying bacteria, also have long
generation times. Some bacteria that are pathogens, such as
Mycobacterium tuberculosis and Treponema pallidum, have especially
long generation times, and this is thought to be an advantage in
their virulence. Generation times for a few bacteria are shown in
Table 4.1.
81
 Table 4.1. Generation times for some common bacteria under Example: What is the generation time of a bacterial
MICROBIAL BIOCHEMISTRY

optimal conditions of growth. population that increases from 10,000 cells to 10,000,000
Generation cells in four hours of growth?
Bacterium Medium Time
(minutes)
Escherichia coli Glucose-salts 17
Bacillus
Sucrose-salts 25
megaterium
Streptococcus
Milk 26
lactis
Streptococcus
Lactose broth 48
lactis
Staphylococcus Heart infusion
27-30
aureus broth
Lactobacillus
Milk 66-87
acidophilus
Rhizobium Mannitol-salts- G= t
344-461 3.3 log N/ N0
japonicum yeast extract
Mycobacterium G= 240 minutes
Synthetic 792-932
tuberculosis 3.3 log 107/104
Treponema G = 240 minutes
Rabbit testes 1980
pallidum
3.3 x 3
G = 24 minutes
b. Calculation of Generation Time
2. Isolation of Pure Bacterial Cultures
When growing exponentially by binary fission, the increase in a
bacterial population is by geometric progression. If we start A. The Streak Plate Technique
with one cell, when it divides, there are 2 cells in the first Most infectious materials such as pus, sputum and urine,
generation, 4 cells in the second generation, 8 cells in the third contain several different kinds of bacteria, so do samples of
generation, and so on. The generation time is the time interval soil, water or food. If these material are plated out onto the
required for the cells (or population) to divide. surface of solid medium, colonies will form that are exact copies
of the original organism. A visible colony theoretically arises
G (generation time) = t(time, in minutes or hours)/n(number
from a single spore or vegetative cell or from group of same
of generations)
microorganisms attached to one another in clumps or chains.
G = t/n Microbial colonies often have a distinctive appearance that
G = generation time (time for the cells to divide) distinguishes one microbe from another. The bacteria must be
t = time interval in hours or minutes distributed widely enough so that the colonies are visibly
separated from each other.
N0 = number of bacteria at the beginning of a time interval
Most bacteriological work requires pure culture, or clones of
N = number of bacteria at the end of the time interval bacteria. The isolation method most commonly used to get
n = number of generations (number of times the cell popula- pure cultures is the streak plate method. A sterile inoculating
tion doubles during the time interval) loop is dipped into a mixed culture that contains more than
N = N0 x 2n (This equation is an expression of growth by one type of microbe and is streaked in a pattern over the surface
binary fission) of the nutrient medium .As the pattern is traced, bacteria are
G = t/n rubbed off the loop onto the medium. The last cells to be
rubbed off the loop are far enough apart to grow in to isolated
Solve for n: colonies ( Fig 4.1). These colonies can be picked up with an
log N = log N0 + nlog2 inoculating loop and transferred to a test tube of nutrient
n = log N - log N0 medium to form a pure culture containing one type of
log2 bacterium.
n = log N - log N0 The streak plate method works well when the organism to be
.301 isolated is present in large numbers relative to the total popula-
n = 3.3 log N/ N0 tion. However, when the microbe to be isolated is present only
in small numbers, its numbers must be greatly increased by
G = t/n
selective enrichment before it can be isolated with the streak
Solve for G plate method.
G= t The assumption is often made that a colony is derived from a
3.3 log N/ N0 single cell ,and, therefore, that the colony is a clone. However,

Copy Right: Rai University

82 2.722
this is not necessarily true. With species in which the cells form a
characteristic grouping during cell division (for example, clumps
of staphylococci or chains of streptococci), the colony may
develop from a group of cells rather than from a single cell.
Although not a clone, such a colony is nevertheless a pure
culture if it contains only one kind of organism.
One should recognize that subculturing a colony from a single
streak plate does not automatically assure purity. The colony
may have been derived from two or more different kinds of
bacteria. For example, when we attempt to isolate slime- or
chain-producing bacteria, contaminants may be found to have
adhered to the slime or to have been enmeshed in the network
of chains, thereby resulting in impure colonies. The use of
selective media can also lead to impure colonies. Although the
growth of contaminants is inhibited on selective media, low
numbers of viable cells may still be present, and such cells can
be subcultured along with a colony. For these reasons, it is
advisable to streak a culture several times in succession, prefer-
ably on nonselective media, in order to ensure purity.
Streaking Bacterial Stocks
Principle
Bacterial cells are streaked onto a medium to obtain an indepen-
dent isolate. This is done to reduce the likelihood of working
with a culture which has become contaminated and/or has
accumulated mutations.
Procedure
1. The cells can be streaked from another plate a stab or from a
frozen glycerol stock. Pick up a slightly visible amount of
cells on the rounded end of a sterile flat toothpick.
2. To transfer the cells to a media plate begin the streak at one
edge of the plate. Press the side of the toothpick containing
the cells to the agar plates surface and quickly streak the
toothpick back and forth across part of the plates surface
(see #1 in the diagram below). The streaks should lie near
one another but should not cross over previous streaks.
3. Turn the toothpick over to the side that did not originally
touch the cells or use a new toothpick for the next streaks.
These streaks should start by crossing over the last streak
then proceeding as before into new areas of the agar plate
(#2 in the diagram below).
4. Repeat in new territory on the agar plate with a fresh
toothpick ( #3 in the diagram). The cells will be distributed
on the plate in decreasing concentration through the streaks
and no matter how many cells were on the toothpick to
begin with there should be an area on the streaked plate that
will produce isolated colonies.
Copy Right: Rai University
2.722
5. Cover the plate invert it and incubate at 37 degrees C
MICROBIAL BIOCHEMISTRY
overnight. The plate should appear to have solid streaks of
cells as well as isolated colonies. Pick a colony well isolated
from the others to use in subsequent work.
Fig 4.1 Plate appearance after overnight growth.
b. The Pour Plate and Spread Plate Technique
In both of these methods the mixed culture is first diluted to
provide only a few cells per milliliter before being used to
inoculate media. Since the number of bacteria in the specimen is
not known beforehand, a series of dilutions is made so that at
least D:!).e of the dilutions will contain a suitably sparse
concentration of cells.
In the pour-plate method the mixed culture is diluted directly
in tubes of liquid (cooled) agar medium (see Fig. 4.2). The
medium is maintained in a liquid state at a temperature of 45C
to allow thorough distribution of the inoculum. The inocu-
lated medium is dispensed intu Petri dishes, allowed to solidify,
and then incubated. A series of agar plates showing decreasing
numbers of colonies resulting from the dilution procedure in
the pour-plate technique is shown in Fig. 8-4. The pour-plate
technique has certain disadvantages. For instance, some at the
organisms are trapped beneath the surface of the medium
when it gels, and therefore both surface and subsurface colonies
develop. The subsurface colonies can be transferred to fresh
medum only by first digging them out of the agar with a sterile
instrument. Another disadvantage is that the organisms being
isolated must be able to withstand temporary exposure (0 the
45C temperature of the liquid agar medium; for instance, the
pour-plate method would be unsuitable for isolating psychro-
philic bacteria.
A
1 loop
1 loop
B
Original bacterial 1 loop ..
suspension
C
Step 1 Step2 Step 3
Figure 4.2. Pour-plate technique is used for isolation of pure
cultures of bacteria. Step 1: One loopful of original suspension
is transferred to tube A (liquid, cooled agar medium). Tube A is
rolled between the hands to effect thorough mixing of
inoculum. Similar transfers are made from A to B to C. Step 2:
83
 Contents of each tube are poured into separate Petri dishes. 3. Preserving Bacterial Cultures
MICROBIAL BIOCHEMISTRY

Step 3: After incubation, plates are examined for the one which
a. Periodic Transfer to Fresh Media
contains well-separat~d colonies. From this plate, pure cultures
of bacteria can be isolated by transferring a portion of a colony Strains can be maintained by periodically preparing a fresh stock
to a tube of sterile medium. culture from the previous stock culture. The culture medium,
the storage temperature, and the time interval at which the
In the spread-plate method the mixed culture is not diluted in
transfers are made vary with the species and must be ascer1ained
the culture medium; instead it is diluted in a series of tubes
beforehand. The temperature and the type of medium chosen
containing a sterile liquid, usually water or physiological saline.
should support a slow rather than a rapid rate of growth so
A sample is removed from each tube, placed onto the surface of
that the time interval between transfers can be as long as
an agar plate, and spread evenly over the surface by means of a
possible. Many of the more common heterotrophs remain
sterile, bent glass rod. On at least one plate of the series the
viable for several weeks or months on a medium like nutrient
bacteria will be in numbers sufficiently low as to allow the
agar. The transfer method has the disadvantage of failing to
development of well-separated colonies (see Fig. 4.3). In
prevent changes in the characteristics of a strain due to the
contrast to the pour-plate technique, only surface colonies
development of variants and mutants.
develop; moreover, the organisms are not required to withstand
the temperature of liquid agar. b. Preservation by Overlaying Cultures with Mineral
Oil
Unlike the streak-plate technique, the pour-plate and the spread-
plate techniques may be performed in a quantitative manner to Many bacteria can be successfully preserved by covering the
determine the number of bacteria (of a particular type) present growth on an agar slant with sterile mineral oil. The oil must
in a specimen . cover the slant completely; to ensure this, the oil should be
about 1/2 in above the tip of the slanted surface. Maintenance
Figure 4.3. Spread plate showing colonies of two different
of viability under this treatment varies with the species (1
bacterial species. A dilution of the mixed culture was spread
month to 2 years). This method of maintenance has the unique
over the surface with a glass rod. The large; dark colonies are
advantage that you can remove some of the growth under the
Serratia marcescens, which has, a brick-red pigment, and the
oil with a transfer needle, inoculate a fresh medium, and still
smaller, fight colonies are Micrococcus luteus, which has a
preserve the original culture. The simplicity of the method
lemon-yellow pigment. (Courtesy of Naval Biological Labora-
makes it attractive, but changes in the characteristics of a strain
tory.)
can still, occur Figure 4.5 illustrates a culture collection
c. Micromanipulator Techniques: A device called maintained by this technique.
micromanipulator can be used in conjunction with a
microscope to pick a single bacterial cell from a mixed culture
Figure 4.4. This micromanipulator permits the operator to
control the movements of micropipette or a microprobe (a
fine needle) so that single cells can be isolated. This skill
requires skilled operators and is reserved for studies in which
clones must be obtained unequivocally.

Figure 4.5. A culture collection maintained by overlaying

Figure 4.4 Isolation of single bacterial cells. (A) Micromanipu-
lator equipment and microscope. The micromanipulator is
equipped with probes that can position on objects a few um in
size. The manipulation of the probes is done while viewing the
specimen through the microscope. (Micromanipulator Com-
pany.)
cultures with mineral oil.
c. Preservation by Lyophilization (Freeze-Drying)
Refrigeration can be used for short-term storage of bacterial
cultures. Two common methods of preserving microbial
cultures for long period are deep-freezing and lyphilization.
Deep-freezing is a process in which a pure culture of microbes
is placed in a suspending liquid and quick-frozen at temperature
ranging from 50oC to 95oC.The culture can usually be thawed
and cultured even several years later.
Most bacteria die if cultures are allowed to become dry, al-
though spore- and cyst-formers can remain viable for many
years. However, freeze-drying can satisfactorily preserve many
kinds of bacteria that would be killed by ordinary drying.

Copy Right: Rai University

84 2.722
During lyophilization (freeze-drying), a suspension of
microbes is quickly frozen at temperatures ranging from 60 to
- 78C and the water is removed by a high vacuum
(sublimation).While under vacuum, the container is sealed by a
high temperature torch. The remaining powder like residue that
contains the surviving microbes can be stored for years. The
organisms can be revived at any time by hydration with a
suitable liquid nutrient medium.
In this process a dense cell suspension is placed in small vials
and frozen at - 60 to - 78C. The vials are then connected to a
high-vacuum line. The ice present in the frozen suspension
sublimes under the vacuum, i.e evaporates without first going
through a liquid water phase. This results in dehydration of the
bacteria with a minimum of damage to delicate cell structures.
The vials are then sealed off under a vacuum and stored in a
refrigerator. One arrangement of equipment employed to
lyophilize cultures is shown in Fig. 4.6. Many species of bacteria
preserved by this method have remained viable and unchanged
in their characteristics for more than 30 years. Only minimal
storage space is required; hundreds of lyophilized cultures can
be stored in a small area. Furthermore, the small vials. can be
sent conveniently through the mail to other microbiology
laboratories when packaged in special sealed mailing containers.
Lyophilized cultures are revived by opening the vials, adding
liquid medium, and transferring the re-hydrated culture to a
suitable growth medium.
Figure 4.2 (A)
Figure 4.6 Lyophilization process for preservation of cultures.
(A) A simple apparatus for lyophilization. Small cotton-
plugged vials containing frozen suspensions of bacteria are
placed in the glass flask, which is attached to a condenser. The
condenser is connected to a high-vacuum pump. The bacteria
become desiccated as the ice in the frozen suspension sublimes
directly to water vapor. The vapor is trapped on the cold surface
of the condenser, thereby preventing it from entering the
vacuum line and contaminating the pump oil
d. Storage at Low Temperatures
The ready availability of liquid nitrogen has provided the
microbiologist with another very useful means for long-term
preservation of cultures. In this procedure cells are prepared as a
dense suspension in a medium containing a cryoprotective
agent such as glycerol or dimethyl sulfoxide (DMSO), which
prevents cell damage due to ice crystal formation during the
subsequent steps. The cell suspension is sealed into small
ampoules or vials and then frozen at a controlled rate to -150C.
The ampoules or vials are- then stored in a liquid nitrogen
Copy Right: Rai University
2.722
refrigerator (essentially a large tank having vacuum-insulated
MICROBIAL BIOCHEMISTRY
walls; see Fig. 8-9) either by immersion in the liquid nitrogen (-
196C) or by storage in the-gas phase above the liquid nitrogen
(-150C). The liquid nitrogen method has been successful with
many species that cannot be preserved by lyophilization, and
most species can remain viable under these conditions for 10 to
30 years or more without undergoing change in their characteris-
tics. However, the method is relatively expensive, since the
liquid nitrogen in the refrigerators must be replenished at
regular intervals to replace the loss due to evaporation
Breifing
Mathematical Calculation
Growth equation:
n = log N - No / 0.301
Streaking solid media plates
bacteria can be introduced onto solid media by a sterile
transfer tool, such as a wire loop (nichrome wire), or
autoclaved toothpick, which has been dipped into a bacterial
culture
such a transfer contains thousands of individual bacterial cells
it is desirable to grow colonies from individual cells rather
than from a large population
This is done to avoid the takeover of the strain by potential
wild-type revertants
streaking is a method to isolate individual cells for growth
on solid media from an inoculation which originally contains
thousands of cells
Questions?
Q1.What is the generation time of a bacterial culture? Would
you expect generation time to be a constant characteristic of a
bacterial species. Explain.
Q2.If a fresh culture medium is inoculated with 500 bacterial
cells per ml, how many cells will be present after seven
generations?
Q3.A bacterium having a generation time of 30 minutes will
undergo_____ cell divisions per hour. During five hours of
growth, the cells would double______ times. Beginning
with 20 cells per ml at time 0, how many cells would be
present at 5 hours?
Q4.An otherwise typical bacterial cell increases from one cell to
256 cells in 10 hours. What is the generation time of this
organism?
Q5. a-c refer to the following set of data.
A standard bacterial growth curve can be plotted from the
following information:
A bacterium was inoculated into fresh broth medium and
incubated at 37 degrees under aerobic conditions. Viable cell
counts (plate counts) over a period of 24 hours resulted in
findings of 100 cells/ml at the time of inoculation, 100 cells/
ml at 2 hours, 100 cells/ml at 4 hours, 103 cells/ml at 6 hours,
104 cells/ml at 8 hours, 106 cells/ml at 12 hours, 108 cells/ml at
16 hours, 109 cells/ml at 18 hours, 109 cells/ml at 20 hours, 109
cells/ml at 22 hours, 108 cells/ml at 24 hours.
85
 a. What coordinates are used to label the graph that plots the
MICROBIAL BIOCHEMISTRY

bacterial growth curve?

A. growth rate vs time
B. growth rate vs generation time
C. optical density vs time
D. number of bacteria vs time
E. log number of viable cells vs time
b. The length of the lag phase of the culture is
A. 2 hours
B. 4 hours
C. 8 hours
D. 14 hours
E. 18 hours
c. The generation time of this bacterial culture is approximately
A. 18 minutes
B. 30 minutes
C. 36 minutes
D. 48 minutes
E. 72 minutes
Notes

Copy Right: Rai University

86 2.722