Download * Growth and Culturing Of Bacteria: * Binary Fission :

Microbiology, pharmacy.
Dr. Amal Bakri.
th
The 6 lecture
Done by: Deema Mohammad.
---------------------------------------------------------------------------------------------------------------------------------------
 At this stage the mother cell will be
able to divide into two daughter cells;
these daughter cells are small
Chapter 6 ….
* Growth and Culturing Of
comparing to the mother cell;
because the mother cell was in the
stage of increase-in-size.
 The time needed for the mother cell to
undergo division is called the
"Generation Time" which is about 20
minutes to 20 hours, it is genetically
defined and it depends on the
environment conditions that the
microorganism grows in.
 The daughter cells that resulted from
the binary fission are independent and
each of them can also undergo binary
fission in order to give another two
daughter cells and these daughter
cells can also divide, so the type of
growth here is an exponential growth
which will give straight line; if drawn
on a semi-log paper.
 But, sometimes total separation will
not occur , if this happened we will
have the phenomena of arrangement
such as : diplococcic , tetracocci,
staphylococci ,streptococci .
Bacteria:
Bacteria is one of the organisms that can
be cultured in the lab , and in order to be
cultured we have to put it in a medium , like
solid medium "Agar" that we mentioned
when we were talking about Robert Koch .
If we bring a fresh nutrient medium (like
source of carbon and nitrogen …); which is
suitable for a certain microorganism and we
put this microorganism in a suitable
environment conditions such as: PH,
temperature, presence of oxygen …. Etc, the
M.O or Bacteria will start to grow.
The concept of growth in human being
means the increase in size , but regarding to
microorganisms it indicates the increase in
number , when we talk about growth in
bacteria we don't mean the process of meiosis
nor mitosis but instead we mean the
processes of Binary Fission or Budding.
* Binary Fission :
Binary fission is the predominant way of
microorganism increase in number. BUT
How does binary fission occur???
 First there is a mother cell, this cell
will undergo a certain type of growth
within a specific time , it doubles in
size and duplicates its contents
(especially the genetic material )
 Then it makes the septum; which is a
partition that grows between the two
daughter cells that will be separated.
1
Microbiology, pharmacy.
Dr. Amal Bakri.
th
The 6 lecture
Done by: Deema Mohammad.
---------------------------------------------------------------------------------------------------------------------------------------
* Budding:
* Traces of Growth:
Binary fission is an equal cell division , the
mother cell will give two daughter cells , but
we have some microorganisms ( mainly the
yeast which is a fungus that is eukaryotic not
prokaryotic ) that have the phenomena of
budding . Budding is an unequal cell division
in which every cell will not give rise to two
daughter cells only, instead it will give many
buds and every bud will separate and starts as
a new microorganism. BUT How does
Budding Occurs ??
If we want to culture a certain type of
bacteria in the lab we will put it in a fresh
nutrient medium and in a favorable physical
conditions, then we will look for an
indication of growth. In fluid medium the
indication of growth is the turbidity,
sometimes we may have turbidity along with
certain odor ,the presence of bubbles in the
gas test tubes and the production of certain
colors may also be an indication of growth .
In solid medium or agar the indication of
growth is the formation of colonies.
Small cells ( buds ) are developed from the
surface of the stem cell and then they will
separate to form new cells that can then
undergo the same method of increasing in
number which is budding . Budding may be
found in certain bacteria but it is basically
found in "yeast".
* Phases of Growth:
If we constructed an experiment to see
how many bacterial cells are increasing with
time, we will notice that:
" Budding "
 The number of bacteria is increasing
significantly with time then we can
conclude that the conditions are
favorable for the growth of bacteria.
 If we took several samples in flasks
from the culture at different times and
enumerated
the
number
of
microorganisms or the amount of
turbidity (any indication for the
quantity of growth) in each sample at
each time.
 Then, by drawing a graph between the
time (x-axis) and the number of
microorganisms or any indication of
the growth like turbidity ( Y-axis )
we will end up by a graph called
growth curve , this curve has four
2
Microbiology, pharmacy.
Dr. Amal Bakri.
th
The 6 lecture
Done by: Deema Mohammad.
---------------------------------------------------------------------------------------------------------------------------------------
phases which are : lag phase , log
phase (exponential phase )
,
stationary phase and decline phase
(death phase) .


 The length of the phase and density of
the microorganisms in each phase are
dependent on:
a) The
microorganism
itself
(genetically).
b) And on the medium where the
microorganism is cultured, if it is
favorable or not >>>> if the
medium is unfavorable then the
rate of growth will be slow, but if
it is favorable we will have the
maximum growth. The same
microorganism
in
different
conditions would give different
growth phases and different
number of cells.
The microorganisms in this phase
are metabolically very active; they
increase in size and incorporate
molecules from the medium, they
synthesize enzymes and produce
large quantities of energy in the
form of ATP in order to help the
microorganism for doubling of the
components and then for entering
the division phase.
Lag phase's length depends on :
1. The microorganism itself.
2. The suitability of the medium
for this microorganism >>>>
when this medium is more
suitable for the microorganism
the length of lag phase will be
shorter , and when it is less
suitable, the length of lag
phase will be longer because
bacteria will need more time
to adapt to the medium.
#2#
Log
phase
(exponential phase) :
 It comes after the lag phase,
once the microorganism has been
adapted to the medium it will
start to duplicate and increase in
number by binary fission.
 At this phase the growth is at
its maximal level, this phase
appears like straight line when
we draw the growth curve on a
semi-log paper (time is a linear
scale on "x-axis" and the
indicator of growth is a
logarithmic scale on "y-axis").
 The Phases :
#1# Lag phase :
 It is an adaptational phase.
 The number of microorganisms
in it doesn't increase or they
increase slightly.

3
Why we want straight line ???
Microbiology, pharmacy.
Dr. Amal Bakri.
th
The 6 lecture
Done by: Deema Mohammad.
---------------------------------------------------------------------------------------------------------------------------------------
In order to be able to calculate the
doubling time or generation time
or the growth rate for certain
bacteria in the log phase.
For example , if we have 200
microorganisms we take the time
needed for them to be 400 from the
curve by making extrapolation ,
then we can calculate the doubling
time (or generation time) which is
genetically determined for this
certain bacteria , some bacteria
have long generation time and
others have short generation time.
Mycobacterium; which is an acid
fast bacteria, has a waxy layer and
this
waxy
layer
impedes
penetration of nutrients, and
because of lack of nutrient the
growth rate will be very low in the
cell , as well as; this bacteria
makes large expenditure for the
amount of ATP in order to
synthesize the waxy layer so the
growth rate of it will be very low .
will be inoculated at the same time (
zero time ) so all of them will be in
the same age. Then when we take
from this medium an inoculum size of
bacteria ( certain number of bacteria )
and put it in a flask ,all of the
bacterial cells will duplicate together
after the generation time finishes .
This Type of growth is called
Synchronous growth.
 Let's take this example to understand
the stair step pattern: Consider that at
zero
time
the
number
of
microorganisms was 10 and after 20
min (the generation or doubling time )
it became 20 , after another 20 min it
would become 40 , then 60 , 80 , 160
… etc. At the period between the
numbers 20 and 40 for example , the
number of microorganisms will be 20
because
the
number
of
microorganisms is constant in the
generation time , so the shape of the
curve will be in a stair step pattern.
We might do this hypothetical
situation in the lab, by applying
freezing for the microorganisms and
then putting them in the medium at
the same time in order to make the
growth of all of them starts at the
same time.
 Remember that; in lag phase the
microorganism have adapted to the
growth medium and at log phase
growth occurs and microorganisms
are at their most rapid growth rate .
we can calculate from log phase the
genetically determined interval which
is called the generation time .
 Synchronous growth: Theoretically if
the microorganisms divide together at
the same rate and the generation time
was exactly 20 minutes the shape of
the curve will be in a stair step
pattern. This means we are assuming
that when we put bacteria in the
medium (we make inoculation for the
bacteria in the medium) all of them
4
Microbiology, pharmacy.
Dr. Amal Bakri.
th
The 6 lecture
Done by: Deema Mohammad.
---------------------------------------------------------------------------------------------------------------------------------------
 Nonsynchronous Growth :In reality
adapt to these conditions so they
will start to die and the culture
will reach to the stationary phase.
o This phase appears as a plateau;
the number of cells in it is not
increasing nor decreasing, it
instead reaches to a steady state,
at this phase cell division
decreases to a point in which the
rate of cell division will be equal
to the rate of cell dying so the
number of cells which are
dividing becomes equal to the
number of cells which are dying
because the growth conditions
start to be not suitable for the
bacteria in the closed flask .
o In exponential phase we have
death but the rate of it is slower
than the rate of growth , but in
stationary phase they become
equal because the conditions will
be unfavorable for the bacteria .
(in life) and even in the lab not all the
bacteria are doubled exactly after 20
min , some of them are doubled in a
shorter duration and some other in a
longer duration. Also not all of them
were at the same age from the
beginning , some of them may be
passed 5 minutes from the generation
time when were put in the flask. This
means that growth in reality is a
Nonsynchronous growth , it is an
exponential growth ( appears as a
straight line on semi-log paper ). So
,the nonsynchronous growth is the
natural situation in which an actual
culture has some cells dividing at
slightly slower rate from the
generation time and some other
dividing at slightly higher rate from
the generation time (there is
differences between cells in the
dividing rate ). for example if the
generation time is 20 min , some
bacteria will divide in 20 min , some
other on 19 min and some on 21 min ,
so there is slight difference between
them and the result is a continuous
growth curve .

#3# Stationary phase:
o If we put the microorganism in a
flask (closed flask); which is
called closed environment or
batch culture , then when the
microorganism starts to grow , it
will consume the nutrients and the
metabolites will be accumulating
within the flask , these
metabolites are toxic ,so the
presence of toxins and the lack of
nutrients will cause the growth
rate to decrease ,and some
microorganisms are not capable to

5
#4# Decline phase ( death
phase ) :
In this phase the growth rate will
decrease very much to a point in
which the number of cells which are
dying would exceed the number of
existing cells , so the total number of
cells is declining.
The number of live cells stayed
constant in the previous phase
(stationary phase) , but because they
stayed in that closed environment ,
the nutrients will continue to deplete
and toxins will keep accumulating
then we will reach to this phase
(decline phase) in which the
conditions of the environment
become less and less supportive for
cell division and cells lose their
ability to divide and thus die. The
number of living cells decreases
Microbiology, pharmacy.
Dr. Amal Bakri.
th
The 6 lecture
Done by: Deema Mohammad.
---------------------------------------------------------------------------------------------------------------------------------------
exponentially; this means if the
decline phase started at 400 bacterial
cells it will become 200 after certain
time and so on …. .
 Now we talked about the growth phases
(lag , log , stationary and decline phases ) ,
and we said that we reaches the stationary
and decline phases; because microorganisms
are growing in a closed
environment ( Batch culture ) which will
result in nutrient depletion and toxins
accumulation.
 We can prevent the microorganism from
reaching the decline phase by using the
chemostat which is a device helps the
microorganism to grow continuously.
 What is the principle of the Chemostat ?
There is a part used for the continuous
adding of the medium to help for the
continuous growing of the microorganism
and there is also another part to remove the
toxins ( remove a part from the medium that
contains toxins ), so we add a part of the
medium and we remove another part . This
means that we are refreshing the medium all
the time by supplying it with nutrients and
removing the toxins from it , so we can help
the microorganism to continue its growing.
we use this device in the fermentation
processes and in the production of penicillin
in industries , so when we want to produce
antibiotics in continuous culture and we don't
want them to die we will use the Chemostat .
Chemostat ,also known as Biostat
6

If we took microorganism from a
culture even if they were in the
stationary phase and we put them in a
suitable medium these microorganisms
would start growing .

Now what are the characteristics of the
microorganisms in decline phase?
The microorganisms in this phase are
under stressful conditions , if the
microorganisms were from the species
of clostridium and bacillus ( G+
bacteria ) they would form endospores,
the formation of endospores will be
mainly in the decline phase because it
is a stressful phase .When we bring a
microorganism from a culture in the
decline phase and we put it in a nutrient
depleted conditions , this microorganism will be more adapted to these
conditions than other microorganisms;
because it was in a decline phase .
Microbiology, pharmacy.
Dr. Amal Bakri.
th
The 6 lecture
Done by: Deema Mohammad.
---------------------------------------------------------------------------------------------------------------------------------------
*
Methods
of
measuring
CFU/ml CFU stands for colony forming
unit.
Bacterial Growth:
 In order to be able to count the colonies
in the plate, the plate must not be
crowded with colonies nor containing
very low number of them . Because if the
plate was very crowded the colonies
would be very close from each other and
we will be confused if this is a one
colony or they are two colonies , so the
recommended number of colonies in the
plate is from 30-300 , more than 300 the
plate will be crowded , less than 30 the
number of colonies will be statistically
not valid .
When we talked about drawing the growth
curve we said that we put the time on x-axis
and the indication of growth on y- axis , and
we said that the indication of growth can be a
measure of how many bacterial cells are
increased in number ( viable bacteria) or can
be the amount of turbidity . Now we will
learn the methods used to assess the number
of bacteria (the viable bacteria ) , sometimes
we need to know the number of viable and
nonviable bacteria. These methods are :
(1) Standard Plate Count.
(2) Counting Bacteria Using a Bacterial
Colony Counter.
(3) Direct Microscope Counts.
(4) Most probable Number (MPN ) Method.
 If we brought a culture and put it directly
on a plate we would have the confluent
growth; confluent growth means that the
plate would be full of the grown bacteria
which would be seen as a layer and no
obvious colonies could be seen.
 Because we don't want confluent growth
and we want discrete colonies (separated
colonies having a number from 30-300)
to enable us for counting them , we have
to make dilution . the standard method of
dilution that is used in microbiology is
called Ten fold serial dilution ; ten fold
means that every dilution process will be
by decreasing the concentration of cells
by ten times , so if we started by 10^4
cells , after the first dilution we would
end up by 10^3 cells and after the
second dilution the number of cells
would be 10^2 …etc.
(1) Standard Plate Count :
 It is a method of measuring bacterial
growth , it is not the sole method , we
have other methods but it is the most
commonly used method .It is medicinal
valuable method , we use in it agar plate
which is a Petri dish containing a
nutrient medium solidified with agar.
 The principle of this method : we bring a
microbial culture and take a volume
from it and put it on agar , the bacteria
will grow in the form of colonies , after
that we count these colonies per the
volume that we took from the culture and
we can then calculate the number of
colonies in the original culture , when we
apply this method we use the unit of

7
How this dilution is performed ?
1. We bring the culture and we put a 9
ml of saline or nutrient growth in a
test tube.
Microbiology, pharmacy.
Dr. Amal Bakri.
th
The 6 lecture
Done by: Deema Mohammad.
---------------------------------------------------------------------------------------------------------------------------------------
2. We take 1 ml from the culture and we
put it on the 9 ml so the 9 ml saline or
nutrient growth becomes 10 ml.
3. Then we make agitation for the
content in the test tube because
bacteria is a suspension and the
medium must be homogenous for the
next dilution process.
4. We repeat these steps again several
times in order to prepare several test
tubes each of them has a different
concentration from the other.
5. Now we need to know which one
contains the appropriate concentration
for counting the colonies (having a
number of colonies between 30 and
300).
6. In order to determine the appropriate
concentration for counting, we have
to make plating out for all test tubes ,
so the determination is by trial and
error , but after a period the
determination of the appropriate
concentration (turbidity) for counting
the colonies will be experienced and
we will be able to know if this amount
of turbidity is appropriate to count
colonies or not without making
plating out for all test tubes.
7. After choosing the appropriate
concentration we calculate the
number of colonies from the test tube
that we selected and we multiply the
result by the dilution factor to know
the real number of colonies in the
original culture.
8. After performing the dilution and
selecting
the
appropriate
concentration we will transfer the
bacteria to a Petri plate, the transfer
can be done by either the pour plate
method or the spread plate method.
"In this figure it is shown that the color differs
according to the difference in concentration but in fact
the color doesn't change the change will be in the
turbidity" .
# Pour Plate Method:
a) It is made by first adding 1.0 ml of a
diluted culture from a serial dilution
to 9 ml of melted nutrient agar.
b) After the medium is mixed, it is
poured into an empty Petri plate.
c) Once the agar medium cools,
solidifies, and is incubated, colonies
will develop both within the medium
and on its surface.
d) Cells suspended in the melted agar
during preparation may be heatdamaged, and then they will not form
colonies.
e) Those that do grow inside the agar
will form smaller colonies than those
growing on the surface.
f) The growing of bacteria inside agar
make the counting of colonies
somehow difficult , so the counting
using spread plate method will be is
easier .
8
Microbiology, pharmacy.
Dr. Amal Bakri.
th
The 6 lecture
Done by: Deema Mohammad.
---------------------------------------------------------------------------------------------------------------------------------------
# Spread Plate Method:
Eliminates such problems because all cells
remain on the surface of the solid medium.
a. 0.1 ml of the diluted sample is first
placed on the center of a solid, cooled
agar medium.
b. The sample is then spread evenly over
the medium’s surface with a sterile,
bent glass rod.
c. After incubation, colonies develop on
the agar surface.
d. In spread plate method we add 0.1 ml
not 1 ml because if we added 1 ml of
the diluted culture on the surface of
agar which is in the solid state , agar
would not absorb all of the fluid
because 90% of agar is water and
there is too much fluid was added ,so
fluid would remain and the colony
would not be grown in the form of
discrete colonies . But in pour plate
method we add the diluted culture
which is a fluid to molten agar , so
incorporation will happen between
these too fluid states and we will not
face a problem such like that.
e. Then we make incubation upside
down , why ?? Because when the
bacteria die respiration happens , and
there will be a fluid on the top and if
this fluid reached to bacteria, colonies
would be damaged .
 Now , after performing any method of these
(pour and spread plate methods ) and after
making incubation ( usually for 18 hours ) ,
the microorganism will be grown in the
form of colonies , we calculate these
colonies and we multiply the result by the
dilution factor to determine the number of
colonies in the original culture , the result
will be in CFU/ml .
 But a single measurement is not very
reliable, so the procedure is repeated at least
three times, and the results are averaged.
The average number of colonies is
multiplied by the dilution factor to ascertain
the total number of organisms per milliliter
of the original culture.
(2) Counting Bacteria Using
a Bacterial Colony Counter:
It is the second method of counting
bacteria, in this method bacterial colonies are
viewed through a magnifying glass against a
colony-counting grid .
Which of these plates would be the correct
one to count ? Why ?
9
Microbiology, pharmacy.
Dr. Amal Bakri.
th
The 6 lecture
Done by: Deema Mohammad.
---------------------------------------------------------------------------------------------------------------------------------------
The second one , because it is not crowded
nor containing low number of colonies .
(3) Direct Microscope Counts
-
It is another way to measure bacterial
growth.
-
It is also used to count blood cells.
-
In this method a known volume of
medium is introduced into a specially
calibrated, etched glass slide called a
Petroff-Hausser counting chamber
also
known
as
a
hemocytometer.Bacterial
suspension
is
introduced onto chamber with a
calibrated pipette.
-
-
We count under the microscope.
-
The accuracy of direct microscopic
counts depends on the presence of more
than 10 million bacteria per milliliter of
culture .This is because counting
chambers are designed to allow accurate
counts only when large numbers of cells
are present. An accurate count also
requires
that
the
bacteria
be
homogeneously distributed throughout
the culture .
-
This technique has the disadvantage of
generally not distinguishing between
living and dead cells. So we can't use it
do
determine
the
number
of
microorganisms exists in a suspension
because the suspension contains many
particle so we can't distinguish between
bacteria and other particles.
When we use this method ??
We use it when we want to count the number
of microorganisms rapidly and directly ,
because it is a rapid method for counting
microorganisms directly without waiting for
18 hours as in the previous methods .
After the bacteria settle and the liquid
currents have slowed, the microorganisms are counted in specific
calibrate areas. Their number per unit
volume of the original suspension is
calculated by using an appropriate
formula we use here the unit of cell/ml
not colony per ml (CFU/ml) .
(4) Most probable Number
(MPN) Method :
10
Microbiology, pharmacy.
Dr. Amal Bakri.
th
The 6 lecture
Done by: Deema Mohammad.
---------------------------------------------------------------------------------------------------------------------------------------
contain an organism will display growth
by producing gas bubbles and/or by
becoming cloudy, when incubated . And
those tubes in which gas bubbles are
visible will be ( labeled + ) and those that
don't contain an organism ( there is no
gas bubbles ) in them will (labeled - ).
Then the number of organisms in the
original culture is estimated from a most
probable number table.
 As the name indicates this method
depends on the probability, so the
number that will result will not be
accurate . So , we will use it only when
samples contain too few organisms to
give reliable measures of population size
by the standard plate count method, as in
food and water sanitation studies, or
when organisms will not grow on agar.
 With this method, the technician
observes the sample, estimates the
number of cells in it, and makes a series
of progressively greater dilutions.
 As the dilution factor increases, a point
will be reached where some tubes will
contain a single organism and others
none.
 To understand that let's take this
example; [ If we have a 10 ml volume of a
culture , and if this culture contains 1000
microorganism , and we took 1 ml from it ,
the probable number of bacterial content
might be 100 or 99 or 98 because we are
talking about suspension , and when this 1
ml is put in a nutrient medium and
incubated for 18 hours we will have a
growth . If the culture contains 100
microorganism and we took 1 ml from it ,
the probable number of microorganisms
might be 10 or 9 or 8 and when we put the
1 ml in a nutrient medium we will have an
indication of growth (turbidity) . If the
number of microorganisms in the culture is
10 and we took 1ml from it , then the
probable number of microorganisms might
be 1 or 0 , so if we put the 1 ml in a nutrient
medium we might have turbidity and we
might not.]
#REMEMBER# that we can use this method
to estimate the number of microorganisms
content in food , because when a certain type
of food contains a very low number of
microorganisms we can't use the previous
methods . for example , we use this method
to determine the presence of salmonella in
food which might exist in a very low number
and cause infection.
 A typical MPN test consists of five tubes
of each of three volumes (using 10, 1,
and 0.1 ml) of a dilution. Those that
Huda
11
®