DISSCUSION

Microbial growth is usually studied as a population not an individual. Individual

cells divide in a process called binary fission where two daughter cells arise from
a single cell. The daughter cells are identical except for the occasional mutation.
Binary fission requires cell mass to increase, chromosome to replicate, cell wall
to be synthesized and cell to divide into two cells. (Hassan Abdullah, Shakinaz
Desa and Zulkaflee, 2009)
The growth of E. coli under ideal condition at 37 . When a small
amount of bacteria is inoculated into a large amount of media the bacteria enter
into a lag phase. During this time, there is little to no growth because cells are
adjusting to their new environment. During the logarithmic (log) phase, cells
grow exponentially and the cell number doubles. During the stationary phase,
cell division matches cell death and the total cell number remains constant. After
an extended period of time or more than 24 hours, cells enter the death phase
and the culture slowly dies due to depletion of nutrients and build-up of wastes.
(Yuyun Tan, 2014)
E. coli are used extensively in molecular biology labs as a means of
manipulating DNA and cloning genes. It is often useful to know the concentration
of cells in a bacterial culture and also to know the growth rate of that culture.
There are several methods by which the number of bacterial cells in a culture
can be determined. One method is to make serial dilutions of the culture, spread
a known volume of the dilutions on agar plates and then incubate the plates at
37 overnight. A single cell will divide many times, giving rise to a colony
that is visible to the unaided eye. The number of colonies are counted at every 1
hour, 20 hours and 24 hours. The total of colony were divided by every specified
hour. Then sum up the total and divided by 3 to find the average. This method is
very accurate, but time consuming. (Reece J.B., Urry L.A., Cain M.L., Wasserman
S.A., Minorsky P.V. and Jackson R.B., 2011)
Then quantitative method is used to measure the microbial growth
because as bacteria are unicellular and divide asexually the growth of the
population can be followed either by the changes in number of cells or weight of
cell mass. Besides, quantitative method is divide into two which is direct method

and indirect method. The example of direct method is gravimeter measurement

and

direct

microscopic

count

while

indirect

method

is

turbidimetric

measurements, method viable count and chemical analysis.

In this experiment, the indirect method which is viable count is used to

measure the growth of E.coli. In viable count you usually pour 0.1 ml of a sample
onto the surface of a nutrient agar plate. In addition nutrient agar plate is a
microbiological growth medium commonly used to assess viable bacterial growth
of a sample. It is usually consist of 0.5% peptone, 0.25% yeast extract, 0.1%
glucose, 1.5% agar and the pH adjusted to neutral at 25 C.
Furthermore, viable count method is one of the most common methods of
determining cell number is the viable plate count. A sample to be counted is
diluted in a solution that will not harm the microbe, yet does not support its
growth. In most cases, a 0.1-1.0 ml portion of this first dilution is then diluted a
further 10-fold, giving a total dilution of 100-fold. Moreover, in the spread-plate
technique some of the highest dilutions which is lowest bacterial density are then
taken and spread with a sterile glass rod onto a solid medium that will support
the growth of the microbe. It is important that the liquid spread onto the plate
soaks into the agar. This prevents left over liquid on the surface from causing
colonies to run together and the need for dry plates restricts the volume to 0.1
ml or less. On the other side, the advantages of the viable count method are it is
sensitivity which is theoretically, a single cell can be detected and it allows for
inspection and positive identification of the organism counted while the
disadvantages are only living cells develop colonies that are counted, clumps or
chains of cells develop into a single colony and colonies develop only from those
organisms for which the cultural conditions are suitable for growth. (Arthur Koch.
1995)
The factors which are essential for bacterial growth are nutrients, pH of
the medium, gaseous requirement and temperature. Nutrients in growth media
must contain all the elements necessary for the synthesis of new organisms. In
general the following must be provided hydrogen donors and acceptors, carbon
source, nitrogen source, minerals such as sulphur and phosphorus.

The pH of the medium is the one of the factor of microbial growth. Most
pathogenic bacteria grow best in pH 7.2-7.4. Vibno cholerae can grow in pH 8.29.0. The gaseous requirement for bacteria to grow also important. When it comes
with the role of oxygen, bacteria may be classified into four groups on oxygen
requirement. There are aerobes and anaerobes. Aerobes are bacteria that cannot
grow without oxygen, for example Mycobacterium tuberculosis. Anaerobes are
the bacteria that only grow in absence of free oxygen, for examples Clostridium
and Bacteroides. Anaerobic bacteria do not grow in the

presence of oxygen. They do not use oxygen for growth and metabolism but
obtain their energy from fermentation reactions. Anaerobic bacteria are killed by
oxygen or toxic oxygen radicals. Multiple mechanisms play role for oxygen. They
do not have cytochrome systems for oxygen metabolism, they may have low
levels of superoxide dismutase and they may or may not have catalase. The
other gaseous that needed by bacteria is carbon dioxide. All bacteria require
carbon dioxide for their growth. Most bacteria produce carbon dioxide. (Reece
J.B., Urry L.A., Cain M.L., Wasserman S.A., Minorsky P.V. and Jackson R.B., 2011)
Bacteria also grow when the temperature is optimum. Mesophilic bacteria
grow best at 30-37C. Optimum temperature for growth of common pathogenic
bacteria is 37C. Bacteria of a species will not grow but may remain alive at a
maximum and a minimum temperature.
After 20 hours, the bacteria grow and make colony then it spread over the
agar. One of the petri dish produce least number of colony of bacteria. There
were some reasons why bacteria grew least than the other two petri dish. Some
of the errors must be overheating effects. Overheating is a common cause of pH
drift, darkening, precipitation, poor gel strength and reduced bacteriological
performance. These effects can also be produced if a concentrated 'pool' of
ingredients at the bottom of the container is heated. All culture media should be
in solution before sterilization. This will reduce the occurrence of Mallard-type
reactions taking place in the medium. Overheating effects will occur if agar
media are allowed to gel in bottles and are later steamed to melt the agar. They
will also occur if molten media are held at 50C for more than 3 hours before
use. Agar media with pH values at or below 5.0 are very sensitive to overheating

in any form because the agar is hydrolysed and the gel strength fails. (Arthur
Koch. 1995)
Most of the difficulties in culture media sterilization occur when large unit
volumes of media must be processed. The best solution to this problem is the
use of a culture medium preparatory. These semi-automatic processors, made by
New Brunswick and other manufacturers overcome the problem of poor heat
penetration of agar by a continuous stirring or agitation of the medium during
the heating phase. They are strongly recommended because of their high
efficiency and minimal damage to culture media. Agar not in solution, poor
mixing and pH too low for agar.

The precautions that must be aware were ccultured bacteria must be

stored at the specified temperature which it was incubated at 37 , under
specified conditions and not longer than the shelf-life periods appropriate to each
product. Loss of moisture from agar plates is a common cause of poor
bacteriological performance. Do not pre-incubate all plates overnight as a
sterility check and only obviously wet plates require pre-inoculation drying.
Ensure that all plates are incubated in a humid environment. Most of the
products supplied have no known risks except those usually associated with fine
powders.
However, to prevent the risk of inhaling fine dust it is recommended that
masks should be worn whilst handling dehydrated media. Lastly, use gloves
when carried out this experiment. It was to prevent from getting a direct contact
between the Bunsen burner and alcohol. (Prescott M. Lansing, Harley P. John and
Klein A. Donald, 2011)