FISIOLOGI

PERTUMBUHAN PADA
MIKROORGANISME
(2)
Pertumbuhan
Mikroorganisme
 Pertumbuhan pada mikroorganisme
uniseluler, sseperti bakteri, khamir atau
protozoa, adalah peningkatan jumlah
populasi.
Hal tersebut menggambarkan
peningkatan biomassa.
Biomassa adalah jumlah total materi
seluler pada suatu sistem.
 Estimasi Jumlah
Mikroba
Pengukuran jumlah bakteri (bisa
juga untuk mikroorganisme uniseluler
lainnya), ada dua kategori
Menghitung jumlah total sel
1.

Menghitung sel-sel hidup saja

2.
 Microscopic Counts
Total cell counts
Secara umum dilakukan dengan
pengamatan mikroskopik
Menggunakan kaca mikroskopo
khusus yang menandai suatu area
tertentu.
Sel-sel yang tampak pada area
tersebut menggambarkan jumlah sel
per unit volume.
 The method may be made more
accurate by the use of a fluorescent
dye such as acridine orange, which
binds to DNA, and hence avoids
confusion with non-cellular debris.
However, such methods cannot
differentiate between living and non-
living cells.
Their usefulness is further limited by
the fact that the smallest bacteria are
difficult to resolve as individual cells
by light microscopy.
Estimation of total cell numbers by direct microscopic
measurement.
 Metode lain untuk menghitung sel
pada
sampel cair dengan mnggunakan flow
cytometer
Flow cytometer merupakan mesin
yang menngunakan sinar laser dan
perangkat alat eletronik untuk
menghitung sel-sel individu.
Flow cytometer banyak digunakan
dalam menghitung dan membedakan
sampel darah untuk sampel klinik.
Dalam ekologi mikrobiologi juga
digunakan untuk membedakan tipe-
tipe sel untuk tujuan isolasi.
Kelemahan:
Tanpapewarnaan, sel-
selmatitidakdapat
dibedakandengan selhidup
Sel-selyang
kecilsulitdiamatidibawah
mikroskop
Ketepatansulit diperoleh
Memerlukanmikroskopfase
kontrasjikasampel tidakdiwarnai
Sel-selyang motil harusdi immobilisasi
sebelum dihitung
Debris pada sampeldapatdisalah
artikansebagai sel-selmikroba
 Viable Counts
A viable cell count, on the other
hand, is a
measure of the number of living cells
in a
sample, or more specifically those
capable of multiplying and
producing a visible colony of cells.
It is most commonly estimated by
spreading a known volume of cell
suspension onto an agar plate, and
counting the number of colonies that
arise after a period of incubation
 The viable count is also called
plate count
we determine the number of cells in
a sample capable of forming colonies
on a suitable agar medium.
The assumption made in the viable
counting procedure is that each
viable cell can grow and divide to
yield one colony
Thus, colony numbers are
reflection of cell numbers
 Ada dua cara untuk melakukan plate
count:
Spread-plate method
Pour plate method
 Spread-plate Method
Pada spread-plate method, volume
(biasanya 0,1 ml) kultur yang telah
diencerkan disebar di atas permukaan
agar cawan menggunakan spreader.
Cawan kemudian diinkubasi
hingga terjadi pertumbuhan koloni.
Total koloni yang tumbuh dihitung.
 Pour-plate Method
Sejumlahvolume kultur(0,11 ml)
dipipetke dalamcawanPetri steril.
Medium agar yang
masihcairkemudian
dituangkedalam cawantersebut.
Agar dankulturdimixed hinggarata.
Cawankemudian diinkubasi hingga
tumbuh koloni.
Koloniyang tumbuh tidakhanyadiatas
permukaanagar tetapijugabisadidalam
agar.
kesalahanpenghitungan.
Untukmenghindari kesalahan-
kesalahan tersebut,
sampelsebaiknyadiencerkan.
 Kesalahan dalam Plate
Counting
Inkonsistensi plating (pemipetan
yang tidak akurat)
Kurang teraduk secara rata
 Turbidimetric
Methods
Turbidimetric methods measure the
change in
optical density or absorbance of the
medium,
that is, how much a beam of light is
scattered by the suspended particulate
matter .
They can be carried out very quickly by
placing a sample in a spectrophotometer.
 Values of optical density can be directly
related to bacterial numbers or mass by
reference to a standard calibration curve.
 Thus, an estimate of bacterial
numbers, albeit a fairly approximate
one, can be obtained almost
instantaneously during an
experimental procedure.
Other indirect methods of measuring
cell density include wet and dry weight
estimations, and the measurement of
cell components such as total nitrogen,
protein or nucleic acid.
THE KINETICS OF MICROBIAL
GROWTH
 Unicellular organisms divide by
binary fission;
each cell grows to full size, replicates
its
genetic material then divides
into two identical daughter
cells.
By

identical means, two cells divide into

four, four into eight and so on, leading
to an exponential increase in cell
numbers:
 If we were to plot the number of
cells in a population against time,
we would get an exponential curve
 It is more convenient when plotting a
growth curve to plot the logarithm of
cell numbers of against time, giving us
a straight line
 Such exponential growth cannot
continue indefinitely, however, and
growth usually slows down due to
either the supply of nutrients
becoming exhausted, or because
metabolism leads to an
accumulation of harmful waste
substances.
Unicellular growth usually occurs in a
series of different phases
A microbial growth curve. The four
phases of a typical growth curve are
shown.
 1 . Lag phase
When an inoculum of bacteria is first
introduced into some growth medium,
it will
probably require a period to adapt to
its new surroundings the less
familiar these are, the longer the
period of adaptation.
If, for example, the carbon source in
the medium is unfamiliar, the cells will
need time to synthesise the necessary
enzymes for its metabolism.
 The length of the lag phase will also
depend on the age and general
health of the cells in the inoculum.
During this period, there is no net
increase in bacterial numbers,
however the cells are metabolically
active.
 2.Log (exponential)
phase.
When the bacteria have acclimatised
to their
new environment and synthesised the
enzymes needed to utilise the
available substrates, they are
able to start regular division by
binary fission.
This leads to the exponential
increase in numbers referred to
above.
 Under optimal conditions, the
population of cells will double in a
constant and predictable length
of time, known as the generation
(doubling) time.
The value for the widely used laboratory
bacterium E. coli is 20 min, and for most
organisms it is less than an hour.
There are some bacteria,
however, whose generation time is
many hours.
Thus, during
exponential growth, the number of cells
can be expressed as:
 3. Stationary phase
As discussed above, the exponential
phase is
limited by environmental factors, and
as the rate
of growth slows down, the culture
enters the next phase.
The levelling out of the growth curve
does not mean that cell division has
ceased completely, but rather that the
increase due to newly formed cells is
cancelled out by a similar number of cell
deaths.
 Eventually, however, as the death rate
increases, the overall numbers fall and
we enter the final phase of growth.
 Death phase.
As cells die off and the culture is
unable to
replace them, the total population of
viable
cells falls.
This is the death (or decline) phase.
BATCH CULTURE
AND
CONTINUOUS
CULTURE
 The phases of growth described
above apply
to a batch culture. In this form of
culture,
appropriate nutrients and other
conditions are provided for growth,
then an inoculum is added and the
culture incubated.
No further nutrients are added and
no waste products are removed, thus
conditions in the culture are
continually changing.
 This results in active growth being
of limited duration for the reasons
outlined above.
 Sometimes it is desirable to keep the
culture in the logarithmic phase, for
example if the cells are being used to
produce alcohol or antibiotics.
In a continuous culture, nutrient
concentrations and other conditions are
held constant, and the cells are held in a
state of exponential growth.
This is achieved by continuously adding
fresh culture medium and removing equal
volumes of the old.
 Parameters such as pH can also be
monitored and adjusted.
The equipment used to do this is
called a chemostat.
It produces a steady-state culture
whose population size is kept
constant by careful control of flow
rates and nutrient
concentrations.
Continuous culture of microorganisms: the chemostat.