INTRODUCTION
In a rather broader perspective the
‘bacteria’ are markedly distinguished by their inherent extreme
metabolic diversity
; whereas, a few of them may conveniently sustain themselves exclusively on
‘inorganic substances’
by strategically making use of such specific pathways which are practically
absent amongst the plant as well as animal kingdoms.
Based upon the aforesaid statement of facts one may individually explore and exploit the various
cardinal factor(s) that essentially govern the
nutrition, cultivation (growth), and isolation of bacteria,
actinomycetes
, fungi and viruses as enumerated under :
BACTERIA
The nutrition, cultivation (growth), and isolation of bacteria shall be dealt with in the sections
that follows :
Nutrition of Microorganisms (Bacteria)
Interestingly, the
microbial cell represents an extremely complex entity, which is essentially
comprised of approximately 70% of by its weight as water, and the remaining 30% by its weight as the
solid components. Besides, the
two major gaseous constituents viz., oxygen (O2) and hydrogen (H2) the
microbial cell
predominantly consists of four other major elements, namely : Carbon (C), nitrogen
(N)
, sulphur (S), and phosphorus (P). In fact, the six aforesaid constituents almost account for 95% of
the ensuing
cellular dry weight. The various other elements that also present but in relatively much
lesser quantum are : Na
+, K+, Ca2+, Mg2+, Mn2+, Co2+, Zn2+, Cu2+, Fe3+ and Mo4+. Based on these
critical observations and findings one may infer that the microorganisms significantly require an exceptionally
large number of elements for its adequate survival as well as growth (
i.e., cultivation).The following displays the various chemical composition of an Escherichia coli cell.
It has been amply proved and established that
carbon represents an integral component of almost
all organic cell material ; and, hence, constitutes practically half of the ensuing dry cell weight.
Nitrogen
is more or less largely confined to the proteins, coenzymes, and the nucleic acids (DNA,
RNA).
Sulphur is a vital component of proteins and coenzymes ; whereas, phosphorus designates as
the major component of the nucleic acids.
It is, however, pertinent to mention here that as to date it is not possible to ascertain the precise
requirement of various elements
viz. C, N, S and O, by virtue of the fact that most bacteria predominantly
differ with regard to the actual chemical form wherein these elements are invariably consumed as
nutrients.
Cultivation (Growth) of Bacteria
The
cultivation (growth) of bacteria may be defined, as — ‘a systematic progressive increase
in the cellular components’
. Nevertheless, an appreciable enhancement in ‘mass’ exclusively may not
always reflect the element of growth because bacteria at certain specific instances may accumulate
enough mass without a corresponding increment in the actual
cell number. In the latest scenario the
terms
‘balanced growth’ has been introduced which essentially draws a line between the so called‘orderly growth’ and the ‘disorderly growth’.
Campbell defined
‘balanced growth’ as — ‘the two-fold increase of each biochemical unit of
the cells very much within the prevailing time period by a single division without having a
slightest change in the rate of growth’
. However, one may accomplish theoretically cultures with a
‘balanced growth’
having a more or less stable and constant chemical composition, but it is rather next
to impossible to achieve this.
Following are some of the cardinal aspects of
cultivation of bacteria, such as :
Binary Fission
It has been established beyond any reasonable doubt that the most abundantly available means of
bacterial cultivation (reproduction) is
binary fission, that is, one specific cell undergoes division to
give rise to the formation of
two cells.
Now, if one may start the process with a
single bacterium, the corresponding enhancement in
population is given by the following
geometric progression :
1 —
→ 2 —→ 22 —→ 23 —→ 2′ —→ 25 —→ 26 —→ 2n
where,
n = Number of generations.
Assuming that there is
no cell death at all, each succeding generation shall give rise to double its
population
. Thus, the total population ‘N’ at the end of a specific given time period may be expressed
as follows :
N = 1 × 2
n ...(a)
Furthermore, under normal experimental parameters, the actual number of organisms N
0 inoculated
at time
‘zero’ is not ‘1’ but most probably may range between several thousands. In such a situation,
the aforesaid ‘formula’ may now be given as follows :
N = N
0 × 2n ...(b)
Now, solving Eqn. (
b) for the value of ‘n’, we may have :
log
10 N = log10 N0 + n log10 2
or
n =
10 10 0
10
log N log N
log 2
−
...(
c)
Substituting the value of log
10 2 (i.e., 0.301) in Eqn. (c) above, we may ultimately simplify the
equation to :
n
= log10 N log10 N0
0.301
−
or
n = 3.3 (log10 N – log10 N0) ...(d)
Application of Eqn. (
d), one may calculate quite easily and conveniently the actual ‘number of
generations’
which have virtually occurred, based on the precise data with respect to the following two
experimental stages, namely :
(
i) Initial population of bacteria, and
Normal Growth Curve (or Growth Cycle) of Microorganisms :
Importantly, one may describe the pattern of
normal growth curve (or growth cycle) of microorganisms
by having an assumption that a
‘single microorganism’ after being carefully inoculated into
a sterilized flask of liquid culture medium aseptically which is incubated subsequently for its apparent
desired growth in due course of time. At this point in time the very
‘seeded bacterium’ would have a
tendency to undergo
‘binary fission’ (see Section 2.2.1), thereby safely plunging into an era of rapid
growth and development whereby the bacterial cells shall undergo
‘multiplication in an exponential
manner’
. Thus, during the said span of rapid growth, if one takes into consideration the theoretical
number of microorganisms
that must be present at different intervals of time, and finally plot the data
thus generated in the following
two ways, namely :
(
a) Logarithm of number of microorganisms, and(b) Arithmatic number of microorganisms Vs time.(
ii) Population after growth affected.
Normal Growth Curve (or Growth Cycle) of Microorganisms :
Importantly, one may describe the pattern of
normal growth curve (or growth cycle) of microorganisms
by having an assumption that a
‘single microorganism’ after being carefully inoculated into
a sterilized flask of liquid culture medium aseptically which is incubated subsequently for its apparent
desired growth in due course of time. At this point in time the very
‘seeded bacterium’ would have a
tendency to undergo
‘binary fission’ (see Section 2.2.1), thereby safely plunging into an era of rapid
growth and development whereby the bacterial cells shall undergo
‘multiplication in an exponential
manner’
. Thus, during the said span of rapid growth, if one takes into consideration the theoretical
number of microorganisms
that must be present at different intervals of time, and finally plot the data
thus generated in the following
two ways, namely :
(
a) Logarithm of number of microorganisms, and(b) Arithmatic number of microorganisms Vs time.
•
Population gets increased regularly,
•
Polulation gets doubled at regular time intervals (usually referred to as the ‘generation time’)
while under incubation, and
•
Exponential growth designates only one particular segment of the ‘growth cycle’ of apopulation.
The Lag Phase of Microbial Growth
In actual practice, however, when one carefully inoculates a fresh-sterilized culture medium with
a stipulated number of cells, subsequently finds out the ensuing
bacterial population intermittently
under the following
two experimental parameters :
(
a) during an incubation period of 24 hours, and
(
b) plot the curve between logarithms of the number of available microbial cells Vs time (inminutes),
Curve A : Lag Phase ; Curve B : Exponential Phase or Log (Logarithmic) Phase ;
Curve C : Stationary Phase ; and Curve D : Death (or Decline) Phase.
From Fig. 5.2. one may distinctly observe the following
salient features :
•
Lag Phase — i.e., at initial stages there exist almost little growth of bacteria,
•
Exponential (or Log) Phase — i.e., showing a rather rapid growth,
•
Stationary Phase — i.e., depicting clearly a levelling off growth of microbes, and
•
Death (or Decline) Phase — i.e., showing a clear cut decline in the viable population of
microorganisms.
Translational Periods Between Various Growth Phases
A close look at Fig. 5. 2 would reveal that a culture invariably proceeds rather slowly from one
particular phase of growth to the next phase. Therefore, it categorically ascertains the fact that all the
bacterial cells are definitely not exposed to an identical physiological condition specifically as they
approach toward the end of a given phase of growth. Importantly, it involves critically the
‘time factor’essentially needed for certain bacteria to enable them catch up with the others in a crowd of microbes.
Synchronous Growth
It has been duly observed that there are quite a few vital aspects with regard to the
internsive
microbiological research
wherein it might be possible to decepher and hence relate the various aspects
of the bacterial growth, organization, and above all the precise differentiation to a specific stage of the
cell-division cycle
. However, it may not be practically feasible to carry out the analysis of a single
bacterium
due to its extremely small size. At this stage if one may assume that all the available cells in
a
culture medium were supposed to be having almost the same stage of the specific division cycle, the
ultimate result from the ensuing analysis of the cell crop might be logically interpreted equivalent to a
single cell
. With the advent of several well elaborated and practised laboratory methodologies one
could conveniently manipulate the on going growth of cultures whereby all the available cells shall
essentially be in the
same status of their ensuing growth cycle. i.e., having a synchronus growth.
Salient Features :
The various salient features pertaining to the aforesaid synchronous growth
are as stated under :
(1)
Synchrony invariably lasts for a few generations, because even the daughters of a single
cell usually get out of phase with one another very much within a short span.
(2) The prevailing population may be synchronized judiciously by carrying out the manipulation
either of the chemical composition of the culture medium or by altering the physical
environment of the culture medium.
Example :
The above hypothesis may be expatiated by subjecting the bacterial cells to a careful
inoculation into a culture medium duly maintained at a
suboptimal temperature. Interestingly, under
these prevailing circumstances after a certian lapse of time the bacterial cells shall
metabolize gradually,
but certainly
may not undergo cell division. However, when the temperature is enhanced from thesuboptimal level to the elevated stage, the bacterial cells shall undergo a synchronized division.
(3) Interestingly, the smallest microbial cells that are usually present in a
specific log-phase
culture
do happen to be those that have just divided ; and hence, lead to the most abundantly
known method of synchronization. Besides, when these cells are duly subjected to separation
either by
differential centrifugation or by simple filtration, they are far better synchronized
with each other explicitely.
Fig. 5.3 illustrates the observed actual growth pattern of a definite population of the available
synchronized bacterial cells as given under.
The steplike growth pattern, as depicted in Fig. 5.3 clearly shows that practically all the cells of
the population invariably undergo division at about the same time.
Effect of Nutrient Concentration
Vs Growth Rate of Bacterial Culture
In order to have a comprehensive understanding with regard to the effect of the nutrient concentration
(substrate) upon the ensuing growth rate of the bacterial culture one should duly take into consideration
the existing relationship between the
exponential growth (R) and the nutrient (substrate)concentration, which eventually does not hold a simple linear relationship
Growth Determining Techniques
As to date there are several both
direct and indirect methodologies whereby one may accomplish
the following
two cardinal aspects with respect to the growth of microorganisms, namely :
(
a) to determine growth of bacteria, and
(
b) to determine growth rates of microorganisms.
In actual practice, however, the
‘choice of the method’ will exclusively depend upon whether
the candidate organism is either
bacteria or fungi ; besides, several inherent characteristic features ofthe microorganisms, for instance : clumping*.