Nutrition, Cultivation and Isolation of Bacteria

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Chapter: Pharmaceutical Microbiology : Nutrition, Cultivation and Isolation of microorganisms : Bacteria-Actinomycetes-Fungi-Viruses

The nutrition, cultivation (growth), and isolation of bacteria shall be dealt with in the sections that follows :



The nutrition, cultivation (growth), and isolation of bacteria shall be dealt with in the sections that follows :


1. 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 excep-tionally large number of elements for its adequate survival as well as growth (i.e., cultivation).


The following Table 5.1 displays the various chemical composition of an Escherichia coli cell.


[Adapted From : Tauro P et al. An Introduction to Microbiology, New Age International, New Delhi, 2004]


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 predomi-nantly differ with regard to the actual chemical form wherein these elements are invariably consumed as nutrients.


2. 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 at all, each succeding generation shall give rise to double its death population. Thus, the total at the end of a specific given time period may be expressed population ‘N’ as follows :

N = 1 × 2n   ...(a)

Furthermore, under normal experimental parameters, the actual number of organisms N0 inocu-lated at time ‘zero’ is not ‘1’ but most probably may range between several thousands. In such a situa-tion, the aforesaid ‘formula’ may now be given as follows :

N = N0 × 2n...(b)

Now, solving Eqn. (b) for the value of ‘n’, we may have :

log10 N = log10 N0 + n log10 2

Substituting the value of log10 2 (i.e., 0.301) in Eqn. (c) above, we may ultimately simplify the equation to :

n = 3.3 ( log10 N - log10 N0 )

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

(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 micro-organisms 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.


one would invariably obtain the ‘Curve’ as depicted in Figure : 5.1.


From Fig. 5.1 one may rightly derive the following three valued and critical informations, such as :


·        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 a population.


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 (in minutes),


thereby obtaining a typical curve as illustrated in Fig. 5.2.

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 manipula-tion 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 gradu-ally, but certainly may not undergo cell division. However, when the temperature is enhanced from the suboptimal 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 syn-chronized 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 concen-tration (substrate) upon the ensuing growth rate of the bacterial culture one should duly take into consid-eration the existing relationship between the exponential growth (R) and the nutrient (substrate) concentration, which eventually does not hold a simple linear relationship as shown in Fig. 5.4.


Growth Determining Techniques


As to date there are several both direct and indirect methodologies whereby one may accom-plish 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 of the microorganisms, for instance : clumping*.


Direct Method. It essentially comprises of the following vital steps :

·        To determine precisely the enhancement of the cell number,

·        Dry weight of bacteiral cell vis-a-vis function of time (minutes/hours), and

·        Enhancement in any other cellular component vis-a-vis function of time (minutes/hours).


Indirect Method : It predominantly involves the inclusion of two important ‘Optical Density’ measurements, such as : (/) optical density, and (/'/') optical turbidity (using Nephelometer).


In short, the direct methods for the determination of the ultimate growth by the aid of cell number are invariably utilized with such organisms as : (a) bacteria - that undergo binary fission ; and (b) yeast - that undergo the ‘budding’* phenomenon.


Summararily, the indirect methods for the precise determination either bacteria or yeast may be duly accomplished by the use of Turbidometers (for translucent liquids), and Colorimeters (for transparent liquids), whereby the observed density of the ensing cell suspension may be measured accurately.

Fig. 5.5 illustrates the kind of curves which one obtains when the ensuing growth is invariably measured in a liquid medium by various methods. It has been amply proved and established that the actual changes which take place in the cell population strategically after the inoculation into the fresh growth medium are represented more accurately and precisely by the dry weight or optical density measurements.


3. Isolation of Bacteria

The isolation of ‘Bacteria’ may be accomplished in several recognized and well-established methods, such as :


(a) Selective and diagnostic media,


(b) Bismuth sulphite agar, and


(c) Selective media for staphylococci.


The aforesaid three methodologies invariably used for the isolation of bacteria shall be treated individually in the sections that follows :


(a) Selective and Diagnostic Media


McConkey’s medium was first and foremost introduced in 1995 so as to isolate Enterobacteriaceae from faeces, urine, foods, water etc. The medium essentially comprises of several nutrients viz., bile salts, lactose, and an appropriate indicator.


Bile salts categorically serve as an important natural surface-active agent which, fails to inhibit the growth of the Enterobacteriaceae, but distinctly inhibits the growth of the specific Gram-positive microorganisms that are probably present in the material to be examined.


Lactose aids in the production of ‘acid’ from E. coli and A. aerogenes upon this culture medium thereby changing the colour of the suitable indicator added ; besides, the said two microorganisms may also adsorb a certain amount of the indicator that may eventually get duly precipitated around the growing cells. Importantly, the bacteria responsible for causing typhoid and paratyphoid fever, and bacillary dysentery fail to ferment lactose ; and, therefore, the resulting colonies produced duly by these organisms appear to be absolutely transparent.

Modifications of McConkey’s Medium — are as stated under :

(1) Synthetic surface-active agent may replace the ‘Bile Salts’,

(2) Selectivity of McConkey’s medium may be enhanced significantly by the incorporation of inhibitory dyes e.g. crystal violet, neutral red. In fact, these dyes particularly suppress the growth of Gram-positive microorganisms viz., Staphylococci.


(b). Bismuth Sulphite Agar


The discovery of the bismuth sulphite agar medium dates back to 1920s for the identification of Salmonella typhi in pharmaceutical preparations, foods, faeces, urine, and water. It essentially comprises of a ‘buffered nutrient agar’ consisting of bismuth sulphate, ferrous sulphate, and an indicator brilliant green.


E. coli gets inhibited at a concentration 0.0025% of brilliant green employed, whereas another organism Salmonella typhi shall grow predominantly. It has been observed that bismuth sulphite does exert certain degree of inhibitory effect upon E. coli.


S. typhi, in the presence of glucose, causes reduction of bismuth sulphite to the corresponding bismuth sulphide (i.e., a black compound), thereby ascertaining the fact that the investigative organism may generate H2S from the S-containing amino acids in the medium, which in turn shall interact with FeSO4 to produce a distinct black precipitate of FeS (ferrous sulphide).


(c) Selective Media for Staphylococci


The presence of the Staphylococci organisms in various specimens viz., pharmaceutical prod-ucts, food items, and pathological specimens, may ultimately cause food poisoning as well as serious systemic infections.


A few typical examples of selective media for various organisms are as follows :


(i) Enterobacteria — a surface active agent serves as the main-selector.


(ii) Staphylococci — NaCl and LiCl serve as the main selectors. Besides, Staphylococci in general are found to be sufficiently tolerant of NaCl concentrations upto an extent of 7.5%.


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