Contents

- 1 Bacterial Growth And Growth Curve
- 2 GROWTH:-
- 3 GROWTH CURVE OF BACTERIA
- 4 MEASUREMENT OF BACTERIAL GROWTH
- 5 1. Determination of cell number:
- 5.1 (ii) Counting chamber method:
- 5.2 (iii) Proportional count method:
- 5.3 (iv) Electronic counter method:
- 5.4 (b) Viable count/Indirect method:
- 5.5 (i) Plate count technique:
- 5.6 (ii) Membrane filter count:
- 5.7 2. Determination of cell mass:
- 5.8 (a) Direct method:
- 5.9 (1) Dry weight measurement:
- 5.10 (ii) Measurement of cell nitrogen:
- 5.11 (b) Indirect method:
- 5.12 Turbidimetric method:
- 5.13 3. Determination of cell activity:

## Bacterial Growth And Growth Curve

**Bacterial Growth And Growth Curve**

**INTRODUCTION**

Bacteria can grow in the variety of media which provide a source of energy and basic nutrients that include **carbon**, nitrogen, **phosphorus**, oxygen, hydrogen and sulphur. There are several environmental factors include temperature, pH, oxygen requirement and, osmotic pressure that control the growth of the microorganisms.**Bacterial Growth And Growth Curve**

The term growth is commonly applied for **biological cell **usually refers to changes in the total population rather than an increase in the size of an individual organism. Prokaryote growth is exponential as cells divide by binary fission. The time require to double the population of cell (generation time), depends on the growth rate of organism, media composition and environmental conditions.

Escherichia coli can grow with a doubling time of approximately 20 min. Bacterial growth in liquid medium follows a typical pattern often called the bacterial growth curve. Continuous bacterial growth can be maintained by adding fresh medium continuously in culture vessel.

The number of cells in a culture can be estimated by counting individual cells, diluting and innoculating the culture on solid medium, measuring the light scattered by a culture, weighing wet or dried cells and using biochemical methods to measure cell components.

Bacteria reproduce asexually as well as sexually. A bacterial reproduction takes place by binary fission’ budding, fragmentation and formation of conidiospores or sporangiospores.

**GROWTH:-**

Growth means an orderly increase in all cellular constituents. Increase of mass may not really reflect growth because there is only increase in the size and weight of the cell. Growth is followed by cell division, resulting in an increase in cell number.**Bacterial Growth And Growth Curve**

All actively growing cells mainly multiply by the asexual process of binary fission where two identic daughter cells are formed from a single cell. Microorganisms grow in a variety of physics and chemical environments.**Bacterial Growth And Growth Curve**

Thus, the population increases geometrically or exponentially

growing cells mainly multiply by the asexual process of binary fission where two identic daughter cells are formed from a single cell. Microorganisms grow in a variety of physics and chemical environments.**Bacterial Growth And Growth Curve**

Thus, the population increases geometrically or exponentially

1 -> 2 -> 2 ^ 2 -> 2 ^ 3 2^ 4 ….2^ n

Where, n is the number of generations.

The total population (N) at the end of a given time period would be expressed as follows by assuming no cell death.

N = 1 x 2n

However, under practical conditions, it is very difficult to innoculate one bacterial cell, so the formula is modified as:

N = No x 2n

Where, No is the number of bacteria innoculated at time zero

Taking logarithm on both sides

log N=log N0 + n log2

n= log(N) -log No/log2

= logN-logN0/0.301

n=3.3( log(N) -log No )

If we know the initial population and the population after growth then we can calculate the number of generations by using the above formula.

The generation time (g) can be determined from the number of generations(n) that occur in a particular time (t). The generation time can be calculated by the following formula:

g = t/n = t/3.3(log N-log No)

**GROWTH CURVE OF BACTERIA**

Normal growth curve of bacteria can be determined by innoculating a small number of bacterial cells into a suitable culture medium and counting the bacteria in aliquot samples at regular intervals. When the logarithms of the viable cells are plotted against time on a graphpaper, it gives a typical curve called as bacterial growth curve or growth cycle of bacteria. The resulting curve has four distinct phases (DIAGRAM).

(1)Lag phase

(2)Log or logarithmic or exponential phase

(3)Stationary phase

(4)Death or decline phase.

**Bacterial Growth And Growth Curve**

**Bacterial Growth And Growth Curve**

**Bacterial Growth And Growth Curve**

**Bacterial Growth And Growth Curve**

**(1) Lag phase:-**

When bacteria are innoculated into a fresh medium, the microbial population remains constant for an initial period. The period between innoculation and the beginning of multiplication is known as the lag phase. In this phase, bacterial cells adjust itself to adopt the new environment.

The enzymes, coenzymes and other essential molecules are synthesized by the bacterial cell during this phase. The cells are metabolically and physiologically very active but do not divide. The length of the lag phase depends upon the nature of medium, species of microorganisms and other various physical and chemical growth factors.

**(2)Log or logarithmic or exponential phase:-**

During this phase the cells divide steadily at a constant rate and the log of the number of cells plotted against time results in a straight line. The bacteria multiply at their maximum rate and their number increases exponentially or by progression with time. The time required for one bacterial division during this phase is known as generation time. The number of bacteria present in each generation period is almost twice that in the previous period. The generation time (g) can be determined from the number of generations (n) that occur in a particular time (t).

g=t/n= t/3.3 (log N-log No)

All bacteria do not have the same generation time. Escherichia coli may have 15 to 20 minutes, Staphylococcus aureus, 25 to 30 minutes and Mycobacterium tuberculosis may have 780 to 940 minutes. Generation time is mainly dependent on type of species, nutrients in the medium and physical conditions.

Growth rate (R, number of generations/hour) is the reciprocal of the generation time (g) It is also the slope of the straight line obtained when the log number of cells is plotted against time.

R = 1/g= 3.3 (log N-log N) / t

**(3)Stationary phase:-**

In this phase a constant high population of cells is maintained by a balance between cell division and cell death. The rate of multiplication is reduced because depletion of nutrients, accumulation of toxic waste products, very high concentration of cells and low partial pressure of oxygen. At the cellular level during stationary phase, reserved food materials get consumed, a proportion of ribosomes may be degraded and enzymes may still be synthesized. A viable population count at this stage shows no change.**Bacterial Growth And Growth Curve**

**(4)Death or decline phase:-**

The death or decline phase is also known as the logarithmic death phase. During death phase, the number of viable cells decreases essentially the inverse of growth during the log phase. A variety of conditions contribute to bacterial death but the most important are depletion of nutrients and accumulation of toxic waste products. Bacteria die at different rates, just as they grow at different rates.

Between each of these phases, there is a small curved portion called the transitional period.

The synchronous growth is the growing of micro-organisms in such a way that they are all in same stage of growth phase and all will divide at same time with respect to each other. Thus, the entire population is kept uniform with respect to growth and division. It is not possible to analyse a single bacterial cell to obtain the information about growth behaviour i.e. organisation, differentiation and macromolecular synthesis.

Synchronous culture provides the entire cell crop in the same stage of growth. Measurements made on such cultures are equivalent to the measurements made on individual cells.

Bacterial growth characterized by two separate phases due to the preferential use of one carbon source over another is called diauxic growth. Glucose is utilised first and after the exhaustion of glucose, lactose is utilised. In between, a short lag period is seen.

Diauxic growth can be obtained by preferentially utilising certain carbon substrates. The technique by which microbial population is maintained in the exponential phase of growth in a constant environment is known as continuous culture technique. It is necessary to maintain a bacterial population in the exponential or log phase for research and industrial processes This condition is known as steady state growth.

**MEASUREMENT OF BACTERIAL GROWTH**

Measurement of microbial growth and physiology is an important parameter of microbial study. This measurement to a great extent depends on the number of microorganisms present in a culture. Growth of microorganisms can be quantitatively measured using various techniques. Microbial growth can be measured either by colony counting or cell counting, by weighing the cell (cell mass measurement) or by cell activity measurement.

**1. Determination of cell number:**

**(a) Total count / direct methods:**

(i) Direct microscopic count/ Breed method.

(ii) Counting chamber method / Haemocytometer method.

(iii) Proportional count method.

(iv) Electronic counter method.

**(b) Viable count/indirect method:**

(i) Plate count technique.

(ii) Membrane filter count.

**2. Determination of cell mass:**

**(a) Direct method:**

(i) Dry weight measurement

(ii) Measurement of cell nitrogen.

**(b) Indirect method:**

Turbidimetric method.

**3. Determination of cell activity:**

Measurement of biochemical activity (indirect method),

**1. Determination of cell number:**

**(a) Total count or direct methods:** Total count of micro-organisms in any given suspension indicates the total number of cells which includes both living and dead. The following methods are used for the determination of total count.**Bacterial Growth And Growth Curve**

**(i) Breed method:** This method is also called as ‘Direct microscopic count’. A known volume of cell suspension (0.01 ml) is spread uniformly over a glass slide within a specific area (1 sq. cm). The smear is then fixed, stained, examined under the oil immersion lens and the cells counted. It is impossible to examine entire area (1 sq. cm), therefore practically only a few microscopic areas are observed. Several microscopic fields are counted and an average is taken. The total cells per square cm are then calculated by determining the number of microscopic feilds per square cm.**Bacterial Growth And Growth Curve**

Area of microscopic field = 𝝅r^{2}

= 3.14 * (0.08)2

= 0.02 sq. mm.

(Radius of oil immersion objective = 0.08 mm)

Area of smear = 1 sq. cm= 100 sq. mm.

Number of microscopic fields =100/0.02 = 5,000

Total number of cells/sq. cm= Average number of cells per field x 5,000

**(ii) Counting chamber method:**

##### Bacteria can be counted easily and accurately with the Petroff – Hausser counting chamber or Haemocytometer. Counting of microorganisms can be made rapidly and simply with a minimum equipments.**Bacterial Growth And Growth Curve**

In the haemocytometer or counting chamber method, a minute drop of the culture is placed in a tiny, shallow, rectangular glass slide called Neubar’s slide. This is a special slide accurately ruled into squares that are 1/400 mm² in area. A suspension of unstained bacteria can be counted in the chamber, using a phase contrast microscope.**Bacterial Growth And Growth Curve**

Total number of bacterial cells/mm³ = Number of cells counted x Dilution Area counted x Depth of fluid

Since cells are counted in five bigger squares and such a square is further divided into 16 small squares.

Each small square is equal to 1/400 sq. mm.

Hence,

Area of 5 x 16 = 80 squares = 80/400 mm² = 1/5mm²

It is a special microscopic slide with a counting chamber 1/10mm deep so that

Volume of liquid over a one square = 1/10mm

Dilution 1:200 = 200

Hence,

Number of bacterial cells counted = = N x 200 / 1/5×1/10 x n x 200×50

= Nx 10,000 cells/mm³

**(iii) Proportional count method:**

A standard suspension of particles (plastic beads, number of particles/volume is known) is mixed with an equal amount of cell suspension. This mixed suspension is spread on the slide, fixed and stained. The particles and cells in the microscopic field are counted. An average count of the particles and the cell is taken from the of fields.

e.g. Suppose an average count of 10 particles and 50 cells per field is obtained. If the number of particles in 1 ml of standard suspension is 25,000.

Then the number of cells/ml of suspension is:

50/10 x 25,000 = 1,25,000 cells/ml

**(iv) Electronic counter method:**

An electronic instrument, Coulter counter can be used for the direct enumeration of cells in a suspension. In this technique, the bacterial suspension is passed through a capillary tube. The diameter of this tube is so microscopic that it allows only one cell to pass at a time. The instrument can count thousands of cells in a few seconds. But this system has a disadvantage, in that the Coulter counter counts even dust particles. Hence, the suspension must be absolutely free of any foreign particles.**Bacterial Growth And Growth Curve**

Direct counting methods are rapid and simple. The morphology of cells can also be observed when they are counted under the microscope. The major disadvantage of this method is that it gives the total cell count which includes both viable and non-viable cells. Accuracy also declines with very dense and very dilute suspensions because of clumping and statistical errors, respectively.**Bacterial Growth And Growth Curve**

**(b) Viable count/Indirect method:**

The principle behind viable count is that all viable cells or spores, under suitable growth conditions multiply and that each cell or spore forms a colony. The number of colonies therefore is the same as the number of viable cells present in the original sample.

**(i) Plate count technique:**

In this method, a measured amount of diluted bacterial suspension is introduced into a Petri plate, after which the agar medium (liquid form 45°C) is added. (Fig. 3.2). Immediately, mix the agar medium with the innoculum by rotating the plate. After the solidification of medium, the plates are incubated at 37°C for 24 hours in an inverted position. A plate having 30 to 300 colonies is selected for counting the number of microorganisms.**Bacterial Growth And Growth Curve**

The plate count technique has certain disadvantages. If the suspension contains different microbial species, then all of them may not grow on the medium used and under the specified conditions of growth. If the suspension is not homogenous and contains aggregates of cells, the resulting colony count will be lower than the actual number of microorganisms because aggregate cells produce a single colony.**Bacterial Growth And Growth Curve**

The plate count technique is used routinely for estimation of bacterial populations in milk, water, foods and other materials. The method is highly sensitive, i.e. extremely high or very low viable populations can be counted.**Bacterial Growth And Growth Curve**

**(ii) Membrane filter count:**

This method follows the same principle as in a plate count technique. A diluted suspension of microorganisms is filtered through a millipore filter. The microbes are retained on the filter disc and this disc is placed in a culture medium in a Petri plate. The plates are incubated and the colonies are counted on the filter disc.

Membrane filter method has many advantages over the plate count. A large volume of the sample can be analysed, especially when the number of organisms is very few. Secondly various types of microorganisms can be detected by using selective media in the plates and under different conditions of growth.**Bacterial Growth And Growth Curve**

**2. Determination of cell mass:**

This is a method in which, the weight or mass of the cells is estimated as an indicator of increased growth.

**(a) Direct method:**

**(1) Dry weight measurement:**

This is a simple and direct method of measuring the cell

mass. The culture suspension is centrifuged and the pellet is repeatedly washed to remove all foreign particles. The residue is then dried and weighed. This method is mainly applicable in research investigation and for measuring growth of molds.

### (ii) Measurement of cell nitrogen:

A major chemical constituent of cell is protein of which nitrogen is an important ingredient. A bacterial population or cell crop can be measured in terms of cell nitrogen. The cells are obtained by centrifugation as mentioned in dry weight measurement and the cell nitrogen is estimated by chemical analysis. This method is useful for dense cell suspensions.

### (b) Indirect method:

**Turbidimetric method:**

A most widely used technique for measuring cell mass is by observing the light scattering capacity of the sample. A suspension of unicellular organisms is placed in a colorimeter or spectrophotometer and light passed through it (Fig. 3.3). This instrument works on Beer and Lambert law i.e. light absorbance is directly proportional to turbidity of medium. The absorbance is measured in terms of optical density (OD).

The measurement of optical density does not give value of cell numbers or cell mass but the cell number can be calculated by plotting ‘calibration curve’ which indicates, the direct relationship of optical density and mass. Cell number of an unknown sample can be determined by taking the optical density and comparing it with the corresponding value on the standard curve.

Turbidimetry is a simple and rapid method used for determination of bacterial growth. However, this method is very accurate only with moderate density. It is not possible to measure cultures grown in coloured media or culture that contain suspended material other than bacteria. It must be recognised that dead as well as living cells contribute to turbidity. Applications of different growth measurement methods are given in Table.

### 3. Determination of cell activity:

Measurement of biochemical activity: This is an indirect method to determine the microbial growth. Measurement of a specific chemical change by metabolic activities of microbes can be correlated with the microbial growth. Cell metabolic activity results into formation of any specific metabolite e.g. lactic acid, H₂S, CO₂, enzymes etc. The measurement of these products forms the principle of measurement of cell activity. The amount of acid produced is proportional to the magnitude of cell suspension.

**Bacterial Growth And Growth Curve**

**Bacterial Growth And Growth Curve**

**Bacterial Growth And Growth Curve**

**Bacterial Growth And Growth Curve**