This document provides information about the course AMBE-101 Agricultural Microbiology which is worth 2 credits and includes a 1 hour lecture and 1 hour lab. It discusses various topics related to microbial growth including bacterial reproduction methods, factors affecting growth, growth curves and phases, synchronous growth, and continuous culture techniques like chemostats which are used to maintain bacterial cultures in exponential phase. Continuous culture using a chemostat works by maintaining a constant dilution rate and limiting nutrient concentration to control bacterial growth rate and keep cell density constant.

1. COURSE: AMBE-101
COURSE TITLE: AGRICULTURAL MICROBIOLOGY
CREDITS: 2(1+1)

2. The variety of microbial organisms that make up most
environments on earth are part of a mixed population.

3. Separating the progeny (offspring) of a single
bacterial cell from all other bacteria.
A culture that contains only a single strain of an
organism

4. A population of cells that arise from a single
bacterial cell in one spot.
About 1 million cells are required for a colony
to be easily seen with the naked eye.

5. 1.Transverse binary fission
1.Fragmentation
Spore germination
1.Budding

6. Common, asexual reproduction
Eg: E. coli

7. Extensive filamentous growth followed by the
fragmentation into a small bacillary and coccoid
cells each of which gives new growth Eg : Nocardia

8. At the tip of filamentous growth each spore
will give new organism
Eg: Streptomyces

9. Parent cell remain intact while new cell buds off which again
grows into a new organism
Eg: Rhodopseudomonas, Hyphomicrobium, Saccaromyces

10. Bacterial growth can be defined as an orderly increase in the cell
size as well as increase in the number of cells.
Growth is an essential component of microbial functions because
any microbial cell has only a finite (limited) life span
Bacteria mainly divide by binary fission when a bacterial cell
reaches a certain size it divides to form a daughter cells

11. Growth of bacteria
Orderly increase in the quantity of all cell
components
Bacterial cell
Size
Shape
Weight
Cell
division
Growth
phases

12. Bacteria are of very small size
Studying individual bacterial cell is
impossible
Bacterial
population
is studied
A growth curve
is determined
Small
number of
inoculum
from old
culture
Suitable fresh
culture media
Growth of cell is
monitored at regular
interval of time
The growth
curve is plotted
by using the
logarithm of
cell number
against time

13.  The time required for the cell to divide or time
required for the population to divide is called as
generation time (g = t/n).

14. Growth rate is the change in cell number or mass per unit time.
It is defined as the number of generations per hour. (R = n/t).
It is expressed as ‘R’ which is the reciprocal of generation time ‘g’.

15.  Balanced growth is a condition where all biochemical
constituents are being synthesized at the same relative rates.
 Growth yield is the mass of cells produced per unit of a
limiting nutrient concentration.

16. Open System
Organisms that grow in nature
Nutrients replenished and wastes removed
Closed System
In the lab (i.e. agar plates, broth tubes)
Nutrients will run out and wastes are not
removed.

17. 1.Lag/Acclimatization Phase
1.Log/Exponential Phase
Stationary Phase
1.Death/ Declined Phase

18. Cell division does not occur immediately
 The population remains temporarily unchange
 The individual cells increase in size beyond
their normal dimension
 Physically they are very active, and they are
synthesizing new protoplasm

19.  The bacteria in this new environment may be deficient in
enzymes or coenzymes
 The organism metabolizes but there is a lag in cell
division
Length of lag phase depends up on a variety of factors
 Age of the inoculum
 Composition of growth media
 Environmental factors: Temperature, pH and Aeration

20. No Lag phase
 Growing culture is inoculated
 Fresh medium under same conditions
 Exponential growth continuous at the same rate
Lag phase – occurs
 Inoculum is taken from old culture
 Inoculated in same medium
 All cell are alive in the inoculum
 The cells are damaged by treatment with heat, radiation or toxic chemicals

21. Cells divide at a constant rate
 The log of no. of cells plotted against time results straight line
 The population nearly uniform in terms of chemical
composition of cell, Metabolic activity and other physiological
activity
 Log phase cultures are usually used in biochemical and
physiological studies. Since the generation time is constant

22. The logarithm of the bacterial mass
increases linearly with Time, and
exponential growth phase because the
number of cells increases as an
exponential function of 2n (i.e. 21, 22,
23, 24, 25 and so on).

23. Bacteria are growing in a constant volume of medium of
batch culture, and no fresh nutrients are added,
 The growth of bacterial population eventually ceases, and
the growth curve becomes horizontal
The cessation of growth may be because of the exhaustion
of available nutrients or by the accumulation of inhibitory end
products

24. The total number of viable cells remains constant because of
no further net increase in cell number and the growth rate is
exactly equal
The various cellular components are synthesized at unequal
rates.
 Growth rate of population is zero
 Cryptic growth was observed in the stationery phase

25.  The number of dying cells begins to exceed the number of
new-born cells and thus the number of viable bacterial
cells present in a batch culture starts declining.
 The population is diminished to a tiny fraction of more
resistant cells, or it may die out entirely.
 Death is also exponential, but inverse, as the number of
viable bacterial cells decreases exponentially

27. The Diauxic Growth Curve of E. coli grown in limiting
concentrations of a mixture of glucose and lactose
During the first
phase of exponential growth,
the bacteria utilize glucose as
a source of energy until
all the glucose is exhausted.
After a secondary lag phase,
the lactose is utilized during a second
stage of exponential growth.

28.  A growth pattern wherein every cell in a culture is in the same
metabolic state and divides at one time is defined as
synchronous growth.
 The growth behavior of individual bacteria can, however, be
obtained by the study of synchronous cultures.
 Synchronized cultures must be composed of cells which are all
at the same stage of the bacterial cell cycle.
 Measurements made on synchronized cultures are equivalent
to measurements made on individual cells.
SYNCHRONOUS GROWTH

29.  Several clever techniques have been devised to obtain bacterial populations at the
same stage in the cell cycle.
 Some techniques involve manipulation of environmental parameters which
induces the population to start or stop growth at the same point in the cell cycle,
while others are physical methods for selection of cells that have just completed
the process of binary fission.
 Theoretically, the smallest cells in a bacterial population are those that have just
completed the process of cell division.
 Synchronous cultures rapidly lose synchrony because not all cells in the population
divide at exactly the same size, age or time.

30. Synchronous cultures can be achieved by inoculating the cells and
maintaining the culture at sub optimal temperatures for some time so that
these cells will metabolize slowly but do not divide. But when the
temperature is raised to optimum, all the cells will undergo a synchronized
division.
Another method to have a synchronous culture is to separate the smallest
cells in log phase culture by filtration or by differential centrifugation,
which are reasonably well synchronized with each other as these cells were
just divided before their separation by filtration.

31. Continuous culture is to keep a culture growing indefinitely. This can be done if:
• fresh nutrients are continually supplied
• accumulated cells and waste products are removed at the same rate
• conditions such as temperature and pH are kept at their optimum values
Continuous culture is important in industrial processes that harvest the primary
metabolites of micro-organisms as their products. (Primary metabolites are produced
in greatest quantities when the organisms are growing at their fastest rate).
HOW TO MAINTAIN CULTURES IN
LOG/EXPONENTIAL PHASE

32.  It is possible to maintain a bacterial culture continuously in exponential phase for
a required period provided that the fresh medium is supplied, and toxic products
or metabolic wastes accumulated in the medium are removed. Such continuous
culturing is possible by devices known as ‘chemostat’ and ‘turbidostat’.
 In a photostat, which is used to get steady state cultures of photosynthetic
organisms, growth rates can be controlled by controlling the light supply

33. It is an apparatus used for the continuous culture of bacteria where the cell density is kept constant
by keeping the dilution rate and flow rate of nutrient medium constant. Chemostat was introduced
in the 1950s by three scientists Monod, Novick and Szilard. The sterile nutrient medium is added
into the reservoir along with the microbial cells.
The concentration of the nutrient medium is, in turn, controlled by the dilution rate. In a
Chemostat, the substrate is continuously added into the reservoir, and the by-products are
continuously eluted from the reservoir. Therefore, the bacterial cells will grow at the constant rate
in the exponential phase because the nutrients are not completely depleted and supplied
continuously to the reservoir.

34. The Chemostat works by two factors like

35. Dilution rate: The dilution rate can define as the flow of the nutrient medium
into the reservoir to the culture volume within the vessel. The change in dilution
rate will change the growth rate of bacteria, i.e., both cell growth and cell
density. Cell density remains constant when the dilution and the flow rate of the
medium are kept constant. If the dilution rate alters, cell density and cell growth
will also change.

36. The concentration of limiting nutrient: In Chemostat, an
essential nutrient or amino acid is added in limited quantity. By
limiting the concentration of an essential nutrient, one can
determine the flow rate of the nutrient medium into the reservoir.
Therefore, this essential nutrient will also determine the growth
rate of the bacteria, i.e., whether the cell density is constant or
not. If the concentration of the limiting nutrient alters, the cell
density will also change.

37.  To perform the continuous culture by the chemostat, there are three elements
like substrate reservoir, culture vessel and the spent culture bottle.
 The sterile nutrient medium is continuously added into the culture vessel
via flow regulator.
 The flow regulator allows the constant flow of the fresh substrate into the
culture vessel.
 In the culture vessel, there is an inoculation port from where the inoculum of
bacteria is added.
 Apart from inoculation port, a culture vessel comprises of magnetic
stirrer at the bottom which allows uniform mixing of the cells with the
nutrient medium.
 The remaining dead cells and the by-products from the culture vessel are
released out from the sampling outlet into the spent culture bottle.

38. It also refers as “Biostat”. A turbidostat is a
device that maintains the constant cell density
by controlling the flow rate of the fresh
medium. Turbidostat is an apparatus which was
first introduced by the two
scientists Bryson and Szybalski in the
year 1952.

39. Photoelectric device: A
photoelectric device like
photodiode performs a key role
in the examination of the cell
density in the culture vessel.
Therefore, it determines the
alternation in the concentration
of the medium.
Optical sensing device: An optical sensing
device plays a pivotal role to measure
the turbidity or absorbance of the culture in the
reservoir. Turbidostat uses a turbidometer which
helps to find out the optical density of the
medium. The flow rate or the dilution rate is
monitored by a turbidostat vigorously to make
the turbidity constant.

40.  A turbidostat is an apparatus which includes a sterile
reservoir, culture vessel, photocell and a light source.
 In turbidostat also the fresh nutrient medium is automatically
regulated which maintains predetermined turbidity.
 The fresh nutrient medium is added to the culture vessel through a
valve which controls the flow of the medium.
 The fresh nutrient medium is added when the optical density
increases.
 The cell density is kept constant is the culture vessel by the help
of a photoelectric device.
 The photoelectric device measures the turbidity of the medium by
absorbing the light source.
 The turbidity is monitored throughout the process.

43. 1. Supply of suitable and retrievable nutrients
2. Presence of a source of carbon or other forms of energy
3. Existence of water
4. Existence of the appropriate temperature
5. Appropriate pH of the environment
6. Optimum levels of oxygen & aeration
7. Presence of sulfur (S) and nitrogen (N)
8. Presence of absence of carbon dioxide (CO2)
9. Presence of trace elements and supplemental factors

44. 1. Define growth. Explain in detail different phases of bacterial growth curve with neat
labelled diagram?
2. Write the difference between
Diauxic growth & synchronous growth
Chemostat & turbidostat
3. How microorganisms will multiply, explain with the help of diagrams and examples?