Pir Mehr Ali Shah

Arid Agriculture University, Rawalpindi

Office of the controller of Examinations
Mid Exam / Spring 2021 (Paper Duration 12 hours)
To be filled by Teacher

Course No.: MICRO-102 Course title: General Veterinary Microbiology

Total Marks: 12 Date of Exam: 29-04-2021
Degree: DVM Semester: 2nd Section: Morning A/B
Marks
Q. No. 1 2 3 4 5 6 7 8 9 10 Obtained/
TotalMarks
Marks
Obtained
Total Marks in Words:

Name of the teacher who taught the course: Dr. Saif-ur-Rehman/Dr. Zahid Manzoor
Signature of teacher / Examiner:

To be filled by Student

Registration No.: 20-ARID-3222 Name: Bisma Aslam

Answer the following questions.

Q. No. 1.
Describe different bacterial structures that are present outside the cytoplasmic membrane
of Gram negative bacteria. Also write note on the functions of each structure.
(Marks 04)
Answer:
Structures outside to the cytoplasmic membranes
The bacterial structures that are present outside the plasma membrane of Gram-negative bacteria
include:
i. Flagella
ii. Axial skeleton
iii. Glycocalyx
iv. Cell wall
v. Pilli
vi. Fimbriae

 Flagella:
A flagellum is a vhip like locomotory organ. It is present in some prokaryotes.
Structure:
A flagellum has three parts.
 Filament: It is long and constant in diameter and is made up of a protein “flagellin”. This
protein is arranged in the form of several chains around hollow structure.
 Hook: It is slightly wider than filament and is made up of a different protein.
 Basal body: attach flagella to the plasma membrane. In gram-negative bacterium two pairs
of rings in basal body are present.
Function:
Flagella functions to propel bacteria, helps in motility.

 Axial skeleton:
These are bundles of filament present beneath “outer sheath”. The rotation of filament produces
a movement of outer sheath that propel spirochete in a spiral movement. These are also known as
endoflagella.
Functions:
They help to move effectively in body fluids. For example: Treponema pallidum.

 Glycocalyx:
Glycocalyx is a sugar coat present on the surface of gram-negative bacterium. It is
viscous and gelatinous in its nature. Glycocalyx is composed of polysaccharide or polypeptides and
sometime it is made up of both of them. Glycocalyx is present in two forms:

 If glycocalyx is firmly attached to the surface of cell then it is called as capsule.

 If glycocalyx is loosely attached to the surface then it is called as slime layer.
Functions of glycocalyx:
i. Capsule:
 Helps in escaping from phagocytes.
 Capsule in certain species contribute in bacterial virulence.
 When energy reserves in a bacterial cell are low, capsule break it down and release sugars that
are used as a source of nutrition.
ii. Slime layer:
 They play an important role in the formation of different variety of Biofilms.
 Helps in attachment to different surfaces.

 Fimbriae:
They occur at the pole of bacterial cell or distributed over the entire surface of cell. They have the
tendency to adhere to each other and to surfaces. They vary from few to several hundred per cell.
Function:
 They are involved in the formation of biofilms and other aggregations on surface of liquids, glass
and rocks.
 They help bacteria to adhere to epithelial surfaces in body. For example: Neisseria gonorrhea,
help microbes colonize mucous membrane.

 Pili:
They are shorter, straighter, and thinner than fimbriae. They consist of pilin protein which is
arranged helically.
Function:
Involve in transfer of genetic material which is also known as conjugation.

 Cell wall:
The cell wall of gram-negative bacteria consists of an outer membrane and one or a very few layers
of peptidoglycan. Between the outer membrane and the plasma membrane a gel-like fluid called
periplasm id present. This periplasm has a high concentration of degradative enzymes and transport
proteins. Cell wall don’t have teichoic acid. Cell wall is more susceptible to mechanical breakage.
Outer membrane of gram negative has lipopolysaccharides (LPS). It is a large complex molecule
having lipids, carbohydrates and three main components.

 Lipid A is the lipid portion embedded in the top layer of outer membrane.
 Core polysaccharide is attached to lipid A and contain unusual sugars.
 O polysaccharides is composed of sugar molecule and extends outward from the core
polysaccharide.
Porins forms channels and permit the passage of molecules such as nucleotides, disaccharides,
peptides, amino acids, vitamin B12 and iron.
Functions:
 When a gram-negative bacterium dies, lipid A from outer membrane of bacteria is released and
functions as an endotoxin.
 Lipid A is responsible for symptoms associated with infections by gram-negative bacterium such
as fever, shock, dilating of blood vessels.
 Outer membrane gives strong negative charge to the gram-negative bacteria.
 Outer membrane protects bacterial cell from different chemicals which can damage gram-
positive bacteria.

Q.No. 2.
What type of physical conditions will you provide to grow bacteria in the laboratory?
Write note on different growth activities of bacterial population in culture medium.
(Marks 04)
Answer:
Bacterial growth refers to increase in number of bacterial cells instead of increase in size. This
growing bacterial cell form colonies (group of bacterial cells that can be seen by unaided eye) of
hundreds and thousands of bacteria. For the growth of a bacterial cell, there are some
requirements. These requirements are divided into two categories:
i. Physical requirements
ii. Chemical requirements

Physical Requirement for bacterial growth:

To grow bacteria on culture medium some physical conditions that we have to provide to bacterial
cell are:

 Temperature:
To grow a bacterial cell well on culture media we must provide bacterial cell a temperature
suitable for its growth. Most of the microorganisms (bacteria) can survive well on a temperature
that is suitable to humans. But there are bacteria, that are capable of survival at extreme
temperature at which most of the eukaryotes are unable to survive. Most of bacteria grow
within a specific range of temperature showing very poor growth at high and low temperature
extreme of their range. Each bacterial species grows at particular optimum (where cell grows
best), minimum (lowest temperature where cell can survive) and maximum temperature
(highest temperature where cell can survive).
On the basis of specific range of temperature, microorganisms are classified as:

 Thermophiles:
These are heat loving microorganisms. They mostly show optimum growth in between 50 to
60 C (temperature of water from a hot water tap). Thermophiles are adapted to live in
 sunlit soil, compost piles and in hot springs. They cannot grow at a temperature less than
45C
Hyperthermophiles: Some bacteria have optimum growth at 80 C or higher fall in this
category. These bacteria are mostly found in ocean and volcanic vents. Members of Archaea
are hyperthermophiles.

 Psychrophiles:
These are cold loving microbes and are capable of growing at 0 C. There are two distinct
groups of these microbes.
True psychrophiles: They can grow at 0C but have optimum growth temperature of about
15C These are sensitive to higher temperature even to room temperature (25 C).
Psychrotrophs: They can grow at 0C and have an optimum temperature of about 20-30 C.
they cannot grow at temperature above 40 C. They are more common and are responsible
for low temperature food spoilage even in refrigerator.

 Mesophiles:
These are the most common type of microorganisms having optimum temperature of about
25-40C. These include most pathogens and common spoilage organisms. They are adapted
to live in the bodies of animals. Incubators for clinical culture of pathogenic bacteria are
usually set at 37C.
Thermodurics: These types can resist brief exposure to a relatively high temperature and
can survive pasteurization temperature.

 Osmotic pressure:
Cells are 80-90% water. Concentration of dissolved substance in environment effects microbial
growth. Plasmolysis is a condition in which large amount of dissolved substance in environment
draw water out of cell causing its shrinkage. That’s why high osmotic pressure prevents cell
growth. Halophiles require high concentration of dissolved salts to grow.
Extreme\obligate halophiles: adapted well to high salt concentration that they actually require
for growth.
Facultative halophiles: higher salt concentration is not required for growth. Able to grow at salt
concentration up to 2%. Some also tolerate 15% of salt concentration.

 pH:
Most bacteria grow at neutral pH (6.5-7.5). these organisms are classified as:
 Acidophiles:
 optimum pH is highly acidic (0.1-5.4). e.g., Lactobacillus produces lactic acid, tolerate mild
acidity.
 Alkaphiles:
Optimum pH is toward basic (7 to 12 or higher). E.g., Vibrio cholerae and Alkaligenes
faecalis has optimal pH 9.
 Neutrophiles:
Optimum pH ranges about pH 7 (±1.5unit pH). Most bacteria are neutrophilic.

Growth activities of bacterial population

Bacteria generally reproduce by binary fission but a few of them reproduce by budding. Some
filamentous species reproduce by producing chains od conidiospores and some simply by producing
fragment. The time required by a bacterial cell to divide is called generation time. Most of the
bacteria have generation time of about 1-3 hours. The generation time for E. coli is 20 minutes.
Phases of growth:

There are four basic phases of bacterial growth.

 Lag Phase:
It is the period of “little or no cell division”. In this phase the cells are not dominant they undergo
a specific period of metabolic activity synthesizing enzymes and various metabolites. The change
in the number of cells is very little and no immediate reproduction is shown by cell in the new
medium. It is the period of adjustment to new situations. i.e., there is tooling-up activity but no
immediate increase in population. May last from 1 hour to several days.
 Log Phase:
In this phase, cells begin to divide and enter a period of rapid growth. Generation time of cell
reaches a constant minimum. This is the time when the cell is most active and is at its highest
metabolic activity. As generation time is constant, so the logarithm plot of growth is straight.
 Stationary Phase:
It is the “period of equilibrium”. Population size begin to stabilize. The rate of growth slows
down and number of deaths of microbial cell balances the number of new cells. The factor that
stops the exponential growth of cells include accumulation of waste material, exhaustion of
nutrients, and harmful changes in pH.
 Death Phase:
When the number of cells dying exceeds the rate of production of new cell, bacteria enter the
phase of death or decline phase. The phase continues the population of bacterial cell reach a
tiny fraction of number of cells or until the entire population dies out.

Q. No. 3.
Give brief answers of the following questions; (Marks 04)
a. What is the logic behind presence of outer membrane in the cell wall of Gram negative bacteria
only?
Answer:
In gram-negative bacteria, due to absence of the teichoic acid, there is no external support or extra
strength to cell which may leads to the mechanical breakage. Thus, to overcome this situation the
outer membrane is present which is not present in gram-positive bacteria. The outer membrane
provides mechanical support to the cell thus decreasing the mechanical stress. The outer membrane
of Gram-negative bacteria invariably contains a unique component, lipopolysaccharide (LPS) in
addition to proteins and phospholipids. The LPS molecule is toxic and is classified as an endotoxin
that elicits a strong immune response when the bacteria infect animals.

b. Describe three reserve materials synthesized by microorganisms.

Answer:
Prokaryotic cells consist of many reserve deposits known as inclusions or inclusion bodies.
Prokaryotic cells accumulate certain deposits of nutrients and use them when they are necessary or
is deficient.
 The large inclusions which sometimes stain red with certain blue dyes are known as
Metachromatic granules. Collectively metachromatic granules are known as Volutin. Volutin
represents the reserved deposits of the inorganic phosphate which can be essential in
synthesis of the ATP.
 The inclusions containing glycogen and starch are known as Polysaccharide granules. The
presence of the glycogen and starch can be determined by applying iodine to the cell.
 The inclusions containing reserved deposits of iron oxide formed by many gram-negative
bacteria such as Magnetospirillum, magnetotacticum that act like magnets are known as
Magnetosomes.
  The inclusions containing reserve deposits of enzyme ribulose 1, 5-diphosphate carboxylase
are known as Carboxysomes.
 Lipid reserved deposits are known as Lipid inclusions.
 Sulfur reserved deposits are known as Sulfur granules which derive energy by oxidation of
the sulfur and sulfur containing compounds.

c. Which molecules are responsible for the negative charge on bacterial cell?
Answer:
The bacteria are categorized into;
1. Gram-positive bacteria
2. Gram-negative bacteria
The negative charge of the cell wall is due to the presence of the teichoic acid which might be linked
to plasma membrane or peptidoglycan. The negative charge is originated from the phosphate group
present in the structure of the teichoic acid. Thus, the teichoic acid is responsible for the negative
charge on the bacterial cell. The Gram-negative bacteria have an outer covering of phospholipids
and Lipopolysaccharides. The lipopolysaccharides impart a strongly negative charge to surface of
Gram-negative bacterial cells.

d. Describe the role of microorganisms in recycling of elements.

Answer:
Microorganism play a significant role in the recycling purpose. Microorganism act as decomposers
by decaying dead organic matter and recycle the essential elements residing the dead matter.
Organic materials include animal dead bodies, fallen leaves and tree trunks. Microbes through a
process called biodegradation, use nutrients found in the environment for their own survival. As a
result of break-down of the products, nutrients that are obtained, which become free to feed pant
and algae, which in turn feed all animals. Thus, microorganism plays an important role in recycling of
the elements from the environment.

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