Pir Mehr Ali Shah

Arid Agriculture University, Rawalpindi

Office of the controller of Examinations
Final Exam (Practical)/ Spring 2020 (Paper Duration 48 hours)
To be filled by Teacher
Course No.: MICRO-102 Course Title: General Veterinary Microbiology
Total Marks: 20 Date of Exam: 07-08-2020
Degree: DVM Semester: 2nd Section: A (MORNING)
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.: 19-ARID-2248 Name: SIDRA ZAMIR

Answer the following questions.

Q. No. 1. What is pure culture? How we isolate pure culture from a sample? Describe in detail the
process for isolation of the pure culture. (Marks 04)
Answer: Pure Culture:

The pure culture is the culture which consists of only a single type of organism. It is the species of
cells that is unadulterated, meaning that it is pure and not mixed with other microorganisms. It is
also called as axenic culture.

Isolation of Pure Culture:

The pure culture bacteria is isolated from a mixed culture sample. Firstly, we decrease the number
of organisms in the inoculum so that the discrete cells can easily spread out on agar medium surface,
thus separating the different species.

Process of Pure Culture Isolation:

We can isolate the pure culture from a sample by using three methods:
a. Streak-plate method
b. Spread plate technique
c. Pour plate technique
❖ Isolation of Pure Culture by Quadrant Streak-plate method:
1. Firstly, divide the agar petri dish into four quadrants by drawing two perpendicular bisecting
lines at the bottom of the plate.
2. The wire of inoculating loop is flamed by Bunsen burner until half of its length turns red-hot.
Then allow it to cool near the Bunsen burner because too hot loop will kill the organisms
showing no growth on the agar plate.
3. Take a tube containing the broth culture and dip the loop in this culture without touching
the sides of the tube. Place back the tube into rack after culture has been taken.
4. The petri dish is protected from aerial contamination by keeping it covered with a lid.
5. The loop is then sweeped over the first quadrant to completely spread out the culture.
6. Then the loop is flamed and cooled by touching it in an un-inoculated area of the medium.
7. After streaking the first quadrant, the loop is smoothly sweeped over 2nd quadrant then
flame and cool the loop.
8. In the same manner, the 3rd quadrant is streaked after which the loop is again flamed and
cooled.
9. Then finally, streak the 4th quadrant.
10. Keep in mind that streaking should be carefully done without overlapping of streaks of
different quadrants.
11. The petri dish is then inverted (because the air-space which is present between the dish lid
and agar surface is saturated with moisture which during incubation condenses on upper lid
and then water drips on the agar surface thereby separating the mixing colonies thus it is
prevented by inversion of agar plate)and incubated at about 37 degrees overnight.

❖ Isolation of pure culture by Four-way Streak-plate Method:

1. Firstly, take an agar plate and label it. In this method we form four sets of streaks.
2. Then we flame an inoculating loop by the Bunsen burner until half of it turns red-hot.
3. Then allow the inoculating loop to cool down near the flame. In order to know that the loop
has cooled touch the loop on the blank area of the agar plate which will sizzle if the loop is
hot.
4. The loopful of culture is then used to make four streaks in the 1st of the total four areas.
This is the primary inoculation.
5. Reflame and cool the loop and rotate the agar plate at an angle of 90 degrees.
6. Secondly, second set of four streaks are made in 2nd area in a way that it does not interfere
with the first streaking area.
7. Then again the loop is reflamed and cooled down and the agar plate is rotated at an angle of
90 degrees.
8. Then a third set of streaks is formed in 3rd area.
 9. Then the loop is reflamed and cooled down again and the agar plate is rotated for the last
time at an angle of 90 degrees.
10. Finally, start streaking in 4th area, where the 3rd area has ended and streak over entire
surface which is left but without interfering with other areas.
11. After this the plate is incubated, and the discrete colonies of have been isolated.

Q. No. 2. What do you understand by the term “cytopathic effect”? Explain in detail different
cytopathic effects of viruses in host cells. (Marks 04)
Answer: Cytopathic Effects:
• The infection of a virus in the host cell produces certain visible effects and these effects are
collectively called cytopathic effects.
• The cytopathic effects which result in the death of a cell are called cytocidal effects.
Whereas, cytopathic effects which only damage the cell instead of its death then these
effects will be called as non-cytocidal effects.
Advantages of Cytopathic Effects:
The cytopathic effects of viruses are important as with the help of these effects the viral infections
can easily be diagnosed. This is because every virus produces its own cytopathic effects.
Cytopathic Effects Produced by Viruses:
The cytopathic effects which a virus produces within a host cell are:
1. Inhibition of Macromolecular Synthesis:
• The virus firstly interferes and stops the synthesis of host cell macromolecules such as
nucleic acid, protein, lipids and carbohydrate synthesis is ceased by the virus.
• Example: Mitosis is irreversibly stopped by some viruses known as Simplex virus.
2. Promotion of Lysozymal Release:
Either the death of the virus itself or the host cell death occurs in the lysosomal compartment when
some viruses promote the release of lysozymes from the host cell.
3. Arising of Inclusion Bodies:
• Viruses also produce some inclusion bodies in the cytoplasm or nucleus of the cells infected
with viruses. These granular structures may be the viral nucleic acid or viral proteins (which
are assembling into virions) showing that these are viral parts.
• At sites, where early viral synthesis is being carried out , some inclusion bodies arise there
which lack the viral viral parts.
• The causative agent of an infection is easily determined by inclusion bodies.
• For Example: Rabies is detected by the presence of Negri bodies which are the inclusion
bodies produced by the rabies virus.
• Apart from Rabies virus, inclusion bodies are also produced by Herpesviruses and
Adenoviruses.
4. Syncytium Formation:
 • When viruses cause infection in cells they become enlarged and then these adjacent and
individual, infected and enlarged cells fuse together, forming a syncytium which is a
multinucleate cell.
• For Example: Common cold, Measles and Mumps viruses form such giant cells.
5. Change in Host Cell Function:
• The function of the host cell is changed by some viruses without causing any visible changes
in the infected cells.
• For Example: The measles virus causes the production of IL-12 cytokine to decrease which
ultimately compromises the ability of the host cell to fight against the infection.
6. Antigenic Changes:
A host-antibody response is initiated when antigenic changes are induced on infected cells surface
by viral infections. This host-antibody response then destroys the infected cells via the immune
system of the host.
7. Host Cell Chromosomal Changes:
• Chromosomal changes such as the breakage of chromosome can be induced in the host cell
as a result of viral infections.
• For Example: The activation of cancer-causing genes known as oncogenes may be due to a
virus.
8. Tumor Formation:
Some viruses like oncogenic viruses change the cell into an abnormal shape such as spindle shape.
Due to this transformation in the host cell's shape caused by viruses, the cells lose their ability to
recognize the contact inhibition and do not stop growing even after the cells have contacted
together. Which results in an uncontrolled cell division and growth.

Q. No. 3. What are the benefits of microbial control? Describe different physical methods for
control of microbial growth along with their mechanism of actions. (Marks 04)
Answer: Benefits of Microbial Control:
1. The contamination of specimens is prevented by microbial control.
2. The spread of infectious agents during the diagnostic procedures can only be controlled if
there is microbial control.
3. The nosocomial infections that are acquired from the hospital area can be prevented if there
is proper microbial control.
4. The microbial control helps to prevent the spread of infectious diseases.
5. The microbial control is also helpful in keeping the environment safe and clean by keeping it
free from the germs.
6. The spoilage of food (food poisoning) can be prevented if microbial population is kept under
control.
 7. By controlling microbes, the contamination of water and soil can be prevented.
8. Microbes also causes diseases in plants thus, microbial control will help to put a full stop on
spread the of plant diseases thus, increasing the total agricultural yield.
9. Decomposition and corrosion of materials is also stopped under microbial control.
Physical Methods for Control of Microbial Growth:
Following are the physical methods which are used to control the microbial growth:
1. Heat
The two types of heat are used to control the microbial growth:
i. Dry Heat
a. Direct Flaming
Method: Direct Flaming is a very effective method of sterilization.
Mechanism of Action: It works by burning and heating the contaminants to ashes.
Used to Sterilize: This method is used to sterilize the inoculating loops.
b. Incineration
Method: It is a method of sterilization which is very effective.
Mechanism of Action: It works by combusting and burning the contaminants to ashes.
Used to Sterilize: This method is used to sterilize the paper cups, contaminated dressings,
animal carcasses, bags and wipes.
c. Hot-air Sterilization
Method: Hot-air Sterilization is a very effective method of sterilization, requiring about 160
degree Celsius of temperature for about an hour.
Mechanism of Action: It works by oxidative destruction.
Used to Sterilize: It is used to sterilize empty glassware, instruments, needles, and glass
syringes.
ii. Moist Heat
a. Free-Flowing Steam under Pressure: (Auto-claving)
Method: This method is accomplished by autoclaving which makes use of an autoclave. The
pressure of about 15 Ibs attains the temperature of about 121degrees Celsius. The whole
sterilization process is completed in 15 minutes in which all vegetative cells along with its
endospores are killed.
Mechanism of Action: Its mode of action involves the denaturation of proteins of organisms.
Used to Sterilize: This method is used to sterilize microbiological media, solutions, linens,
utensils, dressings, equipment, and some other items that are able to bear such high
temperatures and pressure.
b. Free-flowing Steam at 100 degrees Celsius:
 Boiling--The contaminated substance is exposed to the boiling (100 degrees) water for
about 30 minutes. But it only disinfects, killing all the vegetative cells (of bacteria, fungus
and virus) but is not effective for heat resistant spores.
It is used to sterilize dishes, basins, pitchers.
Tyndallization (Intermittent or fractional sterilization)—It requires the material to be
exposed to free-flowing steam at a temperature of 100 degrees for about 20 minutes for
three days consecutively with provision of incubation at about 37 degrees.
All the vegetative cells are killed but the spore are killed after they have germinated due
under continuous exposure to 100 degrees temperature.
Mechanism of action:
Both above process mechanism of action is through protein denaturation.
c. Pasteurization:
Method: It is an effective method of milk treatment with heat, thus killing all the pathogens
and most non-pathogens at a temperature of about 72 degrees for 15 seconds.
Mechanism of Action: It works by protein denaturation.
Used to Sterilize: It is used to sterilize Milk, cream, certain alcoholic beverages (beer,wine).
Types of pasteurization:
• High-temperature short-time (HTST): requires 71 degrees temperature for 15 seconds.
• Low-temperature long-time (LTLT): requires 63 degrees temperature for 30 minutes.
• Ultra-high temperature (UHT): requires 138 degrees temperature for 2 seconds.
2. Cold
This method is used to control microbial growth in a refrigerator by freezing, lypophylization
(known as freeze-drying) and Microbistatic
3. Radiation
It kills microbes by ionizing and non-ionizing radiations.
• Non-ionizing (Infrared and Ultraviolet)
• Ionizing (X-rays and Gamma rays)
4. Filtration
The thermolabile solutions are filtered by using depth filters, membrane filters or air filters
for the removal of particulates and organisms . It is only method used to separate the
microbes and not killed and it is used in culture media.
5. Desiccation
It is done through the removal of water from the organisms.
6. Sonic Vibrations
In this method high frequency sound vibrations are used to control microbial growth.
7. Osmotic Pressure
In this method hypertonicity and hypotonicity are used to control the microbial growth.
 • Hypertonicity: results from increased concentration of salts and sugars.
• Hypotonocity: results from increased concentration of water.

Q. No. 4. Practical performance / practical notebook / viva voce. (Marks 08)