Industrial training in medical microbiology

At Dr. B LAL INSTITUTION

BY VARSHA MEENA

BSc. Biotechnology, AMITY UNIVERSITY

Duration: July – August 2021

Under the supervision of

Mrs. Dr. APOORVA RANA

Mrs. Dr. APARNA DATTA

 Content
Title Page
No.
1. Acknowledgement 4
2. Summary 5
3. Introduction 6
4. Internship Discussion. 7
5. Experiments 8
I. Microbiology lab 9
a) Instruments 9
b) Waste product 11
c) Isolation techniques 11
1. Serial Dilution
2. Spread plate method
3. Pour plate method
4. Streak plate method

d) Culturing media 12

e) Staining 12

1. Grams staining
2. Endospores staining
3. Capsule staining
4. Acid fast stain

g) Biochemical Tests 14
 1. Indole Test
2. Methyl red (MR) Test
3. Voges proskauer (VP) Test
4. Citrate Test
5. Catalyase Test
6. Coagulation Test
7. TSI (Triple Sugar Iron)
8. Urease Test

h) Growth curve study 16

i) Lacto phenol cotton blue mount (LPCB) 17

j) Antibiotic susceptibility Test 17

6. Conclusion. 18
7. Judgment of the Company 19
Acknowledgement

First I would like to thank Dr. Aparna datta mam head of Dr. B Lal
institute for giving me the opportunity to do an internship within the
institution.

I also would like to thank all the people for helping and guiding me
during my training period in B lal institution, with their patience and
openness they created an enjoyable working environment.

It is indeed with a great sense of pleasure and immense sense of

gratitude that I acknowledge the help of these individuals. I am very
thankful to Mrs. APOORVA RANA mam, for the facilities provided
to accomplish this internship.

I thank all the people for their support and advice to get and complete
internship in above said institution. I am extremely great full to my
department staff members, who helped me in successful completion
of this internship.
Summary

This report contains details of industrial training at Dr. B LAL

INSTITUTION in Jaipur, Rajasthan I worked in the Medical
Microbiology Department.

Learning Objectives/ Training Objectives

Origin of the report Internship Program of AMITY UNIVERSITY is

a requirement for completion of degree for the students. The main
purpose of the internship is to meet the students with the job world.
As an intern the main target was to match up the theoretical concepts
into real life experience. The internship program has following
purpose is to obtain practical knowledge. To match up the practical
field with the lessons learned in AMITY. To obtain real-time practical
work experience
INTRODUCTION
Biotech Industrial Training

Dr. B. Lal Institute of Biotechnology offers comprehensive & in-

depth practical hands-on Biotech training, in a real-life environment,
which enables the participants to master the skills in Biotechnology.
The mission of Biotech Industrial Training Programme is to provide
industry-specific training to students, research scholars and faculty
members for skill development and enhancing their job opportunities
in the biotech industry. The Indian Biotechnology sector is one of the
fastest growing knowledge-based sectors and is expected to play a
key role in shaping India’s rapidly developing economy. In
biotechnology industrial sector, a huge importance is given to the
skilled manpower employed and hence, head-hunting has always been
one of the prime priorities. Unfortunately, most of the students of
biotechnology lag behind and fail to satisfy the skill requirement of
these industries even though they may be strong in theoretical
concepts. Hence, it has been marked at various forums that the freshly
graduating students are often not – employable though trainable.

The Biotech Industrial Training Program aims to provide industry

specific training to students for skill development and is specially
designed for students to gain ‘hands-on’ training experience hence
enhancing their job opportunities in biotech industry. Hence, all
possible efforts are made to inculcate research aptitude and good
work culture among the students through well planned, properly
executed and evaluated training.
Descriptions of the Industrial Training

From July to August 2021, I worked in B Lal institution in medical

Microbiology division as a trainee. Microbiology Lab facilitates with
different areas for various dedicated activities and has lab strength of
10 people. Each one of them have allotted to an individual task. I had
the chance to work with each of the members of the lab.

During my summer training I learned about different types of tests

and many of the instruments used in medical microbiology. I have
also made media preparation, and we study about the microbes which
grown in media, on them we tested antibiotic susceptibility test in the
last.

I learned a lot about how to communicate with people in the

community who are very different from me.
MEDICAL MICROBIOLOGY

Medical microbiologists provide services to aid the diagnosis and

management of infectious diseases and help ensure the safety of those
at risk of acquiring infectious diseases, both in hospitals and the
community. Although this role is laboratory-based, the
microbiologist’s role is increasingly clinical.
When a patient is suspected of having an infection, they provide
advice on the likely causes and suggest the best tests to diagnose it.
Tests may involve the identification of parasites under the
microscope, the use of biochemical tests to identify colonies of
bacteria or the use of molecular tests to identify organisms (or even
specific genes) which may govern an organism’s behaviour.
Microbiologists provide advice regarding the interpretation of results
and the appropriateness of further investigations and antibiotic
treatment. The side effects of treatment, along the potential risk of
encouraging further infections (some of which may be antibiotic-
resistant) must be considered, along with any medical problems or
allergies the patient might have.
Medical microbiologists also play a key role in controlling the spread
of infectious diseases. Microbiologists work with hospital infection
control teams to reduce the spread of infections in hospitals (including
hospital ‘super bugs’ such as MRSA and Clostridium Difficile).
They also contribute to the protection of public health by monitoring
the patterns of infectious diseases and reporting new or unusual
occurrences of infections. In their infection control activities,
microbiologists work with nurses and other healthcare professionals,
hospital estates departments and management.
Teaching healthcare workers, both students and qualified staff, is an
important part of the work. Research is undertaken by those with
particular interests.

MICROBIOLOGY LAB
a) INSTRUMENTS:-

1.) AUTOCLAVE:-
The autoclave works on the principle of moist heat sterilization where
steam under pressure is used to sterilize the material present inside the
chamber.
2.) HOT AIR OVEN:-
A hot air oven is a type of dry heat sterilization. Dry heat sterilization
is used on equipment that cannot be wet and on material that will not
melt, catch fire, or change form when exposed to high temperatures.
Moist heat sterilization uses water to boil items or steam them to
sterilize and doesn't take as long as dry heat sterilization.
3.) INCUBATOR:-
An incubator is based on the principle that microorganisms require a
particular set of parameters for their growth and development.
4.) HOT PLATE:-
Heating plate heating material is mainly electric alloy wire, its
working principle is very simple, simply is the electric effect.
5.) WATER BATH:-
A water bath is a device that maintains water at a constant
temperature. It allows the heating of small amounts of fluids over a
period of time without changing the concentration of constituents by
evaporation.
6.) LAMINAR AIR FLOW:-
The working of laminar airflow depends on the high-efficiency
particulate airflow system, eliminating all the air-borne
contaminants to maintain a sterile environment. A laminar hood is
made up of stainless steel avoiding joints and corners to prevent the
accumulations of bacterial spores.
1. Cabinet
• The cabinet is made up of stainless steel with less or no gaps or
joints preventing the collection of spores.
• The cabinet provides insulation to the inner environment created
inside the laminar flow and protects it from the outside
environment.
• The front of the cabinet is provided with a glass shield which in
some laminar cabinets opens entirely or in some has two openings
for the user’s hands to enter the cabinet.
2. Working station
• A flat working station is present inside the cabinet for all the
processes to be taken place.
• Culture plates, burner and loops are all placed on the working
station where the operation takes place.
• The worktop is also made up of stainless steel to prevent rusting.
3. Filter pad/ Pre-filter
• A filter pad is present on the top of the cabinet through which the
air passes into the cabinet.
• The filter pad traps dust particles and some microbes from entering
the working environment within the cabinet.
4. Fan/ Blower
• A fan is present below the filter pad that sucks in the air and
moves it around in the cabinet.
• The fan also allows the movement of air towards the HEPA filter
sp that the remaining microbes become trapped while passing
through the filter.
5. UV lamp
• Some laminar flow hoods might have a UV germicidal lamp that
sterilizes the interior of the cabinet and contents before the
operation.
• The UV lamp is to be turned on 15 minutes before the operation to
prevent the exposure of UV to the body surface of the user.
6. Fluorescent lamp
• Florescent light is placed inside the cabinet to provide proper light
during the operation.
7. HEPA filter
• The High-efficiency particulate air filter is present within the
cabinet that makes the environment more sterile for the operation.
• The pre-filtered air passes through the filter which traps fungi,
bacteria and other dust particles.
• The filter ensures a sterile condition inside the cabinet, thus
reducing the chances of contamination.
•
7.) PH Meter:-
pH meter is a device used is laboratories that measure the Hions
concentration in water based solutions to determine the acidity or
alkalinity of the solution termed as potentiometric pH meter.
8.) Spectrophotometer:-
Based on beer lambert law which states the absorbance of light by a
solution a particulate wavelength is directly proportional to the
concentration of the substance. Difference wavelengths of light are
passed through a solution as difference substances have better
absorbance at different wavelength.
9.) Weighing machine:-
The basis of the rapid and exact working method of our Weigh Cells
is the Principle of Electro Magnetic Force Restoration (EMFR). The
basic principle is comparable to a simple beam balance. The weight is
laid on one side of the beam (coil arm). The result is that the coil
attached to the other side of the beam tries to move out of the
magnetic field of the magnet.
10.) COLONY COUNTERS:-
Colony counters count the number of colonies of microorganisms that
have grown on an agar plate prepared from a sample.

b) WASTE DISPOSAL:-
• Yellow bag: - plastic wares, body organs, gloves masks.
• Red bag:- hazardous waste
• Blue bag: - sharps things like needle blade, glass slide.
 • Black bag:- non-hazardous waste

(C) Isolation techniques

1.) SERIAL DILUTION:-

This method is commonly used to obtain pure cultures of those
microorganisms that have not yet been successfully cultivated on
solid media and grow only in liquid media. A microorganism that
predominates in a mixed culture can be isolated in pure form by a
series of dilutions. The inoculum is subjected to serial dilution in a
sterile liquid medium, and a large number of tubes of sterile liquid
medium are inoculated with aliquots of each successive dilution.
2.) SPREAD PLATE METHOD:-
The spread plate technique involves using a sterilized spreader with a
smooth surface made of metal or glass to apply a small amount of
bacteria suspended in a solution over a plate. The plate needs to be
dry and at room temperature so that the agar can absorb the bacteria
more readily. A successful spread plate will have a countable number
of isolated bacterial colonies evenly distributed on the plate.
3.) POUR PLATE METHOD :-
The bacterial culture and liquid agar medium are mixed together.
After mixing the medium containing the culture poured into sterilized
petri dishes allowed solidified and the incubated after incubation
colonies appear on a surface.
4.) STREAK PLATE TECHNIQUE:-
PRINCIPLE: - Streak plate technique is used for the isolation into a
pure culture of the organisms (mostly bacteria), from a mixed
population. The inoculum is streaked over the agar surface in such a
way that it “thins out” the bacteria.
d) CULTURE MEDIA:-
Principle of media:-
In preparing a culture medium for any microorganism, the primary
goal is to provide a balanced mixture of the required nutrients, at
concentrations that will permit good growth. No ingredient should be
given in excess because many nutrients become growth inhibitory or
toxic as the concentration is raised.

e). STAINING
Principles of different staining techniques
1.) Gram’s staining:
(purple-gram positive) The basic principle of Gram staining is the
properties of certain bacteria cell walls to retain the crystal violet dye.
The cell walls for Gram-positive microorganisms have a higher
peptidoglycan and lower lipid content than Gram-negative bacteria.
RESULT: Gram’s negative: - pink in colour
Gram’s positive: - purple or violet in colour

2. Principle of Endospore Staining

Bacterial endospores are metabolically inactive, highly resistant
structures produced by some bacteria as a defensive strategy against
unfavorable environmental conditions. The bacteria can remain in this
suspended state until conditions become favorable and they can
germinate and return to their vegetative state.
RESULT: - Endospores: Endospores are bright green.
Vegetative Cells: Vegetative cells are brownish red to pink.
3.) Principle of Capsule Staining
Capsules stain very poorly with reagents used in simple staining and a
capsule stain can be, depending on the method, a misnomer
because the capsule may or may not be stained.
Negative staining methods contrast a translucent, darker colored,
background with stained cells but an unstained capsule. The
background is formed with India ink or nigrosin or congo red. India
ink is difficult to obtain nowadays; however, nigrosin is easily
acquired.
A positive capsule stain requires a mordant that precipitates the
capsule. By counterstaining with dyes like crystal violet or
methylene blue, bacterial cell wall takes up the dye. Capsules appear
colorless with stained cells against dark background.
RESULT: - Capsule: Clear halos zone against dark background
No Capsule: No Clear halos zone
4.) Principle of Acid-Fast Stain
When the smear is stained with carbol fushin, it solubilizes the
lipoidal material present in the Mycobacterial cell wall but by the
application of heat, carbol fushin further penetrates through lipoidal
wall and enters into cytoplasm. Then after all cell appears red. Then
the smear is decolorized with decolorizing agent (3% HCL in 95%
alcohol) but the acid fast cells are resistant due to the presence of
large amount of lipoidal material in their cell wall which prevents the
penetration of decolorizing solution. The non-acid fast organism lack
the lipoidal material in their cell wall due to which they are easily
decolorized, leaving the cells colorless. Then the smear is stained with
counterstain, methylene blue. Only decolorized cells absorb the
counter stain and take its color and appear blue while acid-fast cells
retain the red color.
RESULT: - Acid fast: Bright red to intensive purple, Red, straight or
slightly
curved rods, occurring singly or in small groups, may appear beaded
Non-acid fast: Blue color
f). BIOCHEMICAL TESTS (IMViC)
1.) INDOLE TEST :-
Principle:
• Indole test is a biochemical test which differentiates the
coliform from other members of Enterobacteriacee by detecting
their ability to produce the enzyme tryptophanase. This enzyme
hydrolyses the amino acid tryptophan into indole, pyruvic acid
and ammonia. It is the intracellular enzyme (endoenzyme).
Tryptophan + H2O————tryptophanase enzyme——>
Indole + Pyruvic acid + Ammonia
RESULT:-
Indole test positive: cherry red color (E. coli)
Indole test negative: no red color (Klebsiella)
2.) Methyl Red (MR) Test
Principle:-Some bacteria have the ability to utilize glucose and
convert it to a stable acid like lactic acid, acetic acid or formic acid as
the end product.
In the methyl red test (MR test), the test bacteria is grown in a broth
medium containing glucose. If the bacteria has the ability to
utilize glucose with production of a stable acid, the colour of the
methyl red changes from yellow to red, when added into the broth
culture.
If Acid: - RED (positive MR TEST)
If no acid: - YELLOW (negative)

3.)Voges– Proskauer (VP) Test

Principle: - The Voges-Proskauer (VP) test is used to determine if an
organism produces acetylmethyl carbinol from glucose
fermentation. If present, acetylmethyl carbinol is converted
to diacetyl in the presence of ∝- naphthol, strong alkali (40%
KOH), and atmospheric oxygen. The ∝-Naphthol was not part of the
original procedure but was found to act as a color intensifier by Barritt
and must be added first.
Acetoin + Barritt A (∝-Naphthol) in presence of barritt B (KOH) and
atmospheric O2 = compound (reddish brown)
4.) CITRATE TEST:-
Principle:- Bacteria are inoculated on a medium containing sodium
citrate and a pH indicator such as bromothymol blue. The medium
also contains inorganic ammonium salts, which are utilized as sole
source of nitrogen. Use of citrate involves the enzyme citrase, which
breaks down citrate to oxaloacetate and acetate.
(Na2)2+ + Co2 = Na2Co3 (sodium carbonate alkaline) (blue slant)
BLUE SLANT: - POSITIVE
GREEN SLANT: - NEGATIVE
5.) Catalase Test
Principle: - The enzyme catalase mediates the breakdown of hydrogen
peroxide into oxygen and water. The presence of the enzyme in a
bacterial isolate is evident when a small inoculum is introduced into
hydrogen peroxide, and the rapid elaboration of oxygen bubbles
occurs. The lack of catalase is evident by a lack of or weak bubble
production. The culture should not be more than 24 hours old.
2H2O2 = catalase = 2H20+O2 (Gas bubbles)
RESULT: Occurs of bubbles= positive
Occurs of no bubbles= negative
6.) COAGULASE TEST :-
Coagulase is an enzyme-like protein and causes plasma to clot by
converting fibrinogen to fibrin. Staphylococcus aureus produces two
forms of coagulase: bound and free.
Fibrinogen (soluble) =coagulase = Fibrin (insoluble)
RESULT: - if organisms Aggregates (clumps) =positive
No aggregates= negative
GRAM+ve Cocci – catalase positive- staphylococcus
catalase negative – streptococcus, enterococcus
GRAM +ve -coagulase- positive –S aureus
Negative- CONS Coag neg staphylococcus
7.) TSI( TRIPLE SUGAR IRON)
Principle:-
The triple sugar- iron agar test employing Triple Sugar Iron Agar is
designed to differentiate among organisms based on the differences in
carbohydrate fermentation patterns and hydrogen sulfide production.
Carbohydrate fermentation is indicated by the production of gas and a
change in the colour of the pH indicator from red to yellow.
An alkaline/acid (red slant/yellow butt) reaction: It is indicative of
dextrose fermentation only.
An acid/acid (yellow slant/yellow butt) reaction: It indicates the
fermentation of dextrose, lactose and/or sucrose.
An alkaline/alkaline (red slant, red butt) reaction: Absence of
carbohydrate fermentation results.
Blackening of the medium: Occurs in the presence of H2
Gas production: Bubbles or cracks in the agar indicate the production
of gas (formation of CO2and H2)
8.) urease test:
Urea is common metabolic waste product of protein digestion in most
vertebrates that is toxic to most living organism. Urease catalases the
breakdown of urea into ammonia and carbon dioxide. The test
organism is cultured in a medium containing urea and the indicator
phenol red. If the bacterial strain is urease-producing, the enzyme will
hydrolyse the urea to give ammonia and carbon dioxide. With the
release of ammonia, the medium become alkaline shown by change in
color of indicator to reddish pink.
Urea (NH2)2CO + 2H2O———–urease enzyme———
>Ammonia + CO2+ H2O
If the ammonia is alkaline in nature then it will show pink in colour
which is positive test of urease test.
For negative urease test ammonia will show yellow in colour and its
nature is acid.

g) GROWTH CURVE STUDY :-

PRINCIPLE: The increase in the cell size and cell mass during the
development of an organism is termed as growth. To study the
bacterial growth population, the viable cells of the bacterium should
be inoculated on to the sterile broth and incubated under optimal
growth conditions. The bacterium starts utilizing the components of
the media and it will increase in its size and cellular mass.

h). LACTOPHENOL COTTON BLUE MOUNT (LPCB)

PRINCIPLE: - Lacto phenol Cotton Blue (LPCB) Staining method
works on the principle of aiding the identification of the fungal cell
walls.
RESULT: - Fungal spores, hyphae, and fruiting structures stain blue
while the background stains pale blue.
i). ANTIBIOTIC SUSCEPTIBILITY TEST
DISK DIFFUSION (KIRBY BAUER TEST):-
The Kirby-Bauer test, known as the disk-diffusion method, is the most
widely used antibiotic susceptibility test in determining what choice
of antibiotics should be used when treating an infection. This method
relies on the inhibition of bacterial growth measured under standard
conditions. This method is based on the principle that antibiotic-
impregnated disk, placed on agar previously inoculated with the test
bacterium, pick-up moisture and the antibiotic diffuse radially
outward through the agar medium producing an antibiotic
concentration gradient.
CTX: ceftoxin
CIP : Ciprofloxacin
AMX: Amoxycillin
MRP: Meropenem
CAC: Cefazidence/Clav
RESULT OF DISC DIFFUSION:-
Both ciprofloxacin and meropenem inhibit the growth of organism.
1.) CIP5 called ciprofloxacin inhibits the growth of organisms. And its
diameter is 31mm.
2.) MRP10 called meropenem also inhibits the growth of organisms.
And its diameter is 28mm.

Conclusion
In this short period of study, I learned a lot. I learned different types
of instruments and their importance. I also learned about different
types of tests that are performed and their function and importance. I
was a bit weak in microbiology and its related section. But after this
study I know many things regarding medical microbiology. Though it
was for a short period of time, but I tried to acquire as much as
knowledge as possible. In this study I learned about many tests
regarding microbiology. This institute has a superb work culture and
great minds and very high quality of work.

Personal Experience:

Having spent a month there, I received hands on experience on some

of their daily chores. Preparing Media, learning about antibiotic
susceptibility, biochemical tests and staining and the products were
some of the fields where in I was able perform hands on experience.
Even though I was at dr b lal biotechnology institute for only for some
time, I was treated like a part of their students. I was exposed to every
test they carried out.

I not only learnt the procedures of different tests, but I also learnt the
importance of documentation.

Judgment of the institute:

For the time I have spent there, I have come across some of the most
selfless people out there. They were all willing to share tiny details
and problems of every task given in the lab. They shared what the
benefits and cons are to work in a medical microbiology.

I did not only receive my hands-on experience in my field of

education, but I also understood the dynamics of a lab and also
regarding the medical. I would forever be grateful to B lal institute of
biotechnology for the opportunity they provided me. It would be very
unlikely that I can receive the same experience and work environment
at any other company.