DEPARTMENT OF MICROBIOLOGY

INTERNSHIP REPORT

Submitted to

Shri Nehru Maha vidyalaya College of Arts and Science

in the partial fulfilment of the requirements for the award of the Degree of

BACHELOR OF SCIENCE IN MICROBIOLOGY

Internship report submitted by

 CERTIFICATE

This is to certify that the internship Report submitted to Shri Nehru Maha Vidyalaya College of
Arts and Science, Coimbatore in partial fulfilment of the requirements for the award of the Degree
of Bachelor of Science is a record of bonafide work carried out by

Head of the Department

Station: Coimbatore

Date:

Internal Examiner:

External Examiner :
 DECLARATION

I hereby declare that the internship report submitted to Shri Nehru Maha Vidyalaya
College of Arts and Science, Coimbatore, is an original work done by me in partial
fulfilment of the requirements for the award of the Degree of Bachelor of Science.

Signature of the candidate

 ACKNOWLEDGEMENT

I wish to express my sincere gratitude to THE COORDINATOR for providing me a

opportunity to do my internship work in PATHOGENIX LABS. I sincerely thank DR. A.
SANTHOSH for his guidance and encouragement in carrying out this internship work. I
also wish to express my gratitude to the officials and the other staff members of
PATHOGENIX LABS who rendered the help during the period of my internship work. I
would like to express my special thanks and gratitude to our beloved Principal DR.
B.SUBRAMANI and our HOD Dr. R. BHAKYARAJ who gave me the golden opportunity
to do this wonderful work which helped me to know about so many new things.
 TABLE OF THE CONTENT

S.NO TITLE PAGE.NO

1. ANTIBIOTIC SUSCEPTIBILITY TEST

2. ELISA TECHNIQUES

3. ACID-FAST BACILLI (AFB) STAINING

4. TISSUE PROCESSING

5. COMPLETE BLOOD COUNT (CBC)

6. PERIPHERAL BLOOD SMEAR

 MICROBIOLOGY

1. ANTIBIOTIC SUSCEPTIBILITY TEST

INTRODUCTION:

Antibiotics are chemicals that inhibit the growth of or kill microorganisms, including
bacteria. The discovery of antibiotics revolutionized the treatment of bacterial infections and
saved countless lives. However, the overuse and misuse of antibiotics have led to the
emergence of antibiotic-resistant bacteria, making it essential to determine the antibiotic
susceptibility of bacterial isolates

AIM:

The aim of this experiment is to determine the antibiotic susceptibility of a bacterial

isolate using the disc diffusion method.

PRINCIPLE:

The experiment is based on the principle that antibiotics diffuse through the agar
medium at a rate dependent on their concentration and molecular weight. The bacterial isolate
is grown on the agar medium, and antibiotic discs are placed on the surface. The antibiotics
diffuse through the agar, creating a concentration gradient. The bacteria are inhibited or killed
by the antibiotics, resulting in a clear zone around the antibiotic disc, known as the inhibition
zone.

MATERIALS REQUIRED:

- Bacterial isolate (e.g., E. coli, Staphylococcus aureus)

- Antibiotic discs (e.g., ampicillin, tetracycline, ciprofloxacin)

- Nutrient agar plates

- Sterile inoculation loop

- Incubator

- Measuring ruler or caliper

PROCEDURE:

Preparation of Bacterial Isolate:

1. Obtain the bacterial isolate (e.g., E. coli, Staphylococcus aureus) and grow it in nutrient
broth or agar.

2. Incubate the bacterial culture at the optimal temperature (e.g., 37°C for E. coli) for 24
hours.

Preparation Of Antibiotic Discs:

1. Obtain antibiotic discs (e.g., ampicillin, tetracycline, ciprofloxacin) and store them in a
sterile container.

2. Label each antibiotic disc with its corresponding antibiotic name and concentration.

Preparation of Nutrient Agar Plates:

1. Pour nutrient agar into sterile petri dishes and allow it to solidify.

2. Ensure the agar surface is dry and free of condensation.

Inoculation of Nutrient Agar Plates:

1. Inoculate the nutrient agar plates with the bacterial isolate using a sterile inoculation loop.

2. Spread the bacterial inoculum evenly over the agar surface using a sterile spreader.

Placement of Antibiotic Discs:

1. Place the antibiotic discs on the inoculated agar plates, making sure not to touch the discs
to the agar surface.

2. Space the antibiotic discs evenly apart to prevent overlap of the inhibition zones.

Incubation and Observation :

1. Incubate the agar plates at the optimal temperature (e.g., 37°C for E. coli) for 24 hours.

2. Observe the agar plates for inhibition zones around the antibiotic discs.

3. Measure the diameter of the inhibition zones using a measuring ruler or caliper.
OBSERVATION :

Observe the agar plates for the presence and size of inhibition zones around the
antibiotic discs. Record the results, including the diameter of the inhibition zones.

DISCUSSION:

The results of the antibiotic susceptibility test indicate the effectiveness of different
antibiotics against the bacterial isolate. The size of the inhibition zone around each antibiotic
disc corresponds to the susceptibility of the bacteria to that antibiotic. A larger inhibition
zone indicates greater susceptibility, while a smaller inhibition zone indicates reduced
susceptibility or resistance.

RESULT:

The results of the antibiotic susceptibility test are typically recorded as follows:

- Susceptible (S): Inhibition zone diameter ≥ 15 mm

- Intermediate (I): Inhibition zone diameter 11-14 mm

- Resistant (R): Inhibition zone diameter ≤ 10 mm

 SEROLOGY

2. ELISA TECHNIQUES

INTRODUCTION:

Enzyme-Linked Immunosorbent Assay (ELISA) is a widely used laboratory

technique for detecting and quantifying specific antibodies or antigens in a sample. ELISA is
a versatile and sensitive method that has applications in various fields, including
immunology, microbiology, and molecular biology. In this experiment, we will use ELISA to
detect the presence of specific antibodies in a sample.

AIM:

The aim of this experiment is to demonstrate the principles of ELISA and to detect the
presence of specific antibodies in a sample using this technique.

PRINCIPLE:

ELISA is based on the principle of antigen-antibody interactions. The technique

involves the use of an enzyme-linked antibody that binds specifically to the antigen of
interest. The enzyme converts a colorless substrate into a colored product, which is measured
spectrophotometrically. The amount of colored product is directly proportional to the amount
of antigen present in the sample.

MATERIALS REQUIRED:

- ELISA plate (96-well)

- Antigen (e.g., protein, peptide, or virus)

- Antibody (e.g., monoclonal or polyclonal)

- Enzyme-linked antibody (e.g., HRP-conjugated)

- Substrate (e.g., TMB)

- Stop solution (e.g., sulfuric acid)

- Wash buffer (e.g., PBS-T)

- Sample (e.g., serum, plasma, or tissue extract)

PROCEDURE:

Preparation of ELISA Plate:

1. Coat the ELISA plate with antigen by adding 100 μL of antigen solution to each well.

2. Incubate the plate at 4°C overnight to allow the antigen to bind to the plate.

Blocking and Washing:

1. Block the plate by adding 200 μL of blocking solution (e.g., 1% BSA in PBS) to each well.

2. Incubate the plate at room temperature for 1 hour.

3. Wash the plate three times with wash buffer (e.g., PBS-T).

Addition of Sample and Antibody:

1. Add 100 μL of sample to each well.

2. Add 100 μL of antibody solution to each well.

3. Incubate the plate at room temperature for 1 hour.

Addition of Enzyme-Linked Antibody:

1. Add 100 μL of enzyme-linked antibody solution to each well.

2. Incubate the plate at room temperature for 1 hour.

Addition of Substrate:

1. Add 100 μL of substrate solution to each well.

2. Incubate the plate at room temperature for 10-30 minutes.

Stop Solution and Measurement:

1. Add 50 μL of stop solution to each well.

2. Measure the absorbance of each well using a spectrophotometer (e.g., 450 nm).
OBSERVATION :

Observe the color change in each well, which indicates the presence of specific
antibodies in the sample. Record the absorbance values for each well.

DISCUSSION:

The results of the ELISA experiment indicate the presence of specific antibodies in
the sample. The absorbance values are directly proportional to the amount of antigen present
in the sample. A higher absorbance value indicates a higher concentration of antigen.

RESULT:

The results of the ELISA experiment are typically presented as a standard curve,
which shows the relationship between the absorbance values and the concentration of
antigen. The standard curve can be used to determine the concentration of antigen in the
sample.
 MICROBIOLOGY

3. ACID-FAST BACILLI (AFB) STAINING

INTRODUCTION:

AFB staining is a widely used technique in microbiology for the detection and
identification of acid-fast bacteria, such as Mycobacterium tuberculosis. The technique
involves the use of a special stain, known as Ziehl-Neelsen stain, which selectively stains
acid-fast bacteria.

AIM:

The aim of this experiment is to demonstrate the AFB staining technique and to
identify acid-fast bacteria using this method.

PRINCIPLE:

The principle of AFB staining is based on the unique property of acid-fast bacteria to
resist decolorization by acid-alcohol after being stained with a basic dye. This property is due
to the presence of mycolic acid in the cell wall of acid-fast bacteria.

MATERIALS REQUIRED:

- Ziehl-Neelsen stain (carbol fuchsin, acid-alcohol, and methylene blue)

- Acid-fast bacteria (e.g., Mycobacterium tuberculosis)

- Smears or cultures of bacteria

- Glass slides

- Microscope

- Distilled water

- 95% ethanol
PROCEDURE:

Preparation of Smears:

1. Prepare smears of acid-fast bacteria on glass slides.

2. Allow the smears to air-dry.

Fixation:

1. Fix the smears by passing them through a flame or by using a fixative (e.g., methanol).

Staining:

1. Stain the smears with carbol fuchsin for 5-10 minutes.

2. Rinse the smears with distilled water.

3. Decolorize the smears with acid-alcohol for 2-3 minutes.

4. Rinse the smears with distilled water.

5. Counterstain the smears with methylene blue for 1-2 minutes.

Microscopic Examination:

1. Examine the smears under a microscope using oil immersion (100x).

2. Observe the acid-fast bacteria, which will appear red or pink.

OBSERVATION :

Observe the acid-fast bacteria, which will appear red or pink against a blue
background. Note the characteristic morphology of acid-fast bacteria, including their rod-
shaped appearance and beaded or granular staining pattern.

DISCUSSION:

The AFB staining technique is a widely used method for the detection and
identification of acid-fast bacteria. The technique is based on the unique property of acid-fast
bacteria to resist decolorization by acid-alcohol after being stained with a basic dye. The
results of this experiment demonstrate the effectiveness of the AFB staining technique in
identifying acid-fast bacteria

RESULT:

The result of this experiment is the successful identification of acid-fast bacteria using
the AFB staining technique. The acid-fast bacteria appear red or pink against a blue
background, and their characteristic morphology is visible under the microscope.
 HISTOPATHOLOGY

4. TISSUE PROCESSING

INTRODUCTION:

Tissue processing is a critical step in the preparation of tissue samples for histological
examination. The process involves a series of steps that help to preserve the tissue, remove
water and other soluble substances, and infiltrate the tissue with a supporting medium. The
goal of tissue processing is to produce high-quality tissue sections that can be used for
diagnostic and research purposes.

AIM:

The aim of this experiment is to demonstrate the steps involved in tissue processing
and to evaluate the quality of the resulting tissue sections.

PRINCIPLE:

The principle of tissue processing is based on the concept of dehydration, clearing,

and infiltration. Dehydration involves the removal of water from the tissue, clearing involves
the removal of fats and other soluble substances, and infiltration involves the introduction of
a supporting medium into the tissue.

MATERIALS REQUIRED:

- Tissue samples (e.g., mouse liver, rat kidney)

- Fixative (e.g., formalin, ethanol)

- Dehydrating agents (e.g., ethanol, xylene)

- Clearing agents (e.g., xylene, toluene)

- Infiltrating agents (e.g., paraffin wax, resin)

- Embedding medium (e.g., paraffin wax, resin)

- Microtome

- Glass slides
PROCEDURE:

Fixation:

1. Fix the tissue samples in a fixative (e.g., formalin, ethanol) for 24-48 hours.

2. Rinse the tissue samples with distilled water to remove excess fixative.

Dehydration:

1. Dehydrate the tissue samples using a series of ethanol solutions (e.g., 50%, 70%, 90%,
100%).

2. Rinse the tissue samples with xylene to remove excess ethanol.

Clearing:

1. Clear the tissue samples using xylene or toluene.

2. Rinse the tissue samples with xylene to remove excess clearing agent.

Infiltration:

1. Infiltrate the tissue samples with a supporting medium (e.g., paraffin wax, resin).

2. Heat the tissue samples to melt the wax or resin.

Embedding:

1. Embed the tissue samples in a block of paraffin wax or resin.

2. Allow the wax or resin to cool and solidify.

Sectioning:

1. Cut sections of the tissue sample using a microtome.

2. Mount the sections on glass slides.

Staining:

1. Stain the sections using a histological stain (e.g., H&E, PAS).

2. Rinse the sections with distilled water to remove excess stain.

Mounting:

1. Mount the stained sections under a coverslip using a mounting medium (e.g., DPX,
Permount).

2. Allow the mounting medium to dry and harden.

OBSERVATION:

Observe the quality of the tissue sections and evaluate the effectiveness of the tissue
processing steps. Note the presence of any artifacts or defects in the tissue sections.

DISCUSSION:

The results of this experiment demonstrate the importance of tissue processing in the
preparation of high-quality tissue sections. The steps involved in tissue processing, including
fixation, dehydration, clearing, infiltration, embedding, sectioning, staining, and mounting,
are critical for preserving the tissue and removing water and other soluble substances. The
quality of the resulting tissue sections is dependent on the effectiveness of these steps.

RESULT:

The result of this experiment is the production of high-quality tissue sections that can
be used for diagnostic and research purposes. The tissue sections are well-preserved, with
minimal artifacts or defects, and are suitable for histological examination.
 HAEMATOLOGY

5. COMPLETE BLOOD COUNT (CBC)

INTRODUCTION:

A Complete Blood Count (CBC) is a commonly performed blood test that provides
valuable information about the different components of blood, including red blood cells,
white blood cells, and platelets. The CBC test is used to diagnose and monitor a wide range
of medical conditions, including anemia, infection, and blood disorders.

AIM:

The aim of this experiment is to perform a CBC test on a blood sample and to analyze
the results to determine the different components of blood.

PRINCIPLE:

The principle of the CBC test is based on the concept of centrifugation, where the
different components of blood are separated based on their density. The test involves the use
of a centrifuge to separate the blood into its different components, including plasma, buffy
coat, and erythrocytes.

MATERIALS REQUIRED:

- Blood sample

- Centrifuge

- Hemocytometer

- Pipettes

- Microscope

- CBC machine (optional)

- Blood diluent

- Staining solutions (e.g., Wright's stain)

PROCEDURE:

Collection of Blood Sample:

1. Collect a blood sample from a patient or a healthy individual.

2. Use a sterile needle and syringe to collect the blood sample.

3. Transfer the blood sample to a tube containing an anticoagulant (e.g., EDTA).

Centrifugation:

1. Centrifuge the blood sample at 1500-2000 rpm for 10-15 minutes.

2. Separate the blood into its different components, including plasma, buffy coat, and
erythrocytes.

Hemocytometer Count:

1. Use a hemocytometer to count the different components of blood, including red blood
cells, white blood cells, and platelets.

2. Dilute the blood sample with a blood diluent (e.g., saline solution).

3. Load the diluted blood sample onto the hemocytometer.

4. Count the different components of blood using a microscope.

Staining:

1. Use staining solutions (e.g., Wright's stain) to stain the blood smear.

2. Observe the stained blood smear under a microscope.

CBC Machine:

1. Use a CBC machine to analyze the blood sample and determine the different components
of blood.

2. Load the blood sample onto the CBC machine.

3. Run the CBC machine according to the manufacturer's instructions.

OBSERVATION :

Observe the results of the CBC test, including the different components of blood, such
as red blood cells, white blood cells, and platelets. Note any abnormalities or deviations from
the normal range.

DISCUSSION:

The results of the CBC test provide valuable information about the different
components of blood. The test is used to diagnose and monitor a wide range of medical
conditions, including anemia, infection, and blood disorders. The results of the test should be
interpreted in the context of the patient's medical history and clinical presentation.

RESULT:

The result of the CBC test is a comprehensive report that includes the different
components of blood, such as:

- Red blood cell count (RBC)

- Hemoglobin (Hb)

- Hematocrit (Hct)

- White blood cell count (WBC)

- Differential count (e.g., neutrophils, lymphocytes, monocytes)

- Platelet count (PLT)

 HAEMATOLOGY

6. PERIPHERAL BLOOD SMEAR

INTRODUCTION:

A peripheral smear, also known as a peripheral blood smear, is a laboratory test used
to evaluate the different components of blood, including red blood cells, white blood cells,
and platelets. The test involves the examination of a blood smear under a microscope to
identify any abnormalities or deviations from the normal range.

AIM:

The aim of this experiment is to perform a peripheral smear test on a blood sample
and to evaluate the different components of blood.

PRINCIPLE:

The principle of the peripheral smear test is based on the concept of microscopy,
where a blood smear is examined under a microscope to identify the different components of
blood. The test involves the use of staining solutions to differentiate between the different
components of blood.

MATERIALS REQUIRED:

- Blood sample

- Glass slides

- Coverslips

- Staining solutions (e.g., Wright's stain, Giemsa stain)

- Microscope

- Pipettes

- Blood diluent (e.g., saline solution)

PROCEDURE:

Collection of Blood Sample:

1. Collect a blood sample from a patient or a healthy individual.

2. Use a sterile needle and syringe to collect the blood sample.

3. Transfer the blood sample to a tube containing an anticoagulant (e.g., EDTA).

Preparation of Blood Smear:

1. Place a small drop of blood onto a glass slide.

2. Spread the blood drop evenly across the slide using a spreader or a coverslip.

3. Allow the blood smear to air-dry.

Staining:

1. Use staining solutions (e.g., Wright's stain, Giemsa stain) to stain the blood smear.

2. Follow the manufacturer's instructions for the staining solution.

Microscopic Examination:

1. Examine the stained blood smear under a microscope.

2. Use the 100x objective lens to evaluate the different components of blood.

3. Identify any abnormalities or deviations from the normal range.

OBSERVATION:

Observe the different components of blood, including:

- Red blood cells (RBCs): size, shape, color, and distribution

- White blood cells (WBCs): type, size, shape, and distribution

- Platelets: size, shape, and distribution

- Any abnormalities or deviations from the normal range

DISCUSSION:

The results of the peripheral smear test provide valuable information about the
different components of blood. The test is used to diagnose and monitor a wide range of
medical conditions, including anemia, infection, and blood disorders. The results of the test
should be interpreted in the context of the patient's medical history and clinical presentation.

RESULT:

The result of the peripheral smear test is a descriptive report that includes:

- The different components of blood, including RBCs, WBCs, and platelets

- Any abnormalities or deviations from the normal range

- A diagnosis or recommendation for further testing or treatment