ABBREVATIONS

ZN Zeil Neilson

CBC Complete blood picture

HB Haemoglobin

TLC Total leukocyte count

TRBC Total red blood cell

PCV Pack cell volume

HCT Haematocrit

MCH Mean corpuscular haemoglobin

MCHC Mean corpuscular haemoglobin concentration

ESR Erythrocyte sedimentation rate

HBSAG Hepatitis B surface antigen

HCV hepatitis C virus

H.PYLORI helicobacter pylori

ALT Alanine amino transferases

AST Asparte amino transferases

PCR Polymerase chain reaction

HCG Human chorionic gonadotropin

 INTRODUCTION

Internship is an integral part of the program in laboratory medicine and is designed to provide students with an
opportunity to integrate and apply previously acquired knowledge and technical skills in actual clinical settings. Under
the guidance of experienced medical laboratory professionals and other qualified laboratory personnel and health
professionals, students learn more about diagnostic test procedures, quality control methods and instrumentation in the
clinical laboratory. They also gain an understanding of the roles and functions of the medical laboratory professionals.

The internship provides applied learning experiences during which the student should:

1. Acquire and practice clinical laboratory skills.

2. Practice skills in problem-solving.

3. Perform quality control procedures.

4. Adapt and learn new procedures.

5. Operate various laboratory instruments.

6. Understand the responsibilities and functions of the medical laboratory professionals

7. Report accurate and precise results.

8. Learn how to relate tests results to patient clinical diagnosis.

The internship program is conducted in the affiliated hospital laboratories of the program, Where students learn by
participating in the workload of a supervising technologist/specialist/consultant.
ASTHA SEWA SANSTHAN, UPHC ROORKEE

Astha Sewa Sansthan is a Social voluntary Organisation, established in the year 2001 under society registration act
1961, in the state of Uttarakhand.

Astha is derived from the belief & faith in people and trying to disseminate authentic & Scientific information to the
vulnerable community.

Education is the most powerful catalyst for social transformation. A child will go to school only if the family,
particularly the mother, is assured of healthcare and empowered. When an elder sibling is relevantly skilled
to be employable and begins earning, the journey of empowerment continues beyond the present generation.

Education is the most powerful catalyst for social transformation. A child will go to school only if the family,
particularly the mother, is assured of healthcare and empowered. When an elder sibling is relevantly skilled
to be employable and begins earning, the journey of empowerment continues beyond the present generation.

Realizing this. Astha Sewa Sansthan, began its activities & interventions in the area of Education & Health.
It adopted a life cycle approach of development, focusing its interventions on children, their families and the
larger community.

Astha Sewa Sansthan believes that unless members of the civil society are involved proactively in the process
of development, sustainable change will not happen. Following this, model of Civic Driven Change, Astha
Sewa Sansthan sensitizes and engages the civil society, making it an active partner in all its welfare initiatives.

Vision
Holistic Development of the Communities.

Mission
To promote and provide the basic information & services to the weekend section of the community in the state of
Uttarakhand.

Story
This foundation’s vision for developing children has taken roots in the hearts and minds of a small group of people
around the globe as they work together to release communities from poverty.

What started as a short mission trip between a group of young people coming together to make a change, this foundation
continues to exist as an agency to provide medical, social, psychological and education needs of children and their
families.
Our Impact

Today, thousands of children and their families are discovering lives full of promises – making education, clean water,
and opportunities easily accessible. With our global partnership, we have offered financial support to over 15
communities in 9 countries – all thanks to your donations and support.

During this time of uncertainty, our efforts to end poverty need to be stronger than ever and so we launch the global
movement to reach out even further. We have doubled our efforts in making clean water, food and clothing more
accessible to those most at risk during the pandemic.
MICROBIOLOGY
1. COMMON MEDIA IN ROUTINE USE
I. CLED Agar (Cysteine Lactose Electrolyte Deficient): is a non-selective differential plating medium for
the growth and enumeration of urinary tract microorganism.
II. Preparation: 36.0g of medium is suspended in one liter of distilled water, slowly heated and frequently
stirred. Boiled for a minute and sterilized at 121OC for 15minutes and poured into Petri dishes. Plates were
inverted when solidified for storage purposes and to avoid moisture

III. MacConkey Agar. Most commonly used for enterobacteriaceae. It contains agar, peptone, sodium chloride,
bile salt, lactose and neutral red. It is a selective medium:
IV. Selective: as bile salt does not inhibit the growth of enterobacteriaceae but inhibits growth of many other
bacteria.
V. Indicator: Bacteria that ferment lactose take a pink colour due to production of acid. Acid turns the
indicator neutral red to pink. These bacteria are called 'lactose fermenter', e.g. Escherichia coll. Colorless
colony indicates that lactose is not fermented, i.e. the bacterium is non-lactose fermenter, e.g. Salmonella,
Shigella.

a. Preparation: 50g of the Agar was suspended and measured into one liter of purified water and
mixed thoroughly and was heated with frequent agitation, then boiled for one minute to completely
dissolve the powder. The Agar was autoclaved at 121OC for 15minutes.

VI. Chocolate Agar :It is prepared by heating blood agar. It is used for culture of meningococcus,
pneumococcus, gonococcus, and Haemophilus.

VII. Preparation: 2 liters of distilled water was added to 144g of agar powder. Autoclaving was done at 121 OC
for 15minutes and cooled till 45OC then 5% of defribrinated blood was added. Heated slowly and evenly
to 65OC, cooled till 45OC and poured into plates

VI. Blood Agar: Is used most commonly. For blood agar preparation 5-10% defribrinated sheep blood is mixed
with melted agar at 45-50°C. Blood works as an enrichment material and also act as an indicator. Bacteria
when grown in blood agar produce hemolysis around their growing colonies. Some bacteria produce no
hemolysis. Types of changes:
a) Beta haemolysis, There is clear zone around bacterial colony of complete hemolysis, e.g. Streptococcus
pyogenes is a beta hemolytic Streptococci.
b) Alpha haemolysis, When there is greenish discoloration around bacterial colony colony due to
formation of biliverdin, e.g. Viridans streptococci.
c) Gamma haemolysis, or, No hemolysis. There is no change in the medium near the bacterial colony.
2. PREPARATION AND STERILIZATION OF CULTURE MEDIA:
Culture media are available commercially as powder; they require only the addition of water. Nutrient medium is a
general purpose preparation for culturing microorganisms which are not nutritionally fastidious.

The agar prepared has the same composition. The final pH of both media is 7.4.
AUTOCLAVING

Autoclaving is a process that use moist heat and pressure so that all parts of the material to be sterilized reach 121
degree Celsius for 15 minutes. An autoclave is, in essence, a large pressure cooker; a chamber which may be sealed off
against surrounding air.
Objective:

To prepare sterile nutrient agar for culturing microorganisms.

MATERIAL AND REAGENTS:

Commercial nutrient agar, Balance, Distilled water, Scott bottles, Measuring cylinder Beaker, Forceps, Universal
bottles

PROCEDURE
1. Appropriate amount of broth (with agar) powder is weighed into Scott bottles and dissolve with distilled
water. They are mixed well.

Figure 7 Media preparation

2. The bottles are loosely recap and set aside for sterilization.

3. Figure 8 sterilization
4. All the media are sterilized at 121 degree celcius for 15 minutes.

5. After autoclaving, the media is removed. The broth preparation is allowed to cool and the cap of each
bottle is tightened.
GRAM STAINING:

It is the most important and widely used microbiological differential staining technique. It was developed by Dr.
Christian Gram in 1884, and diffentiate bacteria according to their Gram character (Gram positive or Gram negative).
In addition this stain also helps to determine of cell morphology, size, and arrangement.
Principle
The bacteria is stained with primary stain Crystal Violet and fixed by the mordant, certain of the bacteria are able to
retain the crystal violet and some washed off by alcohol.Gram positive bacteria cell wall have a thick layer of protein-
sugar complexes known as peptidoglycan. The action of decolorizer causes cell wall of the cell to dehydrate, causing
the pores in the cell wall to shrink and prevents the stain from exiting the cell. So the ethanol cannot remove the Crystal
Violet-Iodine complex that is bound to the thick layer of peptidoglycan of gram positive bacteria and results in blue or
purple appearance of cell.
In case of gram negative bacteria, cell wall also takes up the CV-Iodine complex but due to the thin layer of
peptidoglycan and thick outer layer which is formed of lipids, CV-Iodine complex gets washed off. When they are
exposed to alcohol, decolorizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine complex to
leach out of the cells. Then when again stained with safranin, they take the stain and appears red in colour.
SMEAR PREPARATION
1. Take a dry slide.
2. Sterilize the inoculating loop on Bunsen burner flame.
3. Transfer culture (or the specimen) by sterile loop and make a smear at the center.
4. Air dry the smear.
5. the dry smear through flame in such a way that smear side is facing up.
PROCEDURE
1. Place the slides above the staining rod.
2. Cover the smear with C-V stain and leave it for 1 minute.
3. Wash it by tap water.
4. Apply Gram’s iodine solution on smera and leave it for 1 minute.
5. Wash the slide again by stream of tap water.
6. Flood the slide with the decolorizing agent then wait for 20-30 seconds. This can also be done by adding a drop by
drop to the slide until the decolorizing agent running from the slides runs clear.
7. Wash the slide by running tap water and drain completely.
8. Stain with safranin and and wait for 30 seconds to 1 minute.
9. Wash slide by indirect stream of tap water until no color appears in the effluent and then blot dry with absorbent
paper.
10. Observe slide under microscope (oil immersion 100x) using a Bright field microscope.

Figure 10 Gram staining

RESULT

The staining results of gram stain are as follows:

• Gram Positive : Appears dark purple

• Gram Negative : Appears pale to dark red

• Yeasts : Appears Dark purple

• Epithelial cells :Appears Pale red

SPUTUM EXAMINATION BY ZN STAINING:

PRINCIPLE
This procedure is used to stain mycobacterium tuberculosis and mycobacterium leprae. These bacteria are also called
acid fast bacilli. They stain with carbol fuschin, which is a red dye. They retain dye when treated with acid, which is
due to presence of mycolic acid in their cell wall.

Zn staining
REAGENTS:
• Carbol fuschin (basic dye)

• 20% sulphuric acid (decolorizer)

• Methylene blue (counter stain) or Malachite green

Zn T.B stain kit

PROCEDURE
1. Spread the sputum evenly over the central area of the slide using a continuous rotational movement. The
recommended size of the smear is about 20 mm by 10 mm.
2. Place slides on dryer with smeared surface upwards, and air dry for about 30 minutes Fig. Heat Fixation of smear
(Upper: using electric heater, lower: using burner)
3. Heat fix dried smear.
4. Cover the smear with carbol fuchsin stain.
5. Heat the smear until vapour just begins to rise (i.e. about 60 degree Celsius). Do not overheat (boil or dry). Add
additional stain if necessary. Allow the heated stain to remain on the slide for 5 minutes.
6. Wash off the stain with clean water.
7. Cover the smear with 3% v/v acid alcohol for 2-5 minutes (or 20% sulfuric acid) or until the smear is sufficiently
decolorized.
8. Wash well with clean water
9. Cover the stain with malachite green stain for 1-2 minutes
10. Wash off stain with clean water.
RESULTS AND INTERPRETATION:
ACID FAST BACILLI:These are Red, straight or curved rods, occurring singly or in small groups.
CELLS: Appear Green (malachite green) or Blue (methylene blue)
BACKGROUND :Appear as Green (malachite green) or Blue (methylene blue).

Slide of Zn staining