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1. MEDICAL PARASITOLOGY
LAB REPORTS
SUBMITTED BY: MARIJ NOOR
REG#: B21F0017BMS005
SUBMITTED TO: DR. FARRAKH JAVED
DEPARTMENT: BIOMEDICAL SCINECES

2. 1
Labtask#01
Protocol for Trypanosoma Sample Collection (Depending on
Species)
The specific method for collecting Trypanosoma samples depends on the parasite species and the
intended diagnostic test. Here's a breakdown for common methods:
1. Blood Collection:
• This is the most common method for detecting Trypanosoma cruzi (Chagas disease) and
Trypanosoma brucei (African Sleeping Sickness).
• Materials: Sterile syringes, needles appropriate for animal size, tourniquet (optional),
cotton swabs with alcohol, disinfectant wipes, microhematocrit tubes (optional) for
capillary blood collection.
• Procedure:
o Restrain the animal securely.
o Clean the puncture site with disinfectant wipes and alcohol swab.
o Apply a tourniquet if necessary (avoid prolonged application).
o Collect blood aseptically according to standard venipuncture techniques.
o Transfer blood to appropriate containers (e.g., EDTA tubes for parasite detection,
serum for serological tests).
2. Lymph Node Aspiration:
• Used for some Trypanosoma species, particularly in cases of lymphadenopathy (swollen
lymph nodes).
• Materials: Sterile syringes, needles appropriate for aspiration, disinfectant wipes, alcohol
swabs.
• Procedure:
o Restrain the animal securely.
o Clean the aspiration site with disinfectant wipes and alcohol swab.

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o Insert the needle into the swollen lymph node and aspirate fluid.
o Transfer aspirate to an appropriate container for analysis (e.g., anticoagulant
tube).
3. Tissue Biopsy:
• Used in specific cases where Trypanosoma infection is suspected in a particular tissue.
• Materials: Sterile surgical instruments, biopsy needle, disinfectant wipes, alcohol swabs,
fixative solution (depending on diagnostic test).
• Procedure:
o This is a surgical procedure requiring a veterinarian.
o Anesthesia and aseptic techniques are essential.
o A small tissue sample is collected and placed in a fixative solution or container
for further analysis.
4. Cerebrospinal Fluid (CSF) Collection:
• Used for Trypanosoma brucei gambiense infections that affect the central nervous
system.
• Materials: Spinal tap needles, sterile collection tubes, disinfectant wipes, alcohol swabs.
• Procedure:
o This is a complex procedure requiring a veterinarian familiar with CSF collection.
o Anesthesia and aseptic techniques are crucial.
o CSF is collected from the spinal cord and placed in sterile tubes for analysis.
5. Other Sample Types:
• Depending on the suspected location of the parasite, other samples like skin scrapings,
mucosal swabs, or body fluids might be collected.
References:
• Clinical Laboratory Science - E-Book: This textbook provides a general overview of
specimen collection for parasite identification, including Trypanosoma [1].
• Molecular Detection of Human Parasitic Pathogens: This book discusses various
methods for detecting human parasitic pathogens, though the specific collection protocols
for Trypanosoma might not be extensively detailed [2].

4. 3
Lab task#02
Protocol for Transporting Trypanosoma Samples
The safe and secure transportation of Trypanosoma samples is crucial to ensure viability for
further analysis and minimize the risk of exposure. Here's a detailed protocol incorporating best
practices:
Preparation:
1. Container Selection: Choose appropriate containers based on the sample type and
intended test.
o Blood: Sealed, sterile tubes containing anticoagulant (e.g., EDTA) for parasite
detection or clot activator (serum separator tubes) for serological tests.
o Tissue Biopsy: Fixative solution-filled container as per the diagnostic test
requirement.
o CSF, Aspirates, Swabs: Sterile leak-proof tubes with appropriate media for
parasite viability (e.g., culture medium for motile trypanosomes).
2. Temperature Control:
o Live Trypanosomes: Maintain a cool temperature (ideally 4°C) using a chilled
container with ice packs (avoid direct contact). Some trypanosomes might require
a specific temperature range depending on the species. Consult specific references
for optimal conditions.
o Fixed Samples: Room temperature is acceptable.
3. Documentation: Prepare a detailed label for each container including:
o Patient/animal identification
o Sample type (blood, CSF, etc.)
o Date and time of collection
o Storage temperature requirements
o Biohazard symbol (if applicable)
Packaging:
1. Primary Container: Securely seal the sample container to prevent leakage.
2. Secondary Container: Place the primary container in a sturdy, leak-proof secondary
container with sufficient absorbent material (e.g., absorbent pads) to contain any potential
spills.
3. Tertiary Packaging: Depending on regulations and shipping distance, a sturdy outer
container might be required.

5. 4
Shipping:
1. Compliance: Adhere to national and international regulations for transporting biological
materials (refer to IATA Dangerous Goods Regulations for air transport [1]).
2. Documentation: Include a properly completed shipping manifest with all necessary
information (refer to regulations).
3. Courier Service: Utilize a reliable courier service with experience in transporting
biological samples. Ideally, choose a service that offers temperature-controlled shipping
if transporting live parasites.
References:
• World Health Organization (WHO) - Laboratory Biosafety Manual: This
comprehensive manual provides guidelines for the safe transport of infectious substances
[2].
• International Air Transport Association (IATA) Dangerous Goods Regulations:
This resource details regulations for transporting dangerous goods by air, including
biological materials [1].

6. 5
Lab Task#03
Devise a protocol To Identify Trypanosoma from the Sample
Here's a protocol outlining different methods for identifying Trypanosoma depending on the
available resources and expertise:
1. Microscopic Examination:
• This is a rapid and readily available method for detecting motile trypanosomes in some
cases.
Materials:
• Microscope with appropriate magnification (10x and 40x objective lenses)
• Thin blood smear or wet mount preparation of the sample (depending on the sample type)
• Giemsa stain (or other suitable stain)
Procedure:
1. Prepare a thin blood smear or a wet mount of the sample depending on the type (blood,
CSF, etc.).
2. Fix and stain the smear with Giemsa stain or a suitable alternative.
3. Examine the stained preparation under a microscope for the presence of motile
trypanosomes. Trypanosomes appear as elongated, slender parasites with a single
flagellum and an undulating membrane.
Limitations:
• This method might not detect low parasite densities.
• It requires trained personnel for accurate identification.
• Not suitable for all Trypanosoma species (e.g., some may not be motile in the sample).
Reference:
• Parasitology: A Conceptual Approach: This textbook provides a detailed overview of
microscopic techniques for parasite identification, including Trypanosoma [1].
2. Rapid Diagnostic Tests (RDTs):
• These immunologically based tests offer a rapid and field-friendly option for
Trypanosoma detection.
Materials:

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• Commercially available RDT kit specific for Trypanosoma species of interest
Procedure:
1. Follow the manufacturer's instructions for the specific RDT kit.
2. Typically, these involve adding a small sample to a cassette containing specific
antibodies against Trypanosoma antigens.
3. The presence of Trypanosoma will be indicated by a visible colored line on the cassette.
Limitations:
• RDTs may have variable sensitivity and specificity depending on the kit used.
• They might not differentiate between different Trypanosoma species.
Reference:
• Point-of-Care Technologies for Infectious Diseases: This book discusses the
application of RDTs for diagnosing various infectious diseases, potentially including
Trypanosomiasis [2].
3. PCR (Polymerase Chain Reaction):
• This highly sensitive and specific method detects Trypanosoma DNA in the sample.
Materials:
• DNA extraction kit
• PCR machine
• Primers and probes specific for Trypanosoma DNA (commercially available or designed
based on the target species)
Procedure:
1. Extract DNA from the sample using a commercially available kit.
2. Set up a PCR reaction using specific primers and probes for Trypanosoma DNA.
3. Amplify the target DNA in a PCR machine and analyze the results for the presence of
Trypanosoma DNA.
Limitations:
• Requires specialized equipment and expertise.
• May not be readily available in all settings.
Reference:

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• Molecular Diagnostics: Applications in Infectious Diseases: This book provides an
overview of PCR techniques used for diagnosing infectious diseases, including potential
applications for Trypanosomiasis [3].
Choosing the Right Method:
• The choice of method depends on factors like:
o Availability of resources and expertise
o Required speed of diagnosis
o Sensitivity and specificity needs
o Suspected Trypanosoma species
Additional Considerations:
• In some cases, a combination of methods might be used for definitive diagnosis (e.g.,
microscopy followed by PCR for confirmation).
• Always consult specific protocols for the chosen diagnostic method and interpret results
in conjunction with clinical signs and symptoms.
References:
1. https://link.springer.com/chapter/10.1007/978-3-030-98427-4_31
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10086565/

9. 8
Lab task#04 (1)
Staining Procedure for Trypanosoma with Giemsa Stain
Giemsa stain is a commonly used method for visualizing Trypanosoma parasites in blood
smears, tissue sections, or other biological samples. Here's the staining protocol:
Materials:
• Air-dried blood smears or fixed tissue sections on slides
• Giemsa stain solution (typically diluted 1:10 in buffered solution at pH 6.8)
• Phosphate buffer solution (PBS, pH 6.8)
• Staining Coplin jars or staining dishes
• Distilled water
• Coverslips
• Methanol (optional)
Procedure:
1. Fixation (Optional): If using fresh blood smears, fix them by immersing the slides in
methanol for 2-5 minutes. This step might be omitted for already fixed tissue sections.
2. Giemsa Staining:
o Place the slides in a Coplin jar or staining dish filled with Giemsa stain solution
diluted 1:10 in PBS (pH 6.8).
o Incubate the slides for 30-45 minutes at room temperature. The optimal staining
time might vary depending on the specific stain and desired results.
3. Washing:
o Gently rinse the slides with distilled water to remove excess stain.
o Alternatively, a brief dip in PBS (pH 6.8) can be used.
4. Differentiation (Optional):
o Briefly differentiate the stain in a solution of distilled water and methanol (e.g.,
50% methanol) for a few seconds to enhance contrast. This step is optional and
needs to be carefully controlled to avoid over-differentiation.
5. Washing:
o Wash the slides thoroughly with distilled water to stop differentiation.
6. Drying:
o Allow the slides to air dry completely.
7. Mounting (Optional):
o Apply a coverslip with a suitable mounting medium (e.g., resinous mounting
medium) for permanent preservation.
Expected Results:
• With Giemsa stain:
o Trypanosoma parasites will appear as elongated, slender organisms with a pale
pink cytoplasm.

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o The nucleus will stain a dark purple or blue.
o The kinetoplast, a DNA-rich structure near the posterior end, will appear as a
bright red dot.
References:
• UK NEQAS Parasitology - Staining of Blood parasites other than malaria
parasites: This website provides a protocol for staining blood parasites, including
Trypanosoma, with Giemsa stain [1].
• Parasitology: A Conceptual Approach: This textbook covers various staining
techniques for parasites, including the use of Giemsa stain for Trypanosoma [2].
Additional Notes:
• The staining time and differentiation steps might require adjustments based on the
specific stain batch and desired results.
• Consider following the manufacturer's instructions for the specific Giemsa stain solution
used.
• Giemsa staining is a relatively simple technique but requires proper handling of
hazardous materials like methanol. Always follow laboratory safety protocols.

11. 10
Lab task#04 (02)
Purpose and Composition of Modified Hematoxylin Staining
Method
Hematoxylin staining is a fundamental technique in histology for visualizing cell nuclei.
However, the standard hematoxylin stain can sometimes lack contrast or specificity, especially
for certain tissues or research applications. This is where modified hematoxylin staining methods
come in.
Purpose of Modified Hematoxylin Staining:
The main purpose of modified hematoxylin staining methods is to:
• Enhance contrast: Modified stains can improve the differentiation between cell nuclei
and other cellular components by intensifying the staining of nuclei or by suppressing
background staining.
• Increase specificity: Certain modifications can target specific components within the
nucleus, such as DNA or RNA, for clearer visualization.
• Highlight specific structures: Modifications can be used to highlight particular
structures within the tissue, such as collagen fibers or basement membranes.
Composition of Modified Hematoxylin Stains:
There are numerous modified hematoxylin staining methods, each with a slightly different
composition. Here are some common modifications and their components:
• Hematoxylin with Mordant: Mordants are metallic salts that act as a bridge between the
negatively charged tissue and the positively charged hematoxylin dye. Examples include:
o Hematoxylin and Eosin (H&E): This is the most common histological stain. It
uses an alum mordant (aluminum potassium sulfate) to enhance nuclear staining
with hematoxylin and counterstains with eosin for cytoplasmic structures.
o Weigert's Hematoxylin: This modification uses an iron mordant (ferric
ammonium sulfate) to achieve a more intense and sharper nuclear stain, often
used for studying the nervous system.
• Hematoxylin with Differentiating Agent: These methods use a solution to remove
excess stain from certain tissue components. Examples include:
o Hematoxylin and Periodic Acid-Schiff (PAS): This stain differentiates between
neutral and acidic carbohydrates. Hematoxylin stains nuclei, while PAS stains
structures rich in carbohydrates (e.g., basement membranes, fungi).
o Hematoxylin and Congo Red: This method uses Congo red to highlight amyloid
deposits in tissues. Hematoxylin stains nuclei as usual.
Choosing the Right Modified Hematoxylin Stain:

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The choice of a modified hematoxylin stain depends on the specific tissue and the structures of
interest. Here are some resources to help you choose the right stain:
• Bancroft's Theory and Practice of Histological Techniques: This comprehensive
textbook details various histological staining methods, including modified hematoxylin
stains [1].
• Armed Forces Institute of Pathology (AFIP) Laboratory Methods in
Histotechnology: This online resource provides protocols for a variety of histological
stains, including some modified hematoxylin methods [2].
Figure 1Trypanosoma cruzi intracytoplasmic amastigotes (arrows). Heart muscle, hematoxylin-eosin
stain, 1000×.