MAY/JUNE 2023
QUESTION 1 [21]
1.1. Explain the purpose of laboratory safety and regulations for handling chemicals.
(3)
The purpose of laboratory safety and regulations for handling chemicals is multi-fold:
1. Protection of Personnel: Safety measures are in place to safeguard the health and
well-being of laboratory workers, minimizing the risk of accidents, injuries, and exposure
to hazardous chemicals. This includes the provision of protective gear, proper training,
and safety protocols.
2. Prevention of Accidents: The regulations are designed to prevent accidents such as
spills, fires, explosions, and other mishaps that could result from mishandling or
improper storage of chemicals.
3. Environmental Protection: Proper handling of chemicals ensures that no hazardous
substances are released into the environment, preventing pollution and harm to
ecosystems.
1.2. Outline the applications of histopathology in medicine. (3)
Histopathology plays a crucial role in medicine:
1. Disease Diagnosis: It helps in the identification and diagnosis of diseases and
conditions by examining tissue samples under a microscope. This is crucial for
identifying cancers, infectious diseases, inflammatory conditions, and more.
2. Treatment Decisions: Histopathology assists in determining the most effective
treatment approaches. For instance, it helps in assessing the stage and characteristics
of tumors, aiding in the selection of appropriate therapies.
3. Research and Understanding: It is fundamental for medical research, providing
insights into the underlying causes of diseases, understanding disease progression, and
discovering new therapeutic targets.
,1.3. List five (5) factors affecting fixation of the cells in a physical and chemical state.
(5)
Several factors impact the fixation of cells in a physical and chemical state:
1. Time of Fixation: The duration for which cells are exposed to fixatives affects their
preservation. Over-fixation or under-fixation can alter cellular structures.
2. Temperature: Fixation can be temperature-dependent; some fixatives work optimally at
certain temperatures. Temperature extremes can alter fixation efficiency.
3. pH of Fixative: The pH level of the fixative can influence the cellular structure
preservation. Changes in pH can disrupt the fixation process.
4. Type of Fixative: Different fixatives (formaldehyde, alcohol, glutaraldehyde, etc.) have
distinct effects on cell preservation. Each type works differently and affects cellular
components uniquely.
5. Size and Type of Tissue: Different tissues might require specific fixatives or fixation
durations based on their composition and structure, affecting fixation efficacy.
1.4. Describe how you would cut a 5µm tissue section for staining. In your answer you
should include (i) information on how to set up your workstation, (ii) microtome and (iii)
any potential troubleshooting you may need to do. (10)
To cut a 5µm tissue section for staining, here's a detailed procedure covering workstation
setup, microtome operation, and potential troubleshooting:
(i) Setting up your Workstation:
1. Clean and Prepare Work Area: Ensure your workstation is clean and free of
contaminants to prevent any interference with the tissue samples.
2. Gather Necessary Materials: Prepare the slides, labeled containers for solutions,
staining materials, and safety gear (gloves, lab coat, goggles).
, 3. Arrange the Microtome: Place the microtome on a stable, level surface, ensuring it's
correctly calibrated and well-maintained for precision cutting.
4. Prepare Tissue Samples: Ensure tissue blocks are adequately fixed and processed,
embedding them in paraffin blocks for stability during sectioning.
(ii) Using the Microtome:
1. Set the Microtome: Adjust the microtome to cut sections at the desired thickness, in
this case, 5µm. This is typically achieved by moving the microtome's cutting blade to the
appropriate settings.
2. Prepare the Block: Mount the tissue block onto the microtome's specimen holder,
securing it properly.
3. Trimming the Block: Trim the tissue block to form a flat surface, enabling the
microtome to cut uniform sections.
4. Sectioning: Use the microtome's wheel or control system to cut thin slices from the
tissue block at the set thickness (5µm in this case). Collect the sections onto labeled
glass slides for staining.
(iii) Potential Troubleshooting:
1. Section Wrinkling or Folding: Adjust the cutting angle or blade sharpness. Wrinkles
might occur due to improper blade angle or dull blades.
2. Uneven Section Thickness: Ensure the tissue block is correctly placed and firmly
secured. Inconsistent thickness can result from the block not being level or not properly
trimmed.
3. Sections Falling Apart: If sections crumble or break, the tissue might be too hard or
too soft. Adjust the temperature during preparation or the block's firmness.
4. Staining Issues: Sections might not stain evenly due to inadequate fixation or improper
cutting. Ensure proper fixation and adjust the cutting parameters if necessary.
Regular maintenance and calibration of the microtome, proper handling of tissue blocks, and
adherence to standard protocols for cutting and handling tissue sections are essential to obtain
, accurate 5µm sections for staining. Regular checks and adjustments should be made to
ensure the equipment is functioning optimally and that tissue sections are being cut accurately.
QUESTION 2 [26]
As the leading scientist in a biomedical science laboratory, it is a requirement to give
advice to your lab assistants when they are having problems with their experiments.
What advice would you give to your assistants that are having the following problems:
After performing a polymerase chain reaction (PCR) and agarose gel electrophoresis to
confirm the presence of the CO1 gene of 750bp
2.1. They observe no band appearing on an agarose gel. What would be your
conclusion? (5)
1. Possible Causes: The absence of the expected band could be due to several factors
such as inadequate PCR amplification, contamination, or issues with the primer design
or DNA template.
2. Suggested Actions:
Reassess PCR Conditions: Check the PCR conditions (annealing temperature,
extension time, and cycles). Optimize these parameters if necessary to enhance
amplification.
DNA Quality: Ensure the DNA template is of good quality and concentration.
Repurify the DNA if needed.
Check Primers: Validate primer sequences and concentrations. Consider
redesigning or acquiring new primers if there are concerns about their efficiency.
Control for Contamination: Implement strict precautions to prevent
contamination during PCR setup. Use fresh reagents and sterile conditions.
Positive Control: Run a positive control in parallel to ensure the PCR setup and
conditions are appropriate.
