SECTION A

QUESTION 1

What is staining and why is it important?

Staining is a micro-biological technique which is used to enhance and contrast a biological

specimen at the microscopic level and study it at a higher magnification for histopathological
studies and diagnostic purposes.

Importance of staining

1. Enhanced Visibility: Staining improves the contrast between cellular structures or

microorganisms and their surroundings, making them easier to observe under a microscope.
2. Cell Differentiation: Staining techniques allow for the differentiation of different cell types
based on their staining properties, aiding in cellular classification and identification.
3. Disease Diagnosis: Staining helps pathologists identify abnormal cells or microorganisms
associated with diseases, aiding in accurate diagnosis and treatment planning.
4. Research Advancement: Staining enables detailed examination of cellular morphology,
function, and interactions, facilitating research in fields such as microbiology, histology, and
pathology.
5. Histological Studies: Staining is essential for studying tissue samples, allowing researchers to
visualize and analyze tissue structures, cellular arrangements, and pathological changes.
6. Microbiological Analysis: Staining techniques are fundamental in microbiology for
visualizing and identifying bacteria, fungi, and other microorganisms, aiding in microbial
classification and research.
7. Education: Staining is a vital tool in educational settings, allowing students to learn about
cellular structures, microbial morphology, and disease pathology through microscopy.
QUESTION 2

List the different types of stains and explain two

Simple Stains:

Examples: Crystal violet, methylene blue, safranin.

Explanation: Simple stains consist of a single dye that stains all microbial cells uniformly. They
are quick and easy to use, providing contrast between the cells and the background, aiding in
visualization under a microscope. However, simple stains do not differentiate between different
types of microorganisms or cellular structures.

Differential stains:

Examples: Gram stain, acid-fast stain.

Explanation: Differential stains utilize multiple dyes to distinguish between different types of
microorganisms or cellular structures based on their staining properties. For instance:

Gram stain: It differentiates bacteria into Gram-positive (retains the crystal violet stain) and
Gram-negative (loses the crystal violet stain but retains the safranin counterstain) based on
differences in cell wall composition.

Acid-fast stain: It distinguishes between acid-fast bacteria (retain the carbolfuchsin stain despite
acid-alcohol treatment) and non-acid-fast bacteria based on their ability to retain the dye in the
presence of acid-alcohol.

These staining techniques are fundamental in microbiology for microbial identification,

classification, and characterization. They provide valuable information about cell morphology,
cell wall composition, and other cellular features essential for understanding microbial behavior
and pathogenicity.
QUESTION 3

List 15 equipment used in microbiology and their uses

1. Microscope: Used for magnifying and observing microorganisms and cellular structures.
2. Incubator: Maintains optimal temperature and conditions for microbial growth.
3. Autoclave: Sterilizes equipment and media by using high-pressure steam.
4. Bunsen burner: Provides a flame for sterilizing tools and creating aseptic conditions.
5. Petri dishes: Used for culturing microorganisms on solid media.
6. Inoculating loop: Transfers microorganisms between different media.
7. Pipettes: Used for precise measurement and transfer of liquids, such as microbial cultures or
reagents.
8. Centrifuge: Separates components of microbial samples based on density through centrifugal
force.
9. Colony counter: Counts individual microbial colonies grown on agar plates.
10. Microbial identification systems: Utilized for identifying microorganisms based on
biochemical or molecular characteristics.
11. pH meter: Measures the acidity or alkalinity of microbial cultures or growth media.
12. Spectrophotometer: Measures the absorbance or optical density of microbial cultures,
indicating microbial growth or metabolic activity.
13. Refrigerator: Stores microbial cultures, media, and reagents at low temperatures to prolong
their shelf life.
14. Freezer: Stores microbial cultures and samples at ultra-low temperatures for long-term
preservation.
15. Incubating shaker: Provides agitation and controlled temperature for microbial cultures,
promoting growth and uniform mixing.
QUESTION 4

Outline the procedures for gram staining

The Gram staining procedure is a differential staining technique used to differentiate bacteria
into Gram-positive and Gram-negative based on differences in cell wall composition. Here's an
outline of the procedure:

Preparation of Bacterial Smear:

Obtain a clean microscope slide and label it with the sample identification. Using a sterile loop
or swab, pick up a small amount of the bacterial culture from a solid agar plate or broth culture.
Spread the bacterial sample onto the slide to create a thin smear. Allow the smear to air dry
completely.

Fixation: Pass the slide containing the bacterial smear over a Bunsen burner flame a few times
to heat-fix the cells. This process helps the cells adhere to the slide and prevents them from
washing off during staining.

Primary Staining (Crystal Violet): Flood the heat-fixed bacterial smear with crystal violet stain
(primary stain). Allow the stain to sit on the slide for about 1 minute.

Washing: Gently rinse the slide with distilled water to remove excess crystal violet stain. Gram's
Iodine (Mordant): Flood the slide with Gram's iodine solution (mordant). Gram's iodine forms a
complex with crystal violet within the cell, enhancing its retention.

Washing: Rinse the slide with distilled water to remove excess iodine solution.

Decolorization: Gently flood the slide with a decolorizing agent, typically ethanol or acetone.
This step removes the stain from Gram-negative bacteria while leaving the stain trapped in the
thick peptidoglycan layer of Gram-positive bacteria. Decolorize until no more color runs off
from the slide (usually around 10-20 seconds).

Washing: Immediately rinse the slide with distilled water to stop the decolorization process.
Counterstaining (Safranin): Flood the decolorized bacterial smear with safranin (counterstain),
which stains the Gram-negative bacteria pink or red. Allow the safranin to sit on the slide for
about 1 minute.

Washing and Drying: Rinse the slide with distilled water to remove excess safranin. Gently blot
the slide with bibulous paper or allow it to air dry.

Microscopic Examination: Examine the stained slide under oil immersion microscopy. Gram-
positive bacteria will appear violet or blue, while Gram-negative bacteria will appear pink or red.
Following these steps meticulously ensures accurate differentiation of bacteria based on Gram
staining, which is crucial for microbial identification and classification in microbiology.

QUESTION 5

Write briefly on the mechanism of staining of acid-fast bacilli

Acid-fastness is a physical property of certain bacterial and eukaryotic cells, as well as some sub-
cellular structures, specifically their resistance to decolorization by acids during laboratory
staining procedures. Once stained as part of a sample, these organisms can resist the acid and/or
ethanol-based decolorization procedures common in many staining protocols, hence the name
acid-fast.

Mycobacterium tuberculosis (stained red) in tissue (blue).

The mechanisms of acid-fastness vary by species although the most well-known example is in
the genus Mycobacterium, which includes the species responsible for tuberculosis and leprosy.
The acid-fastness of Mycobacteria is due to the high mycolic acid content of their cell walls,
which is responsible for the staining pattern of poor absorption followed by high retention. Some
bacteria may also be partially acid-fast, such as Nocardia.

Acid-fast organisms are difficult to characterize using standard microbiological techniques,

though they can be stained using concentrated dyes, particularly when the staining process is
combined with heat. Some, such as Mycobacteria, can be stained with the Gram stain, but they
do not take the crystal violet well and thus appear light purple, which can still potentially result
in an incorrect gram-negative identification.

The most common staining technique used to identify acid-fast bacteria is the Ziehl–Neelsen
stain, in which the acid-fast species are stained bright red and stand out clearly against a blue
background. Another method is the Kinyoun method, in which the bacteria are stained bright red
and stand out clearly against a green background. Acid-fast Mycobacteria can also be visualized
by fluorescence microscopy using specific fluorescent dyes (auramine-rhodamine stain, for
example)
 SECTION B

QUESTION 1

Define the isolation of microorganisms

The isolation of microorganisms refers to the process of separating and culturing individual
microbial species from a mixed population in order to study their characteristics, behavior, and
functions. It is also the process of separating a single species of microorganism from its natural
habitat and growing it by itself, without interference from other organisms, on a sterile
substratum, i.e., in pure culture. This typically involves techniques such as streak plating,
dilution series, and selective media to obtain pure cultures for further analysis.

QUESTION 2

List 5 methods of isolation of microorganisms and describe two

1. Serial Dilution and Plating

2. Spread Plate Technique
3. Streak Plate Method
4. Filtration
5. Selective Media

1. Serial Dilution and Plating: This method is particularly useful when dealing with samples
containing high microbial loads. By diluting the sample in a series of steps, it reduces the
number of microorganisms per unit volume, making it easier to isolate individual
colonies. After dilution, small volumes of the diluted sample are plated onto agar plates
and spread evenly. Colonies that develop on the plates can then be picked and streaked
for further isolation.
 2. Streak Plate Method: The streak plate method is a simple yet effective technique for
isolating bacteria. A loopful of the sample is streaked across the surface of an agar plate
in a pattern that thins out the bacterial cells. With each streak, individual bacterial cells
are deposited onto the agar surface, leading to the formation of isolated colonies. This
method relies on the principle that dilution of the sample on the agar surface results in the
separation of individual colonies, allowing for the isolation of pure cultures.

QUESTION 3

A) What is culture?

In microbiology, a culture refers to the propagation and growth of microorganisms in a

laboratory setting. This process involves providing a suitable environment, such as a nutrient-
rich medium, temperature, and other necessary conditions, for the growth and reproduction of
microorganisms. Culturing microorganisms allows researchers to study their characteristics,
behavior, and interactions under controlled conditions. Cultures can be used for various
purposes, including identification of microorganisms, testing their susceptibility to antimicrobial
agents, and producing useful compounds such as antibiotics or enzymes.

B) List four types of culture media used in microbiology laboratory

1. Nutrient Agar: Nutrient agar is a basic medium containing peptones, beef extract, agar, and
water. It provides essential nutrients for the growth of a wide range of microorganisms and is
often used for general-purpose cultivation and maintenance of bacterial cultures.
2. Blood Agar: Blood agar contains blood (usually from sheep or horse), peptones, agar, and
other nutrients. It is enriched with blood, which provides additional growth factors for
fastidious organisms such as Streptococcus species. Blood agar is commonly used to
differentiate bacteria based on their hemolytic activity (e.g., alpha, beta, or gamma
hemolysis).
3. MacConkey Agar: MacConkey agar is a selective and differential medium primarily used for
the isolation and differentiation of gram-negative bacteria, particularly members of the
 Enterobacteriaceae family. It contains peptones, lactose, bile salts, neutral red dye, and crystal
violet. The bile salts inhibit the growth of gram-positive bacteria, while lactose fermentation
by lactose-fermenting bacteria results in the production of pink/red colonies due to the pH
indicator.
4. Sabouraud Agar: Sabouraud agar is a selective medium used for the isolation and cultivation
of fungi, particularly yeasts and molds. It contains peptones, dextrose, agar, and sometimes
antibiotics to inhibit bacterial growth. The acidic pH of Sabouraud agar and the low water
content make it suitable for the growth of fungi while suppressing bacterial growth.

SECTION C

QUESTION 1

List and state the functions of five components of a microscope

1. Objective lens
2. Eyepiece or ocular lens
3. Stage
4. Condenser
5. Light source

1. Objective Lenses: Objective lenses are responsible for magnifying the specimen.
Microscopes typically have multiple objective lenses with different magnification powers
(e.g., 4x, 10x, 40x, 100x). Users can switch between these lenses to observe the specimen at
various magnifications.
2. Eyepiece or Ocular Lens: The eyepiece, also known as the ocular lens, further magnifies the
image produced by the objective lens. Typically, microscopes have one or two eyepieces,
each with a specific magnification power (e.g., 10x). The combined magnification of the
objective and eyepiece lenses determines the total magnification of the microscope.
3. Stage: The stage is a platform where the specimen is placed for observation. It often includes
clips or a mechanical stage to hold the specimen in place and allow for precise movement.
 Some stages also have controls for adjusting the position of the specimen horizontally and
vertically.
4. Condenser: The condenser is located beneath the stage and is responsible for focusing light
onto the specimen. It contains lenses that converge light rays onto the specimen, enhancing
contrast and resolution. The condenser can be adjusted to control the amount and angle of
light reaching the specimen.
5. Light Source: The light source provides illumination for the specimen. Most microscopes use
either built-in or external light sources, such as halogen bulbs or LEDs. Proper illumination is
essential for obtaining clear and detailed images of the specimen. Users can adjust the
intensity of the light source to optimize the image brightness and contrast.

QUESTION 2:

Differentiate between microscope, microscopy and microscopic

MICROSCOPE:

Definition: A microscope is an instrument used for magnifying and observing small objects or
organisms that are not visible to the naked eye.

Focus: The instrument itself.

Usage: Microscopes are utilized for magnifying and observing small details of objects or
organisms.

Example: Optical microscope, electron microscope, atomic force microscope.

MICROSCOPY:

Definition: Microscopy refers to the study or practice of using microscopes for observing small
objects or organisms.

Focus: Study or practice.

Usage: It denotes the activity or field of using microscopes for research, analysis, or examination
of microscopic specimens.

Example: Techniques such as light microscopy, electron microscopy, and scanning probe
microscopy.

MICROSCOPIC:

Definition: Microscopic refers to objects or organisms that are extremely small and not visible to
the naked eye.

Focus: Describes objects or organisms.

Usage: It indicates the size or scale of objects or organisms, often implying that they are very
small.

Example: Microscopic organisms, microscopic features, microscopic particles.

In summary, a microscope is the instrument used for magnification, microscopy is the study or
practice involving the use of microscopes, and microscopic describes objects or organisms that
are extremely small and require magnification for observation.

QUESTION 3

State 7 care of a microscope

1. Cleaning: Regularly clean the lenses, stage, and other parts of the microscope using lens
paper or a soft cloth to remove dust, oil, and debris.
2. Storage: Store the microscope in a clean, dry, and dust-free environment to prevent
contamination and damage.
3. Handling: Handle the microscope with care, avoiding sudden movements or impacts that
could misalign or damage internal components.
4. Calibration: Periodically calibrate the microscope to ensure accurate magnification and focus
settings.
5. Lubrication: Apply lubricant to moving parts as recommended by the manufacturer to
maintain smooth operation.
6. Covering: When not in use, cover the microscope with a dust cover to protect it from dust
and other environmental contaminants.
7. Inspection: Regularly inspect the microscope for any signs of damage or wear, addressing
issues promptly to prevent further damage.

QUESTION 4

State 10 precautionary measures as a laboratory staff

1. Wear appropriate personal protective equipment (PPE) such as lab coats, gloves, and safety
goggles to protect against chemical splashes, spills, and other hazards.
2. Familiarize yourself with the location and proper use of safety equipment including eyewash
stations, emergency showers, fire extinguishers, and first aid kits.
3. Follow established laboratory protocols and procedures for handling, storing, and disposing
of chemicals and hazardous materials.
4. Never eat, drink, chew gum, or apply cosmetics in the laboratory to avoid accidental
ingestion of chemicals.
5. Be cautious when working with heat sources such as Bunsen burners or hot plates, and use
appropriate handling techniques for hot glassware and equipment.
6. Handle glassware and sharp objects with care to prevent cuts and injuries, and dispose of
broken glass properly in designated containers.
7. Label all containers clearly with the contents, hazards, and date of preparation to prevent
accidental exposure or misuse.
8. Work in a well-ventilated area or use fume hoods when working with volatile or hazardous
chemicals to minimize exposure to fumes and vapors.
9. Keep work areas clean and organized to reduce the risk of spills, trips, and falls, and
promptly clean up any spills using appropriate procedures and materials.
10. Stay informed about potential hazards and safety procedures specific to the experiments and
equipment being used, and seek assistance or clarification if unsure about proper protocols.
 SECTION D

QUESTION 1

List the importance of a laboratory request form

1. Documentation: It serves as an official record of the tests requested by a healthcare provider,

ensuring accurate tracking and documentation of patient care.
2. Communication: It facilitates clear communication between healthcare providers, laboratory
staff, and other healthcare professionals involved in the patient's care.
3. Accuracy: It helps ensure that the correct tests are ordered for the patient based on their
symptoms, medical history, and clinical presentation, reducing the risk of unnecessary or
incorrect testing.
4. Patient Safety: By providing essential patient information such as demographics, medical
history, and allergies, it helps prevent adverse events or reactions during testing.
5. Legal and Regulatory Compliance: It ensures compliance with regulatory requirements and
standards for laboratory testing, helping healthcare facilities maintain accreditation and meet
legal obligations.
6. Billing and Reimbursement: It provides necessary information for billing and reimbursement
purposes, ensuring accurate billing codes and documentation for insurance claims.
7. Laboratory Efficiency: It streamlines the workflow in the laboratory by providing all
necessary information for sample processing, testing, and result reporting, improving
efficiency and turnaround times.
8. Quality Assurance: It supports quality control and quality assurance processes in the
laboratory by documenting test requests and ensuring adherence to standard operating
procedures.
9. Research and Education: It may serve as a source of data for research, quality improvement
initiatives, and educational purposes within healthcare institutions and academic settings.
10. Continuity of Care: It helps maintain continuity of care by providing a comprehensive record
of laboratory testing for the patient's healthcare providers, aiding in diagnosis, treatment
planning, and monitoring of patient outcomes.
QUESTION 2

List the various samples bottles used in medical microbiology laboratory

In a medical microbiology laboratory, various types of sample bottles are used to collect
different types of specimens for testing. Some common sample bottles include:

1. Blood culture bottles: Used to collect blood specimens for the detection of bacterial, fungal, or
other microbial infections in the bloodstream.
2. Sterile containers: Used to collect specimens such as urine, cerebrospinal fluid (CSF), wound
swabs, throat swabs, and other body fluids or tissue samples.
3. Stool containers: Used to collect stool specimens for the detection of bacterial, parasitic, or
viral infections in the gastrointestinal tract.
4. Transport media tubes: Used to collect and transport specimens for culture and sensitivity
testing, including viral transport media, bacterial transport media, and fungal transport media.
5. Sputum collection containers: Used to collect sputum specimens for the detection of
respiratory infections such as tuberculosis, pneumonia, and other bacterial or fungal lung
infections.
6. Viral transport media (VTM) tubes: Specifically designed for the collection and transport of
viral specimens, such as nasopharyngeal swabs or throat swabs, for testing for respiratory
viruses, including influenza and respiratory syncytial virus (RSV).
7. Swab tubes: Used to collect swab specimens from various anatomical sites, including throat
swabs, wound swabs, nasal swabs, vaginal swabs, and rectal swabs, for microbiological
testing.
8. Urine collection containers: Used to collect urine specimens for urinalysis, culture, and
sensitivity testing to detect urinary tract infections and other urinary tract abnormalities.
9. Rectal swab containers: Specifically designed for the collection of rectal swabs for the
detection of gastrointestinal infections, such as Clostridium difficile (C. difficile) and other
enteric pathogens.
10. Genital swab containers: Used to collect genital specimens, such as vaginal swabs, cervical
swabs, urethral swabs, and penile swabs, for the diagnosis of sexually transmitted infections
(STIs) and other genital infections.

QUESTION 3

List 8 parameters found in the microbiology laboratory form

1. Patient Information: Name, age, sex, and other relevant demographic details of the patient.
2. Specimen Source: Details about the origin of the sample, such as site of collection or source
(e.g., blood, urine, sputum).
3. Specimen Type: Description of the specimen received (e.g., swab, aspirate, tissue).
4. Date and Time of Collection: When the specimen was collected from the patient.
5. Test Requested: Specific microbiological tests requested by the healthcare provider.
6. Microorganism Identification: Results of microbiological analysis, including identification of
micro-organisms present (e.g., bacteria, fungi).
7. Susceptibility Testing: Results of antimicrobial susceptibility testing to determine the
sensitivity of microorganisms to antibiotics or other antimicrobial agents.
8. Interpretation/Comments: Any additional notes or interpretations provided by the laboratory
staff regarding the results of the tests.

QUESTION 4

List the disadvantages of wrong sample collection

 Inaccurate Results: Incorrect sampling may lead to inaccurate test results, which can affect
patient diagnosis and treatment decisions.
 Misdiagnosis: Miscollected samples may result in misdiagnosis or failure to detect the
presence of pathogens or other relevant factors.
 Delay in Treatment: Inaccurate results due to wrong sample collection can lead to delays in
appropriate treatment, potentially worsening the patient's condition.
 Waste of Resources: Resources such as time, money, and effort spent on analyzing incorrect
samples are wasted, without providing useful information for patient care.
 Potential Harm to Patient: In some cases, incorrect sample collection may lead to
unnecessary procedures or treatments that could harm the patient.
 False Negative Results: Wrong sample collection techniques can result in false-negative
results, where a test fails to detect the presence of a pathogen or condition that is actually
present.
 Risk of Contamination: Improper sample collection techniques may introduce contaminants
into the sample, leading to false-positive results or confusion in interpreting the findings.
 Loss of Trust: Consistent errors in sample collection can undermine the trust between
patients and healthcare providers, leading to dissatisfaction and reluctance to seek medical
care in the future.

QUESTION 5

State the outcome of improperly filled laboratory request form

 Delayed Testing: Incomplete or inaccurate information on the request form may lead to
delays in processing the sample, as laboratory staff may need to follow up with healthcare
providers to clarify the details.
 Incorrect Testing: Missing or incorrect information on the request form can result in the
wrong tests being performed, leading to inaccurate or irrelevant results.
 Misinterpretation: Incomplete or unclear information on the request form may lead to
misinterpretation of the clinical context or testing requirements, potentially affecting the
accuracy of the laboratory results.
 Wastage of Resources: Incorrectly filled forms may result in wasted resources, including
time and materials, as laboratory staff may need to spend additional time deciphering or
verifying the information.
 Communication Errors: Inadequate information on the request form can lead to
communication errors between healthcare providers and laboratory staff, potentially
impacting patient care and treatment decisions.
 Patient Safety Risks: Inaccurate or incomplete information on laboratory request forms may
pose risks to patient safety, as it may lead to incorrect diagnosis or treatment decisions.
 Legal and Regulatory Compliance Issues: Failure to accurately document test requests may
result in non-compliance with legal and regulatory requirements, potentially leading to
consequences for healthcare providers or institutions.
 Decreased Efficiency: Improperly filled laboratory request forms can disrupt workflow
efficiency in the laboratory, leading to delays in processing other samples and potentially
impacting overall laboratory operations.

SECTION E

QUESTION 1

List seven medical microbiological samples and the anatomical sites for the samples

1. Blood: Anatomical Site - Venipuncture (vein puncture), usually from the arm or hand.
2. Urine: Anatomical Site - Collection of midstream urine sample, usually via clean-catch
method or catheterization.
3. Sputum: Anatomical Site - Collection of respiratory secretions from the lower respiratory
tract through coughing or induced sputum, typically by deep coughing.
4. Stool: Anatomical Site - Collection of fecal matter from the gastrointestinal tract, usually in a
clean container or using a stool collection kit.
5. Wound Swab: Anatomical Site - Collection of material from a wound site using a sterile
swab, typically from the area with visible signs of infection or inflammation.
6. Throat Swab: Anatomical Site - Collection of material from the throat or pharynx using a
sterile swab, often from the tonsils or posterior pharynx.
7. Genital Swab: Anatomical Site - Collection of samples from the genital tract, including the
vagina, cervix, urethra, or penile urethra, using sterile swabs.
QUESTION 2

Outline the steps to obtain the following:

 High vaginal swab

 Urethral swab

 High Vaginal Swab:

Preparation: Explain the procedure to the patient and ensure their consent. Provide privacy and
ask the patient to lie on their back on an examination table with their feet in stirrups.

Glove Up: Wear sterile gloves to maintain aseptic technique.

Examine the Vagina: Use a speculum to visualize the vaginal walls and cervix. Inspect for any
abnormalities.

Swab Insertion: Open a sterile swab package without touching the swab tip. Insert the swab into
the vagina, rotating it gently against the vaginal walls to collect cells and secretions. Avoid
touching the speculum or other non-sterile surfaces.

Swab Collection: Carefully withdraw the swab without touching the vaginal walls or speculum.
Place the swab into a sterile transport container.

Speculum Removal: Remove the speculum gently from the vagina, ensuring no discomfort to the
patient.

Labeling: Label the specimen container with the patient's name, date, and any other required
information.

Documentation: Document the procedure in the patient's medical records, including any relevant
findings.

 Urethral Swab:

Preparation: Explain the procedure to the patient and obtain consent. Provide privacy and ensure
the patient is in a comfortable position, either lying down or sitting with legs apart.
Glove Up: Wear sterile gloves to maintain aseptic technique.

Expose the Urethra: Gently retract the labia minora to expose the urethral opening.

Swab Insertion: Open a sterile swab package without touching the swab tip. Insert the swab into
the urethral opening, being careful not to touch surrounding skin. Rotate the swab gently to
collect cells and secretions.

Swab Collection: Carefully withdraw the swab without touching the surrounding skin. Place the
swab into a sterile transport container.

Labeling: Label the specimen container with the patient's name, date, and any other required
information.

Documentation: Document the procedure in the patient's medical records, including any relevant
findings.

Always follow standard precautions and local protocols for specimen collection to ensure patient
safety and the integrity of the sample.

QUESTION 3

Outline 5 differences between serum and plasma

1. Composition:

Plasma: Plasma is the liquid component of blood that remains after cells (red blood cells, white
blood cells, and platelets) are removed by centrifugation. It contains water, electrolytes, proteins
(including clotting factors), hormones, and waste products.

Serum: Serum is the liquid component of blood that remains after clotting factors are removed
from plasma during the process of blood clotting. It is similar to plasma but lacks clotting factors
such as fibrinogen, which have been used during the clotting process.

2. Clotting Ability:
Plasma: Plasma retains clotting factors, allowing it to clot when exposed to appropriate stimuli. It
is used in coagulation studies and clotting factor assays.

Serum: Serum lacks clotting factors because they have been removed during the clotting process.
It does not clot when left at rest, making it suitable for tests that require a non-clotted sample.

3. Collection Method:

Plasma: Plasma is obtained by anticoagulating whole blood samples with substances like EDTA,
heparin, or citrate to prevent clotting during centrifugation.

Serum: Serum is obtained by allowing a whole blood sample to clot naturally or by using clot
activators like silica particles or thrombin, followed by centrifugation to separate the clot from
the liquid portion.

4. Uses:

Plasma: Plasma is primarily used in tests that require the measurement of clotting factors, such as
prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen assays.

Serum: Serum is used in various laboratory tests, including biochemical assays, hormone assays,
infectious disease testing, and serological testing (e.g., antibody detection).

5. Storage Stability:

Plasma: Plasma has shorter storage stability compared to serum because it contains clotting
factors that may degrade over time, especially if not properly stored.

Serum: Serum has better storage stability compared to plasma because clotting factors have been
removed, reducing the risk of degradation. However, both plasma and serum should be stored
under appropriate conditions to maintain sample integrity.
QUESTION 4

A. List four serological tests in the medical microbiological laboratory

B. Explain the test procedure for any of the serological tests

A. Serological test in the medical laboratory

1. ELISA (Enzyme-Linked Immunosorbent Assay): ELISA is a widely used serological test

for detecting and quantifying antibodies or antigens in a patient’s serum. It involves the
use of enzyme-linked antibodies to detect the presence of specific antibodies or antigens
in a sample.
2. Western Blot: Western blot is a technique used to detect specific proteins in a sample,
often antibodies or antigens. It involves the separation of proteins by gel electrophoresis,
transfer to a membrane, and detection using labeled antibodies that bind to the target
protein.
3. Serum Agglutination Test: Serum agglutination test is a method used to detect antibodies
or antigens by observing their ability to clump together (agglutinate) in the presence of
specific antibodies or antigens. It is commonly used for the detection of bacterial or viral
infections, such as syphilis or certain types of bacteria.
4. Rapid Immunochromatographic Tests: Rapid immunochromatographic tests, also known
as lateral flow assays, are simple and rapid tests used to detect specific antibodies or
antigens in a sample. They typically involve the migration of the sample along a
membrane containing specific capture molecules, resulting in a visible color change if the
 target molecule is present. These tests are commonly used for point-of-care diagnostics
for infectious diseases like HIV, influenza, or COVID-19.

B. Test procedure for ELISA

ELISA (Enzyme-Linked Immunosorbent Assay)

Coating: The first step in ELISA involves coating a microplate with the antigen of interest.
Typically, this is done by adding a solution containing the antigen to the wells of the microplate
and allowing it to adhere to the surface through passive adsorption. The plate is then washed to
remove any unbound antigen.

Blocking: After coating, the microplate is treated with a blocking agent (e.g., bovine serum
albumin or casein) to prevent non-specific binding of antibodies to the plate surface. This step
helps reduce background noise and improve the specificity of the assay.

Sample Addition: Patient serum or plasma samples, along with appropriate controls, are added to
the wells of the microplate. These samples may contain antibodies specific to the antigen coated
on the plate. Typically, samples are diluted in a buffer to ensure that the concentrations fall
within the linear range of the assay.

Incubation: The microplate is then incubated to allow any specific antibodies present in the
samples to bind to the coated antigen. This incubation period usually lasts for a predetermined
amount of time at a controlled temperature (e.g., room temperature or 37°C).

Washing: After the incubation period, the microplate is washed multiple times with a wash
buffer to remove any unbound antibodies or other components from the sample. Proper washing
is critical to minimize background noise and ensure accurate results.

Secondary Antibody Addition: A secondary antibody, conjugated to an enzyme such as

horseradish peroxidase (HRP) or alkaline phosphatase (AP), is added to each well. This
secondary antibody binds specifically to the antibodies captured by the antigen during the
previous steps. This secondary antibody is often raised against the species-specific
immunoglobulin of the primary antibody used in the assay.
Incubation and Washing: The microplate is incubated again to allow the secondary antibody to
bind to any antibodies captured by the antigen. After incubation, the microplate is washed again
to remove any unbound secondary antibodies.

Substrate Addition: A substrate specific to the enzyme conjugated to the secondary antibody is
added to each well. When the enzyme reacts with the substrate, it produces a detectable signal,
typically a color change or fluorescence.

Signal Detection: The signal generated by the enzyme-substrate reaction is measured using a
microplate reader. The intensity of the signal is proportional to the amount of bound antibody in
each well, which allows for the quantification of antibody concentration in the sample.

Data Analysis: The data obtained from the microplate reader are analyzed using appropriate
software or calculations to determine the concentration of specific antibodies in the sample. This
concentration is compared to standard curves generated using known concentrations of control
samples to interpret the results.

SECTION F

QUESTION 1

Write on any of the following staining procedures/ techniques stating their principle and
procedures

 Giemsa Staining technique

 Ziehl Nelson Staining technique
 Field Staining technique
 processes of thin and thick malaria film preparation for Giemsa Staining

 Giemsa Staining Technique:

Principle:

Giemsa staining is a differential staining technique used primarily in microbiology to visualize

and differentiate various microorganisms, especially blood parasites like Plasmodium spp.
(malaria parasites) and Trypanosoma spp. The staining procedure involves using a combination
of methylene blue, eosin, and azure dyes, which selectively stain different cellular components,
allowing for the identification of specific structures within the microorganisms.

Procedure:

Preparation of Smears:

Obtain a thin blood smear by placing a small drop of blood on a glass slide and spreading it
thinly using another slide. For thick blood smears, apply a larger drop of blood and spread it into
a concentrated area without spreading it thinly.

Air Drying: Allow the smears to air dry completely to fix the blood cells onto the slide.

Fixation: Fix the dried smears by immersing them in absolute methanol for 1-2 minutes. This
step helps to preserve the cellular structures and prevent them from washing off during staining.

Staining:

Prepare the Giemsa stain by diluting it with buffered water according to the manufacturer's
instructions.

Cover the fixed smears with Giemsa stain and let them stand for 20-30 minutes at room
temperature.

Alternatively, the smears can be placed in a staining jar containing diluted Giemsa stain and left
to stain for the specified time.

Washing: After staining, rinse the smears with buffered water to remove excess stain.

Drying: Allow the smears to air dry completely before examination under a microscope.

Microscopic Examination: Examine the stained smears under a microscope using oil immersion
objective lenses.
Malaria parasites, if present, will appear as purple-stained structures within or attached to red
blood cells. Different stages of the parasite's life cycle can be identified based on their
morphology.

Interpretation: Interpret the stained smears based on the presence, density, and morphology of
malaria parasites. Quantify the parasitemia by counting the number of parasites per 200, 500, or
1,000 red blood cells, depending on the protocol used.

Reporting: Record the findings, including the species of malaria parasite identified (if applicable)
and the level of parasitemia, in the patient's laboratory report.

Giemsa staining is a valuable tool in the diagnosis of malaria and other blood-borne infections,
providing clinicians with crucial information for patient management and treatment. Proper
staining and interpretation techniques are essential for accurate diagnosis.

QUESTION 2

Mention the applications of the following

 Ziehl Neelson Staining technique

 Giemsa Staining technique

 Ziehl-Neelsen Staining Technique:

1. Diagnosis of Tuberculosis (TB): The Ziehl-Neelsen staining technique is primarily used for
the detection of acid-fast bacilli (AFB) in clinical specimens, aiding in the diagnosis of
tuberculosis. It helps identify Mycobacterium tuberculosis, the causative agent of TB, in
sputum, tissue, or other clinical samples.
2. Surveillance and Screening for TB: The staining technique is employed in surveillance
programs and screening initiatives to detect TB cases in high-risk populations, such as
individuals living in areas with a high prevalence of TB or those with known risk factors for
the disease.
3. Confirmation of Non-Tuberculous Mycobacterial Infections: Apart from TB, the Ziehl-
Neelsen staining technique is used to detect other pathogenic mycobacteria, known as non-
tuberculous mycobacteria (NTM), in clinical specimens. It aids in the confirmation of
infections caused by NTM species, such as Mycobacterium avium complex (MAC) or
Mycobacterium abscessus.
4. Veterinary Medicine: The staining technique finds application in veterinary medicine for the
diagnosis of mycobacterial infections in animals, including bovine tuberculosis and Johne's
disease (caused by Mycobacterium avium subspecies paratuberculosis).
5. Research Studies: Researchers utilize the Ziehl-Neelsen staining technique in microbiology
and epidemiological studies to investigate the prevalence, transmission dynamics, and drug
resistance patterns of tuberculosis and other mycobacterial infections.

 Giemsa Staining Technique:

1. Diagnosis and Quantification of Malaria: The Giemsa staining technique is essential for the
microscopic diagnosis of malaria, allowing for the visualization and identification of
Plasmodium parasites in blood smears. It facilitates the quantification of parasitemia and
determination of the species of Plasmodium involved in the infection.
2. Screening Blood Donors for Malaria: Blood banks and transfusion services use Giemsa
staining to screen donated blood for the presence of malaria parasites. This ensures the safety
of blood transfusions by preventing the transmission of malaria to recipients.
3. Detection of Other Blood-Borne Parasites: Giemsa staining is utilized for the detection of
various other blood-borne parasites, including Trypanosoma species (causative agents of
African trypanosomiasis or sleeping sickness) and Leishmania species (causative agents of
leishmaniasis).
4. Investigation of Vector-Borne Diseases: Researchers and public health professionals employ
Giemsa staining to study vector-borne diseases transmitted by insects, such as malaria
(transmitted by Anopheles mosquitoes) and leishmaniasis (transmitted by sandflies). It aids in
the identification and characterization of parasites in vectors and reservoir hosts.
5. Cytogenetics and Chromosome Analysis: Giemsa staining is used in cytogenetics for
chromosome analysis and karyotyping. It stains chromosomes to produce characteristic
banding patterns, facilitating the identification of structural abnormalities, numerical
aberrations, and other chromosomal rearrangements associated with genetic disorders.
 SECTION G

QUESTION 1

____________ is a instrument used in microbiological laboratory to separate components of

microbial sample based on density.

Answer: A Centrifuge

QUESTION 2

List ten common microbiology laboratory apparatus and their uses

1. Microscope:

Uses: Microscopes are used to observe and magnify microscopic organisms such as bacteria,
fungi, and parasites. They are essential for microbiological research, diagnostic purposes, and
educational applications.

2. Incubator:

Uses: Incubators provide controlled temperature and humidity conditions to promote the
growth of microorganisms. They are used for culturing bacteria, fungi, and other
microorganisms under optimal conditions for growth.

3. Autoclave:

Uses: Autoclaves use steam under pressure to sterilize laboratory equipment, media, and
glassware. They are crucial for ensuring that microbial cultures are free from contaminants
before use in experiments or diagnostic tests.

4. Biosafety Cabinet:

Uses: Biosafety cabinets provide a sterile and enclosed workspace for handling infectious
microorganisms safely. They protect laboratory personnel and the environment from
exposure to hazardous biological materials.
5. Petri Dish:

Uses: Petri dishes are shallow, round, lidded dishes used for culturing microorganisms on
solid media. They provide a flat surface for microbial growth and are essential for isolation,
enumeration, and identification of microorganisms.

6. Inoculating Loop:

Uses: Inoculating loops are metal or plastic wire loops used to transfer small amounts of
microbial cultures onto agar plates or into liquid media. They are essential tools for streaking
agar plates, making bacterial smears, and inoculating broth cultures.

7. Pipettes and Pipettors:

Uses: Pipettes and pipettors are used for precise and accurate measurement and transfer of
liquids in microbiology laboratories. They are essential for dispensing precise volumes of
reagents, media, and microbial cultures.

8. Vortex Mixer:

Uses: Vortex mixers are used to mix and homogenize microbial cultures, solutions, and
reagents by vigorously shaking them. They are essential for resuspending microbial cells and
ensuring uniform distribution of substances in liquid media.

9. Refrigerator and Freezer:

Uses: Refrigerators and freezers are used for storing microbial cultures, media, reagents, and
laboratory samples at low temperatures to slow down microbial growth and preserve their
viability.

10. Incubating Shaker:

Uses: Incubating shakers provide controlled temperature and agitation for culturing
microorganisms in liquid media. They combine the functions of an incubator and a shaker,
allowing for optimal growth conditions and mixing of cultures simultaneously.
QUESTION 3

What are STI and list 5 common sexually transmitted infections

STI stands for Sexually Transmitted Infection. These are infections that are spread through
sexual contact, including vaginal, anal, and oral sex. They can be caused by bacteria, viruses,
parasites, or fungi. Here are five common sexually transmitted infections:

 Chlamydia:

Caused by the bacterium Chlamydia trachomatis.

Often asymptomatic but can cause genital discharge, pain during urination, and pelvic pain.

Can lead to serious complications if left untreated, such as pelvic inflammatory disease (PID)
and infertility.

 Gonorrhea:

Caused by the bacterium Neisseria gonorrhoeae.

Symptoms may include genital discharge, pain during urination, and pelvic pain.

Can lead to complications like PID, infertility, and disseminated gonococcal infection (DGI)
if untreated.

 Syphilis:

Caused by the bacterium Treponema pallidum.

Progresses through several stages, including primary (painless sores), secondary (skin rash,
fever), latent (asymptomatic), and tertiary (serious complications affecting organs).

Can cause severe health problems and complications if not treated, including damage to the
heart, brain, and nervous system.

 Genital Herpes (Herpes Simplex Virus, HSV):

Caused by the herpes simplex virus (HSV), mainly HSV-2.

 Characterized by painful blisters or sores in the genital area, although many people may have
asymptomatic infections.

Symptoms can recur periodically, and the virus remains in the body for life, with potential
for transmission to sexual partners.

 Human Papillomavirus (HPV) Infection:

Caused by the human papillomavirus, which has numerous strains.

Can cause genital warts (low-risk strains) or lead to various cancers, including cervical, anal,
and oropharyngeal cancer (high-risk strains).

Vaccines are available to prevent infection with the most common cancer-causing strains of
HPV.

QUESTION 4

Mention 5 equipment used in the genitourinary clinic and give the full meaning of the ECS,
HVS and US

1. Speculum:

Full Meaning: A speculum is a medical tool used for examining the vaginal canal and cervix.
It consists of a hollow, cylindrical instrument with a blade that can be opened or closed to
allow visualization of the internal reproductive organs.

2. Ultrasound Machine:

Full Meaning: Ultrasound (US) machine is a medical device used to visualize internal organs
and structures using high-frequency sound waves. It is commonly used in genitourinary
clinics for imaging the kidneys, bladder, prostate, and reproductive organs.

3. Cystoscope:

Full Meaning: A cystoscope is a thin, tube-like instrument with a light and camera used to
examine the interior of the bladder and urethra. It is inserted through the urethra into the
 bladder to diagnose and treat conditions such as urinary tract infections, bladder stones, and
tumors.

4. Urine Analyzer:

Full Meaning: A urine analyzer is a diagnostic device used to analyze the chemical and
physical properties of urine samples. It can detect abnormalities such as protein, glucose,
blood, and bacteria in urine, providing valuable information for diagnosing genitourinary
disorders.

5. Endoscope:

Full Meaning: An endoscope is a flexible or rigid tube with a light and camera used to
visualize the interior of hollow organs, such as the urethra, bladder, and ureters. It is inserted
through natural body openings or small incisions to diagnose and treat various genitourinary
conditions.

ECS- Endo cervical swab

HVS- High vaginal swab
US- Urethral swab

QUESTION 5

What are PPE and list 5 in the laboratory

PPE stands for Personal Protective Equipment. These are items worn to minimize exposure to
hazards that may cause serious workplace injuries or illnesses. In a laboratory setting, PPE is
crucial for protecting personnel from chemical, biological, physical, and other hazards. Here are
five common PPE items used in the laboratory:

1. Lab Coats or Aprons:

2. Safety Glasses or Goggles:
3. Gloves:.
4. Face Shields or Masks:
5. Closed-toe Shoes: