PARA311 LABORATORY

First Term

LESSON 2: MICROSCOPE • It enhances resolution and it is used to fill the space

between the objective lens and glass
Microscopy slide.
• It prevents light rays from dispersing and changing
• Is the most common method used both for the wavelength after passing thru the
detection and characterization of microorganisms. samples
Microscope Parts of a Bright Field Microscope

• It is vital to magnify objects too small to be Eyepiece Lenses (Ocular Lenses)

visualized with the naked eye
• Observes specimen, re magnifies the image form by
Anton Van Leeuwenhoek the objective lens

• The first person to observe and describe

• To achieve high magnification with good resolution,
the lens must be small
microorganisms (“animalcules”) accurately.
• He discovered the previously “invisible world of Diopter Adjustment Ring
microorganisms.
• To compensate for observer’s eyesight in the left
Major Types of Microscopes and right eyes.
Light Microscope Interpupillary Distance Scale
• Visible light is passed through the specimen and
then through a series of lenses that reflect the
• It determines the distance between the two eyes.
light in a manner that results in magnification of the Interpupillary Distance Adjustment Seat
organisms present in the specimen.
• It uses glass lenses to bend and focus light rays and • To adjust the interpupillary distance scale
create magnified images of small objects.
• Resolution is determined by numerical aperture and Observation Tube
wavelength of light.
• The total magnification achieved is the product of • A monocular or binocular tube which transmits the
the lenses used image from the objective lens to
ocular
Notes to Remember
Arm
• Human eyes cannot focus on objects nearer than
about 25cm or 10 inches • It is used for holding the microscope
• Ideally, microscope should be parfocal-that is the
image should remain in focus when objectives Revolving Nosepiece
are changed.
• The resolution is increased by decreasing the • It holds the objectives
wavelength of light coming from the illuminator. • Rotating this part easily changes the magnification
of the objectives
Bright Field Microscopy
Objective Lenses
• Is the most commonly used microscope in a clinical
laboratory • Are the primary lenses that magnify the specimen
• It forms a dark image against a brighter background
• It can distinguish between two dots 0.2um apart • Are the most important parts of the microscope
• Magnification: which must produce a clear image
o Ocular lens -10x • The higher the magnification number of an
o Objective lenses objective, the greater the detail seen in
▪ LPO - 10x (100x) specimen parts
▪ HPO - 40x (400x)
▪ OIO - 100x (1000x) Coarse Focus Adjustment Knob

Use of the Cedar Wood Oil • Rotate to obtain coarse focusing by moving the stage
up and down
• The cedar wood oil has the same refractive index as
glass, so the oil becomes part of the Fine Focus Adjustment Knob
optics of the microscope
• It decreases the amount of diffraction or bending of • Rotate to obtain fine focusing by moving the stage
light rays since it has the same refractive index as up and down little by little
glass, resolution must be at 100x
Coarse Focus Adjustment Knob Tension Adjustment
Ring

K.BAGANG 1
 PARA311 LABORATORY
First Term

• Rotate the ring to adjust the tension of the coarse • Slight variations in diffractive index beams of light
focus adjustment knob pass through the specimen and are partially
deflected by the different densities or thickness of
Vertical Feed Knob the microbial cells or structures

• Rotate to move the specimen glass plate in the Fluorescent Microscopy

vertical direction
• It involves excitation of fluorochrome by light
Horizontal Feed Knob
• It use fluorochrome (dyes) to stain microorganisms
• Rotate to move the specimen glass plate in the • Fluorochromes: acridine orange, auramine and
horizontal direction rhodamine, calcofluor white and fluorescein
isothiocyanate (FITC)
Observation Tube Securing Knob
Dark Field Microscopy
• To be loosened when rotating the observation tube
• It uses darkfield condenser that blocks light that
Mechanical Stage would enter the objective directly

• The specimen is placed here • It directs the light to hit the specimen at an oblique
angle, all other light that passes through the
Specimen Holder specimen will miss the objective, thus making the
background a darkfield (organisms appear
• Holds the microscope slide in position extremely bright against a black field

Condenser
• It is used to detect spirochetes (Treponema
pallidum)
• It focuses light through the specimen • It is used to examine unstained microorganisms
• This incorporates a lens which collects illumination
suspended in liquid; living microorganisms that
are invisible in ordinary light.
light on the stage so that the
objectives can perform at full capability Electron Microscope
Condenser Iris Diaphragm Dial
• It uses electrons instead of light to visualize small
• It controls the amount of light entering the
objects.
condenser. • It uses electromagnetic fields instead of lenses to
form an image on a fluorescent screen like a
Coarse and Fine Adjustments television screen

• Can move either the stage or the nosepiece to focus

• Advantages: objects smaller than 0.2um can be
visualized using this type of microscope with
the image 100,000x magnification
Window Lens • It is useful for studying viruses fungi and
microsporidian parasites
• A filter with a diameter of 45mm can be mounted on • It is useful for studying the morphology of bacteria
this type of lens can be well studied
Brightness Adjustment Knob • Resolution: 0.5nm
• Fixatives: glutaraldehyde or osmium tetroxide
• Rotate to adjust the illumination brightness
• Dehydrating agents: alcohol or acetone
Base • Stains: lead citrate and uranyl acetate
• This part supports the entire microscope. Two Types of Electron Microscope
Phase Contrast Microscopy Transmission Electron Microscope(TEM)

• It permits detailed examination of internal structures • Allows visualization of internal structures; greatest
in living organisms resolution
• It is used to identify medically important fungi Scanning Electron Microscope(SEM)
grown in culture.
• The phase differences are seen through the • Scans the surface of objects
microscope as different degrees of brightness
Terminologies
• Staining is not part of phase contrast microscopy.

K.BAGANG 2
 PARA311 LABORATORY
First Term

• Refractive Index - is a measure of the relative • It is recommended that a normal examination for
velocity at which light passes through a material. stool parasites before therapy include three
specimens, consisting of two specimens collected
• Resolving Power - it refers to ability of the lenses from normal movements and one collected
to separate closely distant objects. after the use of a cathartic such as magnesium sulfate
• Contrast - it is necessary to make objects stand out or Fleet’s Phospho-Soda.
from the background. It is achieved by • A cathartic with an oil base should not be used, and
staining techniques a stool softener (taken either orally or as a
suppository) is usually inadequate for obtaining a
Notes purged specimen.
• A series of three specimens should be submitted on
• Total Magnification = Magnification of the separate days; if possible, the specimens
Objective Lens X Magnification of the Ocular Lens should be submitted every other day.
• Liquid specimens should be examined within 30 min
LESSON 3: COLLECTION AND HANDLING
of passage, not 30 min from the time they
OF STOOL SPECIMEN reach the laboratory. If this general time
recommendation of 30 min is not possible, the
Fecal Analysis specimen should be placed in one of the available
• Used in the detection of gastrointestinal (GI) fixatives.
bleeding, liver and biliary duct disorders, • Soft (semi-formed) specimens may contain a
malabsorption syndromes, and infections. mixture of protozoan trophozoites and cysts and
• Routine fecal examination includes macroscopic, should be examined within 1 h of passage.
microscopic, and chemical analyses for the Fixatives/Preservatives for Stool Specimens
early detection of gastrointestinal (GI) bleeding,
liver and biliary duct disorders, Formalin
maldigestion/malabsorption syndromes,
inflammation, and causes of diarrhea and • Has been used for many years as an all-purpose
steatorrhea. fixative that is appropriate for helminth eggs and
• Fluid regulation in the gastrointestinal larvae and for protozoan cysts, oocysts, and spores.
tract. Two concentrations are commonly used: 5%,
which is recommended for preservation of
Specimen Collection protozoan cysts, and 10%, which is recommended
for helminth eggs and larvae.
• Collection of a fecal specimen, frequently called a • To help maintain organism morphology, the
stool specimen. formalin can be buffered with sodium phosphate
• Is not an easy task for patients. Detailed instructions buffers.
and appropriate containers should be
provided. MIF / Merthiolate-Iodine-Formaldehyde
• Patients should be instructed to collect the specimen
in a clean container, such as a bedpan or • Is a good stain preservative for most kinds and
disposable container, and transfer the specimen to stages of parasites found in feces.
the laboratory container. • It is used with all common types of stools and
• Usually collected in plastic or glass containers with aspirates; protozoa, eggs, and larvae can be
screw-capped tops similar to those used for diagnosed without further staining in temporary wet
urine specimens. mounts, either made immediately after
• For quantitative testing, such as for fecal fats, fixation or prepared several weeks later.
timed specimens are required (the most • Some laboratories maintain that a permanent stained
representative sample is a 3-day collection). smear can be prepared from specimens
• Procedures for the recovery of intestinal parasites preserved in MIF, most laboratories using such a
should always be performed before barium is fixative examine the material only as a wet
used for radiological examination. preparation.
• Stool specimens containing barium are SAF /Sodium Acetate -Acetic Acid-Formalin
unacceptable for examination, and intestinal
protozoa may be undetectable for 5 to 10 days after • Lends itself to the concentration technique, the
barium is given to the patient. permanent stained smear, and fecal immunoassays
• The specimens should not be contaminated with for Giardia and Cryptosporidium and has the
water or urine, because water may contain free- advantage of not containing mercuric chloride.
living organisms that can be mistaken for human • Is considered to be a “softer” fixative than mercuric
parasites and urine may destroy motile chloride.
organisms. • The organism morphology is not quite as sharp after
• Every specimen should be identified with the permanent staining as that of organisms
following minimal information: patient’s name and originally fixed in solutions containing mercuric
identification number, physician’s name, and the chloride.
date and time the specimen was collected.
K.BAGANG 3
 PARA311 LABORATORY
First Term

Schaudinn’s Fluid 1. Emulsify a small amount of stool in two

drops of 10% eosin in alcohol.
• Is designed to be used with fresh stool specimens or 2. Coverslip and let stand 3 minutes.
samples from the intestinal mucosal surface. 3. Examine under high power for 5 minutes.
• Provides excellent preservation of protozoan 4. Count the number of undigested fibers.
trophozoites and cysts.
• Not generally recommended for use in concentration Chemical Testing of Feces
procedures.
• Occult blood: Used for early detection of colorectal
PVA / Polyvinyl - Alcohol cancer; old name, guaiac test
o Occult blood most frequently performed fecal
• Is a plastic resin that is normally incorporated into analysis
Schaudinn’s fixative. o Several chemicals used that vary in sensitivity
• Serves as an adhesive for the stool material. o Ortho-toluidine: Pseudoperoxidase activity of
• Can be used to prepare permanent stained smears hemoglobin (Hb) reacts with H2O2 to oxidize a
and perform concentration technique. colorless reagent to a colored product.
• Provides excellent preservation of protozoan o Hb —» H2O2 —> ortho-toluidine —» blue
trophozoites and cysts. oxidized indicator
• Specimens preserved in PVA cannot be used with • Gum guaiac: Least sensitive, most common
the fetal immunoassay kits. • Occult Blood Testing Interference
o False-Positive
Macroscopic Screening ▪ Aspirin and anti-inflammatory
Color and Consistency medications
▪ Red meat
• Black (tarry) stool: Upper GI bleeding; iron ▪ Horseradish
therapy ▪ Raw broccoli, cauliflower, radishes,
• Red stool: Lower GI bleeding turnips
• Steatorrhea: Fat malabsorption ▪ Melons
• Diarrhea: Watery fecal material ▪ Menstrual and hemorrhoid
• Ribbon-like stools: Bowel obstruction contamination
o False-Negative
• Mucus: Inflammation of the intestinal wall (colitis)
▪ Vitamin C 250 mg/d
• Clay-colored, pale: Bile-duct
▪ Iron supplements containing vitamin C
obstruction/obstructive jaundice
Occult Blood
Microscopic Examination of Feces
• Immunological: Use of an antihemoglobin to react
Fecal leukocytes - Determine cause of diarrhea
with the patient's hemoglobin has the advantage of
• Neutrophils: Bacterial intestinal wall infections or not requiring any special diet before sample
ulcerative colitis, abscesses collection. There is the possibility, however, of
• No neutrophils: Toxin-producing bacteria, viruses, hemoglobin degradation (and non-detection by
and parasites antibody), if the gastrointestinal bleed is in the upper
• Red blood cells are also noted intestine
• DNA test detects K-ras mutation, which is
Common Fecal Test for Diarrhea associated with colorectal cancer.
Qualitative Fecal Fat APT Test (Fetal Hemoglobin)

• Detects fat malabsorption disorders by staining fecal • Grossly bloody stools and vomitus are
fats with Sudan III or oil sometimes seen in neonates as the result of
red O; increased fecal fat (>60 droplets/hpf) swallowing maternal blood during delivery.
suggestive of steatorrhea. • Should it be necessary to distinguish between
• Muscle Fibers the presence of fetal blood or maternal blood in
o Look for undigested striated muscle fibers, an infant’s stool or vomitus, the APT test may
which may indicate pancreatic be requested.
insufficiency seen in cystic fibrosis. • The APT test distinguishes not only between
• Methylene Blue Stain Procedure for Fecal fetal hemoglobin and hemoglobin A but also
Leukocytes between maternal hemoglobin AS, CS, and SS,
1. Place mucus or a drop of liquid stool on a and fetal hemoglobin. The presence of maternal
slide. thalassemia major would produce erroneous
2. Add two drops Löffler methylene blue. results owing the high concentration of
3. Mix with a wooden applicator stick. hemoglobin F. Stool specimens should be tested
4. Allow to stand 2–3 minutes. when fresh. They may appear bloody but should
5. Examine for neutrophils under high power. not be black
and tarry, because this would indicate already
• Muscle Fiber Procedure denatured hemoglobin

K.BAGANG 4
 PARA311 LABORATORY
First Term

• APT Test Procedure • Ascorbic acid is strong agent – produces

1. Emulsify specimen in water. anti – oxidant properties inhibiting the
2. Centrifuge. color reaction.
3. Divide pink supernatant into two
tubes. Physical Examination
4. Add 1% sodium hydroxide to one
tube. 1. Color: Brown, Red, Green, Black / tarry, White /
5. Wait 2 minutes. gray
6. Compare color with that in the control 2. Consistency: Formed, Semi-formed, Soft, Watery
tube. • Reflects the level of hydration
7. Prepare controls using cord blood and • Gives an indication as to which organisms
adult blood. are present
Fecal Enzymes • Observe for presence of blood and/or
mucus
Trypsin
Chemical Examination
• Emulsified specimen placed on x-ray paper
determines ability to digest gelatin • Fecal Occult Blood Test (Hemascreen Guiac Slide
o Inability to digest gelatin indicates lack of trypsin Test Kit)
• Principle: Detection of the psuedoperoxidase
Fecal Chymotrypsin
activity of hemoglobin
• Is more resistant to intestinal degradation and is a • Reagent:
more sensitive indicator of less severe o Hema-Screen Slide with on-screen monitor
cases of pancreatic insufficiency. It also remains o Hema-Screen Developer (<6% hydrogen
stable in fecal specimens for up to 10 peroxide)
days at room temperature. Chymotrypsin is capable • Result and interpretation:
of gelatin hydrolysis but is most o POSITIVE: any trace of blue coloration
frequently measured by spectrophotometric method.
o NEGATIVE: no detectable blue
Elastase I coloration

• Is an isoenzyme of the enzyme elastase and is the Procedure

enzyme form produced by the pancreas.
It is present in high concentrations in pancreatic 1. Using applicator stick, apply small amount of
secretions and is strongly resistant to specimen in box 1 forming a thin smear.
degradation. It accounts for about 6% of all secreted 2. Reuse the applicator stick to obtain same specimen
pancreated enzymes. forming 2nd smear on box 2.
• Elastase I can be measured by immunoassay using 3. Allow the specimen to air-dry, then close the cover.
the ELISA kit 4. Open the perforated window at the back of the test
kit,
Fecal Carbohydrates 5. Apply two drops of Hema-Screen Developer on the
• Clinitest back of the box 1 and 2, together with the positive
• Addition of Clinitest tablet to emulsified stool and negative control area.
detects presence of reducing substances 6. Wait for 30 seconds, and read the results within 2
minutes.
*Reaction of 0.5 g/dL reducing substances suggests
carbohydrate intolerance Microscopic Examination

LESSON 4: ROUTINE STOOL • Reporting:

EXAMINATION o NOPS: No ova and/or parasite seen
o NIPS: No intestinal parasite seen
Patient Instruction
Ocular Micrometer
1. Within 3 days, patient should avoid eating red
meats, horse raddish, melons, broccoli, and • Specially designed ocular piece equipped with
turnips. measuring scale
• Prevent presence of dietary • Must be calibrated to ensure accurate measurement
pseudoperoxidase • Expressed in microns (μ or μm) defined as 0.001
2. Within 7 days, prevent aspirin and nonsteroidal [10-3]millimeter, or 10-6 meter
anti-inflammatory agents. • Calibration is aided with the use of a stage
• Prevent GIT irritation micrometer containing a calibrated scale divided
3. Within 3 days, prevent taking Vitamin C into 0.01-mm units.

K.BAGANG 5
 PARA311 LABORATORY
First Term

• The ocular micrometer is a disk equipped with a line

evenly divided into 50 or 100 units

Routine Stool Examination

Direct Fecal Smear

1. Label slide, place one of NSS and one drop of

Lugol’s iodine
2. Place a match head size portion of the stool sample
and make a smooth uniform emulsion by rotary
motion of the applicator stick.
3. Place the coverslip.
4. Examine under the microscope.

Kato – thick

1. Place 50-60 mg of stool specimen (about the size of

2 mango beans) at the center of the glass slide and
cover with a piece of cellophane pre-treated with
glycerin-malachite green solution.
2. Using a test tube press cellophane gently in order to
spread the stool sample
3. The slide is examined after 10-20 minutes or within
one hour after the preparation. Allowing the slide to
stand for long period will cause drying and shells of
hookworm ova will become too transparent and will
be difficult to see.

Examination of Stool Mounts

• Examine the coverslip area with the 10x objective,

focus the objective on the top left-hand corner and
move the slide systematically backwards and
forwards, up and down.
• When suspicious parasite is seen, switch to HPO and
increase the light intensity to observed detailed
morphology. If the mount is not examined
systematically, parasites may be missed.
• Exam Systematically

K.BAGANG 6