Name: PATRICIA NICOLE Y.

SIGUA Date: FEBRUARY 19, 2022

Sections: DASH 5 Group: Score:

EXPERIMENT 1 MICROSCOPY
Upon successful completion the completion of the program the learners
must…
Explain the fundamentals of cells and tissue through observation of
COURSE
commercially prepared tissue and cell specimen. Explain the different
characteristics and function of microscopic structure of human cells tissues, OUTCOMES
and organs using light microscopy. The following discipline: integrity, critical
thinking, honesty, creativity and concern for others

To achieve this unit a learner must:

OUTCOMES

To achieve this unit, the learner must:

UNIT

1. Identify the different parts of a compound microscope.

2. Explain the function of each part of a compound microscope.
3. Properly focus a prepared slide with both dry and oil immersion objectives
4. Demonstrate the proper care and storage of the compound microscope

TEACHING AND LEARNING ACTIVITIES

Laboratory Experimentation
The laboratory scientist will be familiar with the different parts of a compound microscope in order
to execute proper technique in microscope analysis. He will demonstrate this proper technique of
microscopic viewing using a prepared smear in both dry and oil immersion objectives.

PRE-ANALYTICAL PHASE
The site of the experiment should be sterilized. The laboratory scientist should wear personal
protective equipment before the experiment.

The microscope should be placed on a clean, plain and stable surface. The voltage of the power
supply should be inclined to that of the equipment. Proper handling and cleanliness of compound
microscope should be observed at all times. Lens paper is provided in order to clean the objective and ocular
lenses.
 The laboratory scientist MUST be familiar with the following terms and use of the equipment. This
activity provides concise information for light microscopy and the related histological stain appropriate
with the tissue specimen

Light Microscopy is based on the interaction of light and tissue components and can be used to reveal and
study tissue features (Mescher A.L.., 2010). Microscope is an instrument used to observe microorganism
that cannot be seen in the naked eyes.
Histological stains where made to make various tissue components conspicuous, but permit distinction on
the cell organelles. Tissue components without negative charge stain more with basic dyes, known as
basophilic; proteins with many ionized amino group, have the affinity for acidic dyes know as acidophilic
(Mescher A.L.., 2010).

ANALYTICAL PHASE

Materials and Instrument

Compound microscope Lens paper

Prepared slide Xylene
Cedar wood oil Gauze

Procedure

A. Get familiar with a compound microscope.

1. Locate the following parts
Ocular lens Condenser with iris diaphragm
Interpupillary adjustment and scale Condenser focus knob
Diopter ring adjustment Coarse adjustment knob
Revolving nosepiece Fine adjustment knob
Objective lenses Light source
Mechanical stage Light intensity adjustment knob
Stage control knob (X and Y axis) Main switch
Observation tube adjustment Base
Arm

B. Focusing the microscope

1. Plug in the microscope and turn on the microscope using the main switch.
2. Turn on the light source by rotating the light intensity adjustment knob.
3. Place the prepared slide on the mechanical stage. The slide should be centered in the aperture
of the stage.
 4. Use the scanner to locate the specimen for initial observation. Note: Make sure that the
specimen is at the center of the field.
5. Change you objective by rotating the nosepiece to low power objective.
6. Change it high power objective. The higher the objective the higher the magnification. Use the
coarse and fine adjustment knob to adjust the clarity of specimen’s image.
7. Before changing to Oil Immersion, the laboratory scientist must apply a small drop of cedar
wood oil on the specimen. Note: Do not use High power objective with cedar wood oil for this
may damage the objective. If the HPO is used make sure to change it to LPO before adding
cedar wood oil. Then rotate it to scanner then move to oil immersion objective. Use the coarse
and fine adjustment knob to adjust the clarity of specimen’s image.

C. Care and proper storage of microscope after use

1. Remove the excess oil on the objectives by using lens paper with 70% alcohol. This is to
maintain the quality of the objective.
2. Turn off the microscope and unplug it.
3. Rotate the objective into the lowest power objective.
4. Remove the slide and clean the mechanical stage. Make sure all oil, dust or and debris are
removed.

POST-ANALYTICAL PHASE

Storage of Microscope

1. Prepare the microscope for storage and return it properly to storage cabinet
2. Clean and disinfect the work area before leaving.

Critical Thinking

1. Discuss the magnifying parts of a microscope. Give their magnification.

The magnifying part of microscopes are ocular lenses and objective lenses. Ocular lenses are also called
eyepieces. They are the one who form the final image projected into our eyes when using microscope. There
are two ocular lenses and that’s the reason why microscope is also called binoculars. On the other hand,
objective lenses are the most important part of a microscope because they are the first lenses that gather the
light passing through the tissue. Objective lenses come in various magnification powers, with the most
common being 4x, 10x, 40x, and 100x, also known as scanning, low power, high power, and (typically) oil
immersion objectives, respectively.

2. Enumerate and discuss the illuminating parts of the microscope and give their respective functions.

The illuminating parts of the microscope are sub stage condenser, iris diaphragm and illuminator or the
light source. The condenser is located beneath the stage and serves to gather wavefronts from the
microscope light source and concentrate them into a cone of light that illuminates the specimen with
uniform intensity over the entire viewfield. The iris diaphragm controls the angle of illuminating rays which
pass through the condenser, through the specimen and then into the objective. When completely closed, the
diaphragm does not allow any light to enter the microscope. Lastly the illuminator is to provide even, high
intensity light at the place of the field aperture, so that light can travel through the condenser to the
specimen. It's used to direct room light, lamp light, or skylight from below the scope's stage up through the
specimen as transmitted light.

3. Enumerate and discuss the mechanical parts of the microscope and give their respective functions.

The mechanical parts of the microscope are the base, arm and the stage. The base is the support of the
microscope and it’s where the illuminator located. The arm of the microscope supports the body tube. Arm
also connects to the base and supports the microscope head. It is also used to carry the microscope. Lastly
the stage is where the specimen is placed for observation. Stages are often equipped with a mechanical
device that holds the specimen slide in place and can smoothly translate the slide back and forth as well as
from side to side

4. Identify and describe the steps in tissue preparation

Tissue preparation is highly skilled procedure performed by trained histotechnicians in a laboratory.

Specimens are fixed, embedded in wax and sectioned in order to produce material through which light can
pass so the cells and tissues can be observed in a microscope. Tissue preparation g is the technique by which
fixed tissues are made suitable for embedding within a supportive medium such as paraffin.

This are the following steps:

1. Obtaining a fresh specimen
Fresh tissue specimens will come from various sources. It should be noted that they can very easily be
damaged during removal from the patient or experimental animal. It is important that they are handled
carefully and appropriately fixed as soon as possible after dissection. Ideally, fixation should take place at
the site of removal, perhaps in the operating theatre, or, if this is not possible, immediately following
transport to the laboratory.

2. Fixation
The specimen is placed in a liquid fixing agent (fixative) such as formaldehyde solution (formalin). This
will slowly penetrate the tissue causing chemical and physical changes that will harden and preserve the
tissue and protect it against subsequent processing steps.

3. Dehydration
Because melted paraffin wax is hydrophobic (immiscible with water), most of the water in a specimen must
be removed before it can be infiltrated with wax. This process is commonly carried out by immersing
specimens in a series of ethanol (alcohol) solutions of increasing concentration until pure, water-free
alcohol is reached. Ethanol is miscible with water in all proportions so that the water in the specimen is
progressively replaced by the alcohol. A series of increasing concentrations is used to avoid excessive
distortion of the tissue.

4. Clearing
Unfortunately, although the tissue is now essentially water-free, we still cannot infiltrate it with wax
because wax and ethanol are largely immiscible. We, therefore, have to use an intermediate solvent that is
fully miscible with both ethanol and paraffin wax. This solvent will displace the ethanol in the tissue, then
this, in turn, will be displaced by molten paraffin wax.
 5. Wax infiltration
The tissue can now be infiltrated with a suitable histological wax. A typical wax is liquid at 60°C and can
be infiltrated into tissue at this temperature then allowed to cool to 20°C, where it solidifies to a consistency
that allows sections to be consistently cut. These waxes are mixtures of purified paraffin wax and various
additives that may include resins such as styrene or polyethylene. It should be appreciated that these wax
formulations have very particular physical properties which allow tissues infiltrated with the wax to be
sectioned at a thickness down to at least 2 µm, to form ribbons as the sections are cut on the microtome,
and to retain sufficient elasticity to flatten fully during flotation on a warm water bath.

6. Embedding or blocking out

Now that the specimen is thoroughly infiltrated with wax, it must be formed into a “block” which can
be clamped into a microtome for section cutting. This step is carried out using an “embedding centre” where
a mold is filled with molten wax and the specimen placed into it. A cassette is placed on top of the mold,
topped up with more wax, and the whole thing is placed on a cold plate to solidify. When this is completed,
the block with its attached cassette can be removed from the mold and is ready for microtomy. It should be
noted that, if tissue processing is properly carried out, the wax blocks containing the tissue specimens are
very stable and represent an important source of archival material.
Tissue: Tissue:

Organ: Organ:
Illustration: Illustration:

Stain Used: Stain used:

Tissue: Tissue:
Organ: Organ:

Illustration: Illustration:

Stain Used: Stain used:

Tissue: Tissue:
Organ: Organ:

Illustration: Illustration:
 Stain used:

Stain Used:

Tissue: Tissue:

Organ: Organ:

Illustration: Illustration:

Stain used:

Stain Used:

ASSESSMENT CRITERIA FOR PASSING

Outcome To achieve each outcome, the learner must

demonstrate the ability to

1. Identify the different parts of a A. Properly identify and label the different
compound microscope parts of a compound microscope.
2. Explain the function of each part of a B. Utilize the different parts of microscope in
compound microscope. viewing the specimen.
3. Properly focus a prepared slide with C. Handle and store the microscope properly.
both high power objective and oil
immersion objective.
4. Demonstrate the proper care and
storage of the compound microscope

ASSESSMENT STRATEGIES

Focus for assessment

ANECDOTAL NOTES
Observe students as they participate in the activity. Note the extent to which students are able to make and
talk about personal observation.

OBSERVATION CHECKLIST
Create an outcome-based checklist and share with students prior to beginning the activity. Use the checklist
to asses if students are able to make and talk about personal objectives.

RUBRIC
Collaboratively create an outcome-based rubric with students. Use rubric to evaluate how well the
students are able to understand cells.

ASSESSMENT STRATEGIES

Betty A.F., Daniel F.S and Alice S.W.., Bailey and Scott’s Diagnostic Microbiology; Twelfth Edition. Pg
70

Henry R.W: Plastination – Dehydration of specimens. J. Int. Soc. Plastination 6:4, 1992

Mescher, Anthony L (2010). Junqueira’s Basic Histology Text and Atlas, !2th edition, Mc-Greaw Hill
Companies Inc

RUBRICS

Critical 1 2 3 4 5
dimension REFER BEGGINER COMPETENT PROFICIEN EXEMPLARY

Pre-analytical phase
FACTOR 1 No PPE Only 1 PPE is Only 2 PPE is Only 3 PPE is Complete PPE
(Personal worn worn worn is worn
protective
equipment)
Analytical phase
FACTOR 2 There are There are few There are a few Lines are clear
(Quality of several erasures, erasures and not
Drawing) erasures, smudged lines smudged lines smudged. There
smudged lines or stray marks or stray marks are almost no
or stray marks on detract on the paper, erasures or stray
on the paper from the but they do not marks on the
which detract drawing. greatly detract paper. Color is
from the the drawing. used carefully
drawing. to enhance the
drawing.
FACTOR 3 Did not know Only few steps Proper step are Majority of the All steps for
(Proper use how to use are followed done in LPO procedures are microscopic
of microscope. properly. but not in HPO done properly analysis are
microscope) and OIO done properly
FACTOR 4 Was not able Blurred focus of Clear image of
(Focusing) to focus the the specimen. the specimen.
specimen.
FACTOR 5 Less than 85% 86-94% of the 95% or more of
(Accuracy) of the assigned assigned the assigned
structure are structure are structures are
drawn or drawn drawn
labeled accurately and accurately and
accurately. are are
recognizable. recognizable.
All structures
are labeled
correctly
FACTOR 6 Can’t identify Student Student was Student was Able to identify
(Knowledge the parts of the identified 5 able to identify able to identify at least 10 parts
gained) microscope. out of 10 or 6 to 7 out 10 8 to 9 out 10 of the
less parts of parts of parts of microscope
the microscope. microscope.
microscope.

Post-analytical phase
FACTOR 7 Did not return Did not shift Returned Properly
(Returning of the the objective to microscope returned the
microscope) microscope. the scanner and improperly. microscope.
did not clean
the objectives.
FACTOR 8 Left the work Was not able to Dispose waste Properly
(Cleaning of area without dispose waste properly but cleaned and
work are) cleaning. properly. did not disinfect the
Improper disinfect the area.
segregation of area.
waste.