San Pedro College

Department of Medical Laboratory Science

MANUAL OF HISTOPATHOLOGIC TECHNIQUES

LEARNING ACTIVITY NO. 2

The Proper Processing of Fresh Tissue Samples for
Microscopic Examination

SPECIFIC LEARNING OBJECTIVES:

At the end of this activity, the Medical Laboratory Science student will
be able to:

1. Identify the different methods of fresh tissue examination.

2. Explain the theoretical principles of each method.
3. Appreciate the advantages of examining tissues in the fresh state.
4. Achieve a keen awareness of the limitations of the methods.
5. Perform the different methods of fresh tissue preparation.

LEARNING CONTENT

Tissues are generally submitted when histopathological examination will

offer beneficial diagnostic information. It may be examined in its fresh state or
fixed (preserved) state, the method selected being governed by several
factors. These factors include: the structures or inclusions to be studied, the
amount and nature of the tissue to be examined and the urgency of
investigation.

Fresh tissue examination is usually done for a rapid microscopic analysis

of a specimen while surgery is taking place. Protoplasmic activities such as
mitosis and phagocytosis may be observed when tissues are examined in its
fresh state. However, while diagnosis can be rendered in many cases, fixed
tissue processing is preferred in many conditions for more accurate diagnosis.

The methods of preparing fresh (unfixed)tissue mounts include: (a)

teasing or dissociation, (b) squashing or crushing, (c) smearing, and (d) frozen
section. Slides may be viewed stained or unstained.

Teased preparations are done by carefully separating very thin tissue

slices with dissecting needles. Slides are examined via brightfield or phase
contrast microscopy where many details can be studied. Squashed
preparations are done by simply crushing small pieces of tissue between two
glass slides or between a glass slide and a cover slip.

In examining sediments, smearing is usually the method of choice.

Cellular materials are spread lightly over a slide with a wire loop or an
applicator stick (such as in streaking or in spreading) or with another glass slide
such as in the pull apart technique.
When rapid diagnosis is required, tissue sample is frozen and sectioned
using the freezing microtome or cryostat. This procedure is called Frozen
Section. The technical name for this procedure is cryosection. With the
cryostat, the usual histology slice is cut at 5 to 10 µm. The surgical specimen is
placed on a metal tissue disc which is then secured in a chuck and frozen
rapidly to about –20 to –30°C. The specimen is embedded in a gel like medium
consisting of polyethylene glycol and polyvinyl alcohol; this compound is
known by many names and when frozen has the same density as frozen tissue.
Subsequently it is cut frozen with the microtome portion of the cryostat, the
section is picked up on a glass slide and stained. The preparation of the sample
is much more rapid than with traditional histology technique (around 10
minutes vs 16 hours). However, the technical quality of the sections is much
lower.

TIME ALLOTMENT: 3 hours

LEARNING RESOURCES:

Frozen tissue
Body fluid/secretions
Chopping board
Scalpel or knife
Petri dish
Dissecting needles or applicator sticks
Normal saline solution
Frost-ended glass slides
Coverslips
Methylene blue stain

LEARNING STRATEGIES: Pre-lab discussion, Instructor-guided performance

PROCEDURES

Procedure for Teasing and Dissociation:

1. From the frozen block of tissue, cut several small, thin slices of tissue.
2. Immerse the tissue slices in a petri dish filled with saline solution.
3. Using a dissecting needle, carefully tease the tissue slices until they
unfold and spread out.
4. Carefully mount a teased slice on a slide and cover it with a coverslip.

Glass slide
Dissecting needle
Tissue slices
immersed in NSS

Figure 11. Teasing

Procedure for Squash Preparation:

From the 4th step of the previous procedure:

1. Add 1-2 drops of methylene blue stain directly on the tissue slice on the
glass slide.
2. Cover the stained tissue with a cover slip or another glass slide.
3. Applying enough pressure to crush the tissue flat.
4. Label your slide with your name, group number and section and submit
it to your instructor.
 Figure 12. Squash preparation of fresh tissues

Procedure for Smear Preaparation:

Note: Centrifuge body fluid samples and decant the supernatant. Save the
sediments for the procedures below. Avoid making smears that are too thin
or too thick since this will render your smears unsuitable for examination.

A. Streaking:

1. Pour a small amount of the sediments on one end of a clean glass slide.
2. Using an applicator stick, streak the sediments in a straight or zigzag line
throughout the remaining length of the slide. If a wire loop is used
instead, collect a loopful of the material and smear the material on the
slide. A relatively uniform distribution of the material should be obtained.
Cover it with a cover slip.
3. Label your slide with your name, group number and section and submit
it to your instructor.

Figure 13. Streaking

B. Spreading:

1. Transfer a small amount of the sediments on one end of a clean glass

slide.
2. Using an applicator stick, spread the sediments throughout the slide
(similar to how butter is spread on bread). Another technique is to place
the drop of sediments at the center of the slide. Then with a circular
motion, gently spread it into a moderately thick film.
3. Label your slide with your name, group number and section and submit
it to your instructor.

Figure 14. Spreading

C. Pull apart

1. Place a drop of the sediments at one end of a glass slide.

2. Cover it with the end of another glass slide. Allow the sediments to
spread evenly between the attached surfaces of the two slides.
3. With a single, uninterrupted motion, pull the two slides apart in opposite
directions. Cover the smear with a cover slip.
4. Label your slide with your name, group number and section and submit
it to your instructor.

Figure 15. Pull apart technique

D. Touch or Impression smear

1. Retrieve the block of tissue from the first procedure (See Teasing p.18).
Cut the block in half, exposing a fresher surface.
2. Allow the freshly cut surface to come in contact with the surface of a
clean glass slide. Apply gentle pressure, allowing the cells to be
transferred directly to the slide. Cover the smear with a cover slip.
3. Label your slide with your name, group number and section and submit
it to your instructor.

Figure 16. Impression or Touch smearing

 LEARNING ACTIVITY NO. 2

STUDENT LEARNING EVALUATION

The Proper Processing of Fresh Tissue Samples for

Microscopic Examination

Name : _______________________ Score: ______/15

Course/Yr./Sec.:_______________ Date: _________

Guide Questions:

1. What are the advantages of examining tissues in the fresh state?(5pts)

______________________________________________________________
______________________________________________________________
______________________________________________________________

2. What are the disadvantages of examining tissues in the fresh state? (5pts)
______________________________________________________________
______________________________________________________________
______________________________________________________________

3. When are the following methods of fresh tissue preparation especially

recommended to be performed? (5pts)
 a. Streaking ________________________________________________
b. Spreading ________________________________________________
c. Pull apart ________________________________________________
d. Touch Prep _______________________________________________
e. Frozen section ____________________________________________

Rubrics for Scoring Guide Questions

5 pts 4pts 3pts 2 pts 1 pts

Excellent Good Average Fair Needs

The answer The answer The answer The answer improvement
meets all of meets most of meets some of meets only a The answer
the criteria: the criteria: the criteria: few of the does not meet
1.Relevance 1. Relevance 1. Relevance criteria: any of the
2. Clarity 2. Clarity 2. Clarity 1. Relevance following
3.Conciseness 3.Conciseness 3.Conciseness 2. Clarity criteria:
4. Grammar 4. Grammar 4. Grammar 3.Conciseness 1. Relevance
4. Grammar 2. Clarity
3. Conciseness
4. Grammar