Activity 10: Proper Mounting Technique  Mounting media should be colorless.

 It should quickly dry and stick to the

LEARNING OBJECTIVES
slide.
At the end of the activity, the student will be  It should resist contamination
able to: particularly the growth of microbes.
 It should not react with the stain or
 Explain the principle behind the
tissue.
mounting technique
 It should be miscible with clearing agent.
 Appreciate the importance of mounting
 A neutral pH to prevent fading of the
in the routine histopathologic
stain.
techniques
 Enumerate and differentiate the various Mounting Refract Common Advantag Disadvanta
kinds of mounting media medium ive use es ges
 Outline the procedure for mounting index
 Properly perform mounting Canada 1.523 Permanent Well - Yellow
balsam mounting soluble in staining
MOUNTING xylene after some
time
 Once the slides are stained, the - Takes
preparations must be made permanent time to dry
by placing a protective covering over - The basic
the tissue section, usually in the form of dyes are
either a glass coverslip or a plastic film. poorly
 The protective layer is between 1.0 and preserved
2.5 mm thick, and will prevent fading of DPX 1.523 Permanent -Preserve - Retraction
the stain or any further physical mounting the of margin
damage. It is attached to the slide by a standard of coverslip
mounting media. stains
Glycerine 1.47 -Temporary - -Unsuitable
 The mounting media must have the
glycerol mounting Inexpensi for
same refractive index( as the glass slide
-Oil red O ve prolonged
and the coverslip in order to prevent and -Safe preservatio
refraction artefacts. The process of -Sudan -Quick to n, and the
placing coverslip on the slide with the black stain apply coverslip
use of a media is known as mounting. - margin
 In this process, the technologist must Fluorescent should be
ensure that sufficient media is applied stain sealed
in order to prevent shrinkage, and that Polyvinyl 1.5 - Safe and -Unsuitable
excess mountant is not applied in order alcohol Temporary good for
to prevent the coverslip from being mounting alternativ prolonged
encrusted. Air bubbles must also be - Fat stain e to preservatio
avoided. - glycerine- n
Fluorescent glycerol
Properties of a Mounting Medium stain

 The same refractive index of the

coverslip and glass slide. CAUTION!!!!
  Too small amount of mounting
medium:
Air bubbles may appear.
 Too much amount of mounting
medium:
It may spread beyond the edges
of coverslip, and the sample may also
float.
COVERSLIP
The good coverslip should have following
characteristics:
 Clear glass of 0.130-0.170 mm thickness
 Plane surface and straight margin
 Sufficiently wide to cover the smear
Liquid coverslip- incorporated with mounting
media (no need na ug coverslip kay pag dry
mura napud ug mabutangan og coverslip
PROCEDURE
1. Make sure that the slides are not
completely dry prior to mounting.
2. Select an appropriate coverslip for the
section.
3. Apply one to two drops of mounting
media on the middle of the tissue
section over the slide.
4. Incline the cover slip to an angle which
the slide touches the drop. Allow the
drop to spread along the edge of the
cover slip.
5. Let go of the slip and allow the medium
to spread.
6. After the mounting medium has spread,
clean the excess mounting media on the
edges of the cover slip with xylene.
 If many bubbles are present, remove
the slide by immersing to xylene. Do not
pull the cover slip.
 If few bubbles are present, warming the
slide will make the bubbles escape to
the edges of the covers slip. Applying
slight pressure may also be effective.
PAPANICOLAU METHOD (Paps Smear)
 Method of choice for Exfoliative
Cytology
 Diagnosis of malignancy
 Identify stages in the maturation of
exfoliated squamous epithelial cells
 Detect human uterine and cervical
cancer
 Evaluation of sputum, urine, breast and
other secretions
STAINING OF SMEAR PREPARATIONS
STAINING METHOD COMPONENT
 Harris Hematoxylin
 OG 6 Stain
- 0.5% of OG6 + 95% Alcohol
- PTA
 EA 50 Stain
- Light Green SF Yellowish
- Bismarck Brown
- Eosin Y
- PTA - phosphotungstic acid
- Lithium Carbonate
PROCEDURE
1. After fixation, transfer slides directly
from Ether Alcohol mixture to 80%
Alcohol and pass thru 50% and 40%
Alcohol to distilled water.
2. Stain with Harris Hematoxylin: 45
seconds
3. Rinse in distilled water: 3x
4. Rinse in 50% Alcohol
5. Place in a solution of 1.5% Ammonium
Hydroxide in 70% Alcohol: 1 min.
6. Rinse in 70% Alcohol and pass thru 80%
and 95% Alcohol
 7. Stain in OG-6 staining solution: 1.5 mins
8. Rinse in 3 changes of 95% Alcohol
9. Stain in EA-65 or EA-50 staining solution:
3 mins
10. Rinse in 3 changes of 95% Alcohol
11. Dehydrate and Clear
a. Absolute Alcohol
b. Equal parts of Ether and
Absolute Alcohol
c. Two changes of Xylene
MODIFIED PAPANICOLAU STAINING
PROCEDURE
1. Prepare slides and fix immediately in
equal parts of Ether and 95% Ethyl
Alcohol: 30 mins
2. Immerse in 70% Ethyl Alcohol: 10 dips
or until glossy
3. Immerse in 50% Ethyl Alcohol: 10 dips
or until glossy
4. Rinse in distilled water: 10 dips or until
glossy
5. Stain with Mayer's Hematoxylin: 6-8
mins
6. Wash in running water: minimum of 20
mins
7. Dehydrate in graded alcohol: 50%, 70%,
80%, 95% for 10 dips each
8. Stain in Orange G staining solution: 1.5
mins
9. Immerse in 3 changes of 95% Ethyl
Alcohol: 10 dips
10. Stain in EA staining solution: 1.5 mins
11. Immerse in 3 changes of 95% Ethyl
Alcohol: 10 dips each
12. Dehydrate in Absolute Ethyl Alcohol: 10
dips
13. Immerse in equal parts of Absolute
Alcohol and Toluene: 10 dips
14. Clear in 2 changes of Toluene: 10 dips
15. Mount.
Glass cover slip ang ginagamit kay mas tibay and
mas gwapo for storage
Sufficient media must be applied to avoid
shrinkage Properties:
Must have the same refractive index
Mounting media must be clear
Should quickly dry
Should reduce the growth of microbes
Should not react with other forms of tissue
Should be miscible with clearing agents
Coverslip gives protective layer of the smear and
prevents the fading of the stain.
Liquid coverslip- incorporated with the mounting
media
Dapat dili totally dried up ang slide kay mag lain
ang appearance and naay second reaction pag
mag drop ug mounting media na maka interrupt sa
pagbasa
Cotton swab ang ginagamit in order to remove the
excess amount of mounting media
If many bubbles are present, remove the slide by
immersing it into xylene. Do not pull the coverslip.
Then perform mounting again.
Broken slide - possible magmount - have another
slide then make sure to clean it with xylene then
drop a mounting media and place the broken slide
on top. - sufficient for immediate na result
Pap smear - for diagnosis of malignancy - to
identify stages in the maturation of sporulated
epithelial cells
- to detect human uterine and cervical cancer
- for the evaluation of sputum, urine, breast, and
other secretion - universal for exfoliative cytology -
disadvantage: a little complicated
- it does not give acidophilia index because it has
wide spectrum of shades of red in the stain na
magproduce of inaccurate (?)
EA 65- can give lighter or more transparent
staining but less differentiated
 EA 50 - can differentiate well the
structures in the cytoplasm Before mag
perform ug pap smear dapat filtered na
daan ang hematoxylin

The purpose of toluene is to remove alcohol and

excess water

Refractive index in mouting medium 1.52 - 1.54