Sept.

4, 2021
HISTOTECH Seminar Class
Rationale:
1. Carnoy’s fluid recommended for fixing chromosome,
lymph glands & urgent biopsies contain all of the
following, ECEPT:
a. Absolute alcohol
b. Chloroform
c. Glacial acetic acid
d. Picric acid

2. Example of calcium chelating agent which forms

weakly dissociated complex and facilitates removal
of calcium salt
a. Versene
b. Von Ebner’s
c. Ion exchange resin
d. Perenyi’s

3. Graupner’s method is a dehydration procedure

employing what dehydrating agent:
a. Acetone
b. Dioxane
c. Tetrahydrofuran
d. Cellosolve

4. What is added to Mayer’s egg albumin (common

adhesive) to prevent the growth of molds?
a. Glycerin
b. Albumin
c. Thymol
d. Sodium chloride
5. Sections of the ribbons are floated-out on a water
bath set at 45-500C:
a. To avoid contamination
b. For easy location of serial section
c. To flatten the section and easy mounting on the
slide
d. For removal of excess paraffin on the slide

6. Which among the following is true regarding the

correct chronological order of the invention of the
microtome?
a. Freezing - rotary- rocking- sliding
b. Sliding - freezing – rocking – rotary
c. Rocking - rotary – sling – freezing
d. Sliding – rotary – rocking – freezing

7. Dehydration is best done by the use of:

a. Acetone
b. Ascending % of alcohol
c. Xylol
d. Descending % of alcohol

8. Ripening of phosphotungstic acid hematoxylin is

usually accelerated by adding:
a. Dilute nitric acid
b. Gentle heat for 1 hour
c. Potassium permanganate
d. Glacial acetic acid

9. The refractive index of the mounting medium should

be close to the average refractive of tissue which is:
a. 1.730-1.740
b. 1.630-1.640
c. 1.530-1.540
 d. 1.430- 1.440

10. Airholes are found on tissue during trimming. The

most probable reason for this is:
a. Incomplete fixation
b. Incomplete impregnation
c. Prolonged paraffin infiltration
d. Contaminated wax

11. ELLIOT-Bench is an example of:

a. Microtome for tissue sectioning
b. Automatic tissue processor
c. Vacuum embedding machine
d. Disposable embedding mold

12. Technique of paraffin wax impregnation that

gives the fastest result and is generally
recommended for urgent biopsies, dense and hard
fibrous tissues
a. Physical
b. Manual
c. Automatic
d. Vacuum

13. Mordant used in Weigert’s hematoxylin

a. Iron
b. Potassium alum
c. Potassium hydroxide
d. Phenol

14. Clearing agent recommended for CENTRAL

NERVOUS SYSTEM, however becomes MILKY upon
prolonged storage
 a. CCl4
b. Tetrahydrofuran
c. Chloroform
d. Cedarwood oil

15. Process of removing intracellularly and

extracellularly water from the tissue following fixation:
a. Staining
b. Quenching
c. Dehydration
d. Fixation

16. Saturation aqueous solution of ammonium

oxalate is added to the discarded decalcifying fluid.
Cloudiness then appeared. This suggests:
a. Incomplete decalcification
b. Contamination because of wrong choice of
fixative
c. Prolonged decalcification
d. Presence of artifacts

17. Fixation is ENHANCED by all of the following,

EXCEPT:
1. Presence of mucus
2. Agitation
3. Moderate heat
4. Cold temperature
5. Presence of blood
a. 1, 4 and 5
b. 4 only
c. 1, 2 , 3
d. 1 & 3
18. When using Zenker FORMOL (helly solution) in
blood containing organs, brown pigments are
produced due to lysis of RBC. This can be removed
by:
a. Addition of 10% methanol
b. Immersion in saturated alcoholic picric acid or
NaOH
c. Buffering w/ calcium carbonate w/ 10-15%
formalin
d. Wash in running water

19. Which among the following fixative is

recommended for MUCOPOLYSACCHARIDE acting
both as NUCLEAR & HISTOCHEMICAL fixative.
a. BOUINS fluid
b. New comers fluid
c. Osmium tetroxide
d. Heidenhain’s susa

20. MERCURIC CHLORIDE fixative recommended

mainly for TUMOR BIOPSY of skin
a. Heidenhain’s
b. Zenker-Formol
c. Hell’s solution
d. Zenker’s fluid

21. Which among the type of fixative should NOT

have glacial acetic acid because it destroys
mitochondria and golgi bodies?
a. Microanatomical
b. Nuclear
c. Cytoplasmic
 d. Histochemical

22. The following are NUCLEAR FIXATIVE, except:

a. Carnoy’s fluid
b. Orth’s fluid
c. Newcommer’s fluid
d. Bouin’s fluid

23. CHROMATE FIXATIVE recommended for study of

EARLY DEGENERATIVE process and tissue necrosis
a. Orth’s
b. Regaud’s
c. K2C2O7
d. Chromic acid

24. Fixative used at ice cold temperature (-50C to

40C) recommended for the study of H20 diffusable
enzymes & fixing brain tissues for RABIES Diagnosis.
a. Acetone
b. Flemming’s w/o Acetic acid
c. Heat fixation
d. Carnoy’s fluids

25. WASHING OUT is a process of removing excess

fixative from the tissue after fixation. To remove
excessive Mercuric fixative, one would use:
a. Alcoholic iodine
b. 50-70% alcohol
c. Tap H20
d. 70-95% ethanol

26. Process whereby the clearing agent is

completely removed from tissue and replaced by
medium that will completely fill tissue cavity.
 1. Impregnation
2. Infiltration
3. Casting
4. Blocking
a. 1 and 2
b. 3 and 4
c. 1, 2, 3, 4
d. 1 and 4

27. Process of removing calcium or lime solid from

tissue
a. Fixation
b. Decalcification
c. Dehydration
d. Staining

28. The components for the stain for amyloid

include:
a. HCl, H2SO4, I2
b. Gram’s iodine, H2SO4
c. Gram’s iodine, NaCl
d. Alcohol, H2S04
29. How do you remove excess stain in section
particularly H&E stain:
a. Dip the slide in ammonia water
b. Dip the slide in acid alcohol
c. Wash the slide with 95% alcohol
d. Dip the slide in sterile water

30. CARBON DIOXIDE used in freezing microtome

serves for what purpose:
a. To remove the knife towards the tissue block
b. To freeze the tissue
c. To turn the micrometer screw
 d. To demonstrate fat in brain tissues

31. This is used for demonstrating MITOCHONDRIA

during intravital staining:
a. Janus green B
b. Victoria blue
c. Night blue
d. Neutral red

32. A process by which sections are stained with

simple aqueous or alcoholic solution of the dye:
a. Direct staining
b. Progressive staining
c. Regressive staining
d. Counterstaining

33. An apparatus used in fresh tissue microtomy

consisting of an insulated microtome usually
maintained at -200C
a. Vacuum drying
b. Cryostat
c. Microtome
d. Autotechnicon

34. For best result, the amount of fixative used on a

specimen should be:
a. Twice the volume of the specimen
b. 10-15x the volume of the specimen
c. 5-10x the volume of the specimen
d. 20-50x the volume of the specimen

35. Which of the following is NOT a clearing agent?

 a. Xylene
b. Formalin
c. Toluene
d. Benzene

36. Which of the following is NOT a mounting agent?

a. Glycerin jelly
b. Aniline oil
c. Gum Arabic
d. Permount

37. To demonstrate fats in tissuess, the following are

used, EXCEPT:
a. Oil red O
b. Sudan IV
c. Osmic acid
d. Congo red

38. In restaining an old, faded slide, the first step is:

a. Overnight immersion in xylene
b. Rinsing in glacial acetic acid
c. Hydration with water
d. Deparaffinization

39. If the dye has the affinity to only acidophilic

component of the cells, it is:
a. Basic stain
b. Acid stain
c. Neutral stain
d. All

40. BEST CARMINE stain:

 a. Fats
b. Amyloid
c. Glycogen
d. Myelin

41. MASSON trichrome

a. Connective tissue
b. Elastic tissue
c. Nervous tissue
d. Serous fluids

42. Von kossa’s stain:

a. Fats
b. Amyloid
c. Calcium
d. Glycogen

43. Enclosing the tissue in a solid mass of embedding

medium is known as:
1. Embedding
2. Blocking
3. Casting
4. Orientation
a. 1 only
b. 2 and 3 only
c. 1, 2, 3
d. 1, 2, 3, 4

44. Removal of excess primary stain during

regressive staining technique is done using:
 1. Acetone-alcohol
2. Acid alcohol
3. Ether-alcohol
4. Ammonia water
a. 1
b. 1 and 3
c. 2 and 4
d. 1, 2, 3, 4

45. Ripening agents for hematoxylin, EXCEPT:

a. Mercury (II) oxide
b. Potassium permanganate
c. Hydrogen peroxide
d. Manganese oxide

46. A step that renders the tissue transparent or

translucent:
a. Clearing
b. Embedding
c. Fixation
d. Dehydration

47. Paraffin sections are usually cut in _ micron

thickness
a. 11-15
b. B. 15-30
c. 4-6
d. 5-8

48. This type of microtome is invented by Paldwell

Trefall in 1881:
a. Rocking
b. Minot
 c. Rotary
d. None of the above

49. VON EBNER’s fluid recommended as a good

decalcifying agent for TEETH contains what kind of
acid?
a. HCl
b. H2SO4
c. H3PO4
d. H2CrO4

50. The refractive index (RI) of an ideal mounting

medium should have a similar if not better RI than:
a. Water
b. Glycerol
c. Glass
d. Light

51. When using Zenker FORMOL (Helly solution) in

blood containing organs, brown pigments are
produced due to lysis of RBC. This can be removed
by:
a. Addition of 10% methanol
b. Immersion in saturated alcoholic picric acid or
NaOH
c. Buffering w/ calcium carbonate w/ 10-15%
formalin
d. Wash in running water

52. Which of the following stains is recommended

for cell granules and vacuoles of phagocytic cells?
 a. Janus green B
b. Neutral red
c. Night blue
d. Victoria blue

53. Which of the following stains is recommended

for neuroglial cells in frozen section
a. Congo red
b. Victoria blue
c. Sudan IV
d. Night blue

54. Which of the following tissue processing step is

NOT included in an automatic tissue processor?
a. Fixation
b. Clearing
c. Embedding
d. Infiltration

55. A vacuum embedding apparatus is essential

when embedding which media?
a. Paraffin
b. EPON 812
c. OCT
d. Gelatin

56. Fixatives for electron microscopy:

a. Karnovsky’s fixative
b. Osmium tetroxide
c. 2-3% glutaraldehyde
d. All of the above
57. Microtome knife of the ultra-thin microtome is
constructed from:
a. Stainless steel
b. Glass
c. Molybdenum- vanadium steal

58. Eukitt is diluted using

a. Water
b. Xylene
c. Absolute alcohol
d. Acetone

59. Clearing agent/s:

a. Carbon disulfide
b. Clove oil
c. Petroleum ether
d. All of the above

60. Using Gomori’s reticulum stain:

a. Magenta
b. Black
c. Blue
d. Silver

61. Fixative of choice for fine needle aspiration

biopsy (FNAB) smears:
a. 95% ethanol
b. Acetone
c. 10% formalin
d. 2-3% glutaraldehyde
62. The cutting edge of the microtome knife is
cleansed with cloth dampen with:

a. Dist. H20
b. Alcohol
c. Acetone
d. Xylene

63. Picric acid-containing fixative:

a. Regaud’s
b. Bouin’s
c. Zamboni’s
d. Zenker’s

64. Artifact pigment in tissue sections removed after

removed by ACID ALCOHOL treatment is:
a. Formalin hematin pigment
b. Mercury pigment
c. Metallic silver deposits
d. All of the above

65. Formalin-fixed, paraffin embedded tissues can

be used to prepare:
a. Tissue sections for immunohistochemistry
b. Tissue sections for hematoxylin and eosin stain
c. Tissue sections for demonstration of acid-fast bacilli
d. All of the above

66. High protein, high specific gravity, high cellular

content:
a. Transudate
b. Exudates
 c. Both
d. None

67. Which of the following stains is used commonly in

Gram staining procedure?
a. Hematoxyline
b. Crystal violet
c. Acetocarmine
d. Rhodamine

68. Which of the following stain can be used for the

detection of Neutral Lipid?
a. Berlin blue
b. Colloid gold
c. Sudan IV
d. Fuelgens nuclear stain

69. This process serves to provide a clear

visualization of tissue components studied using a
medium with a Rf close to the slide and the tissue.
a. Deparaffinization
b. Dehydration
c. Embedding
d. Dealcoholization

70. Wright stain is used primarily for which of the

following samples?
a. Blood
b. Fats
c. Nervous tissue
d. Elastic fibers
71. Which of the following can be used as a fixative
and decalcifying agent?
a. Nitric acid
b. Tetrahydrofuran
c. Trichloracetic acid
d. Picric acid

72. Histotechnique that collects cells by

spontaneous shed of the body part.
a. Exfoliative cytology
b. FNAB
c. FNAC
d. All of the above

73. Staining procedure that is widely used in the

diagnosis of abnormal cells that are found in
cancerous tumor.
a. Immunoflorence
b. In situ hybridization
c. Immunohistochemistry
d. All of the above

74. Which of the following causes a milky

appearance upon subjecting the tissue to xylene?
a. Incomplete fixation
b. Incomplete dehydration
c. Incomplete decalcification
d. All of the above
75. Tissue processing includes all of the following
except?
a. Fixation
b. Embedding
c. Dehydration
d. Clearing

76. Clearing agent that is recommended for hard

bony tissue?
a. Xylene
b. Benzene
c. Toluene
d. Chloroform

77. Impregnating agent that is suitable for large

hollow cavities.
a. Paraffin wax
b. Gelatin
c. Bees wax
d. Celloidin

78. A vegetable dye extracted from certain lichens

which are normally colorless but produces blue or
violet color when treated with ammonia:
a. Cochineal dye
b. Orcein
c. Hematoxylin
d. Synthetic dyes

79. PERENYI’s fluid used as a decalcifying agent

contains this acid at highest percentage.
 a. Acetic acid
b. Chromic acid
c. Nitric acid
d. Sulfuric acid

80. Characteristic of good adhesive agent for

EXFOLIATIVE CYTOLOGY.
a. Must be permeable to both the fixative and the
stain
b. Must NOT retain the stain
c. Both are correct
d. No answer

81. Fixative for cytologic smear with mercury

a. Carnoy fluid
b. Schauddin fluid
c. Orth fluid
d. Zenker fluid

82. Smear method for vital staining of lymph node

a. Pull apart smear
b. Streaking smear
c. Impression smear
d. Teasing smear

83. Ideal temperature for cryostat?

a. Less than 20 degree Celsius
b. Less than -20 degree Celsius
c. More than 20 degree Celsius
 d. More than -20 degree Celsius

84. Special stain for frozen section

a. Immune-histochemistry
b. Nuclear stain
c. Cytoplasmic stain
d. No answer

85. The most common decalcifying agent is:

a. HCl
b. HNO3
c. HCOOH
d. None of these

86. Decalcification is done:

a. After fixation and after impregnation
b. Before fixation and before impregnation
c. After fixation and before impregnation
d. None of the above

87. These dyes are called “coal tar dyes”

a. Natural
b. Chromium
c. Synthetic
d. All

88. The melting point of paraffin wax is?

 a. 650c
b. 630F
c. 560C
d. 560F

89. Leuckhart’s is issued in:

a. Fixation
b. Dehydration
c. Staining
d. Embedding

90. The microtome invented by Queckett (1881)?

a. Sliding
b. Freezing
c. Rotary
d. Rocking

91. Heel to toe direction is used in:

a. Honing
b. Either
c. Stropping
d. Neither

92. What happens to a tissue specimen if subjected

in an overheated paraffin wax?
a. It becomes hard
b. It becomes brittle
c. It becomes soft
d. It causes the tissue to expand and burst
93. These are characteristic features of a good
mounting medium, EXCEPT:
a. It should not dry quickly
b. It should not dissolve out or fade tissue sections
c. It should not cause shrinkage and distortion of
tissue
d. It should set hard producing permanent mounting

94. Barr bodies are part of:

a. X chromosome
b. Y chromosome
c. Either
d. Neither

95. Part of the microtome that allows the block

holder to move up and down the tissue block in thin
slices is:
a. Knife holder
b. Flywheel
c. Meter
d. Chuck

96. Simple stain for parasites in wet mount:

a. Giemsa
b. Iodine
c. Wrights
d. All

97. Microtome used for cutting serial sections of

large blocks of paraffin embedded tissues:
a. Rocking microtome
b. Rotary microtome
c. Base- sledge microtome
d. Ultra-thin microtome

98. Most dangerous microtome introduce by Adams

1789
a. Rocking microtome
b. Rotary microtome
c. Base- sledge microtome
d. Ultra-thin microtome

99. Which of the following is incorrect regarding

fixation of tissues?
a. Larger tissues require less fixatives and shorter
fixation time
b. Fatty tissues should be cut in the sections and fixed
longer
c. Both
d. Neither

100. Ideal sample for TISSUE PHOTOGRAPHY, except:

a. Renal tissues
b. Fibrinogen
c. Connective
d. Muscles
 Histopathology: By maam Martin

Course outline
Histopathology techniques: Additional notes: histopathology:
 Examination of fresh tissue  Structural and functional organization
 Examination of fixed  Cellular growth and differentiation
tissues-histo pathologic  Abnormalities in cellular growth
techniques/ steps:  Inflammation
 Neoplasia
 Numbering  Cell death
 Fixation  Autopsy
 Decalcification  biopsy
 Dehydration
 Clearing
 Impregnation
 Embedding
 Blocking
 Trimming
 Sectioning
 Staining
 Mounting
 Labelling

Exfoliative Cytology
Definition: it is a branch of cytology which deals with the microscopic study of cells that have been
desquamated from epithelial surfaces.

Purpose:
1. Assessment of malignant or cancerous condition
2. Detection of asymptomatic cancer in women (vaginal cytology)
3. Assessment of female hormonal activity in case of sterility and endocrine disorders
4. Determination of genetic sex
5. Detection of the presence of a possible infection

Specimen for examination

1) Vaginal smears 5) pleural and peritoneal fluids
2) Endometrial and endocervical smears 6) Sputum
3) Prostatic and breast secretions 7) smears of urine sediment
4) Gastric and bronchial secretions 8) CSF
Specimen which require addition of adhesive agent:
1. Urinary sediment
2. Bronchial lavage specimen
3. Specimen that utilizes proteolytic enzymes during processing. Ex: trypsin, concentrated
sputum, GI enzymatic lavage

Characteristic of good adhesive agent

1. Must be permeable to both fixative and the stain

2. Must not retain the stain. EX: EGG ALBUMIN- not recommended as an adhesive agent
because it is intensely stained by basic light green counterstain of PAP’s

Good adhesive for CYTOLOGIC method

 Pooled Human Serum
 Celloidin Ether Alcohol
 Leuconostoc Culture

Fixative For Cytologic Smears

1. 95% ethyl alcohol + ether (equal parts)
2. 95% ethyl alcohol
3. Carnoy’s fluid: Abs. (alcohol + chloroform + HAc)
4. Tertiary butyl alcohol + 95% ethyl alcohol (3:1)
5. Methyl alcohol : dried blood film
6. Schaudinn fluid: HgCl2 + HAc

Methods of Smear Preparation

1) Streaking: used for the preparation of mucoid

Secretions: sputum, gastric secretion, vaginal secretion from posterior fornix

2) Spreading (teasing or dissociation):

- a selected portion of the material is transferred to a clean slide and gently spread into a
moderately thick film by Teasing the mucus strands apart with an applicator stick.

- thick mucoid secretions, fresh sputum, bronchial aspirate

3) Pull apart:
 done by placing a drop secretion or sediment upon one slide and facing it to another clean
slide
 Serous fluid, conc. Sputum, enzymatic lavage from GIT vaginal pool, breast secretion, smears
of urinary sediment, blood smear.
4) Touch or impression smear:

 Special method of smear preparation whereby the surface of a freshly cut piece of tissue is
brought into contact and pressed on the surface of clean glass slide allowing the cells to be
transferred directly to the slide for examination by PHASE CONTRAST Microscopy or after
VITAL Staining.
 Used for the preparation of direct impression made from cut surface of tissue like LYMPH
NODES and other surgical or autopsy specimens.

Examination of Fresh Tissues

 Teasing or Dissociation
 Squash Preparation (crushing)
 Smear Preparation (streaking, spreading, Pull-Apart, Touch or impression Smear
 Frozen section

Frozen Section Indications Limitation in Frozen Section Consideration in Rapid

Frozen section

1. Rapid Diagnosis (guide for 1) Sample: limited section 1) Relevant clinical

intra-operative patient information/ history
management)
2. Optimally process tissues 1. Artifacts: Ice crystal or 2. Type of tissue or location
for special studies: freezing artifact of biopsy
a) Diagnosis
b) Treatment
c) research
3. Confirm (sample 2) Quality of Section: inferior 3) To determine beforehand
adequacy): lesion tissue is quality compared to what information the
present for diagnosis on paraffin sections surgeon requires from the
permanent sections FS and how the
information will be used.
4) Studies: 4. Optimal turn-around time is
lack of special studies = time </= 15 mins
constraint
Special stains = 5. Coordination between lab
immuno-histochemistry culture and OR (personnel involved)
5) Lack of consultation for 6. Check cryostat (-17c)
difficult cases 7. No fixative used
8. Protection of laboratory
personnel
9. Selecting part of the tissue
for FS
Examination of fixed tissues - Histo-pathologic Techniques
/ Steps:
1. Numbering 7) Blocking
2. Fixation 8) trimming
3. Dehydration 9) Sectioning
4. Clearing 10) Staining
5. Impregnation 11) Mounting
6. Embedding 12) Labelling

Fixation
 Procedure adopted to kill, harden and preserve material for microscopic study.

General effects of fixatives:

 Tissue Hardener = harden soft and friable tissue and make the handling and cutting of
sections easier.
 Cell protector = make cells resistant to damage and distortion caused by hypotonic and
hypertonic solutions during tissue processing
 Bacterial inhibitor = inhibition of bacterial decomposition
 Optimal Differentiator = Increase optical differentiation of cells
 Stain Enhancer = Act as mordant or accentuators to promote and hasten staining
 Infection Controller = Reduction of risk of infection during handling and actual processing of
tissues.
 General characteristics of fixatives

1. Cheap
2. Stable
3. Safe to handle
4. Minimum distortion of cell
5. Minimum shrinkage of tissue
6. Inhibit bacterial decomposition and autolysis
7. Rapid and even penetration of tissue
8. Tissue hardener for easy cutting of sections
9. Isotonic (minimal physical and chemical alteration of cell)
10. Permit application of many staining procedure

Main factors involved in fixation

 Hydrogen Ion concentration: pH 6 - 8

 Osmolality = Hypotonic 400-450 mOsm/ 340mOsm
 Temperature = routine- RT, automated tissue processor- 40c, EM and histochemistry- (0-4C)
Formalin 60c/100c/ (TB/microwave)

 Concentration = 10% formalin/ 3% glutaraldehyde

 Duration of fixation = buffer F (2-6 hrs)
Formalin 24hr need washing
Buffer 3hrs EM

 Thickness of section: EM= 1-2 mm2

LM= 2 cm2/ thin no more than 0.4 cm

Practical consideration

1. Speed (autolysis)
2. Penetration 1mm per hour
3. Volume 10-25x / (20x max)
4. Duration of fixation = depends w/ spx

Types Fixatives

 According to composition:
a) Simple fixative = one component substance
b) Compound fixative:
1. Aldehydes: formalin/ glutaraldehyde
2. Metalic: MCL + Heat

I. Mercuric chloride- most common

II. Chromate: Pot dichromate / chromic acid
III. Lead: PAAAO (picric, acetic, acetone, alcohol, osmium tetroxide)
IV. Heat

Types of fixatives according to action

Micro-Anatomical Cytological Histochemical
W/O altering the structural NUCLEAR Cytoplasmic
pattern and normal
intercellular relationship of With HAc, pH <4.6 Without HAc-
tissue (affinity for nuclear (destroys
chromatin, chromosome) mitochondria and
golgi bodies)
1. Flemming’s 1. Formol
1. 10% formol saline 1. Flemming’s fluid fluid without HAC saline(10%)
2. 10% neutral buffered 2. Bouin’s fluid
Formalin 3. Newcomer’s fluid 2. Formalin with 2. Absolute
3. Formol sublimate (formol 4. Carnoy’s fluid (NA) “post-chroming” ethylalcohol
Corrosive) 5. Heidenhain’s Susa
4. Zenker-Formol (helly’s 3. Orth’s fluid 3. Newcomer’s
Solution) 4. Regaud’s fluid fluid
5. Zenker’s solution (muller’s fluid)
6. Bouin’s solution 4. Acetone
7. Brazil’s solution 5. Helly’s fluid

Micro-Anatomical Cytological Histochemical

W/O altering the structural NUCLEAR Cytoplasmic
pattern and normal
intercellular relationship of With HAc, pH <4.6 Without HAc-
tissue (affinity for nuclear (destroys
chromatin, chromosome) mitochondria and
golgi bodies)
5. Formol
F Si F saline(10%)
N Heiden O
F ni Newcomer R 6. Absolute
Heidenhain Susa Sa H ethylalcohol
Z2 (kelly’s solution) Company ni F
BB niya Bouin 7. Newcomer’s
Flemming fluid

8. Acetone
 Fixatives

 Zinc formalin fixation requires special processing

 Bouin’s fixative is quick but it hardens tissues, if fixed for too long, so move specimens to 70%
alcohol in 6 hours

According to composition
Simple - made up only one component substance

Fixative Composition Advantage Disadvantage

a) Aldehyde- 1) Cheap, readily available, 1) Irritating fumes -
routine paraffin 40% formalin easy to prepare and relatively sinusitis, allergic rhinitis,
section, electron diluted 1:10 stable. excessive lacrimation
microscopy, (10%) or 1:20 2) Compatible with many stains 2) Irritating solution -
enzyme studies (5%) 3) Does not over harden tissues allergic dermatitis, tissue
even in prolonged periods of shrinkage
fixation. 3) Soft fixative if
1. Formaldehyde
4) Does not require Washing unbuffered -
(Formalin) Out a. Reduces both
5) Penetrates tissues well basophilic and
6) Preserves Fats, Mucin, eosinophilic staining
Glycogen, Proteins of cells
7) Nervous tissue preservation b. Forms abundant
8) Colored tissue photography brown pigment
9) Frozen tissue preparation granules on blood
containing tissues

Prolonged Fixation -
a) Bleaching- loss of
natural tissue colors
b) Dispersal of fat from
tissues into fluid

1) Even tissue penetration and 1) Same as formalin

a. 10% Formol fixation 2) Slow fixative
Saline 2) Minimum shrinkage and
40% formalin + distortion of tissue 3) Secondary fixation is
[central nervous/ 10% NaCl reduced because of
post mortem tissue] 3) Preserves Fats, Mucin, shrinkage of formol
Enzymes and Nucleoproteins saline fixed tissues
(FM-NPE) during alcohol
dehydration
4) Does not over harden tissue
5) Ideal for most staining 4) Reduced
techniques (silver - impregnation) metachromatic reaction
of Amyloid
6) Restores natural tissue color
upon immersion in 70% alcohol 5) Acid dye stains less
brightly than when fixed
with mercuric chloride
b. 10% neutral 1. Same as to formol saline
buffered 1) Time consuming
40% formalin + 2. Prevents precipitation of Acid 2) PAS reaction of
formalin
NaH2PO4 + Formalin Pigments on Post Mucin - reduced
Na2HPO4 Mortem tissues.
[Surgical / Post
3) Weigert iron
Mortem / Research
3. Best for Iron Pigment hematoxylin reaction of
specimen]
containing tissues Myelin - reduced

4) Gradual loss in
basophilic staining of
cells

5) Inert towards lipids

(neutral fats /
phospholipids)

c. Formol 1) Rapid penetration of small 1) Slow penetration

corrosive / pieces of tissues 2) Mercuric chloride
(HgCl2) deposits
formol
40%formalin + 2) Minimum shrinkage and
sublimate sat. Aq. HgCl2 hardening 3) Not for Frozen tissue
3) Excellent for many staining section
[Routine Post Mortem procedures [silver Reticulum
tissues] methods] 4) Inhibits the
4) Does not require WASHING determination of the
OUT extent of tissue
5) Brightens cytoplasmic and decalcification.
metachromatic stains

6) Well preserved cytological

structures and blood cells

7) Lipid fixative [neutral

fats/phospholipids]

2. Glutaraldehyde 2.5%, 2 -4 1. More stable effect on tissue 1. More expensive

hours, RT = (central nervous) 2. Less stable
[made up of 2 small tissue, 2. Less tissue shrinkage 3. Slow tissue
formaldehyde linked needle biopsy penetration
by 3 carbon chains] 3. Preserves Plasma Proteins 4. PAS positivity of
4%, 6-8-24 and cellular structures better Mucin - reduced
Routine light hours, RT =
microscopy, electron larger tissue 4. Less irritating - does not cause
microscopy, enzyme [<4mm] Dermatitis
histochemistry
 II. Metallic Fixative: Mercuric Chloride
General advantage:

1. Time of Fixation: rapid and tissue hardens well

2. Effect on staining:
a) Nuclear components are shown in fine detail
b) Preferred in lieu of formaldehyde for cytoplasmic staining
c) Trichrome staining - excellent
d) Metachromatic staining - brilliant
e) Fixative of choice for tissue photography

3. Tissue recommended:
a) Renal tissues
b) Fibrin
c) Connective tissue
d) muscles

Metallic Fixative = Mercuric Chloride

Fixative Advantage Disadvantage

Meercuric Chloride stock solution= [HgCl2 + K2Cr2O7 + Na2SO4 + H2O]

Zenker’s Fluid
1. Time of fixation: Rapid 1) Poor penetration
[HgCl2 + K2Cr2O7 + 2) Stability: deteriorates upon
Na2SO4 + H2O + glacial 2. Stability: keeps well without addition of acetic acid
HAc] disintegration
3) Time of Fixation: if prolonged -
Recommended for fixing 3. Uses: hard and brittle tissue
small pieces of liver, a) Trichrome staining
spleen, CT fibers and b) Mordant 4) Effects of tissues:
nuclei a) RBC lysis and removal of
iron from hemosiderin
b) Frozen section is not
possible
c) Mercuric pigment deposits
or precipitates.

Zenker Formol 1. Penetrates and fixes tissues well 1. Same as zenker’s fluid
(Helly’s)
2. Nuclear fixation and staining is 2. Major effect on tissue: brown
[HgCl2 + K2Cr2O7 + better than zenker’s pigment:
Na2SO4 + H2O + 40% a) Due to RBC lysis
formalin] 3. Preserves cytoplasmic granules b) Removed by immersing
well the tissue in saturated
Excellent microanatomic alcoholic Picric Acid
fixative for pituitary gland,
bone marrow, spleen and
liver
 Heidenhain’s Susa
[HgCl2 + NaCl + Trichloro
HAc + H2O + glacial HAc
+ 40% formalin]
Excellent cytologic fixative
mainly for TUMOR
BIOPSY
Metallic Fixative: Chromate fixative
Chromic Acid
[1-2% H2CrO4]
Potassium Dichromate
[3% k2Cr2O7]
Regaud’s (Moller’s) fluid
[3% k2Cr2O7 + 40%
formaldehyde]
Recommended for
demonstration of
chromatin, mitochondria,
mitotic figures, golgi
bodies, RBC and colloid
containing tissues.
1) Time of fixation:
a) Rapid and even penetration
b) Reduced processing time
2) Effect on tissue: minimum
shrinkage and hardening
[counterbalance principle: swell
(acid) and shrink (Hg)]
3) Sectioning of tissue: easier
4) Staining procedure: Silver (Ag)
impregnation
Advantage
1. Precipitates all proteins and
adequately conserves carbohydrates upon prolonged standing
2. Strong oxidizing agent
1. Fixed but not precipitate
cytoplasmic structures
2. Preserves LIPID
3. Preserves Mitochondria [pH = 4.5
- 5.2]
1. It penetrates tissues well
2. Harden tissues better and more
rapidly than Orth’s fluid.
1) Time of fixation:
a) Prolonged - shrinkage,
hardening, bleaching
2) Effect on tissue:
a) RBC preservation: Poor
b) Cytoplasmic granules:
dissolved
c) Mercuric chloride deposits:
alcholic Iodine (I2) to remove
3) Staining: weigert’s method
[elastic fibers] - not possible
Disadvantage
1) Stability: easily decomposed
To prevent: add strong reducing
agent (formaldehyde) to chrome
containing fixative
pH requirement: 4.5 - 5.2
If <4.5 (more acidic): chromatin
bodies + chromosomes [fixed] but
mitochondria [destroyed]
1. Poor penetration: tissue must
be <2-3 mm thick
2. Stability: deteriorates and
darkens on standing due to acidity
3. Time of fixation: prolonged
a) Blackens tissue melanin -
removed by washing
tissue in tap water prior to
dehydration
b) Precipitates of Suboxide -
removed by washing
tissue in tap water prior to
dehydration
4. Effects on tissues:
a) Nuclear staining: poor
b) Fats: not preserved
c) PAS reaction: reduced
d) Hemosiderin: preserved
less
 1. Demonstrates Rickettsiae and
Orth’s Fluid other bacteria

[2.5% k2Cr2O7 2. Preserves Myelin better than

+Na2SO4 + 40% buffered formalin Same as Regaud’s Fluid
formaldehyde]

Recommended fixative
for the study of Early
Degenerative Processes
and tissue Necrosis]

Metallic Fixative: Lead Fixative

Advantage Disadvantage

Lead acetate [4%] Prolonged standing :

a) Lead acetate + CO2
“recommended fixative Fixes connective tissue Mucin Carbonate (insoluble)
for Acid
mucopolysaccharide” To removed:
a) Filtration
b) Add HAc drop by drop to
lower pH and dissolve the
residue

Alcohol Fixative
Effects on tissues:
Alcohol [70-100%] 1. Ideal for small tissue fragments. a) RBC: lysis
2. Fixative and dehydrating agent
“Excellent fixative for 3. Preserve nuclear stain b) Fats (lipids): dissolved
Glycogen preservation” [alcohol containing
fixatives are
contraindicated when
lipids are to be studied]
c) Glycogen Granules:
polarization (movement
towards the end)
1. Dry and wet smears: blood and
Methyl Alcohol bone marrow smears 1. Slow penetration
2. Over fixation (>48 hours): over
2. Fixative and dehydrating agent hardened tissue
 Osmium Tetroxide (Osmic Acid)
Osmic Acid
[6% at 20C]
Fixative used extensively
for neurological tissues
Pale yellow powder
which dissolves in
water to form a strong
oxidizing solution
Trichloroacetic Acid
(TCA)
Acetone
Fixative agent for brain
tissues in the diagnosis of
Rabies
Used at ice cold
temperature ranging from
5c to 4c
Advantage
1) Fixes the following tissues well
a) Conjugated fats and lipids
Fats + osmium tetroxide
Osmium dioxide (black)
b) Cytoplasmic structures 4. Major precautions:
(Golgi body, Mitochondria)
c) Neurological tissues (myelin,
peripheral nerve)
2) Best nuclear staining with
Safranin
3) Application in ultrathin sectioning
for Electron Microscopy
4) No washing out
Adrenal gland + vapor osmium
tetroxide fixation
Other Fixative
1. Precipitating effect: Proteins
2. Counteracting effect: TCA acid
(swell) other (shrink)
3. Decalcification effect: soften
dense fibrous tissues
1. Study of H2O diffusible
enzymes (phosphatases, lipases)
2. Solvent for certain metallic salts
(freeze substitution techniques)
Disadvantage
1. Price: Expensive
2. Stability: extremely volatile
3. Poor penetrating agent: for
small tissues only
a) Prolonged exposure to
acid vapor: wear mask or
gloves, conjunctivitis (eye
irritation).
Blindness (deposition of black
Of osmic oxide in the cornea
b) Fixative exposed to light:
dark colored container -
formation of black that
settles at the bottom of
container.
Osmic acid fixed tissue + Sat.
Aqueous HgCl2 prevent
reduction with formation of black
deposits
Poor penetrating agent, hence
suitable only for small piece of
tissues or bones
1. Stability: evaporates rapidly
2. Effects on tissues: shrinkage
and distortion
3. Fats: dissolved
4. Glycogen: poorly preserved
 Secondary fixation

1st fixative 2nd fixative Stain/ staining tissue

Massons trichrome Connective

10% buffered Zenkers’s solution Mallory’s aniline blue Collagen

neutral formalin
PTAH- phosphotungstic acid hematoxylin Straited muscle

Post - Chromatization

(Aq potassium dichromate) mordant - cytologic preservation of tissue

Washing Out (remove excess fixative)

 50-70% alcohol Picric acid Bouin solution

 Alcoholic iodine mercuric fixative

 Tap water formalin and osmic acid

 Chromates; kelly’s; Zenkers; flemming

Microwave technique

 Physical agent like vacuum, oven (heat) and agitation to increase movement of molecules and
accelerate fixation

 Accelerates staining, decalcification, immunohistochemistry and electron microscopy

 Oscillation frequency 2450 mHz

 Microwave technique

Advantage Disadvantage

Tissue is heated right through the block Penetrates 10-15 mm only

No significant cross linking of protein

Non chemical technique (less interference) molecules, subsequent chemical fixation may
be needed

Rapid Viable spores/ pathogens (alcohol based

fixatives or microwaving alone)

Lesser time for immunohistochemistry and

in-situ hybridization

Special tissue processing

Tissues that must be submitted unfixed

1) Tissues for frozen section evaluation

2) Gout: uric acid dissolves in formalin - may use 100% ethanol instead
3) Tissues submitted for infectious disease and cytogenetic studies
4) Lymph nodes for lymphoma work up
5) Muscle and nerve biopsy
6) Kidney biopsies
7) Tissue submitted for analysis of lipids

Processing bone marrow biopsies

 The fixative used is very important

 Submit entire needle biopsy after fixation in bouin’s fluid overnight, which is mildly acidic and
removes calcium
 Serially number eight slides and cut sections at 4 microns.
 Stain slides 1&5 with H&E, slides 2&6 with reticulin stain and slides 3&7 with Iron
store slides 4&8.
 Factors that affect fixation of tissues

Retarded By: Enhanced By:

1. Size & thickness of tissue specimen: 1. Size & thickness of tissue specimen:

Size: volume of fixation: fixation time Size: volume of fixation: fixation time

2. Presence (FMblood directly related)

2) Agitation:
a) Mucus:
automatic or mechanical tissue processing :
Mucus: penetration of fixative: fixation time Fixation time

b) Fat:

Fat: penetration of fixative: fixation time

c) Blood:

blood: penetration of fixative: fixation time

3. Cold temperature: 3) Moderate heat: (37-56 C)

Temperature: enzyme inactivated: fixation time Temperature: hasten autolytic changes and
enzyme destruction: fixation time

Tissue processor

Technicon - mechanical processor with

An electric motor
 Reagent Processing time

Schedule 1 Schedule 2 Schedule 3

10% buffered formalin 2h 2h 2h

10% buffered formalin 2h 2h 2h

70% alcohol 2h 2h 2h

Absolute alcohol 1 1h 3h 2h

Absolute alcohol 2 1h 3h 2h

Absolute alcohol 3 1h 3h 2h

Xylene / toluene 1 1h

Xylene / toluene 2 1h

Benzene 1 30 min

Benzene 2 1h

Chloroform 1 2h

Chloroform 2 2h

Chloroform 3 2h

Wax 1 2h 3h 2h

Wax 2 3h 3h 2h
 Decalcification- after fixation & before impregnation

 Bones, teeth, calcified tissues - tuberculous lungs, arteriosclerotic vessels

 Poor cutting of hard tissues / knife damage

 Know patient’s case - if too large - use saw

 Change decalcifying agent regularly

 Grating sensation during cutting place block in 10% HCL for 1 hour

 Rapid decalcification - produces effect on nuclear staining - (failure of nuclear chromatin to

take up hematoxylin)

Decalcification

Acids: Ion exchange resins:

HNO3, HCl, formic, TCA, sulfurous, chromic, (Ammonium form of polystrene resin) - (1-14
citric days) spread on bottom of container

 Artifacts produced, usually caused by CO2

bubbles
 slow
Chelating Agents Electrical ionization
EDTA - slow  Electrophoresis - attraction of Ca to
negative electrode
 EDTA (versene)
 Excellent staining  Formic acid, conc HCl, distilled water
 Very slow
 Slight tissue hardening produced
 Completeness of decalcification

 Physical / Mechanical
 X-ray / Radiological
 Chemical

Litmus paper Red if due to acidity; add NH3 drop by drop blue litmus;

if cloudy still with calcium; if clear add ammonium oxalate, 30 mins cloudy if
incomplete

Post Decalcification

 Remove acid by saturated lithium carbonate solution or 5-10%aqueous NaHCO3 for several
hours

 Running tap water

 If EDTA is used - use 70% alcohol

Tissue softeners
 Perenyl’s 12-24 hours
 45 aqueous Phenol 1-3 days
 Molliflex (swollen & soapy appearance)
 2% HCl
 1% HCl in 70% alcohol

Rate of Decalcification
 More concentrated acid solutions - more rapid but more harmful to tissue

 Heat hastens decalcification, but increases damaging effect of acids to tissues

 Mechanical agitation, sonication

 Ideal time 24-48 hours

Dehydration - most critical stage of processing, difficult to correct

 Dehydration
Alcohol - progressively increasing
Dehydrating Agents:
 Ethyl - best, fast, most common, mixes with water and
 Alcohol - most common penetrates easily, not poisonous, cheap
 Acetone
 Dioxane  Methyl - toxic, for blood films
 Cellosolve  Butyl - slow, for plant and animal microtechnique
 Triethyl phosphate
 THF
Disadvantage:
a) Prolonged storage in 70% alcohol - macerates
tissues
b) Directly placed in high grade alcohol - shrinkage &
hardening of tissues.

Cellosolve
Acetone Triethyl Phosphate
 Ethylene glycol
monorthyl ether
 Cheap  Minimum shrinkage
 Rapid acting  Rapid
 Volatile  Minimum distortion and
 Tissue shrinkage  Storage without hardening of tissue
producing hardening or
distortion

Ideal dehydrating solution:

 Rapid acting with no tissue shrinkage or distortion
 Should not evaporate fast
 Can dehydrate even fatty tissues
 Non toxic
 Not fire hazard
 Should not harden tissues excessively

Dioxane (Diethylene Dioxide) Tetrahydrofuran - THF

 Dehydrates and clears
 Excellent dehydrating & clearing agent  Less shrinkage
 Less tissue shrinkage
 Tissues can be left here for a long time  Easier cutting with fewer artifacts
 Ribbons poorly
 Expensive  Non toxic; 6 months exposure -
 Dangerous - vapor - cumulative conjunctival irritation
 Graupner’s method dioxane, paraffin wax
 Offensive odor - use well
 Weiseberger’s method gauge, Ca oxide ventilated room
Clearing / dealcoholization
 Clearing Agents: Clearing:
 Xylene - most  Substances will dissolve wax with
 Toluene which the tissue is to be
 Benzene impregnated
 Chloroform
 Cederwood oil  After staining - transparent tissues
 Aniline oil
 Clove oil  High refractive Index
 CCl4

 Methyl benzoate and methyl salicylate - slow

 THF
a) Dehydrates and clears
b) Non toxic
c) Offensive odor

Clearing Agents:

 Miscible with alcohol and paraffin &/or mounting medium

 Should not produce excessive tissue shrinkage & hardening

 Should not evaporate quickly in a water bath

 Should make tissues transparent

IMPREGNATION / INFILTRATION
Types of Tissue Impregnation/Embedding:

 Paraffin Wax

 Manual – 4 changes of wax 15 mins interval

 Automatic – Autotechnicon, Elliott Bench type

 Vacuum – fastest, negative atmospheric pressure, gives the fastest result

 Celloidin (Collodion)
 a) Wet – bones, teeth, large brain sections

b) Dry – whole eye sections - 70% ROH not used

 Gelatin
 Plastic

IMPREGNATION / INFILTRATION

Paraffin Wax
 Simplest, most common, best, 56 C
 Ease in cutting
 Permanent paraffin blocks
 Good staining results
 Not recommended for fatty tissues
 Overheated paraffin → brittle specimen
 Inadequate impregnation → clearing agent retained

Substitute for Paraffin wax

 Paraplast – more elastic / resilient (56-57C)

a) Embeddol – less brittle (56-58C)

b) Bioloid – for eyes

c) Tissue Mat – with rubber

 Ester Wax – low melting point (46-48C)

a) Harder than paraffin; water insoluble, can be used for impregnation
without prior clearing

 Water Soluble Wax

a) Carbowax – water soluble (no dehydration/clearing); for enzyme
histochemistry; hygroscopic
 Vacuum Embedding - wax impregnation under negative
atmospheric pressure

Time of Impregnation / Size / Type of clearing

Thickness of tissue Benzene/ Toluene Chloroform/ Cedarwood oil

Period in Number of Period in molten Number of
molten wax changes in wax changes in wax
wax
Less than 3 mm 1 hr one 1 1/2 hr two
3-5 mm 1-5 hr two 2-5 hr three
5mm-1cm 3-6 hr two 4-8 hr three
More than 1 cm 6-12 hr three 8-12 hr four

Embedding
 AKA Casting / blocking - Place tissue in a precisely arranged position in a mold
with a medium which is then allowed to solidify (orientation)

 Orientation - on the microtome before cutting and on the slide before staining
 BLOCKING OUT MOLDS
 Leuckhart’s Embedding Mold

 Compound Embedding Unit

 Plastic Embedding Rings & Base Mold

a) Tissue Tek – machine w/ warm plate

 Disposable Embedding Molds:

 Peel Away
 Plastic Ice Trays
 Paper boats

DOUBLE EMBEDDING
 2% Celloidin for 3 days and subsequent paraffin

 for large blocks of dense firm tissues – brain

 Obsolete – due to paraffin waxes with different types of resins

 PLASTIC (RESIN) EMBEDDING
 Epoxy, polyester, acrylic

 For hard tissues, renal and bone marrow biopsies

 For ultrathin sections requiring minimal distortion

 With VCD – vinylcyclohexane dioxide- carcinogenic

TRIMMING

• Process of removing excess wax from the block to expose the tissue surface in
preparation for actual cutting

• Sides, top and bottom of tissue block are trimmed

• Paraffin blocks – require chilling in ice cold water or refrigerator

• Celloidin blocks – do not require chilling or refrigeration

Microtome
 Block holder, knife carrier, knife, pawl, ratchet feed wheel and adjustment screws

 Knife:

 Plane Concave - celloidin embedded, sliding

 Biconcave – paraffin embedded, rotary microtome

 Plane wedge – frozen sections or hard specimens in paraffin blocks with

base sledge/sliding

 Diamond edge – for ultrathin sections

Water bath temperature: 45-50 C or 6-10 C lower the Melting point of
wax
Microtome Inventor Description purpose
Paldwell Trefall Simplest Serial sections of large
1881 paraffin embedded
Rocking
sections, 10-12u

George Adams Most Celloidin embedded

Sliding 1789 Dangerous sections

Charles Most Paraffin embedded

Sedgwick Minot common sections
Rotary
1885

John Quekett For Frozen Unembedded frozen

1881 Section Section, bursts of CO2 gas
Freezing
vs. cryostat

Marfred von Sections for EM, 0.5u

Ultrathin Ardenne
 Base sledge microtome/ sliding microtome:
 Standard sliding - block is stationary; knife is moving
 Most dangerous type of microtome - movable exposed knife

 Bevel <27-32
 Angle 0-15

Fishing Out
 Floaters / pick up

HONING – HEEL TO TOE (SHARPENING)

• Coarse Honing – gross nicks

• Honing Proper – even edge acquired

• Hone – carborundum – 8”x3”

 Belgium Yellow – best result
 Arkansas – more polishing effect
 Fine Carborundum – much coarser – for badly nicked
 Belgium Black or Belgium Blue Green
 STROPPING – TOE TO HEEL (POLISHING)

• Burrs formed during honing is removed

• Cutting edge of knife is polished / sharpened
• Paddlestrop –made of horse leather
• 40-120 double stokes
• Treated with vegetable oil – Castor Oil
• Should never be treated with Mineral oil
• Should not be used 24-48 hours after treatment
• Wax should not come in contact with the strop

Faults Reason Remedy

Prolonged fixation

Prolonged dehydration Tissues may be

Brittle or hard tissue
softened by soaking in
Prolonged clearing a small dish or bowl
containing water with
Prolonged paraffin infiltration detergent, phenol or
Molliflex.
Overheated paraffin oven

Drying out of tissue before

actual fixation
Clearing agent turns milky as Water not completely removed Repeat dehydration with
soon as tissue is placed in it due to incomplete dehydration absolute alcohol, then clear
again
PART 3
1. A step that renders the tissue transparent or
translucent:
a. Clearing
b. Embedding
c. Fixation
d. Dehydration
2. The most common dehydrating agent is:
a. Alcohol
b. Dioxane
c. Acetone
d. Cellosolve
3. Paraffin sections are usually cut in _ micron thickness
a. 11-15
b. 15-30
c. 4-6
d. 5-8
4. This type of microtome is invented by Paldwell Trefall
in 1881
a. Rocking
b. Minot
c. Rotary
d. None of the above
5. Brittle or hard tissues are due to prolonged:
a. Fixation
b. Dehydration
c. Clearing
d. All of the above
 METHODS OF CELL DEMONSTRATION
I. Staining (dyeing)
 Process of artificially giving color to different tissue
constituents to render them more visible and
optically differentiated

REASONS FOR STAINING AFFINITY

1. Physical phenomena
a. Capillary osmosis
b. Solubility
c. Adsorption
d. Absorption
1. Chemical phenomena
a. Nuclear structure- nucleus is acidic in nature therefore has
a greater affinity for BASIC dyes
b. Cytoplasmic structure- cytoplasm is basic in nature
therefore has a greater affinity for ACIDIC dyes

II. IMPREGNATION
 Makes use of salts of heavy metals which are
precipitated with considerable selectivity on certain
cellular and tissue components
 Differs from staining because it consists of an opaque
particulate precipitate

Commonly used agent for impregnation

1. AgNO3 (silver nitrate) – most common
2. AuCl3 (gold chloride)
3. Ammoniacal silver- reduced by Argentaffin cells (Melanin
and Intestinal Gland) forming black deposits
4. Gold sublimate- used for metallic impregnation made up of
gold chloride and mercuric chloride
 APPLICATIONS
1. Nervous tissue
2. Demonstration of Reticulin
3. Outlining fine intracellular structure in case of osmium
tetroxide

STAINING
Natural Dyes
 Hematoxylin
 Cochineal Dyes & its derivatives
 Orcein
 Saffron
Synthetic (Artificial) Dyes / Coal Tar Dyes / Aniline Dyes
 Chromophore Dyes: Acid, Basic, Neutral

Methods of Staining
 Direct vs. Indirect (mordant, accentuator)
 Progressive vs. Regressive
 Differentiation (Decolorization)
 Metachromatic – cations
 Counterstaining
 Microanatomical staining
 Cytoplasmic staining
 Negative staining

GENERAL METHODS OF STAINING

I. DIRECT STAINING
- Staining of tissues by means of simple aqueous or
alcoholic solutions of dyes (methylene blue, eosin)
II. INDIRECT STAINING
- Enhanced staining of tissues by means of a MORDANT
or ACCENTUATOR to intensify the actions of the dye
 ACCENTUATOR POINTS OF MORDANT
DIFFERENCE
- Not essential in the - Combine with the dye
chemical union of remaining valences
tissue and dye - Attach or bind the dye-
- Increases the Main action mordant complex to the
selectivity and tissue components
staining power of the especially to the
dye phosphate groups of
nucleic acid
- “lake” (stain-mordant-
tissue complex)
1. KOH- Loeffler’s 1. K- alum: Erlich’s
methylene blue hematoxylin
2. Phenol- Carbol Examples 2. Fe (iron): Weigert’s
thionine/ Carbol hematoxylin
Fuchsin 3. I2 (iodine): Gram’s stain

III. SPECIFIC STAINING

Basis of HISTOCHEMISTRY
- Identification of certain structures and chemical
substances designed to give a final color at the site or
location of structure or substance in the cells or tissue
Examples:
 Weigert’s elastic stain (elastic fibers)
 Best Carmine (glycogen)
Periodic Acid Schiff (polysaccharides)

IV. PROGRESS STAINING

- Technique whereby stains are applied in a
DIFINITE SEQUENCE with no washing-out or
decolorizing because over staining of the tissue
constituents
V. REGRESSIVE STAINING
- Technique whereby tissues are OVERSTAINED
and the excess dye is then remove selectively
until the desired intensity is obtained
- “differentiation” or “decolorization”
o Process of washing out excess stain until a
specific substance is stained differently from
the surrounding tissue
o Washing is done by:
a. Simple solution (water or alcohol)
b. Acids
c. Oxidizing agents

VI. METACHROMATIC STAINING

- Process of staining the tissues in a color that is
quite different from that of the stain itself
(metachromasia)
- “metachromatic dyes” : cations whose peculiar
metachromatic staining property depends upon
their tendency to polymerize
o 0.1% in 30% ethanol or 0.1-1% in aqueous
solution
- APPLICATION: CAMec
a. Cartilage d. Mast cell granules
b. Connective tissues e. Amyloid
c. Epithelial mucins

- EXAMPLES: A BCMT2 S
a. Azure A, B, C
b. Basic fuchsin
c. Bismarck brown
d. Cresyl blue (for
reticulocytes)
e. Crystal violet
f. Methyl violet
g. Methylene blue
h. Safranine
i. Thonine
 j. Toluidine blue

VII. COUNTERSTAINING
- Application of a different color or stain to
provide contrast and background to the
staining of the structural components to be
demonstrated.
CYTOPLASMIC STAIN NUCLEAR STAIN

Red Yellow Green Red Blue

Phloxine B Picric acid Light Neutral Red Methylene

Eosin Y Orange G Green SF Safranin O Blue
Eosin B Lissamine Carmine Toluidine Blue
Rose Bengal Green Celestine Blue
Hematoxyline

VIII. VITAL STAINING

- Selective staining of living cell constituents by
cytoplasmic phagocytosis of dye particle
- NUCLEUS: not demonstrated because it is
resistant to vital stains; if demonstrated
suggests cellular permeability leading to CELL
DEATH
INTRAVITAL STAINING SUPRAVITAL STAINING
Dye injection to animal Staining living cells
body to produce specific immediately after removal
coloration from the living body
Lithium Neutral Red- best vital dye
India Ink Janus Green- mitochondria
Carmine
 Trypan Blue- toxic after 1
hour
Toluidine Blue
Nile blue
Thionine

CLASSIFICATION OF DYES
I. NATURAL DYES: biological sources (plants and
animals)

A. HEMATOXYLIN: (heartwood of Mexican tree

Hematoxylin campechnianum)
 Most valuable staining reagent because of
powerful nuclear and chromatin staining
capacity
 “hematin”: active coloring agent formed by
oxidation of hematoxylin (ripening)
- Strong oxidizing agents used in ripening process:
a. Hydrogen peroxide
b. Mercuric oxide
c. Potassium permanganate
d. Sodium perborate
e. Calcium hypochlorite
f. Sodium iodate
 “Ripened hematoxylin” is a mixture of
hematoxylin, hematein, active oxidation
products (hematein and hematoxylon) and
inactive ultraoxidation products
 Mordants combined with ripened hematoxylin to
increase tissue affinity:
 1. ALUM HEMATOXYLIN: progressive staining of tissue

 Alum is chemically potassium aluminum sulfate

 Counterstain used: Eosin, Congo red, Safranin
 Blueing: process of passing an alum hematoxylin
stained sections in an alkaline solution to neutralize
the acid and free the OH group and form an
insoluble blue-aluminum-hematin-tissue lake
TYPES OXIDANT PURPOSE
1. Ehrlich’s Sodium iodate Regressive staining
Hematoxylin
2. Harris Mercuric oxide  Routine nuclear
Hematoxylin Mercuric chloride staining
 Exfoliative
cytology
 Staining of sex
chromosome
3. Cole’s Iodine Routine staining
Hematoxylin especially in
sequence with
Celestine Blue
4. Mayer’s Sodium iodate Celestine blue
Hematoxylin Hemalum method
of nuclear staining

2. IRON HEMATOXYLIN: differential or regressive staining

TYPES MORDANT PURPOSE
1. Weigert’s Ferric ammonium Standard iron
Hematoxylin chloride hematoxylin for
demonstrating
muscle fibers and
connective tissue
2. Heidenhain’s Ferric ammonium Cytological stain
Hematoxylin sulfate recommended for
regressive staining
of thin sections

3. COPPER HEMATOXYLIN: study of spermatogenesis

PHOSPHOTUNGSTIC ACID HEMATOXYLIN (PTAH)

 Ripened by the addition of POTASSIUM
 Progressive staining of paraffin, celloidin and
frozen sections

b. COCHINEAL DYES: (female Cochineal Bug, Coccus

cacti)
 “carminic acid”: active principle of carmine
dye, a powerful chromatin and nuclear stain
 PICROCARMINE (carmine + picric acid) :
neuropathological study
 BEST CARMINE (carmine + aluminum chloride) :
Glycogen demonstration

c. ORCEIN: (Lichen-derived vegetable dye)

 Normally colorless but when treated with
ammonia and exposed to air produce blue or
violet colors
 Weak acid, alkali-soluble dye mainly used for
staining ELASTIC FIBERS
 Litmus: lichen derived dye treated with Lime
and Soda and exposed to ammonia and is
mainly used as an indicator

d. SAFFRON: dried stigmata of Crocus sativus

 II. SYNTHETHIC DYES: “ Coal Tar Dyes”
 Derived from the hydrocarbons of benzene
(C6H6) and are collectively known as ANILINE
DYES
 Chromophore: substances with definite atomic
groupings capable of producing visible colors
 Chromogens: simple benzene compounds with
chromophores
 Chromogens: color imparted to tissues is not
permanent
 Chromogens: it becomes a dye if it has color
retention property which can be achieved by
the addition of an auxochrome
 Auxochrome: auxiliary radical that imparts
electrolytic dissociation property for color
retention
COMPONENT ACID DYE BASIC DYE NEUTRAL DYE
ACTIVE Acidic radical Basic radical
(COLORING (picric acid)
AGENT)
INACTIVE Sodium Sulfuric, Acid + basic
acetic or dyes
hydrochloric
acid
GENERAL USE Cytoplasmic Nuclear stain Nuclear and
stain cytoplasmic
stain
EXAMPLE Picric acid Methylene Giemsa /
blue Leishman
FIXATIVE OF Trichloroacetic Mercuric Ethyl alcohol
CHOICE acid chloride Acetic acid
Picric acid Formaldehyde
 Chromium
fixative

EOSIN (acid xanthene or pthalein dyes)

 Routinely used as a counterstain after Hematoxylin
 Commonly used as background for contrasting stains
 Red acid dyes available in 2 shades:
a. Bluish- deeper red color (Eosin B)
b. Yellowish (Eosin Y)- readily soluble in water and less
soluble in alcohol
EXAMPLES:
1. Eosin Y 6. Orange Gall
2. Eosin B 7. Primrose soluble
3. Ploxine 8. Safrosin
4. Erythrosin 9. Rose Bengal
5. Azoploxin 10. Rose B

OTHER STAINS USED

BLUE GROUP: Starplayer of blue team: MAT/PAC/NV
STAIN USE
1. Methylene Blue 1. Stain for plasma cell
2. Cytological
examination of fresh
sputum for malignant
cells
3. Bacterial stain for
evaluation and
grading of milk
4. Vital staining of
nervous tissue
 5. Indicator of
differentiation of colon
and
2. Touidine Blue 1. Substitute for Thionine
in fresh frozen tissue
section
2. Recommended stain
for Nissl granules or
chromophilic bodies
3. Aniline Blue Cytoplasmic stain used for
counterstaining EPITHELIAL
section
4. Celestine Blue Resistant to strong acid
dyes, it gives a good
nuclear definition in
conjunction with ALUM
hematoxylin
5. Prussian Blue (colored Paint manufacturing,
salt of ferric microanatomical color
ferrocyanide) contrast, intra-vital staining
of the CIRCULATORY
SYSTEM
6. Alcian Blue Complex, water soluble
Phthalocyanin dye, similar
to chlorophyll, which stains
ACID
MUCOPOLYSACCHARIDES
7. Victoria Blue Demonstration of
NEURALGIA in frozen
section
8. Night Blue Substitute for Carbol
fuchsin in Acid Fast Staining
II.VIOLET GROUP: gmc
STAIN USE
1. Methyl Violet Metachromatic dye when
methylene blue is heated
with fixed alkali or alkali
carbonate
2. Crystal violet  Amyloid stain in frozen
section
 Platelet stain in blood
films
3. Gentian violet Crystal violet + methyl
violet + dexterin

III. GREEN GROUP (mjm)

STAIN USE
1. Malachite Green  Contrast stain for staining
ascaris eggs and
erythrocytes
 Bacterial spore stain
2. Methyl Green Chromatin stain that gives
a false (+) in MUCIN
secretions
3. Janus Green Intravital staining of
MITOCHONDRIA

IV. RED GROUP (nac)

STAIN USE
1. Neutral Red Cell granules vacuoles of
PHAGOCYTIC CELL

2. Congo Red a. Axis cylinders in embryos

b. 4% aqueous solution in
Krajian method of

3. Acridine Red 3B Calcium deposits and sites
of PHOSPHATASE activity
V. FUCHSIN GROUP
BASIC FUCHSIN
- Main constituent of
Fuelgen’s and Schiff’s
reagent for aldehyde
- Main constituent of Van
Gieson’s reagent for
connective tissue
1. Carbol-fuchsin
2. Coleman’s Fuelgen
3. Schiff’s reagent
4. Mallory’s Fuchsin
5. Gomori’s stain (aldehyde
fuchsin)
VI. MISCELLANEOUS GROUP
STAIN
1. Giemsa stain
2. Bismarck brown
3. Orcein
staining elastic tissues,
amyloid and myelin
ACID FUCHSIN
Demonstration of
CONNECTIVE tissue
1. Van gieson’s stain (acid
fuchsin + picric acid)
2. Masson stain (acid
fuchsin + glacial acetic
acid)
3. Picro-fuchsin (acid
fuchsin + sat.aq.picric
acid)
USE
Stain blood to differentiate
leukocyte
Contrast stain for Gram’s
technique, acid fast and
Papaniculao method; staining
DIPTHERIA organism
Excellent stain for ELASTIC
FIBERS especially
 recommended in
dermatological study
4. Picric acid Fixative, decalcifying agent,
tissue softener, CT Stain

5. Carmine  BEST CARMINE: (carmine +

6. Mayer’s carmalum
7. Acridine orange
8. Rhodamine B
9. Benzidine
aluminum chloride):
GLYCOGEN
 MUCICARMINE: FUCIN
(carminic acid + potassium
alum + water + salicylic acid +
sodium salicylate)
 Mordanted dye acting as
basic dye and staining
acidic substances
a. DNA: green fluorescence
RNA: red fluorescence
b. Basic acridine fluorochrome
which permits discrinination
between dead and living
cells
Used with OSMIC ACID, it fixes
and stains blood and
glandular tissues
Used for staining
HEMOGLOBIN

IODINE STAIN
 Oldest stain originally used for the microscopic stidy
of STARCH GRANULES; also for Glycogen, Amyloid,
Cellulose, Carotene
 Removal of MERCURIC FIXATIVE artifact
 Alter crystal and methyl violet so that bacteria and
fungi can retain the stain
 GRAM’S IODINE
1 g I2 + 2 g Kl + 400 ml distilled
water
Used in WEIGERT method of
staining microorganism and
fibrin
LUGOL’S IODINE
1 g I2 + 2 g Kl + 100 ml distilled
water
Test for glycogen, amyloid,
and corpora amylacea

LYSOCHROMES (Oil Soluble Dyes)

 Non auxochromic dye that gives color to lipids
because of their solubility in lipid medium of the
tissues
 OSMIUM TETROXIDE- fats reduce osmium tetroxide to
osmium dioxide and are stained black
SUDAN BLACK SUDAN IV (scarlach R) SUDAN III

- Slightly basic dye - No secondary amino

containing two groups CNS fat stain
secondary amino - Do not color (central nervous
group per phospholipids and fine system)
molecule lipid droplets
- Non specific stain - Recommended for
for neutral lipids neutral lipids
triglyceride) (triglycerides)

CONNECTIVE TISSUE STAIN: stains connective tissue fibrous

elements like reticulum, collagen, and elastin which are
embedded in a cementing substance known as “ground
substance”
 STAIN FIXATIVE TECHNIQUE RESULT
NUCLEI COLLAGEN ELASTIC RETICULUM
FIBER
1. Masson’s Bouin’s Paraffin Black Blue - -
Trichrome 10% section
(collagen) Formalin
2. Mallory’s Zenker Paraffin Red Intense Pale -
Aniline Blue section Blue yellow
(collagen)

3. Gridley’s Formalin Paraffin Red - Black -

Stain section
(reticulum)

4. Gomori’s 10% Paraffin - - - Black

Stain Formalin section
(reticulum) Celloidin
Section

5. Van Any Paraffin Blue Bright red - Unstained

Gieson’s section Black
Picrofuchsin

6. Ver Hoeff’s Any Paraffin Gray Red Black -

Elastic section to
Method Black

7. Weigert’s Any Paraffin - Blue -

Resorcin- section Black
Fuchsin Celloidin
(elastic Section
fibers)

GLYCOGEN STAIN: Glycogen is a polymer of glucose

manufactured and stored in the LIVER cells but also found
normally in lesser concentration in muscles, parathyroids,
and cartilages. It is soluble in water and insoluble in
alcohol. Therefore, alcoholic fixatives are the BEST.
However, alcohol is slow to penetrate and fix and it
causes glycogen to appear as large granules.
 STAIN FIXATIVE TECHNIQUE RESULT
1. BEST CARMINE - 10% formalin Paraffin Section GLYCOGEN-
METHOD - Carnoy’s brilliant red
(selective stain absolute alcohol
for glycogen)
2. PERIODIC ACID Formalin Paraffin Section PAS +: magenta/
SCHIFF purplish red
LIPID/FAT STAIN: demonstration of neutral fat in tissue is
called in the following circumstances:
a. Fat embolism- skeletal fractures, chronic alcoholics
with fatty livers
b. Identification of lipid deposits
STAIN FIXATIVE TECHNIQUE RESULT
1. SUDAN IV 10% formalin Frozen section FATS: red
STAIN
(Scarlet Red)
2. OIL RED O 10% formalin Frozen section FATS: orange
STAIN red
3. OSMIUM 10% formalin Frozen section FATS: black
TETROXIDE
stain
4. SUDAN Formaldehyde Frozen section FATS: blue
BLACK calcium with black
post chroming
5. NILE BLUE 10% formalin Frozen section FATS: pinkish
SULFATE red
(Lorraine-
Smith)
 MINERALS AND PIGMENTS
STAIN FIXATIVE TECHNIQUE RESULT
1. VON KOSSA Formalin and Paraffin Section Ca: Black
(Ca) alcohol

2. PERL’S 10% Formalin Paraffin Section Hemosiderin:

PRUSSIAN BLUE Blue
(Free Iron)
3. STUN’S BILE Formalin and Paraffin Section Bile pigments:
PIGMENT alcohol emerald green

CHROMAFFIN AND ARGENTAFFIN GRANULES

STAIN FIXATIVE TECHNIQUE RESULT
1. WISSEL’s 10% Formalin Paraffin Section CHROMAFFIN:
METHOD followed by blue
(chromaffin) postchroming with
potassium
dichromate
2. FONTANNA- Formalin
Paraffin Section ARGENTAFFIN:
MASSON black
(ARGENTAFFIN
BACTERIA-FUNGI-INCLUSION BODY
GRANULES)
STAIN FIXATIVE TECHNIQUE RESULT
1. GRAM-WEIGERT Formalin Paraffin Section Gram (-)
(gram (-) bacteria: violet
bacteria and
fibrin)
2. ZIEHL-NEELSEN Neutral Formalin Paraffin Section Tubercle bacilli:
(acid fast Mercuric fixative bright red
bacteria in
tissue)
3. LEVADITI’S Formalin Tissue Spirochete:
(spirochete) Impregnation black
4. GRIDLEY’S Any Paraffin Section Mycelia: deep
(fungi) blue
NERVOUS TISSUE STAIN
 Conidia: deep
rose to purple
Elastic tissue/
mucin: deep
blue
1. NISSL or TIGROID SUBSTANCES
 Coarse granules made up of RNA scattered in
the cytoplasm of nerve cell
 Stained by H and E thionine, toluidine blue or
methylene blue
 Deep blue in toluidine blue against a colorless
background

2. NEURONS, AXONS, NEUROFIBRILS

 Beilschowsky’s technique
 Black on a grayinsh background

3. ASTROCYTES
 Mallory’s phosphotungstic acid hematoxylin
(PTAH)
 Cajal’s gold sublimate method
 Black on a light brownish background

4. NEUROGLIA
 Alkaline colloidal solution of an alkaline silver salt
are reduced by neutral formalin especially in the
presence of light producing SILVER DEPOSIT on
tissue elements

5. MYELIN SHEATH
 Myelin is the fatty insulating sheath surrounding
axons made up of lipid cells
 Osmium tetroxide method
 Black on a grayish background
  Weigert-pal technique of staining normal myelin
sheath
 Blue black
 Sudan Black method for rapid staining of myelin

6. CEREBRAL LIPIDS: Thin Layer Chromatography

CHIEF SOLVENT USED FOR STAINS:

1. Water
2. Alcohol
a. Ethyl alcohol- any concentration
b. Methyl alcohol- absolute
1. Aniline water
2. Phenol

PRECAUTIONS OBSERVED DURING STAINING

Avoid stains on the Failure of section to stain. Failure of staining due
skin. a. Improper and to paraffin. Fixative or
insufficiently ripened decalcifying solution
Failure of sections to Hematoxylin that has not been
remain on slide during b. Impurities in dye or in thoroughly washed
staining. water solvent out and removed.
a. Dirty or greasy slide c. Deteriorated stains
b. Too old albumin (loss of staining Sections do not
adhesive property) appear under
c. Incomplete - Precipitates in the microscope after
dehydration staining solution staining:
d. Paraffin section not - Poor staining results a. Water in the
thoroughly spread absolute alcohol
on slide when b. Moisture in the
mounted cover slip
c. Too much egg
albumin on slide
 d. Acid-alcohol
decolorizer not
completely
removed
e. Film of alkaline
alcohol

RESTAINING OLD, BLEACHED or FADED SECTIONS

 Removal of coverslip by a dissecting needle
a. Immersion in xylene for 24 hours
b. Heating gently the slide until mounting medium starts
to bubble

 Once the coverslip has been removed, the slide is

placed in xylene for 30 minutes to remove remaining
balsam
 Wash in running water
 Place in 0.5 POTASSIUM PERMANGANATE solution for 5-
10 minutes and rinsed in tap water
 Immerse in 5% OXALIC ACID for 5 minutes or until the
section is decolorized
 Wash in running water for 5 minutes
 Stain using the desired technique

TISSUE PIGMENTS AND DEPOSITS
I. ARTEFACTS: highy colored, usually amorphous, granular,
sometimes crystalline stain precipitates seen in section
due to faulty staining techniques
 Acid Alcohol- most common solution in removing
excess stains in sections
FORMALDEHYDE MERCURIC OSMIUM CHROME
DEPOSIT DEPOSIT TETROXIDE DEPOSIT
DEPOSIT
Fine, dark-brown Black, brown or Black deposits Fine brown or
or black crystal- grayish black which have not black deposits
like precipitate granules been properly
washed out
Removed by Removed by Removed by Removed by
saturated saturated BLEACHING ACID ALCOHOL
alcoholic solution alcoholic using hydrogen
of PICRIC ACID IODINE solution peroxide and
70% alcohol

II.ENDOGENOUS PIGMENTS
 These are formed within the tissue by natural means,
many of them formed by the breakdown of
hemoglobin

1. HEMOSIDERIN (free iron)

 Yellowish brown pigment soluble in alcohol, water
and alkalies
 Reacts with acidified potassium ferrocyanide
(Prussian blue)
 Iron containing pigment of hemoglobin which can
be stained by PRUSSIAN BLUE OR TURNBULL’S BLUE

2. HEMATOIDIN (bile pigment)

 Iron-free pigment of hemoglobin found in places
where there is poor oxygenation, participating in
the formation of bile pigments
 3. HEMATIN (hemoglobin minus globin)
 Found in old blood clots
 Malaria, pernicious anemia, toxic hemolysis

4. HEMOZOIN
 Black granules formed by malarial parasite living
in RBC
 Maybe removed by ALCOHOLIC PICRIC ACID
method

5. HEMOFUSCIN (LIPOFUSCIN): “wear and tear” pigment

 Iron free brownish yellow pigment which occurs
with HEMOSIDERIN in HEMOCHROMATOSIS
 Does not react with FERROCYANIDE but stains
intensely with basic dyes
 Yellowish-brown granules in heart muscle, liver,
adrenals and ganglion cells

III.EXOGENOUS PIGMENTS
 Origin is outside the body and consists of minerals
 Identified by the site they occupy in the tissue by
polarized light and by staining method
 Types (Tattoo, asbestos, carbon, silica, silver)

ADVANTAGES OF IMMUNOCHEMISTRY (IHC)

There is probably no other technique that has
revolutionized surgical pathology in the recent years as
the Immunohistochemical technique:
The advantages are:
 Remarkable sensitivity and specificity
 Applicability to routine processed material
 Can be stored for long period of time
 Accurate correlation with traditional morphologic
parameters
 Compatible with fixatives currently in use
 Feasible even if material is decalcified
 Cost effective
 Identify specific/ highly selective epitopes
 Detect organisms
 Epitope demonstrated by direct labelling of AB/
secondary labelling method
 IgG- mostly used

MOUNTING
 Near refractive index of glass 1.518
 Should not dry quickly
 Should not fade
 No shrinkage or tissue distortion
 Permanent

AQUEOUS MEDIA
 Water- low refractive index, temporary
 Glycerin- high refractive index
- Glycerin jelly- 1.47 (gelatin, glycerol, distilled water,
phenol crystals)
 Gum Arabic (Farrant’s medium)-1.43
 Apathy’s medium 1.52 (pure gum arabic, pure cane
sugar or sucrose, distilled water, thymol)
  Brun’s fluid (glucose, glycerin, spirits of camphor,
distilled water)
 Karo corn syrup

RESINOUS MEDIA
 Canada Balsam
 DPX
 Xam
 Permount
 Clarite

RINGING
 Sealing margins of the coverslip
 Kronig cement (2 parts paraffin wax w/ 4-9 parts
powdered colophonium resin)
 Cellulose Adhesives- Durofix

COVERSLIPPING
 The stained section on the slide is covered with a
thin piece of glass to protect the tissue from being
scratched, to provide better optical quality for
viewing under the microscope, and to preserve the
tissue section for years to come.
 The refractive index of the mounting medium
should be close to the average refractive index of
the tissues (1.530-1.540)
 We use commercially prepared EUKITT as mounting
medium
MOUNTING MEDIUM: EUKITT
 It is an adhesive and specimen preservative
(contents: 5% Acrylic resin, 55% Xylene)
 Properties
o Fast- drying (20 min) with minimal air bubbles
o Refractive index close to glass (1.510)
o Chemically neutral, little sensitivity to water
o No reaction to staining
o Non-yellowing, unaffected by cold and UV
o Stays optically clear for over 10 years
o Does not crystallize (no microscopic artifacts)
o Finished slides easily reworked by immersing
previously mounted slide in xylene to remove
coverslip.

MOUNTING TECHNIQUE
 Lay down coverslip on top of a board
 Flood enough amount of Eukitt
 Allow one end of the slide to touch on the other end
of coverslip and gently push the slide down to allow
Eukitt to spread out
 Turn the slide gently, top facing upward and lay
down the slide to allow complete spread out of eukitt
 DO not attempt to forcefully press down the slide on
the coverslip just to spread the mounting media, for it
may produce bubbles.

COVERSLIPPING
 Drain excess xylene
 Place 2-3 drops of mounting medium on slide
 Gently lower coverslip onto slide
  Tease air bubbles from slide
 Dry on flat surface

LABELING
 Slide with frosted edge
 Pencil
 Typewritten- in gum label with hospital’s logo

The original Papanicolaou classification

Class Description
I Absence of atypical or
abnormal cells
II Atypical cytology, but no
evidence for malignancy
III Cytology suggestive of, but
not conclusive for
malignancy
IV Cytology strongly
suggestive of malignancy
V Cytology conclusive for
malignancy