Histology : microscopic study of biological tissues; scientific  Dehydrants – solvents utilized in the removal of water are

study of fine details of biological cells and tissues using graded strength of alcohol
microscope to look at specimens that have been carefully  Ethanol – recommended for routine tissue dehydration
prepared using special process called histological technique  Immersion
 Organization o From lowest conc -> highest conc
 Function o 70-80-90-Absolute alcohol
 Structures  Amount of dehydrating agent = at least 10x the volume of
Tissue : collection of cells with similar structures and functions the tissue or more
that have similar extracellular substances located between
them 5th - Clearing (dealcoholization)
Four Basic Tissue Types  Makes the tissue clear or transparent
1) Epithelial Tissue  Removal of excess alcohol to achieve good staining
2) Connective Tissue  The use of clearing agents makes the tissues translucent
3) Muscle Tissue  Xylene or xylol – routine clearing agent
4) Nervous Tissue  Other clearing agent – toluene, benzene, chloroform,
etc.
Tissue Processing 6th - Paraffin Infiltration / Impregnation
 Process of filling up tissue cavities/ spaces/ holes by the
1st – Numbering use of melted paraffin wax to increase hardening of tissue
 Assigning accession number to the specimen before proceeding to sectioning
 Putting all information to the log book for recording
purposes of the specimen 7th - Embedding
- Accession Number example  Process of placing tissue in a precise position inside a
o UCGH – S – 19 – 0001 tissue mold and allow them to solidify
 UCGH – Initials of Hospital (optional) o Tissue mold – paper boat design made from a glossy
 S – (type of specimen: Surgical/Cytology/Pap’s paper
Smear)  Leuckhart’s embedding mold
 19 – Year (2019)  Compound embedding mold
 0001 – number of recorded specimen (1st  Paraffin - simplest, most common and best
specimen) embedding medium
- Specimen for processing = 3cm x 2cm x 3-5mm  Serial sections are cut easily without
thickness distortion
 Melting point = 56°C
2nd - Fixation  Do not overheat! Not >60°C
 done to stop autolysis or putrefaction  May cause BSH
 Ratio 20:1 (fixative : tissue) 8th – Blocking
 main cause of autolysis  Step that goes hand in hand with embedding if individual
 Lysosomes – responsible for digesting the fats of the mold is used because blocks are produced after
specimen solidification
 preserve the tissue in life-like manner as possible
Two Mechanisms of Action 9th - Trimming
1) Additive Fixation – the fixative becomes part of the tissue  the removal of excess paraffin wax using a cutter
by forming crosslinks and complexes  Truncated pyramid / 4-sided prism
 Mercuric chloride, osmium tetroxide, formalin o At least 2mm of wax should surround the tissue block
2) Non-additive Fixation – the fixative does not form o This is to allow easy sectioning
crosslinks or complexes but rather stabilizing the tissue by
removing water. 10th - Sectioning
 Alcohols (dehydrating agents)  Process of uniformly cutting the tissue blocks into thin
slices (ribbons/ sections) using microtome
3rd - Decalcification (optional) Microtomes
 Applicable for bones, teeth, and teratomas a) Rotary – routinely used microtome invented by Minot
 “demineralization” – the removal of mineral calcium or lime b) Rocking – [Paldwell Trefall] simplest type
salts following fixation by the use of mild acids c) Sliding – [Adams] dangerous (used with knife) for hard
 5-10% HNO3, formic acid, TCA tissue blocks (celloidin)
 Ratio: 20:1 (decal agent : tissue) o Bevel angle – edge of knife 27º-32º
 Other types of decalcifying agents o Clearance angle – between tissue block and knife 0º-
 Acids 15º
 Chelating agents – EDTA o Cutting angle – most desirable cutting angle 15º
 Ion Exchange Resins d) Freezing – [Queckett] use CO2 to freeze specimen
 Electrophoresis *cryostat is used today instead of CO2, rotary microtome
inside; temperature is -5 to -30 degrees centigrade; used
4th - Dehydration for urgent biopsies because there is no need to embed
 process of removing water (intracellular or extracellular)
e) Ultratin – for electron microscopy; will cut 0.5 micrometer; b) Resinous Mounting Media
uses broken plate glass or diamond knife  Canada Balsam
 Paraffin Section = 4-6um  Permount/Clearmount
o To achieve excellent light penetration when examined * Ringing – Sealing of margins to prevent escape of fluid and
under the microscope evaporation; prevents sticking of slides (ex. Kronig Cement,
Durofix)
Minor steps before Staining
1st Subject the tissue on a floatation water bath 13th – Labeling
 Floatation Water bath – a basin with thermostat  Process of indicating the year and specimen number on
 Temp: 10°C below the melting point of wax one end of the prepared slide for proper identification
 Color: black  Permanent labeling using a sticker with barcode/marker
 D 11in x H 4in
 Capacity = 2L always take note of the following...
 Done to remove wrinkles and to flatten the tissue ribbons  The source of the specimen
2nd Gently fish out the tissue ribbon using a slide  Lateral/ cross section/ sagittal section
3rd Deparaffinization  Left or right part of organ (ex. Left breast)
 Removal of paraffin wax  special stain used
o Pass the tissue over a bunsen flame
o Incubator
o Slide blower
o Paraffin oven
o Set the temperature at 56-60°C

11th - Staining
 Application of dyes on tissue sections to allow the
observation of the architectural patterns and physical
characteristics of cells
 Process of promoting optical differentiation of the cell
constituents
Methods
a) Direct Staining – uses aqueous or alcoholic dye solutions
to produce a color
b) Indirect Staining – uses other agent (mordant) to intensify
the action of the dye
c) H & E Staining
i. Hematoxylin
 routine staining technique for histologic studies
 Combined with mordants to form the dye-
mordant-tissue
 Complex
 Stains acidic structures in the cell
 = nuclear staining – blue to blue black nucleus
ii. Eosin
 An acidic dye
 Stains basic structures
 = cytoplasmic staining – red/pink

12th – Mounting
 Use of a medium and a coverslip to facilitate the handling,
storage, protection of the tissue section
 Mounting Medium
 Syrupy/ viscous fluid
o used to prevent movement of the coverslip
o to protect and prevents scratches to the
tissue section
Kinds of Mounting Media
a) Aqueous Mounting Media
 Water
 Glycerin
 Farrant’s Medium
 Apathy’s Medium