used when a rapid diagnosis of a tissue is required.

without use of any chemical fixative

similar to freeze drying

rapid freezing (- 160°C)

removal of water in the form of ice (-40°C vacuum)

Frozen tissue is submerged to________ (1% acetone) and dehydrated using absolute alcohol.

o almost any microtome can be used o uses carbon dioxide

OPTIMUM WORKING TEMPERATURE = -18° to -20°C

a refrigerated cabinet in which a modified microtome is housed.

methods of freezing: best mounting media for cryostat

method of freezing:most rapid

methods of freezing: adequate for freezing small pieces of tissue EXCEPT muscle.

Specimen size for processing

FIRST AND MOST CRITICAL STEP Process of preserving cells and tissue constituents The tissue is preserved by preventing degeneration, putrefaction, decomposition and distortion

to preserve the morphologic and chemical integrity of the cell in as life-like manner as possible.

to harden and protect the tissue from the trauma of further handling.to harden and protect the tissue from the trauma of further handling.

Most important reaction for maintaining tissue morphology.

becomes part of the tissue by formation of cross links or complexes. Ex. Formalin, Hg, osmium tetroxide

10% Formol saline, 10% Neutral buffered formalin (NBF), Heidenhain’s SuSa, Formol sublimate, Zenker’s solution, Zenker’s formol, Bouin’s solution, Brasil’s solution

ex. 10% Formol saline, Absolute Ethyl Alcohol, Newcomers fluid, Acetone (FANA)

contains glacial acetic acid ex. Bouin’s fluid, Flemming’s fluid, Newcomers fluid, Carnoy’s fluid, Heidenhain’s SuSa (BFNCH). <4.6 pH

ex. Helly’s fluid, Orth’s fluid, Regaud’s fluid, Flemming fluid without acetic acid, Formalin with post chroming (HORFF). >4.6 pH

fixatives containing mercuric chloride and potassium dichromate in cryostat section

Alcoholic fixative for glycogen (Rossman’s fluid or cold absolute alcohol)

neutral buffered forma

gas produced by the oxidation of methyl alcohol. Concentrated solutions should not be neutralized (explosion) Stock solution: 37-40% Working solution: 10% (no buffer: unstable)

o White crystalline precipitates o Due to prolonged standing o Removed by: 10% METOH/filtration

Brown/black granular deposits that may obscure microscopic details

diluted in 10% NaCl CNS

Best general tissue fixative Best fixative for tissue containing iron granules w/ double phosphate buffer 1 mm/hr = rate of tissue penetration

for routine post mortem tissues w/ HgCl2

EM

EM: electron histochemistry & electron immunocytochemistry

Mixture w/ formaldehyde/formaldehyde

Lipids (frozen section)

HIGHLY EXPLOSIVE when dry. Excessive YELLOW STAINING of tissues. NEVER WASH IN WATER before dehydration. For glycogen (excellent)

recommended for fixation of embryos and pituitary biopsies

Excellent fixative for glycogen and less messy then Bouin’s solution (excellent)

It is normally used in conjunction with other fixatives to form a compound solution Solidifies at 17°C. Fixes & precipitates nucleoproteins, chromosomes, & chromatin material

for fixing dry and wet smears (PBS and BM tissues)

simple fixative incorporated with compound fixatives for better results, preserves but does not fix glycogen (Disadvantage: polarization)

for fixing touch preparation

for fixing chromosomes, lymph glands and urgent biopsies (MOST RAPID;1-3 hours)

to preserve sputum

for fixing mucopolysaccharides and nuclear proteins. Give better reaction in Fuelgen stain than Carnoys

Pale yellow powder which dissolves in water (up to 6% at 20°C) to form a strong oxidizing solution Inhibits hematoxylin

most common chrome-osmium acetic acid fixative used (FIXATIVE & DECALCIFYING AGENT), permanently fixes fat, for nuclear structures (excellent)

(improve cytoplasmic details) – recommended for mitochondria

Precipitates proteins. Swelling effect counteract shrinkage by other fixatives. Weak decalcifying agent (softening effect)

Used at ice cold temperature from -5°C to 4°C. For diffusible enzymes such as phosphatases and lipases. For fixing BRAIN TISSUE (Rabies Diagnosis)

Thermal coagulation of tissue proteins. For bacteriologic smears. Bacteriologic smears Microwave: 45-55°C

process of placing an already fixed tissue in a second fixative

fixation whereby a primarily fixed tissue is placed in Aqueos solution of 2.5-3% potassium dichromate

Removing excess fixative