MIDTERM LEC 1

Question 1

● For routine HP techniques
● Gas produced by oxidation of methanol
● Usually buffered to pH 7 with phosphate buffer

Answer 1

Formaldehyde (formalin)

Question 2

● Formalin diluted with 10% NaCl
● Saturated formaldehyde + 10% NaCl
● Traditionally, the most common fixative
● Recommended for CNS tissue and general post-mortem
tissues for histochemical examination
● ADV: Ideal for Silver impregnation staining technique
● DADV: Slower; Tissue shrinks during alcohol dehydration
[Remedy: Secondary fixation]

Answer 2

10% formol saline

Question 3

● Formaldehyde + Na Dihydrogen Phosphate + Disodium hydrogen Phosphate
● pH7
● Best general tissue fixative
● Best for iron-containing pigments and elastic fibers which do
not stain well after Susa, Zenker or Chromate fixation,
● DADV: longer to prepare, inert to phospholipids and neutral
fats
● FT: 4- 24 hrs

Answer 3

10% Neutral Buffered Formalin (NBF) or Phosphate Buffered Formalin

Question 4

● Saturated aq. Mercuric chloride + 40% Formaldehyde
● Has HgCl2
● Recommended for routine post mortem tissues; Silver
Reticulum staining methods

Answer 4

FORMOL-SUBLIMATE/CORROSIVE
CHLORIDE)

Question 5

● Has 95% ETOH, Picric acid, and GHAc
● ADV: good for microincineration techniques, fixes sputum

Answer 5

GENDRE’S (ALCOHOLIC FORMALIN)

Question 6

● For gastrointestinal (GI) tissues, prostate biopsies, and bone marrow (BM)

Answer 6

HOLLANDE’S

Question 7

● Made up of 2 formaldehyde resides linked by three carbon chains
● For enzyme histochemistry and electron microscopy
● Container must be refrigerated

Answer 7

GLUTARALDEHYDE

Question 8

● For ultrathin and electron microscopy

Answer 8

Paraformaldehyde

Question 9

● For ultrathin and electron microscopy

Answer 9

Paraformaldehyde

Question 10

● Acrolein in glutaraldehyde or formalin
● Purpose: For Electron Histochemistry and Electron
Immunocytochemistry

Answer 10

KARNOVSKY’S PARAFORMALDEHYDE/GLUTARALDEHYDE

Question 11

● ADV: no smudging of nuclei and distortion of staining compared with formalin
● DADV: reduced staining capacity [Remedy: increase staining time]

Answer 11

40% AQUEOUS GLYOXAL

Question 12

● Most common metallic fixative
● Concentration: 5-7%
● Mostly incorporated in compound fixatives
● Routine fixative of choice for preservation of cell detail in tissue
photography
● Trichrome staining is excellent.
● ADV: penetrates and hardens tissue rapidly

Answer 12

MERCURIC CHLORIDE (HGCL2)

Question 13

➔ HgCl2 + potassium dichromate + glacial acetic acid
➔ Good general fixative for adequate preservation of
all kinds of tissues
➔ Good for Trichrome staining

Answer 13

Zenker’s

Question 14

➔ HgCl2 + potassium dichromate + strong formalin
(40%)
➔ For pituitary gland, BM, blood-containing organs, preserves cytoplasmic granules
➔ Brown pigments are removed with saturated alcoholic picric acid or NaOH

Answer 14

Zenker-formol/Helly’s

Question 15

➔ HgCl2 + NaCl + TCA + glacial acetic acid + formalin
➔ Skin tumor biopsy
➔ ADV: minimum cell shrinkage and tissue hardening
due to counterbalance effect of acids and mercury:

Answer 15

Heidenhain Susa

Question 16

➔ HgCl2 + Anhydrous Na acetate
➔ Recommended for hematopoietic and lymphoid tissues
➔ Bone Marrow biopsies

Answer 16

B5 Fixative

Question 17

● Recommended for making smears of loose cells on slides

Answer 17

Schaudinn’s fluid

Question 18

➔ Conc.: 1-2% aqueous solutions
➔ Preserves carbohydrates; precipitates all protein

Answer 18

Chromic acid

Question 19

➔ For chromatin, mitochondria, mitotic figures, golgi bodies, RBC and colloid-containing tissues
➔ DADV: prolonged fixation may lead to blackening of tissue pigment [Remedy: Wash in running tap water before dehydration]

Answer 19

Regaud’s/Muller’s

Question 20

➔ Study of early degenerative processes and necrosis,
demonstration of Rickettsia and other bacteria

Answer 20

Orth’s

Question 21

➔ E: preserves lipids, mitochondria, at pH 4.5-5.2,
cytoplasm, chromatin and chromosome are fixed
➔ Corrosive, thus avoid skin contact

Answer 21

3% Potassium dichromate

Question 22

● For acid mucopolysaccharides and mucin
● DADV: Prolonged standing → formation of insoluble lead
carbonate [Remedy: add drops of acetic acid to dissolve residue]

Answer 22

4% AQUEOUS LEAD/LEAD FIXATIVE

Question 23

● Normally used in strong saturated aqueous solution (1%)
● For Glycogen demonstration
● ADV: may be used as a stain as yellowing of tissue will prevent
small fragments from being overlooked; suitable also with
Aniline stains

Answer 23

Picric acid

Question 24

● For embryo and pituitary biopsies, and tissues to be stained with Masson’s Trichrome
● ADV: minimum cell shrinkage and tissue hardening due to counterbalance effect of glacial acetic acid (swelling) and picric acid (shrinking)
● DADV: poorly penetrates large tissue, thus limited to small fragments of tissues

Answer 24

Bouin’s

Question 25

● HCHO + picric acid + alcohol + TCA
● ADV: Better and less messy than Bouin’s

Answer 25

BRASIL’S ALCOHOLIC PICROFORMOL

Question 26

● Incorporated in compound fixatives
● Solidifies at 17°C
● Important for nuclear fixatives (precipitates nucleoproteins,
chromatins)
● Destroys mitochondria and Golgi elements, thus not for
cytoplasmic fixation

Answer 26

GLACIAL ACETIC ACID

Question 27

● ADV: good for glycogen
● DADV: never for FATS and LIPOPROTEINS (dissolves); causes polarization of glycogen (granules will move towards the poles or ends of the cells)
● Effect: rapidly denatures and precips CHONs, preserves nuclear stains

Answer 27

ALCOHOL FIXATIVES

Question 28

● Most rapid tissue fixative (1-3 hrs)
● Fixing brain tissues for rabies diagnosis
● Fixes Nissl granules (Tigroid substance) and cytoplasmic
granules

Answer 28

CARNOY’S FIXATIVE

Question 29

● Enzyme studies
● Does not fix but preserves glycogen

Answer 29

ETHANOL (70-100%)

Question 30

● Dry and wet smears, BM smears, bacterial smears

Answer 30

METHANOL/WOOD ALCOHOL (100%)

Question 31

● Touch prep smears to be Wright-stained

Answer 31

ISOPROPYL ALCOHOL/RUBBING ALCOHOL (95%)

Question 32

● Mucopolysaccharides and nuclear CHONs (12-18 hrs)
● Better reaction in Feulgen stain than Carnoy’s

Answer 32

NEWCOMER’S

Question 33

● For sputum (4-18 hrs)

Answer 33

GENDRE’S (ALCOHOLIC FORMALIN)

Question 34

● Pale yellow powder in water (6% in 20°C)
● Ultrathin sections in Electron Microscopy
● E: Fixes and stains conjugated fats and lipids black
● DADV: very expensive, very volatile, inhibits hematoxylin
● Tissue-to-fixative ratio: 1:5

Answer 34

OSMIUM TETROXIDE / OSMIC ACID

Question 35

● Most common osmic acid fixative
● Nuclear fixative (24-48 hrs)
● Effect: permanently fixes fat
● ADV: needs less amount of fixative

Answer 35

FLEMMING’S SOL’N W/ GAC

Question 36

● Cytoplasmic fixative (24-48 hrs)

Answer 36

FLEMMING’S SOL’N W/O GAC

Question 37

● Incorporated into compound fixatives
● Marked swelling effect on tissues
● Poor penetrating agent thus for small pieces of tissues or
bones
● Weak decalcifying agent, thus has softening effect on dense
fibrous tissues

Answer 37

TRICHLOROACETIC FIXATIVES

Question 38

● Use at ice cold temperature (-5 to 4 degree celsius)
● For water-diffusible enzymes (Phosphatase, Lipase)
● For brain tissues (such as in Rabies)
● DADV: Dissolves fat, evaporates rapidly

Answer 38

Acetone

Question 39

● Principle: Involves thermal coagulation of tissue proteins
● For rapid diagnosis: Frozen tissue sections and Bacterial
smear prep
● 10-15 mm thickness of tissue

Answer 39

ACETONE/HEAT FIXATION