Module 2: General staining terms and procedures

Question 1

Progressive staining

Answer 1

staining of different tissue elements in sequence, at a set time. Ie; toluidine blue, counterstains

Question 2

Regressive staining

Answer 2

tissue is first overstained. Excess stain is removed until correct tissue structures are stained – the
other components are lightly colored or have all the stain removed. Ie; hematoxylin in H&E and crystal violent in Gram

Question 3

Direct staining

Answer 3

dye interacts directly with tissue and salt linkages are formed. Ie; eosin staining acidophilic tissue

Question 4

Indirect staining

Answer 4

require additional intermediate substance before color results – involves mordant which has 2 reactive
groups – one which combines with dye to form the lake and the second attaches to the tissue. Ie; nuclear staining
with aluminum or iron hematoxylin

Question 5

Mordants

Answer 5

Mordant and dye may be combined in one solution or applied as two separate solutions in two
separate steps. Iron, aluminum and chromium

Question 6

Accentuators

Answer 6

enhance staining or increase selectivity – not essential for chemical union of dye with tissue (don’t act as
mordants). May act as chemical catalysts by changing pH of staining solution (affects ionization of reactive
groups in tissue). Ie; acetic acid in eosin stains increases number of positively charged groups

Question 7

Accelerators

Answer 7

act the same as accentuators by enhancing staining. Used in metallic impregnation for nervous system

Question 8

Trapping agents

Answer 8

hold a dye in combo with tissue or bacteria. Ie; iodine in Gram’s traps crystal violet in bacteria

Question 9

Differentiation

Answer 9

gradual removal of unwanted excess dye/stain requiring microscopic control

Question 10

Simple solvents (differentiation)

Answer 10

excess dye by simple solubility. Water or alcohol which the dye is dissolved in may
be used. Ie; remove excess eosin by washing in its solvent after staining. Acceptable end product shows variation in staining intensity in acidophilic components

Question 11

Acid differentiation (differentiation)

Answer 11

dyes combining with tissue by firm chemical unions can’t easily be differentiated with
simple solutions but dye-tissue linkages can be broken with acids. Usually used for basic or mordanted dyes – breaks
tissue/mordant bond due to excess hydrogen ions provided

Question 12

Action of other dyes (differentiation)

Answer 12

dyes may be used as differentiators in some trichrome stains for collagen. After initial
staining with primary dye, secondary dye is used which has a stronger affinity for already stained tissue so secondary
displaces some of the primary stain

Question 13

Action of bases (differentiation)

Answer 13

alkaline solutions not used frequently – dilute ammonium hydroxide can be used to remove all
the eosin from the tissue if staining is too intense. Would be followed by washing before restaining. Ie; Luxol fast blue for
myelin – alcohol and lithium carbonate are used as differentiators

Question 14

Action of oxidizers (differentiation)

Answer 14

dye is oxidized to colorless substance – tissues with most dye will remain colored if
oxidization time is controlled carefully. Ie; potassium permanganate

Question 15

Counterstaining

Answer 15

should be of contrasting color, relatively pale and not remove primary stain. Not necessary, but usually
done

Question 16

General staining procedure

Answer 16

Hydration of Paraffin Tissue Sections

1. Remove paraffin wax
2. Remove xylol
3. Water wash to remove alcohol
4. Remove fixation precipitate
5. Postfixation if necessary

Dehydration, Clearing & Mounting

Question 17

Mounting media

Answer 17

• RI should be near to glass (1.518)
• protects tissue from physical harm - protects from oxidization
• nonreactive with stain formed - won’t change in pH or color
• set with no tissue distortion or shrinkage - harden without granularity or cracking
• won’t leach out stain or cause stain loss over time - should be colorless, flow easily and release air bubbles

Question 18

Resinous mounting media

Answer 18

• RI slightly greater than glass - synthetic or natural (Canada Balsam) -in own solvent or toluene or xylene
• should be liquid enough to allow easy mounting with rapid drying and hardening
• should be liquid enough to flow freely between slide and coverslip and allow escape of air bubbles
• should be viscous enough to avoid formation of air spaces under coverslip during drying

Question 19

Aqueous mounting media

Answer 19

• lower RI than resinous (1.40-1.42) - usually contain hardening agent
• glycerine is added to prevent cracking once dried - sugars may raise RI
• thymol, phenol or sodium merthiolate prevents mold growth
• may require ringing media if don’t set hard which will prevent seepage/evaporation, immobilize coverslip and prevent
  sticking of slides when stored

Question 20

Factors affecting tissue staining

Answer 20

• fixative choice determines tissue groups left to bind with stain. Ie; formalin binds amino groups (leaves carboxyl)
  Increase basophilia: formalin, mercuric chloride, osmium tetroxide
  Increase acidophlia: picric acid, potassium dichromate
• dissolved particles/ions (usually from salts) compete with dye for binding sites may decrease staining
• pH - temperature (increases rate of staining)
• increase concentration - dye lot
• variations in stain procedures