Lab Quiz #4: Staining

what is a fixative?

a “permanent preservative” —> allows for best possible preservation of cell components

preservatives prevent …

• autolysis

• evaporation

• swelling and shrinking

• mold and bacteria growth

fixative act on what part of the cell?

protein side chains

• heat —> acts on entire protein structure

• chemical —> acts on selective protein side chains

what are the 4 characteristics of fixatives?

• coagulant

• non-coagulant

• additive

• non-additive

what does coagulant do?

removes water from proteins —> causes shrinkage and rounding

what does a non-coagulant do?

leaves water behind —> causes cellular swelling

is ethanol a coagulant/non coagulant? Additive/non additive?

coagulant and non additive (doesn’t leave any ethanol and water behind)

is formalin a coagulant/non coagulant? Additive/non additive?

non coagulant and additive. (leaves a portion of fixative behind and water is also left behind)

what does an additive do?

leaves a portion of fixative attached to the protein

what does a non-additive do?

does not leave any fixative attached to the protein (fixative is washed away)

what is the fixative used for histology slides?

formalin —> used for tissue and cell blocks

how is formalin made? is it a carcinogen?

• methanol is heated and vaporized —> produced formalin

• gas is added to water to make aqueous formaldehyde (40%)

• one part of aqueous formaldehyde is mixed with 9 parts water —> 10% formalin

• buffer is added

• NOT a carcinogen (even though it contains formaldehyde)

what is the fixative used in cytology?

95% ethanol —> immersion fixation

polyethylene glycol mixed w/ 95% ethanol —> spray fixation (mainly used for FNA procedures)

what was the original “pap” fixative? why was it problematic?

95% ethanol and ether 1:1 —> faster than just 95% ethanol

problematic —> ether explodes 😐

spray fixatives

• acts as a pre-fixative

• protects cells during transport

• slides must be placed in 95% ethanol after procedure to remove waxy film from slide

  • ~ 10 min.

• hold pump 5-7 in. from slide

what can be used as a substitute for carbowax?

cheap hairspray (Aqua Net) —> has polyethylene glycol

why do we have prefixatives?

prevents degeneration, especially if specimen was mailed in or brought over the weekend (during off hours)

types of prefixatives?

• 50% ethanol

• Saccomanno fixative —> 2% polyethylene glycol in 50% ethanol

• mucolexx —> 3% polyethylene glycol, buffers, formaldehyde

final fixation?

95% ethanol for 10 minutes before staining

necessary for specimens that have been —> spray fixed, pre-fixed, treated with coating fixatives

is air drying a type of fixative?

yep :]
can be intentional for rapid stains or unintentional (which causes staining artifacts)

type of rapid stain we use for air drying prep?

Romanowsky style rapid stain —> done during FNA procedures

*air drying is also used for Oil Red O (meant for lipids —> discussed in the staining lecture)

pros and cons to air drying

pros

• allows for rapid staining

• ideal for observing cytoplasm

• used for adequecy assessments and rapid interpretations —> FNAs

cons

• causes nuclear swelling and distortion —> loss of nuclear detail

• loss of cytoplasmic density

• loss of “counter stainability”

• so air drying effect :)

what is done during an FNA procedure?

• each pass produces 2 slides —> one air dried and one wet fixed

• needle rinse in saline or formalin

  • used to make a cell block

  • all needle rinses from the same site (multiple passes) can be combined

spray fixation: what appearance does it create when sprayed onto slide?

produces a “beaded” effect on the slide

at IUHPL, both slides are wet or air dried?

• both are air dried

  • allows for easy and safe transport

  • prevents the fixation artifact that is produced by spray

• one is used for adequacy assessment, other for pap staining

• second one is rehydrated using saline and then final fixed in 95% ethanol before pap stain

types of effusions

• exudate

• transudate

• chylous

from what kind of tree did we get our stains from?

the logwood tree (native to Central America) —> where we get Hematoxylin from

what did Canada and the U.S. form regarding staining?

Biological Stain Commission

the Biological Stain Commission established what?

the color index numbers —> represents standardization of a color of a stain (should be the same color no matter from where the stain is obtained)

what are the 2 bases used for staining?

• water

• alcohol

how do dyes impart color?

• dye: coloring agent —> leaves part of itself in the solution and is then picked up/phagocytosed by cells

• wavelength: determines what color the cell is

• pH: determines which competing dye wins out

• color bearer

• ions

what produces color?

chromophore (AKA color bearer)

what allows chromophore to act as a dye?

ions —> enables the chromophore to bind to target structures in cells, allowing for color retention

ions + chromophore = ?

auxochrome

proteins with a positive charge are …

acidophilic/eosinophilic —> acidic environment and bond to negative charges

proteins with a negative charge are …

basophilic —> basic environment and bond to positive charges

altering the pH can do what regarding staining?

can select for acidophilic or basophilic, allowing for specific selection of which structures are stained

true or false: a chromophore can carry both + and - charges

true :) —> called amphoteric since it can act as an acid or base depending on the pH of the solvent

what are 2 examples of amphoterics?

• light green

• hematoin

what is the primary site for dye attachment?

protein side chains —> allows for selective dyeing

hematoxilin stains what in a cell?

stains nucleus (more specifically DNA)

hematoxylin process

• begins as colorless

• oxidation produces haematin (weak acid dye with a net charge of -1) —> looks pale yellow

• mordant is added —> produces a strong basic dye —> looks red

• bluing agent is added —> looks purple

what are mordants?

salts of various metals —> increases the bond strength of the dyes

dye + mordant = ?

dye lake

strong basic dyes (like hematoxilin) dye what and what charge are they attracted to?

• dye nucleus —> DNA

• attracted to negatively charged anionic structures in the cell

what is the mordant in Gill’s hematoxylin?

ammonium aluminum sulfate

what is ammonium aluminum sulfate?

• salt of aluminum that ionizes water to form hydrated aluminum ions

• process doesn’t require heat

what are the components of Gill’s Hematoxylin?

• water —→ base

• ehylene glycol —→ increases solubility

• sodium iodate —→ oxidizer

• hematoxylin —→ raw dye molecule

• ammonium aluminum sulfate —→ mordant

• glacial acetic acid —→ lowers pH

Gill’s hematoxylin is progressive or regressive?

progressive staining → stain gradually, stains it to where it needs to be stained, and then stop staining it

fill in the blanks: the pap stain is a _____ _____ that has four dyes.

polychrome stain

what are the four dyes in the pap stain?

• hematoxylin —→ nucleus stain

• eosin Y —→ cytoplasmic stain

• light green —→ cytoplasmic stain

• orange G —→ cytoplasmic stain

counterstains in the pap stain

• eosin Y

• light green

• orange G

• referred to as counterstains —→ cytoplasm

• alcohol based

• these dyes compete with one another and the resulting color/intensity is determined by pH of stain and total dye concentration

fill in the blank: cytoplasm is ____, meaning it may react to both acidic and basic dyes

amphoteric

what is the function of orange G?

stains keratin

• orange yellow color

• anionic molecule —→ penetrates dense structures quickly

what is the function of eosin Y?

stains cytoplasm (metabolically inactive cell)

• pink or red (depending on structure)

• small molecule → penetrates quickly

• acidic dye

• “E” in H&E stain

eosin Y: cytoplasm staining colors on different structures

• cytoplasm of superficial cells → pink

• RBCs → red

• nucleoli → red

• granules of eosinophils → red

• cilia → pink

what is the function of light green SF?

stains cytoplasm (metabolically active cells)

• blue/green color

• acidic dye

• large molecule -→ longer time to penetrate

what is eosin azure?

combo of eosin, light green SF, and fast green into one reagent

differences between EA36, EA50, EA65?

• EA36

  • used for gyn specimens

  • most traditional formula → original one made by Papinicolaou

  • good cytoplasmic differentiation

• EA50

  • used for gyn specimens

  • commonly commercially prepared

  • made with METHANOL

• EA65

  • non gyn specimens

  • less light green (about half)

  • works better for thicker specimens

what is phosphotungstic acid used for?

• added to EA

• allows for differential staining of the cytoplasm in cells of different metabolic activity levels → changes the shade of green in EA

what is bismarck brown?

• basic dye used in original pap stain

• not used anymore :(

what should you do with spray fixed slides before staining?

soak in 95% ethanol for 10 min. before staining (removes waxy film)

what is the sequence of steps for the pap stain after fixation? (in full detail)

• hydration → 2 water baths, 10 dips each (prepares for nuclear staining)

• nuclear stain → hematoxylin for 1-2 min. (longer time for thicker ones)

• 2 water baths → 10 dips each (rinses excess off)

• bluing agent → Scott’s tap water for 1 min. (turns hema into purple)

• 2 water baths → 10 dips each (removes excess bluing agent)

• 2 95% ethanol baths → 10 dips each (prepares for counterstains)

• OG counterstain → 30 sec. (stains keratin)

• 2 95% ethanol baths → 10 dips each (removes excess OG)

• EA counterstain → 5-15 min. (time depends on thickness of specimen)

• 3 95% ethanol baths → 10 dips each (removes excess EA)

• 3 100% ethanol baths → 10-20 dips each (final dehydration)

• 1:1 solution of 100% ethanol and xylene → 10 dips (can be optional)

• xylene → 10 min. minimum (clearing agent)

what is the general process for staining? (the simpler version)

final fixation of specimen → nuclear staining → counterstaining/cytoplasmic staining → dehydration → clearing

how does poor leveling affect staining?

light green in EA can’t penetrate through the thick areas of the specimen

results in uneven staining → blue edges and pink middle

what are some solutions to poor leveling affecting staining?

• leave it in EA longer

• try to make specimen thinner

what should you NEVER do with xylene?

pour it down the sink → WILL CONTAMINATE THE WATER SUPPLY :]

routine staining maintenance

• replace water baths after each staining session

• replace scott’s tap water daily or when visibly contaminated

• filter all stains and alcohols daily or when visibly contaminated (replace weekly or when staining a known malignant case)

• replace xylene if cloudy or contaminated

• filter xylene after slides have been coverslipped

• maintain adequate fluid levels → must cover all specimen but not labels)

• store all stains and reagents in covered/closed containers

  • prevents evaporation, contamination from airborne particles, and condensation

Rapid staining in cytology?

• romanowsky stain

• giemsa stain or wright giemsa stain

• diff quik stain → common one in cytology

• hema 3 → similar to diff quik (used at IUH)

Rapid stain process?

• methanol: 10 dips (NOT a fixative)

• solution 1: ~10 dips or until methanol stops beading on slide

• solution 2: 30 sec. - 1min. depending on how thick specimen is

• DI water: ~10 dips or until excess solution 2 has washed off

why do we use Romanowsky stains?

helps us determine cellularity of the specimen and cytoplasm

size of standard slides used in cytology

3 × 1 in.

standard thickness of a slide used in cytology

1mm → the thicker the glass, the greater the light divergence altering the focal length

types of slides

• plain glass → good for slide repairs

• plain glass with frosted end → good for pull smears

• full frosted slides → good for mucoid specimens

  • NEVER use with CSF specimens or suspected lymphoma/leukemia

• charged slides → coated, slight ionic charge

• propriety slides → thinprep and surepath

  • thinprep have marked areas for cell spot

  • surepath have a slight charge

what writing materials can you use to write on slide?

• pencil

• special pens → alcohol and xylene resistant

how do you transport slides?

use a slide tray or slide holders

true or false: when slides are received broken into the lab, it must be documented

true :)

what should you do if a slide is broken?

• large pieces → mount broken pieces onto a blank slide using mounting media

• small pieces → discard slide

what is the recommended thickness for a cover glass used in cytology?

• .13-.17 mm

• AKA “No.1” coverglass

what kind of coverslip does IUHPL use?

automatic coverslipper → plastic tape that bonds to xylene

is mounting media hydrophobic or hydrophilic?

both :] → pap stain requires hydrophobic

what is cornflaking?

when not enough mounting media is used, creating air pockets under the coverslip causing a “brown” artifact

types of mounting media

• krystalon

• histoclad

• permount → the one we use

what should you do when you’re done coverslipping but before placing in slide tray?

• CLEAN OFF EXCESS XYLENE → can stick to slide tray

• make sure labels/case numbers are elegible

what are different methods to remove a coverslip?

• xylene → soak for hours or days depending on how old the slide is

• heat → melts mounting media using a hot plat (careful not to overheat slide since it could also cook the cells)

• freezing → usually comes off in parts (careful with the shards)

• acetone → removes coverslipping tape (usually in seconds)

• blade → pops off coverslip if it is loose and coming off (DON’T DO THIS ONE → dangerous)

what is refractive index?

the amount of light refracted off various components

the refractive index should match all components …

• slide

• coverglass

• mounting media

• stained cell sample

true or false: matching the refractive index of all components maximizes the image quality under the scope

true

what is the refractive index for cytology?

1.52