MLHG 106 - Introduction to Histology and Cytology: Lesson 3 Q&A Flashcards (Staining, cover-slipping, slide labelling and storage)

100 practice flashcards covering H&E staining, staining concepts, reagents, processes, Romanowsky stains, coverslipping, labeling, filing, microwaves, QC, and safety from the provided histology notes.

What does H&E stand for in histology staining?

Hematoxylin and Eosin stain.

From which natural source is hematoxylin derived?

The logwood tree (Hematoxylin campechianum).

Can H&E staining be performed manually, with automated stainers, or both?

Both manual and automated staining.

Name the two staining approaches described for H&E.

Progressive staining and regressive staining.

In progressive H&E staining, when is staining considered complete?

When sufficient dye uptake is achieved (desired intensity).

What is done in regressive H&E staining after overstaining?

Excess dye is removed by decolorization with acid-alcohol.

What constitutes the acid-alcohol used for differentiation in H&E?

A 1% acid-alcohol solution, typically 1 ml HCl in 99 ml 80% ethanol.

What is the purpose of the blueing step in H&E staining?

To blue the nuclei by an alkaline solution (pH ~8.0–8.5).

Name two commonly used blueing methods in H&E.

Scott’s tap water substitute and lithium carbonate (others include ammonia water or tap water).

In H&E, what color does hematoxylin stain nuclei?

Blue to purple.

In H&E, what color does eosin stain cytoplasm?

Pink.

List three tissue components that stain pink in H&E.

Red blood cells, collagen, and muscle.

What is a mordant in hematoxylin staining?

A substance (e.g., potassium alum) that helps fix hematoxylin to tissue.

Which mordants are commonly used with hematoxylin?

Potassium alum, ammonium alum (Alum Hx), or ferric alum (Iron Hx).

What is the process called when hematoxylin is oxidized to hematein?

Ripening.

What is the purpose of ripening in hematoxylin staining?

To oxidize hematoxylin to hematein so it acts as the dye.

Describe the basic composition of an alum hematoxylin preparation.

Potassium alum in water; hematoxylin dissolved in absolute alcohol; mercuric oxide or sodium iodate as oxidizer; heated to boil; cooled; filtered; stain fresh every 2–3 months.

How often should hematoxylin be filtered and how often should fresh stain be prepared?

Filter daily; prepare fresh stain every 2–3 months.

Why is hematoxylin quality monitored during staining?

Because its performance changes over time due to carryover and oxidation, causing day-to-day staining variability.

What is the target pH for eosin staining in H&E?

Approximately pH 5.0.

Why is eosin pH monitoring important?

To maintain optimal staining; alkaline carryover can raise pH and weaken staining.

In the regressive staining protocol, what are the steps after deparaffinization and dehydration?

Hydration (95% then 70% then tap water), staining with hematoxylin, differentiation, blueing, counterstaining with eosin, dehydration, clearing, cover slipping.

What is the purpose of dehydration before clearing in H&E?

To remove water before clearing with xylene.

What is the role of the acid-alcohol in H&E?

Differentiation: to remove excess hematoxylin until the desired intensity is reached.

What is the typical composition of an acid-alcohol differentiation solution.

1% acid alcohol: 1 ml concentrated HCl in 99 ml 80% ethanol.

What is the purpose of the blueing step in H&E staining?

To convert the nuclei color to blue by neutralizing the residual acid (alkaline conditions).

Name one widely used blueing agent for H&E.

Scott's tap water substitute.

What is the function of eosin in H&E staining?

Eosin stains the cytoplasm and acidophilic components pink.

What step follows eosin staining in the regressive H&E procedure?

Dehydration, clearing, and coverslipping.

What is the purpose of a positive control slide in staining practice?

To confirm that the stain can reveal the expected structure/substance.

Why is standardized washing important in staining protocols?

To reduce variability and prevent staining inconsistencies.

What is the consequence of not filtering hematoxylin daily?

Deterioration in stain quality and inconsistent results.

What is the difference between aqueous and resin mounting media?

Aqueous mounts are not usually permanent; resinous mounts (Permount, Canada balsam) are more permanent and have higher refractive indices.

What is the typical refractive index range for tissue and for synthetic mounting media?

Tissue: ~1.3–1.5; mounting media: ~1.51–1.55.

Why should mounting media be free of moisture or water when mounting?

Moisture can cause cloudiness or crystal formation and compromise clarity.

What is the commonly recommended thickness for typical coverslips in labs?

Approximately 1.5 (about 180 microns); #1 coverslips are ~150 microns.

What common artifact is caused by air bubbles under a coverslip?

Obscured microscopic viewing and distorted tissue morphology.

Where should the slide label be placed relative to the coverslip?

The label should not overlap the coverslip; it should be on the label on the slide.

What is a key requirement for labeling slides?

Paper label must match the slide labeling; avoid overlapping with the coverslip.

How long are histology slides and blocks typically stored according to IQMH standards?

A minimum of 20 years.

What is the purpose of a tissue block filing system?

To organize and store tissue blocks for long-term retention and retrieval.

What is the role of a microwave in histology processing?

To speed processing and staining; non-metallic stains around 55–60°C; metallic stains 75–95°C.

What is a major safety concern when microwaving tissue in formalin?

Formalin vapors are hazardous and can be dangerous.

What is recommended instead of consumer-grade microwaves for histology work?

A laboratory-grade microwave with proper venting and containment.

What is the basic sequence of routine tissue processing?

Fixation → dehydration → clearing → infiltration with paraffin wax → embedding.

What safety practices are advised when handling histology samples?

Wear gloves and lab coats; treat all samples as infectious; follow PPE guidelines.

Which fixative is commonly used for surgical pathology specimens and is included in Zenker’s fluid discussions?

Formalin or Zenker’s fluid (depending on protocol).

What is the purpose of a coverslipper in histology labs?

To automate the application of coverslips onto mounted slides, protecting staff from fumes.

What is the typical function of a micrometer/L piece in coverslipping?

Ensure correct cover-slip size and avoid air bubbles.

What is the effect of using unsuitable mounting media in long-term storage?

Crystallization and potential lifting of the coverslip.

What is a reticulin stain used for and which method is mentioned?

Demonstration of reticulin fibers; Gordon & Sweets silver impregnation method.

Which Romanowsky stain is a two-step process using May-Grünwald first, then another stain?

May-Grünwald-Giemsa stain.

Which stain combination is known to brighten cytoplasmic granules in white blood cells?

Wright stain with added Giemsa (Wright-Giemsa) cements red-purple cytoplasmic granules.

What are the typical colors seen in a well-stained Romanowsky smear?

Nuclei blue/purple; cytoplasm blue to pink; granules purple to pink depending on cell type.

What is metachromasia in Romanowsky staining?

The Romanowsky effect; hues produced beyond the original dye colors, enabling differentiation.

Which stains are common examples of Romanowsky-type stains?

Giemsa, Wright, May-Grünwald, Leishman.

What is the role of azure dyes in Romanowsky staining?

Azure dyes (oxidized methylene blue) stain nuclei blue; they are basic dyes.

In Leishman staining, what fixes the preparation?

Methanol.

What is a key step in Leishman staining protocol after applying the stain?

Add distilled water in double volume and rest for 10–12 minutes, then rinse.

What is the difference between Wright and Wright-Giemsa stains?

Adding Giemsa to Wright stain brightens cytoplasmic granules and enhances color.

What is the recommended use of control slides in hematology staining?

To validate that staining yields the expected results before analyzing patient samples.

What equipment maintenance is advised for automated H&E stainers?

Annual preventative maintenance and routine filter and reagent changes.

What is the effect of not cleaning reagents regularly in automated stainers?

Contaminated reagents can lead to inconsistent staining.

Why is it important to filter hematoxylin daily?

To remove precipitates and contaminants that affect staining quality.

What is a common cause of patchy staining in H&E?

Incomplete dewaxing or incomplete dewax removal.

Which step ensures nuclei appear blue after hematoxylin staining?

Bluing (alkaline) step.

What could happen if you skip the differentiation step in H&E?

Excess hematoxylin may obscure detail or cause overly dark nuclei.

What is the primary purpose of cross-contamination risk awareness in histology?

To prevent sample-to-sample contamination during processing and staining.

What is the purpose of labeling slides with accurate matching information?

To ensure correct diagnosis and traceability.

What is the recommended practice for protecting slides during handling?

Avoid drying; mount slides promptly and handle with care to prevent damage.

What are typical temperatures for non-metallic and metallic stains when microwaving?

Non-metallic: 55–60°C; Metallic: 75–95°C.

Why should a fume hood be used for cover slipping?

To contain volatile solvents, vapors, and fumes (xylene) during mounting.

What is the purpose of the stain control in H&E staining?

To ensure the staining is performing as expected and producing interpretable slides.

What is the significance of using a positive control in staining?

To confirm the staining protocol demonstrates the target structure or substance.

What is a key criterion for modern mounting media in histology?

Appropriate refractive index to match tissue optics and long-term stability.

What is the role of a tissue cassette in processing?

To hold tissue samples during embedding, dehydration, clearing, and infiltration.

What is the typical purpose of a water bath in mounting procedures?

To facilitate floating sections onto slides for even adhesion.

Why is zenker’s fluid mentioned in histology notes?

As a fixative used in some histology protocols.

What does 'infiltration' refer to in paraffin processing?

The process of infiltrating tissue with embedding medium (paraffin wax).

What does 'dehydration' refer to in tissue processing?

Removal of water from tissue using ascending alcohol concentrations.

What is the role of 'clearing' in tissue processing?

Replacement of alcohol with a clearing agent (e.g., xylene) to render tissue transparent.

What is the purpose of 'embedding' in histology?

To enclose the tissue in a solid medium (paraffin) for sectioning.

What is the typical fixative used for routine histology specimens?

Formalin (formal-dehyde solution).

What is the difference between formaldehyde and formalin?

Formaldehyde is the chemical; formalin is a 37% solution of formaldehyde used as fixative.

What is 'grossing' in pathology?

The examination and sampling of surgical specimens to select areas for analysis.

What is the purpose of a greyish 'floaters' removal in the water bath?

To remove surface contaminants that can cause artifacts in staining.

What is covered by the term 'optical differentiation' in histology terms?

Differentiation of staining to optimize contrast and visualization of structures.

What are the two primary components of the Romanowsky stains?

Oxidized methylene blue (azure) and eosin Y.

Which color does eosin stain connective tissue in some stains?

Pink to red; in Romanowsky, connective tissue may stain pink to purple depending on the cell type.

What is a key variability factor that affects hematoxylin staining across days?

Oxidation level and carryover from previous runs.

What is 'Scott’s tap water substitute' used for in histology?

A blueing solution option for nuclei in H&E staining.

What is 'Mayers' vs 'Harris' in alum hematoxylin context?

Two common reagents for alum hematoxylin formulations used for staining.

What is a characteristic advantage of automated staining machines?

Consistency of results and time efficiency.

What is a potential disadvantage of automated stainers?

Not easily adaptable to specialized manual techniques or frozen sections.

What is the recommended frequency for updating reagents in histology labs?

Replace reagents as per protocol and when stain quality declines.

Where are mounting media often absorbed?

Xylene or other organic solvents depending on the mounting medium.

What is a common hazard with xylene that necessitates safety precautions?

Volatile organic solvent fumes; use in a fume hood and with protective gear.

What is the typical purpose of 'dewaxing' before staining?

Remove paraffin so the stain can penetrate the tissue.

What is 'graft color' under H&E typically?

Nuclei blue/purple; cytoplasm pink; collagen and other tissue components pink to red.

What does the term 'Zenker’s fluid' refer to?

A fixative used in histology (one of the fixatives sometimes listed in notes).