Evaluate & Troubleshoot

The evaluation of banding quality is a daily quality control (QC) task performed by the laboratory scientist before a case is released for analysis. Because every batch of slides (or even every patient) can react differently to the trypsin enzyme, the “test slide” method is standard practice. The laboratory scientist stains one slide, evaluates it under the microscope, and then adjusts the protocol (troubleshoots) for the remaining slides in the batch

Evaluation Criteria: What is a “Good” Band?

A properly G-banded slide must meet specific visual criteria to allow for accurate karyotyping. The evaluation is done using a brightfield microscope (100x oil immersion objective)

1. Contrast: There must be a sharp distinction between the dark (heterochromatin) bands and the light (euchromatin) bands. The bands should not look “muddy” (low contrast)
2. Definition (Edge Quality): The bands should have crisp, defined edges. They should run horizontally across the chromatid. Fuzzy or blurry edges indicate over-treatment
3. Completeness: The banding pattern should be visible along the entire length of the chromosome, including the telomeres. Often, telomeres are the first structures to degrade if over-trypsinized
4. Stain Intensity: The dark bands should be a rich magenta/purple, not faint pink or pitch black
5. Background: The background of the slide (the glass between cells) should be clear. A purple film or debris (precipitated stain) interferes with automated scanning systems

The Troubleshooting Matrix

When the test slide is suboptimal, the laboratory scientist must diagnose whether the issue is Under-treatment (too little digestion) or Over-treatment (too much digestion) and adjust the variables for the next slide

Scenario A: Under-Trypsinized (Solid Staining)

• Appearance: Chromosomes are uniformly dark purple. Faint bands may be visible, but they look like “shadows.” It looks like a solid stain
• Cause
  • Trypsin exposure was too short
  • Trypsin solution is old/inactive (enzymes degrade at room temp)
  • Slides were “baked” too long or at too high a temperature (chromatin became too hard)
• Corrective Action
  • Increase Trypsin Time: If the test slide was 10 seconds, try 20 seconds for the next slide
  • Increase Temperature: Trypsin works faster in warmer solution. Ensure the jar is at the correct temperature
  • Prepare Fresh Trypsin: If increasing time doesn’t work, the enzyme is likely dead

Scenario B: Over-Trypsinized (Ghosting/Fuzzy)

• Appearance: Chromosomes look “puffy,” “swollen,” or “melted.” The edges are ragged. The dark bands are pale or “chewed up.” In extreme cases, the chromosomes are pale pink outlines (“ghosts”) with no internal structure
• Cause
  • Trypsin exposure was too long
  • Trypsin concentration was too high
  • Slides were too fresh (not aged enough) or “wet”
• Corrective Action
  • Decrease Trypsin Time: If the test slide was 10 seconds, try 5 seconds. Or just “dip” the slide
  • Dilute Trypsin: Add saline to lower the enzyme concentration
  • Age the Slides: Bake the remaining slides for an additional 30–60 minutes to harden the chromatin before staining

Scenario C: Poor Staining (Color Issues)

• Appearance: Red/Pink Chromosomes (Faint)
  • Cause: pH too Acidic (<6.4). Alternatively, the Giemsa stain is too dilute or too old
  • Correction: Check the buffer pH. Remake fresh stain. Increase staining time
• Appearance: Blue/Black Chromosomes (Too Dark)
  • Cause: pH too Basic (>7.2). Or stain is too concentrated
  • Correction: Check buffer pH. Decrease staining time. Rinse slides more thoroughly with water
• Appearance: Precipitate (Black Specks)
  • Cause: A metallic sheen forms on top of the Giemsa stain jar (oxidized dye). If the slide is pulled through this scum, it sticks to the glass
  • Correction: Use filter paper to skim the surface of the stain jar before dipping slides. Filter the stain solution. Do not let slides dry with stain still on them (rinse thoroughly)

Scenario D: Refractility (Shiny/Hollow Chromosomes)

• Note: This is often a Harvest/Slide Dropping: issue, not a staining issue, but it is detected here
• Appearance: Chromosomes look like glass or have a glowing halo. They resist taking up the stain
• Correction: You cannot “fix” the slide with trypsin. You must go back to the fixed pellet, wash it with fresh fixative, and drop a new slide

Destaining & Restaining

• Can you save a slide?
  • Under-trypsinized slides CAN: be saved. The laboratory scientist can destain the slide (using methanol/fixative), rinse it, and then re-dip it in trypsin for a few more seconds
  • Over-trypsinized slides CANNOT: be saved. The protein structure has been digested away. The slide must be discarded and a new one dropped from the pellet