Counting rules, volumes and calculations follow Analytical Methods E-Module 2 — Yeast Biomass Quantification, the Yeast Biomass Quantification Methods Report and the Cell Counter Calculator - Basic.
1. Quick start
- Prepare and load the sample as in E-Module 2: dilute by weight, mix 1:1 with the stain (DF = DF₀ × 2), load both chambers of the Neubauer Improved slide, start counting 2–5 min after stain contact and finish within 10 min.
- Enter the sample: Batch #, Date, Sample, the Dilution factor (including the 1:1 stain) and the Magnification (counting at 40×, 400× total).
- Choose the method: Neubauer Improved — Short (5 Medium Squares), Long (25 Medium Squares) or Custom; Single Use — Grid (5 Grid Squares) or Small Sector (5 Small Sectors). The grid detector reads only the Neubauer Improved ruling, so count Single Use slides with the Cell Counter Calculator - Basic.
- Chamber 1 — load the images: click Choose image… or + Add FOV and select one image or several at once; each becomes a FOV. With After loading: detect grid and anchor ticked (default), the grid of each image is detected as it loads, and with the Short method each image is anchored at the next position. With … and detect cells ticked (default), cell proposals are made for each image. Otherwise use Detect grid and Detect cells on each FOV. With Long, Custom and the Single Use methods, anchor each FOV by clicking the chamber-map square of its top-left Medium Square.
- Count, FOV by FOV: left click = current mode (L/B/D), middle click = budding cell, right click = dead cell; review the proposals and confirm them. Continue until every position of the chamber is covered and counted.
- Chamber 2: set Chamber to Chamber 2 in the Session panel and count it the same way.
- Check and report: the Session totals show both chambers, their agreement (≤ 15 % passes) and the mean. A result is shown only when both chambers are complete and agree; report the mean to two significant figures. If the chambers disagree, repeat the dilution and loading. Export CSV.
2. Counting rules
- Boundary rule: count cells inside the square and cells touching its top and left lines; exclude cells touching its bottom and right lines. A click belongs to the square that contains the point clicked.
- Live cells are unstained; dead cells are uniformly dark blue (methylene blue) or violet (methylene violet).
- Budding cell: a mother with a bud larger than half its diameter (ASBC Yeast-4). A budding mark records the mother as a budding cell and the bud as a live cell (two live cells). A cell with a smaller bud is one live cell: mark it as Live.
- Automatic proposals: B·2 (bud > ½) is confirmed as a budding cell; B·1 (smaller bud) as one live cell; a clump (~n) counts as one cell — mark its cells by hand. The detector does not see the stain: confirm dead cells with a right click. Cells on ruled lines are usually not proposed; add them by hand. Ctrl + click rejects a proposal or removes a mark; any click on a mark removes it.
- Chamber map: green = selected square, orange outline = square in the active image, numbered circles = required positions (✓ when covered). A double click zooms to a square; a double right-click fits the image.
3. Keyboard and mouse
| Key / gesture | Action |
|---|
| L / B / D | Left-button mode: Live / Budding / Dead |
| U or Ctrl+Z | Undo the last mark |
| F | Fit the whole image |
| Esc | Close a window |
| Mouse wheel / Shift+drag | Zoom / pan |
| Left / middle / right click | Live (current mode) / budding cell / dead cell; on a proposal, confirm it |
| Any click on a mark; Ctrl+click | Remove the mark; reject a proposal |
4. Calculations (E-Module 2)
C (cells/mL) = N ÷ (n × V_sq) × DF, V_sq in mL
| Method | Squares × volume | Counted volume | Multiplier | Target per square |
|---|
| Long | 25 × 0.004 µL | 0.1 µL | 10,000 | 25–60 |
| Short | 5 × 0.004 µL | 0.02 µL | 50,000 | 25–60 |
| Grid | 5 × 0.1 µL | 0.5 µL | 2,000 | 60–150 |
| Small Sector | 5 × 0.00625 µL | 0.03125 µL | 32,000 | 25–60 |
N_live = live + budding cells + their buds; Viability (%) = N_live ÷ (N_live + N_dead) × 100
Budding fraction (%) = budding cells ÷ N_live × 100; Live cells/mL = N_live ÷ (n × V_sq) × DF
Chamber agreement (%) = |C_live,1 − C_live,2| ÷ ((C_live,1 + C_live,2) ÷ 2) × 100 ≤ 15 %
Counting precision is 1/√N (25 cells: 20 %; 100 cells: 10 %). Below the target range use more sample or less dilution; above it, more dilution. Concentrations are shown to two significant figures; the CSV keeps four.
5. Image tips
- An image needs at least two triple lines in each direction for grid detection. Focus on the cells with the ruling still visible; keep the illumination even.
- The navigator covers the top-right corner of the canvas; zoom or pan before counting there.
- The grid detector and the calibration readout are built for the Neubauer Improved ruling.
- Images selected together load in the order of the file window; with the Short method, select them in the order of the positions to be covered.
6. References
- ASBC Methods of Analysis — Yeast-3A (Methylene Blue Viability) and Yeast-4 (Microscopic Yeast Cell Counting). American Society of Brewing Chemists.
- FermAxiom LLC. Yeast Biomass Quantification — Methods Report.
- FermAxiom LLC. Analytical Methods E-Module 2 — Yeast Biomass Quantification.
- FermAxiom LLC. Cell Counter Calculator - Basic, and its Instruction Manual.
Printable version: the full Instruction Manual, v5.7 — download FermAxiom_Cell_Counter_Advanced_v5_7_Instructions.docx.