NucleoCounter YC-100 Presentation

The Value of Objective Yeast Count & Viability Same Beer with same taste, same color, same foam, same odors….. To ensure that the same quality is achieved every time: To leave out statistical variance. To leave out personal influence. To leave out interpretation. To trust your method and your final product.

Yeast Management High No. of Dead Cells = Poor Yeast Management Lower Foam Stability Higher Turbidity Lower Flavor Stability Higher work load for filter Correct Pitching of Yeast is Important: Typical Viability Acceptance Criteria is above 90% Non Viable Yeast Cells: Will act as substrate for bacteria = Infection. Will decompose = Filter Load, Turbidity, unwanted taste. Viable Yeast Cells: Too many = waste of resources + variation in production time. Too few = loss of production time + problems with non viable cells.

Base Technology of ChemoMetec: Fluorescence Microscopy Principle: The NucleoCounter is an integrated fluorescence microscope designed to detect signals from the fluorescent dye, propidium iodide (PI) bound to DNA. Results from the NucleoCounter represent either total or nonviable cell concentration, depending on the sample preparation. Emission CCD Filter Lenses Lenses: Magnification 1.4 X. Excitation filter: Sorts out green light. Emission filter: Sorts out long wave length including red light. Fixture LED’s Excitation Filter

Propidium Iodide 1 dye molecule per 4-5 bp Genome 2.5 x 109 – 4.0 x 109 bp. C27H34N42+.2I- Mr 668.4 DNA base pair (bp): A: Adenine T: Thymine G: Guanine C: Cytosine

NucleoCounter® YC-100™ No calibration is a basic advantage of this technique: The PI method looks at the DNA of the cell nuclei rather than the whole cells (or particles of a certain size) Cell sizes differ, whereas cell nuclei size and amount of DNA or number of base pairs are more uniform

Calibration Free No calibration to cell type (size) No calibration to volume, as every single analysis volume is exactly known since: The thickness of the measurement chamber of each NucleoCassette is measured during production and coded with black dots on each Cassette. The NucleoCounter reads the code accurately determining the analyzed volume in each measurement. Durable optical components makes the NucleoCounter calibration free through out its lifetime

A Specific Volume is Analyzed

No Cleaning or Maintenance/Service The NucleoCassette contains the entire flow system (pump, tubing, mixer, reagents and waste container) as well as the measurement chamber. As the NucleoCassette is discarded after use this means neither cleaning nor maintenance of the NucleoCounter is needed. No maintenance as the NucleoCounter is made of very durable and stable components. The NC optics, mechanics and electronics has negligible thermal and aging drift properties. The light source and the linear actuator have been tested to last for >1 x 106 samples. No service is needed as no calibration, cleaning or replacement of components is necessary

Total Cell Count Viable Non-viable Lysis buffer (Reagents Y100) PI Free nucleus PI PI-stained nucleus PI-stained nucleus Viable Non-viable Lysis buffer (Reagents Y100)

Viability Determination Viable Non-viable Viable Non-viable Free nucleus PI PI-stained nucleus PI-stained nucleus Lysis buffer (Reagents Y100) Non-viable cell count Total cell count

Easy Operation

NucleoView™ Software

The NucleoCassette

The NucleoCassette Sensitive parts, moving parts and “wear and tear” parts have been taken out of the instrument and integrated in the disposable NucleoCassette Pump Tubing’s Mixer Precision optics Chemicals Calibration Waste container

Reagent Y100 The lysis reagent Y100 disrupts the yeast cell walls and membranes, rendering the nuclei susceptible to staining with propidium iodide (PI). Dissolves small cell aggregates

NucleoCounter® YC-100™ Key Advantages Easy operation – no preparation To be used in Lab & Production (24 h) To be used by untrained operator 30 sec. analysis time Calibration free No cleaning Maintenance and service free Space saving Safe sample handling & disposal High precision & repeatability Good reproducibility

NucleoCounter® YC-100™ SUMMING UP Gives the head office control of yeast management Insures the same reference standard at all locations The NucleoCassette insures calibrated volume with every measurement Can be used in lab and production (24h) and is not dependent of a computer – can be used as a stand alone unit Easy to implement at all locations Extreme ease of use – very limited training is required Well proven and preferred technology for automatic counting in breweries world wide Robust technology for all environments Handles clustering cells extremely well