Standard plate assay Hemacytometer:
With a counting chamber, mostly called Hemacytometer, one is able to measure cell densities > 10000 ml/L and at a relative small size (~5 – 50 µm, single cells or short chains) under the microscope. As the name suggests it has been developed for counting blood cells but it is now widely established in a huge variety of fields.
The Hemacytometer consists of two chambers, each of which is divided into nine 1.0 mm squares. A cover glass is supported 0.1 mm over these squares; the total volume over each square is 10-4 cm³. Since 1cm³ is approximately equivalent to 1ml, the cell concentration per ml will be the average count per square x 104. In detail there are two different chambers: The one described above is called Levy-Hausser with 0.1 mm and the other one Petroff-Hausser with 0.02 mm in height. Thus the second one is used for higher suspensions, mainly bacteria and the first one mainly for yeasts. The objectives used for the Levy-Hausser is 40x and for Petroff-Hausser the oil-immersion one (100x) (Boulton et al. 1996). Hemacytometer counts do not distinguish between living and dead cell. But there are several stains to make this distinction. For example Trypan blue can be used. The nuclei of damaged or dead cells take up the stain and show the specific color. Viable cells exclude the dye and do not change. In general more than one hundred total cells should be counted to minimize random errors. Very important using this technique is to avoid errors like: unequal cell distribution in the sample, improper filling of chambers, failure to adopt a convention for counting cells in contact with the boundaries lines or with each other. Example: You count 187 particles in five small squares, each square has an area of 1/25 mm-squared and depth of 0.1 mm, so the total volume is 0.04 x 0.1 = 0.004 mm- cubed. Times five squares = 0.02 mm-cubed. => 187 particles / 0.02 = 9350 particles per mm-cubed. Because there are 1000 cubic ml in one cubic cm, the particle count is 9,350,000 per ml.
FMC (flow cytometry):
Rapid and easy to use this technique is a useful alternative to the above method. FCM is an optical system which permits highly sensitive direct estimation of particle or cell numbers present in a suspension. Shape and size of organisms are determined by light scattering while measurement with fluorescent light gives information on cell viability. Furthermore staining cells with fluorescent dyes or fluorogenic substrates allows also the analysis of functional cell parameters including structural properties and biological activities (Malacrino 2001)
Application in Wine Microbiology:
Hemacytometer is the way to go because in comparison to spectrophotometer allows an assessment of cell viability and distinguish cell type as well. With a little practice it is an easy way for a quick determination of the presence or population of yeast a bacteria in liquid suspensions. It is especially helpful in preparation of yeast starter cultures for sparkling wine fermentation and of bacteria starter cultures of MLF. Microscopic cell counting is also useful as a preliminary step for making colony counts. Besides that, counting cells of spoilage organisms during aging is like a effective insurance. If performed on a regular basis the winemaker is always aware of the trend of the microbial load of the wine and he can make decisions based on real facts. Unfortunately the limitations are in the lower cell counts as mentioned and therefore it is hard to give a guaranteed number of lactobacillus for example before reaching harmful levels.
FCM enables real time data especially during fermentative processes. The flipside of this method is mainly the high cost which most wineries prevent from using it plus the lack of specific knowledge in microbial cell labeling and the management of the data obtained. Therefore the potential of FCM has not been largely explored in enological practices. The detection limit was 10³ cells / ml. The detection of spoilage yeasts like Brettanomyces can be successful carried out. Even for MLF this method could work well: The limit for a pure culture was 10000 cell / ml and it is known that the metabolic starts when the population reaches 1000000 cells / ml. On top of that FCM is able to distinguish and measure different parameters like yeasts and bacteria simultaneously (Malacrino 2001).
- Boulton, R. et al. 1996. Principles and Practices of Winemaking. Chapman and Hall, New York.
- Dittrich, H. ; Grossmann, M. 2005. Mikrobiologie des Weines
- Malacrino et al. 2001. Rapid detection of viable yeasts and bacteria in wine by flow cytometry. Journal of Microbiological Methods 45 (2001) 127-134.
- Baena-Ruano, S. et al 2006. Rapid method for total, viable and non-viable acetic acid bacteria determination during acetiﬁcation process .Process Biochemistry 41 1160–1164