Estimation of Population Density

One can approach the task of measuring population density in three ways. Collectively, these may be categorized as either direct or indirect. Direct techniques utilize microscopic counting methods in which viability is estimated by the inclusion of a viable dye or stain (see C.4). By comparison, indirect methods require plating (see C.2, C.3) and, subsequendy, growth of colonies. Additionally, monitoring some specific component unique to the viable cells in the population (i.e., ATP) or a component specific to the cell envelope continues to be of interest. Each has inherent merits, deficiencies, and cost considerations in terms of both equipment and supplies, and personnel that should be evaluated. 

Regardless of the method employed, the microbiologist may need to either dilute (in the case of growing populations) or concentrate (by filtration) the cell population such that the sample examined falls within a range of cells or colonies that can be counted with some degree of confidence. 

Estimating microbiological population density and diversity plays important and often pivotal roles at several junctures in the winemaking process. For instance, it is frequently necessary to determine changes in microbial populations during the preparation of starter cultures, growth and decline phases of malolactic fermentation, or monitoring potential Brettanomyces infections. 

Whenever possible, samples for microbiological analysis (50 to 100 mL) should be removed asepticaUy and placed into sterile containers to reduce the potential for secondary contamination due to non-wine microorganisms. Samples can be removed through sampling ports on tanks or by wine thiefs/pipettes. Sampling devices should be sterilized either by flame or 70% v/v ethanol prior to use. If a steriliant is used, the thief or pipette should be rinsed with sterile water before obtaining the sample. 

Open the sterile swab container, grasping the end of the stick. Do not touch any portion that might be inserted into the 18 X 150 mm test tube containing sterile 9mL 0.1% w/v peptone. 

Aseptically open the sterile diluent, moisten the swab head, and press out excess by rolling the swab on the inside of the diluent. 

Hold the swab handle at a 30° angle and rub the head over a surface of approximately 50 cmV7.7 in three times, reversing direction between strokes. 

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Fig. C-1. Preparation and plating of dilutions for estimation of population density.

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