Standardizing bacterial population in a culture

There are several methods of determining bacterial cells in a culture 1) Direct counts are reliable ways to distinguish biomass of bacteria in natural waters. The usual method is to concentrate the bacteria onto membrane filters, stain them with acridine orange and examined under epifluorescence microscope. 2) Indirect viable count or plate counts involve plating out spreading a sample of culture onto nutrient agar surface. Each colony that can be counted is called colony forming units and the number of cfu is related to the viable number of bacteria in the sample 3) Most probable number (MPN) technique is an important technique in estimating microbial populations in soils, waters, and agricultural products. Many soils are heterogeneous, therefore exact cell numbers of an individual organism can be impossible to determine. The MPN technique is used to estimate microbial population sizes in situations like this. The technique does not rely on quantitative assessment of individual cells, instead it relies on specific qualitative attributes of the microorganism being counted. The important aspect of MPN methodology is the ability to estimate a microbial population size based on a process-related attribute. 4) Turbidity measurements employ a variety of instruments to determine the amount of light scattered by a suspension of cells. Particulate objects such as bacteria scatter light in proportion to their numbers. The turbidity or optical density of a suspension of cells is directly related to cell mass or cell number, after construction and calibration of a standard curve. The method is simple and nondestructive, but the sensitivity is limited to about 107 cells per ml for most bacteria.

For standardizing bacterial population in a culture, make at least three daily transfers in nutrient broth (10µl). Spread a loopful of the sample onto an agar plate with 0.5mm diameter loop and incubate it for 18h. From the agar culture transfer a 1.0 cm x 0.5cm of the culture onto 10mL nutrient broth.