The viable plate count procedure is selective because no one combination of incubation conditions and media allows all types of bacteria to grow.
Direct Count Procedures
Bacteria can also be enumerated by direct countinj procedures. In this procedure, counting is done with out the need to first grow the cells in culture. In om direct count procedure, dilutions of samples are ob served under a microscope, and the numbers of bac terial cells in a given volume of sample are counted These numbers are used to calculate the concentra tion of bacteria in the original sample (FIG. 10-9) Special counting chambers, such as a hemocytometa or Petroff-Hausser chamber, are sometimes employed to determine the number of bacteria. These chambers are ruled with squares of a known area and are so constructed that a film of liquid of known
FIG. 10-9 The direct counting procedure using a Petroff-Hauser counting chamber. The sample is added to a counting chamber of known volume. The slide is viewed and the number of cells determined in an area delimited by a grid. In the counting chamber shown, the entire grid has 25 large squares for a total area of 1 mm2 and a total volume of 0.02 mm3, formed by the spacing of an overlying coverslip. There are 12 cells within the single large grid (composed of 16 smaller boxes) in this example. Assuming the number of cells in this single grid is representative of all the grids, the number of cells within the total area under the grid is 12 cells. The concentration of cells is therefore 300/0.02 mm3.
ENUMERATION OF BACTERIA 295
depth can be introduced between the slide and the cover slip. Consequently, the volume of the sample overlying each square is known.
It is often desirable to stain the cells. This helps in visualizing bacterial cells. Alternatively, a known volume of a sample containing a suspension of bacteria is poured through a filter, such as a nitrocellulose 0.2 |xm pore size filter. The bacteria are stained on this filter, often using a fluorescent stain, and counted under a microscope. Many fluorescent dyes, such as acridine orange, stain all cells, making it impossible to differentiate living from dead bacteria. The difficulty in establishing the metabolic status of the observed bacteria is a major limitation of this procedure.
Date: 2015-02-28; view: 1457