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Terms in this set (26)

(1) Filtration involves passing a fluid such as water or air through a membrane filter.
The pores in the filter are small enough that they do not allow microbes to pass through and
therefore traps them on the surface of the filter. When the filter is placed on a medium containing
nutrients, the cells on its surface grow to form colonies. After incubation, the number of
colonies on the filter is an indication of the number of microbes in the fluid that was filtered.
(We will use this method to analyze water samples in a future lab.)

(2) The most probable number method involves the inoculation of tubes of lactose broth with the
sample and with dilutions of the sample. Coliform bacteria, which are indicators of fecal
contamination, will ferment the lactose in the medium and produce gas. Based on the number of
tubes that are positive for lactose fermentation and gas production, an estimate of the number of
coliforms is obtained using statistical methods.

(3) The direct microscopic count requires the use of a microscope and a special
counting chamber containing a known volume of a sample. This can be done very quickly, but its drawbacks include the tediousness of counting high numbers and the
inability to distinguish living cells from the dead cells that may be present.
(4) The standard plate count involves placing a known volume or mass of a sample in
plates with media and then counting the number of colonies present after a period of
incubation. We will use this method in this lab and with milk samples in a future lab.
We will discuss the theory behind the standard plate count in detail in the following
pages of this lab.
Only plates containing between 30 and 300 colonies can be used in calculations.
The reason for using plates with less than 300 colonies is the difficulty that is encountered in
the counting process with plates containing 300 or more colonies. When there are 300 or more
colonies on the plate, counting becomes very difficult, even with the use of a colony counter. If
colonies are in physical contact with each other, it is sometimes difficult to determine whether
they should be counted as one colony or two colonies. This results in errors in the colony count.
Also the competition for nutrients is too great if the colonies are too close together.
The reason for not using plates that have fewer than 30 colonies is statistical accuracy. Not every
volume taken from a sample will contain exactly the same number of cells. For the sake of illustration, assume that a water sample contains 10 bacteria per milliliter and that the variation between one milliliter samples can vary by as many as 3 bacteria. One milliliter volumes that are removed could contain as many as 13 bacteria or as few as 7 bacteria. The calculated concentration would vary from 13 bacteria/ml to 7 bacteria/ml, which is a huge difference. The variation from the actual concentration could be as high as 30%. Using the same range of variation, one milliliter samples of a culture containing 30 bacteria per milliliter could contain as many as 33 and as few as 27. In this case, the variation from the actual concentration would be 10%. Therefore, plates with less than 30 colonies cannot be used, because sampling error would create unacceptable inaccuracy.
All colonies on the plate must be counted.
When the pour plate method is used, the cells will end up distributed throughout the medium. Those
on the top will form large colonies on the surface, those in the middle will form small colonies
suspended in the middle of the medium, and those on the bottom of the medium will form diffuse colonies at the interface between the plate and the medium. The colonies will differ in size and form, but assuming that there was not contamination, each colony will have come from one colony forming unit in the original
broth. Therefore, all colonies must be counted.