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9-Food and Water

9-Food and Water

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Published by: ankitsaxena123 on Aug 18, 2014
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Food and Water – 1
THE MICROBIOLOGICAL EXAMINATIONOF FOODS & WATER
Foods and water contaminated with pathogenic microorganisms are major avenues for the spread of infectious diseases. In this country, strict state and federal guidelines regulate food and water  processing industries, even so, many outbreaks of diseases linked to contaminated foods or water occur every year.Municipal water and food products are routinely monitored for both the total number of microorganisms and presence of pathogens, but there are many challenges to doing so. Evennonpathogens, if present in large enough numbers, can cause deterioration of food products and distaste in water. And testing for all types of pathogens is not practical since we do not know whichmay be present.The alternative to testing for many different types of pathogens is to test for bacteria that
indicate
the potential presence of pathogens. The most common source of pathogens in food and water is fecal contamination, of either animal or human origin. Feces also contain innumerablenonpathogenic bacteria, and the presence of these other bacteria in food or water indicates that fecalcontamination has occurred (undesirable enough) and the potential presence of pathogens. Themost commonly used “indicator bacteria are fecal coliform and fecal enterococci.
Coliform Bacteria
 
Coliform bacteria can serve as
indicators
 of fecal contamination. They are not themselves pathogenic but are denizens of the digestive systems of animals, and thus abundant in feces.Coliform are defined as “Gram-negative aerobic or facultative anaerobes, nonspore-forming, rod shaped bacteria that ferment lactose with acid and gas production.”
 E. coli
, the most abundant bacterium of the human colon, is the most important indicator of human fecal contamination. However, some coliform bacteria, such as
 Enterobacter aerogenes
, are of non-fecal origin and may be present in uncontaminated samples. Thus testing for coliform bacteria involves several tests tominimize the possibility of false positive results. Bear in mind that the presence of indicator bacteriadoes not mean that human pathogens are definitely present, but their presence suggests that the fecalcontamination has occurred, and that pathogens may be present.
Methods of counting bacteria
There are two standard methods of counting bacteria: the
standard plate count (SPC)
 and the
most probable number (MPN)
methods. Either technique can be used with selective or non-selective technique – the method of choice depends largely upon the number of bacteria to becounted in the sample. The SPC is routinely used for samples that have a relatively large number of bacteria, which can be diluted down and grown as a countable number of colonies in a petri plate.The MPN method is used when the number of bacteria to be counted is so low that the cells could not be detected if a sample were applied to a plate, which is often the case when counting coliform bacteria.
 
Food and Water – 2
Summary of exercise
1. You will count the total number of bacteria in a sample of spoiled milk using the Standard PlateCount technique.2. You will perform a coliform count of a water sample using the Most Probable Number (MPN)technique.
I. Standard Plate Count of Bacteria in Food Products
The
Standard Plate Count
 is the most common method used to quantify bacteria in foods. To perform a standard plate count, the food to be tested is suspended in liquid and a sample is thenspread over the surface of a solid medium in a petri plate. Bacterial cells present will form coloniesthat can be counted to determine the number of cells in the original sample. When the objective isto estimate the total number of bacteria, a complex medium called
Plate Count Agar
is commonlyused since it will support growth of many different types of bacteria. We call the results the number of
Colony Forming Units (PFU)
, not total bacteria. This is because no single culture medium willsupport all different types of bacteria, we can only count those that do grow to form a visible colony.
Serial Dilution of samples
When performing a bacteria count,
between 30 and 300 bacterial colonies
 need to be on the plate. A minimum of 30 assures that the data is statistically reliable; however, if there are more than300 colonies are present, competition for nutrients can suppress growth of colonies. For example,if a sample were to contain 10
6
 cells\ml, a 1 ml sample would contain 10
6
 bacteria – far more thanthe 300 cell limit of a standard plate count – and the sample must be serially diluted:The standard way to dilute a sample in microbiology is through Serial Dilution. As shown inFigure 1, a sample is diluted step-wise in a series of tubes containing sterile diluent. Each step yieldsa particular dilution factor:
Dilution factor for each step
 
=
Final volume in the tube Vol of the sample added to the tubefrom which the total dilution of the sample can be calculated:
Total dilution of a sample plated
 = Product of all dilution steps Vol of the sample plated Since the original concentration of bacteria in the milk sample is unknown, we will plate severaldilutions, and hope that at least one will yield between 30- 300 colonies on the plate.
***Do the practice problems at the end of this lab exercise; these must beturned in with the results of this lab exercise.
***
 
Food and Water – 3
Figure 1. Example dilution series for a sample.
Your dilution series will be longer, and you will plate the 4highest dilutions.
Supplies
sterile 1 ml pipets7 tubes containing 9 ml of sterile distilled water (yellow caps)4 plates containing Plate Count bottom agar.4 tubes containing 4 ml of overlay agar held @ 50
E
C, clear cap
Procedures
First, some helpful hints . . .
When performing your dilutions:1. Be sure to observe aseptic technique, and use a new sterile pipet for each dilution step. Why? 2.
Thoroughly mix
 each tube before preceding with the next dilution step. However, do notmix so violently that the contents splash near the cap.

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