Multiple Tube Methods

The TAC can be conducted using a number of microbiological methods. These are the pour-plate, membrane filtration, and multiple tube methods. The TYMC is conducted by using either the pour-plate or membrane filtration method. The TAC for bioburden is performed by adding 10 g, 10 mL, or 10 units in SCD broth or lactose broth to make 100 mL. Aliquots of this sample preparation are transferred into four standard size (15 x 100 mm) Petri dishes. Into two of the plates 15-20 mL of molten SCD agar is poured, and into the other two the same volume of Sabouraud dextrose agar (SAB) agar is poured. 

The tests used to determine the presence of indicator organisms fall into two groups, multiple tube methods and membrane filtration methods. 

Multiple tube methods are very sensitive to small numbers of micro-organisms due to the powerful amplification of these techniques. However, the methods are not very accurate statistically, and have considerable errors associated with them. In addition, the multiple dilutions needed are very demanding on technician time, sterile glassware and media. Tube methods may be run in conjunction with plate counts. 

Figure 9.1 Water testing procedure for multiple tube method...

One standard test used to determine the presence of the coliform group is called the multiple-tube fermentation technique (sometimes called the presumptive test). If this test indicates the presence of these bacteria, then a confirmed test must be done. If only negative colonies or no colonies develop during this test, it is considered negative otherwise, a completed test must be undertaken. Positive results obtained in the completed test are evidence for the presence of coliform bacteria. Testing methods have been given by the APHA, and the detailed procedures contained therein should be consulted (20). 

Both multiple-tube and membrane-filter methods are also available for testing for the fecal streptococcal group (20). These assays can be used to provide supplementary data regarding the bacteriological quaUty of water. Other fecal indicators should also be used concurrendy because of the survival characteristics of the fecal streptococci. 

Quantitative tests. In some situations, actual enumeration of the number of organisms present may be necessary. This is the case, for example, for limitations imposed on a discharge permit. Generally, two methods are used to enumerate conforms the membrane-filter technique and the multiple-tube technique. 

The other method of enumerating coliforms is through the use of the multiple-tube technique. This method is statistical in nature and the result is reported as the most probable number (MPN) of organisms. Hence, the other name of this method is the MPN technique. This technique is an extension of the qualitative techniques of presumptive, confirmed, and completed tests. In other words, MPN results can be a presumptive, confirmed, and completed MPNs. The number of tubes liberating gases is counted from each of the set of hve tubes. This information is then used to compute the most probable number of organisms in the sample per 100 mL. 

Multiple-tube or MPN technique—A method of estimating the concentration of conforms using the Poisson distribution. 

In microbiology two fundamental types of measurements are used by the analyst. The simplest ones consist in counting colonies on culture media in a Petri dish. Another principle consists in evaluating the most probable number of microbes by inoculating sub-samples into multiple tubes. The result of the latter is given by statistical tables. For both types of methods results are only available after a few days. For the presence of very few microbes, so-called presence/absence tests have been developed by microbiologists. They are mainly used for the detection of pathogenic microbes. For the last two types... 

Both multiple tube and membrane filtration methods are not only qualitative they are also quantitative, thus enabling an assessment to be made of the potential danger to health. 

Multiple-tube fermentation method Three-step method of testing for coliform bacteria in drinking water. 

Most Probable Number Method (Multiple Tube Technique).112... 

Four methods may be used to detect and enumerate total coliforms, fecal coliforms, and E. coli in water samples. These are grouped as (a) multiple tube fermentation technique also known as the MPN technique, (b) membrane filtration technique, (c) presence/absence test, and (d) use of the enzymatic substrates test also known as the chromogenic-fluorogenic substrate test. 

The multiple tube fermentation method requires the use of replicate tubes and dilutions of samples. The fermentation products of lactose include mixed acids and gas, which is usually detectable. Coliforms are reported in terms of MPN of organisms present. MPN tables are based on a random dispersion of coliforms (Poisson distribution). Failure to shake the samples very well will result in a nonrandom distribution that will underestimate the actual density of the bacteria. If testing drinking water, a single bottle containing 100 mL may be used or 5 replicate tubes with 20 mL each or 10 replicate tubes with 10 mL each. If testing nonpotable water such as salt water, brackish water, or sediments, multiples and subsamples of 10 mL (e.g., 0.1 1.0 10 mL) should be used. 

Multiple Tube Fermentation Method (MPN Method) for Detecting Fecal Coliforms... 

International Standard, ISO 9308-2 (1990). Water Quality—Detection and enumeration of coliform organisms, thermotolerant coliform organisms and presumptive Escherichia coli— Part 2 Multiple tube (most probable number) method. International Organization for Standardization, Geneva, Switzerland. 

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