Microsoft Word - Dilutions updated sp07 (1).pdf - Microbiology for Health Science Lab Enumeration and Dilutions Lab Lab 5 Bacterial Enumeration Lab

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Microbiology for Health Science Lab Lab 5 Enumeration and Dilutions Lab - 1 - Bacterial Enumeration Lab written by Dr. Michelle Furlong INTRODUCTION Bacterial enumeration is the measurement of the number of bacterial cells per milliliter, gram, or cubic meter of a sample (the units depends on the nature of the sample). There are a number of methods that can be used to determine the number of cells/unit and these methods can be divided up into different categories. Viable counts involve counting cells that can be cultured and/or are metabolically active. Total counts involve counting all cells including dead or inactive cells. Direct methods of enumeration involve counting actual cells or colonies and indirect methods involve estimating the number of cells based on cell mass, scattering of light through a culture (spectroscopy), or a statistical method called the MPN (most probable number) technique. The table below gives examples of different methods. Category Method of Enumeration Description Indirect and viable MPN Make statistical estimates numbers of cells by their patterns of growth in liquid culture media. Direct and viable Standard plate count Dilute a sample in saline, spread on solid media, and count colonies. Calculate the number of cells in original sample from counts and dilutions. Indirect and total Spectroscopy Measure the amount of light that passes through a liquid culture using a spectrophotometer and estimate the number of cells/ml based on amount of light that passes through culture. Direct and total DAPI or AODC staining and microscopy Stain the cells with fluorescent dyes, which make them visible in raw samples (i.e. soil). Count the number of cells using a fluorescent microscope. In this lab we will be using two methods to enumerate the number of cells per ml of a liquid culture, the spectroscopy and the heterotrophic plate count methods. Standard Plate Count Method: A Viable and Direct Count When healthcare providers (HP) treat infectious disease sometimes it is important for them to know the number of infectious organisms in the tissue or bodily fluid before they prescribe a treatment regime. Since the HP is only interested in determining the number of live pathogens he/she would most likely choose a viable method of enumeration. A popular method used by HPs to enumerate cells is a called the standard plate count (SPC) method. The SPC method is a direct count method used to determine the number of viable, heterotrophic cells per volume (or per milliliter) of fluid. It involves diluting down a sample (if the sample contains > 3,000 cells/ml)
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Microbiology for Health Science Lab Lab 5 Enumeration and Dilutions Lab - 2 - and evenly spreading a small amount across an agar plate. Each cell that ends up on the plate will form a colony overnight. The colonies can then be easily counted and a few calculations can help the HP determine the number of cells/ml of sample.
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