Cult&Ident_Notes&Reading - Cultivating and...

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Unformatted text preview: Cultivating and Identifying Microbes In this lecture, we will answer the questions of WHY would one want to cultivate and identify microorganisms and HOW does one cultivate and identify microorganisms. The first step in cultivating and identifying a microorganism is identifying an environmental sample in which you believe the microorganism of interest to be present (e.g. the spinal fluid of a human or the intestinal fluid of a cow). In the next step, you form a hypothesis about the kind of medium that you can use to grow the microorganism you want and that will also minimize the growth of unwanted microorganisms that might be present. This hypothesis forms the basis of an enrichment culture. Enrichment cultures demonstrate Darwin’s concept of natural selection. Textbook Reading Page 142-143, “Origins of Enrichment Culture Methodology” What you should get out of this text: Be able to explain the basic principle behind an enrichment culture. How does this demonstrate the Darwinian concept of natural selection? Choice of growth medium There are many choices of growth media that can be used to grow microorganisms. This textbook reading will explain the difference between two major classes of growth media – complex and defined. It will also present some of the major environmental conditions that need to be considered when growing microorganisms. Textbook Reading Page 138-139, “A Culture Medium” & “Incubation Conditions” What you should get out of this text: 1. Be able to compare and contrast complex medium and defined medium. 2. Know that growth media can be liquid or solid. 3. Know that temperature and oxygen are the two environmental factors that must be considered when designing a growth medium. How would one decide whether to use a complex or a defined medium? Complex media can be useful when the nonessential nutrients required for growth of an organism are unknown. Nonessential nutrients will be those factors that most organisms need to growth, for example an amino acid like alanine. Some microorganisms are able to synthesize alanine de novo and thus do not required that alanine be added to the growth medium. Other microorganisms cannot synthesize alanine and it must be added to the growth medium of these organisms. Alanine can either be added to a defined medium or obtained via a complex medium, such as one with yeast extract. However, there are times when a complex growth medium can be detrimental to the growth of a desired microorganism. For example, the metals found in complex media, such as 1 yeast extract, typically inhibit the growth of bacteria that live in the open ocean where the metal concentration is low. Liquid or Solid Enrichment Culture After deciding on the growth medium and the environmental conditions to use, the next step is to decide whether to grow the environmental sample on a liquid or solid medium. The textbook explains the pro’s and con’s of each technique. Textbook Reading Page 144, “Aerobic Enrichment” What you should get out of this text: Be able to explain why a greater diversity of microorganisms can be isolated from enrichment on solid media than liquid media. Be able to explain why if you had a growth medium that was very selective for your desired microorganism, you might chose to use a liquid medium rather than a solid medium. Pure culture and Streak Purification After enriching for your desired microorganism, whether on liquid or solid, the next step is to isolate your organism in pure culture. The most common method to accomplish this is streak purification. Textbook Reading Page 139-140, “Streak Plate Procedure” What you should get out of this text: 1. Know what is the goal of the streak plate procedure. 2. Know how a colony arises. 3. Know how the streak plate procedure can be used to assess the purity of a culture. Identifying the Isolated Microorganism Often a first step in identifying a bacterium is to simply ask is it a Gram+ or Grambacterium. Textbook Reading Page 81, Box 4.1 “The Gram Stain” What you should get out of this text: 1. Know that the Gram strain is a differential strain that uses two different stains. 2. Know that cells of a different type stain differently. (Note: The physiological basis of the differential staining will be explained in a later lecture.) Gram strains yield a very rough identification of a microbe. As you will have learned in Lecture 2, a standard way to identify a microorganism is by the sequence of the gene for 16S rRNA. But, this technique will not identify different strains of a bacterium. When this level of detailed information is need, DNA fingerprinting is commonly used. There are currently several different methods for DNA fingerprinting; they all have the goal of 2 distinguishing different entities of DNA based on base pair differences. The method that will be presented in class involves isolating bacterial chromosomal DNA and then digesting it with a restriction enzyme. The fragments of DNA are then separating by gel electrophoresis. A specialized form of gel electrophoresis that is better at separating large fragments of DNA, called pulsed-field gel electrophoresis (PFGE), is used. I do not expect you to know the patterns of current in PFGE or why it is better at separating large DNA fragments. The following link has a nice short explanation of the technique and an animated cartoon of PFGE You will also see that after electrophoresis the DNA is made visible due to staining with ethidium bromide. You will also see how different sequences of DNA yield different patterns on the gel. Think about and be able to answer why different strains of a bacterium would yield different patterns of DNA. Estimating Microbial recovery The procedure outlined above is for isolating a particular microorganism in pure culture from an environmental sample. But another goal one may have is to identify all microorganisms present in an environmental sample. To date we have isolated and grown in pure culture only a small fraction of the microorganisms that are present in the environment. How can we know many microorganisms are present in the environment relative to the number we can culture in the lab? The textbook describes two different methods that are commonly used to estimate the microbial cells present in an environment – a direct microscopic count and a viable cell count on solid growth medium. Be able to explain why the direct microscopic count would yield far greater numbers of cells than the viable cell count. Textbook Reading Page 161-162, “Direct Count of Cells” What you should get out of this text: 1. Know that the number of cells per milliliter of sample can be determined using a microscope and a special glass slide with a grid. 2. Know that this method cannot distinguish between dead and living cells and, sometimes, even debris can look like a bacterial cell. Textbook Reading Page 162, “Viability Count-Living Cells” What you should get out of this text: 1. Understand how the number of viable cells per milliliter of sample can be determined by diluting and plating the sample. 2. Understand why the viability count of a sample can yield much lower numbers of cells that the direct count of cells. 3 ...
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