SCIENCE EDUCATION > Advanced Biology

Microbiology

This collection demonstrates the key tools of microbiological investigation, including proper sterile technique and plating, how to use selective media and enrich samples, and culturing methods for mixed or pure samples. Additionally, it looks at common methods for identifying microbial isolates, as well as procedures for the genetic manipulation of bacteria.

  • Microbiology

    08:07
    Creating a Winogradsky Column: A Method to Enrich the Microbial Species in a Sediment Sample

    Source: Elizabeth Suter1, Christopher Corbo1, Jonathan Blaize1 1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 10301

    The Winogradsky column is a miniature, enclosed ecosystem used for enriching sediment microbial communities, especially those involved in sulfur cycling. The column was first used by Sergei Winogradsky in the 1880s and has since been applied in the study of many diverse microorganisms involved in biogeochemistry, such as photosynthesizers, sulfur oxidizers, sulfate reducers, methanogens, iron oxidizers, nitrogen cyclers, and more (1,2). The majority of microorganisms on Earth are considered unculturable, meaning that they cannot be isolated in a test tube or on a petri dish (3). This is due to many factors, including that microorganisms depend on others for certain metabolic products. The conditions in a Winogradsky column closely mimic a microorganism's natural habitat, including their interactions with other organisms, and allows for them to be grown in a lab. Therefor, this technique permits scientists to study these organisms and understand how they are important to Earth's biogeochemical cycles without having to grow them in isolation. Earth's environments are full of microorganisms which thrive in all types of habitats, such as soils, ocean water, clouds, and deep-

  • Microbiology

    10:53
    Serial Dilutions and Plating: Microbial Enumeration

    Source: Jonathan F. Blaize1, Elizabeth Suter1, and Christopher P. Corbo1 1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 10301

    Quantitative assessment of prokaryotes can be onerous given their abundance, propensity for exponential proliferation, species diversity within a population, and specific physiological needs. Compounding this challenge, is the four-phase nature in which bacteria replicate (lag, log, stationary and death). The ability to accurately estimate microorganism concentration is necessary for successful identification, isolation, cultivation, and characterization (6). As such, microbiologists have employed serial dilution and various plating techniques for over a century to reliably quantify bacterial and viral load in clinical, industrial, pharmaceutical, and academic laboratory environments (2,4,6). Descriptions of this methodology first appeared in 1883 when the German scientist and physician Robert Koch published his work on infectious disease-causing agents (2). Often referred to as the father of modern bacteriology, Koch's forenamed techniques have become the gold standard for enumeration of microorganisms, culturable or otherwise, throughout the world. Serial dilution is a systematic reduction of a known or unknown entity (a solute, organism, etc.) through successive re-suspension of an initial sol

  • Microbiology

    09:35
    Enrichment Cultures: Culturing Aerobic and Anaerobic Microbes on Selective and Differential Medias

    Source: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1 1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 10301

    Prokaryotic cells are able to inhabit nearly every environment on this planet. As a kingdom, they possess a great metabolic diversity, allowing them to use a wide variety of molecules for energy generation (1). Therefore, when cultivating these organisms in the lab, all necessary and specific molecules required to make energy must be provided in the growth media. While some organisms are metabolically diverse, others are able to survive in extreme environments such as high or low temperatures, alkaline and acidic pH, reduced or oxygen absent environments, or environments containing high salt (2,3,4). Termed "extremophiles", these organisms often require these intense environments to proliferate. When scientists look to grow such organisms, the media components as well as any specific environmental conditions all need to be taken into account in order to successfully cultivate the organisms of interest. Scientists are able to grow culturable organisms in the lab because they understand the specific requirements that those species need to grow. However, culturable organisms account for less than 1% of species estimated to be on the planet (5). Organisms that we have detected by gene sequencing

  • Microbiology

    09:03
    Pure Cultures and Streak Plating: Isolation of Single Bacterial Colonies from a Mixed Sample

    Source: Tilde Andersson1, Rolf Lood1 1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund, Sweden

    Seemingly impossible to determine, microbial biodiversity is truly astounding with an estimated one trillion coexisting species (1,2). Although particularly harsh climates, like the acidic environment of the human stomach (3) or the subglacial lakes of Antarctica (4), may be dominated by a specific species, bacteria are typically found in mixed cultures. As each strain may influence the growth of another (5), the ability to separate and cultivate "pure" (consisting only of one type) colonies has become essential in clinical and academic settings alike. Pure cultures enable further genetic (6) and proteomic examinations (7), analysis of sample purity and, perhaps more noteworthy, the identification and characterization of infectious agents from clinical samples. Bacteria have a wide range of growth requirements and there are numerous types of nutrient media designed to sustain both the un-demanding and the fastidious species (8). Growth media can be prepared either in liquid form (as a broth) or in a typically agar-based (a gelling agent derived from red algae) solid form. Whereas direct inoculation into broth carries the risk of generating a genetically diverse or even mixed bacterial population, p

  • Microbiology

    10:56
    16S rRNA Sequencing: A PCR-based Technique to Identify Bacterial Species

    Source: Ewa Bukowska-Faniband1, Tilde Andersson1, Rolf Lood1 1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund, Sweden

    Planet Earth is a habitat for millions of bacterial species, each of which has specific characteristics. Identification of bacterial species is widely used in microbial ecology to determine biodiversity of environmental samples and medical microbiology to diagnose infected patients. Bacteria can be classified using conventional microbiology methods, such as microscopy, growth on specific media, biochemical and serological tests, and antibiotic sensitivity assays. In recent decades, molecular microbiology methods have revolutionized bacterial identification. A popular method is 16S ribosomal RNA (rRNA) gene sequencing. This method is not only faster and more accurate than conventional methods, but also allows identification of strains that are difficult to grow in laboratory conditions. Furthermore, differentiation of strains at the molecular level enables discrimination between phenotypically identical bacteria (1-4). 16S rRNA joins with a complex of 19 proteins to form a 30S subunit of the bacterial ribosome (5). It is encoded by the 16S rRNA gene, which is present and highly conserved in all bacteria due to its essential function in ribosome assembly; however, it also contains

  • Microbiology

    12:14
    Growth Curves: Generating Growth Curves Using Colony Forming Units and Optical Density Measurements

    Source: Andrew J. Van Alst1, Rhiannon M. LeVeque1, Natalia Martin1, and Victor J. DiRita1 1 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America

    Growth curves provide valuable information on bacterial growth kinetics and cell physiology. They allow us to determine how bacteria respond in variable growth conditions as well as to define optimal growth parameters for a given bacterium. An archetypal growth curve progresses through four stages of growth: lag, exponential, stationary, and death (1). Figure 1: Bacterial growth curve. Bacteria grown in batch culture progress through four phases of growth: lag, exponential, stationary, and death. Lag phase is the period of time it takes for the bacteria to reach a physiological state capable of rapid cell growth and division. Exponential phase is the stage of fastest cell growth and division during which DNA replication, RNA transcription, and protein production all occur at a constant, rapid rate. Stationary phase is characterized by a slowing down and plateauing of bacterial growth due to nutrient limitation and/or toxic intermediate accumulation. Death phase is the stage during which cell lysis occurs as a result of severe nutrient limitation. Lag phase is the period of time it takes for

  • Microbiology

    13:38
    Antibiotic Susceptibility Testing: Epsilometer Tests to Determine MIC Values of Two Antibiotics and Evaluate Antibiotic Synergy

    Source: Anna Bläckberg1, Rolf Lood1 1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund Sweden

    Knowledge of the interactions between antibiotics and bacteria is important in understanding how microbes evolve antibiotic resistance. In 1928, Alexander Fleming discovered penicillin, an antibiotic that exerts its antibacterial function by interfering with cell wall regeneration (1). Other antibiotics with diverse mechanisms of action have subsequently been discovered, including drugs that inhibit DNA replication and protein translation in bacteria; however, no new antibiotics have been developed in recent years. Resistance to current antibiotics has been increasing, resulting in severe infectious diseases that cannot be effectively treated (2). Here, we describe several methods to assess antibiotic resistance in bacterial populations. Each of these methods works, regardless of the mechanism of action of the antibiotics used, because bacterial death is the measured outcome. Antibiotic resistance is not only rapidly disseminated specifically through hospital settings, but also throughout society. In order to investigate such means of resistance, different methods have been developed including the Epsilometer test (E-test) and the broth dilution test (3). The E-test is a well-established method and is a cost-effe

  • Microbiology

    11:18
    Microscopy and Staining: Gram, Capsule, and Endospore Staining

    Source: Rhiannon M. LeVeque1, Natalia Martin1, Andrew J. Van Alst1, and Victor J. DiRita1 1 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America

    Bacteria are diverse microorganisms found nearly everywhere on Earth. Many properties help distinguish them from each other, including but not limited to Gram-staining type, shape and arrangement, production of capsule, and formation of spores. To observe these properties, one can use light microscopy; however, some bacterial characteristics (for example size, lack of coloration, and refractive properties) make it hard to distinguish bacteria solely with a light microscope (1, 2). Staining bacteria is necessary when distinguishing bacterial types with light microscopy. The two main types of light microscopes are simple and compound. The main difference between them is the number of lenses used to magnify the object. Simple microscopes (for example a magnifying glass) have only one lens to magnify an object, while compound microscopes have several lenses to enhance magnification (Figure 1). Compound microscopes have an objective lens close to the object which collects light to create an image of the object. This is then magnified by the eyepiece (ocular lens) which enlarges the image. Combining the objective lens and eyepiece allows for

  • Microbiology

    13:00
    Plaque Assay: A Method to Determine Viral Titer as Plaque Forming Units (PFU)

    Source: Tilde Andersson1, Rolf Lood1 1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund, Sweden

    Viruses that infect prokaryotic organisms, called bacteriophages or simply phages, were identified in the early 20th century by Twort (1) and d'Hérelle (2) independently. Phages have since been widely recognized for their therapeutic value (3) and their influence on human (4), as well as global, ecosystems (5). Current concerns have fueled a renewed interest in the use of phages as an alternative to modern antibiotics in treatment of infectious disease (6). Essentially all phage research relies on the ability to purify and quantify viruses, also known as a viral titer. Initially described in 1952, this was the purpose of the plaque assay (7). Decades and multiple technological advancements later, the plaque assay remains one of the most reliable methods for determination of viral titer (8). Bacteriophages subsist by injecting their genetic material into host cells, hijacking the machineries for production of new phage particles, and eventually causing the host to release numerous progeny virions through cell lysis. Because of their minute size, bacteriophages cannot be observed using solely light microscopy; therefore, scanning electron microscopy is required (Figure 1). Additionally, phages cann

  • Microbiology

    10:54
    Transformation of E. coli Cells Using an Adapted Calcium Chloride Procedure

    Source: Natalia Martin1, Andrew J. Van Alst1, Rhiannon M. LeVeque1, and Victor J. DiRita1 1 Department of Microbiology and Molecular Genetics, Michigan State University

    Bacteria have the ability to exchange genetic material (DeoxyriboNucleic Acid, DNA) in a process known as horizontal gene transfer. Incorporating exogenous DNA provides a mechanism by which bacteria can acquire new genetic traits that allow them to adapt to changing environmental conditions, such as the presence of antibiotics or antibodies (1) or molecules found in natural habitats (2). There are three mechanisms of horizontal gene transfer: transformation, transduction, and conjugation (3). Here we will focus on transformation, the ability of bacteria to take up free DNA from the environment. In the laboratory, the transformation process has four general steps: 1) Preparation of competent cells, 2) Incubation of competent cells with DNA, 3) Recovery of cells, and 4) Plating of the cells for growth of the transformants (Figure 1). Figure 1: General steps of the transformation process. The transformation process has four general steps: 1) Preparation of competent cells, 2) Incubation with DNA, 3) Recovery of the cells and 4) Plating cells for growth of the transformants. For transformation to occur, the recipient bacteria must be in a state known as competence. Some bacteria have

  • Microbiology

    12:52
    Conjugation: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

    Source: Alexander S. Gold1, Tonya M. Colpitts1 1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases Laboratories, Boston, MA

    First discovered by Lederberg and Tatum in 1946, conjugation is a form of horizontal gene transfer between bacteria that relies on direct physical contact between two bacterial cells (1). Unlike other forms of gene transfer, such as transformation or transduction, conjugation is a naturally occurring process in which DNA is secreted from a donor cell to a recipient cell in a unidirectional manner. This directionality and the ability for this process to increase the genetic diversity of bacteria has given conjugation the reputation as a form of bacterial "mating," which is believed to have contributed greatly to the recent rise in antibiotic resistant bacteria (2, 3). By using selective pressures, for example the use of antibiotics, conjugation has been manipulated for use in the laboratory setting, making it a powerful tool for horizontal gene transfer between bacteria, and in some cases from bacteria to yeast, plant, and animal cells (4). Apart from applications in the laboratory, bacterium-eukaryote gene transfer by conjugation is an exciting avenue of DNA transfer with a multitude of possible biotechnical applications and naturally occurring implications (5). Conjugation is thought to wo

  • Microbiology

    11:21
    Phage Transduction: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

    Source: Alexander S. Gold1, Tonya M. Colpitts1 1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases Laboratories, Boston, MA

    Transduction is a form of genetic exchange between bacteria that utilizes bacteriophages, or phages, a class of virus that infects exclusively prokaryotic organisms. This form of DNA transfer, from one bacterium to another by way of a phage, was discovered in 1951 by Norton Zinder and Joshua Ledererg, who termed the process "transduction" (1). Bacteriophages were first discovered in 1915 by British bacteriologist Frederick Twort, then independently discovered again in 1917 by French-Canadian microbiologist Felix d'Herelle (2). Since then, the structure and function of these phages have been widely characterized (3), dividing these phages into two classes. The first of these classes are the lytic phages which upon infection multiply within the host bacterium, disrupting the bacterial metabolism, lysing the cell, and releasing progeny phage (4). As a result of this anti-bacterial activity and the increasing prevalence of antibiotic-resistant bacteria, these lytic phages have recently proved useful as a substitute treatment for antibiotics. The second of these classes are the lysogenic phages which can either multiply within the host via the lytic cycle or enter a quiescent state in which their ge

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