DOCSLIB.ORG

Microbial Growth Media & Culturing Protocol

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Microbial Growth Media & Culturing Protocol Microbial Growth Media & Culturing Protocol (Biotech Series) Microbes such as bacteria, fungi, and viruses are like microscopic factories in biotechnology. Bacteria are the key constituent to most biotechnological processes and have revolutionized science and medicine. As part of the DNA revolution, biotechnologies have been developed for introducing DNA (such as a gene) from one species into another. Bacteria are particularly good at accepting foreign DNA, and introducing genes into bacterial cells is now a routine procedure. This procedure is a first step to making bacteria that can do new and useful things for society. In the biotechnology pathway you will routinely be culturing, cloning and transforming bacteria. This protocol details the basic procedures for preparing growth media such as agar plates, nutrient broth and slants. This protocol must be used in conjunction with Aseptic Technique Protocol (click here) and the Autoclave Protocol (click here). Mastering these critical laboratory skills we help to ensure your success as we progress with more advanced biotechnological techniques. Please refer to textbook pages 63-66 for additional details. Media are classified on the basis of composition or application. Usually, a particular medium may fall into two or more categories, which leads to some confusion among students. Thus, for example, a "defined" medium might be also classified as "selective" or "differential" depending upon its specific properties and application in a particular situation. The four principal types of culture media are described below. COMPLEX MEDIA: Pasteur, Koch, and Tyndall typically prepared media by boiling animal or plant materials to extract nutritive molecules. Today, many modern complex media (such as Tryptic Soy Agar) contain extracts of beef (peptone), milk (tryptone), soybean meal (soytone), or yeast. The proteins in these extracts are broken down into small peptides and amino acids. Although the specific amount of these molecules is not precisely determined, their wide assortment allows complex media to support a wide range of bacterial types. Examples may include nutrient agar, lysogeny broth (LB) or Luria-Bertani agar, Miller and Lennox LB, sheep blood, etc. DEFINED MEDIA: Defined media are formulated from pure substances at predetermined concentrations. Thus, unlike complex media, the exact chemical composition of defined media is known precisely. Because the composition is precisely established, defined media are often used to determine the nutritional requirements of bacterial species. SELECTIVE MEDIA: Complex or defined media may also be classified as selective (or enrichment) media, which support the growth of only certain types of bacteria. Media can be made selective through the addition of substances that enhance or inhibit the growth of particular types of bacteria. Media have been developed that are selective for an astonishing diversity of bacteria, and we will be using many of these media throughout the semester. DIFFERENTIAL MEDIA: Any of the above types of media might also be formulated as a differential medium. A differential medium reveals specific metabolic or metabolic characteristics of bacteria grown on it. Differential media are among the most powerful tools available to biotechnologists, revealing a wide range of information about an organism very quickly. Some media are both selective and differential. For example, the medium called MacConkey Agar is selective for gram-negative bacteria and will indicate whether bacteria can ferment lactose. Nutrient Broth, Agar Plates and Slants: The immense amount of variation in recipes for growth media is beyond the scope of this protocol; however, all recipes that will be used in the Biotechnology pathway will be archived and maintained in a separate logbook. A few of the most basic recipes are detailed on the next pages along with several schematic diagrams for inoculating, streaking and culturing microbes. Nutrient Broth: 1. Weigh out 8.0 grams of nutrient broth powder. 2. Add to 1.0 liter of distilled or deionized water in a 2.0 liter flask. 3. Dissolve the powder completely in the water. 4. Dispense into tubes or flasks using a graduated cylinder or a pipette. 5. Sterilize at 121° C for 20-25 minutes. Nutrient Agar Plates: 1. Weigh out 23.0 grams of nutrient agar powder. 2. Add to 1.0 liter of distilled or deionized water in a 2.0 liter flask. 3. Sterilize at 121° C for 20-25 minutes. Figure 1: Streaking an agar plate 4. Cool to 50° C. is done in 3-4 sections. The goal is 5. Swirl thoroughly to mix agar and nutrients. that sections “D” and “E” will 6. Pour 25-35 ml per petri plate. yield isolated, individual colonies. 7. Yields about 35 plates. Nutrient Agar Slants: 1. Place screw cap test tubes in a test tube rack (without the caps). 2. Prepare a nutrient agar medium and boil it with stirring until all agar is melted. You must stir this very well so that the melted agar is distributed throughout the medium. 3. Use a pipette to transfer ~5 ml of molten agar to each test tube. 4. When all tubes contain hot agar, place caps loosely on the tubes. 5. Sterilize the tubes at 121° C for 15 minutes with caps loosely on. Figure 2: Streaking and 6. While the medium is still hot, tilt the rack onto a thick book or other inoculating a slant. solid surface so that the medium in the tubes is slanted. Allow the medium to harden in this position. 7. When the medium is cool, tighten the caps. 8. These tubes can be stored at room temperature or in the refrigerator. Figure 3: Always label the bottom of Figure 4: Technique for streaking a plate the petri before pouring media. in three sections using an inoculating needle. .
Load more

Recommended publications
  • A General Protein O-Glycosylation Gene Cluster Encodes the Species-Specific Glycan of the Oral Pathogen Tannerella Forsythia
    fmicb-09-02008 August 27, 2018 Time: 10:24 # 1 ORIGINAL RESEARCH published: 28 August 2018 doi: 10.3389/fmicb.2018.02008 A General Protein O-Glycosylation Gene Cluster Encodes the Species-Specific Glycan of the Oral Pathogen Tannerella forsythia: Edited by: O-Glycan Biosynthesis and Catherine Ayn Brissette, University of North Dakota, Immunological Implications United States Reviewed by: Markus B. Tomek1, Daniel Maresch2, Markus Windwarder2†, Valentin Friedrich1, Jerry Eichler, Bettina Janesch1, Kristina Fuchs1, Laura Neumann2†, Irene Nimeth1, Nikolaus F. Zwickl3, Ben-Gurion University of the Negev, Juliane C. Dohm3, Arun Everest-Dass4, Daniel Kolarich4, Heinz Himmelbauer3, Israel Friedrich Altmann2 and Christina Schäffer1* Yoann Rombouts, UMR5089 Institut de Pharmacologie 1 NanoGlycobiology Unit, Department of NanoBiotechnology, Universität für Bodenkultur Wien, Vienna, Austria, 2 Division et de Biologie Structurale (IPBS), of Biochemistry, Department of Chemistry, Universität für Bodenkultur Wien, Vienna, Austria, 3 Bioinformatics Group, France Department of Biotechnology, Universität für Bodenkultur Wien, Vienna, Austria, 4 Institute for Glycomics, Griffith University, *Correspondence: Brisbane, QLD, Australia Christina Schäffer [email protected] The cell surface of the oral pathogen Tannerella forsythia is heavily glycosylated with † Present address: a unique, complex decasaccharide that is O-glycosidically linked to the bacterium’s Markus Windwarder, Shire Austria GmbH, Vienna, Austria abundant surface (S-) layer, as well
    [Show full text]
  • Growth Conditions and Experimental Setup for Bacterial Growth and the Fading of Phenolphthalein in Alkaline Solution
    GROWTH CONDITIONS AND EXPERIMENTAL SETUP FOR BACTERIAL GROWTH AND THE FADING OF PHENOLPHTHALEIN IN ALKALINE SOLUTION Bachelor Degree Project in Cell and Molecular Biology 15p Autumn term 2011 Kalle Backlöf Supervisor: Mikael Ejdebäck Examiner: Diana Karlsson Abstract The whole project consisted of two different parts. The first subproject were running optical density measurements to assist in finding optimal growth conditions and experimental setup for use of E.coli BL21 (DE3) bacteria with the PYCARD gene transformed into them. The transformed bacteria will be used for generating data for modeling. Previous laboratory attempts had problems with timing of the exponential phase when several tests were performed simultaneously. The optimizations of a method for displaying growth include trying out using different medium but also different inoculation ratios between culture and medium to see the effects on growth rate. Results have shown that lysogeny broth together with an inoculation ratio of 1:25 results in rapid absorbance increase in the bacterial growth curves. The second part of the thesis project was to study the kinetics of fading of phenolphthalein in sodium hydroxide solution in order to provide a basis for experiments illustrating this in laboratory environment. Phenolphthalein is used in many different applications such as an active ingredient in some laxatives but the perhaps most common use is as an acid-base indicator of pH. The experiments were performed by mixing alkaline solutions of varying sodium hydroxide concentration together with sodium chloride, then after addition of phenolphthalein to study absorbance variations over time to illustrate the rate law which correlates the rate of color fade to sodium hydroxide concentration.
    [Show full text]
  • Segregating Metabolic Processes Into Different Microbial Cells Accelerates the Consumption of Inhibitory Substrates
    The ISME Journal (2016) 10, 1568–1578 © 2016 International Society for Microbial Ecology All rights reserved 1751-7362/16 www.nature.com/ismej ORIGINAL ARTICLE Segregating metabolic processes into different microbial cells accelerates the consumption of inhibitory substrates Elin E Lilja1,2 and David R Johnson1 1Department of Environmental Microbiology, Eawag, Dübendorf, Switzerland and 2Department of Environmental Systems Science, ETH Zürich, Zürich, Switzerland Different microbial cell types typically specialize at performing different metabolic processes. A canonical example is substrate cross-feeding, where one cell type consumes a primary substrate into an intermediate and another cell type consumes the intermediate. While substrate cross-feeding is widely observed, its consequences on ecosystem processes is often unclear. How does substrate cross-feeding affect the rate or extent of substrate consumption? We hypothesized that substrate cross-feeding eliminates competition between different enzymes and reduces the accumulation of growth-inhibiting intermediates, thus accelerating substrate consumption. We tested this hypothesis using isogenic mutants of the bacterium Pseudomonas stutzeri that either completely consume nitrate to dinitrogen gas or cross-feed the intermediate nitrite. We demonstrate that nitrite cross- feeding eliminates inter-enzyme competition and, in turn, reduces nitrite accumulation. We further demonstrate that nitrite cross-feeding accelerates substrate consumption, but only when nitrite has growth-inhibiting
    [Show full text]
  • Genome Sequence of Pseudomonas Stutzeri 273 and Identification of the Exopolysaccharide EPS273 Biosynthesis Locus
    Article Genome Sequence of Pseudomonas stutzeri 273 and Identification of the Exopolysaccharide EPS273 Biosynthesis Locus Shimei Wu 1, Rikuan Zheng 2,3,4, Zhenxia Sha 1,* and Chaomin Sun 2,4,* 1 College of Life Sciences, Qingdao University, Qingdao 266071, China; [email protected] 2 Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; [email protected] 3 College of Earth Science, University of Chinese Academy of Sciences, Beijing 100049, China 4 Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China * Correspondence: [email protected] (Z.S.); [email protected] (C.S.); Tel.: +86-532-82898857 (C.S.); Fax: +86-532-82898648 (C.S.) Academic Editor: Anake Kijjoa Received: 25 April 2017; Accepted: 5 July 2017; Published: 10 July 2017 Abstract: Pseudomonas stutzeri 273 is a marine bacterium producing exopolysaccharide 273 (EPS273) with high anti-biofilm activity against P. aeruginosa PAO1. Here, the complete genome of P. stutzeri 273 was sequenced and the genome contained a circular 5.03 Mb chromosome. With extensive analysis of the genome, a genetic locus containing 18 genes was predicted to be involved in the biosynthesis of EPS273. In order to confirm this prediction, two adjacent genes (eps273-H and eps273-I) encoding glycosyltransferases and one gene (eps273-O) encoding tyrosine protein kinase within the genetic locus were deleted and biosynthesis of EPS273 was checked in parallel. The molecular weight profile of EPS purified from the mutant Δeps273-HI was obviously different from that purified from wild-type P.
    [Show full text]
  • Bacterial Cultures Care Guide SCIENTIFIC Introduction Use the Following Guide to Learn How to Properly Care for Bacterial Cultures
    Bacterial Cultures Care Guide SCIENTIFIC Introduction Use the following guide to learn how to properly care for bacterial cultures. BIO FAX! Safety Precautions After use, agar plates will likely contain viable microbes. Although the bacteria are not likely to be pathogenic, do not open the plates unnecessarily. Use sterile techniques at all times when handling bacterial cultures. When plates are done being used, they should be autoclaved or opened under a 10% bleach solution and soaked for at least one hour. Bleach solution is a corrosive liquid that may dis- color clothing and cause skin burns. Avoid contact of bleach with heat, acids and organic materials; chlorine gas will be generated. Wear chemical splash goggles, chemical-resistant gloves, and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines. Culturing and Maintenance Prior to receiving bacterial cultures it is helpful to be aware of the required conditions for the particular strain of bacteria purchased. Each species has specific conditions that are necessary for optimal growth. Refer to the Bacterial Cultures Table below for incubation temperature and the appropriate general medium. Bacterial Cultures Table Description (Genus species) Incubation Temperature† Medium Bacillus cereus (20–35) 30 °C Nutrient agar Bacillus mycoides 30 °C Nutrient agar Bacillus megaterium (25–35) 30 °C Nutrient agar Bacillus subtilis (25–35) 25–30 °C Nutrient agar Enterobacter aerogenes (30–37) 30 °C Nutrient
    [Show full text]
  • Xylella Fastidiosa Outer Membrane Vesicles Modulate Plant Colonization by Blocking Attachment to Surfaces
    Xylella fastidiosa outer membrane vesicles modulate plant colonization by blocking attachment to surfaces Michael Ionescua,1, Paulo A. Zainib,1, Clelia Baccaria, Sophia Trana, Aline M. da Silvab, and Steven E. Lindowa,2 aDepartment of Plant and Microbial Biology, University of California, Berkeley, CA 94720; and bDepartamento de Bioquímica, Instituto de Química, Universidade de São Paulo, SP 05508-000, São Paulo, Brazil Contributed by Steven E. Lindow, August 8, 2014 (sent for review July 10, 2014) Outer membrane vesicles (OMVs) of Gram-negative bacteria have signaling factors (DSF) with increasing cell concentration in- been studied intensively in recent years, primarily in their role in creases the adhesiveness of the cells, presumably better to enable delivering virulence factors and antigens during pathogenesis. their acquisition by insect vectors, but reduces their ability to However, the near ubiquity of their production suggests that they move and multiply within plants. These observations support the may play other roles, such as responding to envelope stress or hypothesis that XfDSF signaling is used in a context-dependent trafficking various cargoes to prevent dilution or degradation by lifestyle switch that enables a subset of the bacterial cells in a other bacterial species. Here we show that OMVs produced by plant to become more adhesive, and thus able to be acquired by Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, insects, by inducing a phenotype incompatible with the move- block its interaction with various surfaces such as the walls of xylem ment of the more solitary cells throughout the plant (6). vessels in host plants. The release of OMVs was suppressed by the A recent study (16) indicated that an extracellular factor pro- diffusible signal factor-dependent quorum-sensing system, and a duced by X.
    [Show full text]
  • Pseudomonas Stutzeri As an Alternative Host for Membrane Proteins Manuel Sommer, Hao Xie* and Hartmut Michel*
    Sommer et al. Microb Cell Fact (2017) 16:157 DOI 10.1186/s12934-017-0771-0 Microbial Cell Factories RESEARCH Open Access Pseudomonas stutzeri as an alternative host for membrane proteins Manuel Sommer, Hao Xie* and Hartmut Michel* Abstract Background: Studies on membrane proteins are often hampered by insufcient yields of the protein of interest. Several prokaryotic hosts have been tested for their applicability as production platform but still Escherichia coli by far is the one most commonly used. Nevertheless, it has been demonstrated that in some cases hosts other than E. coli are more appropriate for certain target proteins. Results: Here we have developed an expression system for the heterologous production of membrane proteins using a single plasmid-based approach. The gammaproteobacterium Pseudomonas stutzeri was employed as a new production host. We investigated several basic microbiological features crucial for its handling in the laboratory. The organism belonging to bio-safety level one is a close relative of the human pathogen Pseudomonas aeruginosa. Pseu- domonas stutzeri is comparable to E. coli regarding its growth and cultivation conditions. Several efective antibiotics were identifed and a protocol for plasmid transformation was established. We present a workfow including cloning of the target proteins, small-scale screening for the best production conditions and fnally large-scale production in the milligram range. The GFP folding assay was used for the rapid analysis of protein folding states. In summary, out of 36 heterologous target proteins, 20 were produced at high yields. Additionally, eight transporters derived from P. aeruginosa could be obtained with high yields. Upscaling of protein production and purifcation of a Gluconate:H+ Symporter (GntP) family transporter (STM2913) from Salmonella enterica to high purity was demonstrated.
    [Show full text]
  • Plasmid DNA Production in Proteome-Reduced Escherichia Coli
    microorganisms Communication Plasmid DNA Production in Proteome-Reduced Escherichia coli Mitzi de la Cruz 1 , Elisa A. Ramírez 1, Juan-Carlos Sigala 1, José Utrilla 2 and Alvaro R. Lara 1,* 1 Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana-Cuajimalpa, Mexico City 05348, Mexico; [email protected] (M.d.l.C.); [email protected] (E.A.R.); [email protected] (J.-C.S.) 2 Systems and Synthetic Biology Program, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca 62210, Mexico; [email protected] * Correspondence: [email protected] Received: 31 July 2020; Accepted: 11 September 2020; Published: 21 September 2020 Abstract: The design of optimal cell factories requires engineering resource allocation for maximizing product synthesis. A recently developed method to maximize the saving in cell resources released 0.5% of the proteome of Escherichia coli by deleting only three transcription factors. We assessed the capacity for plasmid DNA (pDNA) production in the proteome-reduced strain in a mineral medium, lysogeny, and terrific broths. In all three cases, the pDNA yield from biomass was between 33 and 53% higher in the proteome-reduced than in its wild type strain. When cultured in fed-batch mode in 1 shake-flask, the proteome-reduced strain produced 74.8 mg L− pDNA, which was four times greater than its wild-type strain. Nevertheless, the pDNA supercoiled fraction was less than 60% in all cases. Deletion of recA increased the pDNA yields in the wild type, but not in the proteome-reduced strain. Furthermore, recA mutants produced a higher fraction of supercoiled pDNA, compared to their parents.
    [Show full text]
  • A Mutation in the Promoter of the Pmrd Gene of Shigella Flexneri Abrogates Functional Phopq- Pmrd-Pmrab Signaling and Polymyxin B Resistance
    bioRxiv preprint doi: https://doi.org/10.1101/2021.07.26.453917; this version posted July 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. A mutation in the promoter of the pmrD gene of Shigella flexneri abrogates functional PhoPQ- PmrD-PmrAB signaling and polymyxin B resistance. Raymond Huynh and Joseph B. McPhee Department of Chemistry and Biology, Ryerson University, Toronto ON, M5B 2K3 Running title: Shigella flexneri encodes non-functional pmrD bioRxiv preprint doi: https://doi.org/10.1101/2021.07.26.453917; this version posted July 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract Shigella spp. are the causative agent of bacillary dysentery, a major cause of food-borne morbidity and mortality worldwide. These organisms are recently evolved, polyphyletic pathovar of E. coli, and since their divergence they have undergone multiple cases of gene gain and gene loss and understanding how gene inactivation events alter bacterial behaviour represents an important objective to be better able to understand how virulence and other phenotypes are affected. Here, we identify a frameshift mutation in the pmrD gene of S. flexneri that although it would be predicted to make a functional, full-length protein, no such production occurs, likely due to the non-optimal spacing between the translational initiation site and the Shine-Dalgarno sequence.
    [Show full text]
  • Escherichia Coli Transformation by Heat Shock and Electroporation: a Comparative Study
    Escherichia Coli Transformation by Heat Shock and Electroporation: A Comparative Study Nikita Shahidadpury Undergraduate Research Thesis University of Florida Department of Chemistry Research Advisor: Dr. Charles R. Martin 7/25/2017 Table of Contents Acknowledgements .......................................................................................................... 3 Abstract ............................................................................................................................ 4 Abbreviations .................................................................................................................... 5 Introduction ....................................................................................................................... 6 Bacterial Transformation ............................................................................................... 6 Chemical Transformation .............................................................................................. 6 Transformation via Electroporation ............................................................................... 7 Low-Voltage Electroporation Device ............................................................................. 8 The Chosen Plasmid Vector: pGFPuv .......................................................................... 8 Methods .......................................................................................................................... 11 Preparation of LB Agar Plates ...................................................................................
    [Show full text]
  • Visualizing the Dynamics of Exported Bacterial Proteins with The
    www.nature.com/scientificreports OPEN Visualizing the dynamics of exported bacterial proteins with the chemogenetic fuorescent reporter FAST Yankel Chekli1,2, Caroline Peron‑Cane3,4, Dario Dell’Arciprete3, Jean‑François Allemand3,4, Chenge Li5, Jean‑Marc Ghigo1, Arnaud Gautier5,6,7, Alice Lebreton4,8, Nicolas Desprat3,4* & Christophe Beloin1* Bacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localisation of surface proteins such as adhesins, transporters or hydrolases, monitoring their dynamics in live imaging remains challenging, due to the limited availability of fuorescent probes remaining functional after secretion. In this work, we used the Escherichia coli intimin and the Listeria monocytogenes InlB invasin as surface exposed scafolds fused with the recently developed chemogenetic fuorescent reporter protein FAST. Using both membrane permeant (HBR‑3,5DM) and non‑permeant (HBRAA‑3E) fuorogens that fuoresce upon binding to FAST, we demonstrated that fully functional FAST can be exposed at the cell surface and used to specifcally tag the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study the organization and dynamics of the bacterial cell surface proteins. Te study of protein localisation dynamics using fuorescent reporters has led to major insights into many bio- logical processes. For instance, the use of fuorescent reporters enabled to show that MreB, the actin-like protein found in bacteria, spatially dictates the subcellular sites of cell wall synthesis 1,2. Super folder-GFP fusions with the MinC, D and E proteins, allowed the observation of their oscillation from pole to pole clarifying the role of the Min system in the division septum positioning3,4.
    [Show full text]
  • PBS – Phosphate Buffered Saline Luria Broth (LB)
    Brief Notes on the composition of various biochemical used in bacteria culture PBS – Phosphate Buffered Saline Phosphate buffered saline (PBS) is a buffer solution commonly used in biological research. The buffer helps to maintain a constant pH. Generally a pH of 7.4 is maintained. The osmolarity and ion concentrations of the solution usually match those of the human body. Its ingredients are:- 1. Sodium chloride(NaCl ) 2. Sodium phosphate,(KCl) 3. Potassium chloride(Na2HPO4) 4. Potassium phosphate(KH2PO4) PBS has many uses because it is isotonic and non-toxic to cells. It can be used to dilute substances. It is used to rinse containers containing cells. PBS can be used as a diluent in methods to dry biomolecules, as water molecules within it will be structured around the substance (protein, for example) to be 'dried' and immobilized to a solid surface. The thin film of water that binds to the substance prevents denaturation or other conformational changes. Luria Broth (LB) It is also called as Lysogeny Broth(LB). It is a nutritionally rich medium used for the growth of bacteria. This media has been widely used in medical biology for the preparation of plasmid DNA and recombinant proteins. It is largely used as medium for the cultivation of Escherichia Coli Bacteria. Ingredients of Luria Broth are:- 1. Peptides and casein peptones provided by tryptone 2. Vitamins (including B vitamins) provided by yeast extract 3. Trace elements (e.g. nitrogen, sulfur, magnesium) 4. Minerals Bacto-tryptone is used to provide essential amino acids to the growing bacteria, while the bacto- yeast extractFor is used to provide Vitula a plethora of organic compounds Lab helpful for bacterial Only growth.
    [Show full text]