Lecture 9: Growth and Culturing of Bacteria - 2 PDF

Lecture 9 Chapter 7 in textbook Growth and Culturing of Bacteria - 2 Qiang Chen [email protected] Copyright © 20...

Lecture 9 Chapter 7 in textbook Growth and Culturing of Bacteria - 2 Qiang Chen [email protected] Copyright © 2018 by John Wiley & Sons, Inc. All rights reserved.  Chapter Target  Bacterial Growth and Division  Factors Affecting Bacterial Growth  Culturing Bacteria and Medium Adaptation to Limited Nutrients 1. Some synthesize increased amounts of enzymes for uptake and metabolism of limited nutrients. 2. Others have the ability to synthesize enzymes needed to use a different nutrient. Glucose to Lactose 3. Many organisms adjust the rate at which they metabolize nutrients and the rate at which they synthesize molecules required for growth, no energy is wasted. 4. Some bacteria can form endospores to adapt limited nutrients. Sporulation (產孢)  The formation of endospores  Occurs in Bacillus, Clostridium and a few other Gram-positive genera  Protective and survival mechanism but NOT a means of reproduction for bacteria.  Different from fungal spores  Respond to environmental, metabolic and cell cycle signals. Such as decrease in the level of carbon or nitrogen Core structure RNA, protein and DNA make the core, dipicolinic acid (吡啶二羧酸), calcium ions Cortex (孢子皮層) Against osmotic pressure Spore coat Impervious to chemical Sporulation The vegetative and sporulation cycles Sporulation https://www.youtube.com/watch?v=VbDHV7j5-PQ Factors Affecting Bacterial Growth Bacterial Interactions Organisms live in association with different species: Antagonistic relationships (拮抗) Synergistic relationships (協同) Symbiotic relationships (共生) Fight Cooperation Toxin production Bioluminescence  Bacterial Interactions Quorum Sensing (群體感應)  Quorum: - Meaning in Chinese: 法定人數 - Meaning in English: The minimum number of members of an assembly or society that must be present at any of its meetings to make the proceedings of that meeting valid.  Quorum sensing: - The regulation of gene expression in response to fluctuations in cell-population density. - A way that bacteria communicate with each other Quorum Sensing When enough bacteria (a quorum) are present, the inducer molecules bacteria released will be accumulated sufficiently to activate specific adaptive genes. Biofilms (生物膜) A biofilm is any group of microorganisms in which cells stick to each other and often these cells adhere to a surface. Enclosed a matrix of proteins, DNA and sugars Bacteria within biofilm become antibiotics resistance. Exchange of genetic material between organisms within a biofilm leads to genetic diversity. Bacteria growing near each other “sense” the others’ presence and release lethal factors to kill them. Biofilms  In medicine - Around two-thirds of bacterial infections in humans involve biofilms - Displaying antimicrobial tolerance and immune response evasions  In Industry - An implied cause of premise plumbing corrosion (管道腐蝕)  In aquaculture - Creating microenvironments that are more favorable for biofouling (生物淤積) species, which block nets and cages of the farmed species. https://en.wikipedia.org/wiki/Biofilm#Uses_and_impact Biofilms Major Issue on Public Healthcare https://www.youtube.com/watch?v=fpPWbdK9Dno Biofilms Technological approaches to combat biofilms Nat Rev Microbiol. 2017, 15(12): 740-755. Biofilms Blushwood tree 胭脂樹 Biofilms 泊庫島食烷菌 Microbes reshape oil droplets to speed biodegradation. Science. 2023, 381(6659):748-753. Culturing Bacteria Methods to obtain pure cultures Aseptic technique (無菌技術) prevents contamination of sterile substances or objects. 70% Alcohol Bunsen burner Serial dilution ? Serial dilution was unsuccessful for single 200 μL diluted cell isolation. sample contains one bacteria ? Methods to obtain pure cultures  Streak Plate Method  Pour Plate Method Microaerophiles Aseptic Technique and Streak Plate Method https://www.youtube.com/watch?v=fND5I_A7wNM Methods to obtain pure cultures Some fungi isolated with streak and pour plates Protozoa and motile unicellular algae isolated through dilution of broth cultures For some large microorganisms, single cell can be individually picked and used to establish a culture  Culture Media  Not all bacteria in natural environment can be cultured in laboratory  Types of media State of matter Liquid: nutrient broth is a common liquid medium Solid: agar (2%) is a common addition to make media solid, petri plates and slant tubes Semi solid: 0.5% agar Composition Defined synthetic medium: contains known specific kinds and amounts of chemical substance Complex medium (chemically non-defined medium): contains reasonably familiar material but varies slightly in chemical composition from batch to batch, exact chemical composition is unknown Defined synthetic medium Complex medium 蛋白胨 牛肉浸膏 Culture Media Purposes Purpose  Selective media Encourage the growth of some organisms but suppresses the growth of the others  Differential media Has a constituent that causes an observable changes (color or pH) in medium  Enrichment media Promotes the growth of a particular organism by providing it with the essential nutrients Culture Media Purposes Selective medium Soil samples Culture Media Purposes Differential medium Lactose Broth Durham tube (inverted tube to trap gas) No fermentation Acid fermentation with gas Culture Media Purposes Selective and differential medium MacConkey agar (麥康凱瓊脂培養基) Crystal violet and bile salts inhibit gram-positive bacteria Lactose and the pH indicator neutral red identify lactose fermenters as red colonies and non-fermenters as colorless and translucent Culture Media Purposes The use of MacConkey agar as a selective and differential medium Culture Media Purposes Mannitol salt agar (MSA，甘露醇鹽瓊脂培養基) High concentration (about 7.5–10%) of salt (NaCl) Mannitol and the indicator phenol red Pathogenic Staphylococcus aureus ferments mannitol (甘露醇), producing acid that turns the pH indicator (phenol red) in the medium to yellow. Controlling Oxygen Content  Aeration Provide sufficient oxygen for obligate aerobes  Candle Jar For microaerophiles  Anaerobic Jar/Chamber For obligate anaerobes Bacterial Identification Bacteria  Gram Staining  Morphology  Oxygen Requirement  Biochemical Test Methods of Performing Multiple Diagnostic Tests  Analytical Profile Index (API) 967 bacterial and yeast species 20 biochemical tests Seven-digit profile number Most organisms can not be cultured. Eventually, they may be identified by their DNA (16s rRNA sequencing). Preserving Bacterial Cultures  Refrigeration - Stores for short periods of time  Deep-freezing - A process in which a pure culture of microbes is placed in a suspending liquid and quick-frozen at temperature ranging from -50 ºC to -80 ºC. - Antifreeze: glycerol - Stores for years  Lyophilization (Freeze drying, 凍乾) - A suspension of microbes is quickly frozen at temperature ranging from -50ºC to -80ºC, and the water is removed by a high vacuum. - Keep at room temperature - Stores for decades  American Type Culture Collection (ATCC，美國 典型培養物保藏中心) - Reference