Microbial Nutrition and Growth Chapter 6 Microbial Nutrition and Growth

Metabolism Results in Reproduction Reproduction results in Growth Microbial Growth Metabolism Results in Reproduction Reproduction results in Growth What is microbial growth? an increase in a population of microbes (rather than an increase in size of an individual) Result of microbial growth? a discrete colony – an aggregation of cells arising from single parent cell Animations: Bacterial Growth Overview

Factors affecting growth: Nutritional Factors Nutrients – chemicals taken in and used by organisms for energy, metabolism and growth Water (Hydrogen and Oxygen) Carbon Nitrogen Sulfur Phosphorus Trace Elements Growth Factors

Factors affecting growth: Nutritional Factors Macronutrients - Required in large amounts Carbon Needed for synthesis of cellular material and energy source Nitrogen Needed for protein synthesis, nucleic acids, ATP Sulfur Needed to synthesize amino acids and vitamins (thiamine, biotin) Phosphorus Needed to synthesize nucleic acids, ATP, phospholipids

Factors affecting growth: Nutritional Factors Trace Elements required in trace amounts involved in enzyme function and protein structure Examples: Zn, Cu, Fe Present in tap water and distilled Growth factors Organic compounds that cannot be synthesized by bacteria bacteria are “fastidious” examples: amino acids, purines, pyrimidines, vitamins Fastidious organisms require relatively large amounts of growth factors in the media. Can be used to test samples for presence of growth factors EXP. Euglena granulata reguires Vitamin B12 to grow and the amount of Vitamin B12 affects amount of growth.

Growth Requirements Nutrients: Chemical and Energy Requirements Sources of carbon, energy, and electrons Two groups of organisms based on source of carbon Autotrophs Heterotrophs Two groups of organisms based on source of energy Chemotrophs Phototrophs

Sources of Carbon, Energy, and Electrons Carbon sources Organisms are categorized into two groups: Autotrophs Those using an inorganic carbon source (carbon dioxide) Heterotrophs Those catabolizing organic molecules (proteins, carbohydrates, amino acids, and fatty acids)

Sources of Carbon, Energy, and Electrons Energy sources Organisms are categorized into two groups: Chemotrophs Acquire energy from redox reactions involving inorganic and organic chemicals Phototrophs use light as their energy source

Groups of organisms based on carbon and energy source Figure 6.1

Oxygen sources Oxygen Requirements Found as gaseous O2 or covalently bound in compounds Essential for aerobic respiration Oxygen is the final electron acceptor Deadly for some types of bacteria (anaerobes) Toxic forms of oxygen are highly reactive are excellent oxidizing agents Results in irreparable damage to cells by oxidizing compounds such as proteins and lipids http://www.exrx.net/Nutrition/Antioxidants/Introduction.html

Toxic Forms of Oxygen Singlet oxygen: 1O2 Oxygen boosted to a higher-energy state; extremely reactive Superoxide free radicals: O2 O2 + 2H+ H2O2 +O2 Superoxide Dismutase Peroxide anion: O22 2H2O2 2 H2O + O2 Catalase H2O2 + NADH+H+ 2 H2O Peroxidase Hydroxyl radical (OH) Result of ionizing radiation & incomplete reduction of hydrogen peroxide; extremely reactive but danger averted in aerobes because of catalase & peroxidase

Oxygen and Carbon Dioxide Requirements Aerobes – must use oxygen and can detoxify it Anaerobes- can not use oxygen nor detoxify it Facultative anaerobes- do not require oxygen but can use and detoxify it Aerotolerant anaerobes – can not use aerobic metabolism but have some enzymes to detoxify oxygen’s poisonous forms Microaerophile – requires a small amount of oxygen for growth Capnophile – requires higher CO2 tension (3-10%) than normally found in the atmosphere

Classification of Organisms Based on Oxygen Requirements Microbial Growth is affected by Oxygen Concentration Obligate aerobes Facultative anaerobes Obligate anaerobes Aerotolerant anaerobes Microaerophiles

Nitrogen Requirements Nitrogen Sources Acquired from organic and inorganic nutrients Recycled from amino acids and nucleotides to make other proteins and nucleotides Nitrogen fixation- Nitrogen gas reduced to ammonia Essential to life on Earth because nitrogen is made available in a usable form

Factors that Affect Microbial Growth Temperature – Affects proteins and lipid membranes If too low, membranes become rigid and fragile If too high, membranes become too fluid Categories based on Optimum Temperature Psychrophile – optimum below 15oC Mesophile – optimum between 20oC – 40oC Thermophile – optimum higher than 45oC Hyperthermophiles – optimum above 80oC

Effects of temperature on microbial growth Figure 6.4

Catagories of Microbes Based on Temperature Range Figure 6.5

Temperature

Use of Temperature to Preserve Microbes Preserving Bacteria Cultures: Refrigeration: Storage for short periods of time Deep-freezing: -50° to -95°C Preserves cultures for years Lyophilization (freeze-drying): Frozen (-54° to -72°C) and dehydrated in a vacuum Can last decades

Classification of Microbes based on pH Effects of pH Classification of Microbes based on pH Organisms sensitive to changes in acidity H+ and OH– interfere with H bonding Acidophiles – prefer below 7 Neutrophiles – prefer 7 Alkalinophiles – prefer above 7 Most bacteria grow between pH 6.5 and 7.5 Molds and yeasts grow between pH 5 and 6

Physical Effects of Water Microbes require water to dissolve enzymes and nutrients required in metabolism; to react in many metabolic reactions Some microbes have cell walls that retain water Endospores and cysts stop most metabolic activity to survive in a dry environment for years Two physical effects of water Osmotic pressure Hydrostatic pressure

Osmotic Pressure Osmotic pressure The pressure exerted on the semipermeable membrane by a solution containing solutes, which cannot move across the membrane. Osmosis Diffusion of water across a semipermeable membrane driven by unequal concentration of solutes across the membrane.

Osmotic Variations in the Environment Isotonic External concentration of solutes is equal to cell’s internal environment Diffusion of water equal in both directions No net change in cell volume Hypotonic External concentration of solutes is lower than cell’s internal environment Cells swell and burst Hypertonic Environment has higher solute concentration than cell’s internal environment Cells shrivel (crenate) Halophiles tolerate higher salt concentrations

Osmotic Pressure ISOTONIC Physiologic Saline HYPERTONIC

Hydrostatic Pressure Water exerts pressure in proportion to its depth For every addition of depth, water pressure increases 1 atm Organisms that live under extreme pressure are barophiles Their membranes and enzymes depend on this pressure to maintain their three-dimensional, functional shape

Microbial Growth Binary fission Splitting parent cell to form two similar-sized daughter cells to increase number of cells Generation time Duration of each division Determined by type of bacteria Example: E. coli (20 min)

Exponential Growth by Binary Fission DNA replication Cell elongation Septum formation Septum completion leads to separation or further division Process repeats Figure 6.17a

Bacterial Growth Curve Animation: Bacterial Growth Curve Figure 6.20

Bacterial Growth Curve Graph of a closed bacterial population over time Lag phase Acquire cell mass, no reproduction Log (Exponential growth) phase Cells dividing Stationary phase Cells stop growing, cells dividing and dying at same rate Death phase Cells dying due to lack of nutrients and increased waste products

Methods of Culturing Microbes Specimen Collection Taking a sample of infected material Sterile (aseptic) technique required to avoid introducing unwanted microbes into the sample

Clinical Sampling Table 6.3

Culturing Microorganisms Inoculate Implant microbes onto medium (broth or solid) Inoculum Sample of microbes from specimen Culture act of cultivating microorganisms or the microorganisms that are cultivated

Streak Plate Method Pure Culture Most commonly used. Figure 6.8 - Overview

Mixed Culture Can you see 12 different bacterial colonies? Count colonies. A colony is defined as a population of similar cells that arose from one viable cell/ or one endospore. Can you see 12 different bacterial colonies? Figure 6.9 - Overview

Culture Media Used as solidifying agent for culture media Nutrient preparation for microbial growth Must provide all chemical requirements Physical state depends on amount of AGAR Used as solidifying agent for culture media Composed of complex polysaccharides Advantages of agar vs gelatin: Generally not metabolized by microbes Liquefies at 100°C Solidifies ~40°C Fanny Hesse used agar from seaweed in her jams and jellies, which she learned from a neighbor who had lived in Java (Indonesia).

Chemically Defined vs Complex Media Comparison of Media

Types of Media Used in the Clinical Lab Basic Nutrient Designed to grow broad-spectrum microbes Enriched Add enrichment to encourage growth of microbes Blood, growth factors, serum Selective Suppress unwanted microbes and encourage desired microbes to grow Salt, dyes, alcohol

An example of the use of a selective medium Figure 6.12

Types of Media Used in the Clinical Lab Differential To distinguish colonies of different microbes from one another Dyes, pH indicators Reduced (anaerobic) media Contain chemicals (thioglycollate) that combine O2 Used for anaerobic cultures Transport Maintain and preserve microbes Include atmospheric buffers Prevent drying

MacConkey agar as a selective and differential medium Figure 6.15

Anaerobic Culture Methods Gas Pak Jar Glove Box Figure 6.15 - Overview

Capnophiles require high CO2 Candle jar (3-10% CO2) CO2-packet

Environment to Culture Microbes Incubation Temperature 35-37oC – body temperature 25-30oC – room temperature 4-8oC – refrigerator temperature Atmosphere Aerobic - free oxygen present Microaerophilic – free O2 present; increased CO2 Anaerobic – NO free O2 present Time 18 – 24 hours Longer for slow-growing microbes

Planktonic vs Sessile Bacteria All lab tests use “pure cultures” of suspended cells called planktonic bacteria since they float around in liquid. In fact, pure cultures are virtually absent in nature. Most microbes exist as sessile bacteria– attached to a surface – and they live in communities called biofilms.  Robert Koch Courtesy of the National Library of Medicine

Biofilms Biofilms Complex relationships among numerous microorganisms Develop an extracellular matrix Adheres cells to one another Allows attachment to a substrate Sequesters nutrients May protect individuals in the biofilm Form on surfaces often as a result of quorum sensing Many microorganisms more harmful as part of a biofilm

Ingredients needed for a biofilm : What is a biofilm ? An organized, layered system of microbes attached to a surface Biofilms form when microbes adhere to a surface that is moist and contains organic matter Ingredients needed for a biofilm : Surface Bacteria Aqueous environment Nutrients

How does a biofilm develop? 1. Planktonic cells attach to surface 2. Cells multiply ;Produce glycocalyx 3. Slime layer entraps nutrients, cells, microbes 4. Dynamic pillar-like layers form

How do biofilms communicate? Cell to cell communication - send and receive chemical signaling molecules Quorum sensing - accumulation of signaling molecules - enables a cell to sense the cell density Center for Biofilm Engineering Montana State University–Bozeman http://www.ted.com/talks/lang/eng/bonnie_bassler_on_how_bacteria_communicate.html

Biofilm Behavior Biofilm bacteria “turn on” a different set of genes than planktonic bacteria Examples: Turn off flagellar protein and turn on pili genes Turns on genes for antibiotic resistance Form a "division of labor" by nutrient cycling Some cells turn on metabolic pathways that degrade particulate matter, while other adjacent cells of the same population use the degradation products to produce new cells that are dispersed in the environment

Where are Biofilms Found?

Biofilms Found in Health Care Dental caries Contact lenses Lungs of Cystic Fibrosis patients Indwelling medical devices Endotracheal tube Mechanical heart valves Pacemakers Urinary catheters IV connectors Prosthetic joints Biofilm on a contact lens Staphylococcus biofilm on inner surface of IV connector Rodney M. Donlan, CDC

Where are Biofilms Found? Biofilm on soft, daily-wear, contact lens Biofilm on urinary catheter

Biofilms and Chronic Wounds 60 % of chronic wounds had biofilms Only 6% of acute wounds Found normal flora and pathogens produced different chemicals for their communication.

Medical Importance of Biofilms Are 1000X more resistant to antimicrobial agents than planktonic cells Easily transfer genes to express new and sometimes more virulent phenotypes Are more resistant to host defense mechanisms 80% of nosocomial infections are biofilm associated (NIH) 20% of patients with biofilm-related septicemia die