Source : Prescott et al Microbiology, Microbiology by Pelczar,...
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Source : Prescott et al Microbiology, Microbiology by Pelczar, Brock Et al
Microbiology
Microbial Nutrition
• Purpose
To obtain energy and construct new cellular components
• Nutrient Requirement
The major elements: C, O, H, N, S, P
The minor elements: K, Ca, Mg, Fe
The trace elements: Mn, Zn, Co, Mo, Ni, Cu
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
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
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” (require relatively large amounts of growth
factors in the media. Can be used to test samples for presence of growth
factors )
Examples: amino acids, purines, pyrimidines, vitamins
Micronutrients
Sources of Essential Nutrients
• Carbon – obtain in organic form, or reduce CO2
• Nitrogen – Fix N2 or obtain as NO3-- NO2
-, or NH3
• Oxygen – Atmospheric or dissolved in water
• Hydrogen – Minerals, water, organic compounds
• Phosphorous – Mineral deposits
• Sulfur – Minerals, H2S
• Metal Ions - Minerals
Make it, or eat it?
• Some bacteria are remarkable, being able to make all the organic
compounds needed from a single C source like glucose.
• For others:
– Vitamins, amino acids, blood, etc. added to a culture medium are
called growth factors.
– Bacteria that require a medium with various growth factors or
other components and are hard to grow are referred to as
fastidious.
Nutrient Requirements
• Prototrophs vs. Auxotrophs– Prototroph
– A species or genetic strain of microbe capable ofgrowing on a minimal medium consisting a simplecarbohydrate or CO2 carbon source, with inorganicsources of all other nutrient requirements
– Auxotroph
– A species or genetic strain requiring one or morecomplex organic nutrients (such as amino acids,nucleotide bases, or enzymatic cofactors) for growth
Organisms are categorized into two groups:
Autotrophs
Those using an inorganic carbon source (carbon dioxide)
Heterotrophs
Those catabolizing organic molecules i.e. reduced,
preformed organic molecules (proteins, carbohydrates,
amino acids, and fatty acids)
Carbon sources
Autotrophs Phytosynthetic bacteria:
Few purple sulphur (e.g., Chromatium) bacteria possess pigments, such as, purple pigment, the bacteriopurpurin, and green
pigment, the bacterial chloroyhyll etc. Bacterioviridin occurs hi green sulphur bacteria, e.g., Chlorobium. Such bacteria
synthesize their carbohydrate food in presence of sunlight by photosynthesis and are known as chlorophyll bacteria.
2H2S + CO2 → (CH2O)2 + 2S + H2O
Chemosynthetic bacteria:
These bacteria get their energy for food synthesis from the oxidation of certain inorganic chemicals. Light energy is not used. The
energy obtained from the chemical reactions is exothermic. The Chemosynthetic bacteria are of the following types:
(a) Sulphomonas (Sulphur bacteria): These bacteria get their energy by oxidation of hydrogen sulphide into H2SO4, e.g.,
Thiobacillus, Beggiatoa.
CO2 + 2H2S → 2S + H2O + CH2O + Energy
3CO2 + 2S + 8H2O → 2 H2S04 + 2(CH0) + 3H2O + Energy
(b) Hydromonas (Hydrogen bacteria): These convert hydrogen into water, e.g., Bacillus pantotrophus.
H2 + ½O2 ® → H2O + Energy
(c) Ferromonas (Iron bacteria): These bacteria get their energy by oxidation of ferrous compounds into ferric forms,. e.g.,
Leptothrix.
2Fe(HCO3)2 + H2O + O → 2Fe (OH)3 + 4CO2 + Energy
4FeCO3 + O2 + 6 H2O → 4Fe(OH)3 + 4CO2 + Energy
(d) Methanomonas (Methane bacteria): These bacteria get their energy by oxidation of methane into water and carbon dioxide.
(e) Nitrosomonas (Nitrifying bacteria): These bacteria get their energy by oxidation of ammonia and nitrogen compounds into
nitrates. Nitrosomonas oxidises NH3 to nitrites.
NH3 + ½O2 ® → H2O + HNO2 + Energy
Nitrobacter converts nitrites to nitrates.
NO2 + ½O2 → NO2 + Energy
HeterotrophsSaprophytic bacteria:
These bacteria obtain their food from the dead organic decaying substances such as leaves, fruits,
vegetables, meat, animal faeces, leather, humus etc. They secrete enzymes to digest the food
and absorb it. The breakdown of carbohydrates is fermentation and of proteins the putrefaction.
The former produces alcohols, acetic and other organic acids by fermentation of carbohydrates.
Putrefaction decomposes proteins into ammonia, methane, H2S, carbonic acids. The enzymes
secreted break down the complex compounds into simpler soluble compounds, which are easily
absorbed. Examples are Bacillus acidi lacti, Acetobacter etc.
Parasitic bacteria:
These bacteria obtain their food from the tissues of living organisms, the hosts. They may be
harmless or may cause serious diseases. The disease-producing bacteria are pathogenic which
cause various diseases in plants and animals. Examples are Bacillus typhosus, B. anthracis, B.
tetani. B. diplheriae, B. tuberculosis, B. pneumoniae, Vibrio cholerae, Pseudomonas citri etc.
Symbiotic bacteria:
These bacteria live in close association with other organisms as symbionts. They are beneficial to
the organisms. The common examples are the nitrogen-fixing bacteria, e.g., Bacillus radicicola,
B. azotobacter, Rhizobium, Ctostridium etc. Rhizobium spp.,B. radicicola and B. azotobacter
live inside the roots of leguminous plants and form bacteria nodules for fixation of nitrogen from
the air.
Organisms are categorized into two groups:
Chemotrophs
Acquire energy from redox reactions (oxidation
of chemical compounds) involving inorganic and
organic chemicals
Phototrophs
use light as their energy source
Energy sources
Groups of organisms based on carbon and energy source
Figure 6.1
Electron sources
Lithotrophs
use reduced inorganic substances as their electron
source.
Organotrophs
extract electrons from organic compounds.
Nutritional classes based on primary sources of carbon,
energy and electrons:
• Phtotolithotrophic autotrophs or photoautotrophs or photolithoautotrophs
Source of energy – light energy
Source of electrons – Inorganic hydrogen/ electron
Carbon source - CO2
Example: Algae, purple and green sulfur bacteria and cyanobacteria.
• Photoorganotrophic heterotrophy or photoorganoheterotrophy
Source of energy – light energy
Source of electrons – organic hydrogen/ electron
Carbon source –organic carbon sources (CO2 may also be used)
Example: Purple and green nonsulfur bacteria (common inhabitants of
lakes and streams)
• Chemolithotrophic autotrophs or chemolithoautotrophy
Source of energy – Chemical energy source (inorganic)
Source of electrons – Inorganic hydrogen/ electron donor
Carbon source - CO2
Example: Sulfur-oxidizing bacteria, hydrogen bacteria, nitrifying
bacteria, iron-oxidizing bacteria.
• Chemoorganotrophic heterotrophs or chemoorganoheterotrophy
Source of energy – Chemical energy source (organic)
Source of electrons – Inorganic hydrogen/ electron donor
Carbon source – organic carbon source
Example: Protozoan, fungi, most non-photosynthetic bacteria
(including most pathogens)
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
Mathematics of Population Growth
Mathematics of Population Growth
Number of generations (n) = (log Nt – log No) / log 2
Growth Rate Constant (k) = n/t
It is expressed in units of generations per hours (h-1)
Generation time (g) = 1/k; it is expressed in units of hours (h).
Nt = No + 2n
Exponential Growth by Binary Fission
1. DNA replication
2. Cell elongation
3. Septum formation
4. Septum completion
leads to separation or
further division
5. Process repeats
Generation time (g= t/n)
Duration of each division
Determined by type of
bacteria
Example: E. coli (20 min)
Bacterial Growth Curve
The Population Growth Curve
In laboratory studies, populations typically display a predictable pattern over time – growth curve.
Stages in the normal growth curve:
1.Lag phase – “flat” period of adjustment, enlargement; little growth
2.Exponential growth phase – a period of maximum growth will continueas long as cells have adequate nutrients and a favorable environment
3.Stationary phase – rate of cell growth equals rate of cell death caused bydepleted nutrients and O2, excretion of organic acids and pollutants
4.Death phase – as limiting factors intensify, cells die exponentially in theirown wastes
Stationary Phase
What
–metabolically active cells stop reproducing
– reproductive rate is balanced by death rate
• Why
– nutrient limitation
– limited oxygen availability
– toxic waste accumulation
– critical population density reached
• Starvation Response
–Morphological change
– Decrease in cell size
– Production of starvation proteins
Diauxic growth
• Growth in two phases
• Utilize one carbon source
first
• Utilize the second one until
the first one depleted
• Resulted from inducible
enzyme synthesis
Environmental Effects on Bacterial Growth
• Temperature
• Oxygen
• pH
• Osmotic pressure
TemperatureCardinal temperatures
• Minimum Temperature: Temperature below which growth ceases, or lowest temperature at which microbes will grow.
• Optimum Temperature: Temperature at which growth rate is the fastest.
• Maximum Temperature: Temperature above which growth ceases, or highest temperature at which microbes will grow.
Classification of Microorganisms by Temperature
Requirements
Temperature Classes of Organisms
• Psychrophiles ( 00C-200C)– Cold temperature optima
– Most extreme representatives inhabit permanently cold environments
• Mesophiles ( 200C – 450C)– Midrange temperature optima
– Found in warm-blooded animals and in terrestrial and aquatic environments in temperate and tropical latitudes
• Thermophiles ( 500C- 800C)– Growth temperature optima between 45ºC and 80ºC
• Hyperthermophiles– Optima greater than 800C
– These organisms inhabit hot environments including boiling hot springs, as well as undersea hydrothermal vents that can have temperatures in excess of 100ºC
Temperature
Psychrotrophs and Mesophiles
– “Growth” is generally used to refer to the acquisition of biomass
leading to cell division, or reproduction
– Many microbes can survive under conditions in which they
cannot grow
– The suffix “-phile” is often used to describe conditions
permitting growth, whereas the term “tolerant” describes
conditions in which the organisms survive, but don’t necessarily
grow
– For example, a “thermophilic bacterium” grows under
conditions of elevated temperature, while a “thermotolerant
bacterium” survives elevated temperature, but grows at a lower
temperature
Growth vs. Tolerance
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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
Use of Temperature to Preserve Microbes
Oxygen Requirements
Oxygen sources 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
Classification of organisms based on O2 utilization
• Obligate (strict) aerobes require O2 in order to grow, Ex. Bacillus,
Pseudomonas
• Obligate (strict) anaerobes cannot survive in O2 , Ex. Clostridium sp.
Facultative anaerobes grow better in O2,Ex. E. coli, Staphylococcus
• Aerotolerant organisms don’t care about O2 ,Ex. Lactobacillus sp.
• Microaerophiles require low levels of O2
Capnophile – requires higher CO2 tension (3-10%) than normally found in
the atmosphere, Ex. Neisseria, Brucella, S. pneumoniae
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– “Obligate” (or “strict”) means that a given condition is required
for growth
– “Facultative” means that the organism can grow under the
condition, but doesn’t require it
• The term “facultative” is often applied to sub-optimal condition
– For example, an obligate thermophile requires elevated
temperatures for growth, while a facultative thermophile may
grow in either elevated temperatures or lower temperatures
Obligate (strict) vs. facultative
OxygenToxicity
Hydrogen
peroxide
Superoxide
Hydroxyl radical (OH)
•Result of ionizing radiation & incomplete reduction of hydrogen peroxide;
extremely reactive but danger averted in aerobes because of catalase &
peroxidase
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Oxygen Toxicity
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 Pressure
HYPERTONICISOTONICPhysiologic Saline
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
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
Culture MediaMEDIA
• Nutrient preparation for microbial growth
• Must provide all chemical requirements
• Physical state (Broth-liquid, Agar-Solid)
AGAR
– Used as solidifying agent for culture media (Typically
1.5-2.0%)
– 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(Red Algae) in her jams and jellies,
which she learned from a neighbor who had lived in Java (Indonesia).
Types of Media Used Defined medium : precise amounts of highly purified chemicals
Complex medium (or undefined) : highly nutritious substances.
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
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
Chemically Defined vs Complex Media
Selective medium
MacConkey agar as a selective and differential medium
Anaerobic Culture Methods
Gas Pak Jar Glove Box
Capnophiles require high CO2
• Candle jar
(3-10% CO2)
• CO2-packet
Planktonic vs Sessile Bacteria
Robert Koch
•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.
Biofilms
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
– 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
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
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
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
Quorum Sensing• A mechanism by which members of a bacterial population can
behave cooperatively, altering their patterns of gene expression
(transcription) in response to the density of the population
• In this way, the entire population can respond in a manner most
strategically practical depending on how sparse or dense the
population is.
Mechanism:
• As the bacteria in the population grow, they secrete a quorum signaling
molecule into the environment (for example, in many gram-negative
bacteria the signal is an acyl homoserine lactone, HSL)
• When the quorum signal reaches a high enough concentration, it
triggers specific receptor proteins that usually act as transcriptional
inducers, turning on quorum-sensitive genes