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Microbial growth and nutrition

Growth requirements

Organisms use a variety of nutrients for their energy needs and to build organic molecules and cellular structures

Most common nutrients – those containing necessary elements such as carbon, oxygen, nitrogen, and hydrogen

Microbes obtain nutrients from variety of sources

Carbon is backbone of all organic components present in cell (we are carbon based life forms)

Hydrogen and oxygen are also found in many organic molecules

Electrons play a role in energy production (e.g. electron transport chain) and reduction of molecules during biosynthesis (e.g. CO2 to form organic molecules)

Microbial growth and nutrition

Microbial growth

In microbes, growth is an increase in size and in a population

Result of microbial growth is the formation of discrete colony. A colony is an aggregation of cells arising from single parent cell

Reproduction results in growth in population

Microbial growth and nutrition

Micronutrients (trace elements)

In addition to macroelements (macronutrients), cells also need micronutrients (trace elements) for metabolism and growth.

Manganese (Mn), Zinc (Zn), Cobalt (Co), Molybdenum (Mo), Nickel (Ni), and Copper (Cu).

Required in trace amounts

Often supplied in water or in media components

Ubiquitous in nature

Serve as part of enzymes and cofactors

Some organisms have particular requirements besides the macro and micronutrients.

Growth Requirements

Chemical and energy requirements

Carbon and energy requirements

Two groups of organisms based on source of carbon:

Autotrophs: Those using an inorganic carbon source (carbon dioxide) are autotrophs

Heterotrophs: Those catabolizing reduced organic molecules (proteins, carbohydrates, amino acids, and fatty acids) are heterotrophs

Two groups of organisms based on source of energy

Chemotrophs: Those that acquire energy from redox reactions involving inorganic and organic chemicals are chemotrophs

Phototrophs: Those that use light as their energy source are phototrophs

Two groups of organisms based on based on electron source

Lithotrophs use reduced inorganic substances

Organotrophs obtain electrons from organic compounds

Groups of organisms based on carbon and energy source

Four basic groups of organisms: Based on their carbon and energy sources, most organisms are categorized into one of four basic groups: photoautotrophs, chemoautotrophs, photoheterotrophs and chemoheterotrophs (See table below)

Major groups of organisms based on carbon and energy source

Growth Requirements

Oxygen requirements

Oxygen is essential for obligate aerobes (final electron acceptor in ETC)

Oxygen is deadly for obligate anaerobes. How can this be true?

Neither gaseous O2 nor oxygen covalently bound in compounds is poisonous

The forms of oxygen that are toxic are those that are highly reactive (reactive oxygen species or ROS)

ROS are excellent oxidizing agents

Resulting chain of oxidations cause irreparable damage to cells by oxidizing compounds such as proteins and lipids

Oxygen requirements

Classification of organisms based on oxygen requirements

Aerobes – undergo aerobic respiration

Anaerobes – do not use aerobic metabolism

Facultative anaerobes – can maintain life via fermentation or anaerobic respiration or by aerobic respiration (e.g. E. coli)

Aerotolerant anaerobes – do not use aerobic metabolism but have some enzymes that detoxify oxygen’s poisonous forms (e.g. Lactobacilli)

Microaerophiles – aerobes (e.g. Helicobacter pylori) that require oxygen levels from 2-10% and have a limited ability to detoxify hydrogen peroxide and superoxide radicals

Oxygen requirements
Identifying the oxygen requirements of organisms

Strict aerobes – require oxygen
Strict anaerobes – require no oxygen
Microaerophiles

requires 2–10% O2

Facultative anaerobes

do not require O2 but grow better in its presence

Aerotolerant anaerobes – tolerate presence of oxygen;

grow with or without O2

Growth Requirements

Four toxic forms of oxygen

Singlet oxygen (1O2) – molecular oxygen with electrons boosted to higher energy state, e.g. during aerobic metabolism

A very reactive oxidizing agent used by phagocytic cells to kill invading pathogens

Produced during photosynthesis, so phototropic organisms have carotenoids that prevent toxicity by removing the excess energy of singlet oxygen

Superoxide radicals (O2-) – some form during incomplete reduction of oxygen during electron transport in aerobes (aerobic respiration) and during metabolism by anaerobes (anaerobic respiration) in the presence of oxygen

So reactive that aerobes produce superoxide dismutases (SODs) to detoxify superoxide radicals (O2-)

Anaerobes lack superoxide dismutase and die as a result of oxidizing reactions of superoxide radicals formed in the presence of oxygen

Growth Requirements

Four toxic forms of oxygen (continued)

Peroxide anion (O22–): hydrogen peroxide formed during reactions catalyzed by superoxide dismutase and other metabolic reactions contains another highly reactive oxidant, peroxide anion (O22–); makes hydrogen peroxide an effective antimicrobial agent

Catalase converts hydrogen peroxide to water and molecular oxygen and peroxidase in the presence of a reducing agent (NADH+) breaks down hydrogen peroxide to water without forming oxygen

2H2O2 ↔ 2H2O + O2

H2O2 + 2NADH ↔ 2H2O + 2NAD+

Aerobes contain either catalase or peroxidase to detoxify peroxide anion

Obligate anaerobes either lack both enzymes or have only a small amount of each

Growth Requirements

Four toxic forms of oxygen (continued)

Hydroxyl radical (OH·)

Hydroxyl radical – results from ionizing radiation and from incomplete reduction of hydrogen peroxide:

H2O2 + e- + H+ → H2O + OH·

The most reactive of the four toxic forms of oxygen

Not a threat to aerobes due to action of catalase and peroxidase

Aerobes also use antioxidants such as vitamins C and E to protect against toxic oxygen products

Antioxidants provide electrons that reduce toxic forms of oxygen

Catalase test

Figure 6.2

Growth Requirements

Nitrogen and other requirements

Nitrogen is an essential element contained in many organic and inorganic compounds or nutrients. Anabolism often ceases due to insufficient nitrogen needed for proteins and nucleotides. Often, nitrogen is growth limiting nutrient

All cells recycle nitrogen from amino acids and nucleotides

The reduction of nitrogen gas to ammonia (nitrogen fixation) by certain bacteria is essential to life on Earth because nitrogen is made available in a usable form

Other chemical requirements are:

Phosphorus: required for phospholipid membranes, DNA, RNA, ATP, and some proteins

Sulfur: a component of sulfur-containing amino acids, disulfide bonds critical to tertiary structure of proteins, and in vitamins (thiamin and biotin)

Trace elements: only required in small amounts, but usually found in sufficient quantities in tap water

Growth factors: necessary organic chemicals (vitamins, certain amino acids, purines, pyrimidines, cholesterol, NADH, and heme) that cannot be synthesized by certain organisms)

Growth Requirements

Amino acids

needed for protein synthesis

Purines and pyrimidines

needed for nucleic acid synthesis

Vitamins

function as enzyme cofactors

Heme

for synthesis of cytochromes

Other chemical requirements

Growth Requirements

Physical requirements

In addition to chemical (nutrients) requirements, organisms need temperature, pH, osmolality and pressure for growth.

Temperature

Plays important role in microbial life (growth limiting factor)

At higher temperature, proteins denature and lose their function

Effect of temperature on lipid-containing membranes of cells and organelles

If too low, membranes become rigid and fragile

If too high, membranes become too fluid and cannot contain the cell or organelle

Effects of temperature on microbial growth

Figure 6.4

Effects of temperature on microbial growth

Categories of microbes based on temperature range

Different temperatures have different effects on microbial growth and survival

Based on preferred temperature ranges (minimum, optimum and maximum growth temperature) at which organisms are able to conduct metabolism, microbes are categorized into four groups:

Psychrophiles: grow best at temperatures below about 15ºC (some cause food spoilage in refrigerators)

Mesophiles: grow best in temperatures ranging from 20ºC to 40ºC (include human and animal pathogens)

Thermophiles: these grow best in temperatures ranging from 40 ºC and 80ºC (thermoduric organisms are mesophiles that briefly survive high temperature and cause food spoilage, e.g., pasteurized and canned food stuff)

Hyperthermophiles: grow in water above 80ºC (e.g. archaea) and others more than 100C. Thermophiles and hyperthermophiles do not cause diseases

Categories of microbes based on temperatures for growth

Figure 6.5

An example of a psychrophile

Figure 6.6

Growth Requirements

Chemical requirements

Organisms are sensitive to changes in acidity because, H+ and OH- interfere with H-bonding in proteins and nucleic acids

Most bacteria and protozoa grow best in a narrow range around neutral pH (6.5-7.5) – these organisms are called neutrophiles

Other bacteria and fungi are acidophiles – grow best in acidic habitats (acido-tolerant). Helicobacter pylori grows in the stomach by neutralizing acid by secreting bicarbonate and urease

Acidic waste products can help preserve foods by preventing further microbial growth

Alkalinophiles live in alkaline soils and water up to pH 11.5. Vibrio cholerae grows best at pH 9.0 outside of the body in water

Growth Requirements

Physical effects of water

Microbes require water to dissolve enzymes and nutrients required in metabolism

Water is important reactant in many metabolic reactions

Most cells die in the absence of water

Some have cell walls that retain water (e.g. Mycobacterium tuberculosis has a waxy substance called mycolic acid)

Endospores and cysts can cease most metabolic activity for years

Two physical effects of water on microbes:

Osmotic pressure: Pressure exerted on a semipermeable membrane by a solution containing solutes that cannot freely cross membrane (dissolved molecules and ions in a solution)

Hydrostatic pressure: Water exerts pressure in proportion to its depth. For every additional 10m of depth, water pressure increases 1 atmosphere. Organisms that live under extreme hydrostatic pressure are called barophiles

Growth Requirements

Solutions and physical effects of solutions

Osmotic pressure

The pressure exerted on a semipermeable membrane by a solution containing solutes that cannot freely cross membrane (related to concentration of dissolved molecules and ions in a solution)

Hypotonic solutions: have lower solute concentrations; cells placed in these solutions will swell and burst

Hypertonic solutions: have greater solute concentrations. Cells placed in these solutions will undergo plasmolysis (shriveling of cytoplasm). This effect helps preserve some foods and restricts organisms to certain environments

Two categories of organisms growing under hypertonic environments: Obligate halophiles (grow in up to 30% salt) and facultative halophiles (can tolerate high salt concentrations , e.g. S. aureus does not require salt but can tolerates up to 20% concentration)

Growth Requirements

Ecological associations and relationships

Associations and biofilms

Organisms live in association with individuals of their own or with different species

Antagonistic relationships: when one organism harms or kills another

Synergistic relationships: members cooperate such that each benefits from the relationship

Symbiotic relationships: organisms live interdependently such that they rarely live outside the relationship

Biofilms: Complex relationships among numerous individual microorganisms

Develop an extracellular matrix: matrix adheres cells to one another; allows attachment to a substrate; sequesters nutrients and may protect individuals in the biofilm

Biofilms formation on surfaces is often as a result of quorum sensing

Biofilm-forming organisms have the ability to cause diseases in humans. Salmonella enterica, Pseudomonas aeroginosa and Staphylococcus aureus cause dental plaque on teeth

Plaque on a human tooth

Associations and Biofilms

Biofilms

Complex relationships among numerous microorganisms

Form on surfaces, medical devices, mucous membranes of digestive system

Form as a result of quorum sensing

Many microorganisms more harmful as part of a biofilm

Scientists seeking ways to prevent biofilm formation

Biofilm development

Culturing Microorganisms

Culturing microorganisms

Inoculum: a sample (specimen) introduced into medium (liquid or solid). There are 3 types of specimens (samples):

Environmental specimens

Stored specimens

Clinical specimens (clinical sampling):

Disease diagnosis and treatment depend upon correct clinical specimens collection, transportation and isolation and identification of pathogens

Clinical specimens (e.g. feces, saliva, blood, sputum, cerebrospinal fluid etc.) must be collected in sterile containers and be free of contaminants

Collected specimens must be properly labeled and transported quickly to a lab in transport medium to avoid death of pathogens

Culture: refers to act of cultivating microorganisms or the microorganisms that are cultivated

Culturing Microorganisms

Culture media: Majority of prokaryotes have never been grown in culture media. There are six types of general culture media:

Defined (synthetic) media: one in which the exact composition is known (for fastidious organisms requiring a relatively large number of growth factors such as blood)

Complex media: contain nutrients released by the partial digestion of yeast, beef, soy, or proteins (casein from milk). The exact chemical composition of media is unknown but used to culture organisms whose exact nutritional needs are unknown, including fastidious organisms

Selective media: contain substances that either favor the growth of particular microorganisms or inhibit the growth of unwanted ones. Eosin, methylene blue, crystal violet dyes and bile salts inhibit Gram-positive organisms. High concentration of salt favors the growth of S. aureus and slightly low pH favors the growth of fungi

Differential media: media formulated to either differentiate visible changes in medium or differences in the appearance of colonies. Presence or absence of hemolysis in blood agar by Streptococci

Anaerobic media: anaerobes require culturing media with reducing compounds (e. g. sodium thioglycollate) that chemically combine with free oxygen and remove it with from medium

Transport media: media to transport clinical specimens to labs (maintain ratios among different microorganisms in samples, prevent contamination and keep organisms alive for short period of time

Examples of culture media

Slant tube containing solid media

Culturing Microorganisms

Obtaining pure culture

Cultures are composed of cells arising from a single progenitor

The progenitor from which a particular pure culture (axenic) is derived is called colony forming unit (CFU)

Aseptic technique is used to prevent contamination of sterile substances or objects

Two common isolation techniques:

Streak Plates

Pour Plates

The streak-plate method of isolation

The pour-plate method of isolation

Characteristics of bacterial colonies

Figure 6.8

An example of the use of a selective medium

Figure 6.12

The use of blood agar as a differential medium

Figure 6.13

The use of carbohydrate tubes as differential media

Figure 6.14

MacConkey agar as a selective and differential medium

Figure 6.15

Culturing Microorganisms

Special culture techniques

Techniques developed for culturing microorganisms

Animal and cell culture: technique used for growing microbes for which artificial media are inadequate (e.g. Mycobaterium leprae in armadillos and Treponema pallidum in rabbits)

Low-oxygen culture: carbon dioxide incubators (candle jars) maintain relatively high concentration of carbon dioxide and low levels of oxygen. GasPacks (chemical released combine with free oxygen and create anaerobic atmosphere). Strict anaerobes are studied in labs using large anaerobic glove boxes.

Ideal for growing aerotolerant anaerobes, microaerophiles and capnophiles (e.g. Neisseria gonorrhoeae)

Enrichment culture: enhance the growth of less abundant but potentially important microorganisms

Use of selective media and cold incubation in the refrigerator (cold enrichment)

Culturing Microorganisms

GasPack Candle Jar

Culturing Microorganisms

Preserving cultures

Refrigeration: preserving and storing microorganisms in the cold for short period of time

Deep-freezing: freezing cells at temperatures from -50 Celsius to -95 Celsius and used for long-term (years) storage

Lyophilization (freeze-drying): removal of water from frozen cultures using intense vacuum

Used for long-term preservation and storage (decades)

Growth of Microbial Populations

Growth of microbial populations

Most unicellular microorganisms reproduce by binary fission (divide into two cells and each of these new cells divide in two to make four and so on)

This type of growth is called logarithmic or exponential growth, different from arithmetic growth (simple addition)

Phases of microbial growth

A graph that is used to plot the number of organisms in a growing population over time is known as a growth curve

When bacteria are inoculated into a liquid media, there are four distinct phases to a population’s growth curve (see figure below for phases of microbial growth):

Lag phase

Log phase

Stationary phase

Death phase

Growth of Microbial Populations

Generation (doubling) time

Time required for the population to double in size

Varies depending on species of microorganism and environmental conditions

Range is from 10 minutes for some bacteria to several days for some eukaryotic microorganisms

Binary fission

The arithmetic of generation time

Where n = number of generations

A comparison of arithmetic and logarithmic growth

Figure 6.19

Arithmetic growth

Logarithmic growth

Growth of Microbial Populations

The growth curve

Observed when microorganisms are cultivated in batch culture

Culture incubated in a closed vessel with a single batch of medium

Usually plotted as logarithm of cell number versus time

Time required for a bacterial cell to grow and divide

Dependent on chemical and physical conditions

Has four distinct phases: Lag, exponential, stationary and death

Growth of Microbial Populations

Measuring microbial reproduction

Estimating the number of microorganisms in a sample is important for determining the severity of urinary tract infections, effectiveness of pasteurization, degree of fecal contamination of water and effectiveness of disinfectants and antibiotics

Direct methods for determining the number of microorganisms in a given amount of sample are:

Serial dilution and viable plate counts

Membrane filtration

Most probable number

Microscopic counts

Electronic counters

Growth of Microbial Populations

Viable plate count

Ten-fold serial dilution of samples are made in liquid medium and 0.1ml of each dilution is either directly poured onto a plate and spread or mixed with melted agar medium and poured into plates

After incubation, plates with colonies ranging from 30 to 300 are counted and the number of colonies counted (CFU) is multiplied by the reciprocal of the dilution (dilution factor) to estimate/determine the number of bacteria per ml of the original culture

Membrane filtration

More accurate viable count for samples with few number of microorganisms (e.g. fecal bacteria in a stream or pond)

Samples are filtered and microorganisms trapped on membrane filter are transferred onto solid medium and incubated

The number of bacteria in the original sample is estimated from the number of colonies (CFU) determined on the growth medium multiplied by the volume of sample filtered

Serial dilution and viable plate count

Figure 6.22

Membrane filtration

Microscopic Counts

Microscopic counts

Suitable for stained prokaryotes and relatively large eukaryotes

Sample placed on cell counter (Petroff-Hauser Counting Chamber) and the number of bacteria in 25 large squares is counted and averaged

The number of bacteria per ml of bacterial suspension is calculated by multiplying the mean number of bacteria per square by 1.25X106 (25X50X1X103)

Advantageous when there are more than 10X106 cells ml or when a speedy estimate of population size is required

Method cannot differentiate between dead and live cells and difficult to count motile cells

Microscopic Counts

Most probable number (MPN)

Method used for statistical estimation of the number of microorganisms that will not grow on solid media (e.g. algae seldom form distinct colonies), when bacterial counts are required routinely, and when samples of waste-water, drinking water and food samples contain too few organisms to use a viable plate count

Positive tubes that show turbidity, pH change or gas production in each set of tubes are counted (e.g. 4, 2, 1, figures below) and compared to the numbers in an MPN table to estimate the number of organisms per 100 ml of sample

Electronic counters

Coulter counter (useful for counting larger cells of yeasts, algae and protozoa) and flow cytometry (counts bacteria and other cells differentially stained with fluorescent dyes or tagged with fluorescent antibodies)

The most probable number method (MPN)

Figure 6.24

The most probable number method (MPN)

Growth of Microbial Populations

Measuring microbial growth

Indirect methods

Metabolic Activity: Estimates the number of cells in a culture (whose metabolic rate is established) by measuring changes in such things as nutrient utilization, waste production or pH

Dry Weight: microorganisms are filtered from their culture medium, dried and weighed; method is suitable for broth culture

Turbidity: An indirect method for estimating the growth of microbial population by measuring changes in turbidity using spectrophotometer; easy and rapid results but only useful if the concentration of cells exceeds 1 million per ml; method does not distinguish between dead and live cells

Indirectly measuring population size

Figure 6.26

Growth of Microbial Populations

Measuring microbial growth

Genetic methods

Isolate DNA sequences of un-culturable prokaryotes

Used to estimate the number of these microbes

Microscopy, Staining, and Classification

General principles of microscopy

Wavelength of radiation

Resolution

Contrast

Magnification

Wavelength of radiation

Distance between two corresponding parts of a wave of radiation (from crest to crest or trough to trough)

visible light or electromagnetic, including X-rays, microwaves and radio waves)

The shorter the wave length of radiation, the stronger the resolving power

Microscopy, Staining, and Classification
The electromagnetic spectrum

Microscopy, Staining, and Classification

Resolution (resolving power)

Ability to distinguish between objects that are close together

Resolution is determined by the wavelength of light used and numerical aperture of lens. Resolution distance is dependent on wave length of light, electron beam and/or numerical aperture of the lens

Modern microscopes use shorter wave length radiation and have lenses with larger numerical apertures

Limit of resolution for light microscope is about 0.2 µm.

Contrast

Difference in intensity between two objects or between an object and its background

Important in determining resolution (clarity of an image)

Staining increases contrast

Resolution and contrast determine the magnification of a microscope

Use of light that is in phase increases contrast

Microscopy, Staining, and Classification

Magnification

An increase in size of an object.

Results when a beam of radiation bends as it passes through a lens

Curved lenses refract light and magnetic fields (magnetic lenses) refract electron beams

Lenses refract (bend) radiation because they are optically dense compared to other media (air or water)

Magnification depends on the thickness of the lens, its curvature and the speed of light through its medium (substance such as glass, lens, air or water)

Lenses and the bending of light

When a ray of light passes from one medium to another, refraction occurs (the light is bent at the interface).

The refractive index (n) is a measure of how greatly a substance slows the velocity of light. The direction and magnitude of bending are determined by the refractive indices of the two media forming the interface.

Refraction

Light beam enters head on

Light beam enters glass at angle to normal

Air

n = 1

Air

n = 1

Air

n = 1

Air

n = 1

Glass

n = ~1.5

Glass

n = ~1.5

Dashed line depicts the normal

Light

Bending of light through a rism

Prism

Air

Glass

Normal

Light

n = 1

n = ~1.5

Slowed down

Sped up

Can also say the air is less optically dense than glass.

F = focal point

The Convex Lens

f = focal length

Lens

Air

Glass

strength of lens related to focal length short focal length more magnification

Refractive Properties of Lenses

Flat glass

Convex lens (less round)

Convex lens (more round)

Concave lens

short focal length more magnification

Microscopy, Staining, and Classification
Light refraction and image magnification

Units of Measurement

Range of Light and Electron Microscopes

Light

Electron

Rhodospirillum rubrum

Photoionization microscopy

These are all things you absolutely can not see without microscopes. Know that viruses are smaller than a um and can not be seen with light microscope. Know that bacteria are in the um in sizes and can typically be seen with light microscope. Thin section TEM at bottom right.

Types of microscopes

Light microscopes include:

Bright-field
Dark-field
Phase-contrast
Fluorescence
Confocal

Modern microscopes use visible light to illuminate cells and are compound, meaning that they have two sets of lenses.

Bright-field microscopes aren’t ideal for viewing unpigmented and unstained cells due to lack of contrast. What if you need to see living cells?

These light microscopes are more useful:

Dark-field microscope
Phase-contrast microscope
Differential interference (DIC) microscope.

Types of microscopy

Light Microscopy

Bright-field microscopes
Simple
Contain a single magnifying lens
Similar to magnifying glass
Leeuwenhoek used simple microscope to observe microorganisms

Types of microscopy

Light Microscopy
Bright-field microscopes
Compound
Series of lenses for magnification
Light passes through specimen into objective lens
Oil immersion lens increases resolution
Have one or two ocular lenses
Total magnification = magnification of objective lens X magnification of ocular lens
Most have condenser lens (direct light through specimen)

A bright-field, compound light microscope.

Coarse focusing knob

Moves the stage up and

down to focus the image

Illuminator

Light source

Diaphragm

Controls the amount of

light entering the condenser

Condenser

Focuses light

through specimen

Stage

Holds the microscope

slide in position

Objective lenses

Primary lenses that

magnify the specimen

Body

Transmits the image from the

objective lens to the ocular lens

using prisms

Ocular lens

Remagnifies the image formed by

the objective lens

Line of vision

Ocular lens

Path of light

Prism

Body

Objective

lenses

Specimen

Condenser

lenses

Illuminator

Fine focusing knob

Base

Arm

Routinely used in microbiology to examine both stained and unstained specimens. Specimens are visualized because of differences in contrast (density) between specimen and surroundings.

Named for its ability to form a dark image against a brighter background.

Parfocal – specimen remains in focus as you change objectives.

Multiply objective and ocular magnification to obtain total magnification.

The effect of immersion oil on resolution

Glass cover slip

Slide

Specimen

Light source

Without immersion oil

Lenses

Immersion oil

Glass cover slip

Slide

Light source

With immersion oil

Microscope

objective

Refracted light

rays lost to lens

Microscope

objective

More light

enters lens

Immersion oil redirects light rays by minimizing refraction and prevents reflection, resulting in increased numerical aperture and resolution.

Dark-Field Microscope

Produces detailed images of living, unstained specimens by changing the way in which they are illuminated.

Unreflected and unrefracted rays do not enter the objective.

Object appears bright on black background.

Dark-Field Microscopy

Treponema pallidum (syphilis)

Useful for study of internal structure of eukaryotic microorganisms and for observing motility.

S. Cerevisiae

Microscopy

Light microscopy

Phase microscopes

Used to examine living organisms or specimens that would be damaged or altered by attaching them to slides or staining them

These microscopes treat one set of light rays differently from another set

Light rays in phase produce brighter image, while light rays out of phase produce darker image

Contrast is created because light waves are ½ wavelength out of phase

Two types

Phase Contrast Microscope: produce shapely defined images in which fine structures can be seen in living cells; useful for observing cilia and flagella

Differential Interference Contrast Microscope(Nomarski microscopes): Create phase interference patterns; gives the image a three-dimensional or shadowed appearance

Phase-Contrast Microscope

Four kinds of light microscopy

Fluorescent microscopy

Fluorophores are molecules that absorb energy and emit light, this is the basis of fluorescence microscopy. When some molecules absorb radiant energy, they become excited and release much of their trapped energy as light (emission). Fluorescence light is emitted very quickly by the excited molecule as it gives up its trapped energy and returns to a more stable state.

Explain how antibodies are used in fluorescence microscopy. Mbl is a cytoskeletal protein of Bacillis subtilis.

Immunofluorescence

Staining

Most microorganisms are difficult to view by bright-field microscopy
Coloring specimen with stain increases contrast and resolution
Specimens must be prepared for staining
Thin smear (film) of microorganisms on glass slides is made prior to staining
Smear is allowed to air-dry and then heat-fixed to glass surface

Staining

Principles of Staining
Microbiological stains/dyes used as stains are usually salts composed of cation and anion and contain one colored substance (chromophore)

Acidic dyes (anionic chromophores) stain alkaline structures (positively charged molecules). Acidic dyes are also used in negative staining

Basic dyes (cationic chromophores) stain acidic structures (negatively charged molecules). They are used more commonly in microbiology because most microbial cells are negatively charged.

Types of staining

Simple stains

Differential stains
Gram stain
Acid-fast stain
Endospore stain

Special stains
Negative (capsule) stain
Flagellar stain

Simple Staining

Commonly used and easy.

Fixed smear is covered with a single basic dye such as crystal violet, excess stain is washed off with water, and blotted dry.

Used to determine the size, shape, and arrangement of bacterial and archaeal cells.

Differential Stains

Distinguish organisms based on their staining properties.

For example, the Gram stain, developed in 1884 by the Danish physician Christian Gram, is the most widely employed staining method in bacteriology.

Gram stain divides most bacteria (but not archaea) into two groups – those that stain gram negative and those that stain gram positive.

Acid-fast stain

Mixed stain: Gram positive (purple) Acid-fast stain

and Gram negative stain (pink)

The Gram Staining Procedure

Special stains: Preparation and staining of specimens

Most dyes are used to directly stain the cell or object of interest to make internal and external structures of the cell more visible.

Some dyes (special stains, e.g., India ink) are used in negative staining, where the background but not the cell is stained. The unstained cells appear as bright objects against a dark background.

Negative stain (Capsule stain)

Flagella

Flagellar stain of Proteus vulgaris

Staining and microscopy

Staining

Staining for Electron Microscopy

Chemicals containing heavy metals used for transmission electron microscopy
Stains may bind molecules in specimens or the background
Electrons replace light as the illuminating beam
Wavelength of electron beam is much shorter than light, resulting in much higher resolution
Allows for study of microbial morphology in great detail

Electron Microscopy

The Transmission Electron Microscope (TEM)

Electrons scatter when they pass through thin sections of a specimen

Transmitted electrons are under vacuum which reduces scatter and are used to produce clear image

Denser regions in specimen, scatter more electrons and appear darker

Wavelength of an electron in a TEM can be as short as 2.5 pm as in picometers as in 2.5 x 10-12 m

That’s ~100,000 times shorter wavelength than a light microscope uses.

Transmission electron microscope (TEM)

Specimen is coated with plastic and cut really thin 20-100 nm thick slices.

The Scanning Electron Microscope

Uses electrons reflected from the surface of a specimen that is coated in metal to create detailed image

Produces a realistic 3-dimensional image of specimen’s surface features

Resolution of 7 nm.

Can determine actual in situ location of microorganisms in ecological niches

Scanning Electron Microscope (SEM)

Mycobacterium tuberculosis

Classification and Identification of Microorganisms

Classification and identification of microorganisms

Taxonomy is the science of classifying and naming organisms

Taxonomy consists of:

classification (assigning organisms to taxa based upon similarities)

Nomenclature (rules of naming organisms) and

Identification (determining which individual organism or population belongs to a particular taxa)

Enables scientists to organize large amounts of information about organisms

Make predictions based on knowledge of similar organisms

Classification and Identification of Microorganisms

Linnaeus, Whittaker, and taxonomic categories

Linnaeus

Linnaeus provided system that standardized the naming and classification of organisms based on characteristics they have in common

Grouped similar organisms that can successfully interbreed into categories called species

Used binomial nomenclature in his system

Binomial Nomenclature (assigning two names to every organism)

Linnaeus proposed only two kingdoms: animalia and plantae

Whitaker proposed taxonomic approach based on five kingdoms: Animalia, Plantae, Fungi, Protista, and Prokaryotae (widely accepted)

Classification and Identification of Microorganisms

Taxonomic categories

Linnaeus’s goal was classifying and naming organisms as a means of cataloging them

Today, more modern goal of understanding relationships among groups of organisms

Major goal of modern taxonomy is to reflect phylogenetic hierarchy (derivation from common ancestors)

Greater emphasis on comparisons of organisms’ genetic material led to proposal to add a new, most inclusive taxon, the domain

Classification and Identification of Microorganisms

Domains

Taxonomists compare nucleotide sequences of the smaller rRNA subunits of both prokaryotes and eukaryotes

Carl Woese compared nucleotide sequences of rRNA subunits. rRNA molecules are present in all cells and changes in their nucleotide sequence presumably occur rarely

Proposal of three domains as determined by ribosomal nucleotide sequences: Bacteria, Archaea and Eukarya

Cells in the three domains also differ with respect to many other characteristics

Levels in Linnaean taxonomic scheme

Whittaker’s five-kingdom taxonomic scheme

Classification and Identification of Microorganisms

Taxonomic and identifying characteristics

Main criteria and laboratory techniques used for classifying and identifying microorganisms are:

Macroscopic and microscopic examination

Differential staining

Growth (cultural ) characteristics

Serological tests - microbial interaction with antibodies

Phage typing - microbial susceptibility to viruses

Nucleic acid analysis

Biochemical tests and microbial environmental requirements (temperature and pH).

Two biochemical tests for identifying bacteria

An agglutination test, one type of serological test

Phage typing

Classification and Identification of Microorganisms

Taxonomic Keys

Dichotomous keys

Series of paired statements where only one of two “either/or” choices applies to any particular organism

Key directs user to another pair of statements, or provides name of organism

Use of dichotomous taxonomic key

RICHLAND COLLEGE, Department of Biology,

School of Mathematics, Science & Health Professions

Microbiology syllabus for on-science majors

Instructor Information

Name: Admassu Mitiku

Email: [email protected]

Office Phone: 972-238-6140

Course Information

Course title: Microbiology for Non-Science Majors

Course number: Biol 2420

Section number: 85201

Semester/Year: Summer 2020

Credit hour: 4

Online meeting times

Monday to Friday: Mornings until noon. Meet via college email

Saturday/Sunday: 5:00 8:00pm. Meet via college email

Important dates

Certification Date: 08/06/2020

Drop Date: 07/16/2020

Final exam: Tuesday, August 4, 2020

General course information

Prerequisite:

BIOL 1406 or BIOL 2401 or SCIT 1407. One of the following must be met: Student cannot take both BIOL 2420 and BIOL 2421 to satisfy the Core science credit.

Course Description:

Study of the morphology, physiology, and taxonomy of representative groups of pathogenic and nonpathogenic microorganisms. Emphasis is placed on applications to humans. Pure cultures of microorganisms grown on selected media are used in learning laboratory techniques. Includes a brief preview of food microbes, public health, and immunology. Designed for non-science majors and allied health students. (3 Lecture, 4 Lab.)

Student learning outcomes:

Upon successful completion of this online course lecture and lab parts, students will:

1. Describe distinctive characteristics and diverse growth requirements of prokaryotic organisms compared to eukaryotic organisms.

2. Provide examples of the impact of microorganisms on agriculture, environment, ecosystem, energy, and human health, including biofilms.

3. Distinguish between mechanisms of physical and chemical agents to control microbial populations.

4. Explain the unique characteristics of bacterial metabolism and bacterial genetics.

5. Describe evidence for the evolution of cells, organelles, and major metabolic pathways from early prokaryotes and how phylogenetic trees reflect evolutionary relationships.

6. Compare characteristics and replication of acellular infectious agents (viruses and prions) with characteristics and reproduction of cellular infectious agents (prokaryotes and eukaryotes).

7. Describe functions of host defenses and the immune system in combating infectious diseases and explain how immunizations protect against specific diseases.

8. Explain transmission and virulence mechanisms of cellular and acellular infectious agents.

Upon successful completion of this course lab part, students will:

1. Use and comply with laboratory safety rules, procedures, and universal precautions.

2. Demonstrate proficient use of a compound light microscope.

3. Describe and prepare widely used stains and wet mounts, and discuss their significance in identification of microorganisms.

4. Perform basic microbiology procedures using aseptic techniques for transfer, isolation and observation of commonly encountered, clinically significant bacteria.

5. Use different types of bacterial culture media to grow, isolate, and identify microorganisms.

6. Perform basic bacterial identification procedures using biochemical tests.

7. Estimate the number of microorganisms in a sample using methods such as direct counts, viable plate counts, or spectrophotometric measurements.

8. Demonstrate basic identification protocols based on microscopic morphology of some common fungi and parasites.

Texas core course objectives : Students will be able to describe the morphology, physiology, and taxonomy of representative groups of pathogenic and non-pathogenic organisms, and apply techniques used in growing pure cultures as it relates to humans and public health issues.

Required course materials:

A. Text book: Microbiology with Diseases by Taxonomy, 6thedition by Robert W. Bauman.

B. Mastering Microbiology: Three options to buy required course materials for Mastering Microbiology online work/assignments:

1. Print Textbook + etext + mastering code ISBN: 9780135159927

2. Books a la carte + etext + mastering code ISBN: 9780135204337

3. Mastering code alone + eText (no book) ISBN: 9780135174722

Please make sure that the code you purchase (either from a book store or online) matches the textbook, "Microbiology with Diseases by Taxonomy 6th edition" and NOT “Microbiology with Diseases by Body Systems". Please be advised that access code is mandatory for this course.

A lab manual is available online at this link: https://web.archive.org/web/20190113211746/http://delrio.dcccd.edu/jreynolds/microbiology/RLCmicroindex.html Link also has other resources, such as: hand-outs on safety in microbiology lab, practice questions for lab quizzes and practical exams, graphics/images and videos.

You need to check manual from this link. Link also includes course materials such as, lab practical graphics, practice questions for lab practical exams that go along with the lab manual, Lab. safety handouts and video links for lab procedures.

e-Campus - http://ecampus.dcccd.edu – Please visit this site as often as possible for course materials: Syllabus, PowerPoint lecture notes, study guides for lab quizzes/lecture tests, lab assignments, videos/audios, guide lines for Unknown ID report writing, lab assignments and grades etc. are all posted on e-Campus.

Institutional Policies

Institutional Policies relating to this course can be accessed from the following link: www.richlandcollege.edu/syllabipolicies

Other course policies

Attendance: Attendance is necessary for class/lab participation and course work. There will be no make-up opportunities if a student misses lab practical exams or lecture exams. However, there could be make-up for missed quizzes, tests and assignments if a student can present concrete evidence (example: medical reasons, etc.). Student should contact instructor in advance and at a reasonable time and submit evidence for absence for quiz/test and assignment re-sets.

Plagiarism/cheating: Plagiarism, defined as deliberate use of someone else’s language, ideas, or other original (not common-knowledge) material without acknowledging its source. Plagiarism is not allowed in any online assignments or home works. Cheating in this course, in any manner and circumstance is not allowed. Any student violating any of the above rule(s) will get a ZERO.

Grading and grading scales

Students may earn a maximum of 1000 points for the lecture, lab components and individual/group assignments combined. Table below lists the details of lecture and lab components and point distributions. In addition, a maximum of 50 extra credit points are allowed to count on top of the total grade (1000pts).

Break down of grade components and grading scale for letter grade are assigned as follows:

Course components Grade points

1 Final Exam (Comprehensive) 100 3 Lecture tests (100 pts each) 300 Online Mastering quizzes 100

Practical exam # 1 100

Practical exam # 2 50

3 Lab quizzes (50 pts each) 150

1 Lab assignment 50

Unknown ID (Enteric bacteria) 100

Unknown ID (Staph/Strep) 50

----------------- Total 1000

Grading Scale: Final letter grades are determined following standard procedure (standard grading scale) as follows: 900 - 1000 = A; 800 - 899 = B; 700 - 799 = C; 600 - 699 = D; less than 600 = F

Lab schedule for Biol-2420-85201 Summer 2020

Biology 2421 Microbiology for Science Majors Summer 2020

Week and Unit

Reading assigned in Microbiology text

Lab

Graded assignment

Due date

WEEK 1: short week starts 6/4

Ch1- Introduction to history of Microbiology

Ch2- Microbial chemistry

-Aseptic transfer of bacteria

-Pure culture techniques

-Microscopy use and preparation of specimens

WEEK 2:

Starts 6/8

Ch3- Microbial structures and function

Ch4- Microscopy and staining

-Gram Stain

-Endo-spore stain,

-Acid-fast stain (AFS)

-Capsule Stain

-Flagella stain

-Motility and motility tests

WEEK 3:

Starts 6/15

Lecture exam # 1 (chapters; 1-4)

Ch5- Microbial metabolism

Ch6- Microbial nutrition and growth

July 16 – LAST DAY TO WITHDRAW

-Colony Morphology

-Dilutions & Pipetting

-Counting Bacteria

-Environmental Conditions & Growth

-Effects of Temperature

-Protozoa

-Fungi

Lab assignment (Pipetting and /dilutions) – 50 pts

06/18/20

Lec. Test # 1 opens on Monday, June 15 @10:00am and closes on Monday, June 15 @midnight

WEEK 4:

Starts 6/22

Ch7- Microbial genetics

Ch8- Recombinant DNA technology

Lab quiz # 1

-Antibiotic (Kirby-Bauer) Sensitivity

-Antimicrobial Chemicals

-Ecto-parasites

-Helminths

Lab quiz # 1 opens on Monday June 22 @10:00am and closes on Monday, June 22 24 @midnight

WEEK 5:

Starts 6/29

Ch9- Control of microbial growth in the environment

Ch10 –Control of microbial growth in the human

Lecture exam # 2 (chapters: 5-8)

Practical exam # 1

Unknown (Enteric bacteria) ID

-Oxygen Requirements

-Biochemical tests: IMViC, TTC, Phenol Red broth, Oxidase, Catalase, Nitrate, Decarboxylase, Deaminase, Gelatin, Skim Milk, Lipid, Starch, Urea

Practical exam # 1 opens on Monday, June 29 @10:00m and closes on Monday, June 29@midnight

Lec. Test # 2 opens on Monday, July 6 @10:am and closes on Monday, July 6 @midnight

WEEK 6:

Starts 7/6

Ch13- Viruses and viroids

Ch14- Infection and infectious disease

-API 20E identification

-Complete enteric bacteria unknown ID

WEEK 7:

Starts 7/13

Lecture exam # 3 (chapters: 9, 10, 13 and 14)

Lab quiz # 2

-Staphylococci unknown ID

-Serological Testing

-Complete Staph unknown and submit report

Lec. Test # 3 opens on Friday, July 17 @10:00am and closes on Friday, July 17 @midnight

WEEK 8:

Starts 7/20

Ch17- Vaccines and immunization

-Streptococci unknown ID

-Serological Testing

-Complete Strep unknown and submit report

-Bacteriophages

-Urine culture

Lab quiz # 3

Essay (Extra credit) – 50 pts

Saturday July 24@midnight

Lab quiz # 3 opens on Saturday, July 25 @10:00am and closes on Saturday, July 25 @midnight.

WEEK 9:

Starts 7/27

Practical exam # 2

Practical exam opens on Friday, July 31 @10:00am and closes on Friday, July 31 @midnight

WEEK 10:

Starts 8/3

Final exam (Comprehensive) – Exam opens on Tuesday, August 4 @10:00am and closes on Tuesday, August 4 @midnight

Disclaimer: The instructor reserves the right to amend syllabus, course contents, grading procedures, and/or other related items as conditions dictate. Students will be notified of any changes that are to be made in advance via email (or ecampus announcement).

RICHLAND COLLEGE,

Department of Biology

School of Mathematics, Science & Health Professions

Microbiology syllabus for on

science majors

Instructor Information

Name: Admassu Mitiku

Email:

[email protected]

Office Phone:

972

238

6140

Course Information

Course title: Microbiology for Non

Science Majors

Course number: B

iol

2420

Section number: 852

Semester/Year:

Summer

2020

Credit hour: 4

Online m

eeting times

Monday to Friday:

Mornings until noon. Meet

via

college email

Saturday/Sunday: 5:00 8:00pm. Meet

via

college email

Important dates

Certification Date:

08/06/2020

Drop Date:

07/16/2020

Final exam:

Tuesday

, August

, 2020

eneral course information

Prerequisite:

BIOL 1406 or BIOL 2401 or SCIT 1407.

One of the following must be met: Student cannot take both

BIOL 2420 and BIOL 2421 to satisfy the Core science credit.

Course Description:

Study of the morphology, physiology, and taxonomy of representative groups of pathogenic and

nonpathogenic microorg

anisms. Emphasis is placed on applications to humans. Pure cultures of

microorganisms grown on selected media are used in learning laboratory techniques. Includes a brief

preview of food microbes, public health, and immunology. Designed for non

science maj

ors and allied

health students. (3 Lecture, 4 Lab.)

RICHLAND COLLEGE, Department of Biology,

School of Mathematics, Science & Health Professions

Microbiology syllabus for on-science majors

Instructor Information

Name: Admassu Mitiku

Email: [email protected]

Office Phone: 972-238-6140

Course Information

Course title: Microbiology for Non-Science Majors

Course number: Biol 2420

Section number: 85201

Semester/Year: Summer 2020

Credit hour: 4

Online meeting times

Monday to Friday: Mornings until noon. Meet via college email

Saturday/Sunday: 5:00 8:00pm. Meet via college email

Important dates

Certification Date: 08/06/2020

Drop Date: 07/16/2020

Final exam: Tuesday, August 4, 2020

General course information

Prerequisite:

BIOL 1406 or BIOL 2401 or SCIT 1407. One of the following must be met: Student cannot take both

BIOL 2420 and BIOL 2421 to satisfy the Core science credit.

Course Description:

Study of the morphology, physiology, and taxonomy of representative groups of pathogenic and

nonpathogenic microorganisms. Emphasis is placed on applications to humans. Pure cultures of

microorganisms grown on selected media are used in learning laboratory techniques. Includes a brief

preview of food microbes, public health, and immunology. Designed for non-science majors and allied

health students. (3 Lecture, 4 Lab.)

Lecture prepared by Mindy Miller-Kittrell, University of Tennessee, Knoxville

M I C R O B I O L O G Y WITH DISEASES BY TAXONOMY, THIRD EDITION

Chapter 5

Microbial Metabolism

Basic Chemical Reactions Underlying Metabolism

Metabolism

Collection of controlled biochemical reactions

The ultimate function of metabolism is to reproduce the organism

Basics of metabolic processes

Every cell acquires nutrients necessary for metabolism

Metabolism requires energy from light or catabolism of nutrients

Energy is stored in chemical bonds of ATP

Cells catabolize nutrients to form building blocks (precursor metabolites)

Precursor metabolites, ATP, and enzymes used in anabolic or biosynthetic reactions

Cells build macromolecules using enzymes and ATP from building blocks

Cells reproduce once they have achieved a certain size

Basic Chemical Reactions Underlying Metabolism

Catabolism and anabolism

A series of reactions in metabolism are called pathways. There are two major classes of metabolic reactions:

Catabolic pathways

Break larger molecules into smaller products

Exergonic (release energy)

Anabolic pathways

Synthesize large molecules from the smaller products of catabolism

Endergonic (require more energy than they release)

Metabolism composed of catabolic and anabolic reactions

Figure 5.1

Metabolism

Basic Chemical Reactions Underlying Metabolism

Oxidation - reduction reactions (Redox reactions)

Transfer of electrons from molecule that donates (electron donor) electron to molecule that accepts electrons (electron acceptor)

Metabolic reactions in which electrons are accepted are reduction reactions and reactions in which electrons are donated are oxidation reactions

These reactions are always coupled - always occur simultaneously

Cells use electron carriers to carry electrons (often in H atoms) from one cell location to another

There are three important electron carriers:

Nicotinamide adenine dinucleotide (NAD+)

Nicotinamide adenine dinucleotide phosphate (NADP+)

Flavine adenine dinucleotide (FAD+) → FADH2

Oxidation-reduction (Redox) reactions or redox reactions

Figure 5.2

Oxidation-reduction or redox reactions

Basic Chemical Reactions Underlying Metabolism

ATP Production and Energy Storage

Organisms release energy from nutrients

Can be concentrated and stored in high-energy phosphate bonds of ATP

Phosphorylation – organic/inorganic phosphate is added to substrate (ADP)

Cells phosphorylate ADP to ATP in three ways:

Substrate-level phosphorylation: transfer of phosphate to ADP from phosphorylated organic compound

Oxidative phosphorylation: Energy from redox (respiration) used to attach phosphate to ADP

Photophosphorylation: Light energy used to phosphorylate ADP

Anabolic pathways use some energy of ATP by breaking a phosphate bond

Basic Chemical Reactions Underlying Metabolism

The roles of enzymes in metabolism

There are six categories of enzymes based on mode of action:

Hydrolases: breakdown macromolecules by adding water in hydrolysis reaction

Isomerases: rearrange atoms within a molecule (neither catabolic nor anabolic)

Ligases or polymerases: join two molecules together (anabolic)

Lyases: split large molecule (catabolic)

Oxidoreductases: remove electrons from (oxidized) or add electrons to (reduced) substrates (catabolic and anabolic pathways)

Transferases: transfer functional groups (amino, phosphate) between molecules (anabolic)

Basic Chemical Reactions Underlying Metabolism

The roles of enzymes in metabolism (continued)

Enzymes are organic catalysts – increase the likelihood of a reaction but are not permanently changed

Many protein enzymes are complete in themselves and composed entirely of protein (chains of amino acids, folded into tertiary structure).

Some are RNA molecules called ribozymes

Others composed of protein portions called apoenzymes

Apoenzymes are inactive if not bound to non-protein cofactors (inorganic ions or coenzymes)

Binding of apoenzyme and its cofactor(s) yields holoenzyme

Makeup of a protein enzyme

Figure 5.3

Makeup of a protein enzyme

Enzyme activity

Catalyzes reactions within cells by lowering activation energy, energy needed to trigger a chemical reaction

Enzymes have functional sites (active sites) which are complementary to the shape of their substrates (molecules upon which enzymes act on)

Enzyme-substrate reaction is specific and is critical to enzyme activity

Reaction forms a temporary and intermediate compound called enzyme-substrate complex

During an enzyme-substrate reaction, chemical bonds are either broken to form new products or linked together to form a single product from two reactants

Finally, enzyme disassociates from the newly formed molecules and is ready to associate with another substrate molecule

The effect of enzymes on chemical reactions

Figure 5.4

Effect of enzymes on chemical reactions

Enzymes fitted to substrates

Figure 5.5

Enzymes fitted to substrates-overview

The process of enzymatic activity

Figure 5.6

The process of enzymatic activity

Basic Chemical Reactions Underlying Metabolism

Factors influencing the rate of enzymatic reactions

Many factors influence the rate of enzymatic reactions

Enzyme and substrate concentrations

Temperature

Presence of inhibitors

Inhibitors

Substances that block an enzyme’s active site

Do not denature enzymes

Three types of inhibitors:

Basic Chemical Reactions Underlying Metabolism

Factors influencing the rate of enzymatic reactions (continued)

Three types of inhibitors:

Competitive inhibitors: Fit into an enzyme’s active site and prevent substrate from binding. Binding results in temporary or permanent loss of enzyme activity

Noncompetitive inhibitors: do not bind to active site but prevent enzymatic activity by binding to an allosteric site. Binding at an allosteric site alters the shape of enzyme at the active site so that substrate cannot bind

Feedback (negative) inhibitors: the end-products of a series of reactions is an allosteric inhibitor of an enzyme in an earlier part of the pathway

Factors that affect enzyme activity

Figure 5.7

Effects of temperature, pH, and substrate concentration on enzyme activity

Denaturation of protein enzymes

Figure 5.8

Denaturation of protein enzymes

Competitive inhibition of enzyme activity

Figure 5.9

Competitive inhibition of enzyme activity

Allosteric control of enzyme activity

Figure 5.10

Allosteric control of enzyme activity

Feedback Inhibition

Figure 5.11

Feedback inhibition-overview

Carbohydrate Catabolism

Carbohydrate catabolism

Many organisms oxidize carbohydrates as the primary energy source for anabolic reactions

Glucose used most commonly (also used are: other sugars, amino acids and fats after first converted to glucose)

Glucose is catabolized by either:

Cellular respiration → Utilizes glycolysis, Krebs cycle, and electron transport chain; results in complete breakdown of glucose to carbon dioxide and water; large amounts of ATP produced

Fermentation → Utilizes glycolysis then converts pyruvic acid into organic fermentation products (organic waste products). Lacks Krebs cycle and electron transport chain, thus, fermentation results in the production of much less ATP

Summary of glucose catabolism

Figure 5.12

Summary of glucose catabolism

Carbohydrate Catabolism

Glycolysis (Embden-Meyerhof pathway)

Occurs in the cytoplasm of most cells

Involves splitting of a six-carbon glucose into two three-carbon sugar molecules

Direct transfer of phosphate between two substrates (PEP and ADP) occurs four times – substrate level phosphorylation.

Two ATP molecules invested by substrate level phosphorylation to lyse glucose and 4 molecules of ATP produced

Net gain of two ATP molecules, two molecules of NADH, and precursor metabolite pyruvic acid

Glycolysis is divided into three stages involving 10 total steps:

Energy-Investment Stage

Lysis Stage

Energy-Conserving Stage

Glycolysis

Figure 5.13

Glycolysis-overview

Substrate-level phosphorylation

Figure 5.14

Substrate-level phosphorylation

Carbohydrate Catabolism

Cellular respiration

Resultant pyruvic acid from glycolysis completely oxidized to produce ATP by a series of redox reactions. There are three stages of cellular respiration:

Synthesis of acetyl-CoA

Krebs cycle

Final series of redox reactions which constitute an electron transport

chain (ETC)

Synthesis of acetyl-CoA

Acetyl coenzyme A (Acetyl CoA) formed from pyruvic acid by enzymatic removal of CO2 (decarboxylation) and joining acetate to form coenzyme A

Synthesis results in:

Two molecules of acetyl-CoA

Two molecules of CO2

Two molecules of NADH

Formation of acetyl-CoA

Figure 5.15

Formation of acetyl-CoA

Carbohydrate Catabolism

Cellular Respiration

The Krebs cycle

Great amount of energy remains in bonds of acetyl-CoA

A series of eight enzymatically catalyzed reactions that transfer much of this energy to coenzymes, NAD+ and FAD+. Two carbons in acetate are oxidized and the coenzymes are reduced

Occurs in cytoplasm of prokaryotes and in matrix of mitochondria in eukaryotes. There are eight types of reactions in Krebs cycle:

Anabolism of citric acid (step 1)

Isomerization reactions (steps 2, 7 and 8)

Hydration reaction (Step 7)

Redox reactions (steps 3,4,6 and 8)

De-carboxylations (steps 3 and 4)

Substrate-level phosphorylation (step 5)

The Krebs cycle

Figure 5.16

The Krebs cycle

Carbohydrate Catabolism

Cellular Respiration

The Krebs Cycle

For every two molecules of acetyl-CoA that pass through the Krebs Cycle:

Two molecules of ATP (step 5) and four molecules of CO2 (steps 3 and 4) are produced. A molecule of guanosine triphosphate (GTP), which is similar to ATP serves as an intermediary

Redox reactions produce six molecules of NADH (steps 3, 4 and 8) and two molecules of FADH2 (step 6)

In the Krebs cycle, little energy is captured directly in high-energy phosphate bonds, but much energy is transferred via electrons to NADH and FADH2.

Carbohydrate Catabolism

Cellular Respiration

Electron transport chain (ETC)

The most significant production of ATP occurs through stepwise release of energy from a series of redox reactions between molecules known as an electron transport chain (ETC)

Consists of series of membrane-bound carrier molecules that pass electrons from one to another and ultimately to a final electron acceptor

Energy from electrons used to pump protons (H+) across the membrane, establishing a proton gradient that generates ATP via chemiosmosis

Located in the inner membranes of mitochondria (cristae) of eukaryotes and in the cytoplasmic membrane of prokaryotes

NADH and FADH2 donate electrons as hydrogen atoms (electrons and protons); whereas carrier molecules only pass the electrons down the chain

An electron transport chain

Figure 5.17

An electron transport chain

Carbohydrate Catabolism

Cellular Respiration

Electron transport chain (ETC)

Four categories of carrier molecules:

Flavoproteins: integral membrane proteins that contain a coenzyme derived from riboflavin (vitamin B2): FMN is the initial carrier and FAD is a coenzyme

Ubiquinones: lipid-soluble, non-protein carriers derived from vitamin K; in mitochondria, ubiquinone is called coenzyme Q

Metal-containing proteins: mixed group of integral proteins containing iron, sulfur and copper atoms that can alternate between the reduced and oxidized states

Cytochromes: integral proteins associated with heme, pigmented molecule found in the hemoglobin of blood

Some organisms can vary their carrier molecules under different environmental conditions: In aerobic respiration (aerobes), oxygen serves as final electron acceptor to yield water. In anaerobic respiration (anaerobes), molecules other than oxygen serve as the final electron acceptor

Possible arrangement of an electron transport chain

Figure 5.18

One possible arrangement of an electron transport chain

Carbohydrate Catabolism

Cellular Respiration

Chemiosmosis

Use of electrochemical gradients to generate ATP. Chemicals diffuse from areas of high concentration to areas of low concentration and toward an electrical charge opposite their own. The blockage of diffusion creates potential energy

Membranes maintain electrochemical gradient by keeping one or more chemicals in higher concentration on one side

Cells use energy released in redox reactions of ETC to create electrochemical gradient known as proton gradient, which has potential energy known as proton motive force.

H+ ions (protons) propelled by proton motive force, flow down electrochemical gradient through protein channels called ATP synthases (ATPase) that phosphorylate ADP to ATP

ETC is called oxidative phosphorylation because proton gradient created by oxidation of components of ETC

Total of ~34 ATP molecules formed from one molecule of glucose

Carbohydrate Catabolism

Alternative pathways to glycolysis

Yield fewer molecules of ATP than glycolysis

Reduce coenzymes and yield different metabolites needed in anabolic pathways

Two pathways:

Pentose phosphate pathway

Entner-Doudoroff pathway

Carbohydrate Catabolism

Figure 5.19

Pentose phosphate pathway

Carbohydrate Catabolism

Entner-Douoroff pathway

Carbohydrate Catabolism

Fermentation

Sometimes cells cannot completely oxidize glucose by cellular respiration

Cells require constant source of NAD+ that cannot be obtained by simply using glycolysis and the Krebs cycle

In respiration, electron transport regenerates NAD+ from NADH

Fermentation pathways provide cells with alternative source of NAD+

Partial oxidation of sugar (or other metabolites) to release energy using an organic molecule as an electron acceptor rather than ETC (NADH oxidized to NAD+ while organic molecule reduced)

Carbohydrate Catabolism

Fermentation:

In the simple fermentation reaction, NADH reduces pyruvic acid (from glycolysis) to form lactic acid. Another simple fermentation pathway involves a decarboxylation reaction and reduction results to form ethanol.

Fermentation products and organisms that produce them

Figure 5.22

Representative fermentation products and the organisms that produce them

Carbohydrate Catabolism

Other Catabolic Pathways

Other catabolic pathways

Lipid Catabolism

Protein Catabolism

Lipid and protein molecules contain abundant energy in their chemical bonds. First converted into precursor metabolites, which serve as substrates in glycolysis and the Krebs cycle.

Lipid catabolism

Fats (glycerol and fatty acids) important in ATP and metabolite production

Lipases hydrolyze bonds attaching glycerol to fatty acids

Glycerol converted to DHAP and oxidized to pyruvic acid

Fatty acids degraded by beta-oxidation and converted to acetyl-CoA

NADH and FADH2 generated during beta-oxidation are utilized in the Krebs cycle to produce ATP

Catabolism of a fat molecule

Protein Catabolism

Protein catabolism

Some microorganisms (bacteria and fungi) catabolize proteins as main source of energy and metabolites

Other cells catabolize proteins and fat only when carbon sources are not available

Proteins too large to cross cell membranes; proteases split proteins into amino acids

Amino acids further broken down by deamination and altered molecules enter the Krebs cycle

Amino groups either recycled to synthesize other amino acids or excreted as wastes

Protein catabolism

Figure 5.24

Protein catabolism in microbes

Photosynthesis

Many organisms synthesize their own organic molecules from inorganic carbon dioxide

Most of these organisms capture light energy and use it to synthesize carbohydrates from CO2 and H2O by a process called photosynthesis

Photosynthesis

Chemicals and Structures

Chlorophylls

Important to organisms that capture light energy with pigment molecules

Composed of hydrocarbon tail attached to light-absorbing active site centered on magnesium ion

Active sites structurally similar to cytochrome molecules in ETC

Structural differences cause absorption at different wavelengths

Photosynthesis

Chemicals and Structures

Photosystems

Arrangement of molecules of chlorophyll and other pigments to form light-harvesting matrices

Embedded in cellular membranes called thylakoids

In prokaryotes – invagination of cytoplasmic membrane

In eukaryotes – formed from inner membrane of chloroplasts

Arranged in stacks called grana

Stroma is space between outer membrane of grana and thylakoid membrane

Photosynthesic structures in a prokaryote

Figure 5.25

Photosynthetic structures in a prokaryote

Photosynthesis

Chemicals and Structures

Two types of photosystems

Photosystem I (PS I)

Photosystem II (PS II)

Photosystems absorb light energy and use redox reactions to store energy in the form of ATP and NADPH

Light-dependent reactions depend on light energy

Light-independent reactions synthesize glucose from carbon dioxide and water

Photosynthesis

Light-Dependent Reactions

As electrons move down the chain, their energy is used to pump protons across the membrane

Photophosphorylation uses proton motive force to generate ATP

Photophosphorylation can be cyclic or noncyclic

Reaction center of photosystem

Figure 5.26

Reaction center of a photosystem

The light-dependent reactions of photosynthesis

Figure 5.27

Photosynthesis: photophosphorylation-overview

Photosynthesis

Light-Independent Reactions

Do not require light directly

Use ATP and NADPH generated by light-dependent reactions

Key reaction is carbon fixation by Calvin-Benson cycle

Three steps

Simplified diagram of the Calvin-Benson cycle

Figure 5.28

Simplified diagram of the Calvin-Benson cycle

Other Anabolic Pathways

Other anabolic pathways

Anabolic reactions are synthesis reactions requiring energy and a source of metabolites

Energy derived from ATP from catabolic reactions

Many anabolic pathways are the reverse of catabolic pathways

Reactions that can proceed in either direction are amphibolic

Gluconeogenesis

Figure 5.29

Role of gluconeogenesis in the biosynthesis of complex carbohydrates

Biosynthesis of fat

Figure 5.30

Biosynthesis of fat

Synthesis of amino acids by amination and transamination

Figure 5.31

Synthesis of amino acids via amination and transamination

The biosynthesis of nucleotides

Figure 5.32

Biosynthesis of nucleotides

Integration and Regulation of Metabolic Function

Cells synthesize or degrade channel and transport proteins

Cells often synthesize enzymes needed to catabolize a substrate only when substrate is available

If two energy sources are available, cells catabolize the more energy efficient of the two first

Cells synthesize metabolites they need, cease synthesis if metabolite is available

Eukaryotic cells isolate enzymes of different metabolic pathways within membrane-bounded organelles

Cells use allosteric sites on enzymes to control activity of enzymes

Feedback inhibition slows/stops anabolic pathways when product is in abundance

Cells regulate amphibolic pathways by requiring different coenzymes for each pathway

Integration and Regulation of Metabolic Function

Two types of regulatory mechanisms

Control of gene expression

Cells control amount and timing of protein (enzyme) production

Control of metabolic expression

Cells control activity of proteins (enzymes) once produced

Integration of cellular metabolism

Figure 5.33

Integration of cellular metabolism

Microbial cell structure and function

Cell Structure and Function

The four processes of life

The four processes of life that describe the characteristics of all living organisms:

Metabolism

Growth

Responsiveness

Reproduction

Prokaryotic and Eukaryotic Cells: An Overview

Prokaryotes

Lack membrane-bound nucleus (nuclear material), a cytoskeleton, membrane-bound organelles, and internal membranous structures.

Have simple structures compared

to eukaryotes

Composed of bacteria and archaea

Are typically small in size (~1.0 μm in

diameter

Typical prokaryotic cell

Different morphologic features of bacterial cells

Cells with unusual shapes
Vibrios – Resemble rods but are comma shaped.
Spirilla – rigid, spiral shaped cells. Usually with tufts of flagella at each end.
Actinomycetes – typically form filamentous structures. They lie between bacteria and filamentous fungi.
Pleomorphic - bacteria with variable in shape.

External Structures of Bacterial Cells

Glycocalyces

Gelatinous, sticky substance surrounding the outside of the cell

Composed of polysaccharides, polypeptides, or both

Two types of external structures: capsule and slime layer

Capsules

Composed of organized repeating units of organic chemicals

Firmly attached to cell surfaces

Protect cells from drying out

May prevent bacteria from being recognized and destroyed by host immune and phagocytic cells

Enable bacteria to cause diseases (capsules are virulence factors)

External Structures of Bacterial Cells

Slime layer

Loosely attached to cell surface

Protects cells from drying out

Sticky layer allows prokaryotes to attach to surfaces

Water soluble

Slime layers have little or no medical importance/significance

External Structures of Bacterial Cells

Flagella structure and function

Long, whip-like structures that extend beyond surface of the cell

Are responsible for movement: 360º rotation of flagellum propels bacterium through environment (run or tumble)

Rotation can be clockwise or counterclockwise and reversible

Prokaryotes move in response to stimuli:

Positive (stimulus) taxis – organisms move towards food or light;

Negative (stimulus taxis – organisms move away from danger

Flagella are not present on all bacteria

External Structures of Bacterial Cells

Flagellar arrangements

Monotrichous: Cells with a single flagellum

Lophotrichous: Cells with a tuft of flagella at one end of the cell

Amphitichous: Cells with flagella at both ends

Peritrichous: Cells covered with flagella

External Structures of Bacterial Cells

Fimbriae

Non-motile, rod-like proteinaceous extensions

on cell surfaces

Sticky, proteinaceous, bristle-like projections

Used by bacteria to adhere to one another,

to hosts, and to substances in environment

(e.g., Neisseria gonorrhoeae adhering on mucus

membranes)

May be hundreds per cell

Are shorter than flagella

Serve an important function in biofilms formations (slimy masses of bacteria adhering to one another and to a substrate by means of fimbriae and glycocalyces)

External Structures of Bacterial Cells

Pili

Long, hollow tubules composed of pilin

Longer than fimbriae but shorter than flagella

Bacteria typically only have one or two per cell

Also known as conjugation (sex) pili

Bacteria use pili to move across a substrate or towards another bacterium

Pili mediate the transfer of DNA from one cell to another: join two bacterial cells and help transfer DNA (conjugation)

External Structures of Bacterial Cells

Bacterial cell walls

Give bacterial cells characteristic shapes

Protects cell from osmotic forces

Assists some cells in attaching to other cells or other surfaces

Most bacteria have cell walls composed of peptidoglycan. A complex polysaccharide material that covers the entire surface of the cell and is composed of alternating sugars, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)

Cell walls help in eluding antimicrobial drugs or resisting antimicrobial drugs (certain antibiotics can target cell walls of bacteria, e.g., penicillin attacks cell wall)

A few bacteria lack a cell walls entirely (e.g., Mycoplasma pneumoniae)

Scientists describe two basic types of bacterial cell walls: Gram-positive and Gram-negative

Possible structure of peptidoglycan

External Structures of Bacterial Cells

Gram-positive bacterial cell walls

Relatively thick layer of peptidoglycan

Contains unique polysaccharides called teichoic acids

Some covalently linked to lipids, forming lipoteichoic acids that anchor peptidoglycan to cell membrane

Peptidoglycan retains crystal violet dye and cells appear purple following Gram Staining Procedure

Acid-fast bacteria contain up to 60% mycolic acid, a waxy lipid

Helps cells survive desiccation and resist stain with regular water-based dyes

Gram-positive bacterial cell wall structure

Gram-negative bacterial cell wall structure

Gram-negative bacterial cell walls

Have only a thin layer of peptidoglycan

Have a bilayer membrane (composed of phospholipid bilayers, channel proteins or porins and lipopolysaccharide or endotoxin) outside of peptidoglycan

Lipid portion (called lipid A) - released from dead and disintegrating cell walls may trigger endotoxic shock (fever, vasodilation, hypotension, inflammation and blood clotting in patients)

May be impediment to the treatment of disease

Following Gram staining procedure, cells appear pink

Gram-negative bacterial cell wall structure

Bacterial Cytoplasmic Membranes

Structure of prokaryotic cytoplasmic membrane

Cytoplasmic membrane (also known as cell membrane or plasma membrane) is a phospholipid bilayer composed of lipids and associated proteins

A phospholipid molecule is bipolar (has a hydrophilic and a hydrophobic ends)

Approximately half the cytoplasmic membrane is composed of proteins (integral proteins, peripheral proteins and glycoproteins)

Protein components of cytoplasmic membranes act as recognition proteins, enzymes, receptors, carriers or channels

Proteins and lipids within membranes flow freely (fluid mosaic model or membrane fluidity) and allow easy passage of substance into and out of the cell

Structure of prokaryotic cytoplasmic membrane

Bacterial cytoplasmic membranes

Functions of cytoplasmic membrane

Energy storage

Controls passage of substances into and out of the cell - selectively permeable (allows some substances to cross it, while preventing the crossing of others)

Naturally impermeable to most substances, but proteins (receptors, channels and carriers) allow substances to cross membrane

Membranes maintain a concentration gradient and electrical gradient - chemicals with concentration gradients across membranes have electrical charges and a corresponding electrical gradient

Chemical and electrical gradients collectively are known as electrochemical gradient

Energy found in electrochemical gradient can be used to transport substances across the membrane

Movement of substances across membranes occurs by passive or active processes of

transport

Electrical potential of a cytoplasmic membrane

Bacterial cytoplasmic membranes

Passive processes of transport

Electrochemical gradient provides a source of energy. The cell does not expend its ATP energy reserve for the following three passive processes of transport:

Diffusion

Facilitated diffusion

Osmosis

Diffusion

Net movement of a chemical down its concentration gradient-from an area of high concentration to an area of low concentration

Requires no energy out put by the cell, a common feature of all passive processes

Chemicals that are small or lipid soluble (e.g., oxygen, CO2, alcohol and fatty acids) can diffuse through the lipid portion of the membrane; larger molecules like proteins and glucose cannot – selectively permeable

Bacterial cytoplasmic membranes

Facilitated diffusion

Integral proteins (channels, carriers etc.) facilitate the diffusion of large or electrically charged molecules through phospholipids bilayer of membranes

Cells expend no energy in facilitated diffusion. Electrochemical gradient provides all the necessary energy

Non-specific channel proteins (common in prokaryotes) allow the passage of a wide range of chemicals with the right size or electrical charge

Specific channel proteins (common among eukaryotic cells) carry only specific substrates. These have specific binding site that are selective for one substance

Passive processes of movement

Solutes, solvents and solutions

Concentration of solutes and solutions

Three classes of solutions according to their concentrations of solutes and solvents:

Isotonic solutions: have the same concentration of solutes and water on either sides of selectively permeable membrane; neither side of membrane experience a net loss or gain of water

Hypertonic solution: contains higher concentration of solutes relative to the solvent

Hypotonic solutions: contains lower concentration of solutes in comparison

Hypotonic and hypertonic refer to the concentration of solute, even though osmosis refers to the movement of the solvent, which in cells is water

Osmosis

Diffusion of water across a selectively permeable (not to all solutes such as proteins, salts, amino acids or glucose) membrane

Water crosses from the side of the membrane that contains a higher concentration of water molecules (lower concentration of solute) to the side that contains a lower concentration of water molecules(higher concentration of solute)

When water pressure is at equilibrium, activity of osmosis stops

Like other chemicals, water moves down its concentration gradient from hypotonic solution into a hypertonic solution

The osmotic movement of water out of a cell and shriveling of its cytoplasm is called plasmolysis

Osmosis

Effects of different solutions on cells

Prokaryotic Cytoplasmic Membranes

Active processes of transport

Require the cell to expend energy (ATP) to move materials across cytoplasmic membranes against their electrochemical gradients

Utilizes trans-membrane carrier proteins. When only one substance is transported at a time, the carrier protein is called a uniport

Simultaneous transport of two chemicals, but in opposite directions (one into the cells and the other out of the cell) at the same time is called antiport

When two substance move together in the same direction across the membrane by means of a single carrier protein, the process of transport is termed symport

Active processes of transport in prokaryotes is by means of carrier proteins and a special process called group translocation (where substances are chemically modified during transport)

Mechanisms of active transport

Group translocation

Eukaryotic Cells

Have nucleus and nuclear membrane surrounding their DNA

Have internal membrane-bound organelles (compartmentalize cellular functions that act like tiny organs)

Eukaryote cells are larger compared to prokaryotes (10-100 μm in diameter)

Have more complex structures than prokaryotes

Comprised of algae, protozoa, fungi, animals, and plants

Nucleolus

Cilium

Ribosomes

Cytoskeleton

Cytoplasmic

membrane

Smooth endoplasmic

reticulum

Rough endoplasmic

reticulum

Transport vesicles

Golgi body

Secretory vesicle

Centriole

Mitochondrion

Lysosome

Nuclear pore

Nuclear envelope

Typical eukaryotic cell

External structure of Eukaryotic cells

Glycocalyces

Eukaryotic cells lacking cell walls have sticky carbohydrate, glycocalyces anchored to their cytoplasmic membranes

Never as organized as prokaryotic capsules

Helps animal cells adhere to each other

Strengthens cell surface

Provide protection against dehydration

Function in cell-to-cell recognition and communication

Eukaryotic cell walls

Fungi, algae, and plants have cell walls but no glycocalyx

Composed of various polysaccharides but not peptidoglycan of most bacteria

Cell wall protects cells from the environment and provide shape and support against osmotic pressure

Cellulose found in plant cell walls and fungal cell walls are composed of polysaccharide, including cellulose, chitin, and/or glucomannan

Algal cell walls composed of cellulose, agar, carrageenan, silicates, algin, calcium carbonate or combination of these

Some protozoa have cell walls composed of various polysaccharides (cellulose and glucomannan)

A Eukaryotic cell wall

External structures of Eukaryotic cells

Flagella

Structure and arrangement

Differ structurally and functionally from prokaryotic flagella

Within the cytoplasmic membrane (Flagella are inside the cell, not extensions outside the cell)

Shaft composed of tubulin arranged to form microtubules

Filaments anchored to cell by basal body AND no hook

May be single or multiple (generally found at one pole of cell)

Do not rotate, but undulate rhythmically

Eukaryotic Flagella and Cilia (movement)

External structures of Eukaryotic cells

Cilia

Some eukaryotic cells move by means of hair-like structures called cilia

Shorter and more numerous than flagella (cover the surface of the cell)

In comparison, no prokaryotic cells have cilia

Cilia in multi-cellular eukaryotes are used to move substances in the local environment past the surface of the cell

Coordinated beating propels cells through their environment

Cilia beat rhythmically and this propels cells through their environment

Eukaryotic Cilia

Eukaryotic cytoplasmic membranes

All eukaryotic cells have cell membrane

Is a fluid mosaic of phospholipids and proteins which act as recognition molecules, enzymes, receptors, carriers or channels

Contains steroid lipids (sterols) such as cholesterol in animal cells to help maintain membrane fluidity

Sterols at high temperature stabilize phospholipid bilayer by making it less fluid and at low temperatures they prevent phospholipid packing, making membrane more fluid

Controls movement of materials into and out of cell

Contain regions of lipids and proteins called membrane rafts

Eukaryotic cytoplasmic membranes are used for passive (diffusion, facilitated diffusion, osmosis) and active processes of transport

Eukaryotic membranes do not perform group translocation, but perform endocytosis (also called phagocytosis if solid substance is brought into the cell and pinocytosis if liquid substance is brought into the cell). Exocytosis enables substances to be exported out of the cell

Eukaryotic Cytoplasmic Membrane

Cytoplasm of Eukaryotes

Cytoplasm of Eukaryotic cells

More complex than that of either bacteria or archaea

Most distinctive difference is the presence of numerous membranous organelles in eukaryotic cells (e. g., Gologi body, rough/smooth endoplasmic reticulum)

Non-Membranous organelles

Ribosomes: Larger than prokaryotic ribosomes (80S versus 70S) and composed of 60S and 40S subunits. Many eukaryotic ribosomes are attached to the membranes of the endoplasmic reticulum

Cytoskeleton: composed of extensive internal network of fibers and tubules

Function in cytoplasmic streaming and in movement of organelles within the cytoplasm

Enables contraction of the cell, provides the basic shape of many cells and anchors organelles

Centrioles and Centrosome: Centrioles play a role in mitosis (nuclear division), cytokinesis (cell division), and in the formation of flagella and cilia. Centrosome – region of cytoplasm where centrioles are found

Cytoplasm of Eukaryotes

Mitochondria and chloroplasts

Mitochondria:

Spherical to elongated structures found in most eukaryotic cells

Have two membranes composed of phospholipid bilayer. Inner membrane is folded into numerous crystae, where most of the cell’s ATP is produced

Interior matrix contains small “prokaryotic” 70S ribosomes and circular molecule of DNA (contains genes for some RNA molecules and for a few mitochondrial polypeptides)

Chloroplasts:

Light-harvesting structures found in photosynthetic eukaryotes

Have two phospholipid bilayer membranes, DNA and have 70S ribosomes

Mitochondrion

Chloroplast

Comparison of prokaryotic and eukaryotic organelles

Comparison of prokaryotic and eukaryotic cells

Lecture prepared by Mindy Miller-Kittrell, University of Tennessee, Knoxville

M I C R O B I O L O G Y WITH DISEASES BY TAXONOMY, THIRD EDITION

Chapter 2

The Chemistry of Microbiology

Learning some basic concepts of chemistry will enable us to understand fully the variety of interactions between microorganisms and their environments, including, humans, animals and plants.

Atoms and atomic structure

Matter – anything that takes up space and has mass

Atoms – the smallest chemical units of matter

Electrons – negatively charged subatomic particles circling a nucleus

Nucleus – structure containing neutrons and protons

Bohr model of atomic structure

Figure 2.1

Atoms and atomic Structure

Atoms and atomic structure (continued)

Neutrons – uncharged particles

Protons – positively charged particles

Element – composed of a single type of atom

Atomic number – equal to the number of protons in the nucleus

Atomic mass (atomic weight) – sum of masses of protons, neutrons, and electrons

Isotopes

Every atom of an element has the same number of protons, but atoms of a given element can differ in the number of neutrons in their nuclei

Atoms that differ in the number of neutrons in their nuclei are isotopes. Examples are the three naturally occurring isotopes of Carbon

Carbon-12 (12C) has 6 protons and 6 neutrons

Carbon-13 (13C) has 6 protons and 7 neutrons

Carbon-14 (14C) has 6 protons and 8 neutrons

Stable isotopes (equal ratio of protons and neutrons)

Unstable isotopes (un-equal ratio of protons and neutrons). Unstable isotopes release energy during radioactive decay

Isotopes that undergo radioactive decay are radioactive isotopes

Radioactive isotopes play important roles in microbiological research, medical diagnosis, treatment of disease and sterilization of medical equipment and medical supplies/materials

Isotopes of carbon

Figure 2.2

Atom and atomic structure

Electron configurations

Only the electrons of atoms interact, so they determine atom’s chemical behavior

Electrons occupy electron shells or form clouds in an atom

Each electron shell can hold only a certain, maximum number of electrons (e.g., the first shell can accommodate a maximum of 2 electrons and the second no more than 8 electrons (more on this, please refer to periodic table, Fig. 2.4 page 29 in text)

Valence electrons – electrons in the outermost shell that interact with other atoms

Electron configurations

Figure 2.3

Bohr diagrams of the first 20 elements

Figure 2.4

Chemical Bonds

Chemical bonds

Chemical bonds – atoms combine by sharing or transferring valence electrons

Outer electron shells (valence shells) are stable when they contain eight electrons (except for the first electron shell, which is stable with only two electrons)

When an atom’s outer shells are not filled with 8 electrons, they either have room for more electrons to gain or have extra electrons to lose or are stable when outer electron shells contain eight electrons

Atoms’ outer most electrons are valence electrons and outer most shell of an atom is valence shell

An atom’s valence is its combining capacity and is positive if its valence shell has “extra” electrons to give up, and negative if its valence shell has spaces to fill in (e.g., Calcium with 2 electrons in its valence shell has a valence of +2, whereas Oxygen atom with 2 spaces to fill in its valence shell, has a valence of -2)

Molecule – two or more atoms held together by chemical bonds

Compound – a molecule composed of more than one element

Chemical Bonds

Chemical bonds (continued)

There are three principal types of chemical bonds (plus hydrogen bonds–weak forces that combine with polar covalent bonds)

Covalent bond – sharing of a pair of electrons by two atoms (when more than a pair of electrons are involved, double or triple covalent bonds are formed)

Electro-negativity – attraction of an atom for electrons. The more electronegative an atom, the greater the pull its nucleus exerts on electrons

Elements with more protons in their atoms exert a greater pull on electrons

Elements with increased distance between the nucleus and the valence shell have decreased electro-negativity

Chemical Bonds

Nonpolar covalent bonds

Atoms with similar electro-negativities share electrons equally

Shared electrons spend equal amount of time around each nucleus

No poles exist and the bond between is a non-polar covalent bond

Example: carbon atoms form four non-polar covalent bonds with one another and with many other types of atoms forming very large chains of many organic compounds

Organic compounds: compounds that contain carbon and hydrogen atoms (e. g. proteins and carbohydrates)

Molecules formed by covalent bonds: Hydrogen and oxygen

Figure 2.5a-b

Molecules formed by covalent bonds: Methane and formaldehyde

Figure 2.5c-d

Electronegativity values of selected elements

Figure 2.6

Chemical Bonds

Polar covalent bonds

Unequal sharing of electrons due to significantly different electronegativities (e. g. molecule of water)

Oxygen being more electronegative than hydrogen, electrons spend more time near the oxygen nucleus (partial negative charge, δ-) than the hydrogen nuclei (partial positive charge, δ+)

Thus, the bonds between oxygen atom and two hydrogen atoms are called polar, because they have opposite electrical charges

Most important polar covalent bonds for life processes are those that involve hydrogen because they allow hydrogen bonding to occur

Example of polar covalent bonds: Water molecule with 2 polar covalent bonds

Polar covalent bonding in a water molecule

Figure 2.7

Chemical Bonds

Ionic bonds

Occur when two atoms with vastly different electronegativities come together (e.g., sodium and chlorine ions)

Atom or group of atoms that have either a full negative charge or a full positive charge is called an ion

Positively charged ions are called cation, whereas negatively charged ions are called anion

Cations and anions attract each other and form ionic bonds (no electrons shared, but opposite electrical charges attract each other)

Typically form crystalline ionic compounds known as salts (e.g., sodium chloride and potassium chloride)

When cations and anions dissociate (ionize) from one another and surrounded by water molecules (or are hydrated), they are called electrolytes because they can conduct electricity through the solution

Electrolytes are important in biological/chemical processes - stabilize a variety of compounds, act like electron carriers and allow electron gradients to exist within cells

Interaction of sodium and chlorine to form an ionic bond

Figure 2.8

Dissociation of NaCl in water

Figure 2.9

Chemical Bonds

Hydrogen bonds

Hydrogen bond - Weak forces that combine with polar covalent bonds

Electrical attraction between partially charged hydrogen atom (H+) and a full or partially negative charge on either a different region of the same molecule or another molecule

Hydrogen bonds can be likened to week ionic bonds but are weaker than covalent and ionic bonds but essential for biological processes (for life)

The cumulative effect of weak hydrogen bonds is to stabilize 3-D shapes of large molecules (e.g., DNA)

Because hydrogen bonds are weak, they can be overcome when necessary (e.g., separation of DNA complementary halves during DNA replication)

Hydrogen bonds

Figure 2.10

Chemical Bonds

Table 2.2

Chemical Reactions

Chemical reactions

The making or breaking of chemical bonds

Involve reactants and products (e.g., atoms, ions, molecules)

Reactants and products may have different physical and chemical properties (e.g., hydrogen and oxygen gases react and form water)

The number and types of atoms never changes in a chemical reaction (atoms are neither destroyed nor created, only rearranged)

Biochemistry (biochemical reactions) involves chemical reactions of living things

Three categories of chemical reactions: synthesis, decomposition, and exchange reactions

Chemical Reactions

Synthesis reactions

Involve the formation of larger, more complex molecules. Example: synthesis of glucose by green plants and algae:

6H2O + 6CO2 ↔ C6H12O6 (Glucose) + 6O2

Require energy to break bonds in reactants and to form new bonds to make products (endothermic reactions)

Most common type of synthesis reaction is dehydration synthesis- an important synthesis reaction in which 2 smaller molecules are joined together by a covalent bond to form water molecule (H+ ion from one reactant combines with OH- ion from another reactant to form H2O)

All synthesis reactions in an organism are called anabolism

Dehydration synthesis

Figure 2.11a

Chemical Reactions

Decomposition reactions

Break bonds within larger molecules to form smaller atoms, ions and molecules. Example: aerobic decomposition of glucose to form CO2 and H2O:

C6H12O6 (Glucose) + 6O2 → 6H2O + 6CO2

Decomposition reactions release energy (exothermic reaction)

Hydrolysis: a common type of decomposition reaction in which ionic components of water (H+ and -OH) are added to products

All the decomposition reactions in an organism are called catabolism

Synthesis and decomposition reactions are often reversible in living things

Hydrolysis

Figure 2.11b

Chemical Reactions

Exchange reactions

Have similar features to both synthesis and decomposition reactions

Involve breaking and forming covalent bonds, and involve endothermic and exothermic steps

Involve atoms moving from one molecule to another

A + BC → AB + C or

AB + CD → AD + BC

An important exchange reaction within organisms is the phosphorylation of glucose:

Glucose + Adenosine triphosphate (ATP) → Glucose phosphate + adenosine diphosphate (ADP)

Sum of all chemical reactions in an organisms is called metabolism

Water, Acids, Bases, and Salts

Characteristics of water

Living things depend on organic compounds (those that contain carbon and hydrogen) to survive and reproduce

Also require a variety of inorganic chemicals (lack carbon but contain substances including water, oxygen molecules, metal ions, and many acids, bases and salts) to survive and reproduce.

Water is the:

Most abundant substance in organisms (50-90% of their body mass)

Most of its special characteristics are due to two polar covalent bonds

Cohesive nature of water molecules (tend to stick to one another by hydrogen bonding) – Biologically, creates surface tension, a thin layer on the surface of cells through which dissolved materials are transported into and out of the cell

Characteristics of Water

Characteristics of water (continued)

Excellent solvent - it dissolves salts and other electrically charged molecules because it is attracted to both positive and negative charges

Remains liquid across wide range of temperatures

Can absorb significant amounts of energy without changing temperature; when heated evaporates and molecules take away absorbed energy with them

Participates in many chemical reactions within cells, both as reactants in hydrolysis and as products of hydration synthesis

The cohesiveness of water

Figure 2.12

Water, Acids, Bases, and Salts

Acids and bases

Dissociated by water into component of cations and anions

Acid – dissociates into one or more H+ and one or more anions

Acids can be inorganic molecules (e. g. sulfuric acid, hydrochloric acid) or organic molecules (e. g. amino acids and nucleic acids)

Base – a molecule that binds with H+ when dissolved into water

Some bases (e.g., sodium hydroxide) dissociate into cations and OH- anions which then combine with H+ to form water molecules

Water, Acids, Bases, and Salts

Acids and bases (continued)

Metabolism requires relatively constant balance of acids and bases

When acidity changes (deviation concentration of either hydrogen ions or hydroxyl ions) too far from normal, metabolism stops

Concentration of H+ in solution is expressed using the logarithmic pH (potential hydrogen) scale

Most organisms contain natural buffers such as proteins that prevent drastic changes in internal pH

Microbiological culture media contain pH buffers (e.g. KH2PO4) that prevent a shift in pH as a result of metabolic activity of growing microorganisms

KH2PO4 exists either as a weak acid or a weak base, depending on the pH of its environment

Under acidic conditions, it is a base and combines with H+ neutralizing the acidic environment

In alkaline conditions, KH2PO4 acts as an acid, releasing hydrogen ions

Dissociation of acids and bases

Figure 2.13

The pH scale

Figure 2.14

Water, Acids, Bases, and Salts

Salts

Compounds that dissociate in water into cations and anions other than H+ and OH-

Acids (H+) and hydroxyl (OH-) yielding bases neutralize each other during exchange reactions and form salt and water

Cations and anions of salts are electrolytes

A cell uses electrolytes to:

Create electrical differences between its inside and outside

Transfer electrons from one location to another

Form important components of many enzymes

Organic Macromolecules

Organic macromolecules

Contain carbon and hydrogen atoms

Are larger and much more complex than inorganic molecules, forming branched chains, un-branched chains, and rings - the basic frameworks of organic molecules

Most common elements in organic compounds are: Carbon, hydrogen, oxygen, nitrogen, phosphorus and sulfur

Atoms often appear in certain common arrangements called functional groups

Amino functional group (- NH2) - is found in all amino acids and

Hydroxyl functional group (-OH) - is common to all alcohols

Organic Macromolecules

Organic macromolecules (continued)

Macromolecules – large molecules used by all organisms

Lipids

Carbohydrates

Proteins

Nucleic acids

Monomers – basic building blocks of macromolecules

Polymers: monomers of macromolecules joined together in chains

Organic Macromolecules

Lipids

Not composed of monomers, but are all hydrophobic (insoluble in water)

Four groups: Fats, Phospholipids, Waxes and Steroids

Composed of almost entirely of carbon and hydrogen atoms linked by non-polar covalent bonds

Non-polar bonds have no attraction to the polar bonds of water molecules, i.e., polar water molecules are attracted to each other and exclude the non-polar lipid molecules

Organic Macromolecules

Fats

Made in organisms by dehydration synthesis reaction that form esters between three chain-like fatty acids and alcohol called glycerol

Also called triglycerides-contain three fatty acid molecules linked to a molecule of glycerol

Saturated fatty acids: when every carbon atoms are linked solely by single bonds, with the exception of the terminal ones, covalently linked to two hydrogen atoms (e.g. stearic acid and palmitic acid)

Unsaturated fatty acids: contain at least one double bond between adjacent carbon atoms, and one carbon atom bound to only a single hydrogen atom (e.g., oleic acid)

Polyunsaturated fatty acids: presence of several double bonds between adjacent carbons atoms in a fat molecule (linoleic acid)

Saturated fats (those found in animals) are usually solid at room temperature because their fatty acids can pack close together

Unsaturated fatty acids are bent at every double bond, cannot pack tightly and remain liquid at room temperature (e.g., unsaturated or polyunsaturated fats of most plants)

The primary role of fats in organisms is to store energy in their carbon-carbon covalent bonds

Fats (triglycerides)

Figure 2.15

Organic Macromolecules

Phospholipids

Similar to fats, but contain two fatty acid chains and the third carbon atom of glycerol is linked to a phosphate (PO4) functional group instead of to a fatty acid

The phospholipid “head” is polar and thus is hydrophilic (attracted to water and soluble in water) and the “tail”portion of the molecule is non-polar and is thus hydrophobic (insoluble in water)

Phospholipid bilayers make up the outer membranes of all cells, as well as the internal membranes of plant, fungal and animal cells.

Phospholipids

Figure 2.16

Organic Macromolecules

Steroids

Consist of four rings (containing five or six carbon atoms) that are linked to one another and attached to various side chains and functional groups

Steroids play many roles in human metabolism acting as hormones

Cholesterol is an essential part of the phospholipids bilayer membrane surrounding all animal cells

Sterols, steroids with – OH functional group make membranes fluid and flexible at low temperatures and without them, membranes of cells would become stiff and inflexible in the cold

Steroids

Figure 2.17

Organic Macromolecules

Waxes

Contain one long-chain fatty acid covalently linked to long-chain alcohol by ester bond

Completely insoluble in water

Lack hydrophilic head

Organic Macromolecules

Carbohydrates

Organic molecules composed of atoms of carbon, hydrogen, and oxygen

Most carbohydrates contain an equal number of oxygen and carbon atoms and twice as many as hydrogen atoms as carbon atoms

(CH2O)n, where n indicates the number of CH2O units

Carbohydrates play many important roles in organisms:

Large carbohydrate molecule - long-term storage of chemical energy (e. g. starch and glycogen)

Small carbohydrate molecule - ready energy source (e.g. glucose)

Part of backbones of nucleic acids (RNA and DNA)

Routinely converted into amino acids

Polymers of carbohydrates form cell wall of fungi, algae, plants and prokaryotes

Are involved in intracellular interactions between animal cells (cell surface receptors)

Organic Macromolecules

Types of carbohydrates

Monosaccharides

The simplest carbohydrates (sugars) are monosaccharides

Names are formed from a prefix indicating the number of carbon atoms and the suffix – ose: Example, pentoses and hexoses are sugars with 5 and 6 carbon atoms respectively

Deoxyribose (sugar component of DNA) is a pentose; fructose and glucose are hexoses

Monosaccharides may exist as linear molecules but due to energy dynamics they usually take cyclic forms (e.g., alpha and beta configurations of glucose)

Monosaccharides

Figure 2.18

Organic Macromolecules

Disaccharides

Two monosaccharide molecules link together to form a disaccharide molecule by dehydration synthesis reaction

The linkage of two hexoses, e.g., glucose and fructose forms sucrose (table sugar)

Other examples of disaccharides are maltose (malt sugar) and lactose (milk sugar)

Broken into their monosaccharide components via hydrolysis reaction

Disaccharides

Figure 2.19

Organic Macromolecules

Polysaccharides

Polymers of thousands of monosaccharides covalently linked together in dehydration synthesis reaction

Diverse in monomer configuration (alpha or beta) and shapes (branched or unbranched)

Examples of polysaccharides are: cellulose (constituent of cell wall of plants and algae), amylose (storage starch compound in plants), amylopectin (plant starch) and glycogen (storage molecule in animal liver and muscle cells)

Bacterial cell walls are composed of peptidoglycan (polysaccharide plus amino acids). Lipopolysaccharides (cell wall components of Gram negative bacteria) are formed from polysaccharides and lipids

Polysaccharides

Figure 2.20

Organic Macromolecules

Proteins

Most complex organic compounds, composed mostly of carbon, hydrogen, oxygen, nitrogen, and sulfur

Proteins perform many functions in cells including:

Structure: structural components in cell walls, in membranes and within cells and structural material in hair, nail, skin and muscle

Transportation: certain proteins act as channels and “pumps” that move substances into or out of cells

Enzymatic catalysis - catalysts (typically proteins) that enhance speed of chemical reactions in cells are called enzymes

Regulation - some proteins regulate cell function by stimulating or hindering either the action of other proteins or the expression of genes (e.g., hormones are regulatory proteins)

Defense and offense - Antibodies and complements (proteins) defend our body against microorganisms

Organic Macromolecules

Amino acids

The monomers that make up proteins (protein polymers)

Contain a basic amino group (-NH2), an acidic carboxyl group (-COOH) and a hydrogen atom, all attached to the same carbon atom

Hundreds of amino acids, but most organisms use only 21 amino acids to in the synthesis of proteins

Side groups affect how amino acids interact with one another and how protein interacts with other molecules

A covalent bond (Peptide bond) is formed between two amino acids by dehydration synthesis reaction

A molecule composed of two amino acids linked together by single peptide bond is dipeptide and long chains of amino acids are called polypeptides

Amino acids

Figure 2.21

Organic Macromolecules

Protein structure

Un-branched polypeptides composed of hundreds of amino acids linked together in specific patterns as determined by genes

Every protein has at least 3 levels of structure and some proteins have 4 levels

Primary structure: the sequence of amino acid; sequence vary widely in length; a change in a single amino acid can drastically affect a protein’s overall structure and function

Secondary structure: polypeptide chains of proteins fold into coils (α-helices) or structures called β–pleated sheets as a result of ionic and hydrogen bonding and hydrophobic and hydrophilic characteristics of the proteins

Tertiary structure: protein polypeptides further fold into more complex three- dimensional shapes that are not repetitive like α-helices or β–pleated sheets

Protein structure (continued)

Quaternary structure: some proteins are composed of two or more peptide chains linked together by disulfide bridges or other bonds

Protein structure is directly related to its function; anything that interrupts shape (amino acid substitution, physical and chemical factors such as heat, change in pH and salt concentrations) severely disrupt protein function

The temporary or permanently disruption of structure and function of proteins is called denaturation

Stereoisomers

Figure 2.22

Linkage of amino acids by peptide bonds

Figure 2.23

Levels of protein structure

Figure 2.24

Organic Macromolecules

Nucleic acids and nucleotides

DNA and RNA vital as genetic material of organisms

RNA acting as an enzyme, binds amino acids to form polypeptides

Both are DNA and RNA are un-branched macromolecular polymers that differ primarily in the structures of their monomers

Organic Macromolecules

Nucleic acids and nucleotides

The monomers that make up nucleic acids (nucleotides) are composed of three parts:

A phosphate (PO42-)

A pentose sugar either deoxyribose or ribose

One of five cyclic (ring-shaped) nitrogenous bases

Adenine (A)

Guanine (G)

Cytosine (C)

Thymine (T)

Uracil (U)

Adenine and guanine are double-ringed molecules called purines, whereas cytosine, thymine and uracil are single-ringed and are called pyrymidines.

DNA contains A, G, C and T bases, whereas RNA contains A, G, C and U bases.

Nucleotides

Figure 2.25

Organic Macromolecules

Nucleic acids structure

Nucleic acids are polymers composed of nucleotides linked by covalent bonds between the phosphate of one nucleotide and the sugar of the next

The linear spines of nucleotides is composed of alternating sugars and phosphates, with bases extending from it

Three H bonds form between C and G in DNA and U and A in RNA

Two H bonds form between T and A in DNA or between U and A in RNA

DNA is a double-stranded molecule in most cells and viruses

The two strands of DNA are complementary to one another and also anti-parallel. One strand runs from the 3’ (3 prime) end to 5’ (5 prime) end and its complement runs in the opposite direction from its 5’ end to its 3’ end

General nucleic acid structure

Figure 2.26

Organic Macromolecules

Nucleic acids function

DNA is the genetic material of all organisms and many viruses

Carries instructions for the synthesis of RNA molecules and proteins in all organism

By controlling the synthesis of enzymes and regulatory proteins, DNA controls the synthesis of all other molecules in an organism

Genetic instructions are carried in the sequence of nucleotides of nucleic acid

Cells replicate their DNA molecules and pass copies to their progenies, making sure that each has the instruction necessary for life

Ribonucleic acids play several roles in the synthesis of proteins, including catalyzing the synthesis of proteins

RNA molecules also function as structural components of ribosomes and in place of DNA as the genome (genetic material) of RNA viruses

Organic Macromolecules

Adenosine triphosphate (ATP)

Phosphate - a highly reactive functional group in nucleotides

Forms covalent bonds with other phosphates to make di-phosphates and triphosphates

Molecules made from ribosome nucleotides (AMP, ADP and ATP) are important in many metabolic reactions

ATP is the principal, short-term and renewable energy supply of cells

When the high energy bonds (phosphate-phosphate bond) of ATP are broken, energy is released and ATP is converted to ADP

Energy from ATP is used for life activities such as synthesis reaction, locomotion, and transportation of substances into and out of cells

Adenosine triphosphate (ATP)

Figure 2.27

A Brief History of Microbiology

Prototype monocular microscope

The Early Years of Microbiology

What Does Life Really Look Like?

Antoni van Leeuwenhoek

The birth of Microbiology (study of microbes)

began with the discovery of microscope by

a Dutch lens grinder, Antoni Van Leeuwenhoek

Leeuwenhoek made simple microscopes and examined pond water and other materials to visualized tiny animals such as fungi, algae and single-celled protozoa.

For the first time, he reported the existence of

protozoa in 1674 and bacteria in 1676 and were

called "animalcules“.

By the end of 19th century, these organisms were

called microorganisms. Because of his, Leeuwenhoek is known today as the father of Protozoology and Bacteriology.

The Early Years of Microbiology

How Can Microbes Be Classified?

Carolus Linnaeus developed taxonomic system for naming plants and animals. He grouped similar organisms together into either animal kingdom or plant kingdom.
Leeuwenhoek's microorganisms now grouped into six categories as follows:
Bacteria } Domain Bacteria
Archaea } Domain Archaea
Fungi
Protozoa Domain Eukarya
Algae
Small multicellular animals
Scientists later modified Linnaeus’s scheme by adding more categories to logically reflect the relationships among organisms

The Early Years of Microbiology

How can microbes be classified?

Bacteria and Archaea
Prokaryotic - unicellular microbes

and lack nuclei

Much smaller than eukaryotes
Found everywhere there is sufficient moisture; some isolated from extreme environments
Reproduce asexually
Bacterial cell walls contain peptidoglycan; some lack cell walls
Archaeal cell walls composed of polymers other than peptidoglycan

The Early Years of Microbiology

How Can Microbes Be Classified?
Fungi
Eukaryotic (have membrane-bound nucleus)
Unlike plants, obtain food from dead organic matter or other organisms (saprobes or parasites)
Possess cell walls
Include
Molds – multicellular; grow as long filaments; reproduce by sexual and asexual spores
Yeasts – unicellular; reproduce asexually by budding; some produce sexual spores

The Early Years of Microbiology

How Can Microbes Be Classified?
Protozoa
Single-celled eukaryotes
Similar to animals in nutrient needs

and cellular structure

Live freely in water; some live in animal

hosts

Asexual (most) and sexual reproduction
Most are capable of locomotion by
Pseudopods – cell extensions that flow in direction of travel
Cilia – numerous short protrusions that propel organisms through environment
Flagella – extensions of a cell that are fewer, longer, and more whip-like than cilia

The Early Years of Microbiology

How Can Microbes Be Classified?
Algae
Unicellular or multicellular
Eukaryotic
Photosynthetic
Simple reproductive structures
Categorized on the basis of

pigmentation and composition

of cell wall

(b) diatoms

The Early Years of Microbiology

How Can Microbes Be Classified?
Other organisms that microbiologists study
Parasites

Viruses

The Golden Age of Microbiology

Scientists searched for answers to four questions:

Is spontaneous generation of microbial life possible?

What causes fermentation?

What causes disease?

How can we prevent infection and disease?

The Golden Age of Microbiology

Theory of spontaneous generation (Abiogenesis)

Some philosophers and scientists of the past thought living things arose from three processes:

Asexual reproduction
Sexual reproduction
Nonliving matter

Aristotle proposed spontaneous generation

Living things can arise from nonliving matter
Competition among scientist to answer these questions during the golden age of microbiology drove exploration and discovery and shaped the course of today’s microbiological research

The Golden Age of Microbiology

Does life generate spontaneously?

Francesco Redi's experiments:

When decaying meat was kept isolated from flies, maggots never developed

Meat exposed to flies was soon infested with maggots
As a result, scientists began to doubt Aristotle's theory

Does microbial life spontaneously generate?

Needham’s experiments

Scientists did not believe animals could arise spontaneously, but did believe microbes could

Boiled beef gravy and plant infusions in tightly corked vials to kill all life; later observed vials were cloudy and examinations revealed microscopic animals

Concluded that there must be a “life force” that causes inanimate matter to spontaneously come to life

Needham’s experiments with beef gravy and infusions of plant material reinforced this idea of theory of spontaneous generation

Does microbial life spontaneously generate?

Spallanzani’s experiments

Boiled infusions in vials for almost an hour and sealed/closed the vials by melting their ends

Infusions remained clear unless he broke the seal and exposed the infusion to air, after which they became cloudy with microorganisms

Splallazini concluded that:

Needham failed to heat vials sufficiently to kill all microbes or had not sealed vials tightly enough

Microorganisms exist in air and can contaminate experiments

Spontaneous generation of microorganisms does not occur. All living things arise from other living things

Critics said, sealed vials did not allow enough air for organisms to thrive and that prolonged heating destroyed “life force”

The Golden Age of Microbiology

Does microbial life spontaneously generate?

Pasteur's experiments
When the "swan-necked" flasks filled with boiled infusion remained upright, no microbial growth appeared
When the flask was tilted, dust from the bend in the neck seeped back into the flask and made the infusion cloudy with microbes within a day.

Louis Pasteur

The scientific method

Debate over spontaneous generation led to the development of the scientific method

A group of observations leads a scientist to ask question about a phenomenon

The scientist generates hypothesis (potential answer to question)

The scientist designs and conducts experiment to test hypothesis

Results prove or disprove hypothesis

Accepted hypothesis leads to theory/law

Reject or modify hypothesis

The scientific method

The Golden Age of Microbiology

What causes fermentation?

Spoiled wine threatened livelihood of vintners
Some believed air caused fermentation; others insisted living organisms caused fermentation. This debate is also linked to debate over spontaneous generation
Vintners funded research of methods to promote production of alcohol and prevent spoilage during fermentation
Others thought that yeasts are alive and were spontaneously generated during fermentation
Still others asserted that yeasts were not only living, but also caused fermentation
Some scientists proposed that yeasts observed in fermentation products were nonliving globules of chemicals and gases

Pasteur's application of the scientific method

The Golden Age of Microbiology

What Causes Fermentation?
Pasteur's experiments
Showed that microbes (yeast) are responsible for fermentation
Led to the development of pasteurization
Process of heating liquids just enough to kill most bacteria
Began the field of industrial microbiology
Intentional use of microbes for manufacturing products

Buchner’s experiments on fermentation

Buchner’s experiments on acellular fermentation

Earlier studies of fermentation began with the idea that fermentation reactions were strictly chemical and did not involve living organisms

Idea was supplanted by Pasteur’s work showing that fermentation proceed only when living cells were present

Eduard Buchner resurrected the chemical explanation, and showed that fermentation does not require living cells, but enzymes (cell produced proteins) that promote chemical reactions

His work began the field of biochemistry and the study of metabolism (sum of all chemical reactions within an organism)

The Golden Age of Microbiology

What Causes Disease?
Pasteur developed germ theory of disease: that microorganisms are also responsible for diseases
Robert Koch studied causative agents of disease (etiology)

Today it is known that some diseases are genetic, or caused by allergens or toxins in the environment; thus germ theory applies only to infectious diseases

Anthrax (Bovine disease caused by Bacillus anthracis)

Examined colonies of microorganisms

Robert Koch.

Koch's postulates
Suspected causative agent must be found in every case of the disease and be absent from healthy hosts
Agent must be isolated and grown

outside the host

When agent is introduced into a

healthy, susceptible host, the host must get the disease

Same agent must be found in the diseased experimental host

The Golden Age of Microbiology

Koch's experiments and contributions

Simple staining techniques
First photomicrograph of bacteria
First photomicrograph of bacteria

in diseased tissue

Techniques for estimating CFU/ml
Use of steam to sterilize media
Use of Petri dishes
Techniques to transfer bacteria
Bacteria as distinct species
Because of his achievements, he is considered father of the microbiological laboratory

The Golden Age of Microbiology

How Can We Prevent Infection and Disease?
Ignaz Semmelweis (1818-1865) before germ theory was fully

understood, discovered the benefits of handwashing to prevent

disease in the medical setting.

Joseph Lister (1827-1912) was the first surgeon to advance

the idea of antiseptic surgery with the use of phenol treated surgical instruments.

Florence Nightingale (1820-1910) was an English nurse who introduced cleanliness and other antiseptic techniques into nursing practice.
Edward Jenner (1749-1823) an English physician who spurred the field of immunology by discovering vaccination. He did so by intentionally inoculating a boy with pus collected from a milkmaid’s cowpox lesion. The boy became resistant to cowpox. He named the procedure vaccination after Vaccinia virus.

Florence Nightingale

The Modern Age of Microbiology

Effects of penicillin on a bacterial "lawn" in a Petri dish

The effects of penicillin on a bacterial "lawn" in a Petri dish.

Fungus colony

(Penicillium)

Zone of inhibition

Bacteria

(Staphylococcus)

Alexander Fleming (1881-1955) accidently discovered penicillin,

discovering antibiotics and ultimately saving millions of lives.

Laboratory assignment

The following questions are from two exercises (dilutions/pipetting and counting bacteria) that you read/studied. As reference materials, read lab manual, lab exercise PPts and/or study guides and answer questions given below fully/completely in the spaces provided as instructed. Please submit assignment before or on the due date. Hand written answers are not acceptable, except calculations.

You may submit assignment as an attachment to an email. Alternatively, you may scan assignment or take a photo it and submit as an attachment to an email.

Part One: Pipetting and Dilutions

A. 1. Objectives of this exercise – briefly describe the objectives of this exercise in your own English (refer to lab manual or PPt)

A. 2. Define Solute:

A. 3. Define Solvent:

A. 4. Define dilution:

A. 5. Define solution:

B: Pipetting and Dilutions

B. 1. For each set of dilutions in figure below, calculate the amount of colored substance (dilutions) in the last test tube of each set. Show calculation steps and results in the spaces provided.

image1.emf

B. 2. For questions B. 2. 1—B. 2. 3 in lab manual, first write down each question and then give the corresponding answers.

B. 2.1. What is the “meniscus”?

B. 2. 2. If you transfer 0.1ml of a sample into a 99.9ml saline blank, what is the dilution factor (show calculation steps)?

B. 2. 3. How much fluid is IN the pipette below? _________________________.

image5.emf

B. 3. For the following 3 questions (B. 3. 1 – B. 3. 3) in lab manual, first determine:

· The number of countable colonies (colony forming units, CFUs) that fall within the range of 30-300.

· The dilution factor that gave the count (example: 10-4 or 1/104)

· The amount of diluted sample plated/added (in ml) to each plate that gave the corresponding count.

· Then calculate the number of bacteria in 1 ml of original (undiluted) sample (solid or liquid) using formula given in manual or PPt. Show calculation steps.

Note: If sample is solid (example: hamburger meat), report count as CFUs per gram of meat. If sample is liquid (example: milk), report count as CFUs per ml of milk.

B. 3. 1.

image4.emf

B. 3. 2.

B. 3. 3.

Part two: Counting (enumeration) bacteria

2. 1. Objectives of this exercise - briefly describe the objectives in your own English (see lab manual)

2. 2. What is viable plate count?

2. 3. What do you use to determine the number of bacteria in suspension by the turbidimetric method?

2. 4. For questions 2. 4. 1 -- 2. 4. 6 in lab manual, first write down each question and then give the corresponding answers.

2. 4. 1. Data collection (Insert data table here from PPt)

2. 4. 2. Why do you have to do a standard plate count when running turbidity values the first time?

2. 4. 3. If you have a graph for E. coli, can the same graph also be used for another bacterium, like Staph?

2. 4. 4. How is “transmission” different from “absorbance”?

2. 4. 5. Give the formula for calculating the number of bacteria in 1 ml or 1 gram original sample (example: cheese or fruit juice). (Show calculation steps.

2. 4. 6. Using the formula in above question, calculate the bacterial count per milliliter of E. coli suspension in the original culture tube.

Important: To complete data table (2. 4. 1), enter count (2. 4. 6, above) into y-axis column (first for original E. coli) in data table (question # 2. 4.1.) given in PPt. Then divide count by 2 (or multiply count by ½) to enter count for each corresponding absorbance value under x-axis column. Finally enter zero (0) for both x- and y-axis columns.

2. 4. 7. Finally, using Excel graphic software, plot a standard curve using data from step 2. 4. 1, above. When entering your data in Excel graphic, start with the zero values (X-axis=0 and Y-axis=0) and finish with the highest values. Use absorbance column values for X-axis and the number of E. coli calculated for Y-axis. To plot the graph, follow the steps given in your manual. Please include curve/graph with the rest of report and submit before or due date. Be warned that no assignment is accepted after due date.

Due date: Friday, June 19 @midnight

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RICHLAND COLLEGE, Department of Biology,

School of Mathematics, Science & Health Professions

Microbiology syllabus for on-science majors

Name: Admassu Mitiku

Email: [email protected]

Office Phone: 972-238-6140

Course Information

Course title: Microbiology for Non-Science Majors

Course number: Biol 2420

Section number: 85201

Semester/Year: Summer 2020

Credit hour: 4

Online meeting times

Monday to Friday: Mornings until noon. Meet via college email

Saturday/Sunday: 5:00 8:00pm. Meet via college email

Important dates

Certification Date: 08/06/2020

Drop Date: 07/16/2020

Final exam: Tuesday, August 4, 2020

General course information

1. Describe distinctive characteristics and diverse growth requirements of prokaryotic organisms compared to eukaryotic organisms.

Other course policies

Grading and grading scales

Lab quiz # 2

-Staphylococci unknown ID

-Streptococci unknown ID

B. 3. 2.

B. 3. 3.

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