Lab assignment #1

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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