GENERAL MICROBIOLOGY

Microbial Growth

How do bacteria grow?

Since microorganisms are so small it is usually inconvenient to study individual cell. Therefore microbiologists normally follow changes in the total population. Microbial growth involves an increase in the number of cells.

MICROBIAL GROWTH

Growth of most microorganisms occurs by the process of binary fission(Asexual Reproduction).

This means that the parent cell produces exact copies of itself

MICROBIAL GROWTH

(Two cells arise from one) 1248163264 The required time for growth cycle is highly variable and is dependent on cell type and growth conditions
eg. E. coli has a generation time of 17 min under optimal conditions Most bacteria slower than 20 min Bacteria tends to grow faster than yeast and mould.

BINARY FISSION

MICROBIAL GROWTH TERMS

Doubling time or generation time

refers to the period required for cells in a microbial population to grow divide and produce two cells for each one that existed before.

Doubling time remains constant in a given population until nutrients become depleted or toxic metabolic waste accumulate.

MICROBIAL GROWTH TERMS

Growth Rate: change in cell number/cell population (or mass) per unit time
Growth rate is the doubling time per hour

Generation Time: interval for one cell to become two (or doubling time)

MICROBIAL GROWTH

Microorganisms show a characteristic growth pattern when inoculated into a fresh culture medium in a closed system.

MICROBIAL GROWTH

There is usually a lag phase, then exponential growth commences. As essential nutrients are depleted or toxic products build up, growth ceases, and the population enters the stationary phase. If incubation continues, cells may begin to die (the death phase).

Batch Culture growth curve

LAG of PHASE Phases Growth

Immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged. Adjustment period for organism to adopt to new surroundings. Repair any damage in cell and re-synthesis of essential cell constituents.

LAG PHASE

Occurs when: old cells transferred to fresh medium cells transferred from rich medium to poor Absent when: exponentially growing cells transferred to fresh portion of same media

LAG of PHASE Phases Growth

Although there is no apparent cell division occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity.

LAG of PHASE Phases Growth

The length of the lag phase is apparently dependent on a wide variety of factors including:
1. the size of the innoculum; 2.time necessary to recover from physical damage or shock in the transfer;

LAG of PHASE Phases Growth

3. time required for synthesis of essential coenzymes or division factors; 4. time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium.

LAG PHASE Exponential (log) Phase:

The exponential phase of growth is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression. The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation.

LAG PHASE

Exponential (log) Phase:

The exponential phase of growth is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression.

EXPONENTIAL (LOG) PHASE

The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation.

EXPONENTIAL GROWTH
Exponential growth cannot occur indefinitely One (1) bacterium weight one trillionth of a gram (1/1000000000000) with a generation time of 20 minutes Growing exponentially would produce a population that weighted 4,000 times that of the earth in 48 hours.

STATIONARY PHASE

Exponential growth cannot be continued forever in a batch culture (e.g. a closed system such as a test tube or flask. Population growth becomes limited by one of three factors:
1. Exhaustion of available nutrients; 2. Accumulation of inhibitory metabolites or end products; 3. Exhaustion of space, in this case called a lack of "biological space".

STATIONARY PHASE

The stationary phase, like the lag phase, is not necessarily a period of quiescence. During the stationary phase there is NO NET GROWTH (some cells die while others grow).

STATIONARY PHASE

Bacteria that produce secondary metabolites, such as antibiotics, do so during the stationary phase of the growth cycle
Secondary metabolites are defined as metabolites produced after the active stage of growth.

Endospore producing organisms produced endospores

DEATH PHASE

If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines.

DEATH PHASE

(Note, if counting by turbidimetric measurements or microscopic counts, the death phase cannot be observed.). During the death phase, the number of viable cells decreases geometrically (exponentially), essentially the reverse of growth during the log phase.

CONTINUOUS CULTURE
Bacterial cultures can be maintained in a state of exponential growth over long periods of time using a system of continuous culture. System is designed to relieve the conditions that stop exponential growth in batch cultures. A device called a chemostat, can be used to maintain a bacterial population at a constant density. The conditions in a chemostat more similar to bacterial growth in natural environments.

BACTERIAL METABOLISM Bacterial metabolism is classified into nutritional groups on the basis of three major criteria:
The kind of energy used The source of carbon, and The electron donors used for growth. Additionally respiratory microorganisms are classified according to the electron acceptors used for aerobic or anaerobic respiration.

BACTERIAL METABOLISM

BACTERIAL METABOLISM

Carbon metabolism in bacteria is either: Heterotrophic: organic carbon compounds are used as carbon sources.
All animals, most bacteria and fungi are heterotrophic. Heterotrophic bacteria include parasitic types.

BACTERIAL METABOLISM

Autotrophic: cellular carbon is obtained by fixing carbon dioxide.

Eg. phototrophic cyanobacteria, green sulfur-bacteria and some purple bacteria, but also many chemolithotrophic species, such as nitrifying or sulfur-oxidising bacteria.

MICROBIAL METABOLISM All cells need carbon and energy sources

Chemoorganotrophs
obtain their energy from the oxidation of organic compounds

Chemolithotrophs

obtain their energy from the oxidation of inorganic compounds

Photoautotrophs

contain pigments that allow them to use light as an energy source

BACTERIAL METABOLISM Energy metabolism of bacteria is either based on: Phototrophy: use of light through photosynthesis, or Chemotrophy: use of chemical substances for energy,
The chemical substances are mostly oxidised at the expense of oxygen or alternative electron

BACTERIAL METABOLISM Chemotropes are further subdivided: Lithotrophs: use inorganic electron donors (eg. hydrogen, carbon monoxide, ammonia, ferrous iron and other reduced metal ions, and several reduced sulfur compounds. Organotrophs: use organic compounds as electron donors. Most lithotrophic organisms are autotrophic, whereas organotrophic organisms are heterotrophic.

BACTERIAL METABOLISM Chemotrophic organisms use the respective electron donors for: energy conservation (by aerobic/anaerobic respiration or fermentation) and biosynthetic reactions (e.g. carbon dioxide fixation), Phototrophic organisms use their electron donors only for biosynthetic purposes

BACTERIAL METABOLISM

Aerobic organisms use oxygen as the electron acceptor in the electron transport chain. Anaerobic organisms use other inorganic compounds such as nitrate, sulfate or carbon dioxide as electron acceptors in the electron transport chain.

BACTERIAL METABOLISM

In both aerobic phototrophy and chemolithotrophy, oxygen is used as a terminal electron acceptor, while under anaerobic conditions inorganic compounds are used instead.

BACTERIAL METABOLISM

In the absence of possible electron acceptors chemotrophs carry out fermentation, where the electrons taken from the reduced substrates are transferred to oxidised intermediates to generate reduced fermentation products (e.g. lactate, ethanol, hydrogen, butyric acid).
These organisms do not utilize the electron transport chain.

BACTERIAL METABOLISM

Facultative anaerobes can switch between fermentation and different terminal electron acceptors depending on the environmental conditions in which they find themselves.

BACTERIAL METABOLISM

Combining their nutritional patterns, all organisms in nature can be placed into one of four separate groups:
1. 2. 3. 4. photoautotrophs, photoheterotrophs, chemoautotrophs, and chemoheterotrophs.

BACTERIAL METABOLISM
Nutritional Type
Photoautotrophs

Energy Source
Light

Carbon Source
CO2

Examples
Cyanobacteria, some Purple and Green Bacteria

Photoheterotrophs

Light

Organic Some Purple and compounds Green Bacteria A few Bacteria and many Archaea Most Bacteria, some Archaea

Inorganic Chemoautotrophs or compounds, CO2 Lithotrophs e.g. H2, NH3, (Lithoautotrophs) NO2, H2S Chemoheterotrophs Organic Organic or Heterotrophs compounds compounds

BACTERIAL METABOLISM 1. Photoautotrophs use light as an energy source and carbon dioxide as their main carbon source. Photoautotrophs transform carbon dioxide and water into carbohydrates and oxygen goes through photosynthesis .
They include photosynthetic bacteria (green sulfur bacteria, purple sulfur bacteria, and cyanobacteria), algae, and green plants.

BACTERIAL METABOLISM 2. Photoheterotrophs use light as an energy source but cannot convert carbon dioxide into energy. Instead they use organic compounds as a carbon source. They include the green nonsulfur bacteria and the purple nonsulfur bacteria.

BACTERIAL METABOLISM 3.Chemolithoautotrophs use inorganic compounds such as hydrogen sulfide, sulfur, ammonia, nitrites, hydrogen gas, or iron as an energy source and carbon dioxide as their main carbon source.
They include sulphur bacteria, the iron bacteria, the nitrifying bacteria, the hydrogen bacteria, and the methane bacteria.

BACTERIAL METABOLISM

4.Chemooganoheterotrophs use organic compounds as both an energy source and a carbon source. Saprophytes live on dead organic matter while parasites get their nutrients from a living host.
They include bacteria, and all protozoans, fungi, and animals.

MEDIA TYPES
Defined Media/Synthetic media A defined medium is one in which the exact chemical composition is known.
A defined medium that just has enough ingredients to support growth is called a minimal medium. The number of ingredients in a minimal medium varies depending on the microorganism.

E. coli for instance needs only a simple minimal medium. Minimal medium can only be prepare for microorganisms with known nutritional requirements.
For example Glucose Salt Agar (GSA)

MEDIA TYPES

Minimal media contain the minimum nutrients possible for colony growth, generally without the presence of amino acids, and are often used by microbiologists and geneticists to grow "wild type" microorganisms. Minimal media can also be used to select for or against recombinants. More difficult to prepare

MEDIA TYPES

Minimal medium typically contains:

a carbon source for bacterial growth, which may be a sugar such as glucose, or a less energy-rich source like succinate various salts, which may vary among bacteria species and growing conditions; these generally provide essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow the bacteria to synthesize protein and nucleic acid

MEDIA TYPES
Complex Media A complex medium is one in which the exact chemical composition is not known. It is made from extracts of natural materials like beef, blood, casein, yeast and soybeans. Contain all the elements that most bacteria need for growth and are non-selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory culture collections.
The most common complex media used is nutrient agar. Complex media are easy to prepare and can support the growth of most microorganisms. Eg. Glucose Yeast Peptone Agar, Nutrient Agar, Plate Count Agar

MEDIA TYPES
Selective media By inhibiting unwanted organisms with salts, dyes, or other chemicals, selective media allow growth of only the desired microbes. Some examples of selective media include:
SPS agar. (Sulfite Polymyxin Sulfadiazine) used to detect Clostridium perfringens in food. YM (yeast and mold) which has a low pH, deterring bacterial growth

MEDIA TYPES
Hektoen enteric agar (HE) which is selective for Gramnegative bacteria Mannitol salt agar (MSA) which is selective for Grampositive bacteria and differential for mannitol usage Terrific Broth (TB) is used with glycerol in cultivating recombinant strains of Escherichia coli.

Mannitol Salt Agar (MSA) selects for Staphylococcus

MEDIA TYPES
Differential media Used to distinguish among different organisms according to the appearance of isolated colonies growing on or in the medium. Examples of differential media include:
mannitol salt agar (MSA), which is differential for mannitol fermentation X-gal plates, which are differential for lac operon mutants blood agar (used in strep tests), which contains bovine heart blood that becomes transparent in the presence of hemolytic Streptococcus

MEDIA TYPES
Selective Differential Media Allow growth of only the desired microbes and also distinguish among selected organisms according to the appearance of isolated colonies
Eosin Methylene Blue (EMB) is selective for Gram-negative bacteria and differential for lactose fermentation MacConkey agar is selective for Gram-negative bacteria and differential for lactose fermentation
MacConkey Agar differentiate lactose fermenting and nonfermenting Eosin-methylene blue (EMB) selective for gram-negative bacteria and differentiate between the colonies of lactose fermenting and nonfermenting.

MEDIA TYPES Enrichment Culture An enrichment culture is used to encourage the growth of a particular microorganism, usually in low concentration, from a mixed culture.
Isolation of nitrogen-fixing bacteria using media lacking nitrates Isolation of halophiles using high salt concentration Isolation of thermophiles using high temperatures

NUTRITIONAL REQUIREMENTS
Nitrogen Needed for Protein and nucleic acid synthesis. Obtained from decomposition of proteins or from NH4+ or NH3; a few bacteria are capable of nitrogen (N2) fixation. Sulfur Needed to synthesize sulfur-containing amino acids and certain vitamins. Depending on the organism, sulfates, hydrogen sulfide, or sulfur-containing amino acids may be used as a sulfur source.

NUTRITIONAL REQUIREMENTS
Phosphorus Needed to synthesize phospholipids, DNA, RNA, and ATP. Phosphate ions are the primary source Trace elements Trace elements are elements required in very minute amounts, like potassium, magnesium, calcium, and iron They usually function as cofactors in enzyme reactions.
Cofactors usually function as electron donors or electron acceptors during enzyme reactions.

NUTRITIONAL REQUIREMENTS

Growth factors Growth factors are organic compounds such as amino acids, purines, pyrimidines, and vitamins that a cell must have for growth but cannot synthesize itself. Organisms having complex nutritional requirements and needing many growth factors are said to be FASTIDIOUS.