Chapter 9

FtsZ

FtsZ assembles into a Z ring on the cytoplasmic membrane. The Z ring is anchored by FtsZ-binding proteins and defines the division plane between the two daughter cells. FtsZ is a tubulin-like protein of the prokaryotic cytoskeleton that is important in prokaryotic cytokinesis

Thioglycolate medium

Reducing medium which can accept oxygen

Division septum

Separates new daughter cells in binary fission

Quorum sensing

The ability of bacteria to sense the presence of other bacteria via secreted chemical signals.

Quorum Sensing

The ability of bacteria to sense the presence of other bacteria via secreted chemical signals. Assists in bio-film formation.

What does MSA stand for?

Mannitol Salt Agar (Selective for salt tolerrence, halophiles)

Membrane Filtration

Membrane filtration is used to count live cells in dilute solutions.

Plate count

Method to measure the concentration of viable cells by counting the number of colonies that develop from a sample added to an agar plate

What does TSA stand for?

Trypticase soy agar

Microbial Growth

-Increase in a population of microbes -Due to reproduction of individual microbes -Results in discrete colony or biofilm -Colony: aggregation of cells arising from single parent cell -Biofilm: collection of microbes living on a surface in a complex community

What is a persister cell? Why are they medically important?

-A pathogenic cell within a host that neither grows nor dies when exposed to appropriate antimicrobials; this tolerance is due not to genetic mutation but to the cell's existence in a transient, dormant state. -Why dont persistors respond to antibiotics? A: Some bacteria have protein pumps that throw antibiotics out of their cell. They can also mutate to change their protein receptors

Membrane filtration

-Concentrates microbes by filtration -Filter is incubated on appropriate agar medium

Counting chambers

-Easy, inexpensive, and quick -Useful for counting both eukaryotes and prokaryotes -Cannot distinguish living from dead cells

Biofilms

A surface coating colony of prokaryotic that engage in metabolic cooperation -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 -Dental plaque is a biofilm that can lead to cavities -Scientists seeking ways to prevent biofilm formation

Explain why anaerobic conditions in wounds are dangerous.

Anaerobic bacteria can cause an infection when a normal barrier (such as skin, gums, or intestinal wall) is damaged due to surgery, injury, or disease. Usually, the immune system kills any invading bacteria, but sometimes the bacteria are able to grow and cause an infection. Body sites that have tissue destruction or a poor blood supply are low in oxygen and favor the growth of anaerobic bacteria. Anaerobic bacteria can infect deep wounds, deep tissues, and internal organs where there is little oxygen. These infections are characterized by abscess formation, foul-smelling pus, and tissue destruction.

Biofilms are different from normally growing bacteria because:

Biofilms are the accumulation of thousands of bacteria communicating and working together for the survival of their colony. Normally growing bacteria are just replications of each other that act more independently as their own organisms.

Detoxification of reactive oxygen species

Catalase Test: Test that determines whether or not bacteria has "catalase" enzyme. --- Peroxidase, superoxide dismutase, and catalase are the main enzymes involved in the detoxification of the reactive oxygen species. Superoxide dismutase is usually present in a cell that can tolerate oxygen. All three enzymes are usually detectable in cells that perform aerobic respiration and produce more ROS.

Thioglycolate broth

Complex medium that separates microbes based on oxygen requirements

Sustaining microbial growth

Continuous Culture in a Chemostat: -A Chemostat is a device that allows for the continuous culture of microorganisms with independent control of both growth rate and cell number -Chemostat is used to maintain a microbial population in a particular phase of growth -Open system -Requires addition of fresh medium and removal of old medium -Allows the study of microbial populations in steady but low nutrient levels -Used in several industrial settings

Direct Cell Count

Count all cells present; automated or manual

Direct cell count

Count all cells present; automated or manual Includes: -Counting chambers -Coulter counter -Plate count -Serial Dilution -Membrane filtration

Fermentation and Gas Production

Fermentation can occur without gas production but gas production cannot occur without fermentation.

Turbidity

Indirect cell coount method: can be used to estimate culture density by measuring turbidity of a culture or live cell density by measuring metabolic activity. A measure of how clear water is.

Nutritional requirements for growth

Inoculum introduced into nutrients called media: -Inocula obtained from various sources -Environmental specimens -Clinical specimens -Stored specimens Culture: -Act of cultivating microorganisms or the microorganisms that are cultivated Culute Media: -Majority of prokaryotes have not been grown in culture medium -Nutrient broth is common medium -Agar is a common addition to many media -Complex polysaccharide derived from certain red algae -Produces a solid surface for colonial growth -Most microbes cannot digest agar

Antibiotic resistance

The evolution of populations of pathogenic bacteria that antibiotics are unable to kill

Practice: Suppose you start with 1000 bacterial cells with a generation time of 15 minutes. How long will it take the population to reach 1,024,000 assuming no cell death?

Use a simple number line to solve these problems: 1,000 x 15 minutes = 2,000 2,000 x 15 minutes = 4,000 4,000 x 15 minutes = 8,000 8,000 x 15 minutes = 16,000 16,000 x 15 minutes = 32,000 32,000 x 15 minutes = 64,000 64,000 x 15 minutes = 128,000 128,000 x 15 minutes = 256,000 256,000 x 15 minutes = 512,000 512,000 x 15 minutes = 1,024,000 15 minutes x 10 steps = 150 minutes 150/60 = *2.5 hours*

The most probable cell number

allows estimation of cell numbers in cultures without using solid media.

Binary Fission vs Mitosis

*Binary Fission* don't have nucleus, DNA synthesizes (replicated), fission is formed before it breaks apart prokaryotes cells *Mitosis* eukaryotic cells, much more complicated, more steps involved

Types of Growth Media

*Blood Agar* contains general nutrients and 5% sheep blood. It is useful for cultivating fastidious organisms and for determining the hemolytic capabilities of an organism. Some bacteria produce exoenzymes that lyse red blood cells and degrade hemoglobin; these are called hemolysins. Bacteria can produce different types of hemolysins. Beta-hemolysin breaks down the red blood cells and hemoglobin completely. This leaves a clear zone around the bacterial growth. Such results are referred to as β-hemolysis (beta hemolysis). Alpha-hemolysin partially breaks down the red blood cells and leaves a greenish color behind. This is referred to as α-hemolysis (alpha hemolysis). The greenish color is caused by the presence of biliverdin, which is a by-product of the breakdown of hemoglobin. If the organism does not produce hemolysins and does not break down the blood cells, no clearing will occur. This is called γ-hemolysis (gamma hemolysis). The hemolysins produced by streptococci perform better in an anaerobic environment. Because of this, it is standard procedure to streak a blood plate and then stab the loop into the agar to provide an area of lower oxygen concentration where the streptolysins can more effectively break down the blood cells. *Fermenation Tube* Fermentation tubes are used to verify gas production in fermentation exercises. *MacConkey Agar* a selective and differential medium designed to isolate and differentiate enterics based on their ability to ferment lactose. If a bacteria grows on MacConkey agar it is gram negative. Neutral red is a pH indicator that turns red at a pH below 6.8 and is colorless at any pH greater than 6.8. -Organisms that ferment lactose and thereby produce an acidic environment will appear pink because of the neutral red turning red. Bile salts may also precipitate out of the media surrounding the growth of fermenters because of the change in pH. Non-fermenters will produce normally-colored or colorless colonies.

Measurement of Bacterial Growth:

*Direct methods not requiring incubation* -Microscopic counts -Count microorganisms directly through a microscope -Suitable for stained prokaryotes and large eukaryotes -Electronic counters -Coulter counters -Counts cells as they interrupt an electrical current flowing in front of an electronic detector -Flow cytometry -A light-sensitive detector records changes in light transmission as cells pass through a tube *Direct methods requiring incubation:* -Serial dilution and viable plate counts -Membrane filtration -Most probable number *Indirect Methods* -Turbidity -Metabolic activity -Using changes in nutrient utilization, waste production, or pH to estimate number of cells in a culture -Dry weight -Organisms are filtered from media, dried, and weighed -Genetic methods -Isolate DNA sequences of unculturable prokaryotes

Six types of general culture media

*General Purpose media* designed to grow a broad spectrum of microbes that do not have special growth requirements --- Specialized Medias: *Defined media* media in which the exact chemical composition and amounts are known. *Complex media* media that is made from extracts from plants (soy), animals (beef) or fungi (yeast). The exact chemical composition is not known, it is not uniformed each time its made. *Selective media* favor the growth of some microorganisms while inhibiting others. *Differential media* help distinguish bacteria by the color of the colonies or the change in the medium. *Anaerobic media* media used to grow anaerobic organisms *Transport media* Used to maintain and preserve specimens that need to be held for a period of time *Enriched media* contain added essential nutrients a specific organism needs to grow -Chemically defined media contain only chemically known components.

Bacterial Growth: Oxygen

*Oxygen requirements* -Aerobic and anaerobic environments can be found in diverse niches throughout nature, including different sites within and on the human body. -Microorganisms vary in their requirements for molecular oxygen. Obligate aerobes depend on aerobic respiration and use oxygen as a terminal electron acceptor. They cannot grow without oxygen. -Optimum oxygen concentration for an organism is the oxygen level that promotes the fastest growth rate. -The minimum permissive oxygen concentration and the maximum permissive oxygen concentration are, respectively, the lowest and the highest oxygen levels that the organism will tolerate. -How can this be true? -Toxic forms of oxygen are highly reactive and excellent oxidizing agents -Resulting oxidation causes irreparable damage to cells Four toxic forms of oxygen: -Singlet oxygen — molecular oxygen with electrons in higher energy state -Superoxide radicals — form from the incomplete reduction of O2 -Peroxide anion — formed during reactions catalyzed by superoxide dismutase -Hydroxyl radical — result from ionizing radiation and incomplete reduction of hydrogen peroxide

Bacterial Growth: Water

*Physical effects of water:* -Most bacteria, with few exceptions, require high moisture to grow. -Microbes require water to dissolve enzymes and nutrients -Water is important reactant in many metabolic reactions -Most cells die in absence of water -Some have cell walls that retain water -Endospores and cysts cease most metabolic activity -Two physical effects of water: -Osmotic pressure -Hydrostatic pressure -Photosynthetic bacteria depend on visible light for energy. *Osmotic Pressure* -Pressure exerted on a semipermeable membrane by a solution containing solutes that cannot freely cross membrane -Hypotonic solutions have lower solute concentrations -Cell placed in hypotonic solution swells -Hypertonic solutions have greater solute concentrations -Cell placed in hypertonic solution shrivels -Restricts organisms to certain environments -Obligate and facultative halophiles -Halophiles require high salt concentration in the medium, whereas halotolerant organisms can grow and multiply in the presence of high salt but do not require it for growth. -Halotolerant pathogens are an important source of foodborne illnesses because they contaminate foods preserved in salt. *Hydrostatic Pressure* -Water exerts pressure in proportion to its depth -Barophiles live under extreme pressure -Their membranes and enzymes depend on pressure to maintain their three-dimensional, functional shape

Compare refrigeration, deep freezing, and lyophilization as methods for storing bacterial cultures.

*Refrigeration* best for short peiod of time *Deep freezing* for long term use, involves freezing cells at temperatures from -50C to -95C *Lyophilization* freeze drying, ivolves removing water from a frozen culture using an intense vacuum and can last for decades

Bacterial Growth: PH

-Organisms are sensitive to changes in acidity H+ and OH- interfere with H bonding -Microorganisms grow best at their optimum growth pH. Growth occurs slowly or not at all below the minimum growth pH and above the maximum growth pH. *Neutrophiles* Bacteria are generally neutrophiles. They grow best at neutral pH close to 7.0. *Acidophiles* grow best in acidic habitats, optimally at a pH near 3.0 *Alkalinophiles* organisms that grow optimally between a pH of 8 and 10.5. live in alkaline soils and water -Extreme acidophiles and alkaliphiles grow slowly or not at all near neutral pH. -Many microbes produce acidic waste products that can accumulate and inhibit their growth

Bacterial Growth: Other Factors

-Osmotic and barometric pressure -Salinity (Salt content) -Light -A *capnophile* is an organism that requires a higher than atmospheric concentration of CO2 to grow.

Binary fission

-The most common mechanism of cell replication in bacteria -A form of asexual reproduction in single-celled organisms by which one cell divides into two cells of the same size -Before dividing, the cell grows and increases its number of cellular components. -Replication of DNA starts at a location on the circular chromosome called the origin of replication -Replication continues in opposite directions along the chromosome until the terminus is reached -The center of the enlarged cell constricts until two daughter cells are formed, -Each offspring receiving a complete copy of the parental genome and a division of the cytoplasm (cytokinesis). -This process of cytokinesis and cell division is directed by a protein called FtsZ. -FtsZ assembles into a Z ring on the cytoplasmic membrane --- Other patterns of cell division include multiple nucleoid formation in cells; asymmetric division, as in budding; and the formation of hyphae and terminal spores.

Generation (doubling) time

-The time it takes for the population to double through one round of binary fission. -Most bacterial cells divide by binary fission. Generation time in bacterial growth is defined as the doubling time of the population. -Time required for a complete fission cycle -Time required for a bacterial cell to grow and divide -Dependent on chemical and physical condition -Also referenced as doubling time

Thioglycolate Test

1. *Obligate Aerobes* need oxygen because they cannot ferment or respire anaerobically. They gather at the top of the tube where the oxygen concentration is highest. 2. *Obligate Anaerobes* Obligate anaerobes cannot grow in the presence of oxygen. They depend on fermentation and anaerobic respiration using a final electron acceptor other than oxygen. so they gather at the bottom of the tube where the oxygen concentration is lowest. 3. *Facultative Anaerobes* show better growth in the presence of oxygen but will also grow without it.They gather mostly at the top because aerobic respiration generates more ATP than either fermentation or anaerobic respiration. 4. *Microaerophiles* need oxygen to grow, albeit at a lower concentration than 21% oxygen in air. They cannot ferment or respire anaerobically. However, they are poisoned by high concentrations of oxygen. They gather in the upper part of the test tube, but not the very top. 5. *Aerotolerant Anaerobes* Although they do not perform aerobic respiration, they can grow in the presence of oxygen. Most test negative for the enzyme catalase. They can be found evenly spread throughout the test tube.

Environmental Influences of Biofilms

Bacterial biofilms are found under diverse environmental conditions. Biofilms respond to environmental conditions via a finite number of key regulatory factors and pathways, which affect enzymatic and structural elements that are needed for biofilm formation and dispersal. Among the conditions that affect biofilm development are *temperature, pH, O2 levels, hydrodynamics, osmolarity, the presence of specific ions, nutrients, and factors derived from the biotic environment.* The integration of these influences ultimately determines the pattern of behavior of a given bacterium with respect to biofilm development.

What is a biofilm?

Biofilms are communities of microorganisms enmeshed in a matrix of extracellular polymeric substance. The formation of a biofilm occurs when planktonic cells attach to a substrate and become fixed. Cells in biofilms coordinate their activity by communicating through quorum sensing. Biofilms are commonly found on surfaces in nature and in the human body, where they may be beneficial or cause severe infections. Pathogens associated with biofilms are often more resistant to antibiotics and disinfectants.

Measuring Bacterial Growth

Cells can be counted by *direct viable cell count*. The *pour plate* and *spread plate* methods are used to plate *serial dilutions* into or onto, respectively, agar to allow counting of viable cells that give rise to *colony-forming units*. *Membrane filtration* is used to count live cells in dilute solutions. The *most probable cell number (MPN)* method allows estimation of cell numbers in cultures without using solid media.

Bacterial Growth Curve

Cells in a closed system follow a pattern of growth with four phases: 1) *Lag phase* New enzymes to use available nutrients are induced 2) *Log (Exponential) phase* Binary fission is occurring at maximum rate 3) *Stationary phase* Number of new cells equal to number of dying cells 4) *Death phase* Number of dying cells is higher than the number of cells dividing -Log phase is when you wanna attack with antibiotics becayse all the bacterias resources are being focused on reproduction and not defenses -During the stationary phase, cells switch to a survival mode of metabolism. -As growth slows, so too does the synthesis of peptidoglycans, proteins, and nucleic-acids; -Stationary cultures are less susceptible to antibiotics that disrupt these processes. In bacteria capable of producing endospores, many cells undergo sporulation during the stationary phase. -Secondary metabolites, including antibiotics, are synthesized in the stationary phase. -Sometimes in the death phase there are persisters, which are medically important because they do not respond to antibiotics.

Serial Dilution

Dilution of a substance several times by the same amount each time

Coulter Counter

Electronically scans a culture as it passes through a tiny pipette

Bacterial Growth: Temperature

Microorganisms thrive at a wide range of temperatures; they have colonized different natural environments and have adapted to extreme temperatures. Both extreme cold and hot temperatures require evolutionary adjustments to macromolecules and biological processes. *Psychrophiles* cold-loving microbes grow best in the temperature range of 0-15 °C whereas psychrotrophs thrive between 4°C and 25 °C. *Mesophiles* grow best at moderate temperatures in the range of 20 °C to about 45 °C. Pathogens are usually mesophiles. *Thermophiles* heat lovers *Hyperthermophiles* extremely hot. Thermophiles and hyperthemophiles are adapted to life at temperatures above 50 °C. -Temperature affects three-dimensional structure of proteins -Lipid-containing membranes of cells and organelles are temperature sensitive -If too low, membranes become rigid and fragile -If too high, membranes become too fluid -Adaptations to cold and hot temperatures require changes in the composition of membrane lipids and proteins.

Nitrogen Fixation

Nitrogen fixation is a process whereby bacteria in the soil convert atmospheric nitrogen into a form that plants can use. The reason this process is so important is that animals and plants cannot use atmospheric nitrogen directly. That nitrogen gas is very abundant (78% of the atmosphere) but it must be converted into another form before most organisms can use it. Bacteria convert it into ammonium which then plants can absorb. Animals get their nitrogen by eating plants or other animals. Finally, all organisms need a steady supply of nitrogen because it is a building block of DNA and RNA, and of proteins. These are vital macromolecules that organisms need to build their genetic info (DNA and RNA) and to carry out many of the processes of life (proteins).

The Pour Plate and Spread Plate methods of Serial Dilution

The pour plate and spread plate methods are used to plate serial dilutions into or onto, respectively, agar to allow counting of viable cells that give rise to colony-forming units.

Biofilms are of particular concern in healthcare because:

They are more resistant to antibiotics

Practice: With a doubling time of 30 minutes and a starting population size of 500 cells, how many cells will be present after 2 hours, assuming no cell death?

Use a simple number line to solve these problems: 500 x 30 minutes = 1,000 1,000 x 30 minutes = 2,000 2,000 x 30 minutes = 4,000 4,000 x 30 minutes = 8,000 30 + 30 + 30 + 30 = 2 hours A: 2 hours = *8000 cells present*

Divisome

a complex of proteins that directs cell division processes in prokaryotes

Extracellular polymeric substances

a glycocalyx that helps cells in a biofilm attach to their target environment and to each other