Quiz #2

Question 1

Streptomyces (gram positive)

Answer 1

Streptomyces is a bacterial genus of the Order Actinomycetales, members of which resemble fungi in their branching filamentous structure and are chiefly saprophytic (feed off decaying matter). A number of Streptomyces sp. produce antibiotics, and also play an important role as degraders of biopolymers, such as starch.

Question 2

description of the genus
Streptomyces

Answer 2

a. Look for leathery, raised, colonies that may be pigmented.
b. The surface of the colony may look powdery, rough, or velvety.

Question 3

Antibiotics

Answer 3

Antibiotics are compounds that are produced as secondary metabolites by certain groups of microorganisms, especially Streptomyces, Bacillus, and a few molds (Penicillium and Cephalosporium) that are inhabitants of soils. Antibiotics may have a bactericidal (killing) effect or a bacteriostatic (growth inhibitory) effect on a range of microbes. The range of bacteria or other microorganisms that are affected by a certain antibiotic is expressed as its spectrum of activity. Antibiotics that kill or inhibit a wide range of Gram-positive and/or Gram-negative bacteria are said to be broad spectrum. If effective mainly against a few Gram-positive or a few Gram-negative bacteria, they are narrow spectrum.

Question 4

the Kirby-Bauer disk diffusion technique

Answer 4

This technique is carried out in microbiology laboratories in order to determine optimal antibiotic (or antibiotic concentration) therapy in treating a bacterial infection. It involves paper disks impregnated with known concentrations of a number of antibiotics and placed on top of bacterial plates. The plates are incubated to allow growth of the bacteria and time for the agent to diffuse into the agar. In order to determine if an antibiotic will be effective in treating the bacterial infection, the zone of inhibition must be measured and compared to a standard. If the zone of inhibition is smaller than the predetermined zone for that compound, the organism is considered resistant to that antibiotic. If it is within the range or larger, it is considered sensitive to the antibiotic being tested.

Question 5

Cellular respiration

Answer 5

Heterotrophic bacteria obtain their energy for cell growth and division by means of either respiration or fermentation. Both catabolic systems convert the chemical energy of organic molecules to high-energy bonds in adenosine triphosphate (ATP). In respiration, glucose is converted to ATP in three distinct phases: 1) glycolysis, 2) the tricarboxylic acid cycle (Krebs cycle), and 3) oxidative phosphorylation (sometimes called the electron transport chain, or ETC)

Question 6

Glycolysis

Answer 6

Glycolysis splits the six-carbon glucose molecule into two pyruvate molecules, composed of 3 carbon molecules, with the production of ATP and reduced coenzymes.

Question 7

Krebs cycle

Answer 7

Krebs cycle is the complex pathway in which acetyl-CoA (from the conversion of pyruvate) is oxidized to CO2 and more coenzymes are reduced. ATP is also a product.

Question 8

Electron transport chain

Answer 8

Electron Transport Chain (ETC) is a series of oxidation-reduction reactions that receives electrons from the reduced coenzymes produced during glycolysis and the Krebs cycle. At the end of the ETC is an inorganic molecule called the terminal electron acceptor. When oxygen is the final electron acceptor, the respiration is aerobic. If the terminal electron acceptor is an inorganic molecule other than oxygen (e.g., sulfate or nitrate) the respiration is anaerobic.

Question 9

Lag phase

Answer 9

Lag Phase: Period of little or no cell division.

1. Cells do not immediately reproduce in new medium.
2. The cell density remains temporarily unchanged.
3. Cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and
   increasing in metabolic activity.
   The length of the lag phase is dependent on a wide variety of factors including:
   • the size of the inoculum
   • time necessary to recover from physical damage or shock in the transfer
   • time required for synthesis of essential coenzymes or division factors
   • time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium.

Question 10

Log phase

Answer 10

Log/exponential growth Phase: cells dividing —> period of growth —> increase logarithmically
• Phase when cells are most active metabolically.
• The rate of exponential growth of a bacterial culture is expressed as generation time, also
the doubling time of the bacterial population.

Question 11

Generation time

Answer 11

Generation time (G): defined as the duration time (t) of growth divided by the number of generations (n) G = t/n

The number of generations (n) is calculated using the formula below where Nt is the number of cells at time (t) and No is the number of cells at the start of growth.
Nt = No x 2n
For example if the number of cells at time t is 256 and the number of cells at No is 8, then: 256 = 8 x 2n—> 2n = 32–>n = 5
If the time at Nt is 100 minutes, then G = 100/5, or a generation/doubling time of 20 minutes.

Question 12

Stationary phase

Answer 12

Stationary Phase: When the number of deaths is equivalent to the number of new cells
• The growth rate slows and there is no net change in cell density.
• Population growth is limited by one of three factors:
1. Exhaustion of available nutrients
2. Accumulation of inhibitory metabolites or end products
3. Exhaustion of space: a closed system such as a test tube or flask.
• Bacteria that produce secondary metabolites, such as antibiotics, do so during the stationary phase of the growth cycle.
• During this stationary phase, spore-forming bacteria have to induce or unmask the activity of dozens of genes that may be involved in the sporulation process.

Question 13

Death phase

Answer 13

Death Phase–When the number of deaths exceeds number of new cells formed
1. If incubation continues after the population reaches stationary phase, a death phase follows,
in which the viable cell population declines.
2. During the death phase, the number of viable cells decreases geometrically (exponentially),
essentially the reverse of growth during the log phase.

Question 14

Optimum growth temperature

Answer 14

Pyschrophile: 0-20 Celsius (Pseudomonas fluorescens)

Mesophile: 15-45 Celsius (Serratia marcescens)

Moderate thermophile: 42-70 Celsius (Bacillus stearothermophilus)

Question 15

Characteristics of the Enterobacteriaceae

Answer 15

Members of the Enterobacteriaceae family are found in the gastrointestinal tract of animals, but many are also free living in soil and water. There are a number of Enterobacteriaceae that are important human pathogens, typically causing gastrointestinal illness (e.g., salmonellosis and shigellosis/bacterial dysentery). Since all of the family members look alike after Gram stain, other tests must be used to differentiate them.

Characteristics of the Enterobacteriaceae
Microscopic morphology:
• They are Gram-negative rods and typically 1-5 microns in length.
• They do not produce spores.
• They have peritrichous flagella (exception: Klebsiella and Shigella are non-motile
and have no flagella). Macroscopic colony morphology:
□ Colonies are usually dome shaped, gray, and smooth. Oxygen requirement:
□ They are facultative anaerobic bacteria. They can ferment or respire depending upon the level of oxygen available.
Catalase activity:
□ All members are catalase positive with exception of Shigella dysenteriae.
Glucose fermentation:
□ All members can ferment glucose to pyruvate (pyruvic acid), which is then
converted to different end products depending upon the species. These end
products can be used in identifying the species. Nitrate reduction:
□ Most members reduce nitrate to nitrite. Some members can further reduce nitrite to nitrogen.

Question 16

Bacteria Fermentation

Answer 16

In contrast to respiration, fermentation is the metabolic process by which glucose acts as an electron donor and one or more of its organic products act as the final electron acceptor. Reduced carbon compounds in the form of acids and organic solvents, as well as CO2, are the typical end products of fermentation.

The lactose fermentation ability of the members of Enterobacteriaceae is one of the key characteristics used in identification. Testing for lactose fermentation can distinguish between a lactose negative pathogen and the lactose positive Enterobacteriaceae.

Question 17

Nitrogen fixation

Answer 17

This process converts N2 in the atmosphere into NH3 (ammonia), which is assimilated into amino acids and proteins. It occurs in some free-living bacteria (Azotobacter, Clostridium, and cyanobacteria) in the soil and also in symbiotic bacteria within the roots of leguminous plants, the rhizobia bacteria (Rhizobium and Frankia), within characteristic nodules.
These microorganisms are important in the nitrogen cycle, returning fixed nitrogen to the soil.

Question 18

Examples of nitrogen fixing bacteria

Answer 18

In Azotobacter the nitrogenase enzyme responsible for nitrogen fixation is anaerobic, but the exceedingly high respiratory rate of the Azotobacter species consumes O2 so rapidly that an anaerobic environment is maintained inside the cell.

Rhizobium live endosymbiotically with leguminous plants. These plants include clover, alfalfa, peas, peanuts and soybeans. The plant synthesizes the protein leghemogloben when infected with Rhizobium, which binds to O2 and depletes the levels of O2 in the nodule allowing the Rhizobium to fix nitrogen.

Rhizobia colonies are often slimy, due to synthesis of exopolysaccharide, and pigmented. Note the large size of the cells in the wet mount of an isolated colony. Both cysts (phase bright ovals) and vegetative cells (phase dark bacilli) are visible.

Question 19

Gram negative Rhizobia

Answer 19

The Rhizobia from the nodule are pleomorphic (may have multiple shapes or forms with an inconsistent Gram stain). The Rhizobia grown on a plate and stained will have the typical form of Gram-negative rods.

Question 20

Dual control of the Lac operon

Answer 20

Glucose and lactose high amounts: operon off and CAP/repressor is not bound

Glucose but no lactose: operon off and repressor is bound but CAP is not there

NO glucose and lactose: operon off and CAP and repressor is present

Lactose but no glucose: operon on CAP is present and repressor is not

Question 21

diauxic growth curve

Answer 21

A result of two different exponential growth phases, separated by a time when the culture does not grow. Escherichia coli grown in a medium containing a mixture of glucose and lactose will produce this type of growth curve. During the first few hours the bacteria divide exponentially, using the glucose as the carbon and energy source. When the glucose is used up, there is a brief lag period while the lac genes are switched on before the bacteria return to exponential growth, now using up the lactose.

Question 22

Negative Regulation

Answer 22

Occurs when the DNA-binding form of a protein works to turn a gene off. These proteins work to inhibit the binding of RNA polymerase to the operon. The Lac repressor protein is the protein responsible for inhibiting the expression of the Lac operon. The Lac repressor protein is able to bind to the Lac operon when lactose is absent. Allolactose is an isomer of lactose that binds to the Lac repressor protein and removes it. Removing the repressor protein is one of two necessary steps for the activation/transcription of the Lac operon. The other being the binding of CAP.
The use of both the CAP and the Lac repressor protein allows for the Lac operon to be highly expressed when two conditions are met: lactose must be present and glucose must be absent.

Question 23

Catabolite Control of the Lac Operon

Answer 23

The Lac operon is inducible by lactose to the highest levels when cAMP and CAP form a complex.
1. Under conditions of high glucose, a product of glucose breakdown inhibits the enzyme adenylate cyclase, preventing the conversion of ATP into cAMP.
2. Under conditions of low glucose, there is no product of glucose break down, and therefore adenylate cyclase is active and cAMP is formed.
3. When cAMP is present, it acts as an allosteric effector, complexing with CAP.
4. The cAMP CAP complex acts as an activator of lac operon transcription by binding to a
region within the lac promoter.

If glucose is abundant for the bacteria’s use, it would be a waste of cellular energy for CAP to activate the Lac operon. That being the case, the Lac operon is not just controlled by CAP. Even if CAP is present, the negative control could still repress the gene.

Question 24

Two types of transcriptional controls regulate the Lac operon: Positive and Negative

Answer 24

Positive Regulation - Occurs when the DNA-binding form of a protein works to turn a gene on. These proteins aid RNA polymerase in binding to the promoter region. CAP (Catabolite Activator Protein) is the protein responsible for turning the Lac operon on, leading to gene expression. CAP is used in bacteria to enable the use of alternative carbon sources in the absence of glucose. CAP is able to bind to the Lac operon when cyclic-adenosine monophosphate (cAMP) is present. cAMP binds to CAP and allows the protein to bind to the Lac operon. The levels of cAMP are dependent on whether or not glucose is present. If glucose is abundant cAMP levels are low; therefore, CAP is not in a DNA-binding form because cAMP is not bound to it.

Question 25

Inducible enzyme

Answer 25

In some cases a bacterium will synthesize an enzyme only if the substrate for that enzyme is present.

Question 26

Operon

Answer 26

A set of genes whose expression is coordinated by an operator is defined as an operon.

Question 27

β-galactosidase

Answer 27

The Lac operon codes for proteins required to transport lactose into the cell and break it down to glucose and galactose. The operon is activated in the presence of lactose (and low levels of glucose) and the β-galactosidase enzyme is synthesized following the induction of the lac operon.
In order for bacteria to ferment lactose, they must possess two enzymes: lactose permease, a membrane- bound transport protein, and β-galactosidase, an intracellular enzyme that hydrolyzes the disaccharide lactose into the monosaccharides glucose and galactose. Bacteria that can synthesize both enzymes are active lactose fermenters.

Question 28

ONPG

Answer 28

The compound o-nitrophenyl-β-D-galactopyranoside (ONPG) is a substrate analog of lactose and can be used to measure the induction of the lac operon. Because of its similarity to lactose, ONPG can become the substrate for any β-galactosidase enzyme present. In the reaction that occurs ONPG is hydrolyzed to galactose and o-nitrophenol (ONP), which is yellow. In this experiment ONP is the indicator used to show the presence of B -galactosidase.

Question 29

Isolation of Commensal Bacteria

Answer 29

The genus Staphylococcus is found on normal skin (external surfaces and in hair follicles). The bacteria are facultative anaerobes and chemoorganotrophic, requiring nutritionally rich media. The colonies are opaque and usually pearly white, but may have yellow-gold pigmentation. Clinical tests are used to differentiate the pathogenic Staphylococcus spp. from other Gram- positive cocci. The pathogen most often encountered is S. aureus that causes various infections (e.g., carbuncles, osteomyelitis, bacterial pneumonia, wound infection, food poisoning, toxic shock syndrome). Additionally, S. epidermidis is a common commensal of the healthy skin, but can be an opportunistic pathogen, while S. saprophyticus can be isolated from the vagina and may be responsible for urinary tract infections in females.

Question 30

environmental niches

Answer 30

Bacteria are most numerous in the large intestine (the colon), but they can be found anywhere that is exposed to the outside environment. Bacteria heavily colonize the skin, oral cavity, scalp and nostrils. The human body contains many environmental niches that can vary in regards to the availability of water, salinity, temperature, pH and the presence of fats and oils. Microorganisms are generally well adapted to the niche they occupy on/in the human body. There is a great diversity of bacteria associated with a healthy human (~1,000 different species) although a smaller number of abundant types make up the majority of the bacterial population while much of the diversity is represented by relatively rare species.

Question 31

Catalase Test

Answer 31

Bacterial cells produce hydrogen peroxide (H2O2) during aerobic respiration. If H2O2 accumulates in the cell, it becomes toxic. Therefore, most aerobic and facultative anaerobic bacteria possess an enzyme called catalase, which breaks down H2O2 to water and oxygen. Catalase positive bacteria, such as species of Staphylococcus and Micrococcus, can be identified with bubbly formation (O2) from catalase-negative species of Streptococcus and Enterococcus, which lack this enzyme.

S. epidermidis = positive culture, Streptococcus. = negative culture,

Question 32

Lactic acid bacteria

Answer 32

Important genera of lactic acid bacteria include: Streptococcus, Lactobacillus, Enterococcus, Lactococcus, and Leuconostoc. Some species are part of the normal flora of the human body and are found in the oral cavity, GI tract and vagina. Certain oral lactic acid bacteria are responsible for the formation of dental plaque and the initiation of dental caries (cavities). The initiation of cavities is caused by the extra acidity produced by these bacteria; it dissolves the calcium phosphate in tooth enamel leading to the start of a cavity.

Question 33

Lactobacilli

Answer 33

They are gram positive, rod shaped organisms that grow as single cells or loosely associated chains. Lactobacillus varies from short slender rods to short coccobacilli. They are present in decomposing plant material, milk, and other dairy products, and are found in the microflora of the mouth and the healthy human vagina during childbearing years.

The lactic acid formed from sugars in the food by lactic acid bacteria inhibits the growth of less tolerant microorganisms.

Question 34

Two types of lactic fermentation

Answer 34

Lactic acid fermentation is an anaerobic fermentation reaction, which can be categorized in two ways: The first being homolactic fermentation, in this reaction two molecules of lactic acid are produced from the conversion of one glucose molecule. The second being heterolactic fermentation, in this reaction carbon dioxide and ethanol as well as lactic acid are produced from glucose. The presence of lactic acid bacteria can be determined by their ability to form large amounts of acid. In addition, the formation of vitamins and other substances often increases the nutritional value of the food.

Question 35

Agar for lactic acidosis bacteria

Answer 35

The agar used in this lab is called Yeast Dextrose Calcium carbonate agar (YDC). This is a differential agar that determines whether or not the plated organism produces lactic acid. The CaCO3 in the YDC plates can be broken down to CO2 gas by lactic acid, producing a clearing. This clearing is the indicator for a lactic acid positive bacteria.

Question 36

Durham fermentation tubes

Answer 36

The Durham Fermentation Tubes, used in Part B of this lab, are used to determine whether an organism is a homolactic fermenter or a heterolactic fermenter. The media is a basal media with a high glucose concentration and a pH indicator. The Durham tube is included in order to identify whether or not gas is produced from the metabolic activities of the organism in the media.

Question 37

anoxygenic phototrophs (green sulfur, green non-sulfur, purple sulfur, and purple non-sulfur)

Answer 37

These organisms oxidize H2S or organic material in order to generate reducing power using sunlight. They do not produce O2 and are either obligate or facultative anaerobes and can grow in the presence of O2.

Question 38

Sulfur bacteria

Answer 38

Sulfur bacteria preferentially use H2S while non-sulfur bacteria utilize organic matter as their source of electrons for generating reducing power through NADH. The names green or purple reflect the kind of photosynthetic pigments they possess imparting color to the bacteria. There are also certain structural differences. Green-sulfur bacteria do not have flagella but have gas vacuoles that allow them to float at the surface, they also have extra-cellular S granules. Some purple-sulfur bacteria have flagella as well as gas vacuoles and the S-granules are stored intra- cellularly.

Question 39

oxygenic phototrophs

Answer 39

they generate O2 and use water as their photosynthetic electron donor. They are considered as the primary producers that made the atmosphere change from anoxic (or reducing) to oxygenic. Thus, the current world is more facilitative for aerobic organisms. Members within cyanobacteria include: Merismopedia, Gloeocapsa, Anabaena, Nostoc, Oscillatoria and Lyngbya.

Question 40

Photosynthetic eukaryotic microorganisms

Answer 40

include a large group of algae, specifically the green algae from which higher plants have emerged. The microscopic green algae include unicellular as well as various colonial, coccoid, and filamentous forms of flagellates (usually with two flagella per cell), that all contain chloroplasts. Genera within the green algae include: Euglena, Chlamydomonas, Volvox, Ulothrix, and Spirogyra

Question 41

Wet-Mount Preparation Procedure

Answer 41

The usual wet mount can be performed by putting a small drop of sample onto the clean slide in the center of a waxed circle using Pasteur pipette. Spread the drop evenly and place the cover slip gently over the drop avoiding any air-bubbles.
Use nigrosin for negative stain
Use 10x or 40x
Filaments: trichomes
Heterocysts: nitrogen fixing bacteria in filaments

Question 42

Cyanobacteria

Answer 42

The cyanobacteria deserve special emphasis because of their great ecological importance in the global carbon, oxygen and nitrogen cycles, as well as their evolutionary significance in relationship to plants. Chloroplasts (plastids) are formed by permanent enslavement of cyanobacteria in plants: land plants, green algae (e.g. chlamydomonas) and red algae. Of the 5 major groups of photosynthetic bacteria, only cyanobacteria are oxygenic during photosynthesis. They are often called blue-green algae, even though not all members are blue-green color and they are definitely not algae.

Question 43

Pigments

Answer 43

Most cyanobacteria have a slimy sheath, or coating, which is often deeply pigmented, particularly in species that occur in terrestrial habitats. These pigments impart color to individual cells and colonies as well as to nuisance blooms of cyanobacteria that rise to the surface affecting the other aquatic life. The colors of different species include light gold, yellow, brown, red, green, blue, violet, and blue-black.
Cyanobacteria utilize chlorophyll a, which is responsible for their green coloration. In addition, they have unusual accessory pigments called phycobilins, which absorb wavelengths of light for photosynthesis that are missed by chlorophyll and the carotenoids, thus giving blue- green or reddish color to cyanobacteria.

Question 44

Morphology

Answer 44

Most cyanobacteria have a Gram-negative type cell wall that consists of an outer membrane component, even though they may show a distant phylogenetic relationship with certain Gram-positive bacteria.
The cyanobacteria are morphologically a heterogeneous mixture of bacteria. Some are unicellular, with typical prokaryotic shapes such as cocci rods, and spirals. Such species come in two forms depending on whether they divide by binary fission (unicellular) or multiple fission (colonial). In the latter, a mucilaginous sheath holds individual cells together to produce the colonial forms. Gloeocapsa, for example, secrete individual gelatinous sheaths which can often be seen as sheaths around a recently divided cell, two daughter cells, that remained together temporarily by outer sheaths or they generate mucilaginous sheaths around several cells (colonial). For Merismopedia, cell division is restricted to two directions resulting in a row-like arrangement of cells in flat colonies. The cells are held together by mucilage.
In some species, cytokinesis (division of cytoplasm) is incomplete; thus the dividing cells remained attached through a filament. Such filamentous multi-cellular associations are called trichomes and may or may not be enclosed within a sheath. Cell wall pores allow cells in a filament to communicate with each other, so neighboring cells are not completely separated. As in other filamentous or colonial bacteria, the cells of cyanobacteria may be joined by their walls or by slimy sheaths, but each cell is an independent unit of life.
Further the filamentous forms are either branching or non-branching. In the former, a single cell divides in two directions forming a connected branch (Ex: Stigonematales). The non- branching types are divided into two groups depending on the presence or absence of special nitrogen fixing heterocysts. Anabaena and Nostoc have heterocysts. Oscillatoria and Lyngbya do not.

Question 45

Motility

Answer 45

Some of the filamentous cyanobacteria are motile by means of gliding or rotating around a longitudinal axis. The mechanism for this movement is unexplained but may be connected to the extrusion of slime through small pores in their cell wall, together with contractile waves in one of the surface layers of the wall.

Question 46

Habitat

Answer 46

Cyanobacteria are found in most aerobic environments where water and light are available for growth. They inhabit a wide range of environments, including freshwater and marine habitats, soils, and the surfaces of rocks. They can also be found in the desert where they remain dormant most of the time, taking advantage of the occasional rains. In aquatic environments they often form thick mats. Like many bacteria they have a higher tolerance for heat and low pH than the green plants, so they are often the main autotrophs in hot springs.
Cyanobacteria inhabiting the surface layers of water are part of a complex microbial community called plankton. They adjust their buoyancy by inflating or deflating gas vacuoles, enabling them to adjust their position in the water column, floating near the surface during the day for photosynthesis and sinking deeper at night to harvest nutrients.
When numerous cyanobacteria become unable to regulate their gas vesicles properly (for example, because of extreme fluctuations of temperature or oxygen supply), they may float to the surface of a body of water—where they die and decay— forming nuisance blooms. Some cyanobacteria that form these blooms secrete poisonous substances that are toxic for animals that ingest large amounts of the contaminated water.

Question 47

Nitrogen Fixation

Answer 47

Some cyanobacteria can fix nitrogen. We learned earlier this semester, that nitrogen fixers must avoid oxygen. Cyanobacteria are aerobic and regulate oxygen levels with specialized cells called heterocysts, which appear as enlarged segments in filamentous cyanobacteria. They are surrounded in a thickened, specialized glycolipid cell wall that slows the rate of diffusion of O2 into the cell. Any O2 that diffuses into the heterocyst is rapidly reduced by hydrogen, or is expelled through the cell wall. Heterocysts have intercellular connections to adjacent vegetative cells, and there is continuous movement of the products of nitrogen fixation moving from heterocysts to vegetative cells, and the products of photosynthesis moving from vegetative cells to heterocysts.

Question 48

Algae

Answer 48

Many species live most of their lives as single-cells, other species form colonies or long filaments. Algae employ simple reproductive structures and lack the extensive vascular structures characteristic of higher plants. Eukaryote organisms are capable of oxygenic photosynthesis. They are classified into different groups on the basis of morphology, types of chlorophylls, carbon reserve storage materials, cell wall composition, and habitat. Although many algae, like the higher plants, are non-motile, they may have motile reproductive cells.

Question 49

Euglena

Answer 49

Euglena is a flagellated cell with the same chlorophylls (a and b) as found in higher plants. Euglena is unusual for an alga in that it can lose its chloroplasts when kept in the dark or treated with the antibiotic streptomycin, but it is able to survive as a heterotroph. Thus, this organism has affinity to both algae and protozoa. For this reason, Euglena is claimed by botanists as an alga, but as a protozoan by zoologists.

Question 50

Diatoms

Answer 50

Diatoms belong to a large group called the heterokonts, including both autotrophs (e.g. golden algae, kelp) and heterotrophs (e.g. water moulds). There are more than 200 genera of living diatoms, and it is estimated that there are approximately 100,000 extant species. Diatoms have two hard cell walls (called frustules) composed of silicon oxide. Their yellowish-brown chloroplasts contain pigments such as fucoxanthin. Diatoms are a widespread group and can be found in the oceans, in freshwater, in soils and on damp surfaces. Most live in open water, although some live as surface films at the water-sediment interface, or even under damp atmospheric conditions.

Question 51

Pigments

Answer 51

Although eukaryotic algae all have chlorophyll a pigments, giving them a bright green color. The algae are differentiated by their accessory pigments (such as chlorophylls b, c1, c2, or d, as well as a variety of carotenoid pigments) that catch photons of wavelengths to which chlorophyll a is not sensitive. The specific secondary pigments depend on the color of the light available in the organism’s local environment. Green algae that live at the surface of oceans and lakes use carotene (a yellow molecule) to trap purple and UV radiation. Farther down from the surface of the ocean, red (using phycobilins), brown (using fucoxanthin), and golden algae (using xanthophill) trap the blue-green light that penetrates to deeper levels of the oceans. All land plants appear to have evolved from green algae, perhaps because the pigment combination of green algae is better able to tolerate high light conditions and UV radiation and resist photo bleaching (the destruction of photosynthetic pigments by excess light).

Question 52

T3 phage

Answer 52

Viruses that infect bacteria are called bacteriophages. These can interact with the host cell in two main ways: Lytic and Lysogenic.
Lytic phage- Take over the metabolism of the host cell completely, resulting lysis of the cell. Steps of Infection:
a. Attachment to the bacterial cell wall
b. Injection of viral DNA into the bacterial cytoplasm.
c. The viral genome then commands the cell to produce viral proteins, which
are used for assembly of phages.
d. Once assembly is complete, the cell lyses and releases the phages, which
then attack other bacterial cells and begin the replication cycle anew.
Example: T3 (infects E. coli)

Question 53

Temperate/Latent phage (lysogenic cycle)

Answer 53

Temperate/Latent phage (Lysogenic cycle) - Live in harmony with the host cell and multiply
as the host cell multiplies.
Steps of Infection:
Attachment to the bacterial cell wall
Injection of viral DNA into the bacterial cytoplasm.
Viral nucleic acid becomes integrated into the host cell’s chromosome
- Takeover of cellular processes is not immediate

Replication of phage is delayed
- This process is called lysogeny, which results in a prophage that can remain dormant for long periods of time
Under environmental stressful conditions the lysogenic prophage switches to the lytic
mode to try and escape from the cell before cell death. Example: Lambda (infects E. coli)

Question 54

Switching between cycles

Answer 54

Stimuli that induce the switch from a lysogenic to a lytic phage include exposure to environmental stress factors like radiation and toxic chemicals. Thus, in the lytic cycle, infection by viral DNA leads directly to the multiplication of the virus and lysis of the host bacterial cell. In the lysogenic cycle, a prophage is replicated as part of the host’s chromosome.

Question 55

Lytic bacteriophage: Counting
Plaques

Answer 55

Lysis of bacterial cells growing on
an agar plate produces a clearing that can be viewed with the naked eye. These clearings are called plaques. A plaque assay uses this phenomenon as a means of calculating the phage concentration in a given sample. When a sample of bacteriophage is added to a plate inoculated with enough bacterial host to produce a lawn of growth, the number of plaques formed can be used to calculate the original phage titer or density.

Diluted phage is added directly to a small amount of broth culture. Then this phage-host mixture is added to a tube of soft agar, mixed and poured onto prepared nutrient agar plates as an agar overlay. The consistency of the solidified soft agar is sufficient to immobilize the bacteria while allowing the smaller bacteriophages to diffuse short distances and infect surrounding cells. During incubation, the phage host produces a lawn of growth on the plate in which plaques appear where contiguous cells have been lysed by the virus. At suitable dilutions of phage particles, each plaque that forms represents the point at which a single phage particle was deposited.

Question 56

PFU equation

Answer 56

Pfu/ml = (# of plaques) x (1/ml plated) x (1/dilution)

Examples:
If 162 PFUs were counted after inoculating a plate with 0.1 mL of phage suspension, diluted 1:109, the equation would be set up as follows.
162 pfu / 0.1ml x (1/10-9) = 1.62 x 1012
If 347 plaques were obtained when 0.1 mL of a 10-6 diluted phage suspension was plated, the equation would be set up as follows
347 pfu / 0.1ml x (1/10-6) = 3.47 x 109

Question 57

Total Coliform Rule

Answer 57

Total Coliform Rule was passed and set a both a health goal and legal limits for the presence of total coliforms in drinking water. This rule mandated regular monitoring (by the local government and the Environmental Protection Agency, EPA) of drinking water systems; the frequency of monitoring was set by the size of the population served (<1000 people = once/month, >2.5 million = ~420x/month) less than 5% of the samples could be positive.

Question 58

Water borne diseases

Answer 58

Most water-borne diseases result from transmission through fecal contamination. There are various pathogens, which may be present in low numbers compared to other bacteria making it difficult to specifically detect the pathogens during regular water quality monitoring. Therefore, indicator organisms are used– some typical coliform or group of coliforms as an indication of fecal contamination. The most commonly used such organism is Escherichia coli. E. coli is used because 1) lives longer than most pathogens, 2) is more abundant, and 3) it is less risky to collect and culture. You will perform simple tests to determine the presence of indicator organisms in water samples provided to check the purity of water.

Question 59

YEM agar

Answer 59

YEM Agar which contains mannitol as a carbon source and yeast extract as a source of both nitrogen and growth factors for Agrobacteria. It also poises oxidation - reduction potential of medium in the range favourable for Rhizobia and serves as hydrogen donor in respiratory process

Question 60

EMB agar

Answer 60

Contains peptone, lactose, sucrose, and the dyes eosin Y and Methylene blue. It is selective because the dyes inhibit growth of Gram-positive bacteria. It is a differential media for bacteria that have enzymes to allow lactose fermentation; acid is produced and the change in pH will cause the colony to be colored. EMB agar is used for the
isolation of fecal coliforms. Coliforms are members of the Enterobacteriaceae that produce acid (and gas) from lactose fermentation. Non coliforms are members of the Enterobacteriaceae that do not ferment lactose

1. Strong lactose fermenters are dark purple/green metallic (E. coli).
2. Slower lactose fermenters produce smaller amounts of acid (Enterobacter aerogenes: slow lactose fermenters) that results in a pink coloration of growth. Non-fermenters are colorless—they either retain their normal color or take on the coloration of the medium.

Question 61

MacConkey Agar

Answer 61

MacConkey agar is a culture
medium designed to grow Gram-
negative bacteria and stain them for lactose fermentation

By utilizing the lactose available in the medium, Lac+ bacteria such as E. coli and Klebsiella will produce acid, which lowers the pH of the agar below 6.8 and results in the appearance of red/pink colonies. (Note the precipitated bile salts around the E. coli.) Non-Lactose fermenting bacteria such as Salmonella and Shigella use peptone and excrete ammonia, which raises the pH of the agar. Non-lactose fermenting bacteria appear as white/colorless colonies or retain the color of the media.

Question 62

Phenol red lactose broth

Answer 62

If lactose can be used, the microbe will accumulate acidic byproducts. In a positive test, the pH indicator in the medium changes color from its normal red to yellow, indicating acid production.

Phenol Red Fermentation Tests are used to differentiate members of Enterobacteriaceae and to distinguish them from other Gram-negative rods.

Question 63

Urban Microbiome

Answer 63

1. Environmental samples
2. Genomic DNA extraction
3. PCR amplification of 16S rRNA
4. Next generation sequencing
5. Sequence data analysis

Metagenomic approaches have been introduced
that detect a much greater portion of the actual
diversity in a sample. Metagenomic approaches
are culture-independent, which means that it is not
necessary to cultivate the microorganisms to
detect their presence. Instead, genomic DNA (or
RNA in the case of some viruses) is isolated from
the environmental sample being studied and using
“signature” DNA sequences as a proxy to quantify
and characterize the microorganisms present in the
sample. The metagenomic approach can be used to analyze any set of organisms from the environment, however, for the work in this lab we will begin by focusing our investigation on the bacterial members of the urban microbial community and leave viruses, fungi and other groups microorganisms to future studies. A specific region of each microbial genome is examined to focus on a subset of organisms– in the case of bacteria this is most often the gene coding for the 16S rRNA. The 16S rRNA gene is used because it is present in all bacteria and it has regions of sequence that are shared among all bacteria as well as variable regions in the gene that differ from species to species.

Question 64

Urban microbiome procedure

Answer 64

Procedure - Collect “healthy” soil (unmanaged surface). Add mock bacterial pathogens (E. coli and S. epidermidis) to tubes of healthy soil (autoclaved soil as control). Wait 2 weeks. Extract microbiome community DNA and sequence.

After collecting the environmental sample the DNA from the bacteria needs to be purified away from the bacterial cells and all the other unwanted dirt and debris that is collected. We will use universal PCR primers to amplify a variable region within the bacterial 16S rRNA gene. The universal primers are designed to be variable in several positions (that is, a given position in the sequence of the primer could be A, C, G or T for example, and the resulting primers will not all be identical in sequence). The degenerate primers permit the amplification of 16S rRNA gene from as broad a range as possible of the bacterial species represented in the sample. Each PCR sample will be visually examined by gel electrophoresis to confirm the correct band size and each sample will be quantified using a NanoDrop 2000 spectrophotometer. Ethidium bromide (EtBr) is commonly used as a non-radioactive marker to stain DNA in order to identify and visualize nucleic acid bands in electrophoresis and other gel-based methods of nucleic acid separation. DNA sequencing is next. The sequence data from the 16S rRNA genes can them be compared to a database to determine the bacterial diversity of the sample.

Question 65

Hektoen Enteric (HE) Agar

Answer 65

Hektoen Enteric agar (HE) is another selective/differential agar used for isolation and identification of Gram-negative bacteria that are found in feces (called enteric and/or coliform bacteria).
HE agar is designed to specifically detect and differentiate between Salmonella and Shigella based on 1) the ability to utilize lactose, sucrose or salicin, and 2) the ability to reduce sodium thiosulfate to hydrogen sulfide gas (H2S). Ferric ammonium citrate, present in the media, reacts with H2S to form a black precipitate.
Bromothymol blue and acid fuchsin dyes are added as colored pH indicators that detect acid production from the fermentation of sugars (differential characteristic). Bile salts are included to inhibit most Gram-positive bacteria (selective characteristic).
E. coli produces a yellow color because acid is an end product of its fermentation of lactose (It’s growth moderately inhibited). Shigella is also a lactose non-fermenter and is blue-green; it is not a sulfur reducer. Salmonella enterica does not ferment lactose but does produce a black precipitate from the reaction between ferric ammonium citrate in the medium and H2S from sulfur reduction.

Question 66

Glucose Degradation
Clark & Lubb’s Broth

Answer 66

Clark and Lubb’s, also known as MR-VP Broth, is the combination medium used for both Methyl Red (MR) and Voges-Proskauer (VP) tests. It is a simple solution containing only peptone, glucose, and a phosphate buffer. The peptone and glucose provide protein and a fermentable carbohydrate while the potassium phosphate is included to resist pH changes in the medium. This media is designed to distinguish what pathway an organism uses to breakdown the main product of glycolysis, pyruvate.
The Enterobacteriaceae can be further differentiated based upon other metabolic characteristics, including the end products of glucose fermentation. All members of Enterobacteriaceae can break down glucose into pyruvate. The two major pathways for further break down of the pyruvate are called the Mixed Acid (MR test) and the 2, 3-Butanediol pathways (VP test).
Determination of these two kinds of metabolic patterns, as well as other tests described, have been put to practical use in the sanitary analysis of water supplies in which it is necessary to test for indicators of fecal contamination. For example, tests that detect low acid and acetoin will distinguish between non-fecal enteric bacteria (2, 3-butanediol fermenters, such as Enterobacter, Erwinia, and Serratia) from fecal enterics (mixed acid fermenters, such as E. coli, Salmonella and Shigella).
1) The Mixed Acid Pathway:
Products are a mixture of lactic acid, acetic acid, formic acid, succinate and ethanol. If the bacterium possesses the enzyme formate dehydrogenase, which cleaves formate to generate the gases (CO2 and H2).

Question 67

Voges-Proskauer Test

Answer 67

This test identifies organisms able to produce acetoin from the degradation of glucose during a 2, 3 butanediol fermentation (non-fecal enterics). The Voges-Proskauer test was designed for organisms that are able to ferment glucose, but quickly convert their acid products to acetoin and 2, 3- butanediol.
Addition of VP reagents to the medium oxidizes the acetoin to diacetyl, which in turn reacts with guanidine nuclei from peptone to produce a red color. A positive VP result, therefore, is red. No color change is negative. A copper color is a result of interactions between the reagents and should not be confused with the true red color of a positive result. Use of positive and negative controls for comparison is usually recommended.

Question 68

Motility, H2S Production and Indole Production
SIM (Sulfide-Indole-Motility) Medium

Answer 68

SIM medium is used for determination of three bacterial activities: sulfur reduction, indole production (as opposed to tryptophan production), and motility. The semisolid medium includes casein and animal tissue as sources of amino acids, an iron-containing compound, and sulfur in the form of thiosulfate.
SIM medium is used to identify bacteria that are capable of producing indole, using the enzyme tryptophanase. SIM medium also is used to differentiate sulfur-reducing members of Enterobacteriaceae, especially members of the genera Salmonella, Francisella, and Proteus from the negative Morganella morganii and Providencia rettgeri. In addition to the first two functions of SIM, motility is an important differential characteristic of Enterobacteriaceae.

Sulfur Reduction
Bacteria can accomplish sulfur reduction to H2S in two different ways depending on the enzymes present.
• The enzyme cysteine desulfurase catalyzes the putrefaction of the amino acid cysteine to pyruvate.
• The enzyme thiosulfate reductase catalyzes the reduction of sulfur (in the form of sulfate) at the end of the anaerobic respiratory electron transport chain.
Both systems produce hydrogen sulfide (H2S) gas. When either reaction occurs in SIM medium, the H2S produced combines with iron, in the form of ferrous ammonium sulfate, to form ferric sulfide (FeS), a black precipitate. Any blackening of the medium is an indication of sulfur reduction and a positive test. No blackening of the medium indicates no sulfur reduction and a negative reaction.

Question 69

Nitrate Reduction Test

Answer 69

Sulfanilic acid and alpha-naphthylamine (reagents A and B) are added to the medium to test for nitrate reduction to nitrite (NO-3àNO2).
• Nitrite, if present, will form nitrous acid (HNO2) in the aqueous medium.
• Nitrous acid reacts with the added reagents to produce a red, water-soluble compound.
Red color formation after the addition of reagents indicates that the organism reduced nitrate to nitrite.
If no color change takes place with the addition of reagents, the nitrate either was not reduced or was reduced to one of the other nitrogenous compounds (NO3 —> N2 or NH4). Because it is visually impossible to tell the difference between these two occurrences, another test must be performed.
The test needed to determine if nitrate (NO3) was not reduced or it was reduced to either nitrogen (N2) or ammonium (NH4) involves adding a small amount of powdered zinc to the broth. This addition of zinc will catalyze the reduction of any nitrate (NO3) present to nitrite (NO2).
If nitrate (NO3) is present at the time of zinc addition, the above-described reaction between nitrous acid and reagents will follow and turn the medium red.
Red color after the addition of zinc powder indicates that nitrate was not reduced by the organism.

Question 70

Starch Utilization/Hydrolysis

Answer 70

Starch agar is used to test for the breakdown of starch by amylase. The medium contains beef extract and peptone to support growth, soluble starch and agar. An isolate is inoculated onto a plate with a sterile transfer loop. The plate is flooded with Gram’s iodine, which reacts with starch to produce a purple-blue color throughout the agar medium.

Clearing in the medium = The organism hydrolyzed the starch in the media + providing a clearing

Question 71

Citrate Utilization Test
The citrate utilization test is used to determine the ability of an organism to use citrate as its sole carbon source

Answer 71

In many bacteria, citrate (citric acid) is produced by acetyl coenzyme A from oxidation of pyruvate

Simmon’s Citrate Agar
This agar contains sodium citrate as the sole carbon source and ammonium phosphate as the sole nitrogen source. Bromothymol blue dye is the indicator used in this citrate test. Bromothymol blue is green at pH 6.9 and blue at pH 7.6.
Bacteria that survive in the medium and utilize the citrate also convert the ammonium phosphate ((NH4)2HPO4) to ammonia (NH3) and ammonium hydroxide (NH4OH). Both products tend to alkalinize the agar. As the pH goes up, the medium changes from green to blue. Thus, the conversion of the medium to blue is a positive citrate test result.
Note:
1. Citrate utilization is an aerobic process. As a result, agar slants are used to increase the
surface area exposed to air.
2. A green color can also occur with heavy growth, so be careful not to over inoculate.