UNIT 2 Micro

Petroff-Hausser chamber (Direct Counting Method of a Sample Size)

(a) A Petroff-Hausser chamber is a special slide designed for counting the bacterial cells in a measured volume of a sample. A grid is etched on the slide to facilitate precision in counting. (b) This diagram illustrates the grid of a Petroff-Hausser chamber, which is made up of squares of known areas. The enlarged view shows the square within which bacteria (red cells) are counted. If the coverslip is 0.2 mm above the grid and the square has an area of 0.04 mm?, then the volume is 0.008 mm?, or 0.000008 mL. Since there are 10 cells inside the square, the density of bacteria is 10 cells/0.000008 mL, which equates to 1,250,000 cells/mL.

Types of hemolysin in a blood agar test include:

-Beta: is complete hemolysis. It is a clear (transparent) zone surrounding the colonies. -Alpha: Partial hemolysis. Colonies typically are surrounded by a green, opaque zone. -Gamma: NO hemolysis occurs. There are no notable zones around the colonies.

Many organisms use intermediates from the Krebs cycle, such as..

.. amino acids, fatty acids, and nucleotides, as building blocks for biosynthesis (the production of complex molecules within living organisms or cells.)

Glycolysis using the EMP pathway consists of two distinct phases

1. Energy investment phase: uses energy from two ATP molecules to modify a glucose molecule so that the six-carbon sugar molecule can be split evenly into two phosphorylated three-carbon molecules called glyceraldehyde 3-phosphate (G3P). 2. Energy payoff phase: extracts energy by oxidizing G3P to pyruvate, producing four ATP molecules and reducing two molecules of NAD+ to two molecules of NADH, using electrons that originated from glucose.

Bacterial growth phases (LLSD)

1. Lag phase: no increase in number of living bacterial cells (no bacterial growth) Bacteria adapt themselves to the growing conditions, and prepare for growth. 2. Log phase: exponential increase in number of living bacterial cells. 3. Stationary phase: plateau in number of living bacterial cells; rate of cell division and death roughly equal. At carrying capacity, death rates = birth rates. 4. Death or decline phase: exponential decrease in number of living bacterial cells. At down hill many are dying (running out of food)

If at "0" time there is one organism, and after 1 generation there are 2 ... how can we mathematically express the number of bacteria after n generations?

1. Take the time that would produce that many cells and convert to minutes if it hasn't been done already. 2. Divide the time by the division time to get the number of generations that occurred during that time span. 3. Take 2 to the power of (n) generations to get the amount of bacteria produced. Example: A BACTERIAL CELL HAS A MEAN DIVISION TIME of 30 MINUTES. HOW MANY CELLS WOULD lD IT PRODUCE AFTER 3 HOURS?' 3 hours = 180 min 180/30 =6 6 generations would be 2 to the power of 6 (2^6) which equals total bacteria reproduced within 3 hours = 64 bacteria/cells

Cellular Respiration 4 Stages are? (WATCH VIDEO ON SLIDE 76 to FINSIH)

1. glycolysis (splits sugars in the cytoplasm) 10 step reaction 2. pyruvate oxidation (pyruvate moves into mitochondria) 3. the citric acid or Krebs cycle (carbons, hydrogens and.. 4. oxidative phosphorylation.

What are the outcome after glycolysis

2 ATP, 2 Pyruvate's and 2 HADH

Cellular respiration produces _______ per molecule of glucose across three stages.

32-36 ATP

Method for Bacteria- Coulter counter

A Coulter counter is a direct method that uses an electronic device that counts cells. It measures the change in resistance in an electrolyte solution that takes place when a cell passes through a small opening in the inside container wall. A detector automatically counts the number of cells passing through the opening.

redox reaction

A chemical reaction involving the transfer of one or more electrons from one reactant to another; also called oxidation-reduction reaction. Combinations of OIL and RIG

How do we grow bacteria? What is used to maintain cells' logarithmic (exponential) phase of growth?

A chemostat in which nutrients are supplied at a steady rate, a controlled amount of air is mixed for aerobic processes, and bacterial suspension is removed at the same rate as nutrients flow in to maintain optimal growth environment. -Used to generate antibiotics -Biotech -Make vaccines -Make alcohol -Make bread

Chemostat

A chemostat is a culture vessel fitted with an opening to add nutrients (feed and an outlet to remove contents (effluent), effectively diluting toxic wastes and dead cells. The addition and removal of fluids is adiusted to maintain the culture in the logarithmic phase of growth. If aerobic bacteria are grown, suitable oxygen levels are maintained.

Method for Bacteria- Most Probable Method

A indirect counting method. Used to estimate the number of microbes in dilute samples such as water and food. Changes in color or turbidity to detect growth with results from a reference number.

Glycolysis

A metabolic process that breaks down carbohydrates and sugars through a series of reactions to either pyruvic acid or lactic acid and release energy for the body in the form of ATP

Pour plate method

A method of inoculating a solid nutrient medium by mixing bacteria in the melted medium and pouring the medium into a Petri dish to solidify

Blood Agar Test

A sample blood agar test showing beta-hemolysis. A sample motility test showing both positive and negative results.

What is the septum?

A septum (wall) forms down the middle of the cell, pinching it into two new cells. Each cell has one of the two bacterial chromosome copies.

Pyruvate

A three carbon molecule that (oxidizes) looses a carbon (this carbon makes carbon dioxide), the remaining carbon molecules are Acetyl-CoA

Microorganisms that grow in optimal pH environments less than 5.5 mostly around 3 are known as...

Acidophiles

Define what a catalase enzyme is

An enzyme that detoxifies hydrogen peroxide (HOOH) into water and molecular oxygen (O2 gas) which bubbles

What happens in the log phase?

An exponential increase in number of living bacterial cells.

Anaerobe

An organism that grows without air, or requires oxygen-free conditions to live.

Obligated anaerobes

Bacteria that are killed by oxygen. Can't grow in the presence of oxygen Thrive in oxygen-free environments such as the intestinal tracts of animals, the deep ocean, still waters, landfills, in deep sediments of soil.

Obligated aerobes are?

Bacteria that need oxygen to grow. Bacteria that cannot grow without an abundant (large quality) supply of oxygen. Growth of bacteria will be present towards the surface where oxygen is abundant.

Beta-hemolysis

Beta-hemolysis (complete hemolysis) is a complete lysis of red cells in the media around and under the colonies: the area appears lightened (yellow) and transparent. Streptolysin, an exotoxin, is the enzyme produced by the bacteria which causes the complete lysis of red blood cells.)

What is a concern with indwelling catheters?

Biofilm can develop on them if not changed regularly - communities of bacteria that can come in contact with open wounds. When bacteria assemble together there are no such drugs that can fight against them. Can cause UTI's

What does the flat part of the bacterial growth curve (stationary phase) mean?

Birth rate = death rate Being born and dying at the same rate.

Lypolysis

Breakdown of triglycerides (fat) for energy. Removing fatty acid chains from a triglyceride

What happens in the lag phase?

Cells are synthesizing new components and adapting to there environments, preparing for growth. There is no bacteria growth occurring yet.

Biofilm

Community of microorganisms living within a shared mass of secreted slime.

Types of media- Complex Media

Contain extracts and digests of yeasts, meat, or plants where the precise chemical composition of the medium is not known. Amounts of individual components are undetermined and variable. Nutrient broth, tryptic soy broth, and brain heart infusion are all examples of complex media.

What do we mean when we say that the graph paper is "semi-log"?

Easier way to interpret the growth of bacteria. Uses a straight(linear) line rather than curved graph.

The type of glycolysis found in animals and that is most common in microbes is the ___________.

Embden-Meyerhof-Parnas (EMP) pathway,

When glucose is split into 2 pyruvates, there is a NET gain of only 2 ATP molecules. That represents very little of the potential energy in the glucose. Where is the remaining energy?

Energy is still stored in the remaining bonds of the pyruvate. We still have those two halves and need to continued to be broken down into carbon dioxide and water. Energetic meaningful molecules are stored in mitochondria used later on for anabolic reactions.

What happens to the Carbons during the Krebs Cycle

Eventually fall off making carbon dioxide

Facultative anaerobes

Facultative anaerobes show better growth in the presence of oxygen but will also grow without it. Grows with out oxygen by relying on fermentation or anaerobic respiration, if there is a suitable electron acceptor other than oxygen and the organism is able to perform anaerobic respiration.

Entering the oxidation pathway --> Transition Reaction, Coenzyme A, and the Krebs Cycle.

For pyruvate to enter the next oxidative pathway, it must first be decarboxylated by the enzyme complex pyruvate dehydrogenase to a two-carbon acetyl group in the transition reaction, also called the bridge reaction. In the transition reaction, electrons are also transferred to NAD+ to form NADH. To proceed to the next phase of this metabolic process, the comparatively tiny two-carbon acetyl must be attached to a very large carrier compound called coenzyme A (CoA). The transition reaction occurs in the mitochondrial matrix of eukaryotes; in prokaryotes, it occurs in the cytoplasm because prokaryotes lack membrane-enclosed organelles.

What are other ways Bacteria Replicate other than binary fission?

Fragmentation and budding

Catalase in an enzyme that speeds up the reaction which converts hydrogen peroxide into ______ and ________.

H20(water) + 02 (oxygen gas)

Spread plate method

In the spread plate method of cell counting, the sample is poured onto solid agar and then spread using a sterile spreader. This process is repeated for each serial dilution prepared. The resulting colonies are counted and provide an estimate of the number of cells in the original volume samples.

Method for Bacteria- Spectrophotometer

Indirect method. Commonly used to measure the turbidity of a bacterial cell suspension as an indirect measure of cell density. A spectrophotometer works by splitting white light from a source into a spectrum. The spectrophotometer allows choice of the wavelength of light to use for the measurement. The optical density (turbidity) of the sample will depend on the wavelength, so once one wavelength is chosen, it must be used consistently. The filtered light passes through the sample (or a control with only medium) and the light intensity is measured by a detector. The light passing into a suspension of bacteria is scattered by the cells in such a way that some fraction of it never reaches the detector. This scattering happens to a far lesser degree in the control tube with only the medium. More light=more bacteria Less light= less bacteria

What is the term for the initial bacteria placed into (or onto) a growth medium?

Inoculation

Catalase Test

Is used to identify organisms that produce the enzyme catalase by adding hydrogen peroxide to the medium. If positive (+) for catalase bubbles of oxygen gas (O2) will form. Catalase detoxifies hydrogen into water and releases O2 (bubbles of gas) All anaerobes will be catalase negative

ATP synthase

Large protein that uses energy from H+ ions to bind ADP and a phosphate group together to produce ATP.

One indirect method for counting bacteria includes:

Looking how cloudy the liquid is. This is called spectrophotometry. Then comparing it to other liquids. It will not give you an exact number.

One indirect Method -Spectrophotometry

Looking how cloudy the water is, the more cloudy the more bacteria are within. An analytical method for identifying a substance by its selective absorption of different wavelengths of light. Percent transmittance (%T)(how much light goes through the more bacteria) Absorbance (how much light did not go through because it was absorbed) Can't compare two different bacteria. But can compare between petri dishes (Which one has a denser culture of the same kind, maybe at different temps, environments)

What is a general purpose media?

Media that provides enough nutrients which mostly any microorganism will utilize for growth. Allows for a wide variety of microorganisms to grow (typically agar plus nutrients) Ex: Soy Agar

Bacteria grown in labs are usually temperature organisms called

Mesophiles

The human body temp is around 98.6 Fahrenheit which is 37 Celcius. What are things that grow in are bodies?

Mesophiles

Metabolites are

Molecules left behind by bacteria

All anaerobes will be catalase

Negative

All anaerobes will be catalase positive or negative?

Negative

If an organism grows well at a pH of 6.5, it would be considered a(n)

Neutrophile

Most bacteria have an optimal pH known as...

Neutrophiles (within one or two pH units of the neutral pH of around 7)

OIL RIG stands for what?

OIL= Oxidations is loss of electrons RIG= Reduction is gain of electrons. (Electrons are negative hence reduction. Ading electrons reduces)

Which of the following bacterial types are we UNLIKELY to grow in our laboratories (under ordinary conditions)? A. obligate anaerobes B. obligate aerobes C. facultative organisms

Obligated anaerobes

Substrate-level phosphorylation

One of the two enzymatic reactions in the energy payoff phase of Embden Meyerhof-Parnas glycolysis that produce ATP in this way is shown here.

What is the most favorable pH growth of an organism?

Optimal growth pH.

Mesophiles are organisms that grow:

Organisms that are adapted to moderate temperatures, with optimal growth temperatures ranging from room temperature to about 20 °C - 45 °C.

How to Indirectly Count Bacteria

Percent transmittance (%T)(the percentage of light that passes through a solution) Absorbance (how much light did not go through because it was absorbed)

Plants Vs Animals and ATP

Plants use both cellular respiration and ATP but we only use ATP

What is fermentation and when does it occur?

Process by which cells release energy in the absence of oxygen Fermentation refers to the metabolic process by which organic molecules (normally glucose aka sugar) are converted into acids, gases, or alcohol in the absence of oxygen or any electron transport chain. Fermentation pathways regenerate the coenzyme (NAD+), which is used in glycolysis to release energy in the form of (ATP). Fermentation only yields a net of 2 ATP per glucose molecule (through glycolysis), while aerobic respiration yields as many as 32 molecules of ATP per glucose molecule with the aid of the electron transport chain.

Define binary fission

Process by which prokaryotic organisms divide and reproduce

Division of prokaryotic cells

Prokaryote divide and reproduce every 20 min and strive in warm environments. The parental cell divides and gives rise to two daughter cells. Each of the daughter cells, in turn, divides, giving a total of four cells in the second generation and eight cells in the third generation. Each division doubles (2) the number of cells.

An organism that grows well in a bottle of refrigerated milk would be best classified as a

Psychrophile

Rate of growth of bacteria as a function of temperatures high to low (PMTH)

Psychrophiles (0 °C and below-15 °C) Mesophiles (20 °C-45 °C) Thermophiles (50 °C -maximum of 80 °C) Hyperthermophiles (80 °C - maximum of 110 °C)

Media that inhibit the growth of unwanted microorganisms and support the growth of the organism of interest by supplying nutrients and reducing competition are called?

Selective Media

Mannitol salt agar inhibits the growth of bacteria that do not tolerate salt. Because of this, only salt-tolerant bacteria will grow on MSA. Therefore, it is a ________________ medium.

Selective Medium

Does MacConkey Agar can use differential, selective, or both?

Selective and Differential growth

Direct Counting Method for Bacteria- Serial Dilution

Serial dilution involves diluting a fixed volume of cells mixed with dilution solution using the previous dilution as an inoculum. The result is dilution of the original culture by an exponentially growing factor.

Binary fission in bacteria

Starts with the replication of DNA as the cell elongates. A division septum forms in the center of the cell. Two daughter cells of similar size form and separate, each receiving a copy of the original chromosome.

ATP made during glycolysis is a result of...

Substrate-level phosphorylation

During what phase do cells lyse and release nutrients into the medium, allowing surviving cells to maintain viability and form endospores?

The death phase

Embden-Meyerhof-Parnas glycolysis pathway

The energy investment phase of the Embden-Meyerhof-Parnas glycolysis pathway uses two ATP molecules to phosphorylate glucose, forming two glyceraldehyde 3-phosphate (G3P) molecules. The energy payoff phase harnesses the energy in the G3P molecules, producing four ATP molecules, two NADH molecules, and two pyruvates.

ATP phosphorylation and dephosphorylation

The energy released from dephospho-rylation (to take a phosphate off) of ATP is used to drive cellular work, including anabolic pathways. ATP is regenerated through phosphorylation, harnessing the energy found in chemicals or from sunlight.

What is Fragmentation & what is budding

The new cells often split from the parent filament and float away in a process called fragmentation (Fig. A) Forming a long narrow extension at one poleis a process called budding (Fig. B) The tip of the extension swells and forms a smaller cell, the bud that eventually detaches from the parent cell. The larger cell is the mother cell. Labels indicate the nucleotides (N) and the still-forming nuclear envelope (NE) of the daughter cell.

What happens in the stationary phase?

The population has reached its carrying capacity and the birth rate is equal to the death rate

Exergonic VS Exothermic

The reactions that increase the heat of the surroundings are classified as exothermic reactions, whereas the ones that do not change the temperature of the surroundings are exergonic. Both these reactions do not require additional energy or heat to complete their reactions.

What happens in the death/decline stage?

There is an exponential decrease in number of living bacterial cells. At down hill many are dying (running out of food)

Alkaphiles

These are microorganisms that grow best at a pH of 8.0-10.5 mostly above 9 (Basic or alkaline)

Turbidimetric analysis

This methodology measures light scatter rather than colorimetric absorbance to determine the concentration

The Krebs Cycle (Citric Acid Cycle)

Transfers remaining electrons from the acetyl group produced during the transition reaction to electron carrier molecules, thus reducing them. The Krebs cycle also occurs in the cytoplasm of prokaryotes along with glycolysis and the transition reaction, but it takes place in the mitochondrial matrix of eukaryotic cells where the transition reaction also occurs. The Krebs cycle is named after its discoverer, British scientist Hans Adolf Krebs (1900-1981) and is also called the citric acid cycle, or the tricarboxylic acid cycle (TCA) because citric acid has three carboxyl groups in its structure. Unlike glycolysis, the Krebs cycle is a closed loop: The last part of the pathway regenerates the compound used in the first step (Figure 8.13). The eight steps of the cycle are a series of chemical reactions that capture the two-carbon acetyl group (the CoA carrier does not enter the Krebs cycle) from the transition reaction, which is added to a four-carbon intermediate in the Krebs cycle, producing the six-carbon intermediate citric acid (giving the alternate name for this cycle). As one turn of the cycle returns to the starting point of the four-carbon intermediate, the cycle produces two CO2 molecules, one ATP molecule (or an equivalent, such as guanosine triphosphate [GTP]) produced by substrate-level phosphorylation, and three molecules of NADH and one of FADH2. (A discussion and detailed illustration of the full Krebs cycle appear in Appendix C.) Although many organisms use the Krebs cycle as described as part of glucose metabolism, several of the intermediate compounds in the Krebs cycle can be used in synthesizing a wide variety of important cellular molecules, including amino acids, chlorophylls, fatty acids, and nucleotides; therefore, the cycle is both The Krebs cycle, also known as the citric acid cycle, is summarized here. Note incoming two-carbon acetyl results in the main outputs per turn of two CO2, three NADH, one FADH2, and one ATP (or TP) molecules made by substrate-level phosphorylation. Two turns of the Krebs cycle are required to process all of the carbon from one glucose molecule.

A direct method of counting bacterial population size involves the actual counting of something, whereas an indirect method infers population size via another kind of measurement. Which one of the following would be considered an indirect method?

Turbidimetric analysis

Blood Agar Tests Purpose

Used to detect whether an organism has the ability to produce hemolysin (enzymes that damage red blood cells) Types of hemolysin include: -Beta: is complete hemolysis. It is a clear (transparent) zone surrounding the colonies. -Alpha: Partial hemolysis. Colonies typically are surrounded by a green, opaque zone. -Gamma: NO hemolysis occurs. There are no notable zones around the colonies. Greatest to least hemolysis (BAG)

Anaerobic Jar VS Anaerobic Chamber/Box

Used to study anaerobes by keeping them under conditions without oxygen. (Fig. A) An anaerobic jar (most common): jars include chemical packs that remove oxygen and release carbon dioxide (CO2). (Fig. B) An anaerobic chamber/box is an enclosed box from which all oxygen is removed. Gloves sealed to openings in the box allow handling of the cultures without exposing the culture to air

What's the simplest way to count bacteria?

Using a direct microscopic count. This involves transferring s known volume of a culture to a calibrated slide called a Petroff-Hausser chamber.

Types of media- Chemically defined medium

When the complete chemical composition of a medium is known. For example, in EZ medium, all individual chemical components are identified and the exact amounts of each are known.

Types of media- Differential Media

You can tell the difference between two different types of bacteria ... Make it easy to distinguish colonies of different bacteria by a change in the color of the colonies or the color of the medium. Color changes are the result of end products created by the interaction of bacterial enzymes with differential substrates in the medium or, in the case of hemolytic reactions, the lysis of red blood cells in the medium.

What is a Peroff-Hausser Chamaber?

a special slide designed for counting the bacterial cells in a measured volume of a sample. A grid is etched on the slide to facilitate precision in counting.

Acetyl-CoA

a two-carbon substance found in many cellular metabolisms

Which of the following best describes beta oxidation? A. detaching amino acids from a protein chain B. making smaller sugar molecules from starch chain C. breaking fatty acid chains into 2-carbon units D. removing fatty acid chains from a triglyceride

breaking fatty acid chains into 2-carbon units

Types of media- Enriched Media

contains growth factors, vitamins, and other essential nutrients to promote the growth of fastidious organisms, which are organisms that cannot make certain nutrients and require them to be added to the medium.

NAD+

electron carrier involved in glycolysis. (Vitamins)

pH is a measure of

hydrogen ion concentration

Fermentation occurs during what conditions

in the absence of oxygen

A halophile would grow best in

salt lakes (salt-loving)

Selective media are used to?

select for the growth of a particular "selected" microorganism

intrinsic growth rate

the maximum potential for growth of a population under ideal conditions with unlimited resources