MICRO - Microbial Physiology, Growth & Metabolism

Microbial Physiology

Tackles the mechanisms of vital life processes of an organism

Growth

True measurement is the number of cell rather than size. Orderly increase in the sum of all the components in an organism

Increase in cell number

In microbial physiology of microorganisms like the bacteria, growth is equal to?

Binary fission

How does microorganism reproduce?

Culture

Cell multiplication is a consequence of binary fission that leads to an increase in the number of single bacteria making up a population, referred to as a?

Binary fission

An organism first duplicates its DNA. The cytoplasmic membrane elongates. The cytoplasm divides and forms a septum. Complete separation of the cytoplasm leads to 2 new daughter cells of a prokaryote

Colony

Multiple binary fission will have cells large enough to be seen, even without a microscope

Generation Time

AKA “Doubling time” - interval of time between two cell divisions. The time it takes for one cell to become double. Represented logarithmically and not represented by parabolic and hyperbolic curves 

Growth Rate

How long it takes the bacteria to reach an amount that is actually pathogenic

True

T/F: S. aureus is a rapid grower.

False

T/F: M. tuberculosis is a rapid grower.

Virulence

The more it can give way to account that is already pathologic and will produce effect or a disease like food poisoning

Lag phase

“Flat period” at the beginning of the graph. Cells are metabolically active as they are preparing to divide exponentially. No significant increase in number of bacterial cells noted. Occurs during agar plate preparation.

Log or Exponential Growth Phase

Pertains to the start of cell division where their number increases exponentially. With constant generation time. During this phase, many antibiotics, such as penicillin, are most efficacious

Stationary phase

There is an increase in waste products due to depletion of nutrients and oxygen. Has the greatest population density of bacteria because there is a slow loss of cell balanced by formation of new ones

Chemostat

A laboratory device used to grow microorganisms in a continuous culture under steady-state conditions

Death or Logarithmic Decline Phase

During this period, nutritional support is completely depleted and no longer supports bacterial growth. As a result, the number of living (viable) cells begins to decrease and generation time is already negative

Viable count

Determines the live cells

Non-viable count

Does not distinguish between dead and living cells

Turbidimentry

Indirect, simplest, and practical method for estimating population size and monitoring bacterial growth. Non-specific measure for how much bacteria is in the cell

Turbidity

Referred to as the ‘cloudiness’. Corresponds to the density of microbes grown in inoculated broth

Spectrophotometry

Turbidity can be measured through?

Optical density (OD)

Photons of light passing through turbid area can be used to measure the density of cells within the culture media

Autolytic Catabolism

Refers to the enzymes that pushes the bacteria to death

Colony Forming Unit

Smallest functional unit of colony formation and dispersal in bacterium

Plate Count

Frequently used method for measuring bacterial population. Results reported in CFU. Number of colonies that develop after incubation gives an estimate of the viable count

Serial Dilution Method

Important first step before proceeding to either pour plate or spread plate. Must be done first before going to the next step of the plate method. Goal: Obtain the plates within the ranges of 330 to 300. This is usually done in multiples of 10. As the dilution progresses, colony growth would be expected to decrease and that is normal

Pour plate method

Add 1 mL diluted culture to 9 mL of liquid nutrient melted agar and mix. Pour on a sterile plate. Swirl to mix the sample, allowing it to solidify. Wait until the growth is noticeable. Usually within 45–50 °C and poured within a sterile, controlled environment. If not done properly, it can be destroyed by the heat of agar

Spread plate method

Pour just 0.1 mL of solid agar on the center of the agar plate and then spread it with a bent glass rod. Bacterial colonies grow only on the surface of the agar and are usually easy to count or characterize. The sample again is repeated for each dilution. Either go for pour plate or spread plate method

Streak plate method

More practical way of doing plate method. On the surface of an agar plate, use a wire loop. It must be sterilized. Once the streak is done, the line of course of the streak would be the line of course of the growth. Expect the distribution of the colony growth of the specific organism. Done with the intent to observe if there is growth.

Most Probable Number (MPN)

Another estimate technique to count organisms. Statistically the most probable number of bacteria in a sample. Uses sets of five. 95% of bacteria population falls within the range

Non-Viable Cell Count

Uses the microscope directly to count dead and living cells. The Petroff-Hausser counting chamber is counted in the large center of the square. Count the cells within the larger grid under the microscope and cells would be multiplied by the reciprocal volume of the fluid

Flow cytometer

Counts, measures size, and differentiates between live and dead cells. A rapid method to identify pathogens

Real-time polymerase chain reaction (PCR)

Quantify bacteria and other microorganisms in tissue samples without culturing them

Coulter counter

Automated counting. Uses electronic sensors to detect and count the number of cells

Factors affecting microbial growth

Refers to how microorganisms thrive or survive considering a given factor present in the environment

Physical and chemical factors

What are the two main factors affecting microbial growth?

Nutritional requirements

Refers to various chemical compounds that an organism uses to sustain life. Nutrients as energy source. Organisms need energy, similar to human beings. Energy is used in throughout various enzymatic reactions. Involves absorbing nutrients for metabolism

Carbon

Used in synthesis of carbohydrates, lipids, fats, and energy source

Autotrophs

Use inorganic chemicals for energy source, e.g. CO2, ammonium sulfide

Heterotrophs

Require organic carbon as an energy source, e.g. glucose

Nitrogen

Major component of proteins, nucleic acids, coenzymes. ~5% of the dry weight of bacteria. Used as a terminal electron acceptor in respiration by some bacteria (nitrates)

Assimilation

Make new things out of the basic stuff. e.g. Nitrogen fixation

Sulfur

Constituent of some amino acids in proteins and some coenzymes. Oxidize sulfur to sulfate and reduce sulfate to hydrogen sulfide (H2S)

Hydrogen

Constituent of water and organic cell components. Determines acidity and alkalinity of the environment (pH)

Oxygen

Constituent of water and organic cell components. Serves as an electron acceptor in aerobic respiration

Water

75–85% of bacterial cell composition

Growth Factors

Includes B complex vitamins which serve as component for co-enzymes, and amino acids which are important for protein synthesis. Haemophilus influenzae would need NAD+ or Factor V

Phosphorus

Component of ATP, phospholipids, and coenzymes such as NAD, NADP, and flavins. Particularly important for energy generation and membrane transfer

Mineral Sources

Needed for enzyme function as co-factors for coenzyme reaction. Needed in minute amounts. Facilitate transport of material across cell membrane. Serve as constituents of cytochromes and proteins. Contribute to heat resistance of endospores

Simple diffusion

Random movement from a more concentrated area to a lesser concentrated area

Facilitated Diffusion

Use of carrier-mediated protein, especially when transporting bigger or polar materials like sugar, amino acids, and inorganic ions

Active Transport

Bacteria highly depend on this with the bacteria make more effort to produce ATP so as to acquire molecules to pass through the cell membrane so they can get the nutrition they need. Highly important in moving nutrients present in low concentration to move them into the cell of the microorganism

Group Translocation

AKA vectorial metabolism. Molecules are moved across the membrane while chemically modifying its substance. Exclusively occurs in prokaryotes. Allows molecules to accumulate against the concentration gradient due to the molecules inside being different. Transport of glucose from the periplasm will bring about glucose-6-phosphate in the bacteria’s cytoplasm by virtue of phosphoenolpyruvate (PEP)

Osmotic Pressure

Force with which water moves through the cytoplasmic membrane from a solution of low concentration to that of a high concentration. Most bacteria require an isotonic or hypotonic environment

Isotonic

No net flow of water. Water goes in and out. Most organisms exist in an isotonic solution

Hypertonic

Plasmolysis. Water flows out → Cell shrinks. Some organisms are osmotolerant. Can survive even in high salt concentrations and low water availability. These are called halophiles (salt-loving organisms)

Hypotonic

Hypotonic or hypoosmotic. Water flows in → Cell swells

Hydrostatic Pressure

Pressure exerted on cells by the weight of water surrounding it. High pressure is necessary to keep enzymes in their proper configuration. Without it, enzymes lose their shape and denature, resulting also to the loss of their function

Obligate Aerobes

Require oxygen for growth. Enzymes pertinent in obligate aerobes: Superoxide dismutase, Catalase

Microaerophilic Bacteria

Require oxygen at low levels (2–10%). High amounts of oxygen become toxic. Small amounts of catalase and superoxide dismutase

Facultative Anaerobes

Have both aerobic and fermentation capabilities. Has superoxide dismutase & catalase. It lacks cytochrome C oxidase as the final acceptor of oxygen. It is distinguished from pseudomonas. It can survive in blood and urinary tract systems that do not have oxygen. Host-pathogen interaction would include metabolic flexibility to colonize.

Anaerobes

Need the enzyme superoxide dismutase. Utilize fermentative pathways. Sensitive to inhibition. Find oxygen toxic. Neither catalase nor superoxide dismutase is found in most

Capnophilic Bacteria

High CO2, low oxygen

Aerotolerant Anaerobes

Can survive presence of oxygen but do not use it in their metabolism. Has superoxide dismutase

Incubation

Process wherein Petri dishes are grown in a favorable environment

Danger zone

Where there is rapid growth of bacteria

37ºC

Temperature where virulence factors are released

60ºC - 120ºC

Temperature where Most of the bacteria die. Boiling for 10 minutes is advised to completely eliminate bacterias

Pasteurization

A process used to kill bacteria at 60°C–120°C for 15 secs

<0ºC

Temperature Optimal for keeping bacteria without inducing growth

Psychrophiles

Cold-loving bacteria in <15–20°C

Mesophiles

Most medically important bacteria in >20–40°C

Thermophiles

Heat-loving bacteria in 45–80°C

Hyperthermophiles

Extreme heat-loving bacteria in <80–250°C

30°C

The optimal temperature for many free-living forms

Dessication

Endospores and cysts can survive complete drying process

pH

Acidity or alkalinity. Most pathogenic bacteria grow best between 6.5 and 7.5. Most bacteria are neutrophiles

Microbial Metabolism

Talks about how bacteria (or any microorganism) would deal with energy, anabolism, catabolism, and fermentation.

Bacterial Metabolism

The sum of all chemical processes carried out by living organism needed by bacteria in order to support activity through energy generation and to sustain their lives (e.g. macromolecules and energy biosynthesis)

Glycolytic or Embden-Meyerhof-Parnas (EMP) Pathway

Primary means of converting glucose. Occurs in aerobic and anaerobic conditions. Recall that it has two phases: preparatory and committed where the net product of one glucose molecule are 2 pyruvate, 2 NADH, and 2 ADP

Pentose Phosphate Pathway (PPP)

Provide precursors and reducing power in the form of NADPH used in biosynthesis. Breaks down glucose and other five-carbon sugars. Can be used in nucleic acid synthesis

Kreb’s Cycle (TCA)

Pyruvate first converted to acetyl-CoA through decarboxylation in order to enter the cycle. Occurs during aerobic respiration. End product to be used for ETC

Electron Transport Chain (ETC)

Process: oxidative phosphorylation. Series of membrane-bound carriers. Final electron acceptor: O2. 1 mole of NADH = 3 moles of ATP. 1 mole of FADH = 2 moles of ATP

Entner-Doudoroff Pathway

Can replace glycolysis and PPP if the necessary enzymes are present in the bacterium, such as: 6-phosphogluconate dehydratase, 2-keto-3-deoxy-6-phosphogluconic acid. The main products are pyruvate and glyceraldehyde-3-phosphate, which serve as precursors for entry into the TCA cycle. This pathway is not generally found in all bacteria, especially Gram-positive organisms

Fermentation

Incomplete oxidation of glucose and other carbohydrates in the absence of oxygen. Uses organic compounds as terminal electron acceptors. Yields a small amount of ATP

Alcoholic fermentation

Bacteria can convert pyruvate to ethanol

Acidic fermentation

Varied pathways. Lactic acid bacteria (Lactobacillus) ferment pyruvate to lactic acid

Mixed acid fermentation

Produce a combination of acetic, lactic, succinate, and formic acid

Assimilation

Build-up of macromolecules from smaller precursors. Proteins, lipids, carbohydrates

Calvin cycle

Convert carbon dioxide to organic compounds like carbohydrates

Gluconeogenesis

Biosynthesis of glucose from non-carbohydrate molecules

D. Declining phase

It is the point wherein the GENERATION TIME IS “NEGATIVE”

A. Log phase

B. Preparatory phase

C. Lag phase

D. Declining phase

A. Temperature belonging to mesophile group

Medically relevant bacteria generally grows at which optimal condition?

A. Temperature belonging to mesophile group

B. Absence of Enzyme

C. Extreme Cold Condition

D. Hypertonic Condition

C. ATP is generally required during biosynthetic pathway

Microbial Metabolism refers to biochemical pathway is TRUE?

A. All bacteria are highly dependent to aerobic pathways

B. Channel Translocation is part of the major pathway of metabolism generating ATP

C. ATP is generally required during biosynthetic pathway

D. Pseudomonas sp. in general utilizes PPP as a catabolic pathway

C. It helps estimate the speed of infection progression and timing of treatment

Why is understanding bacterial generation time clinically important?

A. It helps determine the exact antibiotic dosage required

B. It predicts the duration of the lag phase in bacterial growth

C. It helps estimate the speed of infection progression and timing of treatment

D. It allows for the visualization of bacteria under the microscope

B. Stationary phase

In a bacterial culture, the number of viable cells remains constant due to a balance between cell division and cell death. Which phase is this?

A. Lag phase

B. Stationary phase

C. Log phase

D. Death

A. Rapid binary fission

What primarily occurs during the log phase of bacterial growth?

A. Rapid binary fission

B. Bacteria adapt to their environment and synthesize enzymes

C. Nutrient depletion slow's growth

D. Cell Is sis exceeds cell

A. They denature bacterial enzymes

Why do high temperature levels inhibit microbial growth?

A. They denature bacterial enzymes

B. They lower the pH

C. They create a hypertonic environment causing plasmolysis

D. They raise temperature beyond optimal levels

C. Optical density (turbidity)

What does a spectrophotometer measure when assessing bacterial growth?

A. Total viable count

B. Colony-forming units

C. Optical density (turbidity)

D. DNA content