Microbio Lecture 3 Notes

Microbial Physiology

Study of the biological processes of microbes

What are the functions of living microbes, as for every other living organism?

• Metabolism (catabolism and anabolism)

• Interaction with the surrounding environment

• Reproduction and death

Bacteria

Microbes that are well-suited to conduct studies of microbial physiology

• Inexpensive to maintain in a lab, take up little space, reproduce quickly, can be observed easily

Nutrients

• Nutrients are various chemical compounds used by living organisms to sustain life

• Serve as sources of:

  • Energy

  • Major elements (ex. carbon, hydrogen, oxygen, nitrogen, phosphorus, sulfur)

  • Additional elements (ex. sodium, potassium, calcium, magnesium, iron, chlorine)

How are microbes categorized?

Microbes can be categorized based on their sources of energy and carbon

What are phototrophs?

Use light as a source of energy

Oxygenic photosynthesis

Oxygen is produced

Anoxygenic photosynthesis

Oxygen is not produced

What are chemotrophs?

Use chemicals as a source of energy

What are autotrophs?

Use carbon dioxide as their source of carbon to form nutritional organic compounds. Ex. algae

What are heterotrophs?

Use preformed organic compounds (ex. sugars, carbons) as their source of carbon.

Ex. fungi, protozoa, most bacteria

Metabolism

Sum of all biochemical reactions involving different nutrients that occur within any living cell

Catabolism

Degradative process

General term for all processes in which larger molecules or structures are broken down into smaller ones. Ex. hydrolysis of lactose to glucose

Anabolism

Biosynthetic process

General term for all processes in which larger molecules or structures are built from smaller ones

Ex. Synthesis of DNA or RNA from nucleotides

Catabolic reactions

Catabolic reactions release energy

Catabolic reactions involve the breaking of bonds; whenever chemical bonds are broken, energy is released

Anabolic reactions

Anabolic reactions require energy

Anabolic reactions involve the creation of bonds; it takes energy to create chemical bonds

ATP (Adenosine triphosphate molecules)

Major energy-storing molecules within a cell

Metabolic enzymes

• Enzymes are proteins that accelerate metabolic reactions - biological catalysts

• Enzymes are highly specific (each enzyme catalyzes only a particular reaction)

• Substances upon which enzymes act are called - substrates

• Resulting molecules are called - products

• Enzymes themselves are not altered during the reactions they catalyze

How does the shape of enzymes benefit them?

Enzymes have a unique three-dimensional shape which enables the active site of a specific enzyme to fit the combining site of a substrate. They ultimately degenerate and need to be replaced.

What can affect the functioning of enzymes?

• Temperature and pH - Extremes can denature enzymes, ex. break the chemical bonds that are responsible for their three-dimensional shape, so they can no longer react with their substrates

• Substrate concentration - Increases can accelerate the rate of reaction (but only to a certain point)

• Inhibitors - Competitive inhibitors bind to the active sites of enzymes and prevent substrates from binding to those sites, whereas non-competitive inhibitors bind to different sites on enzymes and alter their shape

Endoenzymes

Enzymes produced within a cell that remain within the cell to catalyze intracellular reactions

• Ex. different enzymes involved in metabolic pathways within prokaryotic and eukaryotic cells

Exoenzymes

Enzymes produced within a cell and then released outside of the cell to catalyze extracellular reactions

• Ex. cellulase and pectinase released by saprophytic fungi in the soil (which help degrade cellulose and pectin respectively)

What are the two pathways in which glucose is catabolized within cells?

• Cellular respiration - In aerobic environments

• Fermentation - In anaerobic environments

What is the first phase in each pathway?

Glycolysis

What are the steps of cellular respiration?

1. Glycolysis - occurs in the cytoplasm of both prokaryotic and eukaryotic cells (2 ATP synthesized)

2. Citric acid cycle - Occurs at the inner surface of the cell membrane in prokaryotic cells and in the mitochondria in eukaryotic cells (2 ATP synthesized)

3. Electron transport chain - Occurs at the inner surface of the cell membrane in prokaryotic cells (34 ATP synthesized) and in the mitochondria in eukaryotic cells (32-34 ATP synthesized)

What are the steps in fermentation?

Oxygen does not participate in fermentation

1. Glycolysis - Occurs in the cytoplasm of both prokaryotic and eukaryotic cells (2 ATP synthesis)

2. Conversion of pyruvic acid into an end product - different microbes produce different end products (some of which have commercial importance)

Anabolism

Anabolism requires energy because chemical bonds are being formed

Involves a series of - biosynthetic reactions

• Relatively complex molecules are generated from relatively simple nutrients through a series of enzyme-catalyzed steps

• Two processes through which biosynthesis of organic compounds occur:

  • Photosynthesis - Reactions utilize light energy

  • Chemosynthesis - Reactions utilize chemical energy

What factors influence survival and growth?

• Nutrients

• Moisture

• Temperature

• pH

• Osmotic pressure

• Gaseous atmosphere

• Encouraging growth desirable in - labs, industries

• Inhibiting growth desirable in - nursing homes, hospital wards, operating

Key factors in Microbial Survival & Growth

1. Nutrients - serve as sources of energy, major elements, and additional elements necessary to sustain life

2. Moisture - Water is the most abundant molecule in cells, accounting for > 70% of total cell mass, and is required to carry out normal metabolic processes. Some bacteria form dormant, thick-walled, non-reproductive spores which help them survive a state of extreme dryness.

3. Temperature - Every microbe has a minimum, an optimum, and a maximum growth temperature

4. pH - Refers to the hydrogen ion concentration of a solution (numerical measure of acidity or alkalinity)

5. Osmotic pressure - Refers to the pressure exerted on a cell membrane during osmosis

6. Gaseous atmosphere - Some microbes require increased concentrations of carbon dioxide (usually 5-10%) for optimal growth

Psychrophiles

Microbes that grow best at low temperatures (ex. in glaciers)

Mesophiles

Microbes that grow best at moderate temperatures (ex. human body temperature)

Thermophiles

Microbes that grow best at high temperatures

Hyperthermophiles

Microbes that grow best at extremely high temperatures (ex. lava)

Acidophiles

Microbes that thrive in acidic environments

Alkaliphiles

Microbes that thrive in alkaline environments

Isotonic

• If concentration of solutes in the external environment of a cell = concentration of solutes inside the cell → solution in which the cell is suspended

• Excess water neither leaves nor enters the cell

• No change in pressure occurs within the cell in an isotonic solution

Hypertonic

If concentration of solutes in the external environment of a cell > concentration of solutes inside the cell → solution in which the cell is suspended

Plasmolysis

When a bacterium with a rigid cell wall is placed in a hypertonic solution its cell membrane and cytoplasm shrink away from the cell wall

Crenation

Shrinkage of red blood cells

Hypotonic

Concentration of solutes in the external environment of a cell < concentration of solutes inside the cell → solution in which the cell is suspended

Plasmoptosis

When a bacterium with a rigid cell wall is placed in a hypotonic solution it swells but might not burst; in case it does burst and the cytoplasm escapes

Hemolysis

Bursting or red blood cells

Capnophiles

• Some microbes require increased concentrations of carbon dioxide (usually 5-10%) for optimal growth

• Often grow in candle jars in microbiology labs

In vitro

In a test tube, culture dish, or elsewhere outside a living organism

In vivo

In a living organism

What does bacterial growth refer to?

Bacterial growth refers to an increase in the number of bacterial cells (ex. proliferation) rather than an increase in their size

Bacteria divide by binary fission once they reach their optimum size which means…

Parent cells splits in half to become two daughter cells

What influences binary fission?

• Binary fission is influenced by environmental factors such as moisture, temperature, and pH

• Continues for as long as there is a sufficient supply of nutrients, water, and space

What is culture media?

Bacteria can be grown in different kinds of artificial preparations

What is artificial media?

• Culture media are prepared in labs, i.e. they do not occur naturally, so they are sometimes referred to as artificial media

  • Contain all the basic ingredients that are required for microbial growth

How is media classified?

Classified based on state of matter

Liquid media (or broths)

Contained in tubes, so they are also referred to as tubed media

Solid media

Prepared by adding agar to liquid media, and then pouring the liquid media into tubes or Petri dishes in which they solidify

How else can you classify media?

You can also classify media by whether exact constituents are known

Chemically defined media

All ingredients are known, as they are prepared by combining specific amounts of carbohydrates, amino acids, and salts

Complex media

All ingredients are not exactly known, as they are prepared by combining ground-up extracts from animal organs, fish, and plants

What is Thioglycolate broth (THIO)?

Popular liquid medium used in bacteriology labs

• Concentration of dissolved O2 varies from top to bottom

  • Requires O2 grows at the top, while those that don’t need O2 grow at the bottom

Enriched media

• Contain a rich supply of special nutrients that promote the growth of microbes that have complex nutritional requirements - Fastidious microbes

Selective media

• Contain inhibitors that discourage the growth of certain microbes while selectively allowing for the growth of other microbes of interest

  • E.g. MacConkey agar (inhibits growth of Gram-positive bacteria, therefore selective for Gram-negative bacteria)

Differential media

• Permit differentiating between various microbes growing in them (sometimes also referred to as indicator media)

  • E.g. Mannitol salt agar (pathogenic staphylococci like S. aureus ferment mannitol and turn the medium yellow)

Inoculation

• Initial step in growing bacteria in a medical microbiology lab

• Process of introducing microbes into a culture medium in which they can grow

Streaking

Inoculating loop can be used to apply a portion of the clinical specimen (e.g. pus from a wound) to the surface of the culture medium

Stabbing

Inoculating needle can be used to introduce a portion of the clinical specimen into the culture medium

Incubation

Step after inoculation to grow bacteria in a medical microbiology lab

• Inoculated culture media are placed in an incubator which maintains the appropriate gaseous atmosphere, moisture level, and temperature

What are the 3 types of incubators?

1. Non-CO2 incubators - Contain 20%-21% O2 and less than 1% CO2 (similar to room air)

2. CO2 incubators - Contain less than 20%-21% O2 and 5%-10% CO2

3. Anaerobic incubators - Contain no O2

What is the bacterial growth curve?

Bacterial growth curve represents the growth cycle of a particular species of bacterium in pure culture at a constant temperature

• Samples of the culture are collected at fixed

  intervals and the number of viable (i.e. living)

  bacteria in each sample is determined

• Data are plotted on a graph to obtain a bacterial growth curve

Pure culture

Culture that contains only one species of a microorganism

What is the X-axis (horizontal) on the bacterial growth curve?

Time (minutes or hours)

What is the Y-axis (vertical) on the bacterial growth curve?

Log of the number of viable bacteria

Lag phase

• Bacteria absorb nutrients, synthesize enzymes, and prepare themselves for cell division.

• (No increase in number)

Log (exponential) phase

• Bacteria multiply rapidly.

• (Number doubles with each generational time)

Stationary phase

• Bacteria multiply slowly as the nutrients get used up and toxic wastes accumulate.

• (Number dividing = number dying)

Death phase

• Bacteria die rapidly as the nutrient supply gets depleted

• (Decrease in number)

Chemostat

Species can be cultured in a device that maintains a controlled growth environment

• Fresh culture medium containing nutrients is

  continuously added to the chemostat chamber

• Culture medium containing toxic wastes and excess microbes is continuously removed from the chemostat chamber into a collection vessel

Obligate intracellular parasites

Viruses and certain categories of bacteria (rickettsias and chlamydias) can only survive and multiply within living host cells

• Present a challenge when large numbers are

  needed for diagnostic or research purposes as they cannot be cultured in artificial media

• Grown by inoculation into fertilized chicken eggs, lab animals, or cell cultures (i.e. cultures of living cells from humans or animals)

Aseptic Lab Technique

• Set of routine procedures to prevent sterile

  solutions and cultures from becoming

  contaminated by microbes

• Helps prevent the infection of individuals and

  contamination of the work environment

• Includes the following:

  • Tying back hair and wearing eyeglasses

  • Washing hands and wearing gloves

  • Sterilizing inoculating loops and needles

  • Keeping lids of Petri dishes closed

Inhibiting Microbial Growth

• Necessary to inhibit the growth of microbes in

  healthcare environments to protect patients, staff, and visitors

• Can be accomplished by physical or chemical

  methods

Agents that inhibit the growth and multiplication of microbes have the suffix:

“-static” (ex. Bacteriostatic agents, Virustatic agents)

Agents that kill microbes have the suffix:

“-cidal” (ex. Fungicidal agents, Algicidal agents)

Disinfection

Elimination of most or all pathogens (except bacterial spores) from either nonliving or living surfaces

Disinfectants

Chemicals used to disinfect nonliving surfaces

• Ex. Hydrogen peroxide, bleach

• Cannot be used on living surfaces because they are strong chemical substances

Antiseptics

Solutions used to disinfect skin

• Ex. Ethanol, potassium permanganate

Sterilization

Destruction or killing of all microbes (including cells, bacterial spores, and viruses) on inanimate objects

Sterilants

Physical or chemical agents used to sterilize inanimate objects

What are examples of physical agents?

Dry heat, moist heat, UV rays

What are examples of chemical agents?

Ethylene oxide gas, formaldehyde solution

Can skin be sterilized?

Nope, but it can be disinfected using antiseptic solutions

Physical Inhibitions

1. Heat

2. Cold

3. Desiccation

4. Lyophilization or cryodesiccation (freeze-drying)

5. Radiation

Heat

Kills microbes by altering membranes and denaturing proteins

• Effectiveness determined by temperature and time

• Usually, the higher the temperature, the shorter is the time required to kill a microbe

Thermal death point (TDP)

Lowest temperature at which all organisms of a species (in pure culture) are killed in a specified time period

Thermal death time (TDT)

Length of time needed to kill all organisms of a species (in pure culture) at a specified temperature

What are the categories of heat sterilization?

Dry and moist sterilization

Dry heat sterilization

• Baking glassware or metals in thermostatically

  controlled ovens

• Burning contaminated disposable materials in medical waste incinerators

• Direct application of heat to inoculating loops (flaming or electrical heating)

Moist heat sterilization

• Faster and more effective than dry heat sterilization and can be accomplished at a lower temperature

• Boiling glassware and metals for 30 minutes destroys the vegetative (i.e. growing) forms of most pathogens, but is unable to kill bacterial spores

• Autoclaving (i.e. high-pressure steam) is able to completely destroy all microbes – Increase in pressure raises the temperature to levels higher than boiling water and forces steam into the materials

Cold

Does not kill microbes, but slows down metabolism and inhibits growth

• Freezing places microbes in a state of “suspended animation”, but their biological functions resume once the temperature increases

Desiccation

Does not kills microbes, but slows down metabolism and inhibits growth

• Drying places microbes in a state of “suspended animation”, but their biological functions resume once the moisture increases

Lyophilization or cryodesiccation (freeze-drying)

Does not kill microbes, but slows down metabolism and inhibits growth

• Used to preserve vaccines, blood samples, and

  other biological materials in labs

• Involves freezing a product followed by lowering the pressure to create a vacuum

• Ice changes from a solid state to a gaseous state without passing through a liquid state

  (sublimation), thereby drying out the product

Radiation

UV rays can penetrate microbes, damage their DNA, and result in genetic mutations and death

• UV rays are effective in killing microbes in the air and on surfaces

• UV lamps are used to reduce the number of microbes in hospital wards and operating rooms

Chemical Inhibitions

Chemical agents (disinfectants and antiseptics) can be used to inhibit the growth of pathogens, either temporarily or permanently

How is effectiveness of chemical inhibition determined?

• Concentration of the chemical agent

• Presence of organic matter - feces, pus, blood

• Bioburden - type and extent of microbial contamination

• Contact time - length of time that a chemical agent must remain in contact with microbes

• Physical characteristics of the object