MIC 205: Chapter 6 - Microbial nutrition, growth, biofilms

What does the term growth mean when applied to microbes?

An increase in cell number (reproduction)

What are the 3 microbial growth patterns?

Discrete colony, dispersed cells, complex biofilms

What is discrete colony?

Aggregation of cells visible on the surface of solid media

What is dispersed cells?

Single cells suspended in liquid media

What is complex biofilm?

A community of 1 or more species of bacteria (a biofilm can be a collection of bacteria, fungus, algae, protozoa, and even viruses)

What does an increase in growth also the same as for microorganisms?

An increase in growth is an increase in cellular population

How do bacterial cellular reproduction occur?

Cells use binary fission where it starts off with 1 cell → replicates the DNA and elongate the membrane, and then separate them to have 2 separate cells.

What is the timing it takes for 1 cell to go through binary fission to 2 cells called and how long?

Doubling time - its has a lot of diversity and is species dependent → 2 microorganisms double every 30 minutes (this is doubling time) when 2 cells go through binary fission it is another doubling time, when 4 cells go through binary fission it is another doubling time

What is logarithmic growth?

Bacterial populations can increase to millions of cells in only a few hours - when cells go from 1 cell → 2 cells → 4 cells → 8 cells…

What is the overall best way to describe growth of microbes versus a human?

Microbes do logarithmic growth and humans do arithmetic growth

What are clinical specimens?

Human materials examined or tested for the presence of bacteria and other microbes (like a sample of tissue, blood, urine) AKA: acquiring materials from humans through some type of sample to culture the microbes

What are the considerations of clinical sampling?

Proper specimen collection, technique, timely delivery, proper culture methods

Whose responsibility it is to deliver proper specimen collection to clinical laboratory?

The patient’s physician in a timely delivery

Is there a universal media that grows everything and why?

No because microbes have different nutritional needs and metabolic needs that have a receipe for the specific organisms

What are the different types of culture media?

Defined (synthetic) media, complex media, selective media, differential media, anaerobic media, transport media

What is selective media?

Selecting for certain populations and inhibiting the growth of other populations. You can add some type of selective agent to the media that will inhibit a certain population (for certain populations you are interested in)

What is an example of selective media?

Nutrient agar and dextrose agar plates inoculated with diluted soil samples. The acidic pH of the dextrose agar selects fungi by inhibiting bacterial growth

What is differential media?

Organisms are differentiated by hemolysis to change growth patterns

What is an example of differential media?

Blood agar plates that reveals digestion of red blood cells by bacteria, either being: B-hemolytic (complete lysis), a-hemolytic (partial lysis), and Y-hemolytic (lack of lysis)

What are the 4 phases of microbial growth?

Lag, log, stationary, death phase

What is lag phase?

There is no growth but there is enzymatic activity. This is the surveying phase where microbes are surveying their environment to look for if the nutrients they need are available and the ones they need to generate. Microbes are adjusting to their environment and how long it takes depends on the microbe

What is log phase (exponential phase)?

Lots of binary fission occurs causing exponential or logarithmic growth. There is high enzymatic activity and cells will either 1) grow and cellular population will produce a lot of waste products that begins to slow down/inhibit growth and transition to stationary phase OR 2) nutrients start to run out and lead to stationary phase.

What is stationary phase?

There is no increase in cellular population to stationary phase but there is still growth. You are getting equal growth to equal death

What is death phase?

Cells begin to die off and outweigh population

What phase of growth will microorganisms be most sensitive to antimicrobials?

Log phase because the most growth can be affected here (ex: a microbe will be most sensitive to penicillin during log phase because penicillin inhibits peptidoglycan synthesis and in log phase, it will inhibit the synthesis of peptidoglycan)

How are microbes often classified?

According to their nutritional and physical requirements

What are physical factors requirements required for microbes?

Temperature, pH, osmotic pressure, other microbes present, etc. In the environment, microbes are competing with each other for nutrients

What are the two main sources microbes obtain energy?

Phototrophs and chemotrophs

What is phototrophs?

Acquiring energy from sunlight (photosynthesis) - taking light energy and converting it to chemical energy. (or from Daydiff’s glowing personality)

What is chemotrophs?

Acquiring energy from organic/inorganic molecules by breaking the bonds and harvesting the energy

What 2 main sources do molecules acquire their carbon from?

Autotrophs and heterotrophs

What is autotrophs?

Self feeders that make their own food - like fixing CO2 using calvin-benson cycle

What is heterotrophs?

Different feeders that gets carbon from other organisms - like glucose, proteins, fats

How do humans get energy and carbon?

Chemotroph and heterotroph

How do plants get energy and carbon?

Phototroph and autotroph

Is gaseous oxygen (O2) or oxygen covalently bound in compounds poisonous?

No

What is an aerobes?

Needs oxygen to grow, uses aerobic respiration, can detoxify toxic oxygen

What is an anaerobes?

No oxygen, undergo fermentation, no ability to detoxify

What is aerotolerant anaerobes?

Can grow with or without oxygen, fermentation or anaerobic respiration, and has enzymes to detoxify O2

What is facultative anaerobes?

Can grow with or without oxygen but prefer the oxygen, fermentation or anaerobic respiration or aerobic respiration, and has enzymes to detoxify O2

What is microaerophiles?

Aerobes that require oxygen levels from 2-10%, limited ability to detoxify toxic O2

What are the forms of oxygen that are toxic excellent for?

Oxidizing agents that initiate chains of oxidation reactions that damage a cell’s proteins and lipids

What are toxic forms of oxygen a by-product of (what produces toxic oxygen)?

Aerobic respiration

What are the 4 toxic forms of oxygen

Singlet oxygen, superoxide radicals, peroxide anion, hydroxyl radical

What is a singlet oxygen (¹O₂)

Molecular oxygen with electrons boosted to a higher energy state. Generated during aerobic metabolism and photosynthesis. Excess energy of electrons are removed by carotenoid pigments

What do toxic forms of oxygen do

Cause modifications to the lipids or membrane structure (which means you don’t have selective permeability and ECT).
Cause proteins to misfold and denature (which means metabolism isn’t happening)
Cause altercations to nucleic acid (which mean you can’t replicated DNA and transcription can’t happen/be interpreted)
Ultimately causing the cell to die since reactions aren’t happening

What are superoxide radicals (O₂-)

Formed during incomplete reduction of oxygen in aerobic and anaerobic respiration. Highly reactive

What are peroxide anion (O₂^2-) & (H₂O₂)

Formed during the breakdown of lipids and in reactions catalyzed by SOD (superoxide dismutase)/ Highly reactive

What are hydroxyl radical (OH)

Results from ionizing radiation and incomplete reduction of hydrogen peroxide. Most reactive but not a threat to aerobes due to catalase and peroxide
(the toxic forms are still present but these organisms produce enzymes like superoxide dismutase (SOD) to neutralize the toxic forms)

What is the most reactive form of toxic oxygen

Hydroxyl radical (OH)

How do toxic forms of oxygen get neutralized

The organisms produce enzymes (like superoxide dismutase SOD) and it takes 1 of our oxygen radicals and converts it to hydrogen peroxide. Then, another enzyme called catalase takes the hydrogen peroxide and converts it to water and oxygen gas → hence neutralizing it which is why these organisms can grow in the presence of oxygen due to neutralizing pathway.

How can aerobes process O2

Through detoxifying enzymes that converts O₂

What are the 2 detoxifying enzymes and what do they do

Superoxide dismutase (SOD): (O₂-) → H₂O₂
Catalase and Peroxidase: H₂O₂ → H₂O + O₂

What are aerobes

Utilize molecular oxygen as the final electron acceptor during aerobic respiration. Can detoxify toxic forms of oxygen (have neutralization pathway)

Why do aerobes only grow in the presence of oxygen

1. They metabolism can only do aerobic

2. They have a detoxification pathway to prevent the destruction of their proteins and other molecules due to the toxic forms

What do anaerobes only grow in the absence of oxygen

They do not have a detoxification pathway so they have to live in environments without oxygen and do anaerobic respiration or fermentation

What are anaerobes

Undergo fermentation or utilize other final electron acceptors during anaerobic metabolism. No ability to detoxify toxic forms of oxygen

What are facultative anaerobes

Can survive via fermentation or anaerobic respiration, or by aerobic respiration. Ability to detoxify toxic forms of oxygen by neutralization pathway. In terms of metabolism, they prefer to use oxygen so they do aerobic respiration but if oxygen is not present they switch to anaerobic respiration/fermentation

What is aerotolerant anaerobes

Will use the pathways of fermentation or anaerobic respiration. Possess some enzymes capable of toxic detoxifying forms of oxygen.
they can grow in the presence of oxygen so they must have a detoxifying pathway but they are not using oxygen so they do anaerobic respiration/fermentation

What are microaerophiles

Aerobes that require oxygen levels from 2%-10% (aerobic respiration). They have limited ability to detoxify toxic forms of oxygen and possess a neutralization pathway. (with metabolism, they can only do aerobic respiration)

What does anabolism often ceases

Due to insufficient nitrogen needed for protein and nucleotide synthesis

Where is nitrogen acquired from

Organic and inorganic sources, and recycling unneeded amino acids and nucleotides

What is nitrogen fixation

Some gram- bacteria grown in associated with plant roots, where they convert nitrogen gas (N2) in the atmosphere into ammonia (NH3)

Why do we need nitrogen fixation

Essential to life on earth because most organisms cannot use gaseous nitrogen

What are 2 examples of things in the environment that do nitrogen fixation

Azospirillum: tropical grasses (sugarcane)
Rhizobium: legumes (peas, beans, clover)

What are the four other chemical requirement for microbial growth

Phosphorus: for phospholipid membranes, DNA, RNA, ATP, some proteins
Sulfur: component of sulfur-containing amino acids and vitamins
Trace elements: minerals and inorganic ions found in tap water
Growth factors: organic chemicals that cannot be synthesized by certain organisms (vitamins, certain amino acids, nitrogenous bases, NADH…)

What are the 4 ways microbes can be classified based on growth patterns and temperature

Psychrophile, mesophile, thermophile, and extreme thermophile

What is a psychrophile

-5 to 20 degrees celcius

What is a mesophile

15 to 45 degrees celcius

What is a thermophile

40 to 80 degrees celcius

What is an extreme thermophile

80 to 121 degrees celcius

Can an organism grow over the whole range of 121 and what does thermal energy do

No organism can grow over the whole range. Thermal energy plays a crucial role in structure/function of a cell’s proteins and membranes.

Regarding a temperature graph, what can you replace growth rate with to still get the same graph

Enzymatic activity → higher enzymatic activity equals more growth

Why are most organisms sensitive to changes in acidity

Due to H+ and OH- interferes with the hydrogen bonds in proteins and nucleic acids

What are neutrophiles and examples

Have a neutral pH of 6.5 to 7.5 → most bacteria and protozoa

What are acidophiles and examples

Grow in acidic habitats like some bacteria and many fungi

What are alkalinophiles

Lives in alkaline (basic) soils and water up to 11.5

Why do microbes absolutely require water

To dissolve enzymes and nutrients utilized in metabolism. Water is an important player in many metabolic reactions. Without water, most cells die, but some have cell walls, capsules, or coatings that help cells retain some water and some enter dormant phases where metabolic activity is suspended.

What is an obligate halophile

Grows in up to 30% salt

What is facultative halophiles

Can tolerate high salt concentrations

What is barophiles

Organisms that live under extreme pressure

Who invented methods to isolate microorganisms in pure cultures

Robert Koch

What is a pure culture

Absent in nature (or virtually so in nature)

What is a sessile organisms

Microbes that are attached to a surface

What is a planktonic organisms

Microbes that are free-living

What are the characteristics of a biofilm

Attached to a surface (abiotic - solid substrates or host tissue - soft tissue)
Complex social structure (organisms living together to protect and grow)
1000x resistant to antibiotics (higher doses of antimicrobial need than planktonic)
Biofilm is a molecular filter (reduces drug activity, more resistant to free Cl)
Low metabolism (of cells within biofilm)

Where are a couple common biofilms

Plaque on teeth, water pipes, dental units, dental units, contact lens cases, gel-like film on inside of a vase, slippery slime on river stones

What biofilm infection can be found on pacemakers

Staphylococcus aureus

What biofilm infection can be found on cystic fibrosis pneumonia and burn patients

Pseudomonas aeruginosa

What biofilm infection can be found on urinary catheters

Escherichia coli

What biofilm infection can be found on orthopedic devices/breast implants

Staphylococcus epidermis

What biofilm infection can be found on contact lens

Many gram-positive bacteria

What percentage of all bacterial infections are caused by bacteria in the form of a biofilm

80%

How are biofilms heterogeneous (different in kinds)

They have a great range of microhabitat, gradients of oxygen, pH, and growth factors. The different habitats will lead to different metabolic activity

In a chloramine treatment of biofilm, what does a red and green stain mean

Red stain is alive cells and green stain is dead cells (it takes 120 mins to completely turn green and 90 mins to be half green)

How do bacterial cells know when to produce the expolysaccharide

Quorum sensing

What are the steps of biofilm development

Initial interaction (has flagella/fimbriae) → Stable adhesion (fimbriae attaches to surface) → Microcolony formation (flagella/fimbriae are gone once they make stable adheasion, prokaryotic cells can change their gene expression, and they start to secrete glycocalyx/exopolysaccharide) → Exopolysaccharide synthesis (expo: exporting out and polysaccharide: sugar/made up of sugar units) → Mature biofilm

What are gene regulation in biofilms and what controls it

Change in the gene expression of attached cells
Increased production of capsule
Change in energy metabolism
Gene regulation is controlled by quorum sensing.

What is quorum sensing

Cell to cell signaling - allows an individual bacterium to monitor the cell density of the population and organisms produce autoinducers as signaling molecules.

What do autoinducers do in quorum sensing and example

Allow the regulation of specific genes (autoinducers are a signaling molecule). For example, switching between flagella gene and gene for capsule for the development of a biofilm.
Each bacterial species will synthesize a unique signaling molecule