Microbial Nutrition and Bacterial Growth

• Microbial nutrition and growth involves controlling pathogenic microbial growth, controlling food spoilage microbes, encouraging helpful microbes, and identifying microbes in the lab.

Bacterial Growth via Binary Fission

• Bacteria divide through binary fission, which increases cell numbers rather than cell size.
• Most microbial populations divide very quickly.
• Microbial growth requirements leads to extensive diversity.

Generation time

• Generation time is the time required for a bacterial cell to grow and divide, varying between different bacteria types.
• Generation time is dependent on chemical and physical conditions.
• Bacillus stearothermophilus has a generation time of 11 minutes under optimum conditions.
• E. coli has a generation time of 20 minutes.
• Staphylococcus aureus has a generation time of 28 minutes.
• Lactobacillus acidophilus has a generation time of 60-80 minutes.
• Mycobacterium tuberculosis has a generation time of 360 minutes.
• Treponema pallidum has a generation time of 1980 minutes.

Cell Division

• E. coli doubles in approximately 20 minutes under ideal conditions, therefore growth is rapid.
• A single E.coli cell can grow to 10^23 cells in 24 hours.

Bacterial Growth Phases:

• Bacteria inoculated into liquid growth media has four typical stages of growth.
• Lag Phase sees cells adjusting to a new environment, synthesizing enzymes for the new nutrient media rather than immediately reproducing. The duration varies by microbe type.
• Log Phase results in rapid chromosome replication, growth, and division, leading to a logarithmic population increase.
• Stationary Phase occurs when nutrients deplete and wastes accumulate, reducing the division rate, where the death rate equates to the production rate.
• Death（decline）Phase is when the population reaches a point where death rate exceeds production, with more cells dying than alive.

Metabolic Diversity

• Microorganisms can thrive in various environments with a wide variety of nutritional requirements.
• Nutrients are used for energy and to construct organic molecules and cellular structures.

Organism Classification

• Nutritional patterns are a combination of carbon, energy, and electron sources used to determine the organism classification.
• Autotrophs use inorganic CO2 as a carbon source to make organic compounds, essentially feeding themselves.
• Heterotrophs catabolize organic compounds.
• Phototrophs get energy from light.
• Chemotrophs get energy from redox reactions that involve organic or inorganic compounds.
• Organotrophs acquire electrons from the same organic molecules, providing them with carbon and energy.
• Lithotrophs acquire electrons from inorganic sources like H2, H2S, Fe2+, and NO2-.

Nutritional Classification

• Photoautotrophs like plants, algae, and cyanobacteria use H2O to reduce CO2 producing O2 as a byproduct.
• Photosynthetic green sulfur and purple sulfur bacteria don't use H2O or produce O2.
• Photoheterotrophs include green nonsulfur and purple nonsulfur bacteria.
• Chemoautotrophs include hydrogen, sulfur, and nitrifying bacteria.
• Chemoheterotrophs such as most animals, fungi, protozoa, and many bacteria respire aerobically.
• Some animals, protozoa, and bacteria respire anaerobically.
• Some bacteria and yeasts use fermentation.

Requirements for Microbial Growth

• Organisms are classified by growth requirements.
• These requirements are either Physical (temperature, pH, osmotic pressure) or Chemical (organic molecules, trace elements, oxygen).

Physical Requirements

• Optimal growth occurs at a minimum, optimum and maximum temperature.
• Most bacteria thrive best at a neutral pH, essential for specific enzymatic activity.
• Low pH inhibits growth of many microbes, but acidophiles are able to grow in these conditions.
• Molds and yeasts can grow over a wide range of pH; however, their growth does not continue below a pH of 5.
• A by-product of bacterial growth is acid that protects food from spoilage and inhibits the growth of bacteria to make food like sauerkraut and yogurt.
• Alkalinity can also inhibit microbial growth but this is not usually used in food preservation.

Osmotic Pressure

• Microbes get their nutrients from the surrounding H2O through its plasma membrane.
• Water flows in and out of the cell so that the dissolved molecules inside and outside can be balanced
• Dissolved molecules = salts, carbohydrates, proteins

Plasmolysis

• Plasmolysis occurs in hypertonic conditions in which:
  • Needed H2O leaves the cell
  • The Plasma membrane gets pulled leaving the cell wall
  • Resulting in cell growth being inhibited
• Addition of high [salt] or [sugar] is used to preserve foods - e.g. salted fish and sweetened, condensed milk
• Exception: salt-loving halophiles are able to grow in very high [salt] - e.g., Dead Sea (~30% salt) can support some microbial life.

Chemical Requirements

• Chemical requirements for building blocks of organic materials is carbon, nitrogen, sulfur and phosphorus.
• Proteins, carbohydrates and lipids also are important.
• Needed for tiny amounts for function of important chemical reactions are Trace elements such as iron, copper, molybdenum, zinc.
• Enzymes require Ca2+ ions.

Oxygen

• Aerobes and anaerobes can be determined because it is either essential or deadly for obligate aerobes and anaerobes.
• Aerobic metabolism produces enhanced energy than that of anaerobic.

Organism Classifications Based on Oxygen Requirements

• Obligate aerobes need O2 as the final electron acceptor of the ETC– e.g., Mycobacterium tuberculosis.
• Obligate anaerobes find O2 deadly as they don’t have enzymes to break oxygen molecules - e.g., Clostridium sp.
• Facultative anaerobes: maintain life via fermentation or anaerobic respiration, leading to reduced metabolic efficiency without oxygen- e.g., E. coli.
• Microaerophiles: organisms that are able to grow in low [oxygen] (lower than air) - e.g., Campylobacter jejuni.
• Aerotolerant anaerobes: cannot use O2 in metabolic pathways but can tolerate it by having enzymes that detoxify poisonous forms - e.g., Lactobacilli.

Direct Methods of Measuring Bacterial Growth

• Serial dilution and viable plate counts, Membrane Filtration, and Microscopic counts.

Electronic Counters

• Coulter counter: count cells as they interrupt an electrical current flowing in front of an electronic detector.
• Flow cytometer: a light-sensitive detector records changes in light transmission as cells pass through a tube.

Indirect Methods of Measuring Bacterial Growth

• Use Measurements of dry weight (organisms are filtered from media, dried and weighed).
• Genetic methods are also used to isolate DNA sequences of unculturable organisms.
• Spectrophotometry indirectly measures population size.

Microbial Cultures

• Describes when microorganisms or their growth is witnessed.
• Culture media include liquid or solid nutrient material that helps microorganisms grow.
• Agar makes the media become solid.
• Inoculum involves introducing microbes into culture media to grow by obtaining its specimen from environmental clinical stores.
• Colonies are aggregation of cells that arise from single parent cells, and a colony is produced when one bacterial cell divides to create a "pile" of cells.
• Purpose of culturing is to visualize colony morphology, grow enough bacteria to conduct tests and select specific bacteria.

Pure Culture: Streak Plate

• They are composed of cells arising from a single progenitor
• A progenitor is termed a colony-forming unit (CFU)
• Used to isolate pure cultures.

Biofilms

• Complex communities living on a surface.
• These can form on surfaces, medical devices, mucous membranes of digestive system by the result of quorum sensing.
• Many microorganisms can become harmful as part of a plaque biofilm.

Criteria for a Culture Medium

• Nutrients, moisture, pH, oxygen levels, temperature and sterility are all crucial to consider when culturing microorganisms.
• Taking advantage of certain metabolites and their needs can help identify microorganisms.

Selective Media

• Inhibits the growth of unwanted microbes while encouraging the growth of the desired microbes. Therefore, this increases the amount of a chosen microbe in order for it to be observed better.
• Salmonella typhi are isolated with Bismuth Sulfite agar that inhibits other Gram-positive and most Gram-negative bacteria growth.
• Sabouraud’s Dextrose agar with a pH of 5.6 isolates fungi that can grow well at low pH.
• The acidic pH will also stop bacteria from growing.

Differential Media

• Facilitates the differentiation between one bacteria from another by changing/ altering the microbes, or their surrounding environment.
• Blood agar is an example of differential media because it allows for identifying bacteria that lyse red blood cells.

Types of reactions from Blood Agar

• Beta-hemolysis: bacteria completely lyse RBC, resulting in a clear ring
• Alpha-hemolysis: bacteria damages RBC, resulting in a discoloration
• Gamma-hemolysis: bacteria grow with no effect on media

Combining Selective & Differential Media

• This increases the amount of selective characteristics, increasing the benefits within each media
• Mannitol Salt agar: a high [salt] discourages growth of most organisms.
• If mannitol is fermented, the mannitol metabolism produces acid, and the pH turns red to yellow.
• MacConkey agar: the agar has crystal violet, discouraging gram-positive bacteria growth.
• lactose fermenters lead to red or pink colonies where non-lactose fermenters create colorless colonies.

Anaerobic Microbes Growing

• Obligate anaerobes must be cultured during the absence of free oxygen.
• Petri plates need to be stored in sealed containers.

Biochemical tests

• Check for specific enzymatic activity.
• These are also known as the following exams: Urease, DNase, and Carbohydrate Fermentation tests.

Urease test

• Inoculate media containing urea and pH indicator where a low, high or neutral pH produces different colors i.e yellow, pink or red.
• If urease is the catalyst of ammonia, also described as a base: the increasing pH will have a pink color change.

1. Uninoculated control
2. Proteus vulgaris(pos)
3. E. coli (neg)

DNase test

• DNAse can either be positive or negative, and it helps to identify if there is a presence of precipated DNA.
• Inoculate on agar with DNA and add hydrochloric acid. Once incubated overnight, a clear zone is apparent when DNAse is present with its precipitate form.
• If DNAse is positive, there is a cloudy zone, but if clear, then precipitated DNA is present.
• Many Staphylococci are DNase negative.
• S.aureus is DNAse positive.

Carbohydrate Fermentation test

• If specific carbohydrate and pH indicators are mixed with media, results can tell the acidity of the final product. Color changes mean carbohydrate production exists, producing yellow, red, or neutral colors depending on the reaction.
• There may also be gas production.