Environmental conditions and pH

• Organisms thrive in specific growing conditions, and deviation from the optimum may reduce growth.
• Environmental conditions impact growth, determining optimum conditions and growth ranges.

Laws of Growth

• Law of the Minimum considers that growth is limited by the nutrient in the shortest supply.
• Optimum is where the conditions are most suitable for growth.
• Law of the Maximum considers that growth is influenced by the factor present in excess.

Environmental Factors and pH

• Key factors for testing include pH, temperature, water activity, and oxygen.
• The pH scale ranges from 0 to 14, where 7 is neutral, below 7 is acidic, and above 7 is basic.
• pH is a measure of free protons (H+) or free hydroxyl groups (OH-).
• A pH below neutral has more H+, while a pH above neutral has more OH-.
• Some organisms use proton pumps in acidic conditions to maintain a near-neutral cytoplasm pH.
• Certain organisms can grow at pH values far from neutral.

Optimal Growth Conditions by pH

• Acidophiles optimally grow at a pH lower than 6.
• Neutrophiles optimally grow at a pH around neutral (pH 6-8).
• Alkalophiles optimally grow at a pH at or above 8.

Temperature Adaptations

• Some organisms thrive in very hot environments like hot springs, while others prefer cold environments.
• Adaptations to high temp include lipid monolayers in membranes and heat shock proteins
• Organisms adapted to low temperatures have more unsaturated fatty acids in their membranes for fluidity, along with cold shock proteins.

Optimal Growth Conditions by Temperature

• Psychrophiles optimally grow between -5 °C and +20 °C.
• Mesophiles optimally grow between 20 °C and 50 °C.
• Psychrotrophs, a mesophile, survive and grow slowly at refrigeration temperatures (4 °C).
• Thermophiles optimally grow between 50 °C and 80 °C.
• Hyperthermophiles optimally grow above 80 °C.

Water Activity

• Serratia marcescens produces a red/orange pigment (prodigiosin) under ideal conditions, with primary and secondary metabolites.
• Water is essential for all living organisms, and altering its activity impacts growth due to osmotic pressure.
• Membranes are likely impacted by high osmotic pressure.
• Organisms adapt to environments with limited water activity and high osmotic pressure.
• Water activity is affected by sugar and salt concentrations.

Osmotic Adaptations

• Xerophiles optimally grow in very dry environments like desert soil.
• Osmophiles prefer to grow at high osmotic pressure achieved by high sugar concentrations, such as in jams.
• Halophiles optimally grow at elevated salt concentrations, such as in salt flats.
• Halotolerant organisms optimally grow in the absence of salt but can survive with some salt, like on skin.

Oxygen Requirements

• Oxygen preference is tied to an organism's metabolism.
• Aerobic respiration uses O2 as a terminal electron acceptor to make ATP.
• Anaerobic respiration uses a molecule other than O2 as a terminal electron acceptor.
• Fermentation uses an organic molecule as a terminal electron acceptor.
• TGYA Shake is complex molten media made of Tryptone Glucose Yeast extract Agar, inoculated with a loopful of culture, and rolled in the palms before solidifying.
• FTM is defined semi-solid Fluid Thioglycollate Media, inoculated with a needle of culture.

Oxygen-Based Classifications

• Obligate aerobes must undergo aerobic respiration using O2 as the terminal electron acceptor.
• Microaerophiles require O2 for aerobic respiration but are inactivated unless O2 concentration is reduced to 2-10%.
• Facultative anaerobes prefer aerobic respiration if O2 is available but use anaerobic respiration or fermentation if O2 is not.
• Aerotolerant anaerobes, also known as obligate fermenters or facultative aerobes, cannot use O2 but are not harmed by it and produce ATP through fermentation.
• Obligate anaerobes cannot grow in the presence of O2 and exclusively use anaerobic respiration with an inorganic terminal electron acceptor.
• Capnophiles prefer to grow in a moist environment with high CO2 and low O2.

Gas and Candle Jars

• Gas Pak Jars generate an oxygen-free environment by sealing inoculated Brewer's Anaerobic Agar with a catalyst pack and palladium pellets to convert H2 and O2 to H2O, promoting anaerobe growth.
• Candle Jars generate a low O2, high CO2 environment by sealing inoculated Brewer’s Anaerobic Agar with a lit candle, consuming O2 and releasing CO2 until the flame extinguishes, promoting capnophile growth.

pH Examples

• Staphylococcus aureus grows at pH 6-7 as neutrophiles
• Alcaligenes faecalis grows at pH 7-9 as neutrophiles
• Escherichia coli grows at pH 6.5-7.5 as neutrophiles and at 37 degrees C as mesophiles
• Lactococcus lactis grows at pH 5.5-7.5 as acidophiles
• Saccharomyces cerevisiae grows at 37 degrees C as mesophiles
• Micrococcus luteus grows at 30-37 degrees C as mesophiles
• Leuconostoc mesenteroides grows at 25-30 degrees C as mesophiles
• Bacillus staeropthermophilus grows as thermophiles.
• Halobacterium sp grows in 15-20% NaCl as halophiles.
• Pseudomonas aeruginosa grows as halotolerants.
• Micrococcus luteus grows in 0.5-5% NaCl as halotolerants.
• Staphylococcus aureus grows in 0.5-7.5% NaCl as halotolerants
• Escherichia coli grow as nonhalophiles
• Lactococcus lactis are a facultative anaerobe.
• Escherichia coli are a facultative anaerobe.
• Pseudomonas aeruginosa are a facultative anaerobe.
• Micrococcus luteus are an obligate anaerobes.

Antibiotics

• Antibiotics mean "without life" and are used as antibacterials, with bacteriocidal antibiotics killing bacteria outright, and bacteriostatic antibiotics preventing replication.
• Common Modes of Action of Antibiotics include inhibiting peptidoglycan synthesis, lipopolysaccharide synthesis, DNA replication, RNA synthesis, translation, and damaging the cell membrane outright.
• Antibiotics can be effective whether they are bacteriostatic or bacteriocidal and even bacteriostatic antibiotics are valuable because all effective antibiotics slow or stop bacterial growth by working with the immune system.

Mueller-Hinton Agar Test

• The Mueller-Hinton Agar test involves preparing a lawn, aseptically adding antibiotic discs, incubating at 37 °C for 24-48 hours, measuring zones of inhibition (in mm), and comparing measurements to a table to determine if the organism is susceptible, intermediate, or resistant.
• The media is rich and complex, and chosen because if an antibiotic can slow/stop growth of the bacteria under ideal conditions then in the body with a functional immune system the organism will be controlled even more effectively.
• Lawn in this case means that all surfaces of the agar are covered with a bacterial strain.

Blood Agar

• Blood Agar is differential media because it distinguishes bacteria based on their ability to break down red blood cells with very nutrient rich (Brain Heart Infusion plus 5% defibrinated sheep's blood) and is used to determine hemolysis
• Alpha hemolysis damages red blood cells by converting hemoglobin to methemoglobin, changing the media color to greenish/brown.
• Beta hemolysis destroys red blood cells and shows a clearing around the colonies.
• Gamma hemolysis shows no change in red blood cells with no hemolysis.
• Streptococcus salivarius presents with Alpha.
• Staphylococcus aureus presents with Beta.
• streptococcus pyogenes presents with Beta.
• Streptococcus agalactiae presents with Beta.
• Enterococcus faecalis presents with Beta.
• staphylococcus epidermidis presents with Gamma.
• Staphylococcus saprophyticus presents with Gamma.
• Micrococcus luteus presents with Gamma.
• Neiseseria flava
• Branhammela cattarhalis
• Corynebacterium xerosis

Mannitol Salt Agar

• Mannitol Salt Agar is both differential and selective for bacteria.
• Contains mannitol, 5% salt, and phenol red (pH indicator).
• It inhibits the growth of most bacteria except halotolerant species due to its 7.5% NaCl content.
• Fermentation of sugar by bacteria yields acid end products, lowering the pH and changing the color of the media.
• Media turns red with neutral pH (negative for mannitol fermentation)
• Media turns yellow with acidic pH (positive for mannitol fermentation)
• If there is no growth, no conclusion can be made.
• This test only applies to the family of Micrococceae
• Stapphylococcus aureus is positive.
• Staphylococcus saprophyticus is positive.
• staphyloccos epidermidis is negative.
• Micrococcus luteus is negative.

DNase Agar

• DNase Agar is a differential media as a test to determine an organism's ability to produce deoxyribonuclease (DNase).
• Contains few nutrients.
• Contains methyl green bound to DNA with Positive results mean that there is a clear zone around the growth If DNA is degraded, the dye has nothing to bind to and volatilizes into the air; Negative results mean that there is no clearing or growth.
• This test only applies to the family of Micrococceae Staphylococcus aureus is positive. Staphylococcus epidermidis is negative. Staphylococcus saprophyticus is negative. Micrococcus luteus is negative.

Salt Broth

• Salt Broth is selective for growing halotolerant organisms via high salt concentrations that also inhibit most bacteria.
• Nutrient broth is added with 6.5% NaCl where growth (turbidity) shows a positive result and only applies to Strptococceae and non-strep organisms
• Enterococcus faecalis is positive.
• Neisseria flava is positive.
• Corynebacterium xerosis is positive.
• Streptococcus pyogenes is negative.
• Streptocoocus agalactiae is negative.
• Streptococcus salivarius is negative.
• Branhamella cattarhalis

Bile Esculin Slant

• Bile Esculin Slant is a selective and differential media.
• Bile salts inhibits the growth of gram + bacteria.
• Esculin is a sugar alcohol that when hydrolyzed by certain bacteria and esculatin reacts with ferric citrate to produce a black precipitate, making the test differential.
• Media turns black with a positive result.
• Media turns greenish - yellow means that the result is negative.
• Only applies to Streptococcaceae and non-strep
• Enterococcus faecalis is positive.
• Neisseria flava is positive.
• Streptpcoccus pyogenes is negative.
• Streptococcus agalactiae is negative.
• Streptococcus salivarius is negative.
• Branhamella cattarhalis is negative.
• Corynebacterium xerosis is negative.

Micrococcaea:

• Gram +
• Catalase +
• Grows in clusters or tetrads

Streptococcaceae:

• Gram +
• Catalase -
• Forms in chains

Non-streps

• Neisseria flava and Branhamella cattarhalis are Gram -, catalase +, and form diplococci
• Corynebacterium xerosis is Gram +, catalase +, and forms in rods