Microbial Growth

The Requirements for Growth

n Physical

n Temperature

n pH

n Osmotic pressure

n Chemical

n Carbon

n Nitrogen

n Sulfur

n Phosphorus

n Trace elements

n Oxygen (sometimes)

n Organic growth factors

Physical Requirements

n Temperature

n Cardinal temperatures

n Minimum growth temperature

n Lowest temperature at which an organism will grow

n Below this temp.ànutrient transport difficulty

n Optimum growth temperature

n Temperature at which an organism grows best

n Metabolic enzymes occurring at maximum rate

n Maximum growth temperature

n Highest temperature at which an organism will grow

n Above this temp.àprotein denaturation; membrane collapse, and lysis

Temperature Classes

n Psychrophiles

n Optimum: 10-15 ºC

n RANGE: -10 ºC - 20 ºC

n Requires cold environment

n Mostly found in algae in sea ice and snow fields

n Psychrotroph

n Optimum: 20-25 ºC

n RANGE: 1 ºC-30 ºC

n Grows best at refrigerator temperatures, but can grow at low temperatures

n Found in soils and water and foods in fridge

Food spoilage

Psychrotrophs

Temperature Classes

n Mesophiles

n Optimum:36-38 ºC (body temp)

n RANGE: 10-50 ºC

n Most common

n E. coli

n Thermophiles

n Optimum:58-65 ºC

n RANGE: 40-73 ºC

n Compost, soils, hot water heaters, some hot springs

n Hyperthermophiles

n Optimum:90-100 ºC

n RANGE: 65-120 ºC

n Geothermal vents, hot springs, volcanoes

n Mostly Archaea

Temperature Requirements

Physical Requirements

n pH

n Most bacteria grow between pH 6.5-7.5

n Molds and yeasts grow between pH 5 and 6

n Most bacteria produce organic acids as they grow and metabolize

n Buffers

n Acidophiles

n Grow at low pH (<6) (acidic environments)

n Fungi in general and some bacteria (obligate)

n Thiobacillus and acid mine drainage (pH 1)

n Alkaliphiles

n Grow at high pH (>10-11 pH)

n Soda lakes, high carbonate soils

Physical Requirements- Fig 6.4

n Osmotic pressure

n Osmosis

n Positive water balance

n Plasmolysis

n Caused by hypertonic environments (increase in salt or sugar)

n Use of salt as a preservative

n Halophiles: Can withstand a high salt concentration

n Extreme or obligate halophiles require high salt concentrations (30%)

n Facultative halophiles

n Don’t require high salt, but can survive high salt concentrations (2-15%)

Chemical Requirements

n Nutrients

n Macronutrients

n C, N, P, S,

n Carbon

n 50% of dry weight of cell

n Structural organic molecules, energy source

n Needed for proteins, sugars, lipids to make up cell structures

n Chemoheterotrophs: get C from organic carbon sources such as sugars and lipids

_n Chemoautotrophs: get C from CO₂

n Nitrogen

n 14% of dry weight of cell

n In amino acids and proteins

n Needed for proteins, DNA, RNA

n Most bacteria get nitrogen from decomposing proteins

n Some use nitrates or ammonium

n A few bacteria use N₂ in nitrogen fixing (nitrogen fixing organisms)

Chemical Requirements

n Sulfur and Phosphorus

n In amino acids, thiamine and biotin

n Sulfur for proteins (enzymes) and vitamins

n Most bacteria decompose proteins

n Some bacteria use sulfate ion (SO₄^-2) or hydrogen sulfide (H₂S)

n Phosphorus

n Phosphorus for ATP, DNA, RNA, and membranes

n Phosphate ion (PO₄^-3) is source of phosphorus

n Potassium, magnesium, and calcium

n Other essential nutrients needed for enzymes

n Trace elements (micronutrients)

n Inorganic elements

n Required in small amounts (can be found in tap water)

n Usually as enzyme cofactors

n Organic growth factors

n Things that microorganisms are unable to synthesize

n Organic compounds obtained from environment

n Vitamins, amino acids, purines and pyrimidines

Chemical Requirements

n Oxygen

_n Variation in need to metabolize O₂

n Divided into several groups:

n Obligate aerobes

n Aerobic metabolism (requires O₂to make energy)

n Growth at 21% O₂

n Microaerophiles

n Aerobic metabolism (requires O₂ in small amounts for energy)

n Growth at reduced O₂ levels

n Facultative anaerobe (E. Coli)

n In presence of O₂ uses aerobic metabolism to make energy

n In absence of O₂ will ferment (less energy produced)

n Obligate anaerobe (Clostridium)

n Anaerobic metabolism or fermentation

_nNo O₂ metabolism and killed by O₂

n Aerotolerant

n Anaerobic metabolism or fermentation

_nNo O₂ metabolism, but tolerates O₂

Chemical Requirements

n Oxygen (O₂)

Thioglycollate media

Toxic Forms of Oxygen

n Products of O₂ metabolismàtoxic

n Singlet oxygen: O₂ boosted to a higher-energy state

n Superoxide free radicals: O₂^–

n Peroxide anion: O₂^2–

n Hydroxyl radical (OH·)

Toxic Forms of Oxygen

n Organisms that use aerobic metabolism must detoxify these products

_nCatalase enzyme

n Peroxidase enzyme

n Superoxide dismutase enzyme: detoxifies O₂^-and OH•

n Obligate anaerobes lack these enzymes

Prokaryotic Growth Media

n Nutrient media prepared for microbial growth

n Examples: Tryptic Soy broth or agar (TSB/TSA), nutrient broth or agar

n Contains nutrients prepared for microbial growth

n Energy source

n Essential macronutrients

n Essential micronutrients (trace elements)

n Growth factors

n Importance of sterility

n Sterile: No living microbes

n Inoculate using aseptic technique

n Inoculum: Introduction of microbes into medium

n Culture

n Microbes growing in/on culture medium

Agar vs Broth

n Agar

n Complex polysaccharide

n Used as solidifying agent for culture media in Petri plates, slants, and deeps

n Generally not metabolized by microbes

n Liquefies at 100°C

n Solidifies ~40°C

n Broth

Types of Growth Media

n Chemically defined

n Exact chemical composition is known

n Used to grow specific prokaryotes and determine growth properties

n Complex media

n Extracts and digests of yeasts, meats, or plants

n Nutrient broth and agar

n Used to grow a variety of prokaryotes

Culture Media

Selective Media

n Suppress unwanted microbes and encourage desired microbes.

n Contains components/conditions that suppress the growth of some prokaryotes and allow the growth of others

n Used to select for a specific group from a sample

n Ex. MacConkey media suppresses the growth of Gram + bacteria and stimulates the Growth of enteric bacteria

Selective Media

n Mannitol Salt Agar (MSA)

Differential Media

n Make it easy to distinguish colonies of different microbes.

n Has chemical components that differentiates specific types of prokaryotes

n Ex. Blood agar differentiates hemolytic organisms form non-hemolytic organisms

Enrichment Media

n Encourages growth of desired microbe

n Used to increase the numbers of specific kinds of bacteria from a sample to detectable levels

n Ex. Brain Heart Infusion enriches the growth of salt tolerant streptococci

n May also be selective and/or differential

Pure Culture

n Pure culture

n Come from a single cell or spore

Contains only one species or strain

n Streak plate method

n Used to isolate pure cultures

n Colony made of approximately 10⁶ cells

n Population of cells arising from a single cell or spore or from a group of attached cells

Colony is often called a colony forming unit (CFU)

Streak Plate

Microbial Growth

n Increase in number of cells rather than size

n Binary Fission

n DNA replication

n Double amount of structures

n Growth of new membrane and division

Binary Fission

Population Growth

n Growth rate= change in cell number

n A generation is the interval of two cells from one

n Generation time (doubling time)

n Time it takes to produce two new cells

n Varies greatly

n Norm= 1-3 hours

n Dependent on nutritional and genetic factors

n E. coli= 20 minutes to divide

n Exponential growth

Growth Cycle

n Lag phase: time it takes for cell to start growing once inoculated

n Take in nutrients, synthesize essential components, repair damage, adjust to new media/nutrients, adjust to new concentration of nutrients