I Physical requirements
A. Temperature *Know ranges
1. psychrophiles: an organism that grows best at about 15 C and does not grow at over 20 C.
a. A cold loving microbe.
b. psychrotrophs: an organism capable of growth between 0 and 30 C
1. moderate psychrotrophs
2. facultative psychrotrophs
3. mesophiles: an organism that grows between 10 and 50 C; 35
a. a moderate temperature loving microbe.
b. most common type of microbe
c. includes most common spoilage and disease organisms
4. thermophiles: an organism whose optimum growth temperature is between 50 and 60 C. 40 , 60, 70
a. A heat loving microbe.
b. create endospores
c. Important for compost piles
d. not considered a public health problem.
e. hyperthermophiles: an organism whose optimum growth temperature is at least 80 C. Also called an extreme thermophile. 65, 95, 110
ii. need sulfur
5. Extremophile: a microorganism that lives in environmental extremes of temperature, acidity, alkalinity, salinity or pressure.
6. Minimum growth temperature: The lowest temperature at which a species can grow.
8. Maximum growth temperature: The highest temperature at which a species can grow.
9. Optimum growth temperature: the temperature at which a species grows best.
1. most bacteria grow best near neutrality at 6.5 to 7.5 pH (Sushma 6.3 – 7.3)
2. acidophiles: a bacterium that grows below pH 4
3. buffer: peptones and amino acids which help maintain the proper pH in a growth medium. See page 162 last paragraph, left column.
C. Osmotic pressure
1. plasmolysis: loss of water from a cell in a hypertonic environment Shrinking of the cell.
a. best example: Staphylococcus aureus = halophile
2. Importance: the growth of the cell is inhibited as the plasma membrane pulls away from the cell wall. Thus, the addition of salts can be used to preserve food. The salt keeps the microbes from growing because of plasmolysis. The high salt or sugar concentrations draw water out of the cell and prevent their growth. Effects of osmotic pressure are roughly related to the number of dissolved molecules and ions in a volume of solution.
3. extreme halophiles (obligate halophiles): an organism that requires a high salt concentration for growth
a. add salt to the inoculating loop and medium if growing organisms from the ocean (halophiles)
4. facultative halophiles: a organism capable of growth in, but not requiring, salt. 2% to 15%
5. hypotonic solutions cause swelling of the cell or lysis
6. hypertonic solutions cause plasmolysis
II Chemical requirements
A. Carbon: besides water this is one of the most important microbial requirements for growth
1. structural backbone of living matter
2. needed for all organic compounds that make up a living cell
3. half the dry weight of a typical bacterial cell is carbon
4. don’t know if we need to know about chemo, photo trophs, etc.
1. nitrogen fixation: the conversion of nitrogen (N2) into ammonia.
a. Symbiosis: the living together of two different organisms or populations
2. DNA and RNA synthesis requires nitrogen
3. ATP requires nitrogen
4. used to form the amino group of the amino acid of proteins
E. Trace element: a chemical element required in small amounts for growth.
F. Oxygen see table 6.1 page 166
a. obligate aerobes: an organism that requires molecular oxygen to live. (grow at top of tube)
c. microaerophile: an organism that grows best in an environment with less molecular oxygen than is normally found in air.
d. Microbes that use molecular O2 produce more energy from nutrients than microbes that do not use O2.
a. facultative anaerobes: an organism that can live with or without molecular oxygen. (mostly at top of tube, and throughout)
i. use fermentation or anaerobic respiration when O2 not available.
ii. E. coli
b. obligate anaerobes: an organism that does not use molecular oxygen and is killed in its presence. (bottom of tube)
c. aerotolerant anaerobes: cannot use oxygen for growth, but they tolerate fairly well. (throughout tube evenly)
d. microaerophiles: aerobic (requires O2) only grow in oxygen concentrations lower than that in air. (middle of tube)
3. toxic forms of O2
a. singlet: highly reactive molecular oxygen
b. superoxide free radical: a toxic form of oxygen formed during aerobic respiration.
i. superoxide dismutase: an enzyme that destroys superoxide free radicals
c. hydroxyl radical: a toxic form of oxygen formed in cytoplasm by ionizing radiation and aerobic respiration.
d. peroxide anion
i. Catalase: an enzyme that catalyzes the breakdown of hydrogen peroxide to water and oxygen.
G. Organic growth factor: an essential organic compound that an organism is unable to synthesize.
1. must be obtained from the environment (ex: vitamins for humans)
III Culture Media
A. culture medium: the nutrient material prepared for growth of microorganisms in a laboratory.
1. Sterile: free of microorganisms
B. culture: microorganisms that grow and multiply in a container of culture medium.
C. inoculum: a culture medium in which microorganisms are implanted
D. agar: a complex polysaccharide derived from a marine alga and used as a solidifying agent in culture media.
E. chemically defined medium: a culture medium in which the exact chemical composition is known
1. fastidious: organisms that require many growth factors pg. 168
2. for growth of chemoautotrophs and photoautotrophs
F. Complex media: a culture medium in which the exact chemical composition is not known. Undefined.
1. nutrient broth: A complex medium made of beef extract and peptone. (can be yeast extract)
2. nutrient agar: Nutrient broth containing agar.
3. Major components for growth are carbon and nitrogen. Nitrogen for protein synthesis.
4. for growth of most chemoteterotrophic organisms
G. anaerobic growth media and methods (told to omit this)
1. Reducing: growth of obligate anaerobes
H. special culture techniques (omit)
I. Selective and differential media
1. selective: A culture medium designed to suppress the growth of unwanted microorganisms and encourage the growth of desired ones
a. media that targets only one group
b. Example: add salt for Staphylococcus or increase pH to 5 for fungi.
2. differential: A solid culture medium that makes it easier to distinguish colonies of the desired organism.
a. pH indicators are added to the media.
i. Yellow = acid, pink = base
ii. example: blood agar with alpha, beta, gamma, etc. Differentiated between 3 different Staphylococcus.
J. Enrichment culture (media): a culture medium used for preliminary isolation
that favors the growth of a particular microorganism.
1. similar to selective media but designed to increase numbers of desired microbes to detectable levels.
K. Obtaining pure cultures
1. streak plate method: (omit)
2. Colony: a visible mass of microbial cells arising from one cell or from a group of the same microbes.
IV Preserving bacterial cultures
A. deep freezing: preservation of bacterial cultures at 250 C to 295 C
B. lyophilization: freeze drying. -54 through -72C. water removed by vacuum
V Growth of Bacterial cultures 6.11, 6.12, 6.14
A. bacterial division
1. binary fission
a. cell elongates and dna is replicated
b. cell wall and plasma membrane begin to grow inward
c. cross-wall forms completely around divide dna
d. cells separate
2. budding: asexual reproduction
3. reproductive spores
B. Generation Time
1. generation (doubling) time: the time required for a cell population to divide and double in number.
a. E. coli takes 20 minutes to double in number
C. Phases of growth (Bacterial growth curve: a graph plotting the growth of cells over a period of time.)
1. lag phase: the time interval in bacterial growth curve during which there is no growth. (0-1 hr)
a. The microbial population is undergoing a period of intense metabolic activity.
2. log phase: the period of bacterial growth or logarithmic increase in cell numbers; also called exponential growth phase. (1-4 hr)
a. microorganisms are particularly sensitive during this time as medications can interfere with an important step in the growth process and are thereby harmful to cells during this phase.
b. chemostat: an apparatus that allows this phase to last indefinitely. (continuous culture)
3. Stationary phase: the period in a bacterial growth curve when the number of cells dividing equals the number dying. (4-5 hr)
a. Not dormant during this time, using up all of the nutrients.
b. once nutrients are gone, they start death phase
4. Death phase (logarithmic decline): the period of logarithmic decrease in a bacterial population. (5-10 hr)
D. Measurement of microbial growth
a. plate count: a method of determining the number of bacteria in a sample by counting the number of CFU’s on a solid culture medium.
i. serial dilution: the process of diluting a sample several times
ii. colony forming units (CFU) b/c one bacterium or a chain of bacteria may start a colony.
iii. disadvantages: takes 24 hours for results
iv. advantages: measures number of viable cells
v. Pour plate method: a method of inoculating a solid nutrient medium by mixing bacteria in the melted medium and pouring the medium into a Petri dish to solidify. Disadvantages: melted agar may kill mo’s. Colonies that form best on surfaces are not forming well.
vi. Spread plate method: a plate count method in which inoculum is spread over the surface of a solid culture medium.
b. filtration: 100 ml of water are passed through a thin membrane filter whose pores are too small to allow bacteria to pass. The bacteria remaining on the filter and the filter are transferred to a Petri dish containing a pad soaked in liquid nutrient medium, where colonies arise from the bacteria on the filter’s surface.
i. for E. coli and Klebsiella (coliform bacteria – fecal pollution)
ii. where bacteria quantity is very small such as relatively pure bodies of water
c. most probable number (MPN)
d. direct microscopic count: enumeration of cells by observation through a microscope.
i. disadvantages: cannot tell if alive or dead, must count at least 5 chambers, a high number of cells must be available in order to be counted (10 million per mL)
ii. advantages: no incubation time required
iii. grided slides
iv. uses microscope with a counting chamber.
v. must count at least five chambers
vi. used for milk
a. turbidity: the cloudiness of a suspension. Measure absorbance in a spectrophotometer. (colorimeter)
i. disadvantages: can’t use in the milk industry, can’t tell if alive or dead. More than 1 million cells per mL. Not a useful measure of contamination by liquids with a small amt of bacteria.
b. metabolic activity
c. dry weight
Minimal bactericidal concentration (MBC): The lowest concentration of chemotherapeutic agent that will kill test organisms.
Aerotolerant anaerobe: an organism that does not use molecular oxygen, but is not affected by its presence.
Peroxide anion: an oxygen anion consisting of two atoms of oxygen
Capnophile: an organism that grows best at relatively high CO2 concentrations
Minimal inhibitory concentration (MIC): the lowest concentration of a chemotherapeutic agent that will prevent growth of the test organisms.
Most probable number method (MPN): a statistical determination of the number of coliforms per 100 ml of water or 100 g of food.
Note on refrigeration: preservation of food based on the principle that microbial reproductive rates decrease at low temperatures. Psychrotrophs do not grow well in low temps, but over time they are slowly able to degrade food. A fridge will greatly slow the growth of most spoilage organisms and will entirely prevent the growth (not kill) of all but a few pathogenic bacteria.
Facultative: capable of growth in, but not requiring
Chemoautotrophs: an organism that uses an inorganic chemical as an energy source and CO2 as a carbon source
Photoautotrophs: an organism that uses light as its energy source and CO2 as its carbon source
Chemoheterotrophic: an organism that uses organic molecules as a source of carbon and energy.
autotroph: cell feeders. Carbon is the source. CO2 the end product. Sole carbon source.
heterotroph: saprophytes. Sources other than CO2. sodium citrate.