MB 351 - Lecture 16 Flashcards
Two classes of culture media
Chemically defined, and complex
Chemically defined media
The exact composition of pure chemicals used to formulate the medium is known. -used extensively for well characterized isolates in lab studies (chemoheterotrophs like E.coli) -media must contain organic growth factors, such as glucose, that serves as a source of carbon and energy
Complex media “rich” media
Composed of a mixture of proteins and extracts in which the exact chemical composition is poorly defined, often made of natural source components- red blood cells, yeast extracts, peptone, Casein, etc. Used as a non-specific medium for growing many organisms
When are complex media used?
Complex media usually provide the full range of growth factors that may be required by an organism so they may be more handily used to cultivate unknown bacteria or bacteria whose nutritional requirements are complex (organisms that require a lot of growth factors, known or unknown)
A nutrient material prepared for the growth of microbes in a lab
culture medium
What’s in nutrient broth/agar?
Broth (liquid), agar (solid) -Glucose, peptone (animal, milk, or meat), beef extract, yeast extract, sodium chloride, agar -pH 7.0 to 0.2 -storage 2-8 degC Beef extract added to compliment peptone to add minerals, phosphates, energy sources, things missing from peptone. Yeast provides vitamins an other organic growth factors – rich in vitamin B, also supply organic N and C.
Complex media are easier to prepare than ________
chemically defined media
Three types of culture media used to detect the presence of specific microbes
selective, enriched, and differential media
Selective media
Inhibits growth of unwanted organisms, supports growth of desired organisms, and thereby ‘selects’ for it. -ex. Brilliant green agar isolates gram negative, and prevents growth of gram positive
Enrichment Media
-Usually a complex medium base to which additional nutrients such as serum or whole blood are added. -Contains some important growth factors (vitamin, amino acid, carbon source) needed for the growth of fastidious organisms. -These added nutrients better mimic conditions in the host and are required for successful lab culture of some human pathogens like Streptococcus pyogenes. -ex. blood agar, chocolate agar -the medium for an enrichment culture provides nutrients and environmental conditions that favor the growth of a particular microbe, but not others. In that sense it is also a selective media.
Differential media
-Allows for the separation of organisms by some observable change in appearance of the media. -ex. blood agar -Media that supports the growth of many different organisms, but differentiates between them. -Media will allow identification of microbes based on their growth, color, and appearance on the medium
Sometimes selective and differential media are combined in a single medium ex?
MacConkey agar -bile salts and crystal violet inhibit Gram positive bacteria (selective) and allow growth of gram negative -lactose fermenters produce color change (differential)
Mannitol Salt Agar
????
puncturing suitable medium such as nutrient agar with an inoculating needle that goes deep into the agar, allows growth within the semi-solid media
Stab (deep) culture - long term storage
Inoculating devices
needle or loop
Also suitable for longer term maintenance of bacteria. Agar media is poured into test tubes for and allowed to solidify in an angled position, so that media has a ______ to allow for greater surface area and inoculation on this slanted surface.
Slant cultures
Microbes grow on the surface of an agar _____ . Helps to show a colony, which is a visible mass of microbial cells arising from one cell or from a group of the same microbes.
Plate culture
Use broths for specific assays or for bacteria that will not form colonies on a solid surface. There are two types of broths:
batch cultures and continuous chemostat cultures
Batch cultures
microbes are cultured in an enclosed vessel (tube, flask) with a broth medium that supplies its nutritional requirements
Continuous Chemostat culture
This culture overcomes the limitations of a batch culture. In a chemostat, the growth chamber is connected to a reservoir of sterile medium. Once growth is initiated, fresh medium is continuously supplied from the reservoir.
Chemostat culture helps to prevent this phase…
The stationary phase is often due to a growth-limiting factor such as the depletion of an essential nutrient, and/or the formation of an inhibitory product such as an organic acid. Stationary phase results from a situation in which growth rate and death rate are equal.
Advantage of using chemostat culture is that cell population can be maintained in the ________ phase for days or even weeks.
exponential growth phase
Shaker is used to? For what types of microbes?
Used to supply oxygen to a aerobe
Steps for growing bugs aerobically
- Make culture media -carbon/energy source -nitrogen source -micronutrients/growth factors -buffer -water (add agar for solid medium) 2. Sterilize medium 3. Inoculate liquid or streak plate 4. Wait until culture grows
Anaerobic Culturing: two ways
GasPak jars, and anaerobic chambers (also called a glove box)
GasPak jars - small cultivation
inoculated plates or tubes are placed inside the jar, and anaerobic conditions are created by adding water to a gas generator envelope placed in the jar just before sealing.
Anaerobic chambers - large cultivation
plastic chamber that contains an atmosphere of H2, CO2, and N2. Culture media are placed within the chamber by means of an airlock which can be evacuated and refilled with N2.
inoculated plates or tubes are placed inside the jar, and anaerobic conditions are created by adding water to a gas generator envelope placed in the jar just before sealing.
GasPak jars - small cultivation
plastic chamber that contains an atmosphere of H2, CO2, and N2. Culture media are placed within the chamber by means of an airlock which can be evacuated and refilled with N2.
Anaerobic chambers - large cultivation
Reducing Media
for anaerobic cultures: during preparating of the culture media, it is boiled for severel minutes to removed dissolved oxygen, and a reducing agent (cysteine) is added to further lower the oxygen content. Oxygen free N2 is bubbled through to keep it anaerobic.
