Chapter 6 Flashcards
Facultative Anaerobes
growth with oxygen or with no oxygen
Plate counts
- Count colonies on plates that have 30-300 colonies (CFUs)
- To ensure the right number of colonies, the original inoculum must be diluted via serial dilution
- Counts are performed on baceria mixed into a dish with agar (pour plate method) or spread on the surface (spread plate method)
Physical Requirements for Growth
- Temperature
- pH
- Osomotic Pressure
Direct measurements count microbial _____.
cells
Bacterial Division
Increase the number of cells, not cell size. (Binary Fission)
Optimum growth temperature for Thermophiles
50-60 degrees C
Carbon
- structural backbone of organic molecules
- Chemoheterotrophs use organic molecules as energy
- Autotrophs use CO2 (think of photoautotrophs)
Microaerophiles
require oxygen concentration lower than air
A pure culture contains…
Only one species or strain
Dry weight
Bacteria are filtered, dried, and weighed; used for filamentous organisms
Streak plate method
used to isolate pure cultures
Colony
A population of cells arising from a single cell or spore or from a group of attached cells
CFU
A colony is often called a colony-forming unit (CFU)
Reducing media
- Used for the cultivation of anaerobic bacteria
- Contain chemicals (sodium thioglycate) that combine O2 to deplete it
- Heated to drive off O2
Aerotolerant anaerobes
tolerate but cannot use oxygen
Log phase
Logarithmic or exponential increase in population
Differential Media
- Allow distinguishing of colonies of different microbes on the same plate
- Some media have both selective and differential characteristics
Agar
- Complex polysaccharide
- Used as a solidifying agent for culture media in Petri plates, slants, and deeps
- Generally not metabolized by microbes
- Liquefies at 100 degrees Celsius
- Solidifies at ~40 degrees Celsius
Minimum growth temperature
BSL-3
Biosafety cabinets to prevent airborne transmission
BSL-2
Lab coat, gloves, eye protection
Optimum growth temperature for Hyperthermophiles
Optimum growth temperature >80 degrees C
Filtration
- Solution passed through a filter that collects bacteria
- Filter is transferred to a petri dish and grows as colonies on the surface
Biofilms
- Microbial communities
- Form slime or hydrogels that dhere to surfaces
- Bacteria communicate cell-to-cell via quorum sensing
- Share nutrients
- Shell bacteria from harmful environmental factors
- Found in digestive system and sewage treatment systems (can clog pipes)
- 10000x resistant to microbicides
- Involved in 70% of infections
- Catheters, heart valves, contact lenses, dentail caries
Biosafety levels
Requirements for handling certain cultures
Phosphorous
- Used in DNA, RNA, and ATP
- Found in membranes
- PO4^3- is a source of phosphorus
Lag phase
Intense activity preparing for population growth but no increase in population yet
Obligate anaerobes
unable to use oxygen and are harmed by it
Organic Growth Factors
- Organic compounds obtained from the environment
- Vitamins, amino acids, purines, and pyrimidines
Selective Media
- Suppress unwanted microbes and encourage desired microbes
- Contain inhibitors to suppress growth
Stationary phase
Period of equilibrium, microbial deaths balance production of new cells
BSL-4
- Sealed, negatibbe pressure; “hot zone”
- Exhaust air is filtered twice through HEPA filters
Maximum growth temperature
Mesophiles
Moderate-temperature-loving
What are the phases of growth?
- Lag phase
- Log phase
- Stationary phase
- Death phase
Thermophiles
Heat-loving organisms
Trace Elements
- Inorganic elements required in small amounts
- Usually as enzyme cofactors
- Include iron, copper, molybdenum, and zinc
Hypertonic environments (higher in solutes than inside the cell) cause ________ due to high osmotic pressure
plasmolysis
Methods of direct measurement
- Plate count
- Filtration
- Direct microscopic count
Optimum growth temperature
Culture medium
nutrients prepared for microbial growth
BSL-1
No special precautions; basic teaching labs
How much time is required for a cell to divide?
20 minutes to 24 hours
Direct Microscopic Count
- Volume of a bacterial suspension placed on a slide
- Average number of bacteria per viewing field is calculated
- Uses a special Petroff-Hausser cell counter
of bacteria/ml = (# cells counted)/(volume area counted)
Metabolic activity
Amount of metabolic product is proportional to the number of bacteria
Inoculum
introduction of microbes into a medium
Sterile
no living microbes
Culture
Microbes growing in or on a culture medium
Psychrophiles
Cold-loving organisms
Enrichment Cultures
Encourages the growth of a desired microbe by increasing very small numbers of a desired organism to detectable levels
Usually a liquid
Nitrogen
- Component of proteins, DNA, and ATP
- Most bacteria decompose protein material for the nitrogen source
- A few bacteria use N2 in nitrogen fixation
Binary fission ______ the number of cells each generation
doubles
Facultative halophiles tolerate ______ osmostic pressure
high
Obligate aerobes
require oxygen
Growth curves are represented _________
Logarithmically
Death phase
Population is decreasing at logarithmic rate
Complex media
Extracts and digests yeasts, meat, or plants; chemical composition varies batch to batch
Examples: nutrient broth, nutrient agar
Total number of cells =
2^number of generations
pH needs for growth
- bacteria
- molds and yeasts
- Most bacteria grow between 6.5-7.5 pH
- Molds and yeasts grow between 5-6 pH
Acidophiles
grow in acidic environments
Chemically defined media
- Exact chemical composition is known
- Fastidious organisms are those that require many growth factors provided in chemically defined media
Chemical Requirements
- Carbon
- Nitrogen, sulfur, and phosphorous
- Trace elements
- Oxygen
- Organic growth factors
Extreme or obligate halophiles require _____ osmotic pressure
high
Sulfur
- Used in amino acids, thiamine, and biotin
- Most bacteria decompose protein for the sulfur source
- Some bacteria use SO4^2- or H2S
Turbidity
Measurement of cloudiness with a spectrophotometer
What are the physical requirements of growth?
- Temperature
- pH
- Osmotic pressure
What are the chemical requirements for growth?
- Carbon
- Nitrogen, Sulfur, Phosphorous
- Organic growth factors
- Oygen
- Trace elements
Microorganisms are classified into what four temperature related categories? What is the preference of each?
- Psychophiles
- Cold-loving; grows best around 15ºC, but not above 20ºC
- Psychrotroph
- Cold-loving; can grow in temperatures from 0ºC to 30ºC
- Mesophiles
- moderate temperature loving; can grow between 10º-50º C
- Thermophiles
- high temperature loving; can grow between 50º-60º C
- Hyperthermophiles
- Survive in very high temperatures; >80ºC
What is the best pH for bacterial growth?
A neutral range between 6.5-7.5 pH
Organisms that can tolerate low pH are called _______.
acidophiles
When bacteria grow in the medium they produce ______ which can interfere with their own growth. _______ are added in the medium to neutralize the ______.
acid
Buffers
acidity
_____, _____, and _____ act as buffers.
Phosphate salts
Peptones
Amino acids
_____ osmotic pressure or a _______ environment can cause ______ in bacteria.
High
hypertonic
plasmolysis
Plasmolysis
loss of water from a cell in a hypertonic solution
Mold and yeasts can grow at pH ____.
5-6
Some organisms can grow well in high concentration of salt are called _________.
halophiles
Hypertonic environments or high osmotic pressure is used to _____ ______ such as salted fish, james and pickles.
preserve food
Carbon is required for ______ of _______ in cell.
synthesis
structures
Chemoautotrophs and photoautotrophs use ______ ______ for making sugars.
carbon dioxide
Protein synthesis needs ______ and _____.
nitrogen
sulfur
Chemoheterotrophs use ______ _____ to obtain sugars.
organic materials
DNA and RNA need _____ and ______.
nitrogen
phosphorous
Phosphorous is present in the ATP and the phospholipids of the ____ ______.
cell membrane
Trace elements like iron, copper, molybdenum and zinc are required in minute amount of _______ for _______.
cofactor
enzymes
What are the 4 differences between aerobes and anaerobes?
(not final–need to update)
- Anaerobes
- Do not use oxygen
- Makes lactic acid
- Makes ethanol
- Aerobes
- Uses oxygen
- Uses citric acid cycle
- Uses oxidative phosphorylation
What are biofilms?
- Microbial communities
- Form slime or hydrogels that adhere to surfaces
- Bacteria communicate cell-to-cell via quorum sensing
- Share nutrients
- Shelter bacteria from harmful environmental factors
- Found in digestive system and sewage treatment systems; can clog pipes
- 1000x resistance to microbicides
- Involved in 70% of infections
- Catheters, heart valves, contact lenses, dental caries
Inside biofilms, cell to cell communication is called _____ _____ which allows them to coordinate their activities.
quorum sensing
What are the benefits of growing inside the biofilm to the bacteria?
The biofilm could protect the microbes from environmental factors; these are not good for humans as they resist antiseptics
What is a culture medium and what is inoculum?
Culture medium: nutrients prepared for microbial growth
Inoculum: introduction of microbes into a medium
Why agar is used for preparing growth medium?
Agars have complex polysaccharides which provide nutrients, and are used as a solidifying agent for culture media in petri lates/slants/deeps.
Generally not metabolised by microbes.
(liquifies at 100º C and solidifies at 40º C)
Define chemically defined medium and complex medium.
Chemically defined medium: exact chemical composition is known
Complex medium: Extracts and digests yeasts, meats, or plants; chemical composition varies batch to batch (examples include nutrient broth and nutrient agar)
For the growth of anaerobic organism special media containing _____ ____ (e.g. sodium thioglycolate) is used. The reducing agent reacts with the pxygen and removes it.
reducing agent
BSL 1 precautions
Basic lab safety protocol, basic teaching labs
BSL 2 precautions
Lab coat, gloves, goggles
BSL 3 precautions
biosafety cabinets to prevent airborne transmission
BSL 4 precautions
sealed, negative pressure, “hot zone”
(exhaust air is filtered twice through HEPA filters)
How do you preserve bacteria culture? 2 ways
Deep-freezing (-50ºC - -95ºC)
Lyophilization (freeze-drying): frozen -54ºC to -72ºC) and dehydrated in a vacuum
Selective media
Suppress unwanted microbes and encourage desired microbes
Contain inhibitors to suppress growth
Which method is most common for bacterial growth?
Binary fission
Differential Media
Allow distinguishing colonies of different microbes on the same plate
Enrichment culture
Encourages the growth of a desired microbe by increasing very small numbers of a desired organism to detectable levels
Usually a liquid
The time required for the bacteria to divide is called a _________ ______.
generation time.
[Think of her example of logarithmic growth from generation 1 (1 cell) to generation 21 (~2 million)]
2number of generations
What are the four phases of growth?
Lag phase
Log phase
Stationary phase
Death phase
Lag phase
Different proteins and enzymes are being made during this time to prepare for the division/Log phase. The bacterial cells are adjusting to the environment around them.
Draw a diagram of microbial growth phases and describe each phase
What is the advantage of plate count?
It measures the number of viable cells. Each live bacterium makes a colony called Colony forming unit or CFU
< 30 we do not count
> 300 we do not count
You get aerobic mcirobes
What is serial dilution? Can you provide an example?
When the original inoculum is diluted to ensure the correct number of CFUs.
What are the direct methods of counting the microbial cells?
- Filtration
- Plate count
- Direct microscopic count
Filtration
Solution passed through afilter that collects bacteria
Filter is transferred to a Petri dish and grows as colonies on the surface
Direct Microscopic Count
Volume of bacterial suspension placed on a slide
Average numbber of bacteria per viewing field is calculated
Uses a special Petroff-Hausser cell counter
Niumber of bacter/ml:
Number of cells counted
divided by
Volume of area counted
What are two disadvantages to using direct microscopic count?
Might count dead cells
Microbes which are motile might be counted twice
What is used for indirect method of counting?
Turbidity: measurement of cloudiness with a spectrophotometer
Metabolic activity: amount of metabolic product is proportional to the number of bacteria
Dry weight: bacteria are filtered, dried, and weighed; used for filamentous organisms
Turbidity
Measurement of cloudiness with a spectrophotometer
Indirect method of counting microbes: metabolic activity
Amount of metabolic oroduct is proportional to the number of bacteria
Dry Weight
Bacteria are filtered, dried, and weighted; used for filamentous organisms
