Module 2-week 6 Bacterial Growth And Replication Flashcards
Aside from colony counting, how else can bacteria be quantified?
- Spectrophotometer
- Total Cell Count
- Viable-Count Serial Dilutions
Describe Spectrophotometer as a bacterial quantification method
Spectrophotometer measures CELL DENSITY:
- Record optical density(OD) or Absorbance(A) units
- As population increases, turbidity (density) increases
- Beneficial to monitor growth without disturbing the culture
- Inaccurate in lab and death phase for viable cells
Describe total cell count as a bacterial quantification
Total cell count: microscopic observation
- Beneficial to observe populations in nature rapidly
- Does NOT distinguish between DEAD and LIVE CELLS
Calibrated slide: Petroff-hauler counter
Hemocytometer: RBCs count
- Aliquot of culture under cover slip
- Depth known
- Calculate: Number of cells/unit volume
What are the Viable count Serial Dillutions as a bacterial quantification methods?
- More time and labor intensive
- More prone to pipetting and plating errors
What are the benefits Viable Serial quantification as a bacterial quantification method?
- More accurate for viable bacteria than OD or microscopic counts at high and low concentrations
- Important for testing growth rates and curves, especially in response to growth factors and antimicrobial
Briefly describe Viable count Serial Dilutions as a bacterial quantification method
This method is based on Colony Forming units( CFU)
One bacteria lands on a media plate and replicates to a visible “colony”, which can be counted
What are the drawbacks of Viable Count Serial Dilutions as a bacterial quantification?
Drawbacks
- More time and labor intensive
- More prone to pipetting and plating errors
Give the steps of bacterial quantification
Step 1: Conduct serial dilutions of suspension of bacteria (addition of aliquot to a sterile solution)
If 1 ml of sample added to 9ml of sterile solution
Dilution= 1:10= 10^-1
If 1ml was added to 99 ml
Dilution = 1:100= 10^-2
Step 2: Plate onto growth media (spread or pour)
Describe the spread method of bacterial quantification?
Spread: drop of suspension (known volume ) placed onto center agar plate and spread over surface(sterile glass rod)
- Sample is pipetted onto surface of agar plate (0.1 ml or less)
- Sample is spread evenly over surface of agar using sterile glass spreader
- Incubation
Describe the pour plate method of bacterial quantification
- Sample is pipetted into sterile plate
- Sterile medium is added and mixed well with inoculum
Generation times and growth rates are ___________, dependent on the _________ ________that are present in the environment
Variable
Growth factors
What extremophiles occur in the Dead Sea?
Halophiles
What extremophiles occur in yellow stone hot spring?
Thermophiles
Describe the 3 cardinal temperatures
Minimum -lowest temp. Permits growth+metabolism
Maximum - highest temp. Permits growth + metabolism
Optimum- small range that promotes fastest growth and metabolism rates
Factors: temperature
E. coli at 30 degrees Celsius g time = 60 mins
Optimum at 37 degrees Celsius g time = 30 mins
What are psychrophiles and psychotropics ?
Growth best at cold temperatures below 25 degrees Celsius
Relevant to food pathogens such as milk
What are mesophiles?
Grow best at human body temp. around 37 degrees Celsius
Most human pathogens are in this range
What are thermophiles and hyper thermophiles?
Grow best at high temps over 65 degrees Celsius
Associated with thermal vents
What are neutrophiles?
Most human pathogens live between pH 6-8
What are acidophiles?
Growth at low pH
-ex heliobacter pylori in stomach (pH 2)
What are alkaliphiles?
High pH due to high levels of minerals ( pH 9-11)
What osmotic pressure?
Osmotic pressure is the pressure that is exerted on a cell membrane by solutions both inside and outside the cell
What is a hypertonic solution?
When the concentration of solutes in the external environment of a cell is greater than that of solutes inside the cell., the solution in which the cell is suspended is said to be hypertonic ex. Salt water
What is a hypotonic solution?
When the concentration of solutes outside a cell is less than that of solutes inside a cell, the solution in which the cell is suspended is said to be hypotonic
What are halophiles?
Can grow in high NaCl concentrations
-associated with sea water microbes, like vibrio cholerae
Describe oxygen as a chemical factor affecting microbial growth
- Oxygen plays very important role in Microbial Growth
- terminal electron acceptor in respiration (ETC aerobic)
- limited solubility in water and tissue(can be limiting factor)
- Serves as an important regulatory signal for virulence factors
What is needed foe Oxygen survival ?
Special enzymes are required for oxygen survival
-reduce oxygen to water and non-toxic products
What is produced when oxygen is used for energy?
When oxygen is used for generation of energy, 2 toxic Reactive Oxygen Species ROSare generated :
Superoxide radical (diatomic molecule) O2 -
Hydrogen peroxide H2O2
How do bacteria deal with toxic radicals?
Bacteria that utilize O2 usually produce the following 2 enzymes:
Superoxide dismutase SOD*
2O2 - + 2H+ —> H2O2+ O2
Catalase:
2 H2O2 —> 2 H2O - O2
What is one clinical application for the use of catalase?
By adding hydrogen peroxide to clinical samples, catalase serves as basis for classification for Staphylococcus
What are obligate anaerobes?
O2 necessary, use only aerobic respiration
-Die in absence of oxygen
What are obligate anaerobes ?
- Cannot use Oxygen, die when exposed to oxygen
- Lack SOD and catalase!
- Special consideration for sample collection
What are facultative anaerobes?
- Can use both aerobic respiration(using O2 ) and fermentation /anaerobic respiration (without O2)
- Grow better in aerobic
- Many human pathogens
What are aerotolerant microorganisms?
-Can grow in the presence or absence of Oxygen
What are microaerophiles?
Require lesser amounts of Oxygen - 5% -10%
Describe the SOD and Catalase presence in aerotolerant microorganisms and cultures
Aerotolerant anaerobes
Positive for SOD but negative for Catalase
Cultures evenly dispersed throughout test tube as oxygen concentration is inconsequential to them
Describe presence of cultures, SOD and catalase in a test tube of obligate anaerobes
Cultures stay at the top where oxygen concentration is highest
SOD and catalase both present
Describe the concentration of oxygen throughout a test tube of bacterial cultures
Oxygen concentration highest at the top and decreases as you descend the test tube
Describe the presence of cultures, SOD and catalase in a test tubes facultative anaerobes
Factultative anaerobes mostly at the top, some are dispersed throughout
SOD and Catalase are both present
Describe the presence of cultures , SOD and Catalase for obligate anaerobes
Obligate anaerobes are concentrated at the bottom of the test tube
Negative for both SOD and catalase
Describe the presence of cultures for microaerophiles SOD and catalase
Positive for SOD and can be positive or negative for catalase
Cultures concentrated NEAR the top but not quite to the top
