Microbial Growth & Methods of Measuring Growth Flashcards
List
Kinds of Cell Division
3
- Binary fission
- Budding
- Biofilm
List
Examples of fast growers & their generation times
3
- E. coli (15-18 mins)
- S. aureus (27-30 mins)
- Bacillus subtilis (26 mins)
List
Examples of slow growers & their generation times
2
- Leptospira (6-8 hr)
- Mycobacterium (18-24 hr)
Differentiate
Closed Culture System vs Open Culture System
Closed
* fixed amount of nutrient at the start
* leave for 18-24 hours
* no way to get rid of excess waste
Open
* keep adding fresh medium
* remove waste/excess
* mix for aeration
Identify
What is used to detect turbidity?
spectrophotometer
Define
viable count
Bonus: Where can you find it?
- count of live cells
- found in plates w/ media
List
Growth Phases
4
- Lag Phase
- Logarithmic/Exponential Phase
- Stationary Phase
- Death Phase
Identify
Plots different stages of (bacterial) life
Bacterial Growth Curve
phase with metabolic activity, but no growth
Lag Phase
List
What is the duration of the Lag Phase dependent on?
2
- Nutrients available
- Species
- phase where there is a lot of growth
- ↑ cell population, ↑ rate
- generation time of organism can be determined
Logarithmic/Exponential Phase
Fill in the blank
To stay in the Logarithmic Phase, use an ____ culture system. This system is also called ____.
open, chemostat
Main components of chemostat
3
- inflow of new medium & air
- mixing of medium (aeration)
- outflow of waste
Factors that govern cell density
2
- Dilution rate
- Concentration of limiting nutrient
Why would you want to stay in the Logarithmic Phase?
For extraction purposes; you keep producing more cells & can extract more in a shorter amt of time
Phase where
- Replication Rate = Death Rate
- Limited Nutrients
- ↑ Microbial waste
Stationary Phase
During the stationary phase, the environment becomes ____, with a [higher/lower] pH and a [higher/lower] oxygen level.
toxic, higher, lower
Phase with
- little to no nutrients
- ↑ toxic waste
- Endospores form
Death Phase
What happens to cells during the death phase?
Why do they do that?
Involution: they change shape
- ex. rod → spherical
- to limit cell’s nutrient use
Why do endospores form during the Death Phase?
Endospores are how cells defend themselves in a toxic environment.
Identify
- process that helps reduce bacterial load
- series of tubes with a diluent
Serial Dilution
List
Methods of Measuring Bacterial Growth
7
- Serial Dilutions
- Viable Cell Count
- Direct Cell Count
- Most Probable Number Technique
- Most Probable Count
- Filtration
- Spectophotomoetry
Why do we do serial dilation?
If you don’t, your sample might have too many colonies –> won’t be visible when trying to count them
Kinds of diluent
- Normal Saline Solution (NSS): 0.85% saline solution
- Phosphate Buffer Solution (PBS)
What happens when using a diluent & how do you use it?
- Use it within 10-15 mins
- Non-nutritious: cells don’t grow, but they also don’t die
Explain
Serial Dilution Process
- E. coli (1 mL) in Test Tube 1 (9 mL diluent)
- Vortex (mix)
- Transfer 1 mL TT1 in TT 2 with 9 mL diluent
- Vortex
- Transfer 1 mL TT 2 in TT 2 with 9 mL diluent
- Repeat until 30-300 colonies
- Plate counting
Why should the colony count be 30-300 when plate counting?
What do you declare when the colony count is outside that range?
[< 30] - TFTC (Too Few to Count)
- statistically insignificant
[> 300] - TNTC (Too Numerous to Count)
- won’t see individual colonies, too many layers
Fill in the Blank
The Most Probable Number Technique is used for samples with a [high/low] amount of bacteria
low
Identify
when a microorganism thrives in environments with small amounts of oxygen
microaerophilic
Differentiate
Spread-plate method vs Pour-plate method
Spread-Plate
- sample is spread on agar → surface colonies
Pour-plate
* sample is added first., then medium is added & mixed → colonies grow in agar & on top of agar
where sample is placed for direct cell count
Petroff-Hauser counting chamber / haemocytometer
Explain
The Great Plate Count Anomaly
- no. from direct cell counts > no. from viable plate counts
- Direct cell count: counts both live and dead cells
- Viable plate count: “forgets” non-culturable cells (impossible for all kinds of cells to grow in 1 specific condition)
What are solutions to the Great Plate Count Anomaly?
- Use more more selective/general medium (to target less organisms)
- Vary culture conditions
- Alter experiment
Technique usually used on water (bottled water), food, air samples
Most Probable Number Technique
What are the bases for positive reults in MPNT?
2
- turbidity
- gas production
Describe the setup used in the Most Probable Number Technique.
1) x sets of test tubes, with x tubes per set
- Set 1: 10 mL of sample
- Set 2: 1 mL of sample
- Set 3: 0.1 mL of sample
2) Incubate tubes (37ºC, 24 hr)
3) Compare the number of positive test tubes with what is on MPN table
T/F [Bonus: If false, what is wrong?]
MPN Technique is usually part of a bigger experiment.
T
What are phases of the “bigger experiment” that uses the MPN Technique? Why do we do each phase?
- Presumptive Phase - to observe gas production
- Confirmatory Phase - to confirm whether or not the colonies are fecal and/or harmful
- Completed Phase - identifies actual organism
What other item is used in the presumptive phase, and why do we use it?
Durham tube
- hard to observe gas production without it
- a bubble can be found inside it
Incubation Conditions for Presumptive phase & Confirmatory Phase
Presumptive: 37℃ for 24 hours
Confirmatory: 45 ℃ for 24 hours
T/F
Getting a negative result in the Presumptive Phase requires you to proceed to the Confirmatory Phase
F
you are not required
How long does the Confirmatory Phase take?
What kind of results do you need to proceed to Completed Phase?
2 weeks
positive tubes (in terms of turbidity)
What broth/agar is used in each phase of the MPN Technique?
- Lauryl Sulfate Tryptone Broth (LSTB) - Presumptive phase
- Brilliant Green Lactose Bile Broth (BGLB) - Confirmatory Phase
- Eosine Methyl Blue (EMB) Agar - Completed Phase
What is the size of the filter in a membrane filter? Why do we use that size
0.5 micrometers, so that the target organisms pass through the filter
Explain
Filtration Method
- Use membrane filter
- Place filter on agar
- Leave for 24 hours
measures amount of light unscattered aka light absorbed
Optical Density (OD)
Fill in the blank
[↑/↓] light absorbed = [↑/↓] OD
Spectrophotometry
↑ light absorbed = ↑ OD
[↑/↓] turbidity = [↑/↓] OD
↑ turbidity = ↑ OD
What are the advantages & disadvantages of Spectrophotometry?
Advantage: fast (~10 mins)
Disadvantage: counts dead cells
