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Vibrio natriegens Growth Curve

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by

Andrew Barker

on 28 October 2013

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Transcript of Vibrio natriegens Growth Curve

Growth Curve
Vibrio natriegens Growth Curve
Characteristics
Gram-negative
unicellular
halophile (2% minimum)
marine bacterium

Vibrio natriegens
References
Class Notes
http://www.tgw1916.net/Vibrio/natriegens.html
http://www.tandfonline.com/doi/abs/10.1080/00219266.1973.9653881#preview
Sullivan, Karen. General Microbiology Laboratory Manual. 7th ed. Plymouth, MI: Hayden-McNeil, 2010.

Media
13.8 g nutrient agar powder
9 g NaCl
600 mL water
6.8 + 0.2 pH

2% NaCl
2.5 g dehydrated medium
100mL water
Heart Infusion Broth
Dilution Blanks
13 Dilution Blanks
1500 mL 0.85% NaCl
Dilutions
Spread Plate
Environment
Estuarine muds
Ex: salt marsh mud and coastal seawater
Generation time (doubling)
Under optimal conditions <10mins
Suitable for lab work b/c non-pathogenic and grows rapidly
short lag phase
Has the lowest mean generation time recorded (9-8 mins, Eagon, 1961)
Background
25 SNA Plates
Viable Plate Count
Results
Plate Time (mins) ABS (OD) Colonies Dilution Factor cfu/mL
1 75 0.031 36 10^-5 3.60E+06
2 95 0.096 92 10^-5 9.20E+06
3 115 0.135 4 10^-6 4.00E+06
4 135 0.241 2 10^-7 2.00E+07
5 155 0.516 32 10^-7 3.20E+08
6 175 1.03 10 10^-7 1.00E+08
7 195 1.999+ 55 10^-7 5.50E+08
8 215 1.999+ 117 10^-7 1.17E+09
Generation Time
Time required for cell to divide or for population to double
Number of cells/mL vs time on semi-log paper
Average Generation times:
Bacteria: 1-3 hours
Ecoli: 20 minutes
Vibrio natriegens: 8-10 mins
Procedure
8:30 AM: Innoculated HI +NaCl w/ 1 mL of bacteria
Shaking incubator 37 degrees C
1st OD reading at 9:45 AM
Every 20 minutes:
OD reading
Remove sample from flask
Make serial dilutions
Inoculate SNA plates
Spectrophotometer measures optical density
Blank first!
Optical Density (OD):
amount of light able to pass through liquid culture
used to determine amount of bacterial cells in a liquid culture

Turbidity
100 microliters onto SNA plate
Incubate inverted plates 37 degrees C
pH: start: 7.4,end: 7.0
30-300 colonies
cfu=colony forming units
determine cfu/mL for each time point
new enzymes
Abs (OD) vs. Time
Genertion time: 0.4 x 60 mins = 24 mins

cfu/mL vs. Time
Generation time: 0.25 x 60 mins = 15 mins
Phases of Growth
Lag phase
new enzymes
Log Phase/Exponential Phase
desired for lab work and end products
Stationary Phase
nutrients become limiting or waste products become toxic
death rate=division rate
Death Phase
nutrients depleted or toxic waste build up
death exceeds division

Class Gen Times (cfu/mL vs. time)
12
15
17
15
18
15
20
20
6
12
Average = 15
Absorbance vs. cfu/mL on regular graph paper
Estimate number of viable bacteria present at certain absorbance
Standard Curve
cfu/mL Generation time
Full transcript