Transcript Microbial Methodology

Microbial Methodology
by
E. Börje Lindström
This learning object has been funded by the European Commissions FP6 BioMinE project
What characterize Microbiology?
• The art of the objects
-very small (microscopic size)
• The characteristics
of the objects:
- rapid growth
- asexual growth
- clone
• Methodology:
- indirect
- statistical
- cultivation
- aseptic/ sterile
Cultivation
Nutrition varies:
Main nutrients:
- amount and chemical form important
• energy
• carbon
• nitrogen
• sulphur
m.o.
• phosphor
• hydrogen
• oxygen
The nutrient requirement = k/ the biosynthetic capacity
Classification
By energy source:
• phototropic
• chemotropic
By carbon source:
• autotrophy
• heterotrophic
Growth factors
Three main classes:
• amino acids
- building blocks e.g. for proteins
• purins/ pyrimidines
- building blocks e.g. for nucleic acids
• vitamins:
- building blocks for co-enzymes
Environmental conditions
• pH
-every bacterium has a rather narrow
pH range for optimal growth
acidophiles
neutrophiles
alkalogens
pH
0
• molecular oxygen (O2)
7
14
- strict aerobes
- strict anaerobes
- facultative
• light
- fototrophs
Environmental conditions, cont.
• temperature
psycrophiles
moderate
thermophiles
mesophiles
extreme
thermophiles
Temp, C
0
50
100
Different growth media
Synthetic
-all components are chemically known
Complex
- some components are unknown
Minimal
- a synthetic medium with the fewest nutrients which allows growth
Selective
- contains a substance which do not allow growth of some m.o.
Enriched
- contains nutrients which is not needed but increase growth
Differentiated
- contains a substance that can cause differences in the colony
morphology
Solidified
- a nutrient solution can be solidified by adding e.g. agar, gelatine etc.
Some examples of complex
media
• Beef extract:
- a water solution of carbohydrates, organic
N, vitamins and metal salts
• Peptones:
- hydrolysis (acid or enzymatic) of proteinacious
material, containing organic N
• Yeast extract:
- water extract of yeast cells, containing e.g. B-vitamins
Preparation of a growth medium
• Use stock solutions
• Add the ingredients according to the recipe
• Add the ingredients during stirring
• adjust the pH before autoclaving
Addition of nutrients
stirring
Sterilization
Definitions:
• Sterilization – killing of all m.o.
• Disinfections – reducing m.o.
Kinetics of the killing
• almost always exponential
Let N = amount of bacteria
t = time
dN
 kN
dt
dN
 kdt
N
-the change of bacteria can then be
written
-reorganize
-integrate
Sterilization, cont
N
t
dN
N0 dt  0 kdt
-gives
log N – log N0= -kt
-which can be plotted as
follows
logN
logN
slope = -k = killing rate
constant
N0
N0
t
One-hit curve
t
Multiple- hit curve
Factors that effects the killing
• dose (effects the slope)
-temperature (heat)
- concentration (concentration)
- intensity (irradiation)
• time
• the starting population (N0)
• the growth state of the bacteria
Methods for sterilization
• Physical:
- heat
- irradiation
- ultra sound
- micro waves
- filtration
• Chemical:
- varying compounds
Heat
- most important and used for everything that can
resist heat
• pasteurisation
- a mild method; 71-72 °C –15 sec
• boiling
- not safe
• autoclaving, wet
- 121 °C – 15 min
• owen, dry
- 160 – 180 °C – 2 hr
Irradiation
• UV
- bactericidal at 240-280 nm
- target DNA
- air sterilization
- low penetration
• X-ray
- ionisation
• g- beams
- ionisation
- using membrane filters of different
poor sizes
Filtration
• < 0,45 mm for bacteria
• < 0,01 mm for viruses
Chemical
• Phenols
- dissolve fat; denature proteins
•Alcohols
- dissolve fat; denature proteins
• Jodopax
- I2 + KI + ethanol + detergent
• Ethylenoxid
- alkylating