pH optimum

Report
chapter 6:
measuring microbial growth
microbial growth requirements
binary fission & biotic potential
microbial population growth
direct methods: dilution & plating
The spread plate method
Inoculate plate
containing
solid medium.
100μl
Bacterial
dilution
Spread
inoculum over
surface evenly.
Colonies grow
only on surface
of medium.
direct methods: microscopic counts
indirect methods: most probable number
- control
+ control
pH 11
pH 10
1
pH 8
pH 7
pH 6
growth relative to control
indirect methods: spectrophotometry
pH optimum
0.8
0.6
0.4
0.2
0
growth requirements: temperature
growth requirements: temperature
food spoilage
growth requirements: osmotic pressure
osmoprotectants/compatible solutes
growth requirements: pH
All environments
• DNA/RNA PO4 buffer
• low pH causes tumbling
• membrane gathers H+/OH• ion circuits: Na+/H+ antiport
• surface proteins pH stable
Alkaline environments
• Na+/H+ antiport
• H+ motive force changes
Acidic environments
• not permeable to H+
living with oxygen: anti-oxygen enzymes
oxygen requirements
chemical growth requirements
biological macromolecule synthesis
media: meeting physical & chemical needs
MLS-1 growth optima
2.2
DSIC-II (NH4Cl) - 85 hours
2
DSIC-II (glutamine) - 30 hours
1.8
1.6
OD (600nm)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
24
48
72
96
120 144 168 192 216 240 264 288 312 336 360 384 408
time (minutes)
media
DSIC-II Chemically Defined Medium
EM-II Chemically Defined Medium
80g NaCl (sodium chloride)
70g NaCl
0.5g NH4Cl (ammonium chloride)
OR 14mM glutamine
0.8g NH4Cl
0.6g KH2PO4 (monopotassium phosphate)
0.8g KH2PO4
2.5g K2SO4 (potassium sulfate)
10g Na2SO4
0.1g Na2S2O35H2O (sodium thiosulfate)
0.8g Na2S9H2O (Sodium sulfide) dissolved in
10ml H2O, filter sterilized & added to cooled
media
1ml vitamin solution (10mg biotin, 35mg
nicotinamide, 30mg thiamine dichloride, 10mg
pyridoxyl chloride, 10mg Ca-panthenoate,
5mg vitamin B12, in 100ml dH2O)
No vitamin solution
1ml trace elements (5.2g EDTA, 190mg
CoCl22H2O, 0.1g MnCl24H2O, 1.5g
FeCl24H2O, 6mg H3BO3, 17mg CuCl22H2O,
188mg Na2MoO42H2O, 25mg NiCl26H2O,
70mg ZnCl2, 30mg VOSO42H2O, 2mg
Na2WO42H2O, 2mg NaHSeO3, in 1l dH2O)
1ml SLA trace elements (1.8g FeCl24H2O,;
250mg CoCl26H2O,; 10mg NiCl26H2O,;
10mg CuCl25H20,; 70mg MnCl24H2O,;
100mg ZnCl2,; 500mg H3BO3,; 30mg
Na2MoO42H2O; 10mg Na2SeO35H2O; 1l
dH2O)
0.2g MgSO47H2O (autoclaved separately)
0.1g MgCl26H2O (autoclaved separately)
0.2g CaCl22H2O (autoclaved separately)
50mg CaCl22H2O (autoclaved separately)
20g NaHCO3 (autoclaved separately)
20g NaHCO3 (autoclaved separately)
dH2O up to 1L
dH2O up to 1L, store anaerobically
special media types
differential & selective media
Chapter Six Learning Objectives
1.
How do most bacterial cells reproduce? Why do bacterial cells have tremendous biotic
potential?
2. Discuss what is happening to a bacterial culture during the fourth phases of population
growth. Are they growing, dividing, what is driving the change in population number?
http://professorcrista.com/files/animations/posted_animations/bacterial_growth_curve.html
http://professorcrista.com/files/animations/posted_animations/binary_fission.html
https://www.youtube.com/watch?v=lpI4WCM_9pM&feature=youtu.be (Go to 4:00)
3. Categorize, discuss the pros and cons and understand the mechanism of action for each
of the following methods of counting the number of bacterial cells in culture: serial
dilution and spread plating, microscopic counts, MPN, and spectrophotometry.
http://education.wichita.edu/saltymicro/ecology_interactives/serial_dilution.html
4. How do temperature, osmotic pressure, pH and oxygen changes affect the growth of a
bacterial culture? How are organisms classified according to their needs in regard to
these physical aspects of the environment? What adaptations might various bacterial
species have in order to combat less than desirable environments?
5. Six chemical growth requirements were discussed in lecture. What are these used for in
the bacterial cell? How does the availability of these affect the growth of a bacterial
culture?
6. How are differential and selective media useful in isolating, identifying and enriching for
particular bacterial species?
7. How do complex and chemically defined media differ? When is each useful for the
routine culture of bacterial cultures?
chapter seven:
control of microbial growth
the control of microbial growth
treatment effectivity
•
•
•
•
•
number of microbes/length of exposure
microbial characteristics
environment: moisture & temperature
organic matter
vegetations/biofilms
1. cells populate substrate 2. extracellular polymeric substances (EPS) produced & attaches
3 & 4. biofilm architecture develops and matures 5. cells are released from the biofilm
the benefits of a biofilm
protection from Abx, toxins and immune cells
tobramycin
ciprofloxacin
 planktonic cells
 biofilm cells
 biofilm colony
open symbols are untreated (control)
terminology
• decimal reduction time (DRT)- opposite of decimate
• thermal death point (TDP)
– lowest temperature when all cells killed in 10 min.
• thermal death time (TDT)
– time to kill all cells at given T
heat
•dry heat- oxidation
– flaming
– incineration
– hot-air sterilization
•moist heat- denaturization
– autoclave
– pasteurization
• not sterile
• 63°C for 30 min
• flash (UHST): 72°C for 15 sec
• ultra heat treatment (UHT):
>135°C for <1 sec
•moist vs. dry
– hot air: 170˚C, 2 hr
– autoclave: 121˚C, 15 min
filtration
• physical removal of organisms
• protects heat-labile components
ionizing & nonionizing radiation
ionizing radiation ionizes H2O  OH
OH damages DNA
nonionizing radiation damages DNA
chemical control: use-dilution test
useful for testing bactericidal properties
dip metal ring in bacteria
dry at 37°C
place ring in disinfectant
10 minutes @ 20°C
culture in broth
check survivability
chemical control: disk-diffusion method
filter paper is soaked
with disinfectant
paper placed on
“seeded” agar
plate incubated
examined for zone of inhibition
chemical microbial control
*
for study
Chapter Seven Learning Objectives
1.
Define sterilization, commercial sterilization, disinfection, antisepsis, degerming and
sanitization. Understand what is meant by
“-stat,” “-lytic” and “-cide”.
http://professorcrista.com/files/animations/posted_animations/chemotherapeutic_agents.html
2. How do the following affect the effectivity of a given microbial control agent: microbial
load, exposure length, microbial characteristics, moisture, temperature, organic matter
and vegetations/biofilms?
3. How is a biofilm produced? How are the microbes within it so protected from the
environment?
4. Why is moist heat better than dry heat at killing microbes?
5. Define thermal death point, thermal death time, and decimal reduction time.
6. When is filtration a useful method of controlling microbial growth?
7. How do ionizing and non-ionizing radiation work to control microbial growth?
8. What experimental method discussed in class is useful to determine the bactericidal
properties of a given chemical? Which would you use if you were only concerned with the
bacteriostatic properties?
9. How would you rate the various kinds of microorganisms in terms of their resistance to
the chemical control of microbial growth?
chapter eight:
microbial genetics
the hereditary material
Griffith 1927 & Avery, et al. 1944
the “transforming principle” coined by Griffith, identified by Avery
the hereditary material
Hershey Chase, 1952
the bacterial chromosome
plasmids
• F factor (conjugative plasmid)
– genes for sex pili and plasmid transfer
• dissimilation plasmids
– enzymes to catabolize unusual compounds
• R factors
– antibiotic resistance

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