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• Exercise 9: Aseptic Technique
• Exercise 10: Pure Culture Technique
• Turn in cultures from home for incubation• Finish microscope worksheet if necessary
Lab 2 Goals and Objectives :
Lecture: Chapter 6 (Microbial Growth)Learn aseptic technique and pure culture isolation
• Microbiology is the study of life and organisms that are too small to be seen with the naked eye.
• Microbial growth - an increase in the number of cells in a population , not increase in size.
1. Culture Medium: Nutrients prepared for microbial growth
•Sterile: No living microbes
2. Inoculation: Introduction of microbes into medium
Inoculum: The material used in an inoculation
3. Culture: Microbes growing in/on culture medium
Growing microorganisms
• Nutrient broth- liquid form • Nutrient agar- solid form
– Agar - complex polysaccharide
• Used as solidifying agent for culture media in Petri plates, slants, and deeps
• Generally not metabolized by microbes
• Liquefies at 100°C
• Solidifies ~40°C
Culture Media
Bacterial growth in culture• Bacteria multiply by binary fission• The population grows in geometric progression• 12• 2 4• 3 8• 4 16• 5 32• 6 64• 7 128• 8 256• 9 518• 10 1036• 11 2064• 12 4128• 13 8256• ………
-Lag phase: initial period of little to no cell division as bacteriaacclimate to new media-Log phase: period of exponential growth with a constant generationtime-Stationary phase: cell growth is equal to cell death-Death phase: cell death exceeds cell growth
Generation time
• Generation time - the time required for a cell to divide, to undergo one round of binary fission– If 100 cells growing for 5 hours produced 1,720,320 cells:
• E. coli has a generation time of 20 min• Common bacterial generation times range 1-3 hrs
Quantifying Microbial Growth
• Direct Measurements– Plate Counts– Filtration– Most Probable Number (MPN)– Direct Microscopic Count
• Indirect Estimations– Turbidity– Metabolic Activity– Dry Weight
Microbial Growth• The requirements for microbial growth
• Physical – Temperature– pH– Osmotic pressure
• Chemical• Oxygen• Carbon• Nitrogen• Sulfur• Phosphorus• Trace elements• Organic factors
Physical Requirements -Temperature
– Minimum growth temperature • the lowest temperature at which the species will grow
– Optimum growth temperature• species grow best
– Maximum growth temperature• the highest temperature at which growth is possible.
Physical Requirements - pH
• Most bacteria grow between pH 6.5 and 7.5
• Molds and yeasts grow between pH 5 and 6
• Acidophiles grow in acidic environments
Physical Requirements - Osmotic Pressure• Osmotic pressure is the hydrostatic pressure produced by a difference in
concentration between solutions on the two sides of a surface such as a semipermeable membrane.
• An isotonic environment for a cell is created when the solution outside of the cell is isotonic (having equal accent )with the cytoplasm of the cell.
• Hypotonic environment - the solution outside of the cell is hypotonic in comparison to the cytoplasm of the cell. – cause cell lyses for some cells and nothing to bacteria, because of cell wall .
• Hypertonic environments - hypertonic solution has more dissolved solute than the cytoplasm of the cell ( increase salt or sugar) – cause plasmolysis ( shrink the cells )
– Facultative halophiles tolerate high osmotic pressure – Extreme or obligate halophiles require high osmotic pressure
Figure 4.18 - Overview
• Carbon– Structural organic molecules, energy source– Autotrophs use CO2
– Chemoheterotrophs use organic carbon sources• Nitrogen
– In amino acids, proteins– Most bacteria decompose proteins– Some bacteria use NH4
+ or NO3
– A few bacteria use N2 in nitrogen fixation• Sulfur
– In amino acids, thiamine, biotin– Most bacteria decompose proteins– Some bacteria use SO4
2 or H2S• Phosphorus
– In DNA, RNA, ATP, and membranes– PO4
3 is a source of phosphorus• Inorganic elements required in small amounts- potassium, magnesium and
calcium• Trace Elements - iron, copper, molybdenum and zinc - Usually as enzyme
cofactors
Chemical Requirements
• Organic Growth Factors
– Essential organic compounds an organism is unable to synthesize
– Organic compounds obtained from the environment
– Vitamins, amino acids, purines, pyrimidines
Chemical Requirements
• Singlet oxygen: O2 boosted to a higher-energy state• Superoxide free radicals: O2
• Peroxide anion: O22
• Hydroxyl radical (OH)
Toxic Forms of Oxygen
Clumps of bacteria adhering to surface
Surface
Water currents
Migrating clump of bacteria
Biofilms• Microbial communities
• Form slime or hydrogels
– Bacteria attracted by chemicals via quorum sensing
Culture Media
• Complex Media:
Extracts and digests of yeasts, meat, or plants
• Chemically Defined Media:
Exact chemical composition is known
• Differential Media:
– Make it easy to distinguish colonies of different microbes.
• Enrichment Media
Encourages growth of desired microbe
• Reducing media for anaerobic culture,
Contain chemicals (thioglycollate or oxyrase) that combine O2
Heated to drive off O2
Figure 6.9b
Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates.
Lid with O-ring gasket
Envelope containing sodium bicarbonate and sodium borohydride
Anaerobic indicator (methylene blue)
Petri plates
Clamp with clamp screw
Palladium catalyst pellets
• A pure culture contains only one species or strain
• A colony is a population of cells arising from a single cell or spore or from a group of attached cells
– A colony is often called a colony-forming unit (CFU)
• The streak plate method is used to isolate pure cultures
Obtaining Pure Cultures
• Exercise 9: Aseptic Technique• Each person make 3 inoculations:
1. Broth to broth - Escherichia coli - 37°C 2. Slant to slant - E. coli - 30°C3. Plate to slant - Serratia marcescens - 30°C (changed)
• Exercise 10: Pure Culture Technique– Each person make 2 streak plates: Quadrant Streak Method B,
page 82.– Incubate at 25°C
• Turn in cultures from home for incubation• Finish microscope worksheet if necessary
• Exercise 9: Aseptic Technique• Each person make 3 inoculations:
1. Broth to broth - E. coli - 37°C 2. Slant to slant - E. coli - 37°C3. Plate to slant - S. marcescens -
30°C (changed)
• Exercise 10: Pure Culture Technique– Each person make 2 streak
plates– Quadrant Streak Method B,
page 85.– Incubate plates at 25°C
Each pair will need:1 broth culture Escherichia coli, 1 slant culture Escherichia coli1 plate culture Serratia marcescensEach person will need:1 Nutrient Broth/BHI tubes, 2 Nutrient Agar/BHIA slants
Each pair will need:1 mixed culture (which contains: Escherichia coli, Serratiamarcescens and Micrococcus luteus)Each person will need:2 Nutrient Agar/BHIA plates
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