Chapter 6 Microbial Growth

Lab 2 Goals and Objectives : Lecture: Chapter 6 (Microbial Growth) Learn aseptic technique and pure culture isolation Exercise 9: Aseptic Technique Exercise 10: Pure Culture Technique Turn in cultures from home for incubation Finish microscope worksheet if necessary

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.

Growing microorganisms 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

Culture Media 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

Reproduction of Prokaryotes - Binary fission

Bacterial growth in culture Bacteria multiply by binary fission The population grows in geometric progression 12 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 bacteria acclimate to new media -Log phase: period of exponential growth with a constant generation time -Stationary phase: cell growth is equal to cell death -Death phase: cell death exceeds cell growth

Number of generation

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

Chemical Requirements 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 Some bacteria use SO42 or H2S Phosphorus In DNA, RNA, ATP, and membranes PO43 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 - Oxygen (O2)

Toxic Forms of Oxygen Singlet oxygen: O2 boosted to a higher-energy state Superoxide free radicals: O2 Peroxide anion: O22 Hydroxyl radical (OH)

Biofilms Microbial communities Form slime or hydrogels Bacteria attracted by chemicals via quorum sensing Clumps of bacteria adhering to surface Migrating clump of bacteria Surface Water currents

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

Envelope containing sodium bicarbonate and sodium borohydride Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates. Clamp with clamp screw Lid with O-ring gasket Envelope containing sodium bicarbonate and sodium borohydride Palladium catalyst pellets Anaerobic indicator (methylene blue) Petri plates

Obtaining Pure Cultures 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

Exercise 9: Aseptic Technique Each person make 3 inoculations: Broth to broth - Escherichia coli - 37°C Slant to slant - E. coli - 30°C 3. 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

Streak Plate Flame the loop, cool it Flame the loop, cool it Figure 6.10a, b

Exercise 9: Aseptic Technique Each person make 3 inoculations: Broth to broth - E. coli - 37°C Slant to slant - E. coli - 37°C 3. 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 coli 1 plate culture Serratia marcescens Each person will need: 1 Nutrient Broth/BHI tubes, 2 Nutrient Agar/BHIA slants Each pair will need: 1 mixed culture (which contains: Escherichia coli, Serratia marcescens and Micrococcus luteus) Each person will need: 2 Nutrient Agar/BHIA plates Get help now, today, if you are having any difficulty with oil immersion lens use or specimen measurements using the ocular micrometer!!!