CHAPTER-III R.KAVITHA, M.PHARM, LECTURER, DEPARTMENT OF PHARMACEUTICS, SRM COLLEGE OF PHARMACY, SRM UNIVERSITY, KATTANKULATHUR.
Culture techniques Environment Carbon and Energy Flow Maintains of culture
Culture Techniques Pure Culture -a single strain of microbe grown in isolation Strain - a microbial culture which is the descendent of a single cell originally isolated from the environment Aseptic Technique- method of handling material without contamination from the environment
Bacterial colony Figure 4.1
Inoculation Techniques Two Forms of Medium Broth - a liquid medium Agar - a semi-solid medium agar is chemical from seaweed that melts at 100C and freezes at 45C Agar medium is used for the isolation of microbes- Streak Plates
Figure 4.2 Streak-plate method
Bacterial Growth An Increase in Population Number Not an Increase in Cell Size
Cell Division Most Bacteria Reproduce by Binary Fission The cell doubles in size Replicates the chromosome (DNA) Forms a septum in the center Synthesizes a Cell Wall at the Septum Daughter cells separate.
Binary fission
Bacterial Growth Curve All microorganisms undergo similar growth patterns. Each growth Curve has 4 Phases 1 Lag Phase occurs immediately after inoculation cells do not grow; cells per volume do not increase
Microbial Growth Curve # cells / ml Lag Time
Microbial Growth Curve # cells / ml Log Lag Time
Microbial Growth Curve 2.) Growth Phase Exponential Phase Log Phase During this phase the microbe is growing at the maximum rate possible. Cells per volume increases dramatically Most research is performed on cells during log phase
Microbial Growth Curve Stationary # cells / ml Log Lag Time
Microbial Growth Curve 3.) Stationary Phase Growth levels off. Cells per volume does not increase or decrease Growth Rate = Death Rate Due to Depletion of Nutrients Increase in Waste Products
Microbial Growth Curve Stationary # cells / ml Log Death Lag Time
Microbial Growth Curve 4.) Death Phase Death Rate exceeds Growth Rate Cells per volume decreases Due to: Very low concentrations of Nutrients Very high concentrations of Waste Products
Terms Growth Rate Number of doublings (cell divisions) per hour Doubling Time (Generation Time) Length of time required for a cell to divide during log growth Doubling Time = 1/Growth Rate
Growth Rate Examples If cells double in 15 min. 15 min = 0.25 hr 1/0.25 = 4 If cells double in 300 min 300 min = 5 hr 1/5 = 0.2 Growth Rate = doublings per hour
Two Methods for Culturing Microorganisms BATCH CULTURE the microbe is grown in a closed vessel typically in a test tube or flask CONTINUOUS CULTURE the microbe is grown in vessel which has medium constantly added and spent medium constantly removed performed in a chemostat
Continuous culture Fig. 5.8A (Part 1 of 2) Sterile air inlet Fresh medium Culture flask Spent medium, wastes, and excess microbes Copyright 1996 The McGraw-Hill Companies, Inc.
Nutritional Requirements for Microorganisms WATER ENERGY CARBON ESSENTIAL ELEMENTS
Nutritional Requirements for Microorganisms WATER- water serves as a solvent to carry nutrient to and waste products away from the cell One method preservation of a microbial culture is drying (desiccation).
Energy Sources Phototroph- energy from sunlight Chemotroph- energy from chemicals Chemoorganotrophs [organotrophs]- derive energy from ORGANIC CHEMICALS Chemolithotrophs [lithotrophs]- derive energy from INORGANIC CHEMICALS
Carbon Sources Autotrophs- carbon from carbon dioxide (CO 2 ) - Inorganic carbon Heterotrophs- carbon from Organic Carbon Ex. Carbohydrates, lipids, protein
Essential Elements Hydrogen (H), sulfur (S), Oxygen (O), Phosphorus (P), Nitrogen (N) commonly supplied as ammonia (NH 4 ) some microbes fix atmospheric nitrogen (N 2 ) Trace Elements required in SMALL amounts Copper (Cu), Zinc (Zn), Selenium (Se)
Extra Growth Factors Fastidious Microorganisms microbes which need special organic chemicals added to the medium for growth to occur Ex. Vitamins, amino acids
Vitamin A (Retinol) Vitamin B12 HS-CH 2 -CH 2 -SO 3 - Coenzyme M Biotin
Environmental Requirements for Growth Temperature ph Oxygen Carbon Dioxide Osmotic Pressure Hydrostatic Pressure
Temperature Psychrophiles - optimum less than 20 C Mesophiles- optimum 20-45 C Thermophiles- optimum 45-80 C Extreme Thermophiles- optimum 85+ C
Temperature ranges Figure 4.4
ph -log [H + ] If hydrogen ions increase then ph decreases. Low ph = Acid High ph = Basic or Alkaline
ph Acidophiles- optimum below ph 5.5 Neutrophiles- optimum at ph 6-8 Alkalophiles- optimum above ph 8
Molecular Oxygen (O 2 ) Microbe vary greatly in sensitivity to O 2 Aerobes- microbes which require O 2. Anaerobes- microbes which DO NOT utilize O 2 AND are KILLED by O 2 Facultative Microbes- microbes which can grow in presence OR absence of O 2 Microaerophiles - required 3-15% O 2
GasPak Fig. 5.12B 2 Platinum catalyst Oxygen in jar (O 2 ) O + O H O Culture plates H Water (H 2 O) 2 H H GasPak disposable Hydrogenhydrogen and carbon gas (H dioxide generator 2 ) envelope Clamp with clamp screw Lid with O-ring gasket Catalyst pellets in reaction chamber Flash arrester to prevent explosion GasPak disposable anaerobic indicator strip Copyright 1996 The McGraw-Hill Companies, Inc.
Anaerobes Figure 4.8 Figure 4.9
Molecular Oxygen (O 2 ) Microbe vary greatly in sensitivity to O 2 Aerobes- microbes which require O 2. Anaerobes- microbes which DO NOT utilize O 2 AND are KILLED by O 2 Facultative Microbes- microbes which can grow in presence OR absence of O 2 Microaerophiles - required 3-15% O 2
Oxygen requirements of bacterial isolate plate method / Fig. 5.13A (Part 1 of 2) 1 2 3 4 1 2 3 4 1 2 3 4 Aerobic culture Anaerobic culture Microaerophilic culture Copyright 1996 The McGraw-Hill Companies, Inc.
Oxygen requirements of bacterial isolate deep tube method / Fig. 5.13B Aerobe Obligate (strict) anaerobe Facultative anaerobe Microaerophile Copyright 1996 The McGraw-Hill Companies, Inc.
Carbon Dioxide Capnophiles - optimum 3-10% CO 2 Many microaerophiles are also capnophiles Cultured in a candle jar
Osmotic Pressure Interior osmotic pressures must be balanced against exterior osmotic pressures. Each microorganism displays an optimum salt concentration for growth. Halophiles- require high concentrations of of NaCl Halo-tolerant- will grow in high NaCl concentrations but grow best at lower levels
Hydrostatic Pressure Barophiles- optimum hydrostatic pressure for growth of up to 1000 atm Normal Pressure = 1 atm
Culture Medium Defined Medium- a culture medium in which the amount and the purity of ALL ingredients is known synthetic or minimal Complex Medium- a culture medium which the amounts and purity of ingredients are NOT known.
Specialized Medium Transport Medium- not a true medium; used to transport samples to the lab. Enriched Medium- a complex medium to which more nutritious ingredient is added Ex. Yeast extract to brain heart infusion
Specialized Media Selective Media- supports the growth of some organisms but inhibits the growth of others Ex. Eosin Methylene Blue (EMB) inhibits Gram-positives therefore selects for Gram-negatives
Specialized Medium Differential Media- does not select against a group but allows the differentiation between 2 groups of bacteria Ex. Blood Agar Medium- can distinguish between Alpha-Hemolysis- incomplete RBC lysis Beta Hemolysis- complete RBC lysis Gamma Hemolysis- no RBC lysis
Specialized Media Enrichment Broths encourage the growth of a particular type of microbe; Addition of nutrients enrich for microbial group of interest Ex. Cellulose broth- enriches for microbes which degrade cellulose Ex. Petroleum Broth- enriches for microbes which could eat an oil spill.
Maintenance and Preservation Maintenance Medium- supports slow growth of the culture; typically agar slant. Laborious and costly Preservation - Lyophilization (Freeze-Dry) Dehydrated and Frozen -70C Cultures are viable for decades Low temperatures do not kill most microbes