Growth & Culturing of Bacteria Microbiology 130 Chapter 6

Microbial Growth and Cell Division Microbial Growth Defined: Microbial growth is the increase in number of cells, not cell size Mother cells Daughter cells Cell Division: Binary fission Tetrads Sarcinae Budding

Reproduction in Prokaryotes Binary fission Budding Conidiospores (actinomycetes) Fragmentation of filaments

Binary Fission

Phases of Growth 4 Phases: 1) Lag phase- 2) Log (logarithmic) phase 3) Stationary phase 4) Decline phase or death phase

Log Phase

Log Phase with Calculations If 100 cells growing for 5 hours produced 1,720,320 cells:

Log phase

4 Phases of Microbial Growth

Growth in Colonies 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).

Measuring Bacterial Growth Serial Dilutions Direct Measurements of Microbial Growth Plate counts: Perform serial dilutions of a sample

Standard Plate Count Inoculate Petri plates from serial dilutions 2 methods: Pour Plate Spread Plate

Plate Count After incubation, count colonies on plates that have 25-250 colonies (CFUs)

Direct Measurements of Microbial Growth Filtration

Direct Measurements of Microbial Growth Multiple tube MPN test. Count positive tubes and compare to statistical MPN table.

Direct Measurements of Microbial Growth Direct microscopic count

Most Probable Number Estimate of number MPN 5 test tubes 10, 1, and 0.1 ml Growth is displayed by production of gas bubbles or by becoming cloudy Estimates are from a known chart ( table 6.1 pg 149)

Direct Measurements of Microbial Growth

Other Methods: Estimating Bacterial Numbers by Indirect Methods Turbidity

Measuring Microbial Growth Direct methods Plate counts Filtration MPN Direct microscopic count Dry weight Indirect methods Turbidity Metabolic activity

Factors Affecting Bacterial Growth. The Requirements for Growth: Factors Affecting Bacterial Growth The Requirements for Growth: Physical Requirements Temperature Minimum growth temperature Optimum growth temperature Maximum growth temperature

Temperature Figure 6.1

Psychrotrophs/Psychrophiles Grow between 0°C and 20-30°C Cause food spoilage

Psychrotrophs/Psychrophiles Figure 6.2

The Requirements for Growth: Physical Requirements Most bacteria grow between pH 6.5 and 7.5 Molds and yeasts grow between pH 5 and 6 Acidophiles grow in acidic environments

Mesophiles & Thermophiles grow best between 25 degrees C and 40 degrees C Thermophiles- Heat loving- grow best at 50 - 60 degrees C Obligate thermophiles Facultative thermophiles-

The Requirements for Growth: Physical Factors - 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)

The Requirements for Growth: Physical Factors / Requirements Osmotic pressure Hypertonic environments, increase salt or sugar, cause plasmolysis Extreme or obligate halophiles require high osmotic pressure Facultative halophiles tolerate high osmotic pressure

The Requirements for Growth: Physical Requirements Figure 6.4

The Physical Requirements for Growth Moisture- Hydrostatic Pressure- Radiation-

The Requirements for Growth: Nutritional Factors - Chemical Requirements Carbon Structural organic molecules, energy source Chemoheterotrophs use organic carbon sources Autotrophs use CO2

The Requirements for Growth: Nutritional Factors - Chemical Requirements Nitrogen In amino acids and proteins Most bacteria decompose proteins Some bacteria use NH4+ or NO3– A few bacteria use N2 in nitrogen fixation Sulfur In amino acids, thiamine and biotin Some bacteria use SO42– or H2S Phosphorus In DNA, RNA, ATP, and membranes PO43– is a source of phosphorus

The Requirements for Growth: Nutritional Factors - Chemical Requirements Trace elements Inorganic elements required in small amounts Usually as enzyme cofactors Vitamins- organic substances and growth factors Organic compounds obtained from the environment Vitamins, amino acids, purines, and pyrimidines Nutritional Complexity Locations of Enzymes Adaptations to Limited Nutrients

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

Sporulation / Endospores Formation of endospores in Bacillus, Clostridium and G+ genera Can Survive long periods of drought Axial nucleoid Endospore septum grows Spore coat and Exosporium Germination- 3 stages: 1) Activation, 2) germination, 3) outgrowth

Culturing Bacteria Methods of Obtaining Pure Culture 1) The Streak Plate Method – sterile wire loop and streaked in different patterns on agar 2) Pour Plate Method- serial dilutions using melted agar

Streak Plate

Culture Media Culture medium: Nutrients prepared for microbial growth Sterile: No living microbes Inoculum: Introduction of microbes into medium Culture: Microbes growing in/on culture medium Synthetic media Defined synthetic media Complex media

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 Chemically defined media: Exact chemical composition is known Complex media: Extracts and digests of yeasts, meat, or plants Nutrient broth Nutrient agar

Culture Media Tables 6.2, 6.4

Anaerobic Culture Methods Reducing media Contain chemicals (thioglycollate or oxyrase) that combine O2 Heated to drive off O2

Anaerobic Culture Methods Anaerobic jar Figure 6.5

Anaerobic Culture Methods Anaerobic chamber Figure 6.6

Capnophiles Require High CO2 Candle jar CO2-packet Figure 6.7

Selective Media Suppress unwanted microbes and encourage desired microbes. Figure 6.9b–c

Differential Media Make it easy to distinguish colonies of different microbes. Figure 6.9a

Enrichment Media Encourages growth of desired microbe Assume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteria Inoculate phenol-containing culture medium with the soil and incubate Transfer 1 ml to another flask of the phenol medium and incubate Only phenol-metabolizing bacteria will be growing

Preserving Bacteria Cultures Deep-freezing: –50°to –95°C Lyophilization (freeze-drying): Frozen (–54° to –72°C) and dehydrated in a vacuum

Methods of Performing Multiple Diagnostic Tests Enterotube Multitest API- simultaneous testing To identify enteric bacteria- cause food poisoning, typhoid, shigellosis, gastroenteritis etc Living, But Non-culturable organisms: Some microbs can be observed with microscope, identify their DNA but they can not be cultured