Chapter 6 Microbial Growth Microbial growth increase in

Chapter 6 Microbial Growth

• Microbial growth = increase in number of cells, not cell size

What are • Populations • Colonies

The Requirements for Growth • Physical requirements – Temperature – p. H – Osmotic pressure • Chemical requirements – – – Carbon Nitrogen, sulfur, and phosphorous Trace elements Oxygen Organic growth factor

The Requirements for Growth: Physical Requirements • Temperature – Minimum growth temperature – Optimum growth temperature – Maximum growth temperature

Temperature Figure 6. 1

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

Psychrotrophs Figure 6. 2

The Requirements for Growth: Physical Requirements • p. H – Most bacteria grow between p. H 6. 5 and 7. 5 – Molds and yeasts grow between p. H 5 and 6 – Acidophiles grow in acidic environments

The Requirements for Growth: Physical 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 Requirements for Growth: Chemical Requirements • Carbon – Structural organic molecules, energy source – Chemoheterotrophs use organic carbon sources – Autotrophs use CO 2

• Nitrogen – In amino acids, proteins – Most bacteria decompose proteins – Some bacteria use NH 4+ or NO 3 – A few bacteria use N 2 in nitrogen fixation • Sulfur – In amino acids, thiamine, biotin – Most bacteria decompose proteins – Some bacteria use SO 42 or H 2 S • Phosphorus – In DNA, RNA, ATP, and membranes – PO 43 is a source of phosphorus

• Trace Elements – Inorganic elements required in small amounts – Usually as enzyme cofactors

• Oxygen (O 2) Obligate aerobes Faultative Obligate Aerotolerant Microaerophiles anaerobes

Toxic Forms of Oxygen • Singlet oxygen: O 2 boosted to a higher-energy state • Superoxide free radicals: O 2 • Peroxide anion: O 22 • Hydroxyl radical (OH )

Biofilms • Microbial communities • Form slime or hydrogels – Bacteria attracted by chemicals via quorum sensing Figure 6. 5

The Requirements for Growth: Chemical Requirements • Organic Growth Factors – Organic compounds obtained from the environment – Vitamins, amino acids, purines, pyrimidines

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

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 Table 6. 2 & 6. 4

Anaerobic Culture Methods • Reducing media – Contain chemicals (thioglycollate or oxyrase) that combine O 2 – Heated to drive off O 2

An Anaerobic Chamber Figure 6. 7

Anaerobic Culture Methods • Anaerobic jar Figure 6. 5

Capnophiles require high CO 2 • Candle jar • CO 2 -packet Figure 6. 7

Biosafety Levels • 1: No special precautions • 2: Lab coat, gloves, eye protection • 3: Biosafety cabinets to prevent airborne transmission • 4: Sealed, negative pressure – Exhaust air is filtered twice

Biosafety Level 4 (BSL-4) Laboratory Figure 6. 8

Selective Media • Suppress unwanted microbes and encourage desired microbes. Figure 6. 9 b, c

Differential Media • Make it easy to distinguish colonies of different microbes. Figure 6. 9 a

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

• 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)

Streak Plate Figure 6. 10 a, b

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

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

Binary Fission Figure 6. 11

Figure 6. 12 b

If 100 cells growing for 5 hours produced 1, 720, 320 cells:

Figure 6. 13

Phases of Growth ANIMATION Bacterial Growth Curve Figure 6. 15

Direct Measurements of Microbial Growth • Plate Counts: Perform serial dilutions of a sample Figure 6. 15, top portion

Plate Count • Inoculate Petri plates from serial dilutions Figure 6. 16

Plate Count • After incubation, count colonies on plates that have 25 -250 colonies (CFUs) Figure 6. 15

• Direct Measurements of Filtration Microbial Growth Figure 6. 17 a, b

• Direct Measurements of Multiple Microbial Growth tube MPN test • Count positive tubes and compare to statistical MPN table. Figure 6. 18 b

Direct Measurements of Microbial Growth • Direct Microscopic Count

Direct Measurements of Microbial Growth Figure 6. 19

• Estimating Bacterial Numbers Turbidityby Indirect Methods Figure 620

Estimating Bacterial Numbers by Indirect methods • Metabolic activity • Dry weight