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

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

Temperature Mesophiles (moderate temp. loving microbes) range 20°C - 40°C Thermophiles (heat loving microbes) range 40°C - 100°C

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

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 CO2

The Requirements for Growth: Chemical Requirements 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

The Requirements for Growth: Chemical Requirements Trace Elements Inorganic elements required in small amounts Usually as enzyme cofactors

The Requirements for Growth: Chemical Requirements Oxygen (O2)

Toxic Forms of Oxygen Singlet oxygen(1O2- ): boosted to a higher-energy state Superoxide free radicals: O2 Peroxide anion: O22 Hydroxyl radical (OH)

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 Inoculum: Introduction of microbes into medium Culture: Microbes growing in/on culture medium

Criteria a culture media must meet Contain right nutrients for the growth of microorganisms we want to grow Proper moisture, pH, O2 level etc. It must be sterile – its must initially contain no other organisms The culture should be incubated in proper temperature

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 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 designed to suppress the growth of unwanted bacteria and encourage the growth of the desired microbes Bismuth sulfite agar is one medium used to isolate the typhoid bacterium, the gram-negative Salmonella typhi from feces. Bismuth sulfite inhibits gram positive bacteria and most gram-negative intestinal bacteria (other than S. typhi), as well. Sabouraud's dextrose agar, which has a pH of 5.6, is used to isolate fungi that outgrow most bacteria at this pH.

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

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 6.10a, 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 6.12b

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

Figure 6.13

Figure 6.14

Lag phase The number of cells changes very little because the cells do not immediately reproduce in a new medium Little or no cell division It can last for 1 hour or several days The cells are not dormant The microbial population is undergoing a period of intense metabolic activity involving, in particular, synthesis of enzymes and various molecules.

Log Phase The cells begin to divide and enter a period of growth, or logarithmic increase Also called exponential growth phase Cellular reproduction is most active during this period Generation time reaches a constant minimum. A logarithmic plot of growth during the log phase is a straight line. The log phase is the time when cells are most active metabolically and is preferred for industrial purposes

Stationary Phase If exponential growth continues unchecked, startlingly large numbers of cells could arise A single bacterium (at a weight of 9.5 X 10- 13 g per cell) dividing every 20 minutes for only 25.5 hours can theoretically produce a population equivalent in weight to that of an 80,000-ton aircraft carrier. In reality, this does not happen Eventually, the growth rate slows, the number of microbial deaths balances the number of new cells, and the population stabilizes This period of equilibrium is called the stationary phase. The exhaustion of nutrients, accumulation of waste products, and harmful changes in pH may all play a role.

Death Phase The number of deaths eventually exceeds the number of new cells formed The population enters the death phase, or logarithmic decline phase Phase continues until the population is diminished to a tiny fraction of the number of cells in the previous phase or until the population dies out entirely

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 Microbial Growth Filtration Figure 6.17a, b

Direct Measurements of Microbial Growth Direct Microscopic Count

Direct Measurements of Microbial Growth Figure 6.19

Estimating Bacterial Numbers by Indirect Methods Turbidity Figure 620

Estimating Bacterial Numbers by Indirect methods Metabolic activity Dry weight