GROWTH AND CULTURING OF BACTERIA CHAPTER 6

Growth and Cell Division Growth Definition Binary Fission Budding

Bacterial Growth Curve Lag phase Adjustment to nutrients Enzyme synthesis DNA synthesis Log phase Exponential cell division Stationary phase Nutrient depletion Waste build-up Death phase More die than are produced Always a few survivors

Counting Large Populations Serial Dilutions/ Standard plate counts Very large numbers may be counted by this method Pour plate or spread plate techniques are used

Direct Counts Colony Counts 300 upper limit Hemocytometer

Counting Small Populations Most Probable Number Filtration

Other Measurements Spectrophotometer Tube turbidity

Physical Influences (pH) Bacteria Prefer pH 5.4 – 8.0 Most grow at pH 7.0 – 7.4 Rare acidophiles and alkaliphiles exist Lactobacillus Vibrio cholerae Fungi Can tolerate pH 5-6 Often the only cause of acidic food spoilage Fruit and vegetable juices/products

Physical Influences (Temperature) Psychrophiles 0oC – 20oC Refrigerator spoilage Listeria monocytogenes Mesophiles 10oC – 45oC Food / Disease Thermophiles 40oC – 70oC Soil composters Extreme Thermophiles 70oC – 100oC Archaea

Oxygen’s Influence on Growth OBLIGATE AEROBE (Micrococcus) Must use oxygen OBLIGATE ANAEROBE (Clostridium) Must have anaerobic environment Why? Lack enzymes to degrade oxygen radicals Superoxide dismutase (O2-) Catalase (H2O2) MICROAEROPHILE (Campylobacter) Low oxygen, high carbon dioxide environment Respiration pathway may contain unique electron carriers FACULTATIVE ANAEROBE (Escherichia coli, Staphylococcus) Use oxygen when it is present Can also grow anaerobically AEROTOLERANT ANAEROBE (Lactobacillus) Tolerate oxygen Do not use oxygen

Growth positions in Thioglycollate medium

Physical Influences on Growth Moisture Endospores Fungal preferences Hydrostatic pressure Barophiles Undersea vents Osmotic pressure High salt/sugar used as food preservatives Halophiles – salt tolerant Radiation (Xrays, UV, γrays) Deinococcus radiodurans DNA repair systems

Nutritional Factors Affecting Bacterial Growth C, N, S, P availability Organic versus inorganic Trace elements Minerals for enzyme activity Vitamins Organics not synthesized by the cell Nutritional complexity Determined by the enzyme capabilities of the microbe Fewer enzymes = more nutritional needs (or complexity)

Bacterial Interactions Quorum sensing Biofilms Antibiotic resistances Diseases Tuberculosis Cystic Fibrosis Diabetic Foot Ulcers Toxin production to limit competitor “groups” Sociomicrobiology

Culturing – Plate techniques Streak Plates ¼ “ spacing minimum for well isolated colonies

Culturing Plate Techniques Pour Plates

Culture Media ? Types Defined Precise concentrations Research into new microbes Complex Nutritionally sufficient Unknown molecular composition ? Beef extract

Culture Media Purposes Selective media Suppress unwanted Differential media Distinguishes Enrichment media Favors fastidious microbes Propagation media General growth

Controlling Oxygen Content Brewer jar Anaerobic environment Chemicals absorb oxygen Candle jar Burning a candle creates low O2, high CO2 environment

Applications of Culturing Maintaining cultures Preserved cultures Diagnostic tests Limitations on Culture