Microbial Growth

Microbial Growth • Physical Requirements of Microbes • Temperature (optimal enzyme operation) • Psychrophiles, mesophiles, thermophiles • pH (optimal enzyme operation) • Using buffers in media • Molds & yeasts versus bacteria • Chemical Requirements • Carbon source in medium • Nitrogen, sulfur, phosphorous, trace elements • Oxygen requirements • Obligate aerobes, anaerobes, facultative anaerobes • Free radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and catalase = aerobes • Culture Media for Microbes • Chemically defined vs. complex media • Anaerobes: reducing media/Brewer jar • Other: animals, eggs, tissue culture, CO2 • Media types • Selective, Differential, Enrichment • Bacterial Population Growth • Growth Curve: Lag, Log, Stationary, Death • Quantifying Growth

Characterizing Microbes By Optimal Growth Temperature Figure 6.1

Temperature Growth Ranges and Food Safety “2-40-140” If > 2 hrs at 40-140oF, don’t eat it! Figure 6.2

Physical Requirements: pH Neutrophiles Acidophiles Alkalophiles • Most bacteria grow between pH 6.5 and 7.5 • Molds and yeasts grow between pH 5 and 6

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: 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 • Most bacteria decompose proteins • Some bacteria use SO42 or H2S • Phosphorus • In DNA, RNA, ATP, and membranes • PO43is a source of phosphorus • Trace Elements • Inorganic elements required in small amounts • Usually as enzyme cofactors

How Toxic Forms of Oxygen Are Handled • Singlet oxygen: O2 boosted to a higher-energy state • Handling superoxide free radicals: O22O2- + 2O2- + 8H+ 4H2O2 oxygen radicals hydrogen peroxide • Superoxide Dismutase (SODS) • Handling peroxide anion: O22 2H2O2 2H2O + O2 hydrogen peroxide water oxygen gas Catalase (Peroxidase) Catalase Test: Bacteria + H2O2bubbles

Growth in thioglycollate broth reveals oxygen preferences Resazurin dye is red in the presence of oxygen Oxygen gradient Thyoglycollate binds molecular oxygen, reducing it and removing it: R-SH + O2 R-SO2

Culture Media: Chemically Defined or Complex Table 6.2 & 6.4

Anaerobic and Low O2 Culture Methods Candle jar Brewer or anaerobic jar CO2 packet

Unusual Culture Methods Grows only inside live cells: eggs as culture vessels for influenza virus Grows only in certain cell types: using tissue culture with low O2, enriched CO2 incubators Grows only in certain cell types: using armadillos to culture M. leprae

Selective Media MSA • Goal: To chemically (or physically) suppress unwanted microbes and encourage desired microbes. Mannitol salt agar : selective for halophiles with 7% salt (osmotic challenge) and differential for mannitol fermenters: good for skin bacterial cultures. EMB Agar: kills gram positives with eosin and methylene blue, selective for gram negatives. Differential for lactose fermenters. Good for growing enterics. McConkey Agar: supresses gram positives with crystal violet and bile salts; also differential for EMB MA Figure 6.9b, c

Differential Media Blood agar (sheep’s blood) reveals if hemolytic • Distinguish between different species based on a metabolic ability. Mannitol salt agar contains the pH sensitive dye phenol red (yellow when acidic) Se Sa Figure 6.9a

Enrichment Media • Encourages growth of desired microbe by providing special growth conditions or added growth factors Thioglycollate Anaerobic or Brewer Jar Glucose Salts Agar (enriches for microbes that can growth only on glucose and some inorganic nutrients Lysed red blood cells provide unique nutrients in blood/chocolate agar

Pure Cultures Used To Study Characteristics Of A Particular Species • 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)

Bacterial Growth is Exponential (Logarithmic) Bacterial “growth” means an increase in the number of individuals, not an increase in cell size. Figure 6.12b

Growth Curve for Bacteria (Logarithmic Plot) Figure 6.14

Estimating Bacterial Numbers by Indirect methods • Direct Measures • Plate counts of viable bacterial forming colonies • Counting low viable bacterial numbers by filtration • Counting viable bacteria with Most Probable Number • Counting bacteria per ml in direct microscopy • Indirect Measures • Turbidity/Absorbance with a spectrophotometer • Metabolic activity tracking conversion of colored molecules • Dry weight by weighing a set volume and knowing weight of one cell

Plate Assays: Spread Plate or Pour Plate Methods • After incubation, count colonies on plates that have 30-300 colonies (CFUs) The dilution in a particular tube = ml of fluid added to tube/total volume after addition; e.g. 1ml/(9ml + 1ml) = 1/10 = 10-2 Figure 6.15

Direct Measurements of Microbial Growth Figure 6.19

Direct Measurements of Microbial Growth • Filtration: Good for measuring very dilute samples of bacteria Figure 6.17a, b

Direct Measurements of Microbial Growth • Multiple tube MPN test • Count positive tubes and compare to statistical MPN table • Produces a range of concentrations Figure 6.18b

Estimating Bacterial Numbers by Indirect methods • Direct Measures • Plate counts of viable bacterial forming colonies • Counting low viable bacterial numbers by filtration • Counting viable bacteria with Most Probable Number • Counting bacteria per ml in direct microscopy • Indirect Measures • Turbidity/Absorbance with a spectrophotometer • Metabolic activity tracking conversion of colored molecules/enyzme assay • Dry weight by weighing a set volume and knowing weight of one cell

Estimating Bacterial Numbers by Indirect Methods • Turbidity Figure 620

Metabolic Conversion/Enzyme Assay 1 bacterium produces 4.6 x 1012 NADH/sec/cell under idea growth conditions. In a 1 ml sample of growing cells, 5.2 x 1023 NADH/sec/ml are produced per second (as revealed by a color-based assay of NADH on the sample) Therefore, (4.6 x 1012 NADH/sec/ml) x (5.2 x 1023 NADH/sec/cell) = 2.3 x 1024 cells/ml

Determining dry mass of a fixed volume An E. coli cell has a dry mass of about 7.0 x 10-19 mg. A 1 ml sample with a dry mass of 2 mg therefore has: 2 mg/ml x 1 cell/7 x 10-19 mg = 2.8 x 1020 cells/ml

Microbial Growth