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Microbial Growth Increase in number of cells, not cell size
Populations Colonies
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The Requirements for Growth
Physical requirements Temperature pH Osmotic pressure Chemical requirements Carbon Nitrogen, sulfur, and phosphorous Trace elements Oxygen Organic growth factor
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Physical Requirements
Temperature Minimum growth temperature Optimum growth temperature Maximum growth temperature
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Thermophiles Hyperthermophiles Mesophiles Psychrotrophs Psychrophiles
Figure 6.1 Typical growth rates of different types of microorganisms in response to temperature. Thermophiles Hyperthermophiles Mesophiles Psychrotrophs Psychrophiles
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Figure 6.2 Food preservation temperatures.
Temperatures in this range destroy most microbes, although lower temperatures take more time. Very slow bacterial growth. Rapid growth of bacteria; some may produce toxins. Danger zone Many bacteria survive; some may grow. Refrigerator temperatures; may allow slow growth of spoilage bacteria, very few pathogens. No significant growth below freezing.
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pH Most bacteria grow between pH 6.5 and 7.5
Molds and yeasts grow between pH 5 and 6 Acidophiles grow in acidic environments
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Osmotic Pressure Hypertonic environments, or an increase in salt or sugar, cause plasmolysis Extreme or obligate halophiles require high osmotic pressure Facultative halophiles tolerate high osmotic pressure
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Cell in isotonic solution. Plasmolyzed cell in hypertonic solution.
Figure 6.4 Plasmolysis. Plasma membrane Plasma membrane Cell wall H2O Cytoplasm Cytoplasm NaCl 0.85% NaCl 10% Cell in isotonic solution. Plasmolyzed cell in hypertonic solution.
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Chemical Requirements
Carbon Structural organic molecules, energy source Chemoheterotrophs use organic carbon sources Autotrophs use CO2
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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
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Chemical Requirements
Sulfur In amino acids, thiamine, and biotin Most bacteria decompose proteins Some bacteria use SO42– or H2S Phosphorus In DNA, RNA, ATP, and membranes PO43– is a source of phosphorus
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Chemical Requirements
Trace elements Inorganic elements required in small amounts Usually as enzyme cofactors
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Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria
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Organic Growth Factors
Organic compounds obtained from the environment Vitamins, amino acids, purines, and pyrimidines
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Biofilms Microbial communities Form slime or hydrogels
Bacteria attracted by chemicals via quorum sensing
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Clumps of bacteria adhering to surface Migrating clump of bacteria
Figure 6.5 Biofilms. Clumps of bacteria adhering to surface Migrating clump of bacteria Surface Water currents
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Biofilms Share nutrients Sheltered from harmful factors
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Applications of Microbiology 3.2 Pseudomonas aeruginosa biofilm.
© 2013 Pearson Education, Inc.
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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
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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 at ~40°C
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Culture Media Chemically defined media: exact chemical composition is known Complex media: extracts and digests of yeasts, meat, or plants Nutrient broth Nutrient agar
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Table 6.3 Defined Culture Medium for Leuconostoc mesenteroides
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Table 6.4 Composition of Nutrient Agar, a Complex Medium for the Growth of Heterotrophic Bacteria
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Anaerobic Culture Methods
Reducing media Contain chemicals (thioglycolate or oxyrase) that combine O2 Heated to drive off O2
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Envelope containing sodium bicarbonate and sodium borohydride
Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates. Clamp with clamp screw Lid with O-ring gasket Envelope containing sodium bicarbonate and sodium borohydride Palladium catalyst pellets Anaerobic indicator (methylene blue) Petri plates
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Figure 6.7 An anaerobic chamber.
Air lock Arm ports
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Biosafety Levels BSL-1: no special precautions
BSL-2: lab coat, gloves, eye protection BSL-3: biosafety cabinets to prevent airborne transmission BSL-4: sealed, negative pressure Exhaust air is filtered twice
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Figure 6.8 Technicians in a biosafety level 4 (BSL-4) laboratory.
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Differential Media Make it easy to distinguish colonies of different microbes
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Bacterial colonies Hemolysis
Figure 6.9 Blood agar, a differential medium containing red blood cells. Bacterial colonies Hemolysis
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Selective Media Suppress unwanted microbes and encourage desired microbes
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Uninoculated Staphylococcus epidermis Staphylococcus aureus
Figure 6.10 Differential medium. Uninoculated Staphylococcus epidermis Staphylococcus aureus
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Enrichment Culture 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
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Obtaining Pure Cultures
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) The streak plate method is used to isolate pure cultures
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Figure 6.11 The streak plate method for isolating pure bacterial cultures.
2 3 Colonies
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Preserving Bacterial Cultures
Deep-freezing: –50° to –95°C Lyophilization (freeze-drying): frozen (–54° to –72°C) and dehydrated in a vacuum
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(a) A diagram of the sequence of cell division
Figure 6.12a Binary fission in bacteria. Cell wall Cell elongates and DNA is replicated. Plasma membrane Cell wall and plasma membrane begin to constrict. DNA (nucleoid) Cross-wall forms, completely separating the two DNA copies. Cells separate. (a) A diagram of the sequence of cell division
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Partially formed cross-wall
Figure 6.12b Binary fission in bacteria. Partially formed cross-wall DNA (nucleoid) Cell wall (b) A thin section of a cell of Bacillus licheniformis starting to divide © 2013 Pearson Education, Inc.
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Figure 6.13a Cell division.
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Figure 6.13b Cell division.
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Figure 6.15 Understanding the Bacterial Growth Curve.
Lag Phase Intense activity preparing for population growth, but no increase in population. Log Phase Logarithmic, or exponential, increase in population. Stationary Phase Period of equilibrium; microbial deaths balance production of new cells. Death Phase Population Is decreasing at a logarithmic rate. The logarithmic growth in the log phase is due to reproduction by binary fission (bacteria) or mitosis (yeast). Staphylococcus spp.
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1 ml 1 ml 1 ml 1 ml 1 ml Original inoculum Dilutions 1:10 1:100 1:1000
Figure 6.16 Serial dilutions and plate counts. 1 ml 1 ml 1 ml 1 ml 1 ml Original inoculum 9 m broth in each tube Dilutions 1:10 1:100 1:1000 1:10,000 1:100,000 1 ml 1 ml 1 ml 1 ml 1 ml Plating 1:10 1:100 1:1000 1:10,000 1:100,000 (10-1) (10-2) (10-3) (10-4) (10-5) Calculation: Number of colonies on plate × reciprocal of dilution of sample = number of bacteria/ml (For example, if 54 colonies are on a plate of 1:1000 dilution, then the count is 54 × 1000 = 54,000 bacteria/ml in sample.)
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Plate Counts After incubation, count colonies on plates that have 25–250 colonies (CFUs)
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Figure 6.17 Methods of preparing plates for plate counts.
The pour plate method The spread plate method Inoculate empty plate. 1.0 or 0.1 ml 0.1 ml Inoculate plate containing solid medium. Bacterial dilution Spread inoculum over surface evenly. Add melted nutrient agar. Swirl to mix. Colonies grow only on surface of medium. Colonies grow on and in solidified medium.
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Figure 6.18 Counting bacteria by filtration.
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Grid with 25 large squares
Figure 6.20 Direct microscopic count of bacteria with a Petroff-Hausser cell counter. Grid with 25 large squares Cover glass Slide Bacterial suspension is added here and fills the shallow volume over the squares by capillary action. Bacterial suspension Microscopic count: All cells in several large squares are counted, and the numbers are averaged. The large square shown here has 14 bacterial cells. Cover glass Slide Location of squares The volume of fluid over the large square is 1/1,250,000 of a milliliter. If it contains 14 cells, as shown here, then there are 14 × 1,250,000 = 17,500,000 cells in a milliliter. Cross section of a cell counter. The depth under the cover glass and the area of the squares are known, so the volume of the bacterial suspension over the squares can be calculated (depth × area).
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Light source Spectrophotometer Light Blank Bacterial suspension
Figure 6.21 Turbidity estimation of bacterial numbers. Light source Spectrophotometer Light Light-sensitive detector Blank Scattered light that does not reach detector Bacterial suspension
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Measuring Microbial Growth
Direct Methods Plate counts Filtration MPN Direct microscopic count Indirect Methods Turbidity Metabolic activity Dry weight
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