Microbial Growth Increase in number of cells, not cell size Populations Colonies

The Requirements for Growth Physical requirements Temperature pH Osmotic pressure Chemical requirements Carbon Nitrogen, sulfur, and phosphorous Trace elements Oxygen Organic growth factor

Physical Requirements Temperature Minimum growth temperature Optimum growth temperature Maximum growth temperature

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

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.

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

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

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.

Chemical Requirements Carbon Structural organic molecules, energy source Chemoheterotrophs use organic carbon sources Autotrophs use CO2

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

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

Chemical Requirements Trace elements Inorganic elements required in small amounts Usually as enzyme cofactors

Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria

Organic Growth Factors Organic compounds obtained from the environment Vitamins, amino acids, purines, and pyrimidines

Biofilms Microbial communities Form slime or hydrogels Bacteria attracted by chemicals via quorum sensing

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

Biofilms Share nutrients Sheltered from harmful factors

Applications of Microbiology 3.2 Pseudomonas aeruginosa biofilm. © 2013 Pearson Education, Inc.

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 at ~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

Table 6.3 Defined Culture Medium for Leuconostoc mesenteroides

Table 6.4 Composition of Nutrient Agar, a Complex Medium for the Growth of Heterotrophic Bacteria

Anaerobic Culture Methods Reducing media Contain chemicals (thioglycolate or oxyrase) that combine O2 Heated to drive off O2

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

Figure 6.7 An anaerobic chamber. Air lock Arm ports

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

Figure 6.8 Technicians in a biosafety level 4 (BSL-4) laboratory.

Differential Media Make it easy to distinguish colonies of different microbes

Bacterial colonies Hemolysis Figure 6.9 Blood agar, a differential medium containing red blood cells. Bacterial colonies Hemolysis

Selective Media Suppress unwanted microbes and encourage desired microbes

Uninoculated Staphylococcus epidermis Staphylococcus aureus Figure 6.10 Differential medium. Uninoculated Staphylococcus epidermis Staphylococcus aureus

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

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

Figure 6.11 The streak plate method for isolating pure bacterial cultures. 2 3 Colonies

Preserving Bacterial Cultures Deep-freezing: –50° to –95°C Lyophilization (freeze-drying): frozen (–54° to –72°C) and dehydrated in a vacuum

(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

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.

Figure 6.13a Cell division.

Figure 6.13b Cell division.

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.

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.)

Plate Counts After incubation, count colonies on plates that have 25–250 colonies (CFUs)

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.

Figure 6.18 Counting bacteria by filtration.

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).

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

Measuring Microbial Growth Direct Methods Plate counts Filtration MPN Direct microscopic count Indirect Methods Turbidity Metabolic activity Dry weight