Toxic Forms of Oxygen

  _{ninth edition TORTORA ⏐ FUNKE ⏐ CASE} Microbial Growth

  ƒ Microbial growth is the increase in number of cells,

  M I C R O B I O L O G Y a n i n t r o d u c t i o n not cell size

  6 Microbial _{PowerPoint Lecture ®} Growth _{Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings}  Slide Presentation prepared by Christine L. Case _{Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings} The Requirements for Growth: Temperature Physical Requirements

  6

  ƒ Temperature ƒ

  Minimum growth temperature

  ƒ Optimum growth temperature ƒ

  Maximum growth temperature g p Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.1

Psychrotrophs Psychrotrophs

  ƒ Grow between 0°C and 20-30°C ƒ Cause food spoilage The Requirements for Growth: Physical Requirements

  ƒ pH ƒ Most bacteria grow between pH 6.5 and 7.5 ƒ Molds and yeasts grow between pH 5 and 6 ƒ Acidophiles grow in acidic environments

  ƒ Chemoheterotrophs use organic carbon sources ƒ

  ƒ Trace elements ƒ Inorganic elements required in small amounts ƒ Usually as enzyme cofactors

  ƒ Phosphorus ƒ In DNA, RNA, ATP, and membranes ƒ PO ^{4 3–} is a source of phosphorus

  Most bacteria decompose proteins ƒ Some bacteria use SO ^{4 2–} or H ^{2 S}

  ƒ Sulfur ƒ In amino acids, thiamine and biotin ƒ

  ƒ A few bacteria use N ₂ in nitrogen fixation S lf

  Most bacteria decompose proteins ƒ Some bacteria use NH ^{4 +} or NO ^{3 –}

  ƒ Nitrogen ƒ In amino acids and proteins ƒ

  Autotrophs use CO ² Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings p ² The Requirements for Growth: Chemical Requirements

  Structural organic molecules, energy source

  Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings p g

  ƒ Carbon ƒ

  The Requirements for Growth: Chemical Requirements

  The Requirements for Growth: Physical Requirements Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.4

  ƒ Facultative halophiles tolerate high osmotic pressure

  Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings g p q g pressure

  ƒ Extreme or obligate halophiles require high osmotic

  cause plasmolysis

  ƒ Osmotic pressure ƒ Hypertonic environments, increase salt or sugar,

  The Requirements for Growth: Physical Requirements

The Requirements for Growth: Chemical Requirements

  The Requirements for Growth: Chemical Requirements

  ƒ Sterile: No living microbes ƒ

  ƒ Nutrient broth ƒ

  or plants p

  ƒ Complex media: Extracts and digests of yeasts, meat,

  is known

  ƒ Chemically defined media: Exact chemical composition

  Solidifies ~40°C Culture Media

  ƒ Liquefies at 100°C ƒ

  y y

  ƒ Generally not metabolized by microbes

  plates, slants, and deeps

  ƒ Complex polysaccharide ƒ Used as solidifying agent for culture media in Petri

  Agar

  ƒ Culture: Microbes growing in/on culture medium

  Inoculum: Introduction of microbes into medium Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings

  growth

  ƒ Oxygen (O ₂

  ƒ

  ) Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Table 6.1

  Toxic Forms of Oxygen

  ƒ Singlet oxygen: O ₂

  boosted to a higher-energy state

  ƒ Superoxide free radicals: O ₂ ^–

  Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings

  Peroxide anion: O ₂ ^2–

  ƒ Culture medium: Nutrients prepared for microbial

  ƒ Hydroxyl radical (OH •)

  The Requirements for Growth: Chemical Requirements

  ƒ Organic growth factors ƒ

  Organic compounds obtained from the environment

  ƒ Vitamins, amino acids, purines, and pyrimidines

  Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Culture Media

  Nutrient agar Culture Media Growth in Continuous Culture

  ƒ A “continuous culture” is an open system in which fresh media is continuously added to the culture at a constant rate, and old broth is removed at the same rate. This method is accomplished in a device called a

  ƒ chemostat. ƒ Typically, the concentration of cells will reach an equilibrium level that remains constant as long as _{Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Tables 6.2, 6.4 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings} the nutrient feed is maintained.

  Our Ch em ost at Syst em Basic Chem ost at Syst em Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings

  Anaerobic Culture Methods Anaerobic Culture Methods

  ƒ Reducing media ƒ Anaerobic ƒ Contain chemicals (thioglycollate or oxyrase) that jar

  combine O ₂

  ƒ Heated to drive off O _{2 2} Anaerobic Culture Methods Capnophiles Require High CO

  2

  ƒ Anaerobic ƒ Candle jar

  chamber

  ƒ _{Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.6 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.7} CO ^{2 -packet}

  Selective Media Selective Media

  ƒ Suppress unwanted ƒ Inhibits the growth of some bacteria while selecting for

  microbes and the growth of others encourage desired ƒ Example:

  ƒ microbes.

  Brilliant Green Agar g

  ƒ dyes inhibit the growth of Gram (+) bacteria ƒ selects for Gram (-) bacteria ƒ Most G.I. Tract infections are caused by Gram (-) _{Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.9b–c Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings} bacteria

  Differential Media Selective Media

  ƒ ƒ Make it easy to distinguish colonies of different

  EMB (Eosin Methylene Blue) ƒ dyes inhibit Gram (+) bacteria microbes.

  ƒ

  selects for Gram (-) bacteria

  ƒ G.I. Tract infections caused by Gram (-) y ( )

  bacteria Some media are both selective and Selective and Differential Media differential

  ƒ Mannitol salt agar is both selective and differential

  ƒ

  Mannitol Salt Agar ƒ

  Selective: ƒ Staphylococcus aereus can grow on mannitol salt agar that has a

  ƒ used to identify Staphylococcus aureus

  high concentration of salt; the growth of other organisms will be inhibited inhibited

  ƒ M Mannitol Salt Agar i l S l A

  ƒ Differential:

  ƒ High salt conc. (7.5%) inhibits most bacteria

  ƒ Staphylococcus aureus ferments mannitol and the medium will change color

  ƒ sugar Mannitol

  ƒ Other organisms that grow on high salt will grow on mannitol salt agar but may not ferment mannitol; the media will not change

  ƒ

  pH Indicator (Turns Yellow when acid) _{Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings} colors Selective and Differential Media Enrichment Media

  ƒ

  Encourages growth of desired microbe

  ƒ Assume a soil sample contains a few phenol-degrading ƒ

  MacConkey’s Agar bacteria and thousands of other bacteria

  ƒ used to identify Salmonella ƒ Inoculate phenol-containing culture medium with the

  ƒ MacConkey’s Agar

  soil and incubate soil and incubate

  ƒ Bile salts and crystal violet (inhibits Gram (+) ƒ Transfer 1 ml to another flask of the phenol medium

  bacteria) and incubate

  ƒ

  lactose

  ƒ Transfer 1 ml to another flask of the phenol medium ƒ pH Indicator

  and incubate Many Gram (-) enteric non-pathogenic bacteria can

  ƒ Only phenol-metabolizing bacteria will be growing _{Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings} ferment lactose, Salmonella can not

Streak Plate ƒ A pure culture contains only one species or strain

  ƒ A colony is a population of cells arising from a single cell or spore or from a group of attached cells. ƒ A colony is often called a colony-forming unit (CFU). y y g ( )

  Preserving Bacteria Cultures Reproduction in Prokaryotes

  ƒ Deep-freezing: –50°to –95°C ƒ Binary fission ƒ Lyophilization (freeze-drying): Frozen (–54° to –72°C) ƒ Budding

  and dehydrated in a vacuum ƒ Conidiospores (actinomycetes)

  ƒ Fragmentation of filaments g

  Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Binary Fission

  Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.11 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.12b

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

  cells: PLAY Animation: Bacterial Growth

  1. Lag Phase

  ƒ Bacteria are first introduced into an environment or

  media

  ƒ Bacteria are “checking out” their surroundings ƒ cells are very active metabolically y y ƒ

  # of cells changes very little _{Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.14 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings} ƒ 1 hour to several days

  3. Stationary Phase

  2. Log Phase

  ƒ Death rate = rate of reproduction ƒ Rapid cell growth (exponential growth)

  ƒ cells begin to encounter environmental stress ƒ

  population doubles every generation

  ƒ

  lack of nutrients

  ƒ microbes are sensitive to adverse conditions ƒ lack of water

  ƒ

  antibiotics

  ƒ not enough space ƒ anti-microbial agents

  ƒ metabolic wastes ƒ oxygen ƒ pH _{Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings} Endospores would form now

  4. Death Phase Measuring Microbial Growth

  ƒ Death rate > rate of reproduction Direct methods Indirect methods ƒ Due to limiting factors in the environment ƒ Plate counts ƒ Turbidity

  ƒ Filtration ƒ Metabolic activity ƒ MPN ƒ Dry weight y g ƒ Direct microscopic count ƒ

  Dry weight Direct Measurements of Microbial Growth

  ƒ Plate counts: Perform serial dilutions of a sample

  Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.15, step 1 Plate Count

  ƒ Inoculate Petri

  plates from serial dilutions Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.16

  Plate Count

  ƒ After incubation, count colonies on plates that have

  25-250 colonies (CFUs) Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.15

  Direct Measurements of Microbial Growth

  ƒ Filtration

  Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.17 Direct Measurements of Microbial Growth

  ƒ Multiple tube MPN test. ƒ Count positive

  tubes and compare to statistical MPN table.

  Direct Measurements of Microbial Growth

  ƒ Direct microscopic count

  Estimating Bacterial Numbers Direct Measurements of Microbial Growth by Indirect Methods

  ƒ Turbidity

  Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.19, steps 1, 3 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.20