Copyright © 2010 Pearson Education, Inc. Lectures prepared by Christine L. Case Chapter 6 Microbial Growth

Copyright © 2010 Pearson Education, Inc. Learning Objectives Upon completion of this chapter, you should be able to:  Classify microbes into 5 groups based on their temperature preference  Explain the importance of osmotic pressure in microbial growth  Name a use for each of the four elements (C, N, S, P) needed in large amounts for microbial growth  Explain how microbes are classified on the basis of oxygen requirements  Identify ways in which aerobes avoid damage from toxic forms of oxygen  Describe the formation of biofilms and their potential for causing infection  Distinguish chemically defined and complex media  Differentiate biosafety levels (1, 2, 3, and 4)  Justify the use of the following: anaerobic culture techniques, living host cells, candle jars, selective and differential media, enrichment media  Define colony  Describe how a pure culture can be isolated using the streak plate method  Define bacterial growth, including binary fission  Compare the phases of microbial growth  Explain 4 direct methods for measuring cell growth  Explain 3 indirect methods of measuring cell growth

Copyright © 2010 Pearson Education, Inc. Microbial Growth  Increase in number of cells, not cell size  Populations  Colonies

Copyright © 2010 Pearson Education, Inc. The Requirements for Growth  Physical requirements  Temperature  pH  Osmotic pressure  Chemical requirements  Carbon  Nitrogen, sulfur, and phosphorous  Trace elements  Oxygen  Organic growth factor

Copyright © 2010 Pearson Education, Inc. Physical Requirements  Temperature  Minimum growth temperature  Optimum growth temperature  Maximum growth temperature

Copyright © 2010 Pearson Education, Inc. Figure 6.1 Typical Growth Rates and Temperature

Copyright © 2010 Pearson Education, Inc. Psychrotrophs  Grow between 0°C and 20–30°C  Cause food spoilage

Copyright © 2010 Pearson Education, Inc. Figure 6.2 Food Preservation Temperatures

Copyright © 2010 Pearson Education, Inc. 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

Copyright © 2010 Pearson Education, Inc. 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

Copyright © 2010 Pearson Education, Inc. Figure 6.4 Plasmolysis

Copyright © 2010 Pearson Education, Inc. Chemical Requirements  Carbon  Structural organic molecules, energy source  Chemoheterotrophs use organic carbon sources  Autotrophs use CO 2

Copyright © 2010 Pearson Education, Inc. Chemical Requirements  Nitrogen  In amino acids and proteins  Most bacteria decompose proteins  Some bacteria use NH 4 + or NO 3 –  A few bacteria use N 2 in nitrogen fixation

Copyright © 2010 Pearson Education, Inc. Chemical Requirements  Sulfur  In amino acids, thiamine, and biotin  Most bacteria decompose proteins  Some bacteria use SO 4 2– or H 2 S  Phosphorus  In DNA, RNA, ATP, and membranes  PO 4 3– is a source of phosphorus

Copyright © 2010 Pearson Education, Inc. Chemical Requirements  Trace elements  Inorganic elements required in small amounts  Usually as enzyme cofactors

Copyright © 2010 Pearson Education, Inc. Table 6.1 The Effect of Oxygen (O2) on Growth

Copyright © 2010 Pearson Education, Inc.  Singlet oxygen: O 2 boosted to a higher-energy state  Superoxide free radicals: O 2 –  Peroxide anion: O 2 2–  Hydroxyl radical (OH) Toxic Oxygen

Copyright © 2010 Pearson Education, Inc. Organic Growth Factors  Organic compounds obtained from the environment  Vitamins, amino acids, purines, and pyrimidines

Copyright © 2010 Pearson Education, Inc. Figure 6.5 Biofilms  Microbial communities  Form slime or hydrogels  Bacteria attracted by chemicals via quorum sensing

Copyright © 2010 Pearson Education, Inc. Applications of Microbiology, p. 57 Biofilms  Share nutrients  Sheltered from harmful factors, such as antibiotics

Copyright © 2010 Pearson Education, Inc. Biofilms  Patients with indwelling catheters received contaminated heparin  Bacterial numbers in contaminated heparin were too low to cause infection  84–421 days after exposure, patients developed infections

Copyright © 2010 Pearson Education, Inc. Biofilms  Mucoid form of Pseudomonas aeruginosa was cultured from the CF sputum.  What causes the difference between the original strain and the CF strain?

Copyright © 2010 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

Copyright © 2010 Pearson Education, Inc. 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

Copyright © 2010 Pearson Education, Inc. Culture Media  Chemically defined media: Exact chemical composition is known  Complex media: Extracts and digests of yeasts, meat, or plants  Nutrient broth  Nutrient agar

Copyright © 2010 Pearson Education, Inc.

Anaerobic Culture Methods  Reducing media  Contain chemicals (thioglycolate or oxyrase) that combine O 2  Heated to drive off O 2

Copyright © 2010 Pearson Education, Inc. Figure 6.6 Anaerobic Jar

Copyright © 2010 Pearson Education, Inc. Figure 6.7 An Anaerobic Chamber

Copyright © 2010 Pearson Education, Inc. Capnophiles  Microbes that require high CO2 conditions  CO2 packet  Candle jar

Copyright © 2010 Pearson Education, Inc.  1: No special precautions  2: Lab coat, gloves, eye protection  3: Biosafety cabinets to prevent airborne transmission  4: Sealed, negative pressure  Exhaust air is filtered twice Biosafety Levels

Copyright © 2010 Pearson Education, Inc. Figure 6.8 Biosafety Level 4 (BSL-4) Laboratory

Copyright © 2010 Pearson Education, Inc. Figure 6.10 Selective Media  Suppress unwanted microbes and encourage desired microbes

Copyright © 2010 Pearson Education, Inc. Figure 6.9 Differential Media  Make it easy to distinguish colonies of different microbes.

Copyright © 2010 Pearson Education, Inc. 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

Copyright © 2010 Pearson Education, Inc. 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

Copyright © 2010 Pearson Education, Inc. Figure 6.11 The Streak Plate Method

Copyright © 2010 Pearson Education, Inc. Preserving Bacterial Cultures  Deep-freezing: –50° to –95°C  Lyophilization (freeze-drying): Frozen (–54° to – 72°C) and dehydrated in a vacuum

Copyright © 2010 Pearson Education, Inc.  Binary fission  Budding  Conidiospores (actinomycetes)  Fragmentation of filaments ANIMATION Bacterial Growth: Overview Reproduction in Prokaryotes

Copyright © 2010 Pearson Education, Inc. Figure 6.12a Binary Fission

Copyright © 2010 Pearson Education, Inc. Figure 6.12b Binary Fission

Copyright © 2010 Pearson Education, Inc. Figure 6.13b Cell Division

Copyright © 2010 Pearson Education, Inc. Figure 6.14 Bacterial Growth Curve

Copyright © 2010 Pearson Education, Inc. Figure 6.15 ANIMATION Bacterial Growth Curve Phases of Growth

Copyright © 2010 Pearson Education, Inc. Figure 6.16 Serial Dilutions

Copyright © 2010 Pearson Education, Inc. Figure 6.17 Plate Counts

Copyright © 2010 Pearson Education, Inc. Figure 6.16 Plate Counts  After incubation, count colonies on plates that have 25–250 colonies (CFUs)

Copyright © 2010 Pearson Education, Inc. Figure 6.18 Counting Bacteria by Membrane Filtration

Copyright © 2010 Pearson Education, Inc. Figure 6.19 Most Probable Number  Multiple tube MPN test  Count positive tubes

Copyright © 2010 Pearson Education, Inc. Figure 6.19 Most Probable Number  Compare with a statistical table.

Copyright © 2010 Pearson Education, Inc. Figure 6.20 Direct Microscopic Count

Copyright © 2010 Pearson Education, Inc. Direct Microscopic Count

Copyright © 2010 Pearson Education, Inc. Figure 6.21 Turbidity

Copyright © 2010 Pearson Education, Inc. Figure 6.21 Turbidity

Copyright © 2010 Pearson Education, Inc. Measuring Microbial Growth Direct Methods  Plate counts  Filtration  MPN  Direct microscopic count Indirect Methods  Turbidity  Metabolic activity  Dry weight