16/10/2013

The Requirements for Growth:

Microbial Growth
ninth edition TORTORA  FUNKE  CASE Physical Requirements
MICROBIOLOGY  Microbial growth is the increase in number of cells,  Temperature
an introduction not cell size  Minimum growth temperature
 Optimum growth temperature

Microbial Growth  Maximum growth temperature

PowerPoint® Lecture Slide Presentation prepared by Christine L. Case

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Temperature Psychrotrophs Psychrotrophs

 Grow between 0°C and 20-30°C

 Cause food spoilage

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The Requirements for Growth: The Requirements for Growth: The Requirements for Growth:
Physical Requirements Physical Requirements Physical Requirements
 pH  Osmotic pressure
 Most bacteria grow between pH 6.5 and 7.5  Hypertonic environments, increase salt or sugar,
 Molds and yeasts grow between pH 5 and 6 cause plasmolysis
 Acidophiles grow in acidic environments  Extreme or obligate halophiles require high osmotic
pressure
 Facultative halophiles tolerate high osmotic pressure

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The Requirements for Growth: The Requirements for Growth: The Requirements for Growth:
Chemical Requirements Chemical Requirements Chemical Requirements
 Nitrogen
 Carbon  In amino acids and proteins  Trace elements
 Structural organic molecules, energy source  Most bacteria decompose proteins  Inorganic elements required in small amounts
 Some bacteria use NH4+ or NO3–
 Chemoheterotrophs use organic carbon sources  Usually as enzyme cofactors
 A few bacteria use N2 in nitrogen fixation
 Autotrophs use CO2
 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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The Requirements for Growth: The Requirements for Growth:

Toxic Forms of Oxygen
Chemical Requirements Chemical Requirements
 Oxygen (O2)  Singlet oxygen: O2 boosted to a higher-energy state  Organic growth factors
 Superoxide free radicals: O2–  Organic compounds obtained from the environment
 Vitamins, amino acids, purines, and pyrimidines

 Peroxide anion: O22–

 Hydroxyl radical (OH)

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Culture Media Agar Culture Media

 Culture medium: Nutrients prepared for microbial  Complex polysaccharide  Chemically defined media: Exact chemical composition
growth  Used as solidifying agent for culture media in Petri is known
 Sterile: No living microbes plates, slants, and deeps  Complex media: Extracts and digests of yeasts, meat,
 Inoculum: Introduction of microbes into medium  Generally not metabolized by microbes or plants
 Culture: Microbes growing in/on culture medium  Liquefies at 100°C  Nutrient broth
 Solidifies ~40°C  Nutrient agar

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Culture Media Anaerobic Culture Methods Anaerobic Culture Methods

 Reducing media  Anaerobic

 Contain chemicals (thioglycollate or oxyrase) that jar
combine O2
 Heated to drive off O2

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Anaerobic Culture Methods Capnophiles Require High CO2 Selective Media

 Anaerobic  Candle jar  Suppress unwanted

chamber microbes and
encourage desired
microbes.

 CO2-packet

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Differential Media Enrichment Media

 Make it easy to distinguish colonies of different
microbes.
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 Encourages growth of desired microbe
 A pure culture contains only one species or strain.
 Assume a soil sample contains a few phenol-degrading
 A colony is a population of cells arising from a single
bacteria and thousands of other bacteria
cell or spore or from a group of attached cells.
 Inoculate phenol-containing culture medium with the
 A colony is often called a colony-forming unit (CFU).
soil and incubate
 Transfer 1 ml to another flask of the phenol medium
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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Streak Plate Preserving Bacteria Cultures Reproduction in Prokaryotes

 Deep-freezing: –50°to –95°C  Binary fission

 Lyophilization (freeze-drying): Frozen (–54° to –72°C)  Budding
and dehydrated in a vacuum  Conidiospores (actinomycetes)
 Fragmentation of filaments

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Binary Fission

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

cells:

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Direct Measurements of Microbial Growth

 Plate counts: Perform serial dilutions of a sample

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Plate Count Plate Count Direct Measurements of Microbial Growth

 After incubation, count colonies on plates that have
 Inoculate Petri  Filtration
25-250 colonies (CFUs)
plates from serial
dilutions

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Direct Measurements of Microbial Growth Direct Measurements of Microbial Growth Direct Measurements of Microbial Growth

 Multiple tube  Direct microscopic count

MPN test.
 Count positive
tubes and
compare to
statistical
MPN table.

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Estimating Bacterial Numbers

Measuring Microbial Growth
by Indirect Methods
 Turbidity Direct methods Indirect methods
 Plate counts  Turbidity The Control of Microbial Growth
 Filtration  Metabolic activity Terms to Remember:
 MPN  Dry weight  Sepsis (Greek word) refers to microbial
 Direct microscopic count contamination.
 Dry weight  Asepsis is the absence of significant
contamination.
 Aseptic surgery techniques prevent microbial
contamination of wounds.
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Terminology Effectiveness of antimicrobial treatment

depends on:
 Sterilization: Removal of all microbial life
 When heated or treated with –cidal agents, bacterial  Number of initial
 Commercial Sterilization: Killing C. botulinum endospores with heat (not
populations die at a constant logarithmic rate. microbes
all organisms!)
 Disinfection: Removal of pathogens (not all organisms)  Environment
 Antisepsis: Removal of pathogens from living tissue (not disinfection!) (organic matter-
 Degerming: Removal of microbes from a limited area (such as
fats and proteins
swabbing with alcohol-doesn’t kill bacteria-only removes)
 Sanitization: Lower microbial counts on eating utensils (does not protect;
completely get rid of organisms) temperature,
 Biocide/Germicide: Kills microbes
biofilms-protect)
 Bacteriostasis: Inhibiting, not killing, microbes
 Review table 7.1
 Time of
Figure 7.1a Figure 7.1b
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exposure
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 Microbial
characteristics
(vegetative or
endospore)
Actions of Microbial Control Agents Physical Methods of Microbial Control

 Alternation of membrane permeability  Heat

 Damage to proteins  Thermal death point (TDP): Lowest temperature at

 (denaturation by chemicals and heating) which all cells in a culture are killed in 10 min.

 Damage to nucleic acids  Thermal death time (TDT): Time to kill all cells in a

 (heat, radiation, chemicals) culture

 Decimal reduction time (DRT): Minutes to kill 90% of
a population at a given temperature

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Heat Physical Methods of Microbial Control Physical Methods of Microbial Control

 Moist heat denatures

 Pasteurization reduces spoilage organisms and pathogens  Dry Heat Sterilization kills by oxidation
(coagulates) proteins
 Boiling does not kill (different milk products require different temp and time)- for  Flaming (loops)
endospores or many milk, the industry uses the phosphatase test
 Incineration (paper, bags, dressings)
viruses RAPIDLY
 Equivalent treatments
 Autoclave: Steam  Hot-air sterilization (surgical instruments)
under pressure-  63°C for 30 min
temperature above  High-temperature short-time 72°C for 15 sec
boiling (121-6 C)-15
Hot-air Autoclave
 Ultra-high-temperature: 140°C for <1 sec (can be stored
min –will kill! (but not Equivalent treatments 170˚C, 2 hr 121˚C, 15 min
prions) without refrigeration)
 Thermoduric organisms survive
Steam must contact the surface for this to work
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Physical Methods of Microbial Control Physical Methods of Microbial Control

 Filtration removes microbes (sterilize heat sensitive materials such as
some culture medias, vaccines, and antibiotic solutions) 0.1 mm thick- Effect depends on its wavelength (shorter-more energy),
Intensity and duration
pores 0.22um or 0.45um –mycoplasmas are a problem since they do
not have walls  Radiation damages DNA
 HEPA filters in operating theaters and rooms occupied by burn patients  Ionizing radiation form hydroxyl radicals (X rays, gamma
remove 0.3 um or larger
rays, electron beams) short wavelength
 Low temperature inhibits microbial growth
 Nonionizing radiation (UV) longer wavelength-form
 Refrigeration (Listeria is the exception!)
 Deep freezing thymine dimers (used to disinfect vaccines and other
 Lyophilization (freeze then dry) medical products)
 High pressure denatures proteins  (Microwaves kill by heat; not especially antimicrobial)
 Desiccation prevents metabolism
 Osmotic pressure causes plasmolysis
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Chemical Methods of Microbial Control Chemical Methods of Microbial Control Chemical Methods of Microbial Control
A common problem is that no single disinfectant is appropriate • Evaluating a disinfectant
For all circumstances
 Evaluating a disinfectant • Disk-diffusion method
 Principles of effective disinfection (list the factors  Use-dilution test
related to effective disinfection) 1. Metal rings dipped in test bacteria are dried
 Concentration of disinfectant 2. Dried cultures placed in disinfectant for 10
 Organic matter (protects organisms against its min at 20°C
action) 3. Rings transferred to culture media to
 pH determine whether bacteria survived
 Time treatment
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Types of Disinfectants Types of Disinfectants Types of Disinfectants

• Lister: Phenol  Halogens. Iodine, Chlorine
• Phenolics. Lysol  Biguanides. Chlorhexidine (microbial control on skin and  Oxidizing agents
• Bisphenols.
Hexachlorophene, Triclosan mucous membranes)  Bleach is hypochlorous acid (HOCl) is the most effective form of
(especially effective against
gram +) however, > in  Disrupt plasma membranes chlorine because it is electrically neutral
resistant bacteria  2 drops of bleach in a liter of water for 30 min-ready to drink!
 Combined with a detergent or alcohol, it is used for
• Disrupt plasma  Chloramines are used for water –will kill aquarium fish and so
membranes; especially surgical hand scrubs; high affinity for skin; blocks
mycobacteria must be neutralized
• Suitable for disinfecting enzymes for lipid synthesis  Iodine is effective against all kinds of bacteria, many endospores,
pus, saliva, and feces-
good surface  Biocidal against most vegetative bacteria and yeasts various fungi, and some viruses-impairs protein synthesis and alters
disinfectant
 Endospore, protozoan cysts, mycobacteria resistant cell membranes; available as a tincture or an iodophor (iodine and
organic molecule-Betadine)
 Only viruses affected are certain enveloped types
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Types of Disinfectants Types of Disinfectants Types of Disinfectants

• Alcohols. Ethanol,  Heavy Metals. Ag, Hg, Cu (biocidal or antiseptic)

isopropanol  Oligodynamic action (small amounts are active)  Surface-Active Agents or Surfactants (decrease
• Denature proteins,  Denature proteins (combines with sulfydryl groups)
dissolve lipids surface tension)
 1% AgNO3 was used as an antiseptic for the eyes of newborns to Good Degerming AGENTS
Soap (not antiseptic)
• Kills bacteria and protect against gonorrheal infections (emulsification)
fungi but not Sanitizing (dairy utensils)
 Renewed interested as a result of antibiotic-resistant bacteria Acid-anionic detergents
noncorrosive and fast acting
endospores and
nonenveloped  Applied on the surface or within catheters Bactericidal (gram +), Also
fungicidal, amoebicidal, virucidal
viruses  Mercuric chloride-long history of use as a disinfectant-mainly (enveloped) Denature proteins,
Quarternary ammonium compounds
bacteriostatic Cationic detergents (surface active) disrupt plasma membrane
Pseudomonas can grow in these
 Copper –destroy green algae agents
 Zinc-mouthwashes, on roofs, in paints

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Types of Disinfectants Types of Disinfectants Types of Disinfectants

 Chemical Food Preservatives
 Organic Acids
 Inhibit metabolism
 Sulfur dioxide in wine (Homer’s Odyssey mentions its use)
 Sorbic acid, benzoic acid, calcium propionate
 Control molds and bacteria in foods and cosmetics
 Nitrite prevents endospore germination such as botulism
 (nitrite + amino acids= carcinogen?)
 Antibiotics. Nisin (bacteriocin) and natamycin (antifungal) prevent
spoilage of cheese
 Aldehydes (effective antimicrobial action)  Gaseous Sterilants (large items or items destroyed by heat such as
Petri dishes, mattresses)
 Inactivate proteins by cross-linking with functional
 The proteins’ labile hydrogens are replaced by alkyl groups
groups (–NH2, –OH, –COOH, —SH)  Denature proteins
 Glutaraldehyde (respiratory equipment –used as 2%  Ethylene oxide kills all microbes and endospores but requires a
lengthy exposure period of 4 to 18 hours—toxic and explosive in its
solution-Cidex) bactericidal, tuberculocidal, virucidal
pure form so usually mixed with a nonflamable gas such as
in 10 min and sporicidal in 3 to 10 hours)—one of
nitrogen; highly penetrating (used to sterilize spacecraft send to land
the few agents that is sterilizing on the moon and Mars)
 Formaldehyde-embalming agent  Disadvantage –suspected carcinogen

 Alternative: ortho-phthaldehyde or OPA

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Types of Disinfectants Microbial Characteristics and Microbial Microbial Characteristics and Microbial
Control Sodium hydroxide and 136C
Control
 Peroxygens Autoclaving for 60 min
 Oxidizing agents
 O3, H2O2, peracetic acid Chemical agent Effectiveness against
 Ozone is often used to supplement chlorine in the disinfection of Endospores Mycobacteria
water because it helps neutralize tastes and odors
Phenolics Poor Good
 Hydrogen peroxide may slow wound healing; effectively disinfects
Outer membrane-resistant Quats None None
objects such as packing materials and contact lens
To biocides Chlorines Fair Fair
 Benzoyl peroxide- works against anaerobic bacteria
Alcohols Poor Good
 Peracetic acid-sterilant, sporicide within 30min—disinfect food
Glutaraldehyde Fair Good
processing and medical equipment
Lipid envelopes >
Sensitivity to lipid-
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Soluble Figure 7.11 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings