Microbial Growth - Kuliah 4

ninth edition TORTORA  FUNKE  CASE
MICROBIOLOGY
an introduction
Microbial Growth
PowerPoint® Lecture Slide Presentation prepared by Christine L. Case

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings
Microbial Growth
 Microbial growth is the increase in number of cells,
not cell size

The Requirements for Growth:
Physical Requirements
 Temperature
 Minimum growth temperature
 Optimum growth temperature
 Maximum growth temperature

Temperature
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.1
Psychrotrophs
 Grow between 0°C and 20-30°C
 Cause food spoilage

Psychrotrophs
 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, increase salt or sugar,
cause plasmolysis
 Extreme or obligate halophiles require high osmotic
pressure
 Facultative halophiles tolerate high osmotic pressure

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

 Nitrogen
 In amino acids and proteins
 Most bacteria decompose proteins
 Some bacteria use NH4+ or NO3–
 A few bacteria use N2 in nitrogen fixation
 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
 Trace elements
 Inorganic elements required in small amounts
 Usually as enzyme cofactors

 Oxygen (O2)
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Table 6.1
Toxic Forms of Oxygen
 Singlet oxygen: O2 boosted to a higher-energy state
 Superoxide free radicals: O2–
 Peroxide anion: O22–
 Hydroxyl radical (OH)

 Organic growth factors
 Organic compounds obtained from the environment
 Vitamins, amino acids, purines, and pyrimidines

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 ~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

Culture Media
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Tables 6.2, 6.4
Anaerobic Culture Methods
 Reducing media
 Contain chemicals (thioglycollate or oxyrase) that
combine O2
 Heated to drive off O2

 Anaerobic
jar
 Anaerobic
chamber
Capnophiles Require High CO2
 Candle jar
 CO2-packet
Selective Media
 Suppress unwanted
microbes and
encourage desired
microbes.
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.9b–c
Differential Media
 Make it easy to distinguish colonies of different
microbes.
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.9a
Enrichment Media
 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
 Transfer 1 ml to another flask of the phenol medium
and incubate
 Only phenol-metabolizing bacteria will be growing
 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).

Streak Plate
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.10a–b
Preserving Bacteria Cultures
 Deep-freezing: –50°to –95°C
 Lyophilization (freeze-drying): Frozen (–54° to –72°C)
and dehydrated in a vacuum

Reproduction in Prokaryotes
 Binary fission
 Budding
 Conidiospores (actinomycetes)
 Fragmentation of filaments

Binary Fission
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.12b
 If 100 cells growing for 5 hours produced 1,720,320
cells:

Direct Measurements of Microbial Growth
 Plate counts: Perform serial dilutions of a sample
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.15, step 1
Plate Count
 Inoculate Petri
plates from serial
dilutions
Plate Count
 After incubation, count colonies on plates that have
25-250 colonies (CFUs)
 Filtration
 Multiple tube
MPN test.
 Count positive
tubes and
compare to
statistical
MPN table.
 Direct microscopic count

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.19, steps 1, 3
Estimating Bacterial Numbers
by Indirect Methods
 Turbidity
Measuring Microbial Growth
Direct methods Indirect methods
 Plate counts  Turbidity
 Filtration  Metabolic activity
 MPN  Dry weight
 Direct microscopic count
 Dry weight

The Control of Microbial Growth
Terms to Remember:
 Sepsis (Greek word) refers to microbial
contamination.
 Asepsis is the absence of significant contamination.
 Aseptic surgery techniques prevent microbial
contamination of wounds.

Terminology
 Sterilization: Removal of all microbial life
 Commercial Sterilization: Killing C. botulinum endospores with heat (not
all organisms!)
 Disinfection: Removal of pathogens (not all organisms)
 Antisepsis: Removal of pathogens from living tissue (not disinfection!)
 Degerming: Removal of microbes from a limited area (such as
swabbing with alcohol-doesn’t kill bacteria-only removes)
 Sanitization: Lower microbial counts on eating utensils (does not
completely get rid of organisms)
 Biocide/Germicide: Kills microbes
 Bacteriostasis: Inhibiting, not killing, microbes
 Review table 7.1
 When heated or treated with –cidal agents, bacterial
populations die at a constant logarithmic rate.
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 7.1a
Effectiveness of antimicrobial treatment
depends on:
 Number of initial
microbes
 Environment
(organic matter-
fats and proteins
protect;
temperature,
biofilms-protect)
 Time of
exposure
Actions of Microbial Control Agents
 Alternation of membrane permeability

 Damage to proteins
 (denaturation by chemicals and heating)
 Damage to nucleic acids
 (heat, radiation, chemicals)

Physical Methods of Microbial Control
 Heat
 Thermal death point (TDP): Lowest temperature at
which all cells in a culture are killed in 10 min.
 Thermal death time (TDT): Time to kill all cells in a
culture
 Decimal reduction time (DRT): Minutes to kill 90% of
a population at a given temperature

Heat
 Moist heat denatures

(coagulates) proteins
 Boiling does not kill
endospores or many
viruses RAPIDLY
 Autoclave: Steam
under pressure-
temperature above
boiling (121-6 C)-15
min –will kill! (but not
prions)
Steam must contact the surface for this to work

 Pasteurization reduces spoilage organisms and pathogens

(different milk products require different temp and time)- for
milk, the industry uses the phosphatase test
 Equivalent treatments
 63°C for 30 min
 High-temperature short-time 72°C for 15 sec
 Ultra-high-temperature: 140°C for <1 sec (can be stored
without refrigeration)
 Thermoduric organisms survive

 Dry Heat Sterilization kills by oxidation

 Flaming (loops)
 Incineration (paper, bags, dressings)
 Hot-air sterilization (surgical instruments)
Hot-air
Autoclave
Equivalent treatments
170˚C, 2 hr

Effect depends on its wavelength (shorter-more energy),
Intensity and duration
 Radiation damages DNA
 Ionizing radiation form hydroxyl radicals (X rays, gamma
rays, electron beams) short wavelength
 Nonionizing radiation (UV) longer wavelength-form
thymine dimers (used to disinfect vaccines and other
medical products)
 (Microwaves kill by heat; not especially antimicrobial)

Chemical Methods of Microbial Control
A common problem is that no single disinfectant is appropriate
For all circumstances
 Principles of effective disinfection (list the factors

related to effective disinfection)
 Concentration of disinfectant
 Organic matter (protects organisms against its
action)
 pH
 Time

 Evaluating a disinfectant
 Use-dilution test
1. Metal rings dipped in test bacteria are dried
2. Dried cultures placed in disinfectant for 10
min at 20°C
3. Rings transferred to culture media to
determine whether bacteria survived
treatment

• Evaluating a disinfectant
• Disk-diffusion method
Types of Disinfectants
• Lister: Phenol
• Phenolics. Lysol
• Bisphenols. Hexachlorophene,
Triclosan (especially effective
against gram +) however, > in
resistant bacteria
• Disrupt plasma
membranes; especially
mycobacteria
• Suitable for disinfecting
pus, saliva, and fecesgood
surface disinfectant
 Biguanides. Chlorhexidine (microbial control on skin and

mucous membranes)
 Disrupt plasma membranes
 Combined with a detergent or alcohol, it is used for
surgical hand scrubs; high affinity for skin; blocks
enzymes for lipid synthesis
 Biocidal against most vegetative bacteria and yeasts
 Endospore, protozoan cysts, mycobacteria resistant
 Only viruses affected are certain enveloped types

 Halogens. Iodine, Chlorine
 Oxidizing agents
 Bleach is hypochlorous acid (HOCl) is the most effective form of
chlorine because it is electrically neutral
 2 drops of bleach in a liter of water for 30 min-ready to drink!
 Chloramines are used for water –will kill aquarium fish and so
must be neutralized
 Iodine is effective against all kinds of bacteria, many endospores,
various fungi, and some viruses-impairs protein synthesis and alters
cell membranes; available as a tincture or an iodophor (iodine and
organic molecule-Betadine)

• Alcohols. Ethanol,
isopropanol
• Denature proteins,
dissolve lipids
• Kills bacteria and
fungi but not
endospores and
nonenveloped
viruses
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Table 7.6
 Heavy Metals. Ag, Hg, Cu (biocidal or antiseptic)
 Oligodynamic action (small amounts are active)
 Denature proteins (combines with sulfydryl groups)
 1% AgNO3 was used as an antiseptic for the eyes of newborns to
protect against gonorrheal infections
 Renewed interested as a result of antibiotic-resistant bacteria
 Applied on the surface or within catheters
 Mercuric chloride-long history of use as a disinfectant-mainly
bacteriostatic
 Copper –destroy green algae
 Zinc-mouthwashes, on roofs, in paints

 Surface-Active Agents or Surfactants (decrease

surface tension)
Good Degerming AGENTS
Soap (not antiseptic) (emulsification)
Sanitizing (dairy utensils)
Acid-anionic detergents noncorrosive and fast acting
Bactericidal (gram +), Also
fungicidal, amoebicidal, virucidal
Quarternary ammonium compounds (enveloped) Denature proteins,
Cationic detergents (surface active) disrupt plasma membrane
Pseudomonas can grow in these
agents

 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)

 Inactivate proteins by cross-linking with functional
groups (–NH2, –OH, –COOH, —SH)
 Glutaraldehyde (respiratory equipment –used as 2%
solution-Cidex) bactericidal, tuberculocidal, virucidal
in 10 min and sporicidal in 3 to 10 hours)—one of
the few agents that is sterilizing
 Formaldehyde-embalming agent
 Alternative: ortho-phthaldehyde or OPA
 Gaseous Sterilants (large items or items destroyed by heat such as
Petri dishes, mattresses)
 The proteins’ labile hydrogens are replaced by alkyl groups
 Denature proteins
 Ethylene oxide kills all microbes and endospores but requires a
lengthy exposure period of 4 to 18 hours—toxic and explosive in its
pure form so usually mixed with a nonflamable gas such as
nitrogen; highly penetrating (used to sterilize spacecraft send to
land on the moon and Mars)
 Disadvantage –suspected carcinogen

 Peroxygens
 Oxidizing agents
 O3, H2O2, peracetic acid
 Ozone is often used to supplement chlorine in the disinfection of
water because it helps neutralize tastes and odors
 Hydrogen peroxide may slow wound healing; effectively disinfects
objects such as packing materials and contact lens
 Benzoyl peroxide- works against anaerobic bacteria
 Peracetic acid-sterilant, sporicide within 30min—disinfect food
processing and medical equipment

Microbial Characteristics and Microbial
Control Sodium hydroxide and 136C
Autoclaving for 60 min
Outer membrane-resistant
To biocides
Lipid envelopes >

Sensitivity to lipid-
Soluble Figure 7.11
Microbial Characteristics and Microbial
Control
Chemical agent Effectiveness against

Endospores Mycobacteria
Phenolics Poor Good
Quats None None
Chlorines Fair Fair
Alcohols Poor Good
Glutaraldehyde Fair Good

Microbial Growth - Kuliah 4

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ninth edition TORTORA  FUNKE  CASE

PowerPoint® Lecture Slide Presentation prepared by Christine L. Case

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

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

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

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

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

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

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

 Peroxide anion: O22–

 Hydroxyl radical (OH)

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

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

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

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

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

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

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

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

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

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

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

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

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

 Alternation of membrane permeability

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

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

 Moist heat denatures

Steam must contact the surface for this to work

 Pasteurization reduces spoilage organisms and pathogens

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

 Dry Heat Sterilization kills by oxidation

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

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

 Principles of effective disinfection (list the factors

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

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

 Biguanides. Chlorhexidine (microbial control on skin and

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

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

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

 Surface-Active Agents or Surfactants (decrease

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

 Chemical Food Preservatives

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

 Aldehydes (effective antimicrobial action)

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

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

Lipid envelopes >

Chemical agent Effectiveness against

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

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