Lecture 9: Microbial Growth and Control (Ch7,8,and 9) Flashcards
The reproductive strategies of eukaryotic microbes
– Asexual and sexual, haploid and diploid
Reproductive strategies of bacteria and Archaea
- Haploid only, asexual- binary fission, budding, filamentous - All must replicate and segregate the genome prior to division
most bacteria divide by
binary fission
what are the two pathways that cycle during binary fission
- DNA replication and partition - Cytokinesis
Reproduction in prokaryotes
- Binary fission - Budding - Conidiospores (actinomycetes) - Fragmentation of filaments
What is generation time
(doubling time) is the time it takes for a population to double
A population of microbes that doubles at a constant rate is an example of
exponential growth
What are the 4 phases of the growth curve
- Lag phase - Exponential (log) phase - Stationary phase - Death phase
What occurs during the lag phase of the growth curve
- Cell synthesizing new components
What occurs during the exponential phase of the growth curve
- growth and division is constant and maximal - population is most uniform in terms of chemical and physical properties during this phase
What occurs during the stationary phase of the growth curve
- closed system population growth eventually ceases, total number of viable cells remains constant. (active cells stop reproducing or reproductive rate is balanced by death rate)
Generation (doubling) time varies depending on
species of microorganism and environmental conditions
The range of generation (doubling) time is from ____ minutes for some bacteria to _______ for some eukaryotic microorganisms
10 minutes for some bacteria to several days for some eukaryotic microorganisms
Direct methods for measuring microbial growth
- Plate counts - Filtration - MPN (most probable number) - Direct microscopic count
What are the indirect methods for measuring microbial growth
- Turbidity - Metabolic activity - Dry weight
how to perform viable counting with spread and pour plate techniques
- Spread and pour plate techniques (the difference is one you spread out and the other you add melted nutrient agar and swirl to mix) ( note that in the spread they only grow on the surface and in the pour method they go on and in the medium) (the pour plate method is used with anaerobes because they can grow under the agar) - Diluted sample of bacteria is spread over solid agar surface or mixed with agar and poured into Petri plate - After incubation the number of organisms are determined by counting the number of colonies multiplied by the dilution factor - Results expressed as colony forming units (CFU)
viable counting via the membrane filter technique
- bacteria form aquatic samples are trapped on membranes - membrane soaked in culture media - colonies grow on membrane - colony count determines number of bacteria in sample
If a microbe cannot be cultured on plate media then
dilutions are made and added to suitable media (turbidity determined to yield the most probable number (MPN))
bacterial growth can be measured with a _____, which measures the absorbance at 600nm
spectrophotome
Do bacteria have limited control over their internal environments
Yes. This is why the environmental factors affect microbial growth
Requirements for growth can be divided among ___ and ____ requirements
Physical and chemical requirements
What are some physical requirements that regulate bacterial growth
- Temperature - pH - Osmotic pressure
What some of the chemical requirements that regulate bacterial growth
- Carbon - nitrogen, sulfur, phosphorous - Trace elements - Oxygen - Organic growth factor
Examples of osmotolerant microorganisms
- Staphylococcus aureus - Saccharomyces rouxii
Examples of Halophile (requires high levels of sodium chloried, usually above about 0.2M, to grow)
- Halobacterium, Dunaliella, Ectothirohodospira
examples of Acidophile (Growth optimum between pH 0 and 5.5)
- Sulfolobus - Picrophilus - Ferroplasma - Acontium
Temperature range of psychrophiles
0- 20 degrees C
temperature range of psychrotrophs
0-35 degrees C
Temperature range of mesophiles
20-45 degrees C
Temperature range of thermophiles
55-85 degrees C
Temperature range of hyperthermophiles
85- 113 degrees C
Danger zone for bacterial growth
- 15-50 degrees C - 60-130 degrees F
What are the cardinal temperatures
Minimum, maximum, and optimum temperature
Adaptations of thermophiles
- Protein structure stabilized by a variety of means: more H bonds, more proline, and chaperones - Histones- like proteins stabilize DNA - Membrane stabilized by variety of means (more saturated, more branched and higher molecular weight lipids, ether linkages (archaea like membranes)
Most bacteria grow between pH ____ and ____
6.5 and 7.5
Molds and yeasts how between pH ___ and ___
5 and 6
______ grow in acidic environments
Acidophiles
What is the optimum pH for acidophilus
pH 0 and pH 5.5
Neutrophils growth is optimum between pH ____ and _____
pH- 5.5 and pH 7
Alkaliphiles (alkalophiles) growth optimum is between
pH 8.5 and pH 11.5
Toxicity is a measure of ______
Osmotic pressure (hypertonic, isotonic, hypotonic)
Hypertonic environments, or an increase in salt or sugar, cause _____
Plasmolysis
Halophiles grow optimally at
NaCl concentration greater than 0.2 M
Extreme Halophiles require NaCl concentration
Greater than 2 M
Chemoheterotrophs use ______ carbon sources
Organic
Autotrophs use _____ carbon sources
CO2
Osmotolerant
Able to grow over wide ranges of water activity or osmotic concentration
Halophile
Requires high levels of sodium chloride, usually above about 0.2 M, to grow
Obligate aerobe
Completely dependent at atmospheric O2 for growth
Facultative anaerobe
Does not require O2 for growth but grows better in its presence
Facultative anaerobe
Does not require O2 for growth but grows better in its presence
Aerotolerant anaerobe
Grows equally well in presence or absence of O2
Obligate anaerobe
Does not tolerate O2 and dies in its presence
Microaerophile
Requires O2 levels between 2-10% for growth and is damaged by atmospheric O2 levels (20%)
Growth more rapid at high hydrostatic pressures
Piezophile (basophile)
Nitrogen requirements
- nitrogen is in most amino acids and proteins - most bacteria decompose proteins - Some bacteria use NH4+ or NO3- - A few bacteria use N2 in nitrogen fixation
Sulfur requirements in bacteria
- in amino acids, thiamine, and biotin - Most bacteria decompose proteins - Some bacteria use SO4^2- or H2S
Phosphorus requirements in bacteria
- in DNA, RNA, ATP, and membranes - PO4^3- in a source of phosphorus
______ is a measure of the ability of a microorganism to grow in the presence or absence of oxygen
Aerotolerance
Oxygen easily reduced to ________
- toxic reactive oxygen species (ROS) - Superoxide radical - Hydrogen peroxide - hydroxyl radical
Aerobes produce protective enzymes such as ____. ____ , and ____ to neutralize toxic reactive oxygen species (ROS)
- Superoxide dismutase (SOD) - Catalase - Peroxidase
what is singlet oxygen: O2-
normal molecular oxygen (O2) boosted to a higher-energy state
Superoxide free radicals: O2 - is reduced by _____ to H2O2 + O2
Superoxide dismutase
Peroxide anion can be removed via enzymes
Catalase or peroxidase
all strict anaerobic microorganisms lack or have very low quantities of
- Superoxide dismutase and catalase
Anaerobes must be grown without ____
O2 (work station with incubator, gaspak anaerobic system)
What are capnophiles
organisms that require higher than normal atmospheric levels of CO2
Olbligate aerobes contain what enzymes
- SOD (+) - catalase (+)
Facultative anaerobes contain what enzymes
- SOD (+) - Catalase (+)
Aerotolerant anaerobes contain what enzymes
- SOD (+) - Catalase (-)
Strict anaerobe enzymes
- SOD (-) - Catalase (-)
Microaerophile enzymes
- SOD (+) - Catalase (+/-) (low levels)
Adversely affected by increased pressure, but not as severely as non tolerant organisms
Barotolerant
Barophilic (peizophilic) organisms
- Require or grow more rapidly in the presence of increased pressure - Change membrane fatty acids to adapt to high pressures
Destruction or removal of all viable organisms
Sterilization
Killing, inhibition, or removal of disease causing (pathogenic) organisms
Disinfection
What are some disinfectants
- agents, usually chemical, used for disinfection - usually used on inanimate objects
Reduction of microbial population to levels deemed safe (based on public health standards)
Sanitization
Prevention of infection of living tissue by microorganisms
Antisepsis
Chemical agents that kill or inhibit growth of microorganisms when applied to tissue
Antiseptics
Use of chemicals to kill or inhibit growth of microorganisms within host tissue
Chemotherapy
Agents that kill microorganisms or inhibit their growth
- cidal agents kill - static agents inhibit growth
- suffix indicating that agent kills
-cide
Kills pathogens and many no pathogens but not necessarily endospores
Germicide
Suffix indicating that agent inhibits growth
-static
What is decimal reduction time
Time to kill 90%
What are persisted cells
- Viable but nonculturable (VBNC) condition - once they recover they may regain the ability to reproduce and cause infection
The pattern of microbial death
- Microorganisms are not killed instantly - population death usually occurs exponentially
Conditions influencing the effectiveness of anti microbial agent activity
- Population size (larger populations take longer to kill than smaller populations) - Population composition (microorganisms differ markedly in their sensitivity to anti microbial agents) - Concentration or intensity of an anti microbial agent (usually higher concentrations kill more rapidly but this relationship is not linear) - Duration of exposure (longer exposure means more organisms killed) - Temperature- higher temperatures usually increase killing - Local environment (pH, viscosity, concentration of organic matter, etc. can profoundly impact effectiveness)(organisms in bio films are less susceptible to many anti microbial agents)
Filtration reduces microbial population or sterilizes solutions of _______ by _______. Also used to reduce microbial populations in air
Heat-sensitive materials by removing microorganisms
Porous membranes with defined pore sizes that remove microorganisms primarily by physical screening
Membrane filters
Filtering air by use of
- Surgical Masks - Cotton plugs on culture vessels - High- efficiency particulate air (HEPA) filters- used in laminar flow biological safety cabinets
Physical control methods
- heat - Radiation
Moist heat destroys
- Viruses, fungi, and bacteria - boiling will not destroy spores and does not sterilize - Degrades nucleic acids, denatures proteins, and disrupts membranes
Moist heat will not destroy ___ and does not ____
Spores and does not sterilize
Steam sterilization is carried out above ____ which requires saturated stream under pressure
100 degrees C
stream sterilization uses _____ and is effective against
autoclave, all types of microorganisms (including spores)
Pasteurization is controlled heating at temperatures
Well below boiling
Pasteurization does not ______ but does _____
sterilize but does kill pathogens present and slow spoilage by reducing the total load of organisms present
Dry Heat Sterilization is ____ effective than moist heat sterilization, requiring ____
less, requiring higher temperatures and longer exposure times (items subjected to 160-170 degrees C for 2 to 3 hours)
Dry heat sterilization ____ cell constituents and _____ proteins
oxidizes, denatures
_______ are used to sterilize inoculating loops used in microbiology laboratories
Bench top incinerators
What UV wavelenth is is mot bactercidal
260 (DNA absorbs)
UV radiation cause ____ dimers preventing replication and transcription
thymine
UV limited to surface surface sterilization because
it does not penetrate glass, dirt films, water, and other substances
Gamma radiation penetrates ____ into objects
deep
Ionizing radiation (gamma radiation) destrosy ____ but is not always effective against
bacterial endospores; viruses
Ionizing radiation is used for _____ and pasterization of ____, _____, ____, _____, and ____
sterilization, antibiotics, hormones, sutures, plastic disposable supplies, and food
What to consider when using a disinfectant
- Concentration of disinfectant
- organic matter (this can interfere with a disinfectant)
- pH
- Time
Phenolics
- commonly used as laboratory and hospital disinfectants
- Act by denaturing proteins and disrupting cell membranes
- Tuberculocidal, effective in presence of organic material, and long lasting
- Disagreeable odor and can cause skin irritation
Alcohols
- Among the most widely used disinfectants and antiseptics
- Two most common are ethanol and isopropanol
- Bactericidal, fungicidal, but not sporicidal
- Inactive some viruses
- Denature proteins and possibly dissolve membrane lipids
Ethanol and isopropanol require
Water
Iodine
- Skin antiseptic
- oxidizes cell constituents and iodinates proteins
- At high concetrations may kill spores
- Skin damage, staining, and allergies can be a problem
- Iodophore
- Iodine complexed with organic carrier
- released slowly to minimize skin burns
Chlorine
- Oxidizes cell constituents
- Important in disinfection of water supplies and swimming pools, used in dairy and food industries, effective household disinfectant
- destroys vegetative bacteria and fungi
- Chlorine gas is sporicidal
- Can react with organic matter to form carcinogenic compounds
Heavy metals
- Ions of mecury, silver, arsenic, zinc, and copper
- Effective but usually toxic
- Combine with and inactivate proteins; may also precipitate proteins
Quaternary Ammonium Compounds
- Detergents that have antimicrobial activity and are effective disinfectans (amphipathic organic
- Cationic detergents are effective disinfectants
- Kill most bacteria, but not M. tuberculosis or endospores
- Safe and easy to use, inactivated by hard water and soap
Aldehydes
- Commonly used agents are formaldehyde and glutaraldehyde
- highly reactive molecuels
- Sporicidal and can be used as chemical sterilants
- Combine with and inactivate nucleic acids and proteins
Phenol Coefficient Test
- Potency of a disinfectant is compared to that of phenol
- useful for screening but may be misleading
Dilution test
dilution test
determines rate at which selected bacteria are destroyed by various chemical agents
Method for evaluating the effectiveness fo disinfectants
- Metal rings dipped in test bacteria are dried
- Dried cultures are placed in disinfectant for 10 minutes at 20 degrees C
- Rings are transferred to culture media to determine whether bacteria survived treatment
normal in-use testing
-testing done using conditions that approximate normal use of disinfectant
Streptomycin
- anitbiotic active against tuberculosis
General characteristics of antimicrobial drugs
- Selective toxicity
- Therapeutic dose
- Toxic dose
- Therapeutic index
Selective toxicity
ability of drug to kill or inhibit pathogen while damaging host as little as possible
Drug level required for clinical treatment
Therapeutic dose
Drug level at which drug becomes too toxic for patient (produces side efects)
Toxic dose
Ratio of toxic dose to therapeutic dose
Therapeutic index
