BACTERIAL NUTRITION Major (Macro) elements. Carbon (C), Nitrogen (N), Oxygen (O), Phosphorous (P) and Sulfur (S). Makes up over 95% of cell dry weight

BACTERIAL NUTRITION

• Major (Macro) elements. Carbon (C), Nitrogen (N), Oxygen (O), Phosphorous (P) and Sulfur (S). Makes up over 95% of cell dry weight.

• These 5 macroelements are the elemental components of the 4 macromolecules of life: nucleic acids, proteins, lipids and carbohydrates.

Bacterial Nutrition

• Other Macro Elements: Potassium (K), Iron (Fe), Calcium (Ca),and Magnesium (Mg) all required in the ionic form are used in a variety of critical functions of the cell and are in mg quantities

• Functions: heat resistance, enzyme activity, respiration, maintainance of ionic strength.

Bacterial Nutrition

• Trace Elements: Manganese (Mn), Zinc (Zn), Cobalt (Co),Molybdenum(Mo) Copper (Cu) and Nickel (Ni). Required in ug quantities per liter.

• Required for enzyme function.

Growth Factors

1. Three classes of growth factorsa. Amino acids-protein synthesis

b. Purines and pyrimidines-nucleic acid synthesis

c. Vitamins-usually used as enzyme cofactors

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Table 6.4


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Table 6.2


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Nutritional Types of Organisms

• based on energy source– phototrophs use light– chemotrophs obtain energy from

oxidation of chemical compounds

• based on electron source– lithotrophs use reduced inorganic

substances– organotrophs obtain electrons

from organic compounds


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Classes of Major Nutritional Types

• majority of microorganisms known– photolithoautotrophs (photoautotrophs)– chemoorganoheterotrophs

(chemoheterotrophs) • majority of pathogens

• ecological importance– photoorganoheterotrophs– chemolithoautotrophs– chemolithotrophs


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Table 6.3


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Figure 6.1


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Microorganisms May Change Nutritional Type

• some have great metabolic flexibility based on environmental requirements

• provides distinct advantage if environmental conditions change frequently


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Figure 6.2


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Sources of Nitrogen

• organic molecules

• ammonia

• nitrate via assimilatory nitrate reduction

• nitrogen gas via nitrogen fixation

Forms of Nitrogen

• Preformed organic nitrogen compounds such as amino acids or purines and pyrimidines. Deamination reactions convert the nitrogen groups to ammonia which is then assimilated into biomass

Nutritional Forms of Nitrogen Used by Microorganisms

• Ammonia is the most commonly used for of nitrogen by microorganisms

• NH4+ is incorporated into glutamate by glutamate dehydrogenase when in high concentrations.

• Glutamate synthase-glutamine synthetase system is used when NH4 is in low conc.

Nutritional Forms of Nitrogen Used by Microorganisms

• Many bacteria can use nitrate as a sole nitrogen source

• Nitrate is reduced to ammonia by the assimilatory nitrate reduction process

• ammonia is incorporated as previously described

Nutritional Forms of Phosphorous Used by Microorganisms

• Most use a form of Phosphate (PO4=)

• In growth medium it is usually incorporated as phosphate buffer system

Nutritional Forms of Sulfur Used by Microorganisms

• Most organisms incorporate sulfur as sulfate (SO4-) which is then reduced

• In growth medium it is often incorporated as a salt of ammonia (ammonium sulfate) or magnesium (Magnesium sulfate)

• Used mainly to make the sulfur amino acids methionine and cystiene

Terms relating to nutritional requirements of a microorganism• Prototroph-An organism that requires the

same nutrients for growth as the majority of naturally occuring members of its species.

• Auxotroph-A mutated prototroph that lacks the ability to synthesize an essential nutrient therefore must obtain it or a precursor from its surrounding environment.


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Culture Media• need to grow, transport, and store

microorganisms in the laboratory• culture media is solid or liquid

preparation • must contain all the nutrients

required by the organism for growth• classification

– chemical constituents from which they are made

– physical nature– function

Culture Media

• Defined Medium- All components of the medium are known and in a specific concentration.

• Minimal Salts media are composed of the minimum growth requirements for a given organism


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Defined or Synthetic Media

• all components and their concentrations are known

Table 6.6

Complex media

• Generally an organic rich medium of unknown composition

• routinely used because allows fast growth and supports growth of many different organnisms


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Some Complex Media Components

• peptones– protein hydrolysates prepared by partial

digestion of various protein sources

• extracts– aqueous extracts, usually of beef or

yeast

• agar– sulfated polysaccharide used to solidify

liquid media; most microorganisms cannot degrade it


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Functional Types of Media

Supportive

Enriched

Selective

Differential

Selective Media

• This type of media favors growth of a particular kind of organism and selects against others: eosin methylene blue, endoagar, MacConkey’s all used to detect enterics

Selective media

• Eosin methylene blue (EMB) eosin Y and methylene blue inhibit gram positive organisms and react with enteric end products to give a green sheen to colonies that produce acid from lactose

• MacConkey –selective ingredients are bile salts and crystal violet. Colonies that produce acid are red

Differential Media

• Distinguishes between certain groups of bacteria by a color reaction with a dye or some other characteristics

• Blood agar for hemolytic streptococcus

Fig. 23.18a Streptococcus pyogenes

Beta hemolysis

Fig. 23.18b

Streptococcus pnemoniae

Alpha hemolysis

Fig. 23.18c Staphylococcus epidermidis

No hemolysis

Cardinal Growth Temperatures

For any given organism there is a minimum growth temperature, optimum growth temperature and maximum growth temperatures. These are known as the cardinal temperatures.


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Isolation of Pure Cultures

• population of cells arising from a single cell developed by Robert Koch

• allows for the study of single type of microorganism in mixed culture

• spread plate, streak plate, and pour plate are techniques used to isolate pure cultures

Spread plate techniqueFig. 5.10a

Spread plate results

Fig. 5.10b

Streak plate techniqueFig. 5.11

Pour Plate Technique


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Microbial Growth on Solid Surfaces

• colony characteristics that develop when microorganisms are grown on agar surfaces aid in identification

• microbial growth in biofilms is similar

• differences in growth rate from edges to center is due to – oxygen, nutrients, and toxic products– cells may be dead in some areas


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Figure 6.13

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43

The Influence of Environmental Factors on

Growth• most organisms grow in fairly

moderate environmental conditions

• extremophiles– grow under harsh conditions that

would kill most other organisms


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Table 7.4


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Solutes and Water Activity• changes in osmotic concentrations

in the environment may affect microbial cells– hypotonic solution (lower osmotic

concentration) • water enters the cell• cell swells may burst

– hypertonic (higher osmotic concentration)

• water leaves the cell • membrane shrinks from the cell wall

(plasmolysis) may occur


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Microbes Adapt to Changes in Osmotic Concentrations

• reduce osmotic concentration of cytoplasm in hypotonic solutions– mechanosensitive (MS) channels in

plasma membrane allow solutes to leave

• increase internal solute concentration with compatible solutes to increase their internal osmotic concentration in hypertonic solutions– solutes compatible with metabolism and

growth


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Extremely Adapted Microbes

• halophiles– grow optimally in the presence of NaCl

or other salts at a concentration above about 0.2M

• extreme halophiles– require salt concentrations of 2M and

6.2M– extremely high concentrations of

potassium– cell wall, proteins, and plasma

membrane require high salt to maintain stability and activity


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Effects of NaCl on Microbial Growth

• halophiles– grow optimally

at >0.2 M

• extreme halophiles– require >2 M

Figure 7.24

Solute Concentration and Growth

Halophiles• Require levels of NaCl between 2.8 and 6.2 M to grow.• Extreme halophiles like Halobacterium require 6.2M which

approaches saturation. Many are Archeabacteria• Structure of proteins and membranes have been

significantly altered.• Generally they accumulate huge concentrations of

potassium in order to remain hypertonic to environment.• Enzymes, ribosomes protein structure requires high

concentrations of sodium.

Other Adaptive techniques

• Increase internal K+ concentration in some cases to 7M (Halobacterium)

• Compatible solutes: chemicals that can be kept at high concentrations without interfering with metabolism: glycerol, choline, betaine or proline and glutamate.


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pH

• measure of the relative acidity of a solution

• negative logarithm of the hydrogen ion concentration

Figure 7.25


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pH

• acidophiles– growth optimum between pH 0 and pH 5.5

• neutrophiles– growth optimum between pH 5.5 and pH 7

• alkaliphiles (alkalophiles)– growth optimum between pH 8.5 and pH

11.5


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pH• most microbes maintain an internal pH

near neutrality– the plasma membrane is impermeable to

proton– exchange potassium for protons

• acidic tolerance response – pump protons out of the cell– some synthesize acid and heat shock proteins

that protect proteins

• many microorganisms change the pH of their habitat by producing acidic or basic waste products


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Temperature

• microbes cannot regulate their internal temperature

• enzymes have optimal temperature at which they function optimally

• high temperatures may inhibit enzyme functioning and be lethal


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Temperature

• organisms exhibit distinct cardinal growth temperatures– minimal– maximal– optimal

Figure 7.26


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Temperature Ranges for Microbial Growth

• psychrophiles – 0o C to 20o C

• psychrotrophs – 0o C to 35o C

• mesophiles – 20o C to 45o C

• thermophiles – 55o C to 85o C

• hyperthermophiles – 85o C to 113o C

Temperature and Growth

Psychrophiles- Most belong to genera Psuedomonas,Flavobacterium,Achromobacter and

Alcaligenes.

• Proteins function best at low temperatures• membranes contain high levels of unsaturated fatty

acids

Temperature and Growth

Thermophiles grow at temperatures of 55C or higher

• Obligate thermophiles can only grow above temperatures of 45C. Bacillus stearothermophilus.

• Facultative thermophiles can grow at temperatures lower than 45C but optimal is at 55C. Bacillus coagulans

• Hyperthermophiles grow optimally between 80C and 110 C and usually do not grow below 55C. Pyrococcus abyssi.


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Adaptations of Thermophiles• protein structure inherently heat

stable stabilized by a variety of means – e.g., more H bonds– e.g., more proline– e.g., chaperones

• histone-like proteins stabilize DNA• membrane stabilized by variety of

means– e.g., more saturated, more branched

and higher molecular weight lipids– e.g., ether linkages (archaeal

membranes)

Oxygen and Growth

• Facultative anaerobes prefer growing in the presence of oxygen but can grow anaerobically. E. coli

• Obligate aerobes - require atmospheric oxygen (20%).Azotobacter vinelandi.

• Strict or Obligate anaerobes can only grow in the absence of oxygen and are killed by oxygen. Clostridium botulinum.

• Microaerophilic bacteria are damaged by 20% oxygen but require 2%-10% oxygen.


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Figure 7.28


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Basis of Different Oxygen Sensitivities

• oxygen easily reduced to toxic reactive oxygen species (ROS)– superoxide radical – hydrogen peroxide– hydroxyl radical

• aerobes produce protective enzymes– superoxide dismutase (SOD)– catalase– peroxidase


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Strict Anaerobic Microbes

• all strict anaerobic microorganisms lack or have very low quantities of– superoxide dismutase– catalase

• these microbes cannot tolerate O2

• anaerobes must be grown without O2

– work station with incubator– gaspak anaerobic system


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Figure 7.29


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Figure 7.30

Oxygen and Growth

Oxygen sensitivity

• Oxygen is toxic to all organisms at some concentration

• Toxicity is due to the formation of reactive oxygen molecules or compounds:1) superoxide (O2

-); and 2)hydrogen peroxide (H2O2). These are both powerful oxidizing agents and damage proteins, nucleic acids and lipids.

Oxygen Toxicity

Superoxide• Superoxide is formed during respiration by the univalent

reduction of oxygen which occurs primarily via the reduced flavins of the respiratory chain

O2 + (e-)-----------------> O2-

SUPEROXIDE

• Detoxification occurs through the action of the enzyme superoxide dismutase.superoxide dismutase.

2 O2- + 2H+ ------superoxide dismutase-------->

H2O2

SUPEROXIDE HYDROGEN PEROXIDE

Oxygen Toxicity

Hydrogen Peroxide H2O2

• Hydrogen peroxide is formed primarily through the action of superoxide dismutase

• Detoxification occurs through the action of catalase

2 H2O2------- ------catalase---------------->2 H2O + O2

Oxygen Toxicity

Strict or obligate anaerobes lack or have very low levels of superoxide dismutase (SOD) and catalase and thus are killed by the presence of oxygen.

MICROBE OF THE WEEK

• Clostridium difficile. Obligate anaerobic spore forming rod gram positive bacteria.

• Significance. Cause of 3 million cases of hospital diarrhea and colitis (7% of admitted hospital patients per year).

• In the US 500K infections/15K deaths

MICROBE OF THE WEEK

• Infection usually results because of antibiotic therapy which disturbs normal bacterial flora of colon.

• Cause- C. difficile releases 2 toxins, A and B. A is an enterotoxin and B is a cytotoxin. Both bind to receptors on the intestinal mucal cells compromising fluid absorption + retention

MICROBE OF THE WEEK

• Disposition to: Hospitalization, antibiotic therapy, Age (elderly). Most common antibiotics implicated are chephalosorins, ampicillin/amoxicillin and clindamycin

• Mechanism: Spores of bacteria prevalent in hospitals. Ingestion or surgical contamination of patient. Spores germinate in colon and colonize producing toxins.

MICROBE OF THE WEEK

• Symptoms. Mild to moderate watery diarrhea(rarely bloody)

cramping, anorexia, fever, dehydration, abdominal tenderness.

Diagnosis. Conclusive diagnosis depends on detection of toxin in stool. Fibroblast tissue culture-24-48h(94-100%). Commercial enzyme immunoassay kits (69-87%). Less sensitive but very quick (hours)

MICROBE OF THE WEEK

• Treatment. Usually Vancomycin or Metronidazole. Organism is very susceptible to this vancomycin. It is resistant to cephalosporins, ampicillin/amoxicillin, and clindamycin and aminoglycosides.

• Support therapy. Hydration.

Microbial GrowthBinary FissionFig. 6.1

Fig. 6.2

Process of binary fission

Fig. 6.4

Cytokinesis (septum formation)

Cytokinesis (septum formation)

Key step is the formation of the Z ring created by assembly Of filaments similar to tubulin

Once the Z ring forms the rest of the division machinery is constructed


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Growth

• increase in cellular constituents that may result in:– increase in cell number

– increase in cell size

• growth refers to population growth rather than growth of individual cells


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The Growth Curve• observed when microorganisms are

cultivated in batch culture– culture incubated in a closed vessel

with a single batch of medium

• usually plotted as logarithm of cell number versus time

• has four distinct phases– lag, exponential, stationary,

senescence, and death


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Figure 7.11

Bacterial Growth in Batch Cultures: The Growth Curve

Fig. 6.1 p 114

Bacterial Growth Curve in Batch Culture

The Lag Phase• No cell division occurs• Cells adjusting to medium and new

environment (temp, nutrients, etc)• Synthesis of some new cell components• Varies in length depending on media

shift, temperature shift and age of inoculum


Exponential or Log Phase• Bacteria are actively dividing at the maximum

rate given their genetic potential, nature of medium and environmental conditions

• Cells are most uniform in terms of chemical and phsyiological properties. Log phase cells are commonly used experimentally

Bacterial Growth Curve in Batch CultureStationary Phase

• No net increase in cell numbers thus growth curve levels off

• Total number of viable cells is constant

• Cell death may =cell division or the populations ceases to divide but remains metabolically active


Reasons for Stationary Growth Phase

• Nutrient limitation

• Toxic waste products

• pH (usually becomes acidic)

• Oxygen availability


Death Phase

• The detrimental changes resulting in the stationary phase worsen

• Cells death is faster than cell division

• It is a logarithmic function

Growth Mathematics of the Exponential Phase

Basic Assumptions

• Bacteria multiply by binary fission

• A mathematical equation can be developed specifically for the log growth phase

Mathematics of Cell Growth (Terms)

N0=initial number of bacteria/mlN1=number of bacteria/ml at a given time intervalt=time (h)n=#of cell divisions between N0 and N1

g=generation time (avg time for a cell division)=t/nk=average growth rate (number of cell divisions per

time)k=n/t

• k=log10N1-log10N0 = log10N1-log10N0

(log10 2) x t (0.301) x t

Important Mathematical Relationships

• k=1/g

• g=1/k

• n=kt

Derivation of growth formula

• N1=N0 X 2n -- taking the log of both sides• log10N1=log10N0+nlog102 --solve for n• n= log10N1-log10N0 since k=n/t or n=kt

log102

k= log10N1-log10N0

log102 x t

Generation Time DeterminationFrom Curve

Fig. 6.3 p. 116

Exponential Growth

Fig. 6.2 p.115


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Table 7.3


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Measurement of Microbial Growth

• can measure changes in number of cells in a population

• can measure changes in mass of population

Cell Counts

The Direct Count

Microscopic examination

• -Petroff-Hausser counting chamber for bacteria and the hemocytometer for large eucaryote cells.– Counts live and dead cells– Generally must have at least 106 cells per ml to see in high field

– Quick and easy but takes practice to get reproducibility

Petroff-Hausser counting chamber


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Direct Counts on Membrane Filters

• cells filtered through special membrane that provides dark background for observing cells

• cells are stained with fluorescent dyes

• useful for counting bacteria

• with certain dyes, can distinguish living from dead cells

Cell Counts

Direct CountCoulter counter-electronic counter for

larger eucaryote microorganisms like yeast, algae and protozoa

• Cell suspension passes through a small hole. An electric current flows across the hole and electrodes are placed on each side. Every time a cell passes through the electrical resistance increases and the cell is counted. Counts dead and live cells.

• Fast, easy and accurate. Extensively used in hospitals for WBC and RBC Quantitation.


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Flow Cytometry• microbial suspension forced through

small orifice with a laser light beam• movement of microbe through orifice

impacts electric current that flows through orifice

• instances of disruption of current are counted

• specific antibodies can be used to determine size and internal complexity

Viable Cell Counts

Viable CountsMost commonly used technique for growth

curves or establishing a relationship between turbidity and cell numbers

• Counts only live cells• Accurate and sensitive can readily determine number if 250

organisms per ml or more. However, takes 24-48 hours for results.

• Three common techniques:Spread Plate; Pour Plate and • Membrane Filtration.

Cell Counts

Viable Count Concept• A diluted sample of bacteria is dispersed over a solid

agar growth medium surface contained in a petri dish• Each microorganism or clump of organisms grows on

the surface and develops a distinct, visible colony and counted

• The number can be determined from the amount plated and dilution factor.

• NT = CFU x dilution factor amount plated

Viable Cell Counts

Fig.

Example of viable count quantitation

0.1 ml of a 10-5 dilution plated on nutrient agar and

Incubated 24h. 70 colony forming units grew

N = 70 X 105 = 107 bacteria/ml0.1

Estimating Cell Populations

Fig. 6.6 p. 119

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106

Figure 8.6


107

Figure 8.7

Viable Cell Count

Fig. 6.7 p.119

Growth on filters placed on agar culture media

Turbidity %transmittance vs absorbance

Cell Quantitation

TurbidityConcept of measuring growth by turbidity

is that microbial cells scatter light striking them.

• Since cells in a pure culture are roughly the same size the amount of light scattering is proportional to the concentration of cells (Beer’s Law)

• Fast, easy, reproducible accurate but not sensitive. Must have at least 107cells/ml to see any turbidity.

• Measures live and dead cells

Cell Quantitation

TurbidityMeasurement requires a spectrophotometer of

nephelometer• Spectrophotometer must be set at wave length that does not

measure pigment or other photo absorption. Usually 520-660nm

• Nephelometer measures light reflected at right angles. Klett-Summerson Nephelometer is specifically designed for measuring bacterial growth

• Beer’s law is obeyed only up to about 0.6 absorption units when using a spectrophotometer so if higher than that dilutions must be made.


113

Figure 7.21

Cell Quantitation

Turbidity and Cell number

• Cell number can be obtained directly from turbidity if viable counts have previously been made in the same exact media


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The Chemostat

• rate of incoming medium = rate of removal of medium from vessel

• an essential nutrient is in limiting quantities

Figure 7.22

Continuous Culture

D = f/v

• D= Dilution rate, The rate at which medium flows through the culture vessel (h-1)

• f = Flow rate (ml/h) (in and out same)

• v = Volume of culture vessel (ml)

Nutrient concentration and growth rate

Relationship between cell density/generation time and nutrient concentration

Control of Microbes

Terms• Sterilization-Process by which all living cells, spores,

viruses and viroids are destroyed. Usually done by heat or gas autoclaves

• Disinfection-Killing ,inhibition or removal of microorganisms on inanimate objects that may cause disease

• Antisepsis-prevention of infection by use of chemicals on living tissue

Control of Microbes

Types of Disinfectants or Antiseptics• cides such as germicide-kills pathogens, bactericide-

kills bacteria, algicide-kill algae, fungicide-kills fungi

• statics do not kill but prevents growth, bacteriostatic, fungistatic

Pattern of Microbial Death

Since the effect is on large populations death is generally considered logarithmic

• Factors influencing the effectiveness of agents: 1) population size; 2) population composition; 3) concentration or intensity of chemical or physical agent; 4) duration of exposure; 5) temperature; 6) local environment


122

Conditions Influencing the Effectiveness of Antimicrobial

Agent Activity

• population size– larger populations take longer to

kill than smaller populations

• population composition– microorganisms differ markedly in

their sensitivity to antimicrobial agents


123

More Conditions…• concentration or intensity of an

antimicrobial agent– usually higher concentrations or

intensities kill more rapidly– relationship is not linear

• duration of exposurelonger exposure more organisms killed


124

More Conditions…• temperature

– higher temperatures usually increase amount of killing

• local environment– many factors (e.g., pH, viscosity,

and concentration of organic matter) can profoundly impact effectiveness

– organisms in biofilms are physiologically altered and less susceptible to many antimicrobial agents

Physical Methods of Sterilization

• Moist Heat under pressure: The autoclave. 121C at 15 lb psi is the standard for small volumes of liquid. Larger volumes take longer

• Membrane Filtration• Gamma or ionizing radiation


126

Moist Heat

• destroys viruses, fungi, and bacteria

• boiling will not destroy spores and does not sterilize

• degrades nucleic acids, denatures proteins, and disrupts membranes


127

Figure 8.4


128

Steam Sterilization• must be carried out above 100oC

which requires saturated steam under pressure

• carried out using an autoclave• effective against all types of

microorganisms including spores

• quality control - includes strips with Geobacillus stearothermophilus


129

Table 8.2


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Filtration

• Sterilizes solutions of heat-sensitive materials by removing microorganisms

• also used to reduce microbial populations in air


131

Figure 8.6


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Ionizing Radiation

• gamma radiation penetrates deep into objects

• destroys bacterial endospores; not always effective against viruses

• used for sterilization and pasteurization of antibiotics, hormones, sutures, plastic disposable supplies, and food


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Figure 8.9

Sterilizing Gases

Ethylene oxide (EtO) is both microbicidal and sporicidal. It kills by combining with proteins and can penetrate packing material like plastic wrap.

• Good for sterilizing plastics, heart lung machine components, sutures and catheters

• EtO is very toxic and explosive so usually mixed at 10-20% with CO2

• Decomposes rapidly when exposed to air to non toxic products.


135

Figure 8.12

Heat Pasteurization

• Pasteurization-Process of heating milk and other liquids to destroy microorganisms that can cause spoilage or disease.

• Old method was 63C for 30 min

• New Methods include flash pasteurization or high temperature short term (HTST). 72C for 15 seconds with rapid cooling.

Chemical Antiseptics and Disinfectants

• Phenols: Commonly used in hospitals. Lysol is a penolic.These agents denature proteins and disrupt membranes

• Alcohols: Ethanol, isopropanol. Used at 70%. Denature proteins

• Halogens:fluorine, iodine, chlorine. Uusally at 1-2%. Strong oxidizing agents and react with many proteins.

• Detergents: Both anionic and cationic are organic wetting agents that disrupt cell membranes.

• Aldehydes such as formaldehyde and gluteraldehyde. React with DNA and proteins and inactivate. Sporicidal.

Documents

BACTERIAL NUTRITION Major (Macro) elements. Carbon (C), Nitrogen (N), Oxygen (O), Phosphorous (P) and Sulfur (S). Makes up over 95% of cell dry weight