Transcript The Control of Microbial Growth

Microbial Growth
0 Microbial requirements for
growth:
0 1. Physical
0 A. Temperature
0 Optimal growth temperature
0 Permissible range
0 human pathogens optimal =
37°C
Microbial Growth
0 1. Physical continued:
0 B. pH
0 most organisms optimal pH 6.5-7.5 (near neutral)
0 C. Osmotic pressure- draws the water from the cells
0 Most require isotonic solutions
0 human blood = 0.9% NaCl, isotonic
0 human skin = ~3-6% NaCl, hypertonic
Microbial Growth
0 2. Chemical
0 A. Carbon
0 B. Nitrogen
0 C. Sulfur
0 D. Phosphorus
0 (all above: to build organic molecules)
0 E. Trace elements: K+, Mg2+, Ca2+, Fe2+...
0 cofactors for enzymes
0 F. Oxygen
0 aerobe, anaerobe, facultative
0 G. Organic factors: vitamins, amino acids...
0 (essential organic compound an organism is unable to
synthesize)
Microbial Growth
0 Microbial Growth
0 -for unicellular organisms, growth = increase in cell
number (population) not individual cell size
0 -generation time = time required for a cell to divide
0 -both growth and death of the population is
exponential, graphed on a log scale
Bacterial Growth Curve
0 1. Lag Phase: little to no growth, bacteria acclimate to new
environment
0 2. Log Phase: period of exponential growth with constant
generation time
0 3. Stationary Phase: cell growth is equal to cell death
0 4. Death Phase: cell death exceeds cell growth
Terminology for Microbial
Control
0 Sterilization- removal or destruction of all forms of
microbial life
0 Commercial sterilization- subjects canned food to
only enough heat to destroy the endospores of
Clostridium botulinum
0 Disinfection- is the destruction of vegetative
pathogens on a surface, usually with chemicals
0 Spores and viruses are not necessarily destroyed
0 Antisepsis- is the chemical disinfection of living
tissue, such as skin or mucous membranes
Terminology for Microbial
Control
0 Asepsis- is the absence of pathogens on an object or area,
as in antiseptic surgery
0 Degerming (degermation)- is the removal of transient
microbes from the skin by mechanical cleansing or by
antiseptic
0 Sanitation- is the reduction of microbial populations on
objects to safe public health levels
0 A biocide or germicide- kills microorganisms
0 Fungicides kill fungi, virucides kill viruses
0 Suffix – cide means the killer of a specified microorganism
0 Suffix- stat used in this way indicates only that the substance
inhibits – for example bacteriostasis
Rate of Microbial Death
0 Bacterial populations killed by heat or chemicals tend to die at
constant rates—for example, 90% every 10 minutes. Plotted
logarithmically, these figures form straight descending lines.
Factors that influence effectiveness of
an
antimicrobial treatment:
0 1. Number of microbes
0 -more cells, more time needed to kill all
0 2. Environmental influences
0 -organics often inhibit chemical agents
0 -temperature
0 -pH
0 3. Time of exposure
0 -same agent may need longer on resistant organisms or spores
0 -with heat, lower temps require longer to kill
0 4. Microbial characteristics
0 -resistance genes, protective structures
0 (e.g.capsules) etc. can inhibit action
0 -bioflims prevent penetration
Type and Age of Microbe
0 Bacteria- susceptible to protein denaturing BUT
mycobacteria is not because of its hydrophobic coat
0 Growth cycles- physiologically young bacteria(early in
growth cycle) susceptible to heat
0 Endospores more resistant to heat the older they get
Properties of Materials:
0 Plastic and rubber- not heat tolerant
0 Cutting edges of surgical instruments- no moisture
or corrosive chemicals
0 Edges can become pitted due to rust and corrosion,
microbes can hide there
0 Fabrics- no chemical disinfection; some too fragile
Actions of Microbial Control
Agents
0 1. Alteration of membrane permeability
0 The plasma membrane controls the passage of nutrients
and wastes into and out of the cell. Damage to the
plasma membrane causes leakage of cellular contents
and interferes with cell growth.
0 = Leak
lysis, death
Actions of Microbial Control
Agents
0 2. Damage to proteins and Nucleic Acids
0 denature enzymes (no reactions)
0 prevent replication, transcription, or translation
Physical Methods of Microbial
Control
0 -to disinfect objects, food, and solutions common
methods:
0 -temperature: kill or inhibit growth
0 -filtration: physical removal
0 -desiccation: inhibit growth
0 -radiation: kill
For methods that involve heat:
0 Thermal Death Point (TDP) = lowest temp at which all
microbes in liquid suspension will be killed in 10 min
0 Thermal Death Time (TDT)= minimal length of time for
all microbes in liquid suspension to be killed at given temp
0 *Both are different for different species due to microbial
variation in heat tolerance
0 Concept of equivalent treatments:
0 With any heat treatment, the higher the temperature used the
shorter the exposure time needed to achieve the same effect
0 Moist heat will always kill faster than dry heat at the same
temperature
For methods that involve heat:
0 Moist Heat – coagulation of cell proteins
0 Boiling (100°C) kills vegetative forms of bacterial pathogens,
many viruses, and fungi within 10 minutes.
0 Endospores and some viruses survive boiling for longer times.
0 Steam under pressure
0 allows temperatures above boiling to be reached.
0 Autoclaves, retorts, and pressure cookers are vessels in
which high steam pressures can be contained. A typical
operating condition for sterilization is 15 psi (pounds per
square inch) at 121°C for 15 minutes. Moisture must touch all
surfaces in order to bring about sterilization. Air must be
completely exhausted from the container. An autoclave is
shown in Figure 7.1.
Autoclave
Low Temperature
0 Lower temp inhibits growth,
rapid freezing limits moisture
0 Refrigerator temperatures
(0° to 7°C) slow the
metabolic rate of microbes;
however, psychrotrophic
species still grow slowly.
0 Some organisms grow at
temperatures slightly below
freezing, but microbes at the
usual temperatures of
freezer compartments are
completely dormant.
Filtration
0 Liquids sensitive to heat can be passed through a thin
membrane filter that has carefully controlled pore
sizes to retain microorganisms.
0 Operating theaters and special clean rooms receive air
passed through high-efficiency particulate air
filters (HEPA).
0 Used with solutions for: renal dialysis, heart bypass
machines, I.V.’s
0 Catch particles autoclaves miss
Desiccation
0 Microbes require water for growth, and adequately
dried (desiccated) foods will not support their
growth, therefore inhibiting growth
0 Removing all moisture
0 Lyophilization- rapid freeze drying
0 Used for blood products , serum products, enzymes,
cultures
0 Avoids ice crystal formation; cells burst when water
expands
0 Liquid nitrogen- (-196) degrees C
Osmotic Pressure
0 High salt or sugar concentrations cause water to leave
the cell; this is an example of osmosis. Generally,
molds and yeasts resist osmotic pressures better than
bacteria.
Radiation
0 U.V. light : rearranges proteins and DNA, bases altered to make dimer bonds;
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(T-T or T-C- lethal mutations) BUT limited- does not penetrate surfaces, and
damages human DNA
X-rays, gamma rays, cosmic radiation- microbicidal
Visible light- can destroy some in presence of oxygen, but only if they have
pigments- most pathogens lack pigments
Ionizing radiation- breaks bonds in DNA, creates oxidizing and reducing
agents by reacting with water
Microwave- not effective, limited to use in beer and wine
0 Can be used to disinfect cutting boards, sponges and dish cloths
0 Ionizing radiation such as X rays, gamma rays, and high-energy electron
beams carry high energy and break DNA strands.
0 Ionizing radiation forms reactive hydroxyl radicals. Such radiation is used to
sterilize pharmaceuticals.
0 Nonionizing radiation such as ultraviolet (UV) light has a longer wavelength and
less energy.
0 UV light causes bonds to form between adjacent thymines in DNA chains. Penetration
is low.
0 Sunlight has some biocidal activity, mainly due to formation of singlet oxygen in the
cytoplasm.
Chemical Agents
0 Used for disinfection, not sterilization
0 Protein denaturing- proteins are unfolded and
refolded improperly; irreversible
0 Organic solvents- alcohol, ether, acetone denature all
cell membranes
0 Limited against spores and viruses
0 Alcohols- skin, thermometers, anesthesia equipment
0 Isopropyl- 70 % in water
0 Ethers, benzenes- highly germicidal, BUT irritating and
carcinogenic
Chemical Agents
0 Heavy metals- Hg- toxic, irritating, used to be used in
ointment for newborn eyes to prevent gonorrhea, now
use penicillin and erythromycin
0 Ag- irritating, corrosive, but used in band-aids as an
antimicrobial
0 1% solution of silver nitrate for eye ointment
0 Cu- fungicidal, algicidal
0 Zn- superficial fungal and bacterial infections, diaper
rash
0 As- “606” some protozoal infections
Chemical Agents
0 Oxidizing agents- halogens, hydrogen peroxides, potassium
permanganate, peracetic acid
Oxidize SH groups and NH2 groups of proteins
Cl- and I- used on vegetative(growing) cells, spores, fungi, viruses
Br- is too toxic- but is used to disinfect spas and hot tubs
Iodine solutions used on skin and mucous membranes, suture
materials, thermometers, surgical instruments, eating utensils
0 Chlorine bleach- 5.25% NaClO- floors, bathrooms, linens, dishes,
cutting boards
0 Dakin’s solution- 0.5% Cl-wound cleanser
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0 Cyst and oocysts(parasites) survive chlorine- Giardia
0 H2O2- 3% solution – disinfects spacecraft and contact lenses
0 O2 released quickly in presence of catalase enzyme in human tissue,
causes bubbling- clears wound of dirt, reduces deep tissue anaerobes
0 Lethal to bacteria, viruses, fungi, and reduces # of spores
Chemical Agents
0 Dyes- found in coal tar, have + charge and affinity for
nucleic acids
0 Alkylating agents- formaldehyde- replace H atoms
on NH2, OH, COOH, SH groups
0 Formalin- disinfect instruments, used in gas sterilization
0 Gluteraldehyde- 2% solution- used for respiratory
therapy equipment, unstable at room temp
0 Ethylene oxide- gas sterilization, carcinogenic, explosive
in air,
0 Usually mixed with inert gas, CO2 or N- artificial heart
valves and bedding
Chemical Agents
0 Membrane altering agents-
0 Surface active- reduce surface tension(detergents)
0 Increase penetration of liquid or air surface of molecules
0 Anionic (-) or cationic(+)- cationic example is quaternary salts, or “quats”-alter
charges of phospholipids, they are stable, non-irritating, odorless and cheap- only
effective on some G-, all G+, no opportunistic bacteria.
0 Phenolic derivatives- carbolic acid- toxic, irritating, not effective on spores,
0 5% aqueous phenol solutions used on sputum, urine, feces, contaminated glass slides
0 Hexachlorophene is the main ingredient in pHisoHex and is used in nurseries to
control gram-positive skin bacteria such as staphylococci and streptococci,
surgical scrubs, but excessive use has been linked to neurological damage
0 Chlorhexidine- It is frequently used for surgical skin preparation and surgical
hand scrubs. Betasept, Hibiclens, effective on G- and G+, unaffected by presence of
blood or other organic matter