Microbial Growth and The Control of Microbial GrowthMicrobiology

Microbial Growth0 Microbial requirements for

growth:0 1. Physical

0 A. Temperature0Optimal growth temperature0Permissible range0human pathogens optimal =

37°C

Microbial Growth0 1. Physical continued:

0 B. pH0most organisms optimal pH 6.5-7.5 (near neutral)

0 C. Osmotic pressure- draws the water from the cells0Most require isotonic solutions0human blood = 0.9% NaCl, isotonic0human skin = ~3-6% NaCl, hypertonic

Microbial Growth0 2. Chemical

0 A. Carbon0 B. Nitrogen0 C. Sulfur0 D. Phosphorus0 (all above: to build organic molecules)0 E. Trace elements: K+, Mg2+, Ca2+, Fe2+...

0cofactors for enzymes0 F. Oxygen

0aerobe, anaerobe, facultative0 G. Organic factors: vitamins, amino acids...

0(essential organic compound an organism is unable to synthesize)

Microbial Growth0 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 environment0 2. Log Phase: period of exponential growth with constant generation

time0 3. Stationary Phase: cell growth is equal to cell death0 4. Death Phase: cell death exceeds cell growth

Terminology for Microbial Control0 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 chemicals0 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 microorganisms0 Fungicides kill fungi, virucides kill viruses 0 Suffix – cide means the killer of a specified microorganism0 Suffix- stat used in this way indicates only that the substance

inhibits – for example bacteriostasis

Rate of Microbial Death0 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 influences0 -organics often inhibit chemical agents0 -temperature0 -pH

0 3. Time of exposure0 -same agent may need longer on resistant organisms or spores0 -with heat, lower temps require longer to kill

0 4. Microbial characteristics0 -resistance genes, protective structures0 (e.g.capsules) etc. can inhibit action0 -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 permeability0 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

0 2. Damage to proteins and Nucleic Acids0 denature enzymes (no reactions)0 prevent replication, transcription, or translation

Actions of Microbial Control Agents

Physical Methods of Microbial Control

0 -to disinfect objects, food, and solutions common methods:0 -temperature: kill or inhibit growth0 -filtration: physical removal0 -desiccation: inhibit growth0 -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

0 Moist Heat – coagulation of cell proteins0 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 pressure0 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.

For methods that involve heat:

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.

Filtration0 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

Desiccation0 Microbes require water for growth, and adequately

dried (desiccated) foods will not support their growth, therefore inhibiting growth

0 Removing all moisture0 Lyophilization- rapid freeze drying

0Used for blood products , serum products, enzymes, cultures

0Avoids ice crystal formation; cells burst when water expands

0 Liquid nitrogen- (-196) degrees C

Osmotic Pressure0 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.

Radiation0 U.V. light : rearranges proteins and DNA, bases altered to make dimer bonds; (T-T

or T-C- lethal mutations) BUT limited- does not penetrate surfaces, and damages human DNA

0 X-rays, gamma rays, cosmic radiation- microbicidal0 Visible light- can destroy some in presence of oxygen, but only if they have

pigments- most pathogens lack pigments0 Ionizing radiation- breaks bonds in DNA, creates oxidizing and reducing agents by

reacting with water0 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 Agents0 Used for disinfection, not sterilization0 Protein denaturing- proteins are unfolded and

refolded improperly; irreversible0 Organic solvents- alcohol, ether, acetone denature all

cell membranes0Limited against spores and viruses0Alcohols- skin, thermometers, anesthesia equipment

0 Isopropyl- 70 % in water

0 Ethers, benzenes- highly germicidal, BUT irritating and carcinogenic

Chemical Agents0 Heavy metals- Hg- toxic, irritating, used to be used in

ointment for newborn eyes to prevent gonorrhea, now use penicillin and erythromycin0 Ag- irritating, corrosive, but used in band-aids as an

antimicrobial01% solution of silver nitrate for eye ointment

0 Cu- fungicidal, algicidal0 Zn- superficial fungal and bacterial infections, diaper

rash0 As- “606” some protozoal infections

Chemical Agents0 Oxidizing agents- halogens, hydrogen peroxides, potassium

permanganate, peracetic acid0 Oxidize SH groups and NH2 groups of proteins

0 Cl- and I- used on vegetative(growing) cells, spores, fungi, viruses0 Br- is too toxic- but is used to disinfect spas and hot tubs0 Iodine solutions used on skin and mucous membranes, suture

materials, thermometers, surgical instruments, eating utensils0 Chlorine bleach- 5.25% NaClO- floors, bathrooms, linens, dishes, cutting

boards0 Dakin’s solution- 0.5% Cl-wound cleanser

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 groups0 Formalin- disinfect instruments, used in gas sterilization0 Gluteraldehyde- 2% solution- used for respiratory therapy

equipment, unstable at room temp0 Ethylene oxide- gas sterilization, carcinogenic, explosive in

air, 0 Usually mixed with inert gas, CO2 or N- artificial heart

valves and bedding

Chemical Agents0 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