CONTROL OF MICROBIAL GROWTH

I. INTRODUCTION

A.    IMPORTANCE• 1.  prevent and control infectious diseases in

humans, animals and plants.• 2.  preserve food.• 3.  prevent contaminating microbes from

interfering with certain industrial processes.• 4.  prevent contamination of pure culture

B.   MEANS OF CONTROL• 1.  Destroy or inhibit disease-causing microbes.• 2.  Block the source, route and vector of

transmission of agents.• 3. Protect an infected person from the consequences

of disease by building up body’s defenses and administration of appropriate chemotherapeutic drugs.

DEFINITION OF TERMS

1.       STERILIZATION         - Complete destruction of all living organisms         - Include cells, viable spores and viruses         STERILE---devoid of microbial life         - Done through heat, autoclave, gas, chemicals

and radiation

2.       DISINFECTION         - Destruction or removal of pathogens only

from nonliving objects by physical or chemical methods.

3.       – CIDAL and –STATIC          -cidal------killing agents          -static-----inhibits growth and reproduction

4.       ASEPSIS - absence of pathogens on living tissues

            SEPSIS - growth of pathogens on living tissues

5.       TECHNIQUES          Aseptic technique remove all pathogens          Sterile technique remove all microorganisms

    FACTORS AFFECTING EFFECTIVENESS OF ANTIMICROBIAL PROCEDURES

1.     Length of time2.     Temperature3.     Concentration4.     Nature and number of microbes and spores present (BIOBURDEN)5.     Presence of organic matter

Ex. Feces, blood, vomitus and pus

II. PHYSICAL ANTIMICROBIAL METHODS A.     HEAT·  Advantages: Practical, efficient and inexpensive·   Factors: Temperature and Time·

THERMAL DEATH POINT (TDP) - Lowest temperature that kill all

organisms w/in a specified period of time

THERMAL DEATH TIME (TDT)- Length of time needed to sterilize at a

specified temperature .

• 1.       MOIST HEAT -- Kills microorganisms by protein coagulation -- Method and Effectivity: a. Boiling—10 mins. at 70 C b. Steaming—10 to 30 mins. at 90 to 100 C -- Heat resistant microbes a. Bacterial endospores

(e.g. Anthrax, Tetanus, Gas Gangrene, Botulism) b. Mycobacteria c. Viruses (e.g. Hepatitis) --Affected by altitude (longer boiling time)

• TYNDALIZATION/FRACTIONAL STERILIZATION -- Endospores of bacteria are destroyed

by boiling and cooling 3x

2. PRESSURIZED STEAM (AUTOCLAVE) -- Uses steam under pressure• -- inc Pressure: dec temperature: dec time• -- Effectivity: 15 psi at a temperature

of 121.5 C for 20 mins.• -- Destroys all microbial life

3. DRY HEAT• -- Kills by oxidation effects METHODS:

a. Flaming - hold end of the loop in the yellow portion

of a gas flameb. Incineration (Burning) -- destroy contaminated

disposable materialsc. Hot Air Sterilization —Effectivity: 160 - 165 C for

2 hours OR 170 - 180 C for 1 hour

• 4. PASTEURIZATION• --Mild heating to kill the organism w/o seriously

damaging the taste of the product --At least 72 C for only 15 secs. OR 62 C for 30 secs. B. COLD        --Cold temperature and freezing        -- MICROBIOSTATIC METHOD - slow metabolic

activities

C. DRYING (DESSICATION)       --Water is removed that makes microbes unable

to grow or reproduce but can remain viable

       --LYSOPHILIZATION (FREEZE-DRYING) --Microbes are frozen at temperature -54 to –72

C and the water is removed by high vacuum

D.   RADIATION1.  IONIZING RADIATION

--Gamma rays, x-rays or high electron beams --Radiation ionized water highly reactive

hydrogen radicals destruction of DNA --Not widespread in routine sterilization --Used for sterilizing pharmaceutical, medical, dental supplies and food processing plants

2.  NON-IONIZING --Ultraviolet (UV) light (240 to 280 nm) Ex. Sunlight, UV lamp --Damages DNA (form thymine dimmers) --Used to control microbes in air --MAJOR DISADVANTAGES: a.  Organisms to be killed must be directly exposed b.  Damage to human eyes

c. Burns and skin cancer (prolonged exposure)

E. ULTRASONIC WAVES--Used for delicate equipment--Sound waves mechanically dislodge

organic debris on instruments and glassware

F. FILTRATION--Filters, separates cells, larger viruses, bacteria

and certain microbes from liquid or gases --Cellulose ester membranes

--Pore size: 0.22 micro removes all EXCEPT Chlamydia, Mycoplasma, Rickettsiae, viruses

--FACTORS OF EFFECTIVENESS: 1. Concentration of chemical 2. Contact time

3. pH of Solution 4. Temperature 5. Presence of CHON (blood, pus etc.)

III. CHEMICAL ANTIMICROBIAL METHODS

· Use of chemical agents to inhibit the growth of microorganisms, either temporarily or permanently

· Characteristics of Good Chemical Antimicrobial Agent 1. Kill pathogens within reasonable period and in specified concentrations 2. Non-toxic, non-corrosive and non-destructive 3. Water soluble and easy to apply 4.  Inexpensive and easy to prepare

5.  Stable in dissolved or solid form 6. Stable to pH and temperature changes

A. AGENTS THAT DAMAGE THE CELL MEMBRANE• - Release of small metabolites and interference of

active transport and energy metabolism

• 1. Surface Active Disinfectants (SADS) A.  Quaternary Ammonium Compounds (quats)

-- Cationic agents -- Bactericidal to gram (+) species - Antibacterial activity is reduced in presence

of organic matter        --E.g. Benzalkonium Chloride (Zephiran)        Cetyl pyridinium Chloride (Cepacol)     B. Soap and Detergents          -- Anionic agents dissociate to yield a (-) ion -- most effective against gram (+)

2. Phenolic Compounds --Rapidly bactericidal at low concentration a. Cresols—Ex. Lysol b. Diphenyl Compound—bactericidal at high concentration -- Ex. Hexachlorophene used in germicidal soaps and antiperspirants3. Alcohol --Skin disinfectant (not for instrument

sterilization) --Active against gram (+), gram (-) and AFB --Most effective at 50 to 70% concentration

-- Ex. Aliphatic alcohol (Ethanol), isopropyl alcohol

A. AGENTS THAT DENATURE PROTEINS --Acids, alkalis, alcohol, acetone --Causes unfolding of polypeptide chainchains

become randomly and irregularly coiled --Used as preservatives (benzoic, lactic, Acetic,

Citric)

B. AGENTS THAT MODIFY FUNCTIONAL GROUPS OF PROTEINS AND NUCLEIC ACIDS

-- Bactericidal activity decreased by organic substances

1. Heavy Metals - soluble salts of Hg, Ag, As and other metals

a. Mercurials - Useful antiseptic agents - Unreliable skin disinfectant --Ex. Metaphen, Merthiolate, Mercurochrome b.  Silver Compounds - Bactericidal - Silver Nitrate 1% - prophylaxis for opthalmia neonatorum (Credes prophylaxis) --Silver sulfadiazine -- burns

2.  Oxidizing agents a. Halogens - Bactericidal and effective against sporulating

organism - Iodine - skin disinfection (superior to other

agents) - Chlorine - water disinfectant Ex. Chlorox b.  H2O2 - Weak Antiseptic - For cleansing of wounds - Brief germicidal action 3.  Dyes ·   Stain bacteria and inhibitory at very high

dilutions ·   Basic dyes are the most effective ·   Inactivated by serum and other CHONS ·   Usually used in dermatologic treatment --Ex. Triphenylmethane Dyes, Acridine dyes

4.  Alkylating Agents · Bactericidal in all including spore formers a. Formaldehyde --Formalin - preserving fresh tissue --As a gas - decontaminate rooms, buildings,

fabrics and instruments b. Ethylene Oxide -- Used in gaseous sterilization c. Glutaraldehyde -- Cold sterilant for surgical instruments -- 10x more bactericidal and sporocidal than

formaldehyde

IV. CHEMOTHERAPEUTIC TREATMENT Antimicrobial agents

--- chemotherapeutic agents used to treat infectious diseases

Ideal Antimicrobial agentsA.     Kill or inhibit growth of pathogensB.     Cause no damage to hostC.     Cause no allergic reaction in hostD.     Be stable when stores in solid or liquid formE.     Remain in specific tissues

F. Kill pathogens before they mutate or become resistant

A.  ANTIBACTERIAL AGENTS · Some are bacteriostatic, some are bacteriocidal 1. Inhibition of Cell Wall Synthesis

Penicillin, Cephalosphorin, Bacitracin, Vancomycin

2. Inhibition of Protein Synthesis A. 30s subunit---Aminoglycoside, Tetracycline B. 50s subunit---Chloramphenicol, Lincomycin,

Erythromycin, Clindamycin

3. Disruption of cell membrane—Polymyxin

4. Inhibition of bacterial enzyme activity Sulfonamides, Trimethoprim

5. Inhibition of nucleic acid---Quinolone

B. ANTIFUNGAL AGENTS 1.Bind with cell membrane sterols

(Ex. Nystatin, Amphotericin B) 2. Interfere with sterol synthesis

(Ex. Clotrimazole, miconazole) 3. Block Mitosis or nucleic acid (Ex. Griseofulvin)

C. ANTIPROTOZOAL AGENTS

1. Interfere with DNA and RNA synthesis Ex. Chloroquine, pentamidine, and quinacrine 2. Interfere with protozoal metabolism Ex. metronidazole

D. ANTIVIRAL AGENTS -- Inhibits viral replication within the cell

Ex. Acyclovir, valaciclovir, amantadine, zidovudine, lamivudine, saquinavir