Control of microbial growthControl of microbial growthControl of microbial growth

ByByDr. Carmen RexachDr. Carmen Rexach

MicrobiologyMicrobiologyMt San Antonio CollegeMt San Antonio College

In general• Easier to control microbial growth outside

of body– Semmelweis and handwashing– Lister and aseptic surgery

• Approximately 10% of all patients entering a hospital contract nosocomial infection

• Chemical and physical controls– Needed to prevent infections and food spoilage

Definitions

• Sterilization = process of destroying all microbial life on an object

• Disinfection = reduction or inhibition of microbial growth– destroy organism, not endospores

• Disinfectant = physical or chemical agent which disinfects, used almost exclusively on inanimate objects because destroys animal tissues

Disinfectants

• Bactericides– Kill bacteria

• Fungicides• Germicides

– Broad spectrum• Bacteriostatic

agent– Reduces bacterial

growth

Definitions

• Antiseptics– Agents applied directly to living tissue to

reduce likelihood of infection or sepsis– Sepsis = presence of microorganisms in

blood or tissues• Sanitation

– Any cleaning technique that physically removes microorganisms

• Usually only involves detergent only• Very broad meaning

Conditions• Choose method strong enough to

eliminate most resistant microbes on object

• Rate of disinfection determined by number of microbes present and species

• Susceptibility– Gram positive more susceptible– Pseudomonads– Mycobacteria

Resistance

• Resistant– Endospores– Protozoal cysts– Naked viruses– Cryptosporidium and chlorinated water

• Increased susceptibility during log phase• Environmental conditions (pH, heat)• Organic material

– Vomit, feces can interfere with actions of detergent

How do microbial control agents work?

• Permanent alteration of plasma membrane– Cell contents leak out

• Damage to proteins and nucleic acids– Denature proteins by interfering with

hydrogen bonds and disulfide bridges– Damage DNA and RNA which alters

function or destroys cells

Physical methods of control

• Heat• Pasteurization• Dry heat• Filtration• Cold• Desiccation• Osmotic pressure• Radiation• Heat and chemical

vapor

Heat• Economical, widely applicable sterilizing

agent• Definitions

– Thermal death point (TDP): lowest temperature at which all microbes in a liquid suspension are killed in 10 minutes

– Thermal death time (TDT): minimal length of time in which all bacteria in liquid killed at a given temperature

– Decimal reduction time (DRT): number of minutes in which 90% of population of bacteria are killed at given temperature

Moist heat

• Used for >90% all medical or lab products• Mechanism

– Denatures enzymes, coagulation• Boiling

– 100oC at sea level kills vegetative forms, most viruses, fungi, and fungal spores in 10 minutes

– Does not effect resistant viruses or endospores

Moist heat• Autoclave

– Most effective when organisms in direct contact with steam or in aqueous solution

– 15psi (1.1kg/cm2), 121oC kills all organisms and endospores in 15 minutes

– Longer for solids– Flash autoclaving: 135oC

(2.2kg/cm2) for 3 min.• Pressure cooker (canning)

Pasteurization• Utilizes mild heat to kill microbes while

preserving product taste• Pasteur

– 63oC for 30 minutes• HTST (high temperature short time)

– 72oC for 15 minutes• UHT (ultra-high temperature)

– 140oC for 3 seconds– Does not require refrigeration (coffee

creamers)

Filtration• Passage of liquid or gas through filter with

pores small enough to impede microbes• HEPA (high efficiency particulate air

filters)– Removes microbes larger than 0.3μm from air

• Nitrocellulose or Cellulose acetate membrane filters– Filters out bacteria, viruses, large proteins– Pore size varies from 0.22-0.01μm

• Used when chemicals don’t work– Giardia lamblia = resistant spores– Cryptosporidium parvum = chlorine resistance

Filters

Cold• Bacteriostatic to most microbes

– Slows down metabolism– Inhibits reproduction– Inhibits production of toxins

Desiccation• Effectiveness varies depending on

organism– Viruses remain viable– Bacteria generally effected

• Restoration of water returns microbes to vegetative state

Osmotic pressure• Use salt and water to preserve food

– Same idea as desiccation• Organisms remain susceptible to mold

and yeast

Radiation• Effects depend on wavelength,

intensity, duration• Two types kill microbes

– Ionizing– Non-ionizing

Ionizing radiation

• Wavelength < 1nm– Gamma, x-rays, high electron beams

• Mechanism– Forms reactive OH- radicals which disrupt

DNA & other organic molecules by ionizing the water

– High penetration• Applications

– Food preservation, sterilization of pharmaceuticals & disposable medical and dental supplies

Non-ionizing radiation• Wavelength > 1nm

– UV light– Most lethal wavelength = 260nm

• Mechanism– Induces dimer formation between adjacent

thymines in DNA– Inhibits DNA replication

• Applications– Germicidal lamp, destroys microbes in air,

drinking water, vaccines, serum, etc.• Disadvantages

– Poor penetration = requires direct exposure– Damage to eyes, skin cancer

Heat and chemical vapor• Chemiclave• Used in dentistry

– Alcohol/formaldehyde vapor at 1.1kg/cm2 and 132oC

– Short cycle time– Because no water vapor is

formed, cutting surfaces remain sharp

Chemical methods of microbial control

• Antiseptics = Use on living tissues• Disinfectants = Inanimate objects• Very few achieve sterility• Factors to consider

– Nature of microbe to be controlled– Type of material being disinfected

• Organic matter may interfere– Temperature

• More effective in high temperatures

Classification of organisms by susceptibility to disinfectants

• Group A– Easily killed by disinfectants– Most vegetative forms of bacteria– Enveloped viruses

• Group B– More difficult to kill– Tuberculosis bacillus– Naked viruses

• Group C– Highly resistant bacterial endospores

Evaluating disinfectants• Phenol coefficient test

– Standard for many years• American Official Analytical Chemist’s use-

dilution method– Test three strains: S. aureus, Salmonella

choleraesuis, Ps. aeruginosa– Effectiveness determined by number of

surviving bacteria after standardized heat treatment & exposure to disinfectant per manufacturer’s recommendations

• Filter paper method– Paper discs soaked in disinfectant on inoculated

plate– Zones of inhibition

Types of disinfectants• Phenol and phenolics• Chlorhexidine• Halogens• Alcohols• Heavy metals and their compounds• Surfactants

Phenol and phenolics• Lister = carbolic acid• Mechanism

– Damages plasma membrane, inactivates enzymes, denatures proteins

– Ex) hexachlorophene (pHisohex: excessive use damage to neurological system of infants)

• Application– Main ingredient in lysol– Especially effective against gram +

Staph/Strep– Not commonly used due to odor and tissue

damage

Chlorhexidine• Mechanism

– Damages plasma membrane• Application

– Sterilization of skin and mucous membranes

– Surgical hand scrubs– Not sporicidal– Can effect some enveloped viruses

Halogens• Mechanism

– Interferes with protein structure• Example = Iodine, Chlorine

– Effective against bacteria, many endospores, fungi, some viruses

– Iodine = antiseptic– Chlorine = disinfectant– Tincture of iodine = alcoholic solution of iodine

Alcohols• Mechanism

– Denatures protein, disrupts lipid membrane structure

• Examples– Ethanol, isopropanol– Effective against bacteria, fungi– Not effective against spores or naked

viruses

Heavy metals and their compounds

• Mechanism– Oligodynamic action = denatures

proteins• Examples

– Silver nitrate• Protects against gonorrheal eye infections in

infants– Copper sulfate

• Algicide in swimming pools

Surfactants• Detergents and soaps• Decrease surface tension =

mechanical removal of microbes• Two types

– Quats = quaternary ammonium compounds

– Aldehydes

Surfactants• Quats

– Unknown mechanism– Cationic detergent– Especially effective

against gram positive organisms

– Fungicidal, amoebacidal, enveloped viruses

– Ineffective against spores or M. tuberculosis

– Pseudomonas lives in this!

• Aldehydes– Mechanism: inactivates

proteins– Example: formaldehyde– Used for embalming,

preserving specimens– Bactericidal,

tubericidal, virucidal, sporicidal

– Only liquid that can be considered a sterilizing agent!