Control of Microbial Growth

Chapter 7

Terminology• Sepsis refers to microbial contamination.• Asepsis is the absence of significant

contamination.

• Antisepsis: Removal of pathogens from living tissue

• Degerming: Removal of microbes from a limited area

• Sanitization: Lower microbial counts on eating utensils

Terminology cont’d

• Decontaminated

- item that has been treated to reduce # of disease causing organisms

• Preservation

- delaying spoilage of foods

• Sterilization, Disinfection, Antiseptic, Bacteriocidal, Bacteriostatic

Terms used:

Sterilization vs. Disinfection

• Sterilization: destroying all forms of life

• Disinfection: destroying pathogens or unwanted organisms

Disinfectant vs. Antiseptic Disinfectant: antimicrobial agent used on inanimate

objects Antiseptic : antimicrobial agent used on living tissue

cidal vs. static• Biocide/Germicide: Kills microbes• Bacteriostasis: Inhibiting, not killing, microbes• Examples:

- Bactericidal - kills bacteria- Bacteriostasis (Bacteriostatic)- inhibits bacterial growth- Fungicidal- Fungistatic- Algacidal- Algastatic

Methods of control

• Physical or chemical?– physical control includes heat, irradiation,

filtration and mechanical removal

– Chemical control involves the use of microbial chemicals

– Depends on the situation

– degree of control required

Methods of control cont’d

• Daily life

- Cooking

- refrigeration

- cleaning

- soap water

mechanical

Mechanical and chemical

• Hospitals– Important to minimize nosocomial infection

(hospital acquired infection) due to

- weakened patients’ condition

- breaching of intact skin

- high concentration of pathogens from patients and workers

– Sterile condition

Methods of control cont’d

• Microbiology lab utilizes– Sterile equipment

– Aseptic technique

– And possesses workers who takes care of the nature of of microbiologists (GLP)

Methods of control cont’d

• Foods/food production industry

- physical removal

- adding chemicals

- may result in toxicity

- clean surface/ machinery

Methods of control cont’d

Selection of Control Method

• Antimicrobial procedure used for control of microbial growth is based on – Types of microbe

– Extent of contamination

– Environmental conditions

– Potential risk

Selection of Control Method cont’d

• Types of microorganism

- some organisms are more resistant and require stronger measures for control

- endospores require chemical treatment for 10 hours

- Mycobacterium’s waxy cells are resistant to chemicals

Mycobacterial cell wall: 1-outer lipids, 2-mycolic acid, 3-polysaccharides (arabinogalactan), 4-peptidoglycan, 5-plasma membrane, 6-lipoarabinomannan (LAM), 7-phosphatidylinositol mannoside, 8-cell wall skeleton

• Extent of microbial population

- larger population take more time to destroy

- usually 90% of the population is destroyed in

a given period

e.g if in 1st 3 minutes 90% of the population is destroyed, then 90% of the remaining population gets destroyed in the next 3 minutes and so on

Selection of Control Method cont’d

• Environmental conditions- pH, temperature- presence of

- organics: blood- dirt- grease

- the potential risk of transmitting infectious agents- critical items- semicritical items- non-critical items

Selection of Control Method cont’d

Must be cleaned first, then controlled

• Critical items have

- indirect contact with body tissues

- needles, scalpels

• Semicritical items have contact with

- mucous membranes but it does not penetrate

endoscopes, endotrachial tubes

Selection of Control cont’d

Selection of Control cont’d

• Non-critical items have

- indirect contact with unbroken skin

- countertops, stethoscopes

Methods to Control Microbial Growth

Physical Microbial Controls: Heat

• Heat as a microbial control- fast, reliable, inexpensive- does not introduce potential toxic substances

• Types of heat control include- moist heat- pasteurization- pressurized steam- dry heat

• Moist heat

- causes irreversible coagulation of proteins found in microorganisms

- 10 minutes of boiling

- most microbes and viruses will be destroyed except endospores and few

others which can survives hours of boiling

Physical Microbial Controls: Heat cont’d

• Pasteurization

- reduces number of heat sensitive organisms

- widely used in milk and juices

increases shelf life and does not alter quality

- original pasteurization was 62ºC, 30 mins

- now: UHT-shorter time 72ºC, 15 secs

Physical Microbial Controls: Heat cont’d

• Pressurized steam

- pressure cooker or autoclave

- higher air pressure increases the temperature

at which steam forms

- 15 psi (lbs/square inch) at 121ºC for 15 mins

-effective to kill endospores

Physical Microbial Controls: Heat cont’d

The autoclave: Moist heat and pressure

• 15psi, 121ºC, 15 minutes

• Thermal death point (TDP): Lowest temperature at which all cells in a culture get killed in 10 mins

• Thermal death time (TDT): time to kill all cells in a culture

• Decimal reduction time (DRT): Minutes to kill 90% of a population at a given temperature

The autoclaving machine

Temperature of steam and Pressure at sea level

The autoclaving machine

• Dry heat- without moisture e.g. flaming- burns cell constituents- object is oxidized to ash- irreversibly denatures proteins- takes longer (200ºC, 1.5 hrs dry=121ºC, 15 min moist)- advantages are for powders, does not corrode metals and blunt sharps- e.g flasks, tubes, pipettes in microbiological laboratories.

Physical Microbial Controls: Heat cont’d

Physical Microbial Controls: Filtration

• Used for heat sensitive fluids

• air

• Fluids– solutions of antibiotics, vitamins,

tissue extracts, animal serum, etc.– Depth filters

–able to retain microorganisms while allowing fluids to pass through

–Membrane Filter- The use of graded pore size 0.2-0.4µm

Physical Microbial Controls: Filtration cont’d

• Air- HEPA (High Efficient Particulate Air) filter and laminar air flow are commonly used

- filter incoming air and outgoing air respectively

- HEPA filter prevents the income of 0.3µm and large size particles to enter.

Physical Microbial Controls: Filtration cont’d

• “Cold sterilization“ for disposable materials made up of plastics, wool, cotton, etc without altering the material.

• Radiation damages DNA• Ionizing radiation (X-rays, gamma rays, electron

beams) • Non-ionizing radiation

- UV- Microwaves kill by heat not especially

antimicrobial

Physical Microbial Controls: Radiation

• Gamma irradiation

- penetrate deeply

- for heat sensitive materials

- causes biological damage to microorganisms

- does not alter food flavor (meat)

Physical Microbial Controls: Radiation cont’d

• UV light– damages the structure and function of nucleic acids

– Penetrate poorly- cannot penetrate even into liquid.

– Used to disinfect surfaces

– Can cause damage to human cells

– Germicidal lamps -kill or reduce the number of viable microorganisms to sterilize microbiological laboratories hospital operating rooms, and specific filling rooms in various industries

Physical Microbial Controls: Radiation cont’d

• Microwave– Kills by heat

– Does not affect microorganisms directly

Physical Microbial Controls: Radiation cont’d

Physical Methods used to control Microbial growth

Chemical Microbial Control

• Grouped according to potency– Sterilants

– High-level

– Intermediate level

– Low level

Chemical Microbial Control cont’d

Chemical Control

sterilants

• destroy microorganisms, endospores and viruses

• used for critical equipment-scalpels

chemical control cont'd

• high level

- destroy viruses and  vegetative microorganisms (no endospores)

- used for semicritical equipment: endoscopes

• intermediate level

- destroy vegetative microorganisms, some viruses

- used for non-critical equipment: sthetoscopes

• low level

-destroy fungi, vegetative microorganisms

- used for general purpose disinfectants

Selecting Germicidal Chemical

• Germicide: An agent capable of killing pathogens and non-pathogens but not necessarily endospores

• toxicity to human or environment?

- weigh the benefits vs the risks

• presence of organic material

- hypochlotrite is inactivated by the presence of organic matter

• compatibility

-electrical equipment with a liquid??

• residue

- some have to be rinsed with sterile water

• cost and availability

Selecting germicidal Chemical cont'd

• storage and stability

- may come in concentrated form for ease in storing

- those have to be mixed

• environmental risk

- is neutralization necessary before disposal?

Classes of Germicidal Chemicals

• alcohols alcohols

• aldehydes

• biguanides

• ethylene oxide 

• halogens: oxidize proteins

Classes of Germicidal Chemicals

• alcohols

- coagulated enzymes and proteins

- damage lipid membranes

- on-toxic

- inexpensive

- no residue

Classes of Germicidal Chemicals cont’d

• aldehydes

- inactivate proteins and nucleic acids

- toxic to humans

• Peroxygens

- oxidizing agents

- hydrogen peroxide

- leaves no residue

Classes of Germicidal Chemicals cont’d

• biguanides

- extensive antiseptic use

- adheres and persists on skin, mucous membranes

- low toxicity

• Phenolic compounds– Hitorically important

– Irritant, unpleasant odor

– Destroy cytoplasmic membrane and denatures protein

Classes of Germicidal Chemicals cont’d

• ethylene oxide 

- reacts with proteins

- gas: penetrable 

- mutagenic

• Metal compounds– Interfere protein function

– Toxic

– pollutants

Classes of Germicidal Chemicals cont’d

• halogens: oxidize proteins

-chlorine

-irritating to skin

-organic compounds consume free chlorine

-iodine

-tincture

- Iodophore

Effect of germicidal activity on Microbes

Effect of germicidal activity on Microbes

Chemical methods of microbial control

• Evaluating a disinfectant

- Disk diffusion method

Preservation of Perishables

• Extends shelf life– Slow or halts microbial growth thus delaying

spoilage

• Chemical preservatives– Some chemical preservatives are used in non-food

items

– Food preservatives must be non-toxic to humans• Benzoic acid, propionic acids, nitrate are commonly

used

• Nitrate– Inhibits germination of C. botulinum endospores

Preservation of Perishables

Preservation of Perishables

• Low temperature storage– Temperature dependent• most microorganisms do not reproduce in ordinary

refrigerator (0-7ºC)

– Freezing• ice crystals can cause irreversible damage to many

microorganisms (kills up to 50% growth)

• Freezing stops all growth, but may start to reproduce again once food is thawed

• Reducing water– Salt/sugar

– Draw water out of cell

– Less available for microorganisms

• Drying– Desiccation• Removing water such as milk powder

Preservation of Perishables

Salt cured meat

• Lyophilization– Freeze drying• Freeze food first

• followed by putting in vacuum

Preservation of Perishables

Factors that influence effectiveness

• Number of microbes

• Environmental influences

• Time of exposure

• Microbial characteristics

Microbial death curve

Limitations: Microbial Characteristics

Actions of Microbial Control Agents

• Alteration of membrane permeability

• Damage to proteins

• Damage to nucleic acids

Questions????

Sand rich in salts n nitrates preserved mummy