Microbial Control Methods (lec 4)

Physical, Mechanical, Chemical (disinfectant and antiseptic)

3 categories of sterilization

Sterilization

Removal of life (microorganisms of every kind)

- 3 categories: physical, mechanical, chemical (disinfectant and antiseptic)

Disinfectant

A chemical substance to kill microorganisms on surfaces but are too toxic to be applied directly to tissue

- may cause irritation or damage to the tissue

- may have carcinogen that causes mutation on the cells that can cause cancer

formalin / formaldehyde

Example of Disinfectant

Antiseptic

inhibits or eliminate microorganisms in the living tissue

Septic

Presence of microorganisms on living tissue

Antiseptic

Absence of microorganisms on living tissue

Sterile

Free of life of every kind

Bactericidal

Power or ability to kill the total population of bacteria

Bacteriostatic

Power not to kill but inhibit the growth of bacteria

Sporicidal

Ability to kill both vegetative and spore of a bacteria

Non-sporicidal

Only the vegetative is damaged but not the spores

Dry heat

no presence of water in the system

- principle: oxidation of cellular components

Flaming

- sporicidal

- direct flame (blue flame)

- sterilize inoculating wires, tip of forceps, mouth of flasks

- use of alcohol lamp or Bunsen burner

Incineration / Cremation

burning to ashes irreversibly

Incineration

used specifically for contaminated materials (870-980 °C)

Cremation

burning of human body to ashes (300-400 °C)

Dry oven / Hot air oven

- sporicidal and bactericidal

- oxidation of cellular component

- intended to sterilize materials that are made up of metal or glass but not with rubber or plastic

- 160-180 °C for 1.5-3hrs

Moist Heat

Principle: coagulation of proteins

Boiling

- Non-sporicidal (only kill the vegetative form of bacteria)

- Kill bacteria but cannot kill spore-forming bacteria

- 100 °C at 1-3 hours for complete sterilization

Pasteurization

- Bacteriostatic

- Used in milk, fruit juices, wines

- Preservation of nutritional value

- Prevent the nutritional component to be destroyed

Holding Method

62-63 °C for 30 min

Flash Method

72 °C for 15 sec

Ultra High Temperature

74-140 °C for 5 sec

Autoclave

- Sporicidal

- Principle: moist under pressure

- Most effective and efficient under moist heat

- Used for pre-disposal treatment

Temperature: 121 °C

Pressure: 15 psi (pound per square inch)

Time: 15 mins

* (for pre-disposal/loaded, 20 mins)

* For medical waste: 132°C, 20 PSI, 30 mins

Parameters of Autoclave

Inspissation

Sporicidal

- Fractional sterilization

- Instrument: inspissator

70-80°C for 2 hours for 3 consecutive days

Tyndallization

- Sporicidal

- Invented by Arnold Tyndall

- Fractional sterilization

- As temperature increases, the exposure decreases

100°C for 30 mins for 3 consecutive days

Radiation

Principle: alkylation of nucleic acid

- The alkyl part of DNA will be removed and destroyed

Ionizing radiation

- May cause possible cell mutation if too much exposure

- Not advised to use routinely

- May cause cancer

- Used for industrial facilities to sterilize disposable plastics and rubbers

Non-ionizing radiation

- Limited sterilizing power

- Poor penetration to materials being sterilized

- More commonly used because it is less toxic

- Use of UV light to sterilize such as operating rooms, laboratories

- Biosafety cabinets also have UV light

Filtration

Removal of microorganisms in the air or in a solution

(antibiotics, sera, sugars)

- Air and solutions are typically contaminated with microorganisms, thus they use membrane filters

Ex: sintered glass filter, asbestos discs

Pore filter

SOLUTIONS: 0.22-0.45um

High Efficiency Particulate Air (HEPA) Filter

AIR: 0.3um

Biosafety cabinet

A laboratory workplace for safety working

- Where specimens are processed safely

- The circulating air is filtered by HEPA filter; UV light as an added feature for microbial control

BSC Class 1

- Open fronted

- air circulating is 100% exhausted out from the system and then filtered by the HEPA filter

- inflow velocity of 75 fpm

- it will protect the personnel but not the specimen

biological level 2 and 3

BSC Class 2-A

- fixed opening

- 30% exhaust, 70% re-circulate

- Negative pressure plenum

- Inflow velocity of 75 fpm minimum

- Both personnel and specimen are protected

biological level 2 and 3

BSC Class 2-B

- Variable opening

- 100% exhaust

- Negative pressure

- No air recirculated

- Faster inflow velocity of 100 fpm

- The personnel and the product are protected

biological level 2 and 3

BSC Class 3

- “glovebox”

- For highly contagious specimens

- 100% exhaust

- Negative pressure

- Double HEPA filter

- Personnel and product protection

- PPE must be complete and all must be disposable

Biological level 4

High Osmotic Pressure

- Bacteriostatic

- In a form of salting and sugaring

- More on preservation than sterilization

- High concentration of salt or sugar to prevent growth of microorganisms

- Ex: jam, pickles, dried fish

- Principle: plasmolysis (destruction of plasma membrane)

Dessication / Dehydration

- Removal of water

- More on preservation of edible foods

- Usual in dry foods

- Inhibit growth of microorganisms such as bacteria and fungi

- Ex: cereals, pasta

Low temperature

- Bacteriostatic

- More on preservation than sterilization

- Either refrigeration or freezing

Refrigeration

- For storage of days

- 2-8°C

- Can be used for chemicals and solutions

- cleaned periodically (once a month) to free from possible psychrophilic contaminants and prevent spoilage

Freezer

- -20°C

- Longer shelf life (Weeks to months)

- In a suspension of culture medium, from the colony, transfer it in another medium then put it in the freezer. The bacteria will not die nor grow.

Type of organism

Number of organism

Concentration of disinfecting agents

Presence of organic material

Nature of surface to be disinfected

Contact time

temperature

pH

biofilms

Compatibility of disinfectants

Factors that influence the degree of killing of disinfection

Bacillus stearothermophilus (colorless – black)

Biological indicator in autoclave

Bacillus subtilis var. niger

Biological indicator in dry heat

Bacillus subtilis var. globigii

Biological indicator in Ethylene oxide

Bacillus pumilis

Biological indicator in radiation