8. CONTROL OF MICROORGANISMS

STERILIZATION

complete removal or destruction of all forms of life, including bacterial spores

DISINFECTION

process that eliminates a defined scope of microorganisms

DISINFECTION

process of killing or removing microorganisms on inanimate surfaces through the use of chemical agents (e.g., use of alcohol)

DISINFECTION

destroys pathogenic organisms, but not necessarily all microorganisms or spores

STERILE

free of life of every kind

BACTERIOSTATIC

having the property of inhibiting bacterial growth or multiplication

BACTERICIDAL

having the property of killing or destroying bacteria

False

cannot be used to human tissues

T or F

bactericidal substances can be used to human tissues

GERMICIDE OR DISINFECTANT

chemical substance used to kill infection-producing microorganisms on the surface, but too toxic to be applied directly on tissues

SEPTIC

characterized by the presence of pathogenic microbes in living tissue

ASEPTIC

characterized by the absence of pathogenic microbes

ANTISEPTIC

chemical substance which opposes sepsis or putrefaction either by killing microorganism or preventing their growth

DECIMAL REDUCTION TIME (DRT/D/D)

time in minutes to reduce the bacterial population or spores by 90% at a specified temperature

PRIONS

misfolded protein that is most resistant to the actions of heat, chemicals, and radiation

True

T or F

pH can neutralize or remove the effectiveness of disinfectants

1 to 2

alcohol and iodine preparations (betadine)- requires at least ______ minutes to kill microorganisms

deactivate / neutralize

combination of disinfectants might ___________ their effectivity

Prions

Bacterial spores

Mycobacteria

Nonlipid viruses

Fungi

Bacteria

Lipid viruses

[Pres. Bong Marcos No Fuck BiLat]

ENUMERATE

different types of organisms and their resistance to killing agents

(from most resistant → least resistant)

HEAT

most common method used for the elimination of microorganisms

Moist Heat

fastest and simplest physical method of sterilization

Moist Heat

sterilization method of choice for heat-stable objects

100 C for 15-30 minutes (20 minutes minimum)

working principle of boiling

Boiling

PHYSICAL METHOD - HEAT

kills all vegetative organism but not all spores or viruses

○ for disinfection only, not for sterilization

Autoclaving

PHYSICAL METHOD - HEAT

sterilize biohazardous trash and heat-stable objects

• example: materials used in bacteriology

15

in AUTOCLAVING all microorganisms (except for prions) and their endospores are destroyed within approximately ___ minutes of exposure

chamber which is filled with hot Steam Under Pressure

working principle of autoclaving

• 121 C (250 F), 15 psi for 15 minutes

• 132 C (270 F), 15 psi for 30-60 minutes

AUTOCLAVING

two common sterilization temperatures:

121 C (250 F), 15 psi for 15 minutes

TWO COMMON STERILIZATION TEMP (AUTOCLAVE)

for media, liquids, and instruments

132 C (270 F), 15 psi for 30-60 minutes

TWO COMMON STERILIZATION TEMP (AUTOCLAVE)

infectious medical waste

Geobacillus stearothermophilus (B. stearothermophilus) (56 C)

BIOLOGIC INDICATOR for AUTOCLAVING w temp

Gravity Displacement Type

most commonly used steam sterilizer in the microbiology laboratory

Flowing Steam

PHYSICAL METHOD - HEAT

sterilizing heat-sensitive culture media containing carbohydrates

Fractional/Intermittent Sterilization/Tyndallization

PHYSICAL METHOD - HEAT

sterilizing heat-materials are exposed for 30 minutes for 3 successive days

Arnold Sterilizer

instrument for Fractional / Intermittent / Sterilization / Tyndallization

100 C for 30 minutes

temp for Fractional / Intermittent / Sterilization / Tyndallization

○ Day 1: Vegetative Cells

○ Day 2: Sporulated Cells

○ Day 3: Remaining Cells

[VaSa Recta]

TYNDALLIZATION

materials are exposed for 30 minutes for 3 successive days

○ Day 1: ______________

○ Day 2: ______________

○ Day 3: ______________

Fractional/Intermittent Sterilization/Tyndallization

PHYSICAL METHOD - HEAT
destroys vegetative cells and spores after three consecutive days

thickening through evaporation

working principle of Inspissation

Inspissation

PHYSICAL METHOD - HEAT

used for sterilizing high protein containing media that cannot withstand high temperature

LJ, Leoffler’s and Dorsey egg medium

ex of inspissation

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

temp for inspissation

Pasteurization / Partial Sterilization

PHYSICAL METHOD - HEAT

Partially sterilizing organic solutions by heat without altering their chemical properties

Pasteurization / Partial Sterilization

PHYSICAL METHOD - HEAT

● eliminates food-borne pathogen and organisms responsible for food spoilage

● cannot eliminate bacterial endospores

A. Low Temperature Holding (LTH) / Batch Method

B. High Temperature Short-Time (HTST) / Flash Pasteurization

C. Ultra-high Temperature

ENUMERATE

Three Types of Pasteurization

• Batch method: 63 C for 30 minutes

• Flash pasteurization: 72 C for 15 seconds

• Ultra-high temp: 140 C for 3 seconds

GIVE TEMP

• Batch method: _____

• Flash pasteurization: ______

• Ultra-high temp: ______

RT ; 2 months

when using ultra-high temp pasteurization: milk can be stored at ___ for ___ without affecting its flavor

Low Temperature Holding (LTH) / Batch Method

TYPES OF PASTEURIZATION

destroys milk-borne pathogens

High Temperature Short-Time (HTST) / Flash Pasteurization

TYPES OF PASTEURIZATION

quick heating and immediate cooling

Ultra-high Temperature

TYPES OF PASTEURIZATION

cooled very quickly in a vacuum chamber

1.5 to 3 hours ; 160 C- 180 C)

dry heat requires longer exposure times (__________) and higher temperatures than moist heat (__________)

• protein denaturation

• oxidative damage

• toxic effects of elevated levels of electrolytes

lethal effects of dry heat

● Flaming with a Bunsen Burner- flaming the mouth of culture tubes or slides

● Burning with a Bunsen Burner- wire loops, forceps, and straight wire

● Flaming with a Bunsen Burner- ________________

● Burning with a Bunsen Burner- ________________

Hot Air Oven

PHYSICAL - DRY HEAT

most widely used type of dry heat

Bacillus subtilis var. Niger (Bacillus atrophaeus) (35 C- 37 C)

biological indicator for hot air oven w temp

burning of materials into ashes at 300 C- 400 C

working principle of incineration

Incineration

PHYSICAL - DRY HEAT

● most common method of treating infectious waste and infected laboratory animals

● destruction of sputum cups, garbage and used dressings

870 C - 980 C

incineration temp for hazardous material

LOW / COLD TEMPERATURE

considered bacteriostatic- reduces the rate of metabolism

2 C for 72 hours

how to kill syphilis (T. pallidum)?

• protein denaturation,

• toxic effects of increased intracellular electrolyte concentration

lethal effects of freezing

Desiccation

disruption of metabolism that involves removing of water from microbes (bacteriostatic)

Lyophilization

this is the changes in proteins and chemical reactions

• Bacillus and Clostridium

• Neisseria gonorrhoeae

• Mycobacterium tuberculosis

[BC NaMan = kaya dry]

ENUMERATE

bacteria which remain active in dry environment

• Bacillus and Clostridium: 10 years

• Neisseria gonorrhoeae: 1 hour

• Mycobacterium tuberculosis: several months

BACTE THAT REMAIN ACTIVE IN DRY ENV’T

• Bacillus and Clostridium:

• Neisseria gonorrhoeae:

• Mycobacterium tuberculosis:

FILTRATION

method of choice for antibiotics solution, toxin chemicals, radioisotopes, vaccines and carbohydrates, serum, plasma, urea (heat-sensitive solutions)

Depth Filters

TYPE OF FILTER

made up of fibrous or granular materials

Membrane Filter (Circular Filter) ; 0.1 um

TYPE OF FILTER

● porous membranes (almost ______ thick)
● composed of cellulose acetate or polycarbonate

● sterilize pharmaceuticals, ophthalmic solutions, culture media, antibiotics and oil products

Air Filtration ; 0.3 um

● uses high-efficiency particulate air filters

● remove microorganisms larger than ______ from isolation rooms, operating rooms, and biologic safety cabinets (BSCs)

0.45 and 0.80 um

Filtration of Bacteria, Yeasts, and Molds SIZE of pores of membrane filter

0.2 to 0.45 um

FILTRATION OF BACTERIA, YEASTS, AND MOLDS

__________ in diameter- remove most bacteria as well as fungi but not viruses

0.01 um

FILTRATION OF BACTERIA, YEASTS, AND MOLDS

_______ are capable of retaining small viruses

0.22 um

Critical Sterilizing Filter SIZE

when radiation passes through the cells, free hydrogen and hydroxyl radicals and some peroxidase are created which in turn cause different intracellular damage

working principle of RADIATION

Bacillus pumilus

biologic indicator for radiation

Ionizing radiation (Cold Sterilization)

● short wavelength and high energy

● used for plastic syringes, catheters, sutures, gloves, hormone solutions and antibiotics

● causes mutation in the DNA and produces peroxidase ● destroys vegetative cells and endospores

Ionizing radiation (Cold Sterilization): gamma rays

Non Ionizing Radiation : ultraviolet rays

Ionizing radiation (Cold Sterilization): ________ rays

Non Ionizing Radiation : ________ rays

Thymine dimers

Non-ionizing radiation damages the cellular DNA by producing __________

Non Ionizing Radiation

● poor penetrability

● used to disinfect exposed surfaces, operating rooms, nursery rooms

● control of airborne infection

Chemosterilizers

Chemical agents used to sterilize (eg. glutaraldehyde)

Silver nitrate (AgNO3)

used as a prophylactic agent in the eye for newborns with mothers suspected of being (+) N. gonorrhoeae

False

much active in lower conc

T or F

iodine / betadine is much active in higher conc

2% glutaraldehyde

acts as Pseudomonacidal, Tuberculocidal, Fungicidal and Virucidal)

10 minutes

GLUTARALDEHYDE

2% solution germicidal in approximately _____

3 to 10 hours

GLUTARALDEHYDE

2% solution sporicidal in _____

25°C to 30°C ; 100%

GLUTARALDEHYDE

2% solution at ______ is ____% tuberculocidal

PHENOL COEFFICIENT

standard reference material for the evaluation of disinfectants

>1= disinfectant better than phenol

<1= phenol is much better

Equal to 1= same efficiency as that of phenol

PHENOL COEFFICIENT

>1= ______

<1= ______

Equal to 1=______

END POINT

lowest concentration that kills the test organisms in 10 minutes at 20°C