MICROBIOLOGY LECTURE (Sterilization and Disinfection)

Definition
Sterilization is the process of killing or removing all viable organisms
achieved by: physical & chemical
Disinfection
is the process of removing or killing most but not all, viable organisms

Methods:
1. Chemical
the substance is called a disinfectant it kills pathogens but may not kill
viruses or spores
2. Physical process
boiling or low pressure steam reduces only the bioburden

Antiseptics:
A particular group of disinfectants used to reduce the number of viable
organisms in the skin.
act differentially on organism and host tissue
1. Germicide
chemical agent capable of killing microbes
2. Sporicide
a germicide capable of killing bacterial spores



Antiseptics on normal skin flora


Microbes in decreasing order of resistance to germicidal chemicals
Pasteurization:
reduces the total number of viable microbes in bulk fluids such as
milkand fruit juices without destroying flavor and palatability.
Decision whether to use Sterilization or Disinfection:
o Cost
o Damage involved
Low bioburden is a prerequisite for cost-effective sterilization.
Uses
1. Prevention of hospital infection depends on
o sterile equipments, instruments and dressings
o isolation facilities
o safe disposal of infected materials
2. Microbiologists
o production of sterile media and the laboratory activities
3. Central to almost all areas of medical practice like surgery(from hand
washing to needles and prosthesis)
Process: The rate of killing of microorganisms depends upon the
concentration of the killing agent and time of exposure.
N = 1/CT
N number of survivors
C concentration of agent
T time of exposure to the agent


Mechanisms of action of antimicrobial agents:
1. Damage cell membrane
2. Denature proteins
3. Modify functional groups of proteins and nucleic acids
Activity of a particular disinfectant may result from one or combination
of pathways.

Factors affecting efficacy:
1. Physical environment
2. Presence of moisture
3. Temperature and Ph
4. Concentration of the agent
5. Hardness of water
6. Bioburden and the object
7. Mature and state of microbes in bioburden
8. Ability of microbes to inactivate the chemical agent

Techniques for Sterilization
A. Heat preferred choice
ease of use
controllability
cost
efficiency
Types:
1. Dry heat
o Incineration
o use of bunsen burner
o hot air oven (one hour)
2. Moist heat under pressure
o autoclave - most effective for sterilization (121
o
C, 15 mins.)
o pressure aids in penetration
o derived from thermal death curves for pathogens like Clostridium
o sterilization of surgical instruments, dressing and heat resistant
pharmaceuticals
3. Boiling Water
o for a few minutes can be used as a rapid emergency measure to
disinfect instruments
o kills vegetative bacteria but not all spores
4. Pasteurization
o done at 62.8 65.6C for 30 minutes
o used for fluids
o reduce the number of bacteria
o eliminate pathogen present in small numbers
o improve shelf-like of milk
o Flash pasteurization
71.7
o
C for 15 secs.
after the process, the fluid should be kept at a
temperature below 10C to minimize, subsequent bacterial
growth.

B. Irradiation
Gamma irradiation used for sterilizing large batches of small volume
items such as:
1. Needles, syringes, catheters, gloves
2. Vaccines
3. To prevent food spoilage
capital cost is high but process is 100% efficient
killing mechanism involves production of free radicals that break the
bonds in DNA
sporocidal at higher doses (4.5 mega rads)

C. Filtration
used to produce particles and pyrogen-free fluid.
composed of nitrocellulose
work by electrostatic attraction and physical pore size
purify drinking water
to recover very small number of organism from large volumes of fluid
can be used for quantitating bacteria in fluids





D. Chemical agents (liquid or gas phase)

1. Alkylating agents
a. Gases
i.

ethylene oxide (toxic and explosive; prosthetic heart
valves)
ii. formaldehyde (extremely unpleasantodor; and irritant to
mucous membrane decontaminate rooms and
exhaustprotective cabinets)
b. Liquid
i. Glutaraldehyde - disinfects heatsensitive articles
(endoscopes and inanimate surfaces)
2. Oxidizing agents
a. Hydrogen peroxide
3 6% kills most bacteria
10-25% kills all including spores
(disinfects plastic implants, contact lenses and surgical
prosthesis)
b.

Halogens I
2
or Cl
2
containing:
. Iodine compounds
most effective, virtually effective among all organisms
including spore- formersand mycobacteria.
effective in acid pH because more free I
2
is liberated
acts more rapidly than other halogens and quarternary
NH
4
compounds
effectivity reduced by serum,feces, bodyfluids,Na
thiosulfate and NH
3


Povidone iodine used to disinfect metalsurfaces and tissues.
ii. Chlorine compounds
used extensively
3 forms available
Cl
2

HOCl( hypochlorous acid)
OCl (hypochlorite)
good germicide activity, though sporesare resistant to it.
2. Phenolic compounds
o rarely used as disinfectant nowadays
o historical interest it was used as comparative standard for
assessing the activity of germicidal compounds
o Phenol coefficient - ratio of germicidal activity by a test
compound compared to that by a specified concentration of
phenol

o sporocidal at T approaching 100C but not at room temperature
o effective on resistant mycobacteria
o Activity potentiated by halogenation and aromatic group into
their nucleus
o Hexachlorophene
4. Quarternary Ammonium compounds
o organic groups covalently linked to nitrogen (Benzalkonium
chloride and Cetyl pyridinium chloride. Bacteriostaticat low
concentrations, bactericidal at high. Not effective against
Pseudomonas,Mycobacteria and Trichophyton)
5. Alcohols
o germicidal activity increases with increasing chain length (5-8
carbons)
o most commonly used ethanol and isopropanol
o no activity against spores
o activity is more active in the presenceof water hence 70% alcohol
is more active than 95% alcohol.
o common disinfectants used for skinsurfaces
o extremely effective when followed bytreatment with iodophor
6. Heavy metals
o soluble salts of Hg, arsenic, silver and other heavy metals
o by forming mercaptides with sulfhydrylgroups of cysteine residue
o mercurials merthiolate, mercurochrome
o silver compounds irritant and caustic effect; silver nitrate
(propylaxis);sulfadiazine cream; wound, culloidal silver
compounds used in ophth

E. Others freezing, lysis, dessication, ultrasonication, use of electrical
discharges