Dr Shireen RafiqMBBS, M.Phil, Ph.D

PURPOSE

CULTURE MEDIA

INSTRUMENTS AND GLASSWARE

DISPOSAL OF MATERIALS

STERILIZATION CHECKS

Basic terms

sterilization disinfection Antisepsis Bacteriostasis Asepsis

Sterilization The process of destroying all

microbial forms both pathogenic and non pathogenic. A sterile object is one free of all microbial forms, including bacterial spores.

Disinfection The reduction or elimination of

pathogenic microorganisms in or on materials, so they are no longer a health hazard.

Antisepsis

Use of chemical agents on skin or other living tissue to inhibit or eliminate microbes; no sporicidal action is implied.

Bacteriostasis Inhibits the growth of micro-

organisms.

Asepsis No living microorganisms exists.

Risk Groups (WHO)

Risk Groups OrganismsGroup I Low risk Food spoilage bacteria moulds and yeasts

Group II Moderate risk

Staph, Strep, vibrios, Enterobacteriaceae, adeno, polio, coxsackie V Toxoplasma, Leishmania

Group III High Risk

Brucella, MTB, S typhii, Plague bacteriaHIV, Rickettsiae,

Group IV Viruses

Smallpox, Hemmorhagic fevers, Encephalitis, Arbo Viruses

Physical Chemical

Methods of Sterilization and Disinfection

Heat

Radiation

Filtration

Disinfectants

Antiseptics

HEAT

DRYMOIST

RED HEATHOT AIR

OVEN

PASTEURIZATIONBOILING

STEAMINGWASHING MACHINES

High Temperature

static action cidal action

DRY HEAT

>Red Heat Flaming (Metallic

objects) >Hot Air Sterilizer 160-1800C Glassware and instruments Oils, Powders, Waxes

MOIST HEAT advantage

Pasteurization 56C for 1 hour (Heat below 100C) Heat labile fluids (serum, body fluids) Juices, milk Washing machines (washing+heat) (3mins at 71C or 1min at 80C Linen, bedpans, protective clothing,

MOIST HEAT Below 100 Pasteurization Vaccine bath At 100 Boiling Steaming Tyndalization Above 100 Autoclave

MOIST HEAT

Boiling at 100C for 5mins (water bath) >Advantage HBV, all vegetative bacteria, some spores Useful in emergency conditions >Disadvantage Not for heat labile /porous articles

MOIST HEAT cont…

Steaming at 100C (for 5 mins) >Advantage: prevents cracking of glassware Heat labile culture media Destroys most vegetative bacteria >Disadvantage: Spores, thermophiles not destroyed

fractional sterilization or Tyndallization: 1) Steam heating to 100 °C for 30 min——

Vegetative cells are destroyed but endospores survive

2) Incubate at 30 °C -37 °C overnight Most bacterial endospores germinate

3) Second heat treatment, 100 °C, 30 min Germinated endospores are killed.

4) Second incubation at 30°C-37 °C overnight Remaining endospores germinate

5) Third heat treatment, 100 °C, 60 min Last remaining germinated endospores are killed

MOIST HEAT cont…

AUTOCLAVES Produces saturated steam at higher

than atmospheric pressure Latent + sensible heat 121C for 15mins at 15lbs pressure Denaturation of all microbial protein

Penetration time Thermocouples

Hold time Bacillus stearothermopilus

Condensation of steam

Saturated steam

1670 vol steam =1vol condensate

Uses Culture media and other lab supplies Fluids in sealed bottles Glassware and reusable equipment Waste materials Grade 2-4 pathogens

Autoclave Efficiency testing MC

Bowie Dick Test

Browns Indicator Tube or TST indicator

STERILIZATION by GAS

Uses Heat labile equipment LTSFEthylene OxideGas plasma

Filters

Membrane filters Diameters 13-239mm Pore sizes 0.015-12umSyringe filtersVacuum filtersPressure filtersAir filters HEPA

Air filters

Hepa

Vacuum filter

Sterilization by Radiation Ionizing Radiation

UV Radiation

Disinfection

Definition Destruction of microorganisms

but not usually their spores British Standards Institution

Disinfectants: Chemical agents capable of disinfection

GROUPS OF DISINFECTANTS PHENOLICS Disruption of plasma membrane. Denaturation HALOGENS Inhibition of protein function. Strong oxidizing agents

BIGUANIDES Disruption of plasma membrane.

ALCOHOLS Protein denaturation and lipid dissolution

QUATS Disruption of plasma membrane.

Use of Disinfectants

Decontamination of surfaces Medical devices Large surface areas Heat labile objects

Activity

Gram positive easily killed Gram negative more resistant

(Pseudos) AFB even more resistant Viruses variable Spores very resistant

Disadvantages

Disinfection of Surfaces and Spillages

Body Fluids, Blood, Virus – 10,000ppm Hypochlorite 2% Phenol 2% Glutaraldehyde

Phenolics : Gram positive and negative bacteria and Mycobacteria, Not suitable for endospores, or viruses

Glutaraldehyde (Cidex) Pre-cleaned surfaces Small surfaces areas with suspected

viral contamination Useful against Mycobacteria and

spores

Ethyl Alcohol Bactericidal Not active against spores and many viruses Poor activity in presence of organic

material. WIPING WITH ALCOHOL IS DANGEROUSLY INEFFECTIVE

Sterilization by Gas

Ethylene Oxide Alkylation of protein, DNA and RNA

Formaldehyde

Uses: Devices and materials that cannot stand high temp/humiditiy

Critical and semi critical items