Physical and Chemical Methods of Sterilization: Techniques and Controls

1. Physical methods of sterilization  

Dry heat

Mechanism (C O D E)

• Charring.
• Oxidative damage.
• Denaturation of proteins.
• Elevation of electrolytes

Direct contact with flames or sunlight:

• Candle
• Flame burners
• Sunlight
• Hot air oven
• Incinerator

Incineration

There are 2 chambers.

• 700° C - 800° C
• 1000° C - 1100° C

Used for

• Hospital waste.
• Human anatomical waste.
• Animal carcasses.
• Soiled bedding & dressings.
• Expire cytotoxic drugs.

Hot Air Oven

• Temperature and duration
  • 160° C for 2 hr
  • 170° C for 1 hr
  • 180°C, 30 mins
• Uses

• Glassware
• Cotton swabs
• Gel, oil, wax, dusting powder.
• Greasy paraffin wax
• Metallic instruments (sharp and non sharp).

Controls: To check whether the machine is working or not

Also read: Staphylococcaceae : Staphylococcus aureus and Clinical Implications

Methods for Sterilisation Control

For Dry Heat

• Physical: Temperature monitoring by thermocouples
• Chemical: Browne's tube (green spot).

→ If the green spot shows, then sterilization is complete.

→ Kill spores: sterilization is done.

Moist Heat: Mechanism

• Coagulation and denaturation of proteins
• Moist heat is considered better

    ↓

Steam

   ↓

Now act as “Latent Heat.”

   ↓

Steam will settle on surface

Below 100°

Pasteurisation

• Milk is pasteurized.
• Holder Method: 63 oC for 30 minutes
• Flash Method
  • 72° C for 20 seconds
  • Ultra-high-temperature method.

→ 149° C for 1-2 secs

Test for Efficacy of Pasteurization

• Phosphatase test

→ Milk has an enzyme called phosphatase.

→ Pasteurization leads to denaturation of enzymes.

→ On adding the substrate, there is no color change.

• Coliform test

→ If milk is pasteurized, there is no growth on MacConkey agar

Inspissation

• 80° C for 20-30 minutes for 3 consecutive days.
• Used for
  • LJ media for TB
  • LSS for C. Diphtheriae

Vaccine water bath

• 60° C for 60 minutes
• Bacterial vaccines are heat inactivated

Also read: Coccidian Parasites: Toxoplasma Gondii

At 100°

Boiling

• At 100°C for 15 min
• Does not kill spores.

Tyndallization

• 100° C x 20 min x 3 days.
• Kills spores

Used for media with gelatin and sugar.

TCBS, XLD, DCA, Selenite F Broth.

Above 100°

Autoclave

• 121° C for 15 min at 15 psi (pressure)
• To kill prions: 134° C for 1.5 hrs

Control

• Geobacillus Stearothermophilus

Uses

• Non-sharp instruments
• Aprons
• Sutures except Catgut sutures.
• Sputum: Prior to discarding

Filtration (Average Pore Size: 0.22m

Types of filters

• Depth filters:
  • Candle filters: Water purifier
  • Sintered Glass filters
  • Seitz filters: Asbestos sheets for filtration
• Membrane filters: 

• Cellulose acetate
• cellulose nitrate

• Uses:
• Heat-sensitive substances.

• Vaccines
• Antibiotics
• Serum
• Toxins
• Sugar solutions

HEPA: High-Efficiency Particulate Air Filter (99.97% efficient).

ULPA: Ultra Low Particulate Air Filter (99.99% efficient).

Also read: Miscellaneous Pathogenic Bacteria

MCQs on Physical method of sterilization

Q. Identify?

A. Autoclave

B. Incineration

C. Seitz Filter

D. Water Bath

Q. Which one of the following organisms is used as a control for filtration?

A. Bacillus pumilus

B. B.diminuta

C. Bacillus globigii

D. Clostridium tetani

Q. Incineration is done for

A. Human body parts

B. Syringe

C. Body fluids

D. Gloves

Q. Grease, dusting powder, and paraffin are sterilized by?

A. Gamma radiation

B. Sunlight

C. Dry heat sterilization

D. Autoclave

Q. Method of choice for sterilization of liquid paraffin?

A. Flaming

B. Moist heat

C. Autoclave

D. Hot air oven

Q. Pasteurisation of milk is done at?

A. 63 degrees for 30 minutes

B. 63 degrees for 20 minutes

C. 72 degrees for 30 minutes

D. 72 degrees for 20 minutes

Q. Cold sterilization is done by?

A. Steam

B. Ionizing radiation

C. Infra-red

D. UV

Also read: NEET PG High Yield Questions for Microbiology

2. Chemical Methods of sterilization

Alcohols

• Ethyl alcohol (70%) : Spirit
• Isopropyl Alcohol: 70-80%
  • Stethoscope and thermometer

Aldehyde

• Mechanism: cross-linking and alkylating the molecules.
• Formaldehyde
  • Gas: OT fumigation.
  • Formalin: All human specimens stored.
    • Animal wool is treated with formaldehyde.

• Glutaraldehyde
  • Mechanism: cross-linking and alkylating the molecules.
  • CIDEX: bronchoscope, laryngoscope
  • Activated by alkalinization—active for 14 days.
• Ortho-phthalaldehyde (opa-cidex)
  • 0.55% solution
  • Cystoscopes etc
  • No activation needed and more stable
  • More bactericidal activity.

Phenols

• Antiseptic Surgery: Joseph Lister.
  • Phenol (5%).
  • Cresol (1-3%).
  • Lysol (2-5%)
• Modified phenol.
  • Chlorhexidine + Cetrimide = Savlon
  • Chloroxylenol = Dettol

Halogens

• MOA-oxidizing agents
• Iodine
  • Tincture iodine (2% iodine) for skin antiseptic
  • Povidone iodine-Betadine (IODOPHOR)
    • I2 absorbed on the neutral carrier (poly vinyl pyrrolidone)
    • Has residual action.

Chlorine

• 1% sodium hypochlorite- Dilution depends on how much blood has spilled.
• <10ml spill 1:100.
• >10ml spill 1:10.

Also read: INI-CET High Yield Questions for Microbiology

Plasma Sterilization or Sterrad

• Plasma
  • Gaseous state containing ions and electrons.
• Process
  • UV photons are used to create plasma states.
  • Vacuum
  • Low temperature
  • H2O2 chemical present
• Advantage
  • No toxic residues
  • Short cycle time: 75 minutes
• Used
  • Sterilization of Surgical Instruments: Eg., Arthroscopes, Ureteroscopes.
• Control
  • Bacillus sterothermophilus (autoclave, plasma sterilization).

Gas Sterilization

• Ethylene oxide (ETO)
• MOA
  • Microbicidal
  • Sporicidal
• Side effects
  • Highly inflammable.
  • Highly irritant.
  • Highly explosive.
• Factors affecting ETO.
  • Concentration
  • Humidity
  • Temperature
  • The entire cycle might take 18–24 hours.
• Used
  • Cardiopulmonary machines.
  • Pre-packed syringes
• Control
  • Bacillus Globigii.

Also read: Hemoflagellates: Developmental Stages, Types, Clinical Features

Other Sterilization Methods

Heavy Metal Salts

• Ophthalmia neonatorum: (Crede's method) 1-2% AgNO3
• Silver sulfadiazine: for burns
• Antisera, Vaccines Mercury

Surface Acting Agents

• MOA: Lower the surface tension.
• Anionic
  • Detergent-like action.
  • Weak antimicrobial activity.
  • Eg : Soap
• Cationic
  • Quaternary ammonium compounds
  • More effective against GP than GN bacteria
  • Eg: Cetrimide savlon
• Non-ionic
• Amphoteric (TEGO compounds)
  • Anionic plus cationic.
  • Detergent + antimicrobial activity.
  • Used as an antiseptic in dental procedures.

Dyes

• Aniline dye
• More active against GP.
• Not active against MTB.
  • Crystal Violet
  • Gentian violet
  • Malachite green
    • Culture media for TB is LJ media.
    • Malachite green will damage gram-positive organisms and it allows MTB to grow.
• Acridine dyes
  • More active against GP
  • Can act in presence of organic waste
    • Acriflavine
    • Proflavine

Also read: Chemical Regulation of Respiration: Role of Chemoreceptors and Reflexes

MCQS

Q. Which of the following tests is used for testing the capacity of a disinfectant?

A. Rideal walker test

B. Chick martin test

C. Kelsey sykes test

D. Kelsey Maurer test

Q. Glutaraldehyde is used for all of the following except?

A. Bronchoscope

B. Thermometer

C. Proctoscope

D. Endoscopic tubes

Q. Control for ethylene oxide (chemical) disinfection is?

A. Bacillus pumilus

B. Bacillus stearothermophilus

C. Bacillus globigii

D. Clostridium tetani

Also read: How to Treat Infectious Diseases?

Q. All are true about plasma sterilization except?

A. Produces less toxic products than Eto

B. Uses bacillus stearothermophilus as control

C. Can be utilized for sterilization of products which are heat sensitive

D. Chamber used for carrying out plasma sterilization uses ambient air as its medium

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