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Physical Agents

  1. Heat

    1. Dry heat Kills by oxidation

    2. Red heat: indicating wires, loops, points of forceps
    3. Flaming: Scalpel blades, glass slides, mouth of culture tubes
    4. Hot Air: 160°c for 1hr
      1. Sterilization of glassware: Glass syringe, test tube, petri dishes, pipettes, flasks
      2. Metal instruments: Forceps, scissors, scalpels
      3. Oils, jellies, powders
      4. Controls: Biological: Bacillus subtilis subspecies niger 106 spores (filter paper strip)
      5. Chemical control: Browns tubes (redgreen)
    5. Moist heat:
      1. ​​Kills by denaturation and coagulation of proteins
  1. Temperature below 100°c
    1. Heat labile fluids disinfected
    2. Pasteurization - 63°C 30 min (Holder)
      -  72°C 20 sec (Flash)
    3. Serum bath 56°C , 1hr
    4. Vaccine bath 60°C, 1hr
    5. LTSF (Low Temperature Steam- Formaldehyde sterilization)- Method of sterilization
    6. Steam at subatmospheric pressure at Temp: 75°C
  2. Moist heat, temp at 100°c
    1. Boiling at 100°C: 10-30 minutes, Some bacterial spores not killed, Metal, glass, rubber items
  3. Free steam at 100°c
    1. Latent heat
    2. Culture media containing sugar, gelatin
    3. Some spores not killed
    4. Tyndallization/ intermittent sterilization: 100°c, 20 minutes for three consecutive days
  4. Moist heat, temp >100°c
    1. Steam under pressure (autoclaving)
    2. 15 psi, 121°c, 15-20 min
    3. Dressing material, linen, gloves
    4. Culture media, aqueous solution
    5. Controls: Bacillus stearothermophilus 106 spores
    6. Chemical: brownes tube no 1
  1. Filtration
    1. Liquids such as sera, solutions of heat labile substances - sugars, urea sterilized by filtration
    2. Mycoplasma, viruses cannot be kept back by the bacterial filters (0.45MICRON)
    3. Controls: Serratia marcescens, Pseudomonas diminuta
    4. Earthenware filters
      1. (Kieselguhr) fossil diatomaceous earth
      2. Chamberland made of unglazed porcelain
    5. Asbestos (seitz) filters
      Disc of magnesium trisilicate
    6. Sintered glass filters-reusable discs
    7. Membrane filters : cellulose esters (0.015- 12μm), most commonly used filters.
    8. Air filters: HEPA remove particles more than 0.3μm
(Extra Edge) :


  1. Distilled water should have conductivity < than given by 1ppm NaCl
  2. Deionized water (demineralized water) should be equivalent to double
  3. Glass-distilled water and should have very low conductivity



Recent Advances

  1. In Ion exchange resins used to deionize water:
    1. Cation exchanger column is regenerated by 10% aqueous HCl
    2. Anion       ,,                      ,,                   ,,     10% aqueous NaOH
  2. McFarland standards – are opacity standards used to prepare a bacterial suspension of a standard concentration eg 0.5 McFarland standard opacity corresponds to bacterial concentration of 150 millions/ml = 100 x 1000,000
  3. Gnotobiotic animals- are ‘germ free’ animals reared in sterile isolator are abnormal animals ( structurally & physiologically) but are valuable in some kinds of microbiological experiments
  4. Hydroclave is more sophisticated method of autoclaving in which there is no direct contact of steam with waste. Moisture content of waste turns into steam which builds required pressure in the chamber
  5. HEPA filters in biological safety cabinets are sterilized by hydrogen peroxide
  6. Sodium dichloroisocyanurate (NADCC)- solid chlorine releasing granules for direct application to spillages
  7. NEW methods/agents of sterilization
    1. Surfacine - Antimicrobial coating containing silver iodide
    2. Sterilox - Superoxidized water
    3. Endoclens - Liquid sterilization system for endoscopes
      Attest   - Rapid read out ethylene Oxide biological indicator producing fluorescent change on failure  
  1. Radiation
    1. Non-ionizing : UV radiation 250-260 nm, spores resistant, HIV not inactivated. Induce thymidine/ pyrimidine dimers, disinfection of clean surfaces.
    2. Ionizing: α rays, gamma rays, cosmic rays, sterilization of pre packed disposable items (cold sterilization) Control: Bacillus pumilis 

Recent Advances :

NIOSH approved N95 disposable particulate respirators (Filtering face pieces/masks) are designed to help provide respiratory protection for the wearer. They have filter efficiency level of 95% or greater against particulate aerosols free of oil when tested against a 0.3 micron particle. The “N” means “Not resistant to oil”. The “95” refers to a 95% filter efficiency


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