Disinfection, Antiseptic & Sterilization| GPAT | Drug Inspector Pharmacist

Disinfection, Antiseptic & Sterilization | Pharmacy Notes| GPAT | Drug Inspector Pharmacist

Sterilization

it is a process by which an article, surface for the medium is made free of all microorganisms (MOs) either in the vegetative or spore form.

Disinfection:

It means the destruction of all pathogens or organisms capable of producing infection but not necessarily spores (the number is reduced to a level that is no longer harmful to health).

Antiseptic:

these are chemicals disinfectant which can safely be applied to living tissue and are used to prevent infection by inhibiting the growth of microorganisms.

Asepsis:

It is the technique by which the occurances of infection into an uninfected tissue is prevented.

Methods of Sterilization:

Methods of Sterilization (Physical & Chemical methods) Thepharmapedia
Diagram: Methods of Sterilization

PHYSICAL METHOD OF STERILIZATION

  1. Sunlight (265nm UV rays)
  2. Heat (Most reliable & commonly used)
    1. Dry Heat (Oxidative damage & Denaturation of protein)
      1. Red hot
      2. Flaming
      3. Incineration-ashes
      4. Hot air oven
    2. Moist heat (denaturation & Coagulation of protein)
      1. Temperature below 100 
        • Pasteurisation
          • Holder method: 63ºc for 30 min
          •  Flash method:  72 ºc for 20 sec followed by cooling quickly to 13 ºc or lower.
        • Inspissation (80-85 ºc for 30 min for 3 consecutive days). Serum or egg media
        • Vaccine bath (bacterial vaccine are sterilised in vaccine bath (serum/body fluid at 56-60 ºc for one hr for several successive days.
        • Low-temperature steam formaldehyde (LTSE)– (Steam at subatmospheric pressure at the temperature of 75 degree centigrade with formaldehyde vapour is used.  Biological indicator- bacillus stearothermophilus)
      2. At temperature  100
        • Boiling: (Bowling for 10 to 30 minutes kill most of the  vegetative  formed.
        • Tyndallisation: Steam   at    100   degree   centigrade    for 20 minute   on  three successive days. First  exposure   kills all the  vegetative forms,  and in the interval between the heating, the remaining spore   germinate  into vegetative  from   which are killed on the subsequent   heating.  Tyndallization or intermittent sterilization is used for the sterilization of egg, serum and sugar containing media which are damaged at Higher temperature of autoclave.
        • Steam sterilization @ 100 for 90 min/koch’s or arnolds steriliser
      3. Temperature above 100 under pressure (Atm. Pressure increased- also boiling temp increases). Eg. Autoclave
Factors influencing heat sterilization
  • Nature of heat: Dry or moist
  • Temp &duration: time for sterilization inversly proportional to temp.
  • Characteristics of MOs & Spore
  • Spore are killed by moist heat 121 for 15 min
  • Most vegetative  bact., fungi and viruses are killed in 30 min at 65 by moist heat
  • Type of materials: high conc. organics matters tend to protect the vegetative  form and spore form

3) Ozone

Ozone sterilizer uses oxygen, water and  electricity  to  produce  Ozone  within the sterilizer low temperature 25 to 35-degree centigrade.  Ozone provides Sterility Assurance of 10-6 in approximately 4 hours.

4) Filtration:

Filtration method is Used for the substances

  • A substance which get damaged by heat process like Sera, Sugars, antibiotic solution etc.
  • Separation of toxin and bacteriophages from bacteria; 
  • to obtain bacteria-free filtrate for virus isolation; 
  • sterilization of hydatid fluid;
  • testing of Water sample for cholera vibrio for Typhoid bacilli  bye retaining the organism on filter disc.
  • Purification of water

Limitations of filtration method: virus and mycoplasma pass through member filter

Type of filters-
  1. Candle filters-Diatomaceous earth
  2. Asbestos disc filters/Seitz filter: made of magnesium silicate (asbestos)
  3. Sintered glass filters: made by choosing finely powdered glass particle & available in different pore sizes
  4.  Membrane filter: made of cellulose Ester (acetate & nitrate)
  5. Syringe filters
  6. Air filter: HEPA  filter: used to deliver clean bacteria free air to a room. HEPA filter can remove particles of 0.3 micrometre or larger.

5) Radiation (Cold sterilization)

  1. Ionization radiation:  high penetration power; include Gamma Rays(Co-60,), x-ray and Cosmic rays.
  2. Non-ionization radiation: IR & UV radiation(240 to 280 NM): act by denaturation of protein and interference with DNA replication.

CHEMICAL METHOD OF STERILIZATION

  1. Alcohol (Ethyl Alcohol 60-70%, Isopropyl alcohol 35)
  2. Aldehyde (Formaldehyde, glutaraldehyde,  ortho- phthaladehyde)
  3. Phenols
  4. Halogens (chlorine & iodine)
  5. Oxidizing agent (Hydrogen peroxide,  peracetic acid, plasma)
  6. Salt (MERCURIC CHLORIDE, THIOMERSAL & COPPER SALTS)
  7. Surface active agent/surfactant
  8. Dyes
  9. Vapour phase disinfectants  (Formaldehyde gas, ethylene oxide, beta propio-lactone)
  1. Alcohol
  • Ethyl alcohol and isopropyl alcohol most frequently used
  • Act by denaturing the bacterial protein
  • Ethyl Alcohol 60-70%, Isopropyl alcohol 35
  • No sporicidal/virucidal  activity (But HIV is susceptible)
  • Mainly used for skin antiseptic
  • Methyl alcohol vapors is toxic and inflammable and effective against fungal spore

2) Aldehyde (Formaldehyde, glutaraldehyde,  ortho- phthaladehyde/OPA)

  • Formaldehyde:- Bactericidal, sporicidal, virucidal. Used to sterilize bacterial vaccine,  to prepare toxoid from toxin, preservation of tissue for histological examination
  • Glutaraldehyde(cidex; 2% buffered solution): Effective  against  bacteria, Fungi and Virus and also killed spore. Sterilization   of cytoscopes (DI CG), endoscope, bronchoscope, endotracheal tube,   rubber anaesthetic tubes etc.
  • Ortho- phthaladehyde/OPA:- 0.5% OPA, used same as glutaraldehyde

3) Phenols(Carbolic acid)

  • Cause cell membrane damage & lysis
  • Phenol derivatives like cresol, chlorhexidine, chloroxylenol  &   hexachlorophene are commonly used as antiseptic.
  • Cresol (Lysol)-not inactivated  in the presence of organic matter
  • Chlorhexidine(savlon)– (chlorhexidine and cetrimide)
  • Chloroxylenol( dettol)-inactivated by presence of organic matter

4) Halogens (chlorine & iodine)

  • Bactericidal and virucidal
  • Chlorine compounds in  the  form of  bleaching  powder,  Sodium  hypochlorite  and chloramine are used.
  • Hypochlorite with water, chlorine is released and form hypochlorous acid with water which is a strong oxidizing agent and effective disinfectant.
  • Hypochlorite  solution is the most widely used for human immunodeficiency  virus-infected materials, H1N5, Covid 10 virus.
  • Hypochlorite has bactericidal,  fungicidal, virucidal.
  • Iodine in alcoholic and aqueous  solution used as a skin disinfectant.  The compound of iodine with surface-active agent known as iodophors are claimed to be more active than the aqueous & alcoholic solution of Iodine. Eg Betadine

Chorine Disinfectant: Chlorine is an effective disinfectant where water is not turbid (cloudy) and the pH of the water to be treated is not alkaline, for instance not above pH 8.0.

Find complete details here (reaction of chlorine in water which result in the formation of hypochlorous acid & availability of chlorine in various forms, including calcium hypochlorite, sodium hypochlorite and as pure chlorine gas in cylinders etc)

5) Oxidizing agent (Hydrogen peroxide 3&6%,  peracetic acid, plasma)

  • H2O2: Liberation of free hydroxyl radical on the decomposition of hydrogen peroxide. Free radicals are oxidizing
  • Peracetic acid: oxidising agent and effective in the presence of organic matter

6) Salt (MERCURIC CHLORIDE, THIOMERSAL & COPPER SALTS)

  • Protein coagulation by binding with SH (Sulphydryl) group of bact. protein

7) Surface active agent

  • Surface active agent  are classified  into an Ionic,  Cationic, Non Ionic &  Amphoteric Compound.
    • Cationic surfactant are most important antibacterial agent.  They act on the phosphate group of the cell membrane and also enter the cell.  This results in loss of membrane semi permeability and the cell protein are denatured.
    • Quarternary ammonium compound: acetyl trimethyl ammonium bromide (cetrimide or cetavalon)   & benzalkonium chloride-activite in  alkaline  pH,   inactivate in acid and anionic surfactant render them inactivate.
    • An ionic compound  (soaps)  have a strong  detergent but weak microbial properties & most active at acidic pH.
    • An amphoteric compound known as ‘Tego’  posses  detergent property  of  anionic and antimicrobial activity of cationic compound.

8) Dyes (Aniline Dyes & Acridine dye)

  • Aniline dyes: includes Crystal Violet, brilliant green and Malachite green;  more active against Gram-Positive Bacteria & and no activity against Mycobacterium tuberculosis, hence the use of malachite green in the Lowenstein Jensen medium makes it selective for isolation of tubercular bacilli.
  • Acridine dyes: acriflavine,  euflavine, proflavine & aminacrine. These dyes interfere with the synthesis of nucleic acid and protein in a bacterial cell
  • Gentian violet and acriflavine are widely used for skin disinfectants.

9) Vapour phase disinfectants  (Formaldehyde gas, ethylene oxide, beta propio-lactone)

  • Formaldehyde gas
    • Fumigation of operation theatres ward Laboratories
    • Door should be sealed and left unopen for 48 hours.After completion of disinfection,  the effect of irritant vapour should be nullified by exposure to ammonia vapour.
  • Ethylene oxide: colourless gas, BP 10.7 degree centigrade
  • Beta propiolactone: Condensation product of Ketan and formaldehyde, have carcinogenic activity & Used for sterilization of vaccines

The concentration of Common disinfectants

DisinfectantConcentration
Ethyl alcohol70 %
Sodium hypochlorite1 %, 0.1%
Betadine (Iodophore)2 %
Dettol (Chloroxylenol)4 %
Savlon ( Chlorhexidine & Cetrimide)2%, 5 %
Prepared by ThePharmapedia.comThePharmapedia.com
Bleaching powder (Calcium hypochlorite)14 gm in one liter of water
Lysol2.5 %
Glutaraldehyde2 %
Table: 1 Concentration of disinfectants

Choice of Sterilization’s method & Disinfection of some Important Materials

MaterialsMethod of Sterilization
MilkPasteurisation
SkinTincture iodine, 70% Alcohol, Savlon
Culture mediaAutoclaving
Culture media containing serum, eggTyndallisation
Inoculating wires or loopRed heat
Glasswares: syringes, Petri dishes, test tubes, flasks, universal container, oily fluids like paraffinHot air oven
Disposable syringes & other disposable itemsGamma radiation
Toxin, Serum, Sugar & Antibiotic solutionsFiltration
Cystoscope & EndoscopeGlutaraldehyde
Operation theatreFormaldehyde
Infective material like soiled dressings, beddings, animal carcassesIncineration
Aprons, Gloves, catheters, surgical instruments except sharp instrumentsAutoclaving
Sharp instruments5 % cresol
Rubber, plastic & polythene tubesGlutaraldehyde
Table2 : Methods of Sterilization & Disinfection of Important Materials

Evaluation/Testing of Disinfectants

  1. Rideal Walker test
  2. Chick Martin test
  3. Kelsey sykes test
  4. In-use test

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