Disinfection & antiseptics in microbiology , bpharm 3rd semester 2nd year

Disinfection & antiseptics in microbiology , bpharm 3rd semester 2nd year

• 1.
  DISINFECTION & ANTISEPTICS 1. Factorsinfluencing disinfection 2. Antiseptics and their evaluation For bacteriostatic and bactericidal actions 3. Evaluation of bactericidal & Bacteriostatic.
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  Disinfectants A disinfectant isan antimicrobial agent that can be applied to the surface of non-living objects to destroy or reduce the number of microorganisms residing on them. Antiseptics Antiseptics are antimicrobial agents applied to the living tissues of living organisms (such as skin or mucous membranes) to inhibit the action of microbes. Examples: Hydrogen Peroxide, Chlorhexidine, Povidone Iodine & Chloroxylenol (Dettol). Asepsis It refers to the state of being free from disease-causing microorganisms such as pathogenic bacteria, viruses, fungi, and parasites. Also known as clean technique, medical asepsis aims to reduce the number of microorganisms and prevent their spread. Practices include hand hygiene, wearing gloves, and maintaining a clean environment.
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  FACTORS INFLUENCING DISINFECTIONAND ANTISEPTICS 1. Chemical Structure of Disinfectant 2. Interfering Substances in the Environment 3. Surface Tension 4. Types and Number of Microbes Present 5. Temperature 6. Potentiation and Antagonism of Disinfectants 7. pH of the Surrounding
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  1. Chemical Structureof Disinfectant: The chemical structure of disinfectants significantly influences their efficacy.  For instance, adding an alkyl chain to the para position of phenol enhances its activity. However, excessive carbon atoms in the chain reduce solubility and disinfecting effectiveness.  Halogenation of phenol enhances its antibacterial properties, while nitration reduced its effect. 2. Interfering Substances in the Environment:  Organic elements like pus, blood, and bodily fluids at the site of disinfection limit the disinfectant’s effectiveness.  Fats and oils present at the phenol’s site of action hinder its efficacy.
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  3. Surface Tension: Lowering surface tension in an aqueous solution increases the adsorption of disinfectants on microbial cells.  Soap, which reduces surface tension, enhances the disinfection effect when combined with phenol. 4. Types and Number of Microbes Present:  Disinfectants are most effective against microorganisms in their vegetative phase (rather than spores).  Formaldehyde, a sporicidal aldehyde, is effective against bacterial spores. 5. Temperature:  Disinfectant activity declines beyond a specific temperature, depending on the material.  The “Temperature coefficient” (Q10) indicates the influence of temperature on antibacterial activity.  In order to determine the Q10, use the following formula. Time to kill at T0 Q10 = ----------------------------- Time to kill at (T+10)0
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  6. Potentiation andAntagonism of Disinfectants:  Some disinfectants’ efficacy depends on interactions with others, while others may be diminished. 7. pH of the Surrounding:  Acidic disinfectants are most effective at an acidic pH (bacteria thrive best at pH 6-8).
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  BACTERIOSTATIC VS. BACTERICIDAL ACTIONS Bacteriostatic: Action: Halts bacterial growth without necessarily killing the microorganisms.  Reversibility: Its action is reversible; normal bacterial growth resumes upon removal of the antibiotic.  Examples: Tetracyclines, chloramphenicol, and macrolides. Bactericidal:  Action: Directly kills bacteria.  Irreversibility: Its action is irreversible.  Examples: Beta-lactam antibiotics (e.g., penicillin), which inhibit cell wall synthesis, leading to bacterial death.
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  EVALUATION OF DISINFECTANTS The evaluation of disinfectants to ensure their potency and efficacy. Disinfectants play a vital role in maintaining hygiene and preventing the spread of infections.  Here are some methods commonly used for evaluating disinfectants: 1. Tube dilution and agar plate method. 2. Cup plate method or Filter paper method. 3. Ditch- Plate method. 4. Gradient plate technique. 5. Phenol coefficient method. 6. Kelsey Sykes Method.
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  TUBE DILUTION METHOD Most commonly used method.  In this method, a series of tubes is set up, each containing the same quantity of a standard growth liquid medium (Commonly Mueller-Hinton broth) and a gradually increasing concentration of the disinfectant to be tested.  The tubes are then inoculated with the same quantity of cell suspension of the test microorganism and incubated for 2-3 days @ 30-35℃.  The first tube in the series where there is complete absence of growth of the test microorganism denotes the minimal inhibitory concentration (MIC) of the disinfectant.
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  AGAR PLATE METHOD It is very similar to the tube dilution method.  In this method, Petri plates containing a standard growth agar medium (usually Mueller-Hinton agar) are taken at the place of lubes containing liquid medium.  Disinfectants of various concentrations are inoculated on the agar surface of plates already inoculated with the same quantity of the test microorganism.  Plates are incubated and then examined for growth.  The first plate in the series where there is complete absence of growth of the test microorganism denotes the minimal inhibitory concentration (MIC) of the disinfectant.
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  CUP PLATE METHODOR FILTER PAPER METHOD  These methods are known as “Agar Diffusion Tests”.  The agar is melted and cooled @ 45℃, inoculated with test microorganisms and poured in a sterile petri plate.  In case of “Cup Plate Method” when the agar is solidified the holes of 9 mm diameter were made by using sterile cork borer and the disinfectant is directly placed in it.  In case of the Filter Paper method the filter paper disks soaked in disinfectant solution are placed in the holes.  The zone of inhibition is observed after incubation @ 30-35 ℃.  During incubation, the disinfectant diffuses into the agar.  The diameter of the zone of inhibition gives indication of activity of the disinfectant, larger the diameter greater is the efficiency of the disinfectant.  A zone of Inhibition is proportional to the amount of the antimicrobial agent added to the filter paper disc, the solubility of the agent, the diffusion coefficient, and the overall effectiveness of the agent.
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  DITCH- PLATE METHOD Agar is melted and then solidified in a petri plate.  A ditch is made in the petri plate by cutting the soliodified agar.  The disinfectant solution is made to run through the ditch carefully.  The test organisms are streaked outwards from the ditch.  The Petri plate is incubated at desired temp. And time period.  The microorganisms which are resistant to the disinfectant grow even near the start at the ditch itself.  The sensitive organisms show a zone of inhibition near the ditch or at center of the petri plate.  The width of the zone of inhibition is an indication of activity against the test organism.
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  GRADIENT PLATE TECHNIQUE Principle:A gradient of antibiotic concentration is established on an agar plate. Procedure:  A bottom layer of plain nutrient agar is solidified at an angle.  A top layer of nutrient agar containing the antibiotic is added.  This creates a gradient of antibiotic concentration from high to low across the plate. Application: It’s used to isolate and identify mutants resistant to antibiotics. Advantages:  Allows observation of bacterial growth in varying concentrations of antibiotics.  Helps in studying the resistance patterns of bacteria
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  PHENOL COEFFICIENT METHOD In this method the efficacy of the disinfectant in use is rated by comparing it with the activity of Phenol taking as a standard.  The test is carried out by adding an increasing amount of phenol and disinfectant in test tubes containing microorganisms.  In UK the test organism used is Salmonella typhi while the USA uses Salmonella typhi, Staphylococcus aureus and Pseudomonas aeruginosa.  The official phenol coefficient tests include, 1. Rideal-Walker Test (RW Test). 2. Chick-Martin Test. 3. United States FDA Test for Phenol Coefficient. (FDA Test) 4. The US Association of Official Agricultural Chemists Test (FDA Test)
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  RIDEAL-WALKER TEST  Introducedby Rideal and Walker the British chemists in 1903 are still in use.  The test uses Rideal, Walker Broth and Salmonella typhi as a test organism.  Different dilutions of the phenol and test disinfectants are made and 5 ml of each is inoculated with the 0.5 ml of the 24 hr broth culture of the test microorganism.  All the test tubes are placed in a water bath at 17.5 ℃.  Subcultures from each test tube are taken and transferred to 5 ml sterile broth after 2.5, 5, 7.5 and 10 minutes.  The broth tubes are incubated at 37 ℃ for 2 - 3 days and are examined for the presence or absence of the growth and the Phenol coefficient is calculated using the formula,  The Phenol Coefficient for phenol is considered as “1” any value for disinfectant coming below one is considered as less while above it is more.
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  CHICK-MARTIN TEST  TheChick-Martin Test is a method used to determine the phenol coefficient of a disinfectant, particularly in the presence of organic matter. Purpose: To evaluate the efficacy of a disinfectant in conditions that simulate real-world use, where organic matter is present. Procedure:  The test is conducted by adding a standardised quantity of bacteria (such as Salmonella typhi or Staphylococcus aureus) to sterilised faeces or a yeast suspension.  Various dilutions of the disinfectant are then compared with the efficacy of phenol under the same conditions. Phenol Coefficient:  The phenol coefficient is calculated by dividing the dilution of the test disinfectant that is effective in the presence of organic matter by the dilution of phenol that is effective under the same conditions.  A coefficient greater than 1 indicates that the disinfectant is more effective than phenol, while a coefficient less than 1 indicates it is less effective
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  Advantages of PhenolCoefficient Test:  Quick.  Inexpensive.  Reproducible results.  Useful to eliminate useless products.  Sets standard for the preparations. Disadvantages of Phenol Coefficient Test:  Most tests use only one test organism i.e. Salmonella typhi which represents inadequate information.  Compares the activity of disinfectants at only one concentration at fixed conditions.  Presence of organic matter at action time is not considered.  Does not give information about tissue toxicity of the disinfectant.  Sampling errors are large.
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  KELSEY SYKES METHOD Developed in 1969, this test overcomes many drawbacks of the RW Test.  The test uses several test organisms viz. Staphylococcus aureus, Proteus vulgaris, Eschericia coli and Pseudomonas aeruginosa.  Tests can be carried out in a clean as well as dirty environment.  In both clean and dirty cases the final bacterial concentration should be about 109/ml.  Clean conditions are simulated by using a clean broth while dirty conditions are simulated by using a yeast suspension or inactivated horse serum.  The samples are taken at 8, 18 and 28 minutes are then incubated at 30 to 32 ℃ and observations of growth of microorganisms in test tubes are recorded. Result interpretation: A disinfectant is considered satisfactory if,  No growth in 2 or 5 tubes of 18 min sample or,  No more than 5 colonies from 5 drops on the agar plate.