Evaluation Methods For Antimicrobial Agents

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Evaluation Methods For Anti-microbial Agents
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Evaluation Methods For Anti-microbial Agents

• Evaluation of non antibiotic
  Sensitivity testing Antibiotic assay

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Evaluation of non antibiotic

• Effect of anti-microbial agents on microbial
  growth
• Cidal
• Kill all M.O
• Higher conc. Of the agent, shorter extinction
  time
• Static
• Prevent or inhibit growth
• Growth rate is zero
• Prolonged static effect-----cidal
• Sub-static
• Conc. below static
• Prolonged generation time
• Any compound may exert cidal, static or
  sub-static effect depend on conc.

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Evaluation of non antibiotic

• Evaluation is not for conc. But it is for the
  activity of the agent under certain conditions
  and uses
• Disinfectant
• Cidal
• Dead objectives
• Wide variety of M.O
• Antispetic
• Static or cidal
• Living tissue
• Pyogenic M.O

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Factors affecting the disinfection process

• Effect of temperature
• Bacteriologist Koch had noted that anthrax spore
  were more readily killed by the same
  concentrations of phenol if the temperature was
  elevated.
• The fate of a bacterial population when
  inoculated into
• Nutrient medium, normal growth curve.
• Bacetriostatic environment. No change in viable
  population after a prolonged time-interval the
  viable population will probably begin to fall.
• Bactericidal environment. A sigmoid death curve
  is shown.

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Factors affecting the disinfection process

• The effect of temperature on bactericidal
  activity may be expressed quantitatively by means
  of a temperature coefficient, either the
  temperature coefficient per degree rise in
  temperature, denoted by ?, or the coefficient per
  10 degrees rise, the Q10 value.
• ? may be calculated from the equation
• Where t1 is the extinction time at T1C, and t2
  the extinction time at T2C.
•  
• it can be calculated easily by determining the
  extinction time at two temperatures differing
  exactly by 10C.
•  Effect of dilution
• It was realized that there was an exponential
  relationship between potency concentration.
•  
• We found HgCl2 which it's conc. exponent 1 gm
  will decrease the activity of it by power of 1 on
  dilution.

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Factors affecting the disinfection process

• Effect of pH
• Any change in PH will affect
• Rate of growth of bacteria gtgt growth is optimal
  at pH (6-8).
• Potency of antimicrobial agent gtgt due to
  ionization at pH.
• Ability of drugs to combine with cell surface
  sites of bacteria.
•  
• Effect of surface activity
• The addition of low concentrations of surface
  active compounds may potentiate the biological
  effect of an antibacterial agent.
•  
• Presence of interfering substances
• The presence of other material may reduce the
  effect of such an agent by adsorbing or
  inactivating it and thus reducing the amount
  available for combining with the cells it is
  desired to kill.

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Evaluation of biocidal action

• Mainly for disinfectant
• Principle contact and removal method
• Removal to recovery media in the absence of
  anti-microbial agent
• Sterility ----no living M.O (extinction time
  method)
• Change in viable count (counting methods)
• Extinction time methods
• Phenol coefficient test (suspension tests)
• Qualitative, disinfectant compared with phenol
• Rideal Walker test (R.W)
• Test organism Salmonella typhi
• Different conc. of disinfectant and phenol
• Inoculate specific conc. of M.O in each tubes,
  incubate at 17-18 oC
• Subculture in recovery medium at times (2.5, 5,
  7.5 and 10 min)
• RW phenol coefficient dilution of disinfectant
  having growth at 5 min but not at 7.5 min /
  dilution of phenol have the same effect

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Evaluation of biocidal action

• Chick-Martin (C.M)
• Organism mixture of S. typhi dried yeast
  (organic load)
• Temp 20oC
• Recovery media after contact time 30 min
  (loopful)---incubate at 37oC for 48hr
• Growth or no growth
• C.M phenol coefficient mean of lowest conc. of
  phenol prevent growth in subculture/ unknown
  disinfectant

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Evaluation of biocidal action

• Association of Official Analytical Chemists
  (AOAC)
• 3 organisms
• Select suitable media
• Limitation of phenol coefficient
• Single organism (AOAC)
• Ignore effect of organic matter (C.M)
• Loopful (not accurate)
• Phenol as standard (non phenolic)
• Contact time is short
• Recovery medium not suitable for partially damage
  M.O
• Fixing temp., (ignore temp. coefficient)
• Fixing conc., (ignore conc. coefficient)

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Capacity use dilution test

• Kelsey-Sykes (KS) test
• Organisms 4 organisms (Staphylococcus aureus,
  E.coli, P. aeruginosa and Proteus vulgaris)
• Three successive loads of bacteria (additions)
  (0, 1, and 5 min)
• Temp. 20oC
• Calibrated pipette for subculture rather than
  loop
• Clean and dirty conditions
• Assessment (kill or not) (no phenol coefficient)

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Viable counting technique

• Inoculums from reaction mixture on solid media
  and incubation
• Viable cells count (CFU/ml)
• Bactericidal, fungicidal and sporicidal
• 1- Percentage kill calculations
• Suitable time
• Achieve 99 or 99.999 kill
• Semi-quantitative test
• 2- Comparison of death rates
• Plot log survivors against time and compare
• Organic matter (albumin)
• Arrest disinfections by neutralizing, filtration,
  dilution

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Viable counting technique

• Evaluation of fungicidal activity
• Evaluation of sporicidal activity
• Modified bactericidal test
• Bacillus cereus suspension in water heated at
  80oC for 1 min
• Suspension mixed with sporicidal for 5 min
• Reduce the count one log
• AOAC carrier test
• Bacillus subtilis suspension on porcelain
  carriers
• Not fixed time
• At least 59 out of 60 replicates do not show
  growth

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Evaluation of Mycobactericidal activity

• Presumptive test
• Organism Mycobacterium smegmatus
• The test is performed as bactericidal action if
  ve continue
• Confirmative test
• Organism Mycobacterium tuberculosis
• As porcelain carrier, immerse in 10 ml of test
  solution for 10 min, followed by immerse in 10 ml
  serum for neutralization, sub-culturing
• Safe use dilution kills T.B in 10 carriers

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Evaluation of virucidal activity

• Obligate intra-cellular parasites
• Cytopathic effect
• Plaque formation in specific tissue
• Disinfectant can abolish or modify this effect
• Neutralization if infectivity
• Abolish the infectivity of virus

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Evaluation of Bacteriostatic effect

• Determination of minimal inhibitory concentration
  (MIC)
• Determination of the MIC by serial dilution test
• Two fold dilution
• Broth tubes
• Inoculated with test organism
• Incubated
• The least dilution prevent growth (MIC)
• Serial dilution in solid medium
• In agar plates, microorganisms inoculated on the
  surface
• Advantage
• More than one organisms can be tested
• Turbid solution can be tested
• Fungi are best tested
• Detect resistance cells
• Disadvantage
• No one medium can support growth
• Test microorganisms did not grow in the same rate

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Evaluation of Bacteriostatic effect

• Agar diffusion tests
• Qualitative
• Cup plate techniques
• Cork bore
• Disk
• Zone of inhibition
• Plotting log conc, diameter of inhibition zone
• Straight line
• Extrapolated to the cup diameter
• ---MIC conc
• Gradient plate techniques
• First layer contain AMA and solidified
• In wedge position
• Second layer in flat surface
• M.O streak on direction running from high to low
  conc of AMA
• MIC C. Y/X mg /ml
• Y total length of actual growth, X total length
  of streak

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Evaluation of Bacteriostatic effect

• Ditch-plate technique
• Cut at N.A plate
• AMA placed on the ditch
• M.O streak at right angle to the ditch
• Inhibition zones
• Use during assessment of new AMA
• Determination of synergism and antagonism
• Two strips of filter paper (each contain one AMA)
• Placed at right angle on the surface of
  inoculated agar plate
• Pattern of inhibition zone at intersection
• Synergism (inhibition zone at intersection)
• Antagonism (band of growth at intersection)

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Evaluation of Bacteriostatic effect

• MIC of each compound is determine alone
• A series of combination of AMA and then determine
  their MIC
• Plot graph (isobologram)
• Evaluation of antiseptics
• MIC (solid, liquid, organic matteretc)
• Biocidal activity
• Index ratio (I.R)
• Elongation of generation time in absence (G) and
  presence (G) pf AMA
• IR G/ G
• If one ---no effect
• More than one----inhibition
• Phase tolerance curve
• Plotting IR of different conc of the same agent
• Same slop -------same mode of action alone
  different conc.
• Abrupt change in slop------different mode of
  action

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Determination of permeability

• Surface contact inhibition test
• Antiseptic is impregnated onto semi-permeable
  cellophane membrane
• Placed on the surface of previously inoculated
  agar plates
• Squares (membranes) are removed at time intervals
  (0, 5, 10, 15..etc)
• Plate is incubated---inhibition zone recorded
  (first are show no growth).contact time
• Surface contact lethal test
• Antiseptic impregnated onto semi-permeable
  cellophane membrane
• Placed on the surface of agar plate on which a
  test M.O grown
• At time intervals, sub-culturing of M.O below
  membrane in tubes
• Record growth (first tube show no growth)
• Toxicity tests
• On vivo test on leukocytes, egg membranesetc for
  toxicity and irritation
• Skin test
• M.O placed on skin (hand), compound in the same
  areaafter timeswab and incubated in suitable
  media----viable count
• Evaluation of oral antiseptics
• Mouth wash, gargles
• Extinction time technique in presence of saliva,
  Staph. aureus

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Evaluation of Preservatives

• Prevent microbial contamination of pharmaceutical
  prep. and cosmetics
• Evaluation
• MIC
• Viable count
• Formula ingredient may affect preservative
• Challenge test
• Preservative product inoculated with suitable
  test organism
• Incubated
• Examine if growth or not/ reduction from initial
  numbers
• Capacity testing
• Remain its activity in presence of increasing
  load of bacteria
• Carrier testing
• Max. dilution that still effective
• Practical testing
• Simulate the actual conditions of challenge
  towards disinfectant
• In use testing
• Re-evaluation of disinfectant

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Anti-microbial susceptibility (sensitivity)
testing

• Kill or inhibit M.O
• 1- Agar diffusion methods
• Sensitive
• Resistant
• Intermediate
• 2- Serial dilution methods
• MIC determination

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Antibiotic Assay

• Why
• During manufacture (potency and QC)
• Pharmacokinetics
• Monitoring chemotherapy
• Microbiological methods
• Agar diffusion assay
• Turbidimetric assay
• Agar diffusion assay
• Factors affecting the size of the inhibition
  zone
• Law of diffusion
• Viscosity, type of agar, adsorption of
  antibiotics
• Diffusion coefficient (MIC at agar is 3-4 time
  greater than in liquid medium

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Antibiotic Assay

• Factors affecting MIC for an antibiotics
• Conc. of cells
• Factors affecting growth rate
• Conc. of antibiotics
• Factors affecting the availability of active
  molecules
• Adsorption
• pH
• Laws of growth
• Critical time (time of antibiotics to reach edge
  of growth zone) is independent of the conc. of
  antibiotics
• Pre-diffusion
• Low temp (affect growth (starts)) but not affect
  diffusion -----zone of inhibition increases
• Pre-incubation
• Incubation of M.O containing plates before
  addition of antibiotics---reduced inhibition
  zones

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Antibiotic Assay

• Factors affecting the response fall in 4
  categories
• Errors in measurements
• Errors in standardization (pH, sovlent, depth of
  the medium,cupetc)
• Errors cannot be controlled (position in
  plates----use large plates)
• Errors cannot be controlled and unknown
• Use base and seed layer method------uniformity of
  depth

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Turbidimetric Assay

• Sub-MIC conc. usually extend generation time and
  hence, growth rate is reduce
• Different conc. of log sub-MIC against
  turbidimetry (indicate growth) ---straight line
• Assay of antibiotics in serum and body fluids
• Antibiotics taken orally or systemically
• After certain time, samples taken for assay
• Assay of mixture of antibiotics
• M.O sensitive to one but not for the others
• Chemical or enzyme inactivity one antibiotic
• Differences in solubility
• Dilution, one antibiotics may become inactive

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Assay of accessory or growth factors

• Heterotrophic bacteria require specific growth
  factor
• Plot amount of growth factor and growth yield
• Other methods
• 1-HPLC
• 2- Urease assay
• Proteus mirabilis produce ammonia (increase pH)
  upon hydrolysis urea
• Aminoglycoside inhibit protein synthesis thus no
  urea hydrolysis
• Change in pH proportion to conc.
• 3- Lucipherase assay
• Intracellular ATP and luciferase enzyme
• Aminoglycosides ----decrease ATP
• 4- Radiotransferase
• Rediolable acetyl Co A or ATP
• Used to radiolabel the antibiotics
• 5- Immunoassay

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Thank you ..