Evaluation Methods For

Anti-microbial Agents
Evaluation Methods For Anti-microbial Agents

– Evaluation of non antibiotic Sensitivity testing Antibiotic assay

 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.
 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
 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.
: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 θ, or the coefficient per 10 degrees rise, the Q 10 value.
• θ may be calculated from the equation; t
( T2  T1 )
• 
Where t1 is the extinction time at T­1˚C, and t2 the extinction time  at
1

t 2 T2˚C.
 

• it Q Time to kill at T 
10 
can be calculated easily by determining the extinction time at two temperatures differing
exactly by Time
10˚C. to kill at ( T  10)
•  Effect of dilution;
• It was realized that there was an exponential relationship between potency & concentration.
 

We found HgCl2 which it's conc. exponent =1, will decrease the activity of it by power of 1 on
dilution. Threefold dilution means the disinfectant activity will be reduced by the value 3 1, or to a
third of (log death time at concentration C )  (log death time at concentration C1 )
 its
 original activity. Phenol, however, has2a concentration exponent of 6, so a threefold
.dilution in this case will mean a decrease inlog C1 of
activity log3 6Cor2 729 times less active than the original
Concentration exponents, η, η (conc. Coefficient) Antimicrobial agent
for some 0.5 Hydrogen peroxide
disinfectant substances
0.9–1.0 Silver nitrate
0.03–3.0 Mercurials
0.9 Iodine
0.9 Crystal violet
2 Chlorhexidine
1 Formaldehyde
0.8–2.5 QACs (quaternary ammonium compound)
0.7–1.9 Acridines
0.8–0.9 Formaldehyde donors
0.7 Bronopol
1.5–1.6 Polymeric biguanides
2.5 Parabens
2.6–3.2 Sorbic acid
2.3 Potassium laurate
2.6–4.6 Benzyl alcohol
6.0–12.7 Aliphatic alcohols
5.8–6.4 Glycolmonophenyl ethers
6.4–15.9 Glycolmonoalkyl ethers
4.0–9.9 Phenolic agents
 Factors affecting the
:disinfection process
• Effect of pH;
• Any change in PH will affect:
• Rate of growth of bacteria >> growth is optimal at pH (6-8).
• Potency of antimicrobial agent >> 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.
 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
 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
 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)
 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, M.O recover at 32oC
– Calibrated pipette for subculture rather than loop
– Clean and dirty conditions
– Assessment (kill or not) (no phenol coefficient)
 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

Neutralizing agents for some
antimicrobial agents
Other appropriate enzymes -
can be considered—e.g.
inactivating or modifying
enzymes for chloramphenicol
and aminoglycosides,
.respectively
Filter microorganisms onto -
membrane, wash, transfer
.membrane to growth medium
.Tween 80 (polysorbate 80) -
Neutralizing and/or inactivating) Antimicrobial agent
agent
β-Lactamase from Bacillus cereus Amoxycillin
None (dilution, membrane filtration, resin Antibiotics (most)
adsorption)
Thioglycollic acid (-SH compounds) Mercurials
Lubrol W and egg lecithin or Tween 80 Chlorhexidine
and lecithin (Letheen)
Dilution or Tween 80 Benzoic acid
β-Lactamase from Bacillus cereus Benzyl penicillin
Ammonium ions Formaldehyde
Lubrol W and lecithin or Tween 80 and QACs (quaternary ammonium
lecithin (Letheen) compound)
Glycine Glutaraldehyde
p-Aminobenzoic acid Sulphonamides
Cysteine hydrochloride Bronopol
Sodium thiosulphate Halogens
Tween 80 Hexachlorophane
Tween 80, saponin, histidine and lecithin Alcohol-based hand gels
None (dilution) Alcohols
Dilution or Tween 80 Phenolic agents
 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
 Evaluation of Mycobactericidal
activity
• Presumptive test:
– Organism: Mycobacterium smegmatus
– Mycobacterium terrae can be substituted in tests (as representative of
M. tuberculosis), non-pathogenic fast growing saprophytic.
– 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
 Evaluation of sporocidal activity
• Incubation for several days to allow for germination and
growth.

• Biofilm forming bacteria

– Can be modified to involve biofilms produced on small pieces of an appropriate glass or
metal substrate, or on the bottom of microtitre tray wells.
– After being immersed in, or exposed to the disinfectant solution for the appropriate time
interval, the cells from the biofilm are removed, e.g. by sonication, and resuspended in a
suitable neutralizing medium.
– Viable counts are then performed on the resulting planktonic cells.
• Intracellular parasites of other microbes, e.g. Legionella pneumophila
within the protozoan Acanthamoeba polyphaga.
– Conducted on both planktonic bacteria and on suspensions involving amoebae-
containing bacteria.
– Lysis amoebae and then do viable count.
 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
 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
 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
 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)
 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 matter…etc)
– 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
 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 membranes…etc for toxicity and irritation
– Skin test:
– M.O placed on skin (hand), compound in the same area—after time—swab
and incubated in suitable media----viable count
– Evaluation of oral antiseptics:
– Mouth wash, gargles
– Extinction time technique in presence of saliva, Staph. aureus
 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
 Anti-microbial susceptibility
:(sensitivity) testing
• Kill or inhibit M.O
• 1- Agar diffusion methods
– Sensitive
– Resistant
– Intermediate
• 2- Serial dilution methods
– MIC determination
 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
 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
 Antibiotic Assay
• Factors affecting the response fall in 4 categories:
• Errors in measurements
• Errors in standardization (pH, sovlent, depth of the medium,cup…etc)
• 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

 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
 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
..… Thank you