evaluating activity of disinfectants Flashcards
what are the ideal properties of a disinfectant
broad spectrum, fast acting, not affected by environmental factors, non toxic, surface compatibility, residual effect on treated surface, odorless, economical, soluble, stability, cleaner, environmental friendly
what are the factors linked to microorganisms that affect activity of disinfectants
nature of microorganisms, physiologic state of microorganisms, microbial communities and biofilms, natural and acquired resistance
what are the factors linked to antimicrobial agent that affect activity of disinfectants
conc, temp, electrolytes (can cause precipitation), presence of organic matter, additives and components of excipients
what are the stages of bacteria growth
lag, log/exponential, stationary, death
what is the lag phase of bacteria growth
cells are not dormant but are starting the processes for cell replication like RNA and enzyme synthesis
what is the log/ exponential phase of bacteria growth
cells are doubling at constant rate (slope represents speed of process)
what is the stationary phase of bacteria growth
growth limiting factors affect replication, making growth and death rate comparable
what phase is affected if agent is bacteriostatic
increases lag phase
what are the kinetic parameters
rate constants, temp coeff, electric field
what are the factors that can affect efficacy of disinfectants
number of microbes, innate resistance of microorganisms, conc and potency of disinfectant, chemical and physical factors (pH, temp, water hardness), organic and inorganic matter, duration of exposure, biofilms
what is the process of evaluation tests
- preparation of carriers (usually stainless steel, PVC)
- preparation of test organism and inoculum
- inoculation, drying and transfer of the carrier
- exposure of the dried inoculation to the test substance or control fluid
- neutralisation of the test substance and elution of test organism
- dilution and recovery of test organism
- counting the surviving test organism and assessing the performance of the test substance
how is the representativeness and reliability of test strains determined by
determined by 1. exposure time (time, pH, temp, number and type of microorganism)
2. interfering factors (use of bovine aka cow serum to simulate organic contamination which is usually the most difficult to eliminate, safe to use)
3. interpreting criteria (using log reduction in a certain amount of time)
what is the alternative to using bovine serum for testing
two step method where test substance is strained in solution (aq suspension of a mixture of bacteria and water), bacteria and dirt aka interfering substance dried onto stainless steel discs and disinfectant tested
what is the log reduction you should aim for
4-6log reduction
what is MIC
minimum inhibitory conc - lowest conc of an antimicrobial agent that is bacteriostatic (inhibit growth)
what is MEC
minimum effective conc - lowest conc of an antimicrobial agent that shows bactericidal effect
what is MRC
minimum recc conc - takes into account imperfect processes (MRC > MEC)
what is MBC
minimum bactericidal conc - lowest conc of an antibacterial agent to show at least a 3lg reduction after comparison with initial preincubation conc after incubating for 18-24h
what is MSC
minimum selective conc - for biofilms
what is an inoculum
population of microorganisms or cells that is introduced in the fermentation medium or any other suitable medium
how do you prepare an inoculum
putting selected strain onto carrier and growing it to a certain extent -> now known as initial microbial load
what is the alternative to neutralisation
washing by membrane filtration (preferred over neutralisation)
what is the advantage of membrane filtration
permits processing of entire eluate volumes and more efficient removal of any residue of the test substance
when is counting of survivor done
after cultivation and growth of test organism in suitable medium
how to ensure no other bacteria is counted during counting of survivor
use of propanol to eliminate almost all potential contamination during washing step
how to quantitatively calculate recovery of microorganisms
known inoculum of microorganisms is spotted onto stainless steel surface (carrier) -> dried under heat and ventilation to obtain simulated contaminated surface -> contact plate applied to recover microorganisms spotted -> compare number of CFU on contact plate to number of CFU spotted to calculate recovery rate
what does sample size affect
affect reloiability
how are survivors revived
microbed recovered and plated on a suitable medium like agar plates, saline etc and growth conditions must be assessed such that it follows the guidelines
what are the types of test that can be used to evaluate efficacy of disinfectants
suspension test, carrier test, in-use and field test
what is a suspension test
one step quick test which does not need any interfering substances, involves mixing fixed volume of known dilution of disinfectant with fixed volume of inoculum
what is a carrier test
disinfectants directly dropped on the carrier surface at room temp/ carrier immediately immersed in disinfectant dilution/ disinfectant dispersed by spraying or aerosol
what is a in-use and field test
either suspension or carrier test done in field of where disinfectants will be used
what is the disadvantage of in-use and field test
no end point: not quantitative approach to the reduction of flora, last choice as not quick enough of a test
what are the specific tests under standard use
AOAC test, AFNOR, DGHM, dutch standard/ EN13727, EN1276, WI216028
what is the procedure of AOAC test
carrier test - soaking stainless steel carrier in bacteria, treating them with disinfectant then determining if there are any surviving bacteria after placing carrier into growth media
what is the procedure of AFNOR test
big plate used instead of carrier and spraying of disinfectant instead of applying drop by drop - disinfectant sprayed onto a test surface at a predetermined distance, germs are applied to sample plates located on the surface
what is the procedure of DGHM test
simulates swabbing surface of skin for contaminants - dirty surfaces exposed to disinfectant and swabbed with damp swab and frictional surface of swab plated out on nutrient agar
what is the procedure of dutch standard
performed by introducing a test sample into a bacterial suspension in the interfering substance solution following prior dilution, maintained at ambient conditions for a defined time interval, mixture is then neutralised and number of surviving organisms measured
what are the two types of washing steps for EN1276
dilution-neutralisation, membrane filtration (if neutralisation failed)
what are the strains used in EN1276
p. aeruginosa, e. coli, s. aureus, e. hirae
what is the inoculum for EN1276
adjusted suspension of agar culture obtained under specified conditions, number of viable cells in presence of antiseptic or disinfectant is 1.5-5 x10^7 CFU/ml
what is the incubation criteria for EN1276
37degC, 48h
how to interpret results for EN1276
general application: 5lg reduction within 5 mins at 20degC in clean or dirty condition with each of the four ref strains
specific application: 5lg reduction within 5 mins at 20deg C for each of the four ref strains (more if needed) and tested in additional experimental conditions (exposure time, temp, interfering substances)
what type of test is WI 216028
surface test without mechanical action
what strains are used for WI 216028
p. aeuruginosa, e. coli, s. aureus, e. hirae
what is the inoculum for WI 216028
adjusted suspension of agar culture obtained at specified conditions, number of viable cells in presence of antiseptic or disinfectant 1.5-5 x10^8 CFU/ml, adjusted suspension mixed with an equal volume of chosen interfering substance -> 0.05ml of mixture dropped on carrier and dried at 37 degC
what is the incubation criteria for WI 216028
37degC for 48h
what is the diluent and tested conc for WI 216028
standardised hard water used, at least three-dilutions including one in active range and one in inactive range, 0.1ml of product dilution applied on contaminated carrier
what is the contact time for WI 216028
5min (additional times are 1,15,30,60mins)
what is the temperature for WI 216028
18-25 degC (additional temps 4,10,40degC)
what are the interfering substances used for WI 216028
- bovine serum - 0.3g/L in clean conditions, 3g/L in dirty conditions
- skimmed milk, yeast extract, sucrose, buffers, SLS
how is counting of survivors carried out for WI 216028
survival count in neutralised mixture (undiluted and diluted) by inclusion in agar medium under specified conditions
how to interpret results for WI 216028
general application: 4lg reduction within 5 mins at 20degC in clean or dirty conditions with each of the four ref strains
specific application: 4lg reduction within 5mins at 20degC in clean or dirty conditions with each of the four ref strains and in additional experimental conditions (exposure time, temp, interfering substances)
what is the difference between hygienic handrub and handwash
hand rub: treatment of hands with antiseptic hand rub, broad spectrum and fast acting, persistent activity not necessary
handwash: treatment of hands with antiseptic hand wash and water, broad spectrum but less efficacious and slower acting than hand rub
what are the factors which limit comparison between protocols testing hygienic hand rub/ hand wash
contamination of hand with a test organism before test, to contaminate fingers or hands, amount of product applied, contact time
what are the standardised tests for hand washes
EN 1499, ASTM E-1174
what is the process of testing of eucalyptus against e. coli and s. aureus
(tested without interfering substance) oil extraction by steam distillation followed by decantation and isolation via rotavapor -> place strain on agar plate -> (agar diffusion method) filter paper discs soaked in oil -> placed on inoculated plates -> dry for 15 mins -> incubate at 37degC for 24h -> inhibition zone diameter measured in mm -> stock solutions and controls of oil diluted in ethanol -> tested culture strains added to nutrient broth -> maintained in Marie bath at 37degC for 24h under stirring -> streak surface of agar medium and incubate at 37degC for 24h
what is the process of testing of eucalyptus against e. coli and s. aureus
(tested without interfering substance) oil extraction by steam distillation followed by decantation and isolation via rotavapor -> place strain on agar plate -> (agar diffusion method) filter paper discs soaked in oil -> placed on inoculated plates -> dry for 15 mins -> incubate at 37degC for 24h -> inhibition zone diameter measured in mm -> stock solutions and controls of oil diluted in ethanol -> tested culture strains added to nutrient broth -> maintained in Marie bath at 37degC for 24h under stirring -> streak surface of agar medium and incubate at 37degC for 24h
is gram neg or gram pos bacteria more sensitive to eucalyptus oil
gram neg due to presence of lipoproteins and lipopolysaccharides in their cell wall structure that form barrier to hydrophobic compounds
what is the testing for formaldehydes
EN 14667
what is the process of EN 14667
0.8ml of aliquot supplemented with 0.1ml of interfering substance -> 1.4% of formaldehyde control supplemented with 0.1ml of interfering substance -> both test product and formaldehyde control inoculated with viral suspension and held for duration of contact time -> virus recovery control prepared using inert substance like buffered saline or cell culture media in order to determine initial viral titer used to challenged test substance -> neutralisation and cytotoxic controls performed to determine effectiveness of selected neutralisation method and impact of test product on host cell -> neutralisation of test product and controls -> serially diluted and plated -> incubated for 7d -> assay scored using spearman-kaber method to quantify viral titer and any cytotoxicity observed noted accordingly -> to have 4lg reduction
what are the advantages of EN 14667
can compare results if used this standardised testing, more reproducible than most hard surface carrier testings, several active ingredients conc can be evaluated efficiently over various contact times, original method accounts for product dilution occurring from application of interfering substances and viral inoculum which avoids artificial reduction of product efficacy
what are the disadvantages of EN 14667
only meant to simulate one specific environment, data generated for suspension viruses may not translate to dried virus films on hard non porous surfaces
