IC7 Evaluating disinfectants Flashcards
What are two ways to count microorganisms?
- Direct flow spectrometry (laser scattering) - expose sample to laser ray
- Indirect countring - observe turbidity (affected by density)
Describe the 4 phases in bacterial growth
- Lag phase
- Cells are not dormant, starting process for cell replication such as RNA and enzymes synthesis
- *Bacteriostatic disinfectants affect this phase, cause bacteria to be unable to replicate - Log or exponential phase
- Binary fission, cells double at constant rate
- Slope indicates speed of doubling
- *Some bacteria double at slow rate, process can vary from hours to days depending on the strain => therefore incubation time should be sufficient - to accurately determine if no growth is due to effect of disinfectant OR due to slow growing/forming colonies - Stationary phase
- Growth-limiting factors affect replication
=> when nutrient in medium starts to dwindle
=> Accumulation of waste and toxins released when the cell dies can affect growth of other cells - Death phase
What are the 3 kinetic parameters that can be used to determine the inactivation of microorganisms?
- Rate constant - change of microbial popln over time
Log[N/No] = -t/D
Reduction in microbial popln is a constant - Temperature coefficient - influence of temp on microbial popln inactivation rate
Log[D/Dr] = - (T - Tr) / Z
Z is the thermal resistance constant: temp incr required for 1 log cycle reduction in D - Electric field
Log[D/Dr] = - (E - Er) / Z
Z is the electric resistance constant: electric field intensity incr required for 1 log cycle reduction in D
What are the 7 consecutive steps for disinfectant test method?
- Preparation of carriers
- surface/object to be inoculated with the test organism, e.g., stainless steel, PVC - Preparation of the test organism and inoculum
- selection of test organism strain is one of the key parameters to test activity of disinfectant - Inoculation, drying, transfer of carriers
- *drying phase may affect microbial load (know initial load to ascertain load after drying) - Exposure of dried inoculum to test substance or control fluid
- Factors: exposure time, contact area (used standardized carrier)
- Incubation - Neutralization of the test substance and elution of the test organism
- quench antimicrobial activity by dilution OR adding chemical neutralizer
OR
- wash away disinfectant via membrane filtration with eluent (collect eluate to perform recovery step to regrow test organism) - Dilution and recovery of test organism
- Recover and regrow organism at a specific rate in a suitable medium, to have a proper counting via back calculation (step 7)
=> e.g., recover from carrier and include in agar medium under specified conditions
- Growth conditions must be assessed before the testing in order to assess recovery rate - Counting the surviving test organisms and assessing performance of test substance
In step 2, when preparing test organism and inoculum, how do we ensure that the correct strain is selected from the working stock culture for seeding and inoculation?
- Avoid picking variant strains from the agar plate - observe under microscope, do not pick strains that are diff in morphology etc.
- Ensure carriers are clean before physically seeding the selected strain of bacteria, to ensure no other strain exist on the carrier
*Can wash with isopropanol (2-propanol) to eliminate possible contamination
In step 2, when preparing test organism and inoculum, what are the considerations for selection of test strain?
Representativeness and reliability
- Exposure time
- time, pH, concentration, temperature
- type and amount of microorganisms - Broad spectrum of efficacy according to in-use environment
- consider the diff mechanisms and activity of the microbes present in the environment
In step 2, when preparing test organism and inoculum, interfering substance can be added, explain the purpose of interfering substance.
Interfering substance is added to simulate practical conditions
- representative of organic contamination, present test substance wit challenge to overcome chemical demand from soil load + physical shielding of test organism
E.g., dirt, cow bovine serum, cow bovine albumin, skim milk (simulate protein/content in stomach), yeast extract, SLS (surfactant excipient)
*If disinfectant product has SLS as excipient, add SLS into test to prevent underestimation of disinfectant activity
What should be considered when choosing interfering substance?
Why is it important to determine substance/interfering material ratio?
Consider whether disinfectant activity is affected by organic/inorganic material
E.g., biguanides (chlorhexidine), hypochlorite, iodine, QAC
If increase interfering substance, then test substance might be inhibited
Interfering substance is only added in the second step of the two step method.
Explain the two step method (*both part of Step 2 Preparation of the test organism and inoculum)
Two step method:
In step 1 (suspension test) - aq suspension of bacteria + water
In step 2 (hard surface carrier test) - bacteria + interfering substance fried onto stainless steel discs
Disinfectant is added to reduce bacterial numbers
*Not always necessary to have step 2, because step 1 itself is more reproducible
What is the interpretation criteria for the efficacy of the disinfectant?
Log reduction of microbial popln in a certain amount of time
*To test efficacy, 1 log reduction is insufficient, should go much lower (4-6 log reduction)
What are the criteria for activity to consider when testing disinfectant?
*These criteria determine how much reduction in microorganism is required to conclude whether disinfectant if active or not
- Minimum inhibitory concentration (MIC)
- lowest conc. to inhibit growth (bacteriostatic action)
- determined visually after standard incubation period (18-24h) - Minimum effective concentration (MEC)
- arbituary, lowest conc. at which product is still effective - Minimum recommended concentration (MRC)
- how much to dilute disinfectant for use (recommendation higher than actual to account for improper filution, cleaning etc.) - Minimum bactericidal concentration (MBC)
- lowest conc. to achieve at least 3nlog reduction after standard incubation period (18-24h) (bactericidal action)
- minimum lethal conc. => can be directed towards any microbe - Minimum selective concentration (MSC)
- At MSC, resistance strains have a competitive advantage
- Goal is to go above MSC ensure strains do not become resistant
In “Step 7 - Counting the surviving test organisms and assessing performance of test substance”, how is counting of the surviving test organisms performed?
Counting of colony forming units (CFU)
*CFU/mL or CFU/mm2
- EIther manually, or using software like imageJ
- Colony morphology should be used to identify microorganism presnet, ensure no other bacteria present
Quantitative recovery of microorganisms is necessary to calculate the recovery rate after inoculum on stainless steel surface is dried under heat and ventilation. How is recovery rate calculated?
After dried under heat and ventilation,
A contact plate is applied to recover microorganisms spotted on the stainless steel surface,
Number of CFU recovered on contact plate is compared to Number of CFU spotted to calculate the recovery rate.
What is the significance of the recovery rate after drying?
Not possible to achieve 100% survival in drying step, some microbials will be lost.
Hence, important to know recovery rate in order to determine how many microbials survived (before moving to next step).
What are the 3 classifications of tests?
- Suspension test
- mix fixed volume of known dilutions of disinfectants with fixed volume of inoculum
- Evaluation: contact time (incubation) and culture for counting - Carrier test
- disinfectant drop directly onto carrier surface OR carrier immersed in disinfectant dilution OR disinfectant dispersed by aerosol spray
- Evaluation: activity varies according to application method - in-use and field test
- can be suspension of carrier test
- involves taking sample from the field/environment
- Evaluation: no end point, not a quantitative approach
What is classification based on standard use?
E.g., AOAC test, EN13727 test…
These test use different methods (e.g., carriers, suspension, swabbing surface) to evaluate activity of disinfectant against microorganism
What is the purpose of hygienic handrub or handwash?
Reduce transient flora, without affecting the resident skin flora
*eliminate transient pathogens only
What is the difference between hygienic handrub and handwash?
Both should be broad spectrum antiseptics, HOWEVER
Handrub - fast acting, persistent activity not necessary
Handwash - less efficacious and acts more slowly
How to test activity of hygienic handrub/handwash?
Hands must be experimentally contaminated with the test organism, and then apply hand hygiene product, evaluate reduction
Considerations: *variations in protocol
- which parts of hands to contaminate
- volume of hand hygiene product
- contact time with hand hygiene product
What is the general time needed for incubation of bacteria
Dependent on growth cycle of the strain
Fast-replicating bacteria: 24h
But subsequently may test again after additional 72h to ensure no slow forming colony (confirm effect of disinfectant vs slow bacteria growth)
When using plant-based disinfectant (essential oils from the leaves of Eucalyptus globulus) against E. coli and S. auerues, which was it more effective against?
More effective against gram-negative E. coli, showed greater rate of inhibition of growth
Presence of lipoproteins and lipopolysacharides in gram-negative bacteria that form a barrier to hydrophobic compounds
What is a method to quantify the viral titer after incubation?
Assay is scored using the Spearman-Karber method
How to evaluate virucidal efficacy success criteria?
Staritng viral inoculum must have sufficiently high titer for the test to show at least a 4 log reduction relative to the virus recovery control
*Viral recovery control is a control prepared using inert substance such as buffered saline in order to determine the initial viral titer
Should there be cytotoxicity observed, 4 log reduction must be demonstrated past the level of cytotoxicity
What are the strengths of standard tests?
- Data generated will conform to guideline, efficacy data required for disinfectant claims
- Reproducible
- Comparable results
- Several active ingredients can be evaluated efficiently over various contact times
- Accounts for product dilution (occurs with application of interfering substances, viral inoculum etc.) => therefore prevent artificial reduction of product efficacy
