Challenges Associated with Food Microbiology Tests (with focus on requirements for good laboratory practice)

Transcription

1 Challenges Associated with Food Microbiology Tests (with focus on requirements for good laboratory practice) Christina Oscroft Microbiology Department, Campden BRI, Chipping Campden Glos GL55 6LD, UK Tel:

2 Important decisions made on food safety, quality and compliance with customer specifications/legislation based on microbiological results The Challenge for Food Microbiology Laboratories is the ability to demonstrate they deliver RELIABLE ACCURATE RESULTS This can be achieved by the adoption of Good Laboratory Practices copyright Larson the wild side

3 Training An Overview Of GLP In Operation (ISO17025) Organisation Management Control Personnel Sample Handling Equipment Calibration Competence data Bench Practice ANALYSIS Methods Validation Document Control Facility Result Media Chemicals Quality Control NCW Corrective Action Records Reports Audits Management Review Improvement

4 RELIABLE Result Reflects True Level Of Micro-Organism In Test Sample (not from external contamination/poor handling practices) Laboratory Location/Containment/Access Separation of Work/Activities Environmental Control/Monitoring Hygiene Practices Sample handling Timeliness of testing Aseptic techniques/manipulations

5 ACCURATE Ability To Get Right Result First Time Methods - standard methods (BIS/ISO); validated commercial methods (AOAC,AFNOR/ISO16140); recognised industry norm Data held to show fitness for use and lab competence in test Staff trained and competent in conduct of test Use of good bench practices Working practices ensure plates/broths/confirmations read/subbed on the due date/time including weekends/public holidays Media Performance Good performances in External Proficiency Tests Establishment of Internal Method Performance/Staff Proficiency Programme

6 GLP Environmental Control Size Adequate for volume of work/staff numbers - too cramped increases risk of contamination from poor separation of work Windows - not opened (risk of air-bourne contamination to work) Temperature and humidity - control so do not compromise tests or operation of equipment (incubators, fridges etc) Separate Activities (space /time) - reduces cross contamination Examples Media preparation Waste decontamination Sample Receipt Sample Preparation Plating/Subcultures Plate Reading Handling pathogens Confirmation work Handling cultures

7 GLP Environmental Control Formal Cleaning/Disinfection Schedule to keep facility suitable for conduct of tests to cover facility, surfaces + equipment records Promptly treat culture/bio-hazard spillages Disinfectant and concentration to suitable for nature of spillage. Allow appropriate contact time (to effect kill) Verify effectiveness - post treatment check (contact plate/ swab affected area)

8 GLP - Environmental Monitoring To verify cleaning and ensure hygiene + working practices do not introduce sources of contamination Air quality non selective agar for defined time (eg PCA/15min) selective agar for target organism sought (eg Moulds) Carry out when sample preparation/plating on going Surface contact plates/swabs (eg benches, floors, LFC/SC, incubator/fridge interiors, stomacher interior, door handles/pushes, key boards, taps/handles, staff hands) TVC, infective pathogens relevant to laboratory (eg Salmonella, Listeria, Campylobacter) Contact plate or broth to moisten/dilute swab to contain suitable neutralisers for the sanitizers used by lab (eg lecithin, Sodium thiosulphate, Tween 80)

9 GLP - Environmental Monitoring Review - trend results/set criteria to appraise results Take action if spikes/results out of specification Pathogens isolated: take immediate action (disinfect and recheck area) Escalate screening (check more areas to verify if isolated, or, more wide spread contamination problem) Investigate source/cause and take action to prevent reoccurrence appriase impact on reported results and ongoing work, document outcome and take action if results/work affected

10 GLP Hygiene Practices No eating/drinking/smoking. Long hair tied back, beard snoods, minimal jewellery, keep nails short Hand-washing on entry/exit using bactericidal soap before starting work, regularly during work and in between different activities, after handling pathogens/reference cultures Laboratory Coats visually distinct, not worn outside lab, long sleeved, tight cuffs, regularly changed and laundered coat changes for different areas/work (eg pathogen lab, waste decontamination, handling/preparing heavily contaminated samples (or documented risk assessment to support no coat change) Sterile Gloves: for specific activities, regularly disinfect/change to prevent being a contamination source

11 GLP Sample Transportation Timely Packaging to protect from damage/contamination Conditions to minimise abuse, change in condition and/or microbial status (important for refrigerated/frozen samples, water and environmental swabs do not use loose ice, but ice packs/cool blocks) If transport time considerable + temperature control a concern, transport temperature monitoring, or, sacrificial sample included to check on receipt. If shows significant temp abuse it may not be appropriate to test (If tests progress details of temperature to be included in report/test certificate)

12 GLP - Sample Receipt Sample receipt to be away from other work/activities. If matrices with different microbial loadings received - consider separate benches, or, order of registration, or, disinfect work surface between different deliveries. Promptly register into lab system and check condition suitable for testing abuse, leakages, splits/damage to packaging that may question suitably for analysis/affect results (If tested record damage/concern in certificate of analysis)

13 GLP - Storage of Samples Pre-analysis - condition to minimise change in microbial condition (ambient/dry, refrigerated, frozen) Ambient/refrigerated samples not to be stored frozen before analysis (affects microbial level) Frozen samples not be held refrigerated for longer than required to temper for testing Separate samples according to risk (and other materials if storage not dedicated) to reduce cross contamination Post Analysis keep under appropriate conditions until results reported (to allow for retests, further investigations)

14 GLP Timeliness of Testing Refrigerated food samples (except end of shelf life) test on day of receipt, or within 24h of receipt (to ensure results reflect true condition of sample and not due to change associated with age) Water/Environmental Swabs/Hygiene Swabs ideally should test within 4h of being taken/lifted at source, but must be within 24h of being taken/lifted Requests for Microbiology + Chemistry tests best practice to ask client to send separate samples if not possible microbiology to test samples first, or if appropriate, aseptically take subsample for micro or chemistry.

15 GLP - Sample Preparation Avoid contamination at sample preparation stage Separate dedicated rooms or separate areas and equipment for raw/highly contaminated samples If not possible prepare raw/highly contaminated samples after all other samples full clean down of equipment/surfaces in between different batches of samples needed. Clinical samples, soil, effluent should not be tested in same room where food materials handled/analysed

16 GLP - Hygienic Precautions During Analysis (Aseptic Technique) Working Area Clean/disinfect work area to remove/reduce sources of contamination. No draughts and keep people movement to minimum. Ensure everything required for work is ready/in place eg media ready/cooled, pre-poured plates dry, plates/tubes labelled. Carry out work without delay (time between sample dilution and plating <45min) team work approach or process samples in small batches Mop up spillages (eg material impregnated with 70% alcohol/appropriate disinfectant

17 GLP - Hygienic Precautions During Analysis (Aseptic Technique) Working Practices Avoid talking/coughing/sneezing over work Aerosols - major causes of environmental contamination. Sources: emptying pipettes to vigorously flaming wet inoculation loops opening ampoules of freeze dried cultures mixing dilution series, using shakers, centrifuges sloppy practices associated with pipette discard/disposal of liquids Use techniques/practices that minimise aerosols

18 GLP - Hygienic Precautions During Analysis (Aseptic Technique) Sample Handling Consider handling dehydrated powdered samples in separate room/area or in protective hood (to avoid contamination of environment/other samples/work) safety cabinet for handling products likely to contain pathogenic bacteria, (if required by national regulations). Before opening samples, swab area of packaging to be opened (70% alcohol/equivalent and allow to evaporate).

19 GLP - Hygienic Precautions During Analysis (Aseptic Technique) Sample Handling Implements to open packaging and remove test portion should be sterile (sterilised in hot air oven, autoclaved + dried, or immersed and flamed in alcohol). Protected from contamination before/during use Ensure instruments are placed in suitable container for disposal/cleaning so do not pose contamination risk Clean, dry and re-sterilise before re-use

20 GLP - Hygienic Precautions During Analysis (Aseptic Technique) Pipette Technique ensure required volumes dispensed Do Not allow tip to touch outside surface of container when removing pipette; touch outside of stomacher bags, or lip/neck of dilution tubes, outer surfaces of plates/tubes handle area of pipette that comes into contact with sample/test material. dispense pipette content by running down inside of tubes (ie pipette vertically into tubes) Automatic pipettes - avoid splash back onto pipette barrel, which can be cross contamination risk (consider using filtered tips), avoid barrel contacting inside of bags

21 GLP Good Bench Practice Sample Preparation Specific Guidance on Diluents/Sample Preparation ISO 6887 pt 1: preparation of initial suspension/dilutions ISO 6887 pt 2: preparation of meat/meat products ISO 6887 pt 3: preparation of fish/fish products ISO 6888 pt 4: preparation of other products ISO 6887 pt 5: preparation of milk/milk product Representative sample ; correct weight, appropriate diluent Homogenise solid/particulate samples - release microorganisms into suspension Decimal dilutions- mixed, pipette change between dilutions Duplicate plates per dilution; 2 or more consecutive dilutions if counts expected with chosen dilutions to give countable number of colonies (eg up to 300 non selective; 150 selective/spread or as in method)

22 GLP - Good Bench Practice Plate Pouring Pour within 15min of plate being inoculated (to avoid colony aggregation). Lift lid just high enough to insert pipette without touching sides Dry agar bottle after removal from water bath (to prevent water contaminating plates) Add correct volume of agar and ensure over layers are complete Do not pour agar directly onto inoculum (to avoid heat stress) Thoroughly mix plates to evenly distribute incoulum and subsequent colonies over the entire plate Avoid splashes to lid underside (better to mix plate on table top more stable )

23 GLP - Good Bench Practice Surface Inoculation/Spread Plates Depth of agar (at least 3mm or volume as stated in method) Surface level and free from air bubbles. Dry plates before use (to aid absorption of inoculum, esp. if spreading growth expected) - LFC, drying oven lid removed/ agar side down Spread quickly after inoculum applied to plate Ensure spreader sterile, cool + changed in between dilutions. Avoid spreading to edge of plates (makes colony counting difficult) Allow inoculum to absorb before inverting for incubation (prevent inoculum dripping into lid and possible under recovery)

24 GLP - Good Bench Practice Streak plate technique to give well isolated colonies (change loop in between streaks) ensure correct size loop; sterile (sterile disposable, or flame wire loop/stem + cool without contamination, use wire loops in rotation to help cooling/increase efficiency) Track work in progress and monitor incubation times label shelves, plates, racks, bottles with date/time in and/or date/time out Or, keep records of date/time in, date/time due out and date/time removed Plate Assessment (ISO 7218:2007 +A1:2013) Accurate recognition/counting of typical colony morphologies Check ~10 fold difference in counts between dilutions; and similar counts obtained on duplicate plates at each dilution If not question results (consider voiding and repeat test) Retain positive plates + purity plates until confirmations complete In case problems with conduct of confirmations or further work needed if anomalous results obtained

25 GLP- Media Preparation/Performance (ISO11133: 2014) Stock control Sterility ph Productivity Preparation/ Sterilisation MEDIA PERFORMANCE Growth Selectivity Storage/ shelf life Enhances Optimal Recovery/Detection of Target Microorganism Physical Appearance Morphology

26 GLP - Media Storage/Handling Agar plates In dark, chilled for defined time that has been validated (eg 2 weeks). Do not dry before storing Storage in sealed plastic bags can prolong shelf life. Bottled media (without supplement addition) in sealed tubes/bottles ideally refrigerated (eg 3-6 months) Melting Agar Media melt bottled solidified agar only once boiling bath or free steam in autoclave (not re-autoclave) temper to 47-50C use within 4h (monitor agar cooling + use by times)

27 GLP - Media Performance (QC) This Takes Time and Resource! Check each batch of media prepared by laboratory Visual appearance ph post supplement addition on cool portion not used in test Sterility Volume check - critical vols dispensed pre-sterilisation 90ml/9ml Growth properties Productivity: level of recovery of target microorganism Selectivity: degree of inhibition of a non target microorganism Qualitative, Quantitative ISO11133 gives guidance on how to do, inoculation levels, criteria to appraise results

28 GLP - Traceability of Work All data from each stage of test to be recorded (traceability, to verify /defend final result) Dates, staff, media batches / kit batch #s Dilutions; colony counts from all plates; biochemical reactions # colonies taken for confirmation, individual confirmation results Results of +/-ve controls run with method/confirmation tests Correct Calculation of Results ISO 7218:2007+A1:2013 Enumerations take into account dilution factor, inoculation volume (if not 1ml), % colonies confirmed positive, weighted mean calculations (if based on 2 dilutions) correct reporting when no colonies recovered (<1 x dilution factor taking into consideration inoculation volume if not 1ml) correct reporting when TNTC colonies recovered (> 300/150 x dilution factor taking into consideration inoculation volume if not 1ml) Detections D/ND in correct sample weight/volume

29 GLP - Assuring Quality Of Results Some of the most important data a lab will generate Ongoing Performance of Method Ongoing Staff Competence Media/Commercial Kit Performance Carry out to defined plans + frequencies Use suitable technically approaches Define criteria to appraise results/performance Review results/performances REACT to poor performances/trends (treat as Non Conforming Work) APPRAISE IMPACT on Reliability of Routine Results

30 GLP - Assuring Quality of Results Analysis of External PT samples Independent, compares performance with other labs Ability to consistently obtain satisfactory results Z score +/-2 Z score between +/- 2 and +/-3 Z score greater than +/-3 good marginally acceptable poor Respond to marginally acceptable results, keep records (eg monitor next performance, if still marginal, not good, consider taking action) Immediately respond to poor/unsatisfactory results, keep records:- investigate, correct, verify actions, appraise impact on routine results, keep records. Look for bias/trends (eg consistent under recovery) and improve

31 GLP - Assuring Quality of Results Regular analysis of spiked samples reflective of those handled by lab artificially contaminate with known levels of target organism levels to be appropriate for organism/method and on occasions challenge LofD Set criteria appraise recovery/detection, trend results, take action for any failures Daily method controls (positive, negative, sterility) Do last/after analysis of samples completed to minimise cross contamination risk QC Check commercial biochemical galleries/sera etc (each use/each batch code)

32 An Overview Of GLP Training Organisation Management Control Personnel Sample Handling Equipment Calibration Competence data Bench Practice ANALYSIS Methods Validation Document Control Facility Result Media Chemicals Quality Control NCW Corrective Action Records Reports Audits Management Review Improvement

33 Thank you for your attention Any Questions?