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

email: christina.oscroft@campdenbri.co.uk

Tel: + 44 1386 842087

copyright Larson the

wild side

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

An Overview Of GLP In Operation

(ISO17025)

Personnel

Facility

Equipment

Media

Chemicals

Training

Document

Control

Quality

Control

Records

Reports Audits

Management Review

Improvement

ANALYSIS

Calibration

Methods Validation

Sample

Handling

Result

Bench

Practice Competence

data

Organisation Management Control

NCW

Corrective

Action

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

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

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

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)

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)

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

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

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)

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)

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)

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.

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

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

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

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).

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

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

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)

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 )

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)

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

GLP- Media Preparation/Performance (ISO11133: 2014)

Preparation/

Sterilisation

Stock control

Storage/

shelf life

Sterility

pH

Growth

Physical

Appearance

Productivity

Selectivity

MEDIA

PERFORMANCE

Morphology

Enhances Optimal

Recovery/Detection of

Target Microorganism

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)

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

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

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

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 good

Z score between +/- 2 and +/-3 marginally acceptable

Z score greater than +/-3 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

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)

An Overview Of GLP

Personnel

Facility

Equipment

Media

Chemicals

Training

Document

Control

Quality

Control

Records

Reports Audits

Management Review

Improvement

ANALYSIS

Calibration

Methods Validation

Sample

Handling

Result

Bench

Practice Competence

data

Organisation Management Control

NCW

Corrective

Action

Thank you for your attention

Any Questions?