8/19/2018

QUALITY CONTROL AND QUALITY

ASSURANCE IN BACTERIOLOGY

• QUALITY ASSURANCE and QUALITY CONTROL both

represent the procedures that laboratories establish
to ensure that the patients results reported are
accurate , timely, reliable, and relevant

• Microbiological investigations are important in the:

• Diagnosis
• Treament
• Surveillance of infectious diseases
• Policies regarding the selection and use of antimicrobial
drugs

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QUALITY
 According to ISO quality is defined as totality
of characteristics of an entity that bear on its
ability to satisfy stated and implied needs

 A diagnostic test in Clinical Microbiology to be

of good quality , must be clinically relevant
i.e. it must help in the prevention or treatment
of disease

 To state a quality, should answer;

- Is the result correct? = Reliability
- Is the same result obtained when test is
repeated?= Reproducibility
- Is the test rapid enough to be of use to the
doctor in prescribing treatment ? =Speed

FACTORS INFLUENCING QUALITY

• Quality is not only achieved through improving and

controlling the analytical process alone without
parallel improvement and control of pre-analytical
and post analytical stage
 Pre - Analytical stage
- Patient preparation
- Proper sample selection
- Proper collection and transportation
- Details of the patient and specimen identification
- Knowledge of the normal range of the results and
abnormalities
- selection of right test method

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ANALYTICAL STAGE - ACTUAL LABORATORY TESTING

POST ANALYTICAL STAGE

 Final report writing

 Interpretation of report by clinician
 Instruction of appropriate therapy

 Others-
-Accuracy

-Bias

-Error
The difference between an observed or measured value and the best
obtainable estimates of its true value

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• It is now important to realize that

preanalytic, analytic, and postanalytic
activities all affect quality. An outcome can
be interrupted or destroyed at any point in
the process.

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TOTAL QUALITY MANAGEMENT(TQM)

 TQM means all those activities in the laboratory which controls

the every variable that could possibly affect the quality of the
test results. eg. In Gram stain

 Ideal situation of TQM may not be possible but a level of quality

assurance that can control most of the factors which are likely to
affect the test results can be attained

Good Laboratory Practices(GLP)

Performance of all the activities of the laboratory in the best possible
way so that the results obtained are of the highest possible accuracy
•Proper collection of samples

• Appropriate identification of specimens with special labels on hazardous

specimens

• Prompt transportation to laboratory at appropriate temperature

• Collection and storage under conditions which prevent deterioration of the

sample before the performance of Test (e.g for anaerobic culture)

• Accurate performance of test

• Release of reports

• Delivery of reports to the correct destination in the shortest possible time

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STANDARD OPERATING PROCEDURES

(SOPS)

QUALITY CRITERIA IN MICROBIOLOGY

 Clinical relevance - contribution of test to the prevention or cure of

infectious diseases

• If Streptococcus pyogens is isolated, a full antibiograms has no clinical

relevance since benzyl penicillin is the drug of choice, and this is
always active in vitro.

• If Escherichia coli is isolated from a sporadic case of non-bloody

diarrhoea, identification of the serotype is of no clinical relevance,
since there is no clearly established correlation between serotype and
pathogenicity

Good quality test is one that is accurate and gives useful results for the prevention
and cure of infection

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 Reliability: correct result

• For tests that give quantitative results, reliability is
measured by how close the results are to the true
value

For test quantitative results:

1. measurement of minimal inhibitory concentration
(MIC) values of antibiotics in vitro;
2. Antibiotic assay of serum
3. Serum antibody titrations

• For tests that give qualitative results, reliability is

measured by whether the result is correct. Some
examples of tests of this kind are:
1. identification of pathogens;
2. Antibiotic susceptibility testing

 Reproducibility: or precision
- same result with repetition of the test i.e. precision:

- lack of homogeneity

- Lack of stability

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 Efficiency:
• Measured by two criteria

For example, the sensitivity of MacConkey agar is poor for the

isolation of Salmonella Typhi from stool. This important enteric
pathogen is often missed because of overgrowth by nonpathogenic
intestinal bacteria.

For example:
• Ziehl–Neelsen staining of sputum is highly specific for diagnosing
tuberculosis, because it gives only a few false-positive results

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QUALITY ASSURANCE

 QUALITY ASSURANCE
is the sum of all those
activities in which the
laboratory is engaged to
ensure that test results are of
good quality:

 Comprehensive

 Rational

 Regular

 Frequent

GOOD-QUALITY LABORATORY SERVICES MEAN GOOD-

QUALITY MEDICINE

 INTERNAL QUALITY CONTROL - Quality Control

 EXTERNAL QUALITY CONTROL - Quality Assessment

 Internal quality assurance:-Each laboratory has

programme to check the quality of its own test
 Ideally , involves:

1. Continuous monitoring of test quality

2. Comprehensive checking of all steps

INTERNAL QUALITY ASSURANCE IS ABSOLUTELY ESSENTIAL FOR

GOOD OPERATING PROCEDURE

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QUALITY CONTROL(QC)

 Ensures that information generated by a

laboratory is accurate, reliable and
reproducible

 Accomplished by
 assessing the quality of specimens; QUALITY
 monitoring the performances of test MANAGEMENT
procedures, reagents, media, instruments,
and personnel;
 reviewing the test results; and
 documenting the validity of test methods

INTERNAL QUALITY CONTROL CONTD….

- Set of procedures undertaken to ensure quality from

the collection of specimens, the performance of the
test up to analytical results, and the procedure being
planned, ordered and followed up

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Aspects of Internal Quality Control

 Cleaning of the working space,
 Personal hygiene,
 Safety precautions,
 Designated eating and smoking areas
located outside the laboratory,
 Handling and disposal of infected
material,
 Appropriate vaccinations for workers,
e.g. hepatitis B,
 Care of equipment,
 Collection of specimens,
 Registration of specimens,
 Elimination of unsuitable specimens,
 Processing of specimens,
 Recording of results,
 Reporting of results

QUALITY CONTROL OF EQUIPMENT

 Good quality tests cannot be performed if the equipment is either
of poor quality or poorly maintained

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Inoculating loop Remove deposits by dipping in sand or clean by Calibrate each new
wetting with alcohol and igniting loop / or after 1 week

Hot air oven Autoclave

• Physical: Temperature • Physical -thermocouple and
chart recorder and temperature chart recorder.
thermocouple
• Chemical -Browne’s tube
and succinic acid (whose
• Chemical: Browne’s tube melting point is 121oC) and
(green spot, color Bowie Dick tape. Bowie Dick
changes from red to tape is applied to articles
green) being autoclaved. If the
process has been
satisfactory, dark brown
• Biological: spores of stripes will appear across the
Bacillus subtilis or non- tape.
toxigenic strain of
Clostridium tetani on • Biological-paper strip
paper strips containing spores of Bacillus
stearothermophilus.

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BROWNE TUBE- GLASS TUBE

CONTAINING HEAT SENSITIVE DYE

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QUALITY CONTROL OF CULTURE

MEDIA
 Prepared form basic ingredients or from commercially available
dehydrated powders or purchased ready

ORDERING AND STORAGE OF DEHYDRATED MEDIA

 Order quantities that will be used up within 6 months or at most 1

year and working media should be used up within one month
 Record date and amount on receiving the container
 Store in dark, cool ,well ventilated place and protect against sunlight
 Discard all dehydrated media that are either caked or darkened
 Keep written records of media in stock

Storage of Prepared Media

 Protect against light

 Protect against heat
 Media containing blood, other organic additives, or antibiotics
should be stored in the refrigerator

 Typical shelf-lives of media are:

o Tubes with cotton-wool plugs,= 3 weeks;
o Tubes with loose caps,= 2 weeks;
o Containers with screw-caps, =3 months;
o Petri dishes, if sealed in plastic bags, =4 weeks

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 pH testing:
 Sterility testing:
 Performance testing:

Standard Strains for quality control

Staphylococcus aureus (ATCC 25923)
Escherichia coli (ATCC 25922)
Pseudomonas aeruginosa (ATCC
27853)
Enterococcus faecalis (ATCC 29212)

Performance tests on commonly-used media

Medium Incubation Control Organism Expected Result
Blood Agar 24h, CO2 S. aureus Growth and beta-haemolysis

S.pneumoniae Growth and alpha-haemolysis

Chocolate agar 24h, CO2 H.influenzae Growth

MacConkey agar With crystal 24h E.coli Red colonies

violet
P.mirabilis Colourless colonies (no swarming)

E.faecalis No growth

Methyl red/Voges-Proskauer 48h E.coli Positive/negative

broth
K.pneumoniae Negative/positive

Mueller-Hinton agar 24h E.coli ATCC 25922 Acceptable zone sizes

P.aeruginosa ATCC 27853 Acceptable zone sizes

Peptone water (indole) 24h E.coli Positive

K.pneumoniae Negative

Simmons citrate (incubate with 48h E.coli No growth

loose screwcap)
K.pneumoniae Growth, blue colour

Thiosulfate citrate bile salt 24h Vibrio spp. (non agglutinable Yellow colonies
(TCBS) agar

Thayer Martin Agar 24h, CO2 N.meningitidis Growth

N.gonorrhoeae Growth

Staphylococci No growth

E.coli No growth

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Quality control of Reagents and Tests

Procedure/result Test Control organism Expected reaction

Catalase S. aureus + Bubbling reaction

Streptococcus spp. – No bubbling

Coagulase S. aureus + Clot formation in 4 hours

Indole E. coli + Red ring at surface

E. aerogenes – Yellow ring at surface

Methyl red E. coli + Instant red colour

E.. aerogenes – No colour change

Oxidase P.aeruginosa + Purple colour in 20 seconds

E. coli – No color in 20 seconds

Voges E. aerogenes + Red colour

Proskauer E. coli – No colour change

Bacitracin disc Streptococcus group A + Zone of inhibition

E. faecalis – No Zone of inhibition

Optochin disc S. pneumoniae + Zone of inhibition

S. viridans – No zone of inhibition

Oxidase disc P.aeruginosa + Purple colour in 30 seconds

E. coli – No change in colour

QUALITY CONTROL IN ANTIBIOTIC SUSCEPTIBILITY

TESTING

 Discs should be of correct diameter (6.35mm) and of correct potency

 Stock supply should be stored frozen (-20 C)

 Working supply should be kept no longer than 1 month in a refrigerator (2–8 C)

 Only Mueller–Hinton agar of performance-tested quality and pH (7.2–7.4) should be

used

 Inoculum should be standardized against the turbidity standard

 Zone sizes should be measured and interpreted by referring to a table of critical

diameters
 Tests should be carried out with the three standard strains:
- when a new batch of discs is put into use;
- when a new batch of medium is put into use;
-once a week, in parallel with the routine antibiograms

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Factors influencing zone size in antibiotic susceptibility testing

Factor Influence

Inoculum density Larger zones with light inoculum and vice versa

Timing of disc If after application of disc, the plate is kept for longer time at room temperature, small
application zones may form
Temperature of Larger zones are seen with temperatures < 35oC
incubation
Incubation time Ideal 16-18 hours; less time does not give reliable results

Size of the plate Smaller plates accommodate less number of discs

Depth of the agar Thin media yield excessively large inhibition zones and vice versa
medium
Proper spacing of the Avoids overlapping of zones
discs
Potency of antibiotic Deterioration in contents leads to reduced size
discs
Composition of Affects rate of growth, diffusion of antibiotics and activity of antibiotics
medium

Factors (contd.)
Acidic pH of medium Tetracycline, novobiocin, methicillin zones are larger

Alkaline pH of medium Aminoglycosides, erythromycin zones are larger

Incubation in the presence of CO2 Increases zone size of tetracycline and methicillin

Addition of thymidine to medium Decreases activity of trimethoprim

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MAINTENANCE AND USE OF STOCK CULTURES

Selection and origin- strains of maximum characters established
Preservation
But the best method are lyphilization (freeze-drying) stored at -70C in
dry freezer or liquid nitrogen
1. Long term preservation
• Glycerol at -20C
• Mineral oil at room temperature
• Stab cultures at room temperature
• Stab cultures in cystine trypticase agar (CTA)(for Neisseria and streptococci)
• Cooked-meat medium for anaerobes

2. Short term preservation

 Rapid growing organisms : in TSA slants in screw capped bottles
 Streptococci: in Blood agar slats in screw capped bottles
 Meningococci and Haemophilus: In chocolate agar slants/Plates
 Gonococci: in Chocolate agar

DIAGNOSTIC ANTIGENS AND ANTISERA

 Store at the recommended temperature, avoid repeated freezing

and thawing

 Discard when the manufacturer’s expiry date is reached

 Always include a serum control of known reactivity in each batch

of tests
 Each batch of serological tests should include:

-A negative serum (specificity control);

-A weakly reactive serum (sensitivity control);
-A strongly reactive serum (titration control)

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 EXTERNAL QUALITY ASSURANCE - Quality Assessment

 Laboratory performance is controlled by external professional agency

 In some countries, participation is mandatory(regulated by
government) and required for licensure

 Involves:
• Periodic monitoring of test quality
• Spot checking of identification tests, and sometimes of isolation
techniques

EXTERNAL QUALITY ASSESSMENT

 Assessment of Quality in a schematic way through an external
agency using material of known but undisclosed results is called
EQA
 Powerful tool that challenges the internal Quality Control measures
that are being adopted by the laboratory

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Objectives of External Quality Assessment

 Monitor laboratory performance and evaluation of quality control

measures

 Establish inter-laboratory comparability

 Influence reliability of future testing

 Promote high standards of good laboratory practices

 Encourage use of standard reagents/ methodology and trained personnel

 Identify common errors

 Provide mechanisms to remedy identified deficiencies

 Education through exercises, reports and meetings

WHAT IS SENT

• Cultures(pure or mix)
• Serum (for syphilis, rubella,brucellosis,typhoid
fever,etc)

• In the philippines RESEARCH INSTITUTE OF TROPICAL

MEDICINE (RITM) is the one who facilitate for
National External Quality assesment Scheme
(NEQAS) for the bacteriology, parasitology,
mycology and virology.

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SUMMARY

 QA = QC +IQA+ EQA

 QC must be practical, achievable and affordable

 QA is important in the diagnosis, treatment, and surveillance

of infectious diseases and policies regarding the selection and use
of antimicrobial drugs

 Trained technical staff and clinical microbiologists are important

resource persons in providing the establishment of essential
microbiology services

 Regular use of Standard operating procedures (SOPs) is essential

to maintain the quality of results in between lab

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