Specific measures to control

laboratory investigations in
microbiology

Suhair.A.A./ Quality Assurance / 3rd year 1

Test Cycle
Preanalytical
(sampling , labelling , transport , storage , specimen clinical information

↓
Analytical
( macroscopic evaluation , microscopy , culture , identification ,
interpretation , antibiogram , .. )

↓
Post analytical
( Final report to physician )

Suhair.A.A./ Quality Assurance / 3rd

year 2
QUALITY ASSURANCE PROGRAMME QA
◼ A quality assurance (QA) programme:……..
◼ The bacteriology and immunology laboratory is responsible for
providing accurate and relevant information that is of use for clinical
diagnosis of a patient or in support of a public health activity. The
accuracy and clinical value of the laboratory analysis of the clinical
specimen and microbial isolates are dependent upon a QA
programme that
◼ assesses the quality of the specimen,
◼ documents the validity of the test method,
◼ monitors the performance of test procedures, reagents,
media, instruments and personnel,
◼ and reviews test results for errors and clinical relevance
An effective QA programme is dependent on a continuous process of
assessment and improvement. It has two broad components:
◼ Quality assurance = Internal quality control + External
quality assessment
3
 Internal quality control
◼ Internal quality control (IQC) is the set of procedures
undertaken by the staff of a laboratory for continuously
and concurrently assessing laboratory work and the
emergent results, to decide whether they are reliable
enough to be released.
◼ These results may be required for a wide variety of
purposes; e.g. support of clinical decision making or
epidemiological surveillance or research. Internal quality
control procedures have an immediate effect on the
laboratory’s activities and should actually control – as
opposed to merely examining – the laboratory’s output.

Suhair.A.A./ Quality Assurance / 3rd

year 4
Internal Quality Control
IQC

Practical
Realistic
Economical

Suhair.A.A./ Quality Assurance / 3rd

year 5
Sources of error that effect the reliability and
reproducibility
:
Sampling , transport,storage, processing Specimens

Environmental factors: Inadequate space working :

ventilation, temperature, excessive noise, unsafe :work conditions,..
Personnel:
Education, training , experience, condition of employment
Laboratory materials
Quality or reagents, stains, chemicals, culture media , ,
Test method ; some methods are more reliable

:
Substandard or poorly maintained instruments Equipments
Examination and reading :Hurried Reading of results , failure to examine
sufficient number of microscopic fields,…
Reporting
6
 Procedures
◼ Begins with proper laboratory operations based on SOPS for:
Collection of specimen
Registeration of specimen
Processing of specimen.
Care of equipment
Preparation and storage of culture media , stains , reagents and
antisera
Antibiotic susceptibility tests
Stock cultures
Safety precautions
Personal hygiene
Handling and disposal of infected material

Suhair.A.A./ Quality Assurance / 3rd

year 7
Parameter Guidelines

Specimen collection Provide instructions for collection and transport

and transportation
Establish criteria for acceptable specimens

Establish rejection criteria for unacceptable specimens

Personnel Use sufficient qualified personnel depending upon volume and complexities of work

Provide continuous technical education

Provide written performance standards

Evaluate annually

QC records Record all QC results on prescribed forms

Report all out-of-control observations to supervisor

Note corrective actions on QC form

Review QC records monthly

Patient reports Report only to authorized personnel

Notify test requester of important values immediately

Provide normal ranges where appropriate

Correct errors in patient’s reports in timely fashion

Retain records for at least two years

8
Information should be filled

Patient name
Hospital or laboratory number
Ordering physician
Whether the patient receiving antimicrobial
therapy
Suspect agent or syndrome

Suhair.A.A./ Quality Assurance / 3rd

year 9
Written collection instruction
Test selection criteria
Patients selection criteria
Timing of specimen collection (e.g.,
Before antimicrobial are administration)
Optimal specimen collection site
Approved specimen collection method
Specimen transport medium

Suhair.A.A./ Quality Assurance / 3rd

year 10
Continue…
Specimen transport time and temperature
Specimen holding instructions if it cannot be
transported immediately (e.g. hold at 4C for 24
hours)
Availability of test (on –site or sent to reference
laboratory )
Hours test performed (daily or batch)
Turn-around time
Result reporting procedures
Suhair.A.A./ Quality Assurance / 3rd
year 11
Criteria for unacceptable
specimens
Unlabeled or mislabeled specimens
Use of improper transport medium
Excessive transport time
Improper temperature during transport or
storage
Improper collection site for test request
Specimen leakage out of transport container

Suhair.A.A./ Quality Assurance / 3rd

year 12
Personnel
It is laboratory director's responsibility to employ
sufficient qualified personnel for the volume and
complexity of the work performed.
Document competency and training twice a year
Continuing education program should be provided
All documentation should maintained in personnel
file

Suhair.A.A./ Quality Assurance / 3rd

year 13
External quality assessment
◼ External quality assessment (EQA), The assessment is
retrospective and periodic in clinical microbiology unlike
industrial microbiology such as production of vaccines and
immunobiologicals which, as per the laws of the land, require
testing by an agency independent of the manufacturer before
their release for use.
◼ The main objective of external quality assessment is to
establish inter-laboratory comparability. This will influence
the reliability of future testing. In contrast, the main objective of
internal quality control is to ensure day-to-day consistency.
Hence, both internal quality control and external quality
assessment are complementary in ensuring the reliability of
procedures, their results and finally the quality of the product

Suhair.A.A./ Quality Assurance / 3rd

year 14
quality control of equipment
◼ Maintenance records should ➢ Biological safety
be retained in the laboratory for cabinets
the life of instrument. Specific
guidelines regarding periodicity ➢ Autoclave
of testing for autoclaves, ➢ Oven
biological safety cabinets
➢ Incubator
,centrifuges ,incubators,
microscope, refrigerators ➢ Refrigerator
,freezers, water bathes , heat ➢ Water- bath
blocks and other microbiology
laboratory can be found in ➢ Microscope
reference books
Suhair.A.A./ Quality Assurance / 3rd
year 15
Culture media
Ordering and storage of dehydrated media
Order quantities for 6 months or at most 1 year.
The overall quantity should be packed in containers that
will be used up in 1-2 months.
Write date of receipt on each container
Store in a dark , cool, well ventilated place
When a container is opened , write the date of opening on
it
Discard all dehydrated media that are either caked or
darkened

Suhair.A.A./ Quality Assurance / 3rd

year 16
 Storage of prepared media

◼ Distilled or demineralized water should be used for

preparation of media
◼ Protect against sunlight
◼ Protect against heat. Media containing blood, antibiotics
,… should be stored in refrigerator
◼ The shelf-life of prepared media when stored in cool , dark
place depend on the type of container used.
◼ Tubes with cotton – wool plugs 3 weeks
◼ Tubes with loose caps 2 weeks
◼ containers with screw – caps 3 months
◼ Petri dishes , sealed in plastic bags 4 weeks
Suhair.A.A./ Quality Assurance / 3rd
year 17
pH testing
▪ Prepared media should not be checked
routinely.
▪ If it is prepared in house from basic ingrediente it
should be allowed to cool before the pH tested.
▪ If pH differs 0.2 units from the specification,
adjust with acid or alkali or prepare a new
batch.

Suhair.A.A./ Quality Assurance / 3rd

year 18
Sterility Check
A representative sample of each media which
blood or their components are added after
autoclaving should be test for sterility; 3-5% of
any batch is tested.
Sterility is routinely checked by incubating the
medium for 48 hours at the 35 C temperature .
More than 2 colonies per plate if seen , discard
the whole batch..

Suhair.A.A./ Quality Assurance / 3rd

year 19
Performance testing
When medium dose need to quality controlled
because it was prepared “in house (in the
laboratory) or because it is complex, several
basic rules must be followed :
All media must be tested before use
Each medium must be tested with organisms
expected to give positive reaction as well as with
organism expected either not to grow or produce
a negative reaction

Suhair.A.A./ Quality Assurance / 3rd

year 20
Continue..
Performance tests for different commonly used media are
Performed by different microorganisms ( stock culture )

Blood agar
:Performance test *( hemolysis and growth )
s.Pyogenes
s.Pneumoniae
Medium Negative Bacitracin disc positive

s. pyogenes (zone) E.faecalis blood agar

Suhair.A.A./ Quality Assurance / 3rd

year 21
User-Prepared and Noncommercially prepared
Media
QC forms for user-prepared media should
contain:
The amount of prepared
The source of each ingredient
The lot number
The preparation date
The expiration date (Usually 1 month for agar plate
and 6 month for tube media)
Sterlization methods:
All user prepared colures media also should
checked for : Suhair.A.A./ Quality Assurance / 3rd
year 22
Continue.
Proper color
Depth
Smoothness
Clarity
Hemolysis
Excessive bubbles
Contamination

Suhair.A.A./ Quality Assurance / 3rd

year 23
Stain and Reagents
Containers of stains and reagents should be
labeled as to contents, concentration ,storage
requirements, date prepared (or received) date
placed in service ,expiration date, source
(commercial manufacture or user prepared) and
lot number .All stains and reagents should be
stored according manufacture's
recommendations and tested with positive and
negative controls before use.

Suhair.A.A./ Quality Assurance / 3rd

year 24
Antisera
The lot number, date received, condition
received ,and expiration date must be
recorded for all shipments of antisera.In
addition ,the antisera should be dated
when opened .New lots must be tested
concurrently with previous lots, and testing
must include positive and negative
controls.
Suhair.A.A./ Quality Assurance / 3rd
year 25
Use of Stock Cultures

To operate a quality control program, stock culture must be

maintained by all laboratories .They are available from
many sources.
Commercial sources
Clinical specimens
Proficiency testing
Reference laboratories
Official Culture Collection (ATCC)

Suhair.A.A./ Quality Assurance / 3rd

year 26
Preservation

Long –term
Lyophilization
Storage at -70C TSB (Tryptic Soy Broth) with 15% Glycerol,
CTA for Neisseria and streptococci
Cooked-meat medium for anaerobes

Short-term
TSA slants
Store in refrigerator
Transfer every 2 weeks.

Suhair.A.A./ Quality Assurance / 3rd

year 27
Reference Strains
▪ E. coli ATCC 25922
▪ S. aureus ATCC 25923
▪ P. aeruginosa ATCC 27853

QC organisms must be obtained from

reputable source
Use specific QC organisms to test different
groups of “drug-bug” combinations

QC Antimicrobial Susceptibility Testing -

Module 8 28
28
Reagents for the Disk Diffusion Test

1-Selection of Antimicrobial Disks (source of

infection and the isolated pathogen )

2- Mueller- Hinton Agar medium

-prepared from commercially dehydrated powder
-4mm depth ( 25-30 ml for plates with100mm diameter )
-Store in 2-8 C within 7days

0.5 McFarland ) from isolated colony ) 3-

StandardTurbidity

Suhair.A.A./ Quality Assurance / 3rd

year 29
Selection of a Colony to Test

30
30
MacFarland 0.5 and
Adjusted Test Organism

31
31
Use of Disk Dispensers
◼ Advantages
 practical,rapid
 increase reproducibility

◼ Risks:
 contamination
 reducespersonal
judgment skills

32
32
Disk Susceptibility Testing Problems

33
33
Disk Susceptibility Testing Problems

34
Mueller Hinton Agar Medium

Preparation of Mueller Hinton Agar

Moisture
PH
Effects of Thymidine or Thymine
Effects of Variation in Divalent Cations
Testing Strains That Fail to Grow
Satisfactorily

Suhair.A.A./ Quality Assurance / 3rd

year 35
Turbidity Standard

0.5 McFarland standard

( 0.5 ml of 0.048 mol/ml BaCl2 added to
99.5 ml of 0.18 mol/L H2SO4(1% v/v)
density in 625 nm = 0.08 - 0.1

Keep in 4-6 ml aliquots in screw cap

tubes in dark , room temperature
Replace or verify density month
Suhair.A.A./ Quality Assurance / 3rd
year 36
Suhair.A.A./ Quality Assurance / 3rd
year 37
 Suhair.A.A./ Quality Assurance
38 / 3rd year
Common sources of error

Inhibitory substance thymine and thymidine:

Enterococcus faecalis ATCC 29212 or 33186 tested by SXT disc . An
inhibition
. zone ≥ 20 mm and free of colony.
Control zones too small may be due to :
Discs with low potency for bad storage or expired discs.
Too dense inoculum.
Too thick culture media.
small zones for tetracyclines and beta- lactams indicate pH is too high.
zones for aminoglycosides which are too small indicate pH is too low

Suhair.A.A./ Quality Assurance / 3rd

year 39
Patient Report
The laboratory should established a system for
supervisory of all laboratory reports. This review
should involve checking the specimens workup
to verify that the correct conclusion were drawn
and no clerical errors were made in reporting
results. Reports should be given only given only
authorized by law to receive them.

Suhair.A.A./ Quality Assurance / 3rd

year 40
Continue..
Clinician should be notified about ‘’panic
values’’ immediately. Panic values are
potential-threatening results, for example
positive Gram stain for CSF or a positive
blood culture. All patients records should
be maintained for least 2 years.

Suhair.A.A./ Quality Assurance / 3rd

year 41
 Comments?

Questions?

Suhair.A.A./ Quality Assurance / 3rd year 42