QUALITY CONTROL IN MICROBIOLOGY

WHAT IS QUALITY?

Quality means meeting the pre-determined requirements of users for a

particular substance or service.

Quality includes the following :

Total Quality Management (TQM)

Continuous Quality Improvement (CQI)

Quality Assurance (QA)

 QUALITY ASSURANCE

• Quality assurance has been defined by WHO as:

“The total process whereby the quality of laboratory reports can

be guaranteed.”
 Right result

Right price Right time

QUALITY
ASSURANCE
Interpretaion
based on
correct Right
reference specimen
data
Right patient
 QUALITY ASSURANCE

IQA EQA QC QUALITY

ASSURANCE
QUALITY CONTROL(QC)

• The term QC covers that part of QA, which primarily concerns the control of
errors in the performance of tests and verification of test results.

• QC must cover all aspects of every procedure within the department.

• The laboratory director is responsible for both QA& QC.

Affordable Achievable
Practical
 QUALITY CONTROL AND ASSURANCE

• QC is associated with internal activities that ensure diagnostic test accuracy.

• QA is associated with the external activities that ensure positive patient outcomes.

Reduced length of stay

Reduced cost of stay

Reduced turn around time for diagnosis of infection

Change to appropriate antimicrobial therapy

Customer (physician or patient satisfaction)

THE DIAGNOSTIC CYCLE

Pre-
analytic
phase

Post
Analytic
analytic
PRE ANALYTIC PHASE

Specimen collection

Criteria for rejection

of specimen Cost
Specimen transport
effectiveness

Specimen receipt &

preliminary
observation
 SPECIMEN COLLECTION

SITE OF COLLECTION

• Must be from the actual site

• Minimum contamination from adjacent tissues, organs

or secretions

• Swabs are inferior in the collection of most specimens

• Use of aspiration needles and catheters should be

encouraged

• A patient information sheet should be given to patients

for collection of urine specimens.
SPECIMEN COLLECTION

TIME OF COLLECTION

• Optimal time of collection.

• Pathophysiology of the infectious disease should be known

• Blood cultures are usually positive in the first week

• Urine and stool culture positive during 2nd and 3rd week of illness

• 24 hours collection of sputum and urine should not be done.

SPECIMEN COLLECTION

• QUANITY OF THE SPECIMEN

• Must be sufficient

• Guidelines should be established to define a sufficient volume.

• If quantity is low

• Tubes containing holding broth such as physiologic saline(non nuntrient) or

phosphate yeast glucose (PYG) should be provided.
SPECIMEN COLLECTION

DEVICE FOR COLLECTION

• Sterile containers should be used.

• Wide mouthed

• Tightly fitted caps to prevent leakage and

contamination

• Swabs tipped with Dacron or Rayon

polyester are better choices.
SPECIMEN COLLECTION

• Specimen should not remain in contact with swab for long duration.

• Swabs should be placed in transport media to prevent drying (upto 48 hours.

• Skin scrapings and nail clipping for recovery of dermatophytes should be

submitted dry in a clean container
SPECIMEN COLLECTION

ASPIRATED CULTURE VS SWAB CULTURE

 SPECIMEN COLLECTION

SPECIMEN BEFORE ANTIBIOTIC ADMINISTRATION

Obtain cultures before administering antibiotics

Ideal for organisms highly susceptible to antibiotics

SPECIMEN COLLECTION

• SMEARS SHOULD BE PERFORMED

• Provide clues to the inflammatory nature of

the condition.

• Indicates whether the result of culture are

meaningful or not

• A wound swab which contains no PMNs but

yields mixed bacterial growth cannot be
considered valid
SPECIMEN COLLECTION

PROPER LABELLING

• Identification number

• Name/Age and gender

• Source of specimen

• Clinician in charge

• Date/Hour collected

• Diagnosis

• Any antibiotics given

• Investigation required
 SPECIMEN TRANSPORT

• Primary objective is to maintain sample in its original state.

• Adverse environmental conditions such as

Extremes of heat and cold

Rapid changes in pressure(during air transport)

Excessive drying should be avoided

• For prolonged delay (>4 days) specimens should be frozen at

-70 deg.

• Samples for recovery of mycobacteria and fungi should be shipped

immediately
SPECIMEN RECEIPT & PRELIMINARY OBSERVATION

• Area should be designated for receipt of

specimens.

• Initial observation and handling in the biosafety

cabinet.

• Personnel should wear protective clothing

SPECIMEN RECEIPT & PRELIMINARY OBSERVATION

1. ENTRY OF DATA

Essential data should be entered into a log book or

computer data base.

2. GROSS EXAMINATION

Visual examination & determination whether all

criteria for acceptance are met.
[Link] EXAMINATION

Direct mounts or stained smears to establish a

presumptive diagnosis
CRITERIA FOR SPECIMEN REJECTION

Any specimen received in formalin

• 24 hour sputum collection

• A single swab submitted for multiple

requests Eg: aerobes,anaerobes,fungus
and tuberculosis

• Improper collection site like stool for

respiratory syncytial virus
 CRITERIA FOR SPECIMEN REJECTION

• Leaking container

• Unlabelled/wrongly labelled/mismatched samples

• Quantity Not Sufficient For Testing (QNS)

• Prolonged transport

• Dried specimen

• Haemolysed blood

• Wrong tube
ANALYTIC PHASE

[Link]
[Link] [Link]
identification of
examination specimens bacterial isolates

[Link]
[Link] [Link]
susceptibility
effectiveness of cultures
testing
 MICROSCOPIC EXAMINATION

• [Link] and percentage of segmented neutrophils

Indicate the magnitude and type of inflammatory

response.

Quality of specimen can be validated

• [Link] of bacteria, mycelial elements, yeast gives

an immediate presumptive diagnosis

• [Link] evidence that species of anaerobic

bacteria are present.
 QC OF STAINS

• All stains and reagents must be clearly

labelled, dated, and stored correctly.

• Should not be used beyond their expiry

date

• Should not be used when they show

signs of deterioration like abnormal
turbidity and decolouration.

• At regular intervals and whenever a

new stain is prepared, control smears
should be stained

• Smear should not be too thick.

• Decolourization is often incomplete

which can result in gram negative
organisms being reported as gram
positive
QC OF STAINS
 PROCESSING THE SPECIMEN

• [Link] the primary culture media for the specimen

• [Link] the temperature and conditions for incubation.

• [Link] which of the following isolates recovered on primary

media require further characterization

• [Link] whether antimicrobial susceptibility tests are required

 PROCESSING SPECIMENS

• Panculture- Indiscriminate ordering of cultures from all

accessible body sites in hope of recovering a pathogen
should not be done

• Non selective media free of inhibitors support the growth

of most bacteria

• 5% sheep blood agar is the most commonly used

• Chocolate agar should be used where Haemophilus and

Neisseria species are suspected [Link] sputum, CSF and
semen.
PROCESSING SPECIMENS

• TRANSFERING SPECIMEN ON A CULTURE PLATE:

• Should be carried out in a biosafety cabinet

• Rubber gloves should be worn

• Appropriate charts and instructions posted on a bulletin board

• Or included in the bench manual for those who are new to the lab.
 SEEDING A CULTURE PLATE

• Essential to learn the skills of inoculating specimens.

• Instrument for seeding media selected according to the

nature of the medium and inoculum.

• Platinum or nichrome wires of different gauges are

used.
 SEEDING A CULTURE PLATE

• This wire is sterilized by holding it vertically in the flame

of the burner so that the whole length of wire becomes
red hot.

• It is allowed to cool down before it touches any

material suspected to be having bacteria to avoid the
heat killing the organisms.

• Pre sterilized disposable loops are now available

commercially.

• The wire can be used as a:

STRAIGHT WIRE

• To stab the culture

• Picking of single colonies

• Inoculating the liquid media

SEEDING A LIQUID MEDIUM

THICK WIRE /LOOP

• For lifting viscid material such as sputum

• Seed a plate of medium as the straight wire usually

cuts the agar

FOR LIQUID MEDIA

• If the tubes have got cotton plugs

The mouth of the tubes should be heated in flame

before and after any handling of tube

• Not required when metal caps and screw-capped tubes

are handled
PROCESSING SPECIMEN
 SEEDING A MEDIUM

• SUBCULTURE FROM A SOLID MEDIUM TO SOLID MEDIUM

Using a sterile wire or loop, a representative colony is

touched and sub cultured onto appropriate solid medium by
touching the wire or loop onto SURFACE OF THE MEDIA

➢When more than one medium is inoculated, follow a particular

order.

Inoculate media without inhibitors, followed by indicator and then

selective media.

➢While processing fluid specimen inoculate liquid media first to

reduce the chances of carry over from contaminated solid media.
 ASEPTIC TECHNIQUES

• Open caps and lids of containers for the briefest

period.

• Do not keep lids on the workbench.

• Inoculating loops should be put through the flame

• While working on the infectious material, keep the

specimen away from the face.
ASEPTIC TECHNIQUES

• Loops should not contain fluid or large

particles of matter that may splatter when
placed in the flame.

• Open the caps slowly to minimize aerosol

production.

• Homogenization & grinding of tissue or

biopsy specimen should be done in safety
cabinet.
 ASEPTIC TECHNIQUES

• Keep all specimens in racks to reduce the

risk of accidental spillage.

• Mop up the workbench clean with

disinfectant at the start and close of work.

• Wash hands with soap and water before

and after handling infectious specimens
 INTERPRETATION OF CULTURES

• Interpretation of primary cultures should be

done after 24-48 hrs

• Requires considerable skill.

• By assessing the colonial characteristics the

microbiologist can make a preliminary
identification of bacteria.

• This is one of the cornerstones of diagnostic

microbiology.
PRELIMINARY TESTS & THEIR QC
ANTIMICROBIAL SUSCEPTIBILITY TESTING

Salient features of quality assurance in

antibiotic susceptibility testing

• Use antibiotic discs of 6 mm diameter.

• Use correct content of antimicrobial agent

per disc.

• Stock the supply of antimicrobial discs at -

20 Deg C.

• Use Mueller-Hinton medium for antibiotic

susceptibility testing.
 ANTIMICROBIAL SUSCEPTIBILITY TESTING

• Use appropriate control cultures.

• Use standard methodology for the

test.
 ANTIMICROBIAL SUSCEPTIBILITY TESTING

• Space the antibiotic discs properly to avoid overlapping of inhibition zone.

• Use inoculum size that produces near confluent growth.

• Ensure an even contact of the antibiotic disc with the inoculated medium.

• Measure the zone sizes precisely.

• Interpret the zone sizes by referring to standard charts.

• Keep the antibiotic discs at room temperature for one hour before use.

• Incubate the sensitivity plates for 16-18 hours before reporting.

• Incubate the sensitivity plates at 35 deg C.

QC OF EQUIPMENTS

BALANCE
MICROSCOPE Keep the balance and weights clean and
Wipe lenses with lens paper at the end of each dry
days work
Always use a container or weighing
Protect the microscope from dust, moisture and paper
vibrations.
Check alignment of condenser once a month Do not put material directly on the plate

REFRIGERATOR HOT AIR OVEN

Keep at least 10 inches away from the Air flow should be checked
wall
Clean and defrost every 2 months Drying process should be
monitored by use of moisture
Record temperatures daily and temperature probes
QC OF EQUIPMENTS

WATER BATH GLASSWARE

Check water level daily Discard chipped glassware
Check temp before and during use Ensure these are free of detergents
Clean monthly Do no store sterile glassware for more than 3
Technical inspection once in 6 months weeks before it is used

EQUIPMENTS

CENTRIFUGE
Check revolution by tachometer INSPISSATOR
Wipe inner walls with antiseptic solution Check temperatures daily
weekly Clean after each batch of culture media prepared
Check brushes and bearings every six months
QC OF EQUIPMENTS
QC OF CULTURE MEDIA
POST ANALYTIC

Reporting
results

Maintenance POST
of sample & Interaction with
records ANALYTIC epidemiologist
PHASE

Analysis
 REPORTING OF RESULTS

• Should be quick

• Terminology should be understandable

• Reports maybe designated as Urgent or important

• Urgent reports must always be telephoned to the caregiver

• Confidentiality of the patient data must be ensured

• Timely ,preliminary reports should be issued

• Reference range should be given where appropriate.

• To avoid time and labour reports that are negative should be stamped

• A copy of the report dispatched should be kept in the lab

INTERACTION WITH EPIDEMIOLOGIST

• Certain infectious agents must be

reported to the public health authorities.

• They are different for different regions.

• Microbiologists should remain alert for

unusual patterns of isolates.
 MAINTENANCE OF SAMPLES & RECORDS

• Local and National guidelines must be followed.

• All patient records should be maintained for atleast 2 years and ideally for 10 years.

• Sterile body fluids should be maintained at room temp until culture and other procedures have
been performed

• Blood culture isolates should be maintained for 30 days.

• All positive cultures should be kept for 7 days for further evaluation like molecular typing.
MAINTENANCE OF SAMPLES & RECORDS

• Viruses maybe frozen to -70 deg in a solution containing a cryoprotectant like 10%
dimethyl sulphoxide (DMSO).

• Tissue should be frozen at -70 deg for potential future use.

• CSF should be stored at room temp because of lability of Neisseria meningitides at 4

deg C
MAINTENANCE OF SAMPLES & RECORDS

• Long-term storage of aerobes and anaerobes can be accomplished by lyophilisation

(freeze drying) or freezing at -70 deg.

• Frozen, non fastidious organisms should be thawed, re isolated and refrozen every 3
years.
• Stock isolates may be maintained by freezing them in :--

10% skim milk

TSB with 15% glycerol

10 % horse blood in sterile vials

MAINTENANCE OF SAMPLES & RECORDS

• Yeasts should be stored like non fastidious bacteria

• Moulds can be stored on potato dextrose agar (PDA) slants at 4 deg for 6
months -1 year

• Long term storage in PDA slants overlaid with sterile mineral oil at room temp

• AFB may be kept on LJ agar slants at 4 deg for 1 year.

• Or frozen at -70 deg in 7H9 broth with glycerol

 STANDARD OPERATING PROCEDURE(SOP)

• Essential component of QC

• Should be written in format of CLSI guidelines.

• Must be reviewed and signed annually or biannually by the laboratory director

• FUNCTION

• Improve and maintain the quality of laboratory service to patients

• To provide laboratory staff with written instructions on how to perform tests

consistently to an acceptable standard in the laboratory.

• To help avoid short-cuts being taken when performing tests.

• To provide safety in laboratory

 STANDARD OPERATING PROCEDURE(SOP)

Each laboratory should have Standard Operating Procedure Manuals (SOPMs) which should include the
following information about the

• Infrastructure of a laboratory • Processing of specimens

• Biosafety precautions • Maintenance of equipment

• Disposal of infectious waste • Recording of results

Collection, transport and storage of • Reporting of results

specimens
• Tolerance of limits
Reagent preparation
• Procedure of quality control
• Criteria of rejection of samples
• Referral
 FACILITIES IN A LABORATORY

Each laboratory must possess space for :

Sample collection

Sample analysis

Storage of samples

Reagents, chemicals

Stationary& record etc.

 FACILITIES IN THE LABORATORY

• Washing

• Media preparation

• Autoclaving

• Seminar room

• Library

• Staff room and

• Toilets
FACILITIES IN THE LABORATORY

• Laboratory must be well lit with dust-free

• Air conditioned environment.

• Uninterrupted power supply.

• The laboratory must monitor, control and

record environmental conditions

• Biological sterility

• Humidity

• Temperature.
FACTORS IN PREVENTING THE ESTABLISHMENT OF ESSENTIAL MICROBIOLOGY
SERVICES IN DEVELOPING COUNTRIES

• High cost of culture media and reagents

• Lack of rational approach to the selection and use of microbiological investigations

• Shortage of trained technical staff

• Shortage of clinical microbiologists.

TTTT

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