This document discusses quality control in microbiology. It covers various aspects of quality control including the pre-analytic, analytic and post-analytic phases of diagnostic testing. In the pre-analytic phase, it discusses proper specimen collection, transport and processing. The analytic phase covers microscopic examination, culture processing, identification and antimicrobial susceptibility testing. It also discusses quality control of equipment, reagents and culture media. The post-analytic phase involves reporting of results and interaction with epidemiologists. Maintaining accurate records and samples is also emphasized.

1. QUALITY CONTROL IN MICROBIOLOGY
PRESENTER:DR TABEEN MANSOOR
1st year PG
MICROBIOLOGY
Sheri Kashmir Institute Of Medical Sciences

2. 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)

3. QUALITY ASSURANCE
• Quality assurance has been defined by WHO as:
“The total process whereby the quality of laboratory reports can
be guaranteed.”

4. QUALITY
ASSURANCE
Right result
Right time
Right
specimen
Right patient
Interpretaion
based on
correct
reference
data
Right price

5. QUALITY ASSURANCE
IQA EQA QC QUALITY
ASSURANCE

6. 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.
Practical Affordable Achievable

7. 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)

8. Pre-
analytic
phase
Analytic
Post
analytic
THE DIAGNOSTIC CYCLE

9. PRE ANALYTIC PHASE
Cost
effectiveness
Specimen collection
Specimen transport
Specimen receipt &
preliminary
observation
Criteria for rejection
of specimen

10. 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.

11. 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.

12. 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.

13. 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.

14. 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).
EXCEPTIONS :
• Skin scrapings and nail clipping for recovery of dermatophytes should be
submitted dry in a clean container
Prevents overgrowth of bacteria.
• Swab for recovery of Streptococcus pyogenes
Bacteria that colonize dry off.

15. SPECIMEN COLLECTION
ASPIRATE YIELDED A PURE CULTURE OF STAPHYLOCOCCUS AUREUS.STAPH WAS ALSO RECOVERED IN
THE SPECIMEN BY SWABBING BUT IT WAS MIXED WITH OTHER CONTAMINATING FLORA

16. SPECIMEN COLLECTION
SPECIMEN BEFORE ANTIBIOTIC ADMINISTRATION
Obtain cultures before administering antibiotics
Ideal for organisms highly susceptible to antibiotics
B-haemolytic streptococci from throat specimens
Neisseria gonorrhoea from genitourinary samples
H.influenza or Neisseria meningitis from CSF

17. 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

18. 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

19. 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

20. 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
• Lab Coats
• Rubber gloves and
• In some cases custom fitted masks

21. 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.
3.MICROSCOPIC EXAMINATION
Direct mounts or stained smears to establish a
presumptive diagnosis

22. CRITERIA FOR SPECIMEN REJECTION
Any specimen received in formalin
• 24 hour sputum collection
• Smears of secretions from uterine cervix,
vaginal canal or anus for Gram’s stain
detection of Neisseria gonorrhoea.
• A single swab submitted for multiple
requests Eg: aerobes,anaerobes,fungus
and tuberculosis
• Improper collection site like stool for
respiratory syncytial virus

23. CRITERIA FOR SPECIMEN REJECTION
• Leaking container
• Unlabelled/wrongly labelled/mismatched samples
• Quantity Not Sufficient For Testing (QNS)
• Prolonged transport
• Dried specimen
• Haemolysed blood
• Lipemic serum
• Wrong tube

24. SPECIMENS UNSUITABLE FOR ANAEROBIC CULTURE:
• Gastric washings
• Midstream urine
• Prostatic secretions collected transurethrally
• Faeces(except for recovery of C.difficle,
C.perfringens, C.septicum)
• Ileostomy or colonostomy swabs
• Throat ,nose or other oropharyngeal
specimens(except specimens obtained from
deep tissue during oral surgery)
• Superficial skin and environmental cultures

25. ANALYTIC PHASE
1.Microscopic
examination
2.Processing
specimens
3.Preliminary
identification of
bacterial isolates
4.Interpretation
of cultures
5.Antimicrobial
susceptibility
testing
6.Cost
effectiveness

26. MICROSCOPIC EXAMINATION
• 1.Number and percentage of segmented neutrophils
Indicate the magnitude and type of inflammatory
response.
Quality of specimen can be validated
• 2.Observation of bacteria, mycelial elements, yeast gives
an immediate presumptive diagnosis
• 3.Presumptive evidence that species of anaerobic
bacteria are present.

27. 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

28. QC OF STAINS

29. PROCESSING THE SPECIMEN
• 1.Select the primary culture media for the specimen
• 2.Select the temperature and conditions for incubation.
• 3.Determine which of the following isolates recovered on primary
media require further characterization
• 4.Determine whether antimicrobial susceptibility tests are required

30. 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 ie.in sputum, CSF and
semen.

31. PROCESSING SPECIMENS
• PRIMARY CULTURE MEDIA
• Inhibitory agars should not be used alone
May inhibit use of organisms of interest.
• Broth cultures are done in special situations like
Spontaneous bacterial peritonitis
Peritoneal infections in patients of peritoneal dialysis
Septic arthritis

32. 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.

33. 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.
• Nichrome has oxidizing properties
• Tests where this property of bacterium is to be tested
(e.g. oxidase test), platinum wire, instead of nichrome
should be used.

34. 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

35. 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

36. PROCESSING SPECIMEN

37. 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.

38. 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.

39. ASEPTIC TECHNIQUES
• Loops should not contain fluid or large
particles of matter that may splatter when
placed in the flame.
• Avoid vigorous shaking of the specimen prior
to opening.
• Open the caps slowly to minimize aerosol
production.
• Homogenization & grinding of tissue or
biopsy specimen should be done in safety
cabinet.

40. 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

41. 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.

42. PRELIMINARY TESTS & THEIR QC

43. 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.

44. ANTIMICROBIAL SUSCEPTIBILITY TESTING
• Use appropriate control cultures.
• Use standard methodology for the
test.
Use coded strains from time to time
for internal quality control.

45. 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.

46. QC OF EQUIPMENTS
MICROSCOPE
Wipe lenses with lens paper at the end of each
days work
Protect the microscope from dust, moisture and
vibrations.
Check alignment of condenser once a month
BALANCE
Keep the balance and weights clean and
dry
Always use a container or weighing
paper
Do not put material directly on the plate
REFRIGERATOR
Keep at least 10 inches away from the
wall
Clean and defrost every 2 months
Record temperatures daily
HOT AIR OVEN
Air flow should be checked
Drying process should be
monitored by use of moisture
and temperature probes

47. QC OF EQUIPMENTS
WATER BATH
Check water level daily
Check temp before and during use
Clean monthly
Technical inspection once in 6 months
GLASSWARE
Discard chipped glassware
Ensure these are free of detergents
Do no store sterile glassware for more than 3
weeks before it is used
CENTRIFUGE
Check revolution by tachometer
Wipe inner walls with antiseptic solution
weekly
Check brushes and bearings every six months
INSPISSATOR
Check temperatures daily
Clean after each batch of culture media prepared
EQUIPMENTS

48. QC OF EQUIPMENTS

49. QC OF CULTURE MEDIA
• Water used for the media should be free of Copper ions
• The pH of the water should be slightly on the acidic side, but should not be
less than 5.5.
• The conductivity should be less than 15 µS (microsiemens).
• Media prepared should be free from excessive bubbles or pits.
• Should be free of cracks, freezing or crystallization.
• Thickness of the medium should be 4mm
• Should be checked at four points being at right angles to each other
• Only borosilicate glassware should be used because soda glass can leach
alkali into the media.

50. QC OF CULTURE MEDIA

51. MICROBIOLOGICAL QC OF MEDIA
• The control organism is inoculated in soyabean casein digest (SCD) broth
• Incubated for 4 hours to get a cell density comparable to 0.5 McFarland’s
standards.
• Standard suspension should give a colony count of 107- 108 cfu/mL (0.08-
0.1 absorbance at 625nm).
• A 10 μL quantity of inoculum of 1in 10 and 1 in 100 dilution in normal saline
or in SCD broth should be used for selective and nonselective

52. MICROBIOLOGICAL QC OF MEDIA
• These diluted inocula are used to ascertain the growth
supporting capacity of the media.
• The inoculation is done in duplicates for each type of
inoculum.
• After inoculation, the plates are incubated at 37°C for 24
hours
• Their growth and colony characteristics are observed.
• The results can be reported by mentioning presence or
absence of growth and the growth characteristics in a
tabular form as shown in Table 1.

53. COST EFFECTIVENESS
RULE OF THREE:
As a guideline Schreckenberger and Miller have
suggested the “rule of three”
1 or 2 potential pathogens should be evaluated even in
the presence of commensal flora BUT
3 or more pathogens should not be evaluated

54. POST ANALYTIC
POST
ANALYTIC
PHASE
Reporting
results
Interaction with
epidemiologist
Analysis
Maintenance
of sample &
records

55. 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 and interim 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

56. 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.

57. 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.

58. 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

59. MAINTENANCE OF SAMPLES & RECORDS
• Non fastidious aerobic bacteria can be saved upto 1 year on TSA slants
• 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

60. 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
• Fungi can also be maintained as water cultures at room temperature.
• AFB may be kept on LJ agar slants at 4 deg for 1 year.
• Or frozen at -70 deg in 7H9 broth with glycerol

61. 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
• Identify problems associated with poor work performance.
• 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

62. STANDARD OPERATING PROCEDURE(SOP)
• Infrastructure of a laboratory
• Biosafety precautions
• Disposal of infectious waste
Collection, transport and storage of
specimens
Reagent preparation
• Criteria of rejection of samples
• Processing of specimens
• Maintenance of equipment
• Recording of results
• Reporting of results
• Tolerance of limits
• Procedure of quality control
• Referral
Each laboratory should have Standard Operating Procedure Manuals (SOPMs) which should include the
following information about the

63. FACILITIES IN A LABORATORY
Each laboratory must possess space for :
Sample collection
Sample analysis
Storage of samples
Reagents, chemicals
Stationary& record etc.

64. FACILITIES IN THE LABORATORY
• Washing
• Media preparation
• Autoclaving
• Seminar room
• Library
• Staff room and
• Toilets

65. 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.

66. 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.

67. STAFF QUALIFICATIONS
• SUPERVISORY PANEL:
Small and medium laboratory
May be manned by an MBBS or an MSc in
concerned speciality with at least 5 years
experience in laboratory medicine.
Large and superspeciality laboratory:
Shall be manned by a medical person with
postgraduate qualification in Microbiology
in their respective division.

68. STAFF QUALIFICATIONS
• TECHNICAL PERSONAL
• 1 technologist per 500-1000 specimens per year.
• Should have one of the following qualifications:
• a) Graduate in medical laboratory technology.
• b) Science graduate with one-year experience in a medium-sized laboratory.
• c) Diploma in medical laboratory technology with 2 years experience in a medium-
sized laboratory.
• d) A laboratory may appoint up to 25% of staff without experience but with requisite
qualifications or with more than 10 years of laboratory experience with at least
matriculation in science.