Quality Assurance in the Microbiology

Laboratory
Quality Assurance in the Microbiology Laboratory

• A component of the Quality Management System (QMS)

• A comprehensive proactive approach that ensures every aspect of
laboratory operations, from personnel training to equipment
maintenance and method validation, meets established standards for
quality.
• By implementing effective QA systems, laboratories can ensure accurate,
consistent, and reliable test results, improve patient outcomes, meet
regulatory standards, and maintain operational efficiency.
Key Elements of Quality Assurance in Microbiology
Key Elements
1. Establishing Quality Standards and Policies

2. Standard Operating Procedures (SOPs)

3. Validation of Testing Methods
4. Maintenance of Equipment
5. Personnel Training and Competency
6. Quality Control (QC) Integration a) Internal Quality Control (IQC)
b) External Quality Assessment (EQA)
7. Sample Handling and Storage
8. Error Reporting and Corrective Actions a) Identification of Problems
b)Root Cause Analysis
c) Corrective and Preventive Actions
9. Accreditation and Compliance
10. Quality improvement (part of QMS)
Biosafety vs Biosecurity

Biosafety and biosecurity measures are usually complementary but

different objectives:

• Biosafety – Protects people from dangerous pathogens.

• Biosecurity – Protects pathogens from dangerous people.
Biosafety

• The laboratory minimizes the occupational risk of exposure to

infectious agents through the use of appropriate work practice
controls in accordance with current recommendations on the
biosafety levels (BSL) for working with different organisms
Biosafety levels
• Biosafety level 1 (BSL-1) Special containment equipment is not required
• Biosafety level 2 (BSL-2) BSCs (risk assessment), other appropriate PPE
• Biosafety level 3 (BSL-3) BSCs & special engineering and design features
• Biosafety level 4 (BSL-4) BSCIII cabinet or BSCII & positive pressure suit,
special engineering and design features

Each Biosafety Level builds upon the previous BSL requirements

Most clinical microbiology laboratories and hospital-based microbiology

laboratories are BSL-2
High Risk Microbes

BSL4
Exotic organisms, Aerosol formation, Fatal,
No treatment or vaccine

BSL3
Indigenous or exotic, potentially lethal disease
through inhalation

BSL2
Pathogenic / infectious organisms — that
pose a moderate health hazard

BSL1
Organisms not associated with disease

Low Risk Microbes

 BSL1
• Controlled access
• Hand washing sink
• Sharp hazards warning policy
• Laboratory bench
• Personal protective equipment
• Autoclave

BSL2
• Controlled access
• Hand washing sink
• Sharp hazards warning policy
• Physical containment device
• Personal protective equipment
• Laboratory bench [Link]

• Autoclave
BSL3 (with risk-based enhancements)
• Airtight when disinfecting
• Self-closing, double-door access
• Controlled access
• Sharp hazards warning policy
• Hand washing sink
• Sealed penetrations
• Physical containment device
• Laboratory bench
• Autoclave

Risk-based enhancement:
• Personal shower out [Link]

• Exhaust HEPA filter

• Effluent decontamination system
• Powered air purifying respirator
• Due to the risk of laboratory accidents by highly pathogenic organisms, cultivation of
colonies from cerebrospinal fluid (CSF), blood, and lymph nodes should be performed in a
biological safety cabinet (BSC) while wearing protective gloves.
• This practice should continue until pathogens known to cause laboratory-acquired
infections (e.g., Brucella, Francisella, and Neisseria meningitidis) are ruled out.
• Any unidentified gram-negative or gram-variable rods or coccobacilli that grow only on
blood and chocolate agars, and not on MacConkey agar, must be handled with extreme
caution until highly pathogenic species are excluded.
• Gram stains and wet mounts should be prepared in the BSC, with microbiologists wearing
gloves.
• Procedures known to produce aerosols (e.g., catalase test, microdilution tray inoculation)
should be performed in a BSC with additional care.
Risk Assessment
• Identification of hazards connected to an infectious agent.
• Recognize the activities that might cause exposure.
• Review the competencies and experience of laboratory personnel.
• Assess and prioritize risks
• Implement appropriate controls to minimize risk and evaluate
periodically.
Risks are evaluated according to the likelihood of occurrence and
severity of consequences

A) Likelihood of occurrence
5. — Almost certain: expected to occur
4. — Likely: could happen sometime
3. — Moderate: could happen but not likely
2. — Unlikely: could happen but rare
1. — Rare: could happen, but probably never will
Consequences may depend on duration and frequency of exposure
and on availability of vaccine and appropriate treatment.
Following are examples of consequences for individual workers:

B) Severity of consequences:

1. — Colonization leading to a carrier state 1. — Negligible

2. — Asymptomatic infection 2. — Minor
3. — Toxicity, oncogenicity, allergenicity 3. — Moderate
4. — Infection, acute or chronic 4. — Major
5. — Illness, medical treatment
5. — Catastrophic
6. — Disease and sequelae
7. — Death
• When the risk level is determined, controls to minimize the risk
for exposure may be developed, implemented, and evaluated, like:
• Engineering controls
• Administrative and work practice controls
• Monitoring effectiveness of controls (at least annually) KPIs
Quality Control (QC)

• In a clinical microbiology laboratory, essential quality control (QC)

processes are crucial to ensure accurate, reliable, and timely results for
patient care.

• Quality Control (QC) Integration

a) Internal Quality Control (IQC), Control of Reagents and Media (Control organisms)
b) External Quality Assessment (EQA)
Quality Control Organisms
• Known strains of microorganisms used in a microbiology
laboratory to verify the accuracy, reliability, and consistency of
laboratory procedures and diagnostic tests.

• These organisms serve to monitor and maintain the integrity of

microbiological testing by ensuring that diagnostic methods,
media, and instruments are working as expected

• Obtained through ATCC or the American Type Culture

Collection (nonprofit organization) which provides authenticated
strains, or ATCC-derived cultures from commercial sources & PT
programs
Bacterial Authentication

• ATCC performs numerous quality control (QC) assays to

guarantee that the product is of the highest standard

• All bacterial strains undergo thorough phenotypic and

genotypic examinations to ensure that:
• Strain identification is accurate
• The culture is pure
• All biochemical results are consistent
Preservation of QC Strains
• Most bacterial strains can be stored through:
• Freeze-dried cultures (lyophilization)
• At temperatures below -130°C (cryopreservation).
• Refrigerated-Frozen (user)
Refrigerated-Frozen (user)
• Each manufacturer should supply a list of QC organisms
recommended for use with their Microbial identification systems
MISs, accessible from either culture collections or commercial
sources

• The user should follow the manufacturer’s recommended

specifications for QC organism selection to ensure optimal and
expected reactions.
Processing/Preparation of Stock and Working Quality Control Cultures

At least once a year, prepare a Label as stock QC culture,

Inoculate one to two slants
stock culture of each QC Incubate under conditions then the name of the
of an appropriate
organism. Use lyophilized or favorable for growth. organism and the date of
nonselective medium.
frozen organisms. inoculation.

Moulds and certain

Tightly seal and store the fastidious bacteria, such as
slant(s) for no longer than 12 Neisseria gonorrhoeae, may From the stock QC culture, Store the working QC culture
months at 2 °C to 8 °C, or require 25 °C to 35 °C prepare a second subculture at 2 °C to 8 °C for less than
as recommended by the storage, and along S. (working QC culture). four weeks.
manufacturer. pneumonia may require more
frequent subculture

Prepare a fresh working QC culture at least once

per month, or as recommended by the
manufacturer, from the refrigerated stock QC
culture.
Maintenance and Testing of Quality Control Strains

• For long-term storage, maintain stock cultures at:

1. −20°C or below (preferably at −60°C or below or in liquid nitrogen) in a

suitable stabilizer (eg, 50% fetal calf serum in broth, 10% to 15%
glycerol in tryptic soy broth, defibrinated sheep blood, or skim milk)
2. In a freeze-dried state.

• Some QC strains, with plasmid-mediated resistance, have been shown

to lose the plasmid when stored at temperatures above −60°C.
Internal Quality Control (IQC)
• The laboratory uses appropriate quality control organisms or reference
cultures to check stains, reagents and susceptibility test methods

• The laboratory tests control specimens in the same manner and by the
same personnel as patient samples

• For direct antigen tests performed on patient specimens, positive and

negative controls are tested and recorded at least daily, or more
frequently if specified in the manufacturer's instructions
ATCC Commonly Used in AST by CLSI
• The recommended bacterial strains commonly used include:
Aerobic controls ATCC
Escherichia coli ATCC 25922
Staphylococcus aureus ATCC 25923
Pseudomonas aeruginosa ATCC 27853
Enterococcus faecalis ATCC 29212
Haemophilus influenzae ATCC 49247 & ATCC 49766
Neisseria gonorrhoeae ATCC 49226
Streptococcus pneumoniae ATCC 49619
Anaerobic Bacteria ATCC
Bacteroides fragilis ATCC25285
Bacteroides thetaiotaomicron ATCC 29741
Clostridioides (formerly Clostridium) difficile ATCC700057
Eggerthella lenta (formerly Eubacterium lentum) ATCC 43055b
Smear Preparation and Stain Quality
• The quality of smear preparation and staining is satisfactory for all
microbiology stains (ie, proper smear thickness, free of precipitate,
proper cell distribution, appropriate staining reactions, etc.)
Gram stain

• The laboratory must have policies for the interpretation of the

Gram stain, including the quantification, stain reaction, and
morphotypes of organisms and cells (eg, neutrophils or squamous
epithelial cells).
• Laboratories may correlate Gram stain results with the final culture
results as a component of the quality management system.
Gram Stain QC
• Quality control of Gram stain reagents is performed for intended
reactivity for each new batch or lot, and shipment of stains and at least
weekly against known gram-positive and gram-negative quality control
organisms.
• Personnel who perform Gram stains infrequently must run a gram-
positive and gram-negative control each day of testing.
• Control testing is not required during periods when patient testing is not
performed.
AFB Stain

• Acid-fast bacillus stains are checked each day of use with

appropriate positive and negative controls.

Courtesy of MedLabs Consultancy Group

Morphologic Observation Evaluation

• The laboratory evaluates consistency of morphologic observation among

personnel performing Gram stain and other organism stains at least
annually.
• The laboratory must ensure the description of bacteria and other
organisms is reported consistently amongst all personnel performing the
microscopic analysis.
1. Circulation of a pre-graded set of organisms with defined staining characteristics.
2. Multi-headed microscopy
3. Use of photomicrographs with referee and participant identifications (eg, previous EQC)
4. Use of digital images
5. Enrollment and participation of all personnel in an external assessment program for
morphologic observation for Gram stains
Reagent QC

• Positive and negative controls are tested for each new batch, lot
number, and shipment of reagents, disks/strips and stains.
• Reagents subject to this requirement include (but are not limited
to):
• catalase, coagulase (including latex methods), oxidase and indole
reagents; bacitracin, optochin, streptococcal latex agglutination
grouping reagents, ONPG, X, V, and XV disks/strips.
Identification System QC

• Appropriate positive and negative control

organisms are tested for each new lot and
shipment of reagents used in bacterial
identification systems.

[Link]
• Antisera QC
• Positive and negative controls are tested for each new batch,
lot number and shipment of antisera when prepared or
opened and once every 6 months thereafter
(eg, Salmonella/Shigella antisera).

• Beta-Lactamase QC
• Positive and negative controls are tested for beta-lactamase
(other than Cefinase) tests on each day of use.
• Beta lactamase tests using Cefinase need be checked only
with each batch, lot number and shipment.

[Link]
o-antisera-quality-control-antigens-35/11758153
• Anaerobic Incubation Conditions QC
• The laboratory checks the adequacy of anaerobic incubation conditions
through the use of methylene blue strips, fastidious anaerobic organisms
or other appropriate methods.

• CO2 Incubator Levels

• The laboratory checks CO2 incubators daily for adequate CO2 levels.
• It is acceptable to monitor and record CO2 levels from digital readouts;
however, the laboratory must verify that the readout is accurate (by initial
calibration, Fyrite, or other calibrated CO2 meter).
• The frequency of verification of the digital readout must be defined and
should be performed, at minimum, at the frequency recommended by the [Link]
combustion/Analyseurs/Analyseur-de-gaz-Fyrite-
Classic/p/FyriteClassic?locale=en

manufacturer.
Bacteriology Susceptibility Testing Verification

• Implementing any new diagnostic test requires verification with a minimum of 20 samples.

• Each laboratory that introduces a new AST system or adds a new antimicrobial agent to an
existing AST system must verify that the system meets performance specifications before
reporting patient test results.

• Verification generally involves testing patient isolates with the new AST system and
comparing results to those obtained with an established reference method or a system that
has been previously verified and following manufacturer instructions & recommendations.

• Testing patient isolates may be done concurrently with the two systems. Alternatively,
organisms with known MICs or zone sizes may be used for the verification.
Susceptibility Testing QC

• Antimicrobial susceptibility testing of isolates must be performed using pure

isolates or colonies

• A purity check must be performed by subculturing an aliquot of the inoculum

onto a blood agar plate or other non-selective media at the same time the
inoculum is used for susceptibility testing with the following exceptions:
1. A separate purity plate is not required for disk diffusion testing from isolated
colonies, as long as the disk diffusion plate is carefully examined for a mixed culture.
2. When testing by gradient diffusion methods, the manufacturer’s instructions must be
followed.
Susceptibility Test QC Frequency

• For antimicrobial susceptibility testing by either disk, gradient diffusion strips, or

dilution (MIC) methods, quality control organisms are tested with:
1. Each new lot number
2. Shipment of antimicrobials or media before or concurrent with initial use
3. Each day the test is performed thereafter.

• The frequency of QC testing may be reduced to weekly (including the testing of new
lots or batches of antimicrobials or media) if
1. An individualized quality control plan (IQCP) is implemented
2. The laboratory has records of satisfactory performance with daily QC tests as suggested by CLSI
Standards.

• If multiple instruments are used for automated MIC testing, QC testing should be
rotated equally among all testing instruments.
Satisfactory performance criteria are
defined either by:
1. Records must show that all QC organisms are tested for 20 or 30
consecutive test days.
• For each drug/microorganism combination, no more than 1 of 20 or 3 of the 30 values may
be outside the accepted QC ranges.

2. Records must show that all QC organisms are tested in triplicate

(separate inoculum suspensions) for 5 consecutive test days.
• For each drug/microorganism combination, no more than 1 of the 15 values may be outside
the accepted QC ranges.
• If 2 or 3 values are outside the accepted QC range during testing of 15 replicates, daily QC
testing must be continued and performed in triplicate (using separate inoculum suspensions)
for another 5 consecutive test days
• Weekly quality control testing could be performed once the
satisfactory performance of daily testing has been documented.

• The results of daily quality control should be documented

• If any of weekly quality control results are out of the acceptable range,
corrective action should be done.
The “Individualized Quality Control Plan” (IQCP)
• It is no longer acceptable for your laboratory to follow CLSI AST
guidelines alone for converting from daily to weekly testing of QC
strains.

• The laboratory is required to develop an IQCP regardless of when

weekly QC of AST was implemented

• Developing an AST IQCP must take into consideration all the

activities that are in place to ensure quality AST results. Including all
steps in “Path of Workflow”

• The IQCP may demonstrate that daily QC is not necessary and less
frequent QC (e.g., weekly QC) is sufficient to ensure quality of AST
• A laboratory path of workflow consists of the sequential processes:
• Pre-examination
• Examination ordering
• Sample collection
• Sample transport
• Sample receipt
• Examination
• Examination
• Results review and follow-up
• Interpretation
• Post-examination
• Results reporting and archiving
• Sample management

[Link]
Disk Diffusion AST IQCP

• Appropriate documentation must be provided to

justify any QC testing schedule other than the CLIA-
mandated daily schedule
• For many laboratories, historical records will likely
justify your current QC testing
• If a laboratory wishes to perform daily QC, no IQCP
is required
• QC cannot be less than that recommended by the
manufacturer, and must be supported by the risk
assessment and QC data
Courtesy of MedLabs Consultancy Group/Micro Dept.
Courtesy of MedLabs Consultancy Group/Micro Dept.
Corrective Action For The Out-of-control Result:

• Immediate Corrective action:

• Test the out-of-control antimicrobial agent/organism combination
on the day the error is observed and monitor for a total of five
consecutive test days.
• If all five zone diameter measurements for the antimicrobial
agent/organism combination are within the acceptable ranges, No
additional corrective action is necessary.
• If any of the five zone diameter measurements are outside the
acceptable range, additional corrective action should be done, and
the daily control test must be continued until final resolution of the
problem is achieved.
Additional Corrective action:

• The following should be investigated and verified:

1. Correct measuring and transcribing zone diameter.
2. The turbidity standard is not expired, stored properly, and was adequately mixed well
prior to use.
3. All material used are within their expiration dates and stored at the proper
temperature.
4. The incubator was at proper temperature and atmosphere.
5. The control strain has not changed and was not contaminated.
6. Inoculum suspension were prepared and adjusted correctly
• Once the problem is corrected, documentation of satisfactory
performance for another 20or 30 consecutive days is required to
return to weekly quality control testing.

• Reporting patient results when out-of-control tests occur:

• The reporting of patient results should be made on an individual patient basis.
• The items that should be considered include suppressing the results for an individual
antimicrobial agent
• Retrospectively reviewing individual patient, or cumulative data for unusual patterns
• Using an alternative test method or a reference laboratory until the problem is resolved
Source of Disks and Quality Specifications

• Disks should be purchased from a reliable commercial vendor.

• The disks should be accompanied, at minimum, by:
• A certificate of analysis stating the content of the disks
• Lot number
• Expiration date
• Assurance that they were tested and performed according to established QC
specifications.
Cumulative Susceptibility Data

• Cumulative susceptibility data in a microbiology laboratory refers to the collection

and analysis of antimicrobial susceptibility testing (AST) results over a period of
time.

• This data provides valuable insights into trends in antimicrobial resistance (AMR)
patterns within a specific population, hospital, or community, helping to:
1. Guide treatment decisions (empiric)
2. Monitor resistance trends

• For hospital-based microbiology laboratories, cumulative antimicrobial susceptibility

test data are maintained and reported to the medical staff at least yearly.
 Steps to Create Cumulative Susceptibility Data
• Collection of AST Data: (≥30 organism-antibiotic combination)
1. Organism (e.g., E. coli, S. aureus)
2. Antimicrobial agents tested
• Interpretation (susceptible, intermediate, resistant)
• Categorize Results:
1. Group by Organism
2. Group by Antibiotic
• Calculate Cumulative Susceptibility (% )
• Create a Cumulative Report (tables or graphs)
• Interpret the Data:
1. Identify any emerging resistance patterns or changes in susceptibility over time.
2. Use the data to make informed decisions about antimicrobial use, surveillance, and infection control.
3. Contribute to larger-scale antimicrobial stewardship efforts
External Quality Assessment (EQA)

• The laboratory participates in the appropriate required proficiency

testing (PT)/external quality assessment (EQA) program for the patient
testing performed.
• The laboratory integrates all proficiency testing (PT) and alternative
performance assessment specimens within the routine laboratory
workload,
• PT specimens are analyzed by personnel who routinely test patient
specimens
• If unable to participate for a given test, the laboratory must implement
an alternative performance assessment procedure for the affected analytes
at least semiannually

• Appropriate alternative performance assessment processes include:

• Participation in an ungraded/educational PT program
• Split sample analysis with another laboratory
• Split sample analysis with an established in-house method
• Use of assayed materials

• Evaluated by defined criteria for successful performance in accordance

with good clinical and scientific laboratory practice
• There is ongoing evaluation of proficiency testing (PT) and alternative performance
assessment results by the laboratory director or designee with appropriate corrective action
taken for each unacceptable result

• Each unacceptable PT or alternative performance assessment result must be evaluated in a

timely manner to determine the impact on patient test results and correct problems
identified.

• It is recommended that the laboratory investigate acceptable results that show significant
bias or trends
Instruments and Equipment Quality Control

• The laboratory verifies the performance of all instruments and

equipment prior to initial use, after major maintenance or service, and
after relocation to ensure that they run according to expectations

• Prior to clinical use of each unmodified FDA-cleared or approved test,

the laboratory has performed a verification study and prepared a written
assessment of each of the following test method performance
specifications, as applicable, using a sufficient number of characterized
samples (20 samples):
1. Analytical accuracy
2. Analytical precision
• The laboratory performs and records appropriate maintenance and function
checks for all instruments (eg, analyzers) and equipment (eg, centrifuges) following
a defined schedule, at least as frequent as specified by the manufacturer

• Operators Daily/periodic start-ups, background reading, function or calibration

checks are done, documented on the designated form, & in accordance with
manufacturer recommendations

• Temperature readings and recordings: All temperature dependent devises including

refrigerators, freezers, incubators and others must be read at least two times daily
or whenever in use and/or as per manufacturer recommendations.

• Include but not limited to, centrifuge calibration or checks, pipette calibrations,
thermometers check, and others as applicable and as per manufacturer
requirements
• Equipment scheduled maintenance/preventive maintenance
according to a scheduled time frame and as per
operators/maintenance equipment manual or at least annually.

• If the laboratory uses more than one instrument/method to test for

a given analyte, the instruments and methods are checked against
each other at least twice a year for comparability of results, and
acceptability criteria are defined
Biological Safety Cabinet
• A biological safety cabinet (BSC) or
hood is used for handling specimens or
organisms considered highly contagious
by airborne routes

• For mycobacterial, mycologic, and

virology work
• Exhaust air from a class I or class II BSC must be
filtered through high efficiency particulate air
(HEPA) filters.
• Air from Class I and IIB cabinets is hard-ducted
to the outside.
• Air from Class IIA cabinets may be recirculated
within the laboratory if the cabinet is tested and
certified at least annually.
• The biological safety cabinet (BSC) is certified at least annually to
ensure that filters are functioning properly and that airflow rates
meet specifications.
• Airflow Velocity Test
• HEPA Filter Integrity Test
• Inward Airflow Test (Sash Containment Test)
• Microbiological Test
Centrifuge Safety

• For TB testing, sealed screw-capped tubes are

enclosed in sealed safety centrifuge carriers
(ie, a double closure system) to minimize
aerosol hazards when centrifuging specimens.
Microscope Maintenance
• Microscopes are clean, adequate (eg, low,
high dry and oil immersion lenses as
appropriate for the intended use),
optically aligned, and properly
maintained with records of preventive
maintenance at least annually.

• Koehler illumination must be maintained

for optimal resolution
• Ocular Micrometer is calibrated for the
microscope and the specific objective with
which it is used.

• Calibrations must be checked against a

calibrated stage micrometer slide or other
object(s) of known dimensions appropriate to
the use of the ocular micrometer.

• Any change in the optics of the microscope

(eg, change in objective or ocular lens) requires
recalibration.

• If there are no changes to a particular

microscope's optical components, there is no
need to recheck calibration
Courtesy of MedLabs Consultancy Group/Micro Dept.
Autoclaves

• Biochemical indicators of autoclaves are tools used to validate the

sterilization effectiveness of an autoclave by confirming that it achieves
conditions sufficient to destroy microorganisms.

• Biological Indicators (BI):

• Contain spores of highly resistant microorganisms like Geobacillus
stearothermophilus.
• After sterilization, the spore-containing vial is incubated. A change in the medium's
color (e.g., pH shift) or turbidity indicates microbial growth, signifying
sterilization failure.

• Chemical Indicators (Supplementary to Biological Indicators):

• Often used alongside biochemical indicators, chemical strips change color based on
heat, pressure, or time conditions. [Link]
indicator-for-steam/
Media Quality Control

• For culture media prepared by the laboratory, an appropriate sample

of each medium (1-5%) is checked before or concurrent with initial
use for each of the following:
1. Sterility
2. Ability to support the growth of organisms intended to be
isolated on the media by means of stock cultures
3. Biochemical reactivity (where appropriate)
• Media Visual Examination
1. All media are in visibly satisfactory condition prior
to use (within expiration date, plates smooth,
adequately hydrated, uncontaminated, appropriate
color and thickness
2. Tubed media not dried or loose from sides.

• pH Check of each batch of MHA

• The exact method used depends largely on the type of
equipment available in the laboratory.
• Check the pH by one of the following means:
• Macerate enough agar to submerge the tip of a pH
electrode.
• Allow a small amount of agar to solidify around the tip of Courtesy of MedLabs Consultancy Group/Micro Dept.

a pH electrode in a beaker or cup.

• Use a surface electrode.
 MH QC

• The MH agar medium should have a pH between 7.2 and 7.4 at room temperature and
must therefore be checked after solidifying.

• Check the pH after aseptic addition of the blood to the autoclaved and cooled medium.
The final pH should be the same as un-supplemented MHA

• If the pH is less than 7.2, certain drugs will appear to lose potency (eg, aminoglycosides,
macrolides), whereas other antimicrobial agents may appear to have excessive activity (eg,
tetracyclines).

• The CLSI and EUCAST specify that the depth of the agar should be maintained at 4 ±
0.5 mm. This corresponds to approximately 25 mL of agar in a standard 90 mm petri
dish.
Effects of Thymidine or Thymine:

• MHA containing excessive amounts of thymidine or thymine can reverse the

inhibitory effect of sulfonamides and trimethoprim, thus yielding smaller and
less distinct zones, or even no zone at all, which may result in false-resistance
reports.

• Use MHA that is as low in thymidine content as possible.

• To evaluate a lot of MHA, Enterococcus faecalis ATCC 29212 or, alternatively,

E. faecalis ATCC 33186, may be tested with trimethoprim-sulfamethoxazole
disks.
• Satisfactory media provide essentially clear, distinct zones of inhibition ≥ 20 mm.
• Unsatisfactory media produce no zone of inhibition, growth within the zone, or a zone of < 20 mm.
QC bacterial strains for media inspection and
biochemical testing
• When inspecting media hemolysis and
performing biochemical testing in a clinical
microbiology laboratory, it is important to
use well-characterized ATCC control strains
to ensure the accuracy and reproducibility of
the tests

Courtesy of MedLabs Consultancy Group/Micro Dept.

 Bacterial Name ATCC Number Hemolysis Biochemical Tests

Lactose fermenter (pink on MacConkey agar), Catalase

Escherichia coli ATCC 25922 None ( γ-hemolysis)
positive, Indole positive, Oxidase negative.
Catalase positive, Coagulase positive, Mannitol
Staphylococcus aureus ATCC 25923 ß-hemolysis
fermentation (yellow on MSA).
Oxidase positive, Non-fermenter, Pigment production
Pseudomonas aeruginosa ATCC 27853 None ( γ-hemolysis)
(blue-green), Catalase positive.
Catalase negative, Optochin susceptible, Bile solubility
Streptococcus pneumoniae ATCC 49619 α-hemolysis
positive.
Catalase negative, Bile esculin positive, Growth in 6.5%
Enterococcus faecalis ATCC 29212 None ( γ-hemolysis)
NaCl, PYR positive.
Lactose fermenter (pink on MacConkey agar), Catalase
Klebsiella pneumoniae ATCC 13883 None ( γ-hemolysis)
positive, Indole negative, Oxidase negative.
Oxidase positive, Requires X and V factors, Growth on
Haemophilus influenzae ATCC 49247 None ( γ-hemolysis)
chocolate agar.
Spore-forming, Catalase positive, Motile, Non-lactose
Bacillus subtilis ATCC 6633 None ( γ-hemolysis)
fermenter.
Catalase negative, Bacitracin susceptible, PYR positive,
Streptococcus pyogenes ATCC 19615 ß-hemolysis
Gram-positive cocci in chains.
Salmonella enterica serovar Lactose non-fermenter, H2S producer (on XLD agar),
ATCC 14028 None ( γ-hemolysis)
Typhimurium Oxidase negative, Catalase positive.
Key Performance Indicators (KPIs)

• Measurable values that indicate how effectively an individual, team, organization, or system is
achieving its objectives. KPIs are essential tools for tracking progress.

• In the context of a microbiology lab or any other organization, KPIs serve as benchmarks for
performance, helping to monitor progress and identify areas that need improvement.

• Including all steps in “Path of Workflow”

1. Pre-analytical
2. Analytical
3. Post-analytical
KPIs
• LIMS (Lab Information Management Systems) is used for automated
tracking.
• Excel sheets or dashboards may be used for visualization and reporting.
• Review of KPIs monthly or quarterly to identify trends and make
improvements.
• The threshold may be established through peer group benchmark in
collaboration with other relevant institutional groups (eg, infection
prevention).
• The laboratory must perform and record corrective action if the
threshold is exceeded.
KPI dashboard
KPI Current Value Target Status Trend (Last Month)

Turnaround Time (Urine) 24 ≤24 hrs On Target Improving

Contamination Rate (Blood) 3 <3% On Target Stable

Sample Rejection Rate 6 <5% Needs Attention Worsening

Blood Culture Volume 91 ≥90% On Target Improving

Equipment Downtime 5 ≤10 hrs On Target Stable

Critical Value Reporting within 30 min 90 ≥95% Below target Worsening

Customer Satisfaction 92 ≥90% On Target Improving

Disposal of Infectious Materials

• All bacterial cultures, stocks, and potentially infectious materials need to

be properly decontaminated prior to disposal.
• SOPs for proper decontamination should be available in the laboratory.
• Microbiology specimen residuals and contaminated media are
disinfected, sterilized, and disposed of in a manner to minimize
infectious hazards to personnel after completion of testing and storage
period.
• If waste material is transported before treatment, it must be placed into
a leak-resistant rigid container, and appropriately labeled.
• Proper waste disposal in a microbiology
laboratory is critical to ensure safety,
compliance with regulations, and
environmental protection
• Staff has always to wear appropriate PPE
while handling waste.
• Recommended PPE:
• Gloves
• Lab coat or gown.
• Face shield or goggles if there’s a risk of splashes.
A typical workflow for microbiology lab waste:
[Link]: At the point of generation.
[Link]: In designated bins and containers.
[Link]: Autoclaving or chemical treatment.
[Link]: Temporarily store in secured areas, following guidelines for
duration.
[Link] Disposal:
1. Biohazard waste: Incineration or burial in a regulated landfill.
2. Sharps: Incineration or autoclave and shred before disposal.
3. Chemical waste: Disposed of through licensed hazardous waste handlers.
Waste Segregation
• Proper segregation at the point of generation is essential for safe
handling and disposal.
• Key Practices:
• Use Color-Coded Bins:
• Red bags: Biohazardous waste.
• Yellow bins: Infectious waste
• Black bins: General waste.
• Separate Sharps: [Link]

• Dispose of in puncture-resistant, leak-proof sharps containers.

• Training and Awareness: Regular training ensures staff
handle waste safely and efficiently.
• Training Topics:
• Waste segregation and labeling.
• Decontamination procedures.
• Emergency protocols for spills or exposure.
• Use and maintenance of autoclaves and sharps containers.
• Emergency Preparedness: prepared for accidental spills or
exposure.
• Response Plan:
• Spill Kits: Include absorbent materials, disinfectants, and PPE.
• Incident Reporting: Document and investigate all incidents to prevent
recurrence (root cause analysis).
[Link]
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• [Link]
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