Quality control in

Clinical microbiology

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 Summary of Content

• Summary
• Content:
• IQC
• EQC
 Overview of Quality Control

• Quality control is the operating procedure

system established by clinical microbiology
laboratory in order to ensure that the test
results reflect the objective existence factually.
 Overview of Quality Control
• Including :
• Internal quality control (IQC)
Core and Foundation of Quality Assurance
• external quality control (EQC)
A quality assessment of laboratories conducted by
organizations or institutions outside the laboratory.
 IQC in clinical microbiology
• Microbiology technician and Their Training and Education

• Basic Requirements for Microbial Testing Process

• Quality Control of Instruments and Equipment

• Quality Control of Culture Medium

• Reagent quality control

• Quality Control of Antimicrobial Drugs

• Rectification and control

• Quality Control of Antimicrobial

 Microbiology technician and Their Training and
Education
(1) Training staff in clinical bacteriological laboratory
to establish the concept of quality control
• What is the quality in bacterial testing: The results
really show the pathogenic bacteria and their
biochemical characteristics and drug resistance in
specimens.
• What is quality control in bacterial testing: to
establish the quality level of bacterial testing and
to adopt monitoring means, this process is called
quality control.
Microbiology technician and Their Training and
Education
• Quality Assurance: Measures to Achieve the
Due Quality Level

• Quality Assessment: The process of assessing

whether a test in a laboratory or laboratory
meets the criteria by an objective and
recognized standard.
 Microbiology technician and Their Training and Education

• 2) Establishment of operation manual

• Rules and regulations

• Inspection items
• Minimum Requirements for Bacterial Identification and
Antimicrobial Sensitivity Tests
• Medium and its preparation
• Preparation of reagents and dyes
• Biosafety protection
• Reference Data and Books
• IQC and EQC
 Basic Requirements for Microbial Testing Process

• Collection and transportation of specimens

• After the laboratory receives the clinical
specimens, the acceptance of the specimens
should be done well, and the rejection criteria of
the specimens should be strictly enforced (e.g.
direct smears, clinical specimens except blood
specimens, especially sputum specimens).
• Reasonable selection of culture medium and
timely inoculation according to different
cultivation purposes
Characters of common sputum specimens
 Laboratory judgment of sputum smear quality
Class WBC Squamous cells
6 <25 <25
Unit 5 >25 <10
Individual/Low 4 >25 10~25
power lens
3 >25 >25
2 10~25 >25
1 <10 >25
• 1-3类标本不做培养，需要重新采集标本
• Class 1-3 do not need to be cultured, requiring to reserve specimens
• 4、5类为合格标本
• Class 4 and 5 are qualified specimens
• 6类为气管穿刺液时，如未见白细胞，而鳞状上皮细胞>10个/低倍镜，亦应重新留取
• If no white blood cells were found in the 6 class of tracheal puncture fluid, and squamous cells >
10/low power microscopy, they should be re-retained.
Qualified sputum from clinical samples
 Selection of culture medium
Disk Scope of Application
Columbia blood agar Suitable for growth of various aerobic and
facultative anaerobic bacteria
Chocolate agar mainly used for the isolation of Haemophilus spp and
enrichment culture of Neisseria spp
Anaerobic blood agar Mainly used for anaerobic bacteria culture

Sabouraud Dextrose agar Primary medium for isolation of Fungi

MacConkey Agar Moderate selectively medium for isolation of Gram’s Negative

bacteria
BCYE(buffered charcoal Optimal growth for Legionella spp
yeast extract)
Roche medium Optimal growth for Mycobacterium
 Basic Requirements for Microbial Testing Process

• The positive culture should be isolated and purified,

and then be subdivided into groups, species
identification and drug susceptibility test.
• Faithfully record the phenomena and problems
found in the process of inspection, so as to facilitate
systematic analysis of experimental results, draw
correct conclusions and issue credible reports.
• For difficult strains, we should consult the literature,
organize consultation, and not make diagnosis easily.
Identification and AST report of rare bacteria
Identification Suggestions：
Resistance information
MIC
Possible therapeutic plan

Explanation of ID result：
epidemiology，
Common site of infection
Susceptible population
 Quality Control of Instruments and
Equipment

• With the continuous development of laboratory

medicine, clinical microbiological testing has
entered the era of micromation and automation,
which makes the cultivation, identification and
drug sensitivity of microorganisms more rapid and
accurate. The quality control of these instruments
can be done according to the method
recommended by the manufacturer to ensure the
accuracy and precision of the test system.
Quality Control of Instruments and Equipment
Name Allowed scope Monitoring methods and frequencies

optical microscope —————— Clean and debug 4 times a year or as needed

Constant temperature 35 ± 1 ℃ Observe and record temperature every day
incubator
CO2 incubator 35 ± 1 ℃；5% CO2 Observe and record temperature, [CO2] every
day
Refrigerator refrigerate 2-8℃ Observe and record temperature every day
Freeze -15- -25℃
Biosafety cabinet —————— Regular annual check, filter calibration once
every 3 months, after qualified use
Pressure Steam Sterilizer —————— Sterilization efficacy was tested weekly with
Bacillus thermophilus or chemically every time

Automated blood Regular Calibration and Annual Inspection

culture system
ID/AST system Weekly QC, Regular Calibration,
Annual Inspection
 Quality Control of Culture Medium

• 1) General traits

• The liquid medium is transparent and clear, and its color meets the
requirements.
• The surface of solid medium is wet but without water vapor,
smooth and uniform, and its thickness is generally 3 cm (the
thickness of M-H plate is 4 cm).
• The slope length of the inclined medium shall not exceed 2/3 of
the inclined medium, and the vertical section of the basal layer
shall be at least 1 cm.
• The PH value should be within the range of the specified value
(+0.2)
 Quality Control of Culture Medium

• (2) Sterile test

• Each batch of medium should be sampled after high pressure
or filtration to verify that there is no bacterial contamination.
 Quality Control of Culture Medium

• Generally, 5%~10% of the sterilized medium was randomly

selected and incubated in incubator at 35 C for 24 hours, which
proved that the aseptic growth was qualified.

• The medium for sterile procedure subpackage should be

incubated at 35 C for 24 hours, and the sterile growth is qualified.

• Selective medium should be diluted with 10-20 times non-

inhibitory broth medium and incubated in incubator at 35 C for 24
hours, which proves that aseptic growth is qualified
 Quality Control of Culture Medium

• (3) Growth inhibition test

• When the non-selective medium is prepared, standard
quality control strains are required to be inoculated by
sampling and grow well after routine culture.
• Selective medium should select at least one standard
strain which can grow and one inhibited strain for
inoculation and culture. The growth bacteria should
grow well and the inhibited strain should not grow or
grow partially
 Quality Control of Culture Medium

• (4) complete set of quality-controlled strains should be kept in the

Evolution Laboratory to observe the evolution characteristics of
various properties of the medium (e.g. Enterobacteriaceae,
microbiochemical identification tubes of non-fermenting bacteria). At
least one positive and one negative strain should be selected in the
biochemical reaction medium to confirm the proper biochemical
reaction.
 Reagent quality control

• (1) In the process of dye quality control,

• quality control strains should be used to control both
negative and positive quality. The most common dyes used
in clinical microbiology lab are Gram stain and acid-fast
dyes.
• The self-made dye solution should be prepared in strict
accordance with SOP regulations. It is required that the
operation steps of the whole preparation process be
recorded and preserved. The name of the dye solution, the
date of preparation, the maker and the expiry date should
be indicated on the bottle label
 Different stain for different pathogens

Staining Method Scope of application Results interpretation

Gram stain Bacteria Red G-/ Purple G+
India ink stain Cryptococcus neoformans White
Acid-fast stain Mycobacterium & Nocardia Red
GMS Fungi Dark brown
Wright's staining Bacteria & Fungi Blue or purple

GMS: Gomori’s methenamine silver stain

 Reagent quality control

• Commercial dyeing solution shall be specified

by the manufacturer, name of reagent and
storage condition of batch number, and the
manufacturer shall be required to provide
quality assurance certificate.
• Dyes that exceed their shelf life or have
become turbid, precipitated or discolored
should be disposed of and discarded in time.
 Reagent quality control

• 2) Test paper and reagent

• Whether purchased or self-made, be sure to indicate
the opening time and expiration date when using it.
• The test paper or reagent for the determination of
metabolites should be tested with known positive
and negative quality control strains, and the test
records should be made to prevent bacterial
contamination.
 Reagent quality control

Quality Identification of Common Biochemical Reagents

Reagent name Positive control strain Negative control strain Frequency of QC

3% H2O2 S.aureus S.pyogenes Daily
Coagulated plasma S.aureus S. epidermidis Daily
Oxidase P. aeruginosa E.coli Daily
Bacitracin S.pyogenes S. agalactiae Batch；weekly
Optochin slips S. pneumoniae —————— Batch；weekly
Vp reagent E. aerogenes E.coli Batch；weekly
 Reagent quality control

• (3) Quality control of antiserum

• Sources must be reliable and should be

purchased from qualified units.
• Use and Preservation of Antiserum
• The titer of antiserum must be controlled
by positive reaction quality control strains.
Only qualified strains can be used
 Quality Control of Antimicrobial Drugs

• The main purpose of quality control of Antimicrobial

sensitivity test is to monitor the precision and
accuracy of test steps, the quality of reagents used
in test and the proficiency of staff in test operation
and result interpretation
 Quality Control of Antimicrobial Drugs

• In order to achieve these objectives, the laboratory usually

uses standard quality control strains with stable genetic
performance to monitor the quality of culture medium, the
titer of antimicrobial drugs and paper, and the test operation
of staff in the course of drug sensitivity test, and find out the
factors affecting the results of drug sensitivity in time
 Quality Control of Antimicrobial Drugs

• 1. Standard quality control strains for Antimicrobial

susceptibility test

• CLSI has selected and recommended some strains as

quality control standard strains from ATCC.
 Quality Control of Antimicrobial Drugs
Classification and Application of Common Standard Strains
Name ATCC code Functions
S.aureus ATCC 25923 QC of Disk-fusion
S.aureus ATCC 29213 QC of MIC
E.coli ATCC 25922 QC of MIC and Disk-fusion

P. aeruginosa ATCC 27853 QC of MIC and Disk-fusion

E. faecalis ATCC 29212 Thymine determination

N. gonorrhoeae ATCC 49226 AST of Haemophilus

K. pneumoniae ATCC 700603 ESBL (+)
K. pneumoniae ATCC BAA1705 KPC(+)
K. pneumoniae ATCC BAA2146 NDM(+)
 Quality Control of Antimicrobial Drugs

• Refrigerated once a month to recover, planted in soybean casein

digestive broth (anaerobic bacteria can use GAM broth, etc.) as a
working strain. The working plants should be preserved at 4-8 C and
transplanted once a week. Usually, the working plant must be
abandoned after 4 to 5 times of transplantation. In the process of
quality control, if the result of the working plant is questionable, it
should be replaced.
 Quality Control of Antimicrobial Drugs

• 2. Antimicrobial Sensitivity Test Method and

Influencing Factors

• Introduction to Methodology
• Disk-Diffusion Method,
• Broth and Agar Dilution
• E-test
• Instrument
 Quality Control of Antimicrobial Drugs

• influence factor
• Normality of operation steps
• Quality of Operators
• Contamination or variation of quality control strains
• Non-uniformity or deterioration of 0.5 McDonald standard tubes
• Concentration of bacterial suspension
• Quality Problems of Culture Medium, Drug-sensitive Paper and
Drug-sensitive Slab for Test
• Incorrect incubation time, temperature or gas
• Misinterpretation of results
• Writing errors in data entry
 Quality Control of Antimicrobial Drugs

• Quality control method

• Establishment of Drug Sensitivity Quality Control System
• Of the 30 data for each drug/bacterium combination, three or less
data are allowed to exceed the prescribed range.
• To meet the above data, we can change the quality control to once a
week.
• If there is a data beyond the allowable range during the weekly
quality control, find out the reason.
• The 5-day data show that the reasons are found
• Five outcomes, once out of control, should be restored to daily quality
control
• There should be another 30 days of continuous quality control data
before weekly quality control can be restored
 ATCC strains used for QC in TDR-300B

Card type Code Strain

One-64 ATCC 25922 E.coli
NF-64 ATCC 27854 P. aeruginosa
STR-64 ATCC 29212 E.faecalis
NH-64 ATCC 49217 H. influenzae
STAPH-64 ATCC 29213 S. aureus
Yeast-64 ATCC 29019 C. parapsilosis
 Group Stains ATCC* Resin Resin Resin

ATCC strains used

aerobic Anaerobic Peds

Clostridium histolyticum 19401 X N/A X

for QC in TDR blood Clostridium perfringens 13123

25825
X

X
N/A

N/A
X

cultures
Bacteroides fragilis

Anaerobic bacteria Bacteroides vulgaris 8482 X N/A X

Streptococcus 6305 X N/A X

pneumoniae

Escherichia coli 25922 X N/A X

25923 X N/A X
X: indicated that the strain was
Staphylococcus aureus

13090 N/A X N/A

not used for performance testing
Neisseria meningitidis

19418 N/A X N/A

in the flask, but did not mean that Haemophilus influenzae
Aerobic bacteria

6305 N/A X N/A

it could not grow in the flask Streptococcus

pneumoniae

N/A indicated that the strain was streptococcus pyogenes 19615 N/A X N/A

used for performance testing in Pseudomonas aeruginosa 27853 N/A X N/A

the culture flask Staphylococcus aureus 25923 N/A X N/A

Escherichia coli 259220 N/A X N/A

Alkalinobacter faecalis 8750 N/A X N/A

Yeast Candida albicans 18804 N/A X N/A

 Common problems

• Identification • AST

• cotton swab
• Strains
• Options
• ···········

• Contaminated by Grams-
negative bacteria
 Quality Control of Antimicrobial Drugs
• 3. Quality control methods (continued)

• Quality control test should be done when using new

batches of drug-sensitive slips, M-H medium and
drug-sensitive kit.
• Quality control should be done when participating
in the competency comparison test.
• Quality control should be carried out in testing
• Once the drug susceptibility test data are found out
of control, no report should be issued to the public
 Correct control
• Identification is carried out from the following
aspects (operating procedures)
• Collection and transportation of specimens
• Selection of medium for culture and
identification
• Identification process (including smear staining)
• Culture conditions
• Points for Attention in Instrumental Method
 Correct control
• Antimicrobial sensitivity tests are carried out in the
following aspects:
• Quality control strain
• Preparation of Culture Medium (Formula,
Thickness)
• Selection of antibiotics (duration, dose)
• The process of inoculation and sticking
• Culture conditions
• Interpretation
 Correct control
• Instrument

• Maintenance and Maintenance of Hardware Equipment

• Update and Upgrade of Software Program

 EQC in clinical microbiology
• Originated in the United States in 1966, the
National Bacteriological Quality Control Work
was formally organized by the Medical
Inspection Center of the Ministry of Health in
1985. The quality control work has made great
progress in the accuracy and standardization
of identification and drug susceptibility test in
clinical bacteriological laboratories in China,
and has gradually been in line with
international standards.
 EQC in clinical microbiology
• Finding System Errors Promotes and Improves the
Theoretical Level and Technical Level of the
Controlled Laboratory
• External quality control is an evaluation of internal
quality control
• Promoting communication among clinical
bacterial rooms
• It is conducive to the standardization of operation
procedures and methods and the unification and
commercialization of diagnostic reagents.
 EQC in clinical microbiology
• Requirements of the Ministry of Health Industry
Standards for the External Quality Assessment of Clinical
Bacteria:

• Quality control substance (>50%) is mixed sample

• Operators are required to identify pathogens to be
reported based on demographic data
• The specimens contain a wide range of bacteria,
including normal flora, conditioned pathogens and
pathogens
Thank you