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1
Introduction to Microbiology Laboratory Practice
Retno Kadarsih S
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Clinical problems
Microbiologic Examination
Request
Specimen handling
Microbiological
diagnostic tests
Clinical Interpretation of microbiology tests
Report to clinicians
Solutions
Working diagnosis
Microbiology test’s results
Case management
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Introduction
Respiratory tract diseases and pathogens
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How to build provicional diagnosis ?
Clinical symptoms: anamnesis physical examination
Radiologic Examinations Laboratory Examinations
MicrobiologyCulturable m.oNon culturable m.o
Non microbiology
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Microbiology Examination
Microscopic morphology stainingMacroscopic colony charactersBiochemical-based tests Immunological-based testsMolecular-based tests
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Specimens
Upper Respiratory Tract Infections Pharyngitis :
Group A β hemolytic streptococci Acute cases Specimen : Posterion pharynx Culture and direct antigen detection tests
Bordetella pertussis : fragile m.o immediate culture Specimen : posterior nasopharynx mucus
Corynebacterium diptheriae : swab of posterior nares and posterior pharynx
Viral : Nasal Swab/washings viral transport medium
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Laryngitis Primary caused by viruses Specimen : swab/nasal washing with viral transport
medium Epiglottitis
Culture not indicated Touching the inflamed epiglottis may precipitate complete
obstruction of the airway Specimen of choice : blood culture
Sinusitis Needle aspirate of sinuses after decontamination of the
nasal cavity No specimen other than an aspirate is recommended
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Lower Respiratory Tract Infections Pneumonia
Specimen : Sputum Aspirate/lung biopsy Bronchial lavage Transtracheal aspirate Blood
In bacterial pneumonia sputum may not be the specimen of choice for determining etiologic agent
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Specimen handling
Type : swab, sputum or blood ? How much : adult VS pediatric ? How many : 1 or more ? When : morning VS spot ? Colleting : proper specimen ? Labeling : critical item to be added ? Transportation : time needed, packaging? Storage before handling in laboratory :
RT vs. COOL vs. immediate handling ?
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Throat s w ab • Using a tongue blade
to hold the tongue down, look at the back of the throat and the tonsillar area for localized areas of inflammation and exudates (theses areas are the most productive for producing cultures of the etiologic agents of acute pharyngitis)
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Label the swab container with patient identification data, including the time of collection
Transport the swab to the laboratory as soon as possible (if transport is to be delayed beyond 1 hour, refrigerate the swab)
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SPUTUM – collection
Good Container Sterile (except for tuberculosis)
Clean Widemouth Clear Tight screwcap Leak proof
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SPUTUM – collection
Explain the patient how to collect sputum properly
Patient should be given time to produce bronchial sputum from deep in the chest
Never collect sputum specimens inside the clinic or laboratory or in a closed circulation room
Collect the sputum outdoor and far from other people
Put the container to the lower lip, collect the sputum and close the container immediately
(please read your practice guide book)
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Sputum quality
MucoidMucoid SalivaSalivaPurulentPurulent Bloody SputumBloody Sputum
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SPUTUM – Labeling Container labeling Patient’s data : name, age, sex
Time and date of specimen collection, Specimen No, Physician’s name
A request form must accompany each specimen.
The information on the form must exactly match the information on the container.
Always label the container on the side, never on the lid.
Using an indelible marking pen, write the name of the TB suspect, the date of collection and health institution
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SPUTUM – Labeling Request Form Patient’s Data :
name, age, sex, addressPhysician : name, address, hospital, phone no, Specimen:Special procedureDate and time of specimen collectionAntibiotic administrationsWorking DiagnosisType of laboratory examinationOther relevan data post operation, imunodefisiency statusSpecial case : HIV, urgent case, etc
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SPUTUM – Delayed transport
Send clinical specimen as soon as possible to microbiology laboratoy
For delayed transport ( > 2 hours), store in 4°C
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SPUTUM – additional note
For bacterial examination other mycobactera and mycology ,single specimen is enough
For fungi 3 morning sputum should be performed.
For Mycobacteria spot-morning-spot sputum
Anaerobic bacteria examination in sputum is uncommon
Direct detection from sputum for molecular examination can be performed in some referral microbiology laboratory
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Making a smear slide for staining …
Always use new slides especially when preparing AFB smears. Never reuse sputum smear slides for TB work!!!
They should be cleaned with alcohol and wiped dry…or passed briskly through a flame. (This will remove any residue of oil that could interfere with staining.)
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Properly identify each slide with patient’s name, date and specimen number
Use a lead pencil to write these numbers on the frosted end of the slide
Make a 3x2cm2 oval zone in the middle of the slide
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For AFB staining : Wet slides can create aerosols if disturbed. Place them in a protected area where they can air dry for 15-30 minutes. Avoid direct sun drying DO NOT flame them to induce drying. This can also produce aerosols.
For other staining : Flaming slides direclty is allowed
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Proper thickness of the smear
Newsprint can usually be read behind a dried smearwhen it is the proper thickness
If it is too thick, the smear may wash off the slide during the staining procedure and also may yield false negative results
If it is too thin, the specimen may yield false negative results.
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Fixing the smear by heating
When dried, fix the s lides us ing the blue flame of a B uns en burner.
With forc eps , pas s the s lide briefly throug h the flame 3 times , s mear s ide up
Heat fixation ensures that the sputum will stick to the glass slide
E xc es s ive heating c ould damag e bac teria l s truc ture
U nder heated fixing w ill make ins uffic iently fixed and parts of the materia l w ill be w as hed off during s ta ining
Over and under heated fix ing w ill make fa ls e neg a tive res ults
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Gram staining
To examine sputum microorganism microscopically
To evaluate a good specimen ( SPUTUM and NOT SALIVA)
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1. PM N > 25 (40X 10 mic ros c opic field)
1. N o epithelia l c ells
1. S ing le m ic roorg anis m found
Sputum Gram Stain Good Quality
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In 40 x 10 m ic ros c opic field :
1. E pithelia l c ells > 25
2. Polymorphonuc lear (PM N ) < 10
3. Polymic robia l found (B artlett, 1987)
4. WH O : PM N < 10 per 1 epithelia l
c ell
Sputum contamination with mouth flora
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Sputum Gram Stain Unacceptable
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Ziehl Neelsen Staining Staining reagents, Clock/Timer Forceps Cotton/alcohol holder
or bunsen burner Tray containing fixed
slides to be stained. A slide rack should be
placed in the sink.
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Standard reagents (according to WHO and IUATLD)
0.3% Carbol fuchsin
3% Acid alcohol/HCl Alcolhol (decolorizing solution)
0.3% Methylene blue (counterstain)
All reagents must have expiration dates. Discard all expired dates
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.
Using forceps, place slides on a staining rack with the smear side up.
Place all slides in uniform orientation.
Slides must not touch each
other. This avoids the possibility of stain running over and causing cross-contamination.
Include a + and - control slide daily for quality control purposes.
Never stain more than 12 slides at a time
33Filter carbol fuchsin prior to use.
If precipitate is still detected, discard the stain.
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Cover the entire surface of each slide with carbol fuchsin.
Using a Bunsen burner or cotton and alcohol flame, gently heat the slides until vapor rises.
Do not allow them to boil or dry.
Boiling will alter the shape of the TB bacilli and could result in a false negative reading.
Allow the stain to remain on the slides for 5 minutes.
Maintain heat throughout this period.
Adequate time is required for the carbol fuchsin to penetrate and stain the cell wall.
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Rinse slides again carefully with water and tilt each slide to remove excess water.
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Cover each slide with decolorizing solution such as acid alcohol.
Leave this on the slides until no remaining carbol fuchsin solution out of the slides for 2 seconds (or 3 minutes maximum)
If under-decolorized, sputum contents other than the TB bacilli may remain stained. This could lead to a false positive result.
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Rinse slides again carefully with water and tilt each slide to remove excess water.
If the slide is still pink, an additional amount of decolorizing solution can be reapplied for 1 to 3 minutes.
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counter-stain with methylene blue for 1-2 minutes
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Rinse slides again carefully with water and tilt each slide to remove excess water
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Finally, tilt each slide and place in a slide block to air dry
DO NOT blot After smears are stained, clean
off the back of each slide if necessary with some alcohol on a paper towel
Do not examine slides until they are thoroughly dried.
Cover the stained slides to protect them from sunlight which can fade acid-fast bacilli.
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Always wash hands with soap and water after handling specimens and containers.
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Good stained smear
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Bad Smears
Too thick
Large/uneven
Large/unevenpoor destained
Bad smearing
Too small
Small/Slough-off
Slough-off
Too thin
Uneven/Poor decolorized
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Before examining the next slide, wipe the immersion lens with lens paper. This will protect from carry over of organisms that could create a false positive reading.
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Use the 40X objective to focus and determine a suitable reading area of the slide.
Place a drop of immersion oil
on the stained smear. Let the drop fall freely onto the slide.
Never touch the slide with the oil applicator. This could lead to carry over of AFB in the immersion oil to the next slide.
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Turn the nosepiece to bring the 100X objective into place.
Now, gently lower the 100X objective. It should barely touch the oil. Never allow the lens
to touch the slide. This can damage the lens and possibly break the slide.
While looking through the eye-piece, adjust the immersion
lens slowly and focus until the image on the smear appears.
To fine focus, turn the fine adjustment knob carefully.
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Polymorphonuclear cell
Epithelial cell
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Ensure to have a good specimen : Sputum not saliva PMN >25 and Epithelial cell < 10 per 100 fields
Acid-fast bacilli (AFB ) typically appear as slender, rod-shaped bacilli but may appear curved or bent and retain the first stain color (red)
P olimorphonuclear cells and other bacterias appear as counterstain color (blue)
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At least 100 microscopic fields should be examined before reporting as a negative smear. Examining fewer than 100 fields may cause a false negative report. Fewer than 100 fields can be read if the slide is positive for AFB.
Read systematically to avoid overlap, moving the slide lengthwise so that the next field to the right can then be examined.
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The WHO and IUATLD recommended method for reporting results The WHO and IUATLD recommended method for reporting results is as follows:is as follows: Negative: Report: “Negative for acid-fast bacilli” where no organisms have been observed in 100 fields. Positive: Report: “Positive for acid-fast bacilli.” Provide AFB quantization. The number of AFB found is an indication of the degree of infectivity as well as severity of disease. Results must be quantitated.
Reporting
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Recording & Reporting by WHO/IUATLD recommended method
Acid Fast Bacilli (AFB) appear red or pink colored rod and the background is stained in blue colour
More than 10 AFB per field in 20 fields ( +++ )
1 – 10 AFB per field in 50 fields ( ++ )
10 – 99 AFB per 100 fields ( + )
1 – 9 AFB per 100 fields Exact number
Request repeat specimen
No AFB found in at least 100 fields ( - )
(Never report as M. tuberculosis by smear result only)
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After completing the reading, remove the oil from the slide with organic solvent.
When dried, place the slides carefully in a slide storage container for transfer to a referral laboratory for quality
control. This must be done on a regular basis as determined by the National Tuberculosis Programme.
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Handle specimens carefully! All sputum specimens are
considered potentially infectious.
Save all specimens until the smears have been examined and recorded; then sterilize and discard them.
Disposable containers must be used only once
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False Positive
Errors in specimen handling or recording information Re-use of containers or positive slides Unfiltered fuchsin Contaminated immersion oil Inadequate decolorization
If a False positive result is reported, a patient will be placed on treatment unnecessarily.
If it is a Follow-up, treatment is lengthened. Valuable medication is wasted and, sadly It can cause emotional trauma to patients and their families. Patients may lose confidence in the program itself.
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False Negative
Errors in specimen handling or recording information
Poor quality sputum Excessive decolorization Reading less than 100 microscopic fields
A false negative can result a catastrophic set of events A patient with TB is not treated If the sample was a follow-up specimen, the intensive phase of therapy would not be extended causing inadequate treatment. This can lead to additional suffering, spread of TB to family and community, and even end in patient death.
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Other respiratory tract disease’s pathogens
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Classification aerobic Streptococcus species
based on haemolytic activity
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Classification aerobic Streptococcus species based on antigenic activity :
Lancefield grouping of beta hemolytic streptococci
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Identification and classification of aerobic Staphylococcus species
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Identification and classification of Haemophillus species
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Identification of Klebsiella pneumoniae
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Identification of Corynebacterium diphteriae
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Identification of Pseudomonas aeruginosa
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Identification and classification of Mycobacteria
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Identification of yeats and molds
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Drug Susceptibility Testing for M. tuberculosis
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Drug Susceptibility Testing
DSCP (drug susceptibility culture plate)
Proportion method (Lowenstein Jensen/solid media
Proportion method (MGIT/liquid media)
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Molecular-based detection system of Mycobacteria
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PRINCIPLE
1. dNTP2. PCR buffer3. Taq Pol.4. Primers
DNA Thermal-cyclerDenaturation stepAnnealing stepExtension step
1 cycle
2n
(n = number of cycle)
rDNA
DNAPhysicalrupture tech.
DNAextraction
Bacterial cell
MTB PCR
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PCR-RFLP Analysis to detect species of Mycobacteria
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Reverse Line probe Hybridization
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Unique Spacer 1Direct Repeat
PCR primers
Example of possible PCR products
PCR products hybridized to a prefabricated membrane with the sequence of each unique spacer covalently bonded to it in columns.
2 4Unique Spacer: 10 11 13 141 3 9 125 76 8
Isolate #1Isolate #2etc
etc
Unique Spacer 2 Unique Spacer 3 Unique Spacer 4
Excess PCR product removed, autoradiography film exposed and developed.
Spoligotyping
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Spoligotyping
Spacer
DR locus
BCG
H37Rv
BCG
H37Rv
X
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Molecular detection of H5N1
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Specimens Throat swab Nasal swab/wash Nasopharingeal swab/aspirate Bronchoalveolar lavage
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Primer
Primer
PC NC #1 #2 #3
Neg Pos Neg
Electrophoresis analysis
PCR
Conventional PCR
Template
Conventional PCR
Positive band
None-specific bandsg
Sample PC : Positive control
NC : Negative control
Judge
(1) Principle of Conventional PCR and Real-time PCR
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Run: 07-12-05, BioRad PCT100Gel: 07-12-05
1 kb
Lad
der
054
TS
068
BS
483
TS
484
NS
485
TS
486
TS
052
TS
053
NS
476
TS
477
NS
478
P
479
TS
1 kb
Lad
der
480
NS
481
TS
482
TS
RN
A St
d 1
RN
A St
d 2
RN
A St
d 3
RN
A St
d 4
RN
A St
d 5
NTC
452 bp306 bp
452 bp306 bp
Mutiara Firdaus Herdi Setiawan In Juju Vervanya
Maya
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Q
F Q
QF
QF
1) Denature
2) Annealing
3) Extension
Primer
Probe
Fluorescence Quencher
PolymeraseHybridize
Cleavage
Real-time PCR chemistry
F
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×Mutation
ProbePrimer
Primer
Probe is cleavedIncreased fluorescence
Probe is not cleavedNone fluorescence
Negative
Positive
Real-time monitoring Real-time monitoring
False-negative
PCR cycle number
fluo
resc
ence
inte
nsity
PCR
Real-time PCR
Template
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Hasil rRT-PCR . A12-A15 pengenceran RNA 10 3, 10 4, 10 5 dan 10 6
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Summary of Laboratory Summary of Laboratory Tests for Influenza TestingTests for Influenza Testing
Test Target Specimen Senstvy Specty Time
RT-PCR RNA Swabs, Tissue
+++++ +++++ <5 hr
ViralIsolt
Virus Swabs, Tissue
+++++ +++++ 3-5 dy
RapidTest
Antigen Swabs ++ ++ <1 hr
HI Antibody Sera +++ ++++ 48 hr
MicroNeut
Antibody Sera ++++ +++++ 72 h
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Practice for today ….
1. Throat swab continued by Gram staining2. Ziehl Neelsen staining from tuberculosis
patient’s sputum3. Learn some microbiology examinations in
respiratory tract infections (display)
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