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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)