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WORK
INSTRUCTIONS

MICROBIOLOGY

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COLLECTION OF CLINICAL SPECIMENS

GENERAL GUIDELINES
 Collect specimen before antibiotic treatment
 Collect from appropriate site
 Practice proper and aseptic collection technique
 Ensure sufficient quantity
 Follow recommended time of transport
 Label specimen accordingly
 Accompany specimen with complete request form

CLINICAL SPECIMEN FOR AEROBIC CULTURE

I.BLOOD

Blood Culture is requested when

a. Acute illness
b. Fever of unknown origin
c. Acute infective endocarditis
d. Suspected bacterial endocarditis

Collection Method:
1. Find puncture site
2. Clean site using 70% alcohol, followed by an iodine solution
3. Sterilize the rubber stopper using alcohol swab
4. Collect the required amount of blood
5. Replace the needle of the syringe with a new needle
6. Inoculate collected blood into the blood culture broth (Volume required; Adult = 1:10,
Pediatric = 1:5)

Incubation Temperature: 35 to 37 degree Celcius

Incubation Period: 7 days
Subculture: 24 hours (Preliminary result)
3 days
5 days
7 days (Final Result)

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Microscopy: Gram stain (as requested)

Culture Media: BAP, CAP, MAC

II. CEREBROSPINAL FLUID (CSF)

1st tube – Chemistry ( Protein, Glucose, LDH)

2nd tube – Gram stain and Culture

3rd tube – Cell count

Volume: 0.5 – 5ml

Time of Collection: Within 1st week of illness
Subculture: 24 hours (Preliminary Result)
Incubation Period: 3 days (Final Result)
Microscopy: Gram Stain and India ink
Culture Media: BAP, CAP, MAC
Enrichment Broth: BHI

Insufficient Volume
1. Culture
2. Serological Exam
3. Microscopy
4. Cell Count
5. Other clinical pathology tests

III. EFFUSION

a. Synovial Fluid
b. Pleural Fluid
c. Peritoneal Fluid
d. Pericardial Fluid
e. Hydrocoele

Incubation Temperature: 35 to 37 degree Celcius

Incubation Period: 3 days
Subculture: 24 hours (Preliminary result)

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Microscopy: Grams stain

Culture Media: BAP, MAC, CAP
Enrichment broth: BHI

IV: RESPIRATORY TRACT

4.1 UPPER RESPIRATORY TRACT

A. NASOPHARYGEAL SWAB/ ASPIRATE

Incubation Temperature: 35 – 37 degree Celcius

Incubation Period: 24 hours
Microscopy: Gram Stain (as requested)
Culture Media: BAP
Transport Media: Amies(general), Regan-Lowe (for pertussis)
Type of sawb: Calcium alginate

B. THROAT SWAB

Incubation Temperature: 35 – 37 degree Celcius

Incubation Period: 24 hours
Microscopy: Gram stain
Culture Media: BAP
Transport Medium: Amies

4.2 SPUTUM

Transport device: Sterile, Leak- proof container

Incubation Temperature: 35 – 37 degree Celcius
Incubation Period: 48 hours
Microscopy: Gram satin
Culture Media: BAP, CAP, MAC

Suitability Criteria for Culture

Classification of Sputum in the basis of Leukocyte and Squamous Epithelial cell densisties

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Cell numbers per x 100 (LPO)

GROUP LEUKOCYTE CELLS EPITHELIAL

6 <25 <5
5 >25 <10
4 >25 10 – 25
3 >25 >25
2 10 – 25 >25
1 <10 >25

*Only Sputum samples in categories 4 – 6 should be cultured.

4.3 ENDOTRACHEAL ASPIRATE/BRONCHOALVEOLAR LAVAGE

Transport Device: Sterile, Leak-proof container

Incubation Temperature: 35 – 37 degree Celcius
Incubation Period: 48 hours
Microscopy: Gram Stain
Culture Media: BAP, MAC, CAP

V. URINE

Methods:
1. Clean catch midstream
2. Cytoscopy or Catheterization
3. Suprapubic aspiration

Transport Device: Sterile, wide mouth, Leak Prood container

Incubation Temperature: 35 – 37 degree Celcius
Incubation Period: 48 hours
Microscopy: Gram stain
Culture Media: BAP, MAC

VI. TRANSUDATES/EXUDATES

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Collection Method
General: Remove surface exudate by wiping with sterile saline or 70% alcohol
Open Wound: Aspirate or swab deep into the lesion to firmly sample the lesion’s “fresh border”
Close Wound: Aspirate

6.1 ABSCESS/WOUND

Incubation Temperature: 35 – 37 degree Celcuis

Incubation Period: 48 hours
Microscopy: Gram stain
Culture Media: BAP, MAC
Enrichment broth: Thioglycholate

2. EAR/EYE DISCHARGE

Collection Method:
1. Inner Discharge: Aspirate (if ear drum is intact) or swab
2. Outer ear discharge: Moistened swab
3. Eye Discharge: moistened swab (Conjunctiva)
Scrapings (corneal scrapings)
Aspirate (vitreous fluid)

*DO NOT USE SYRINGE FOR TRANSPORT!

Incubation Temperature: 35 – 37 degree Celcius

Incubation period: 48 hours
Microscopy: Gram stain
Culture Media: BAP, GBA, BCA, MAC
Enrichment broth: Thioglycholate

3. URETHRAL/VAGINAL/PENILE/ANORECTAL DISCHARGE

Collection Method: Swab

Incubation Temperature: 35 – 37 degree Celcius
Incubation Period: 48 hours

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Microscopy: Gram stain

Culture Media: BAP, CAP, MAC, MTM

4. TISSUE

Transport Device: Sterile, screw capped container

Incubation Temperature: 35 – 37 degree Celcius
Incubation Period: 48 hours:
Microscopy: Gram stain
Culture Media: BAP, CAP MAC
Enrichment broth: Thioglycholate

VII. STOOL

Container: Sterile, Leak-proof container

Amount: 1- 2 grams
Transport Media: Carry-Blair
*If there is a delay place it in a Selenite F at room Temperature
CCDA: Incubation for 2 days at 42 degree Celcius
Candle Jar: 3 days

TRANSPORT TIME

BY TYPE OF SPECIMEN

SPECIMEN TIME RECOMMENDED

Respiratory 1 hour
Gastrointestinal 1 hour
Blood 1 hour
CSF Immediately
Other body fluids Immediately
Urine 1 hour
Exudates/transudates 30 minutes

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BY TYPE OF BACTERIOLOGIC EXAMINATION

TYPE OF EXAM TIME RECOMMENDED

Anaerobic culture 30 minutes
Fungal culture 1 hour
Atypical bacteria culture 1 hour
TB/MOTT culture 1 hour
Leptospirosa culture 1 hour

PROCESSING OF SPECIMENS

General
1. Label the slides, plates and tubes with laboratory number, abbreviation of the name of the
patient and date.

I.WOUND DISCHARGE

2 WOUND SWABS

1ST SWAB 2ND SWAB

Roll the sides and tip of the swab onto Make a smear and perform Gram stain
the upper corner of the 1st quadrant of
BAP and MAC

Submerge the swab in the Thioglycholate

broth

Perform isolation streak technique onto

the BAP and MAC

Incubate inverted plates and

thioglycholate broth at 35- 37 degree Celcius
for 18 – 24 hours

Subculture broth with or without turbidity

when no growth at original plate(when no

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growth after subculture after 24 hours, discard)

II. THROAT SWAB (T/C TONSILLOPHARYNGITIS)

SWAB

Roll the sides and tip of the swap onto the upper corner of the 1st quadrant of BAP

Perform isolation streaking onto the BAP

Incubate inverted plate under 5 – 10% CO2 at 35 degree Celcius incubator for 18 – 24 hours

Note: If the colonies are still too small to recognize re-incubate the plate to 48 hours

III. THROAT SWAB (T/C Diphtheria)

SWAB

Roll the sides and tip of the swap onto the upper corner of the 1st quadrant of BAP and CTBA

Perform isolation streaking onto the BAP

Perform continuous streaking onto CTBA

Incubate inverted plate under 5 – 10% CO2 at 35 degree Celcius incubator for 18 – 24 hours if
no growth is observed, re-incubate the plates up to 48 hours

Prepare microscopic slides and make smear for Gram stain and LMB stain

IV. URINE

Mix thoroughly the urine specimen, if sample container is small invert 3-4 times or swirl if the
sample container is large

Get a loop of urine aseptically by submerging vertically only the loop portion (shaft not
included)

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Inoculate the BAP by continuous streaking; spread the urine downward from the first quadrant
and streaking up to the 4th quadrant without changing loop

Use another sterile calibrated loop and get another loopful of urine to inoculate MAC agar and
do the isolation streaking

Incubate the plates at 35 to 37 degree Celcius for 16 – 24 hours

IF NO GROWTH AFTER 24 HOURS OR IF THE COLONIES ARE STILL TOO SMALL TO RECOGNIZE
RE-INCUBATE THE PLATES UPTO 48 HOURS

V. BLOOD

BLOOD CULTURE BROTH

Mix the blood culture bottle 2 – 3 times

By using a forceps, get sterile cotton ball with 70% alcohol and sterilize the rubber stopper then
gently pass into the flame

With Sterile disposable syringe and needle aspirate at least 0.5 ml of blood from the bottle

Place a drop of the sample on each agar plate (BAP, CAP, and MAC)

Perform isolation streak

Incubate: BAP and CAP in 5 – 10% CO2 at 35 to 37 degree Celcius; MAC at 35 – 37 degree
Celcius, ambient air for 18 – 24 hours
Reincubate the BHIB

Subculture Blood Culture Broth on the 3rd, 5th until 7th day if there is no growth

VI. CEREBROSPINAL FLUID

Perform gross examination of CSF and record.

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About 1 ml is preferred (if large volume is submitted, centrifuge is recommended and use the
sediment) for the bacteriological investigation. If volume is less than 1 ml, no centrifugation is
required.

With sterile pasteur pipette, place 1 drop of CSF onto the first quadrant of each plate (BAP,
MAC, CAP), 3 – 5 drops in BHIB, one drop for gram stain and one drop for india ink

Prepare CSF smear for Gram stain and India Ink.

Perform isolation streak onto each agar plate.

Incubate: BAP and CAP, in 5 – 10% CO2 at 35 – 37 degree Celcius; MAC at 35 – 37 degree
Celcius, ambient air for 18 – 24 hours.

VII. SPUTUM or ENDOTRACHEAL ASPIRATE/ BRONCHOALVEOLAR LAVAGE

Get loopful of purulent part of the specimen to be tested and make an evenly thin smear on a
slide for Gram stain. Air dry (Before sterilizing the loop, dip the loop rubbing in sand alcohol jar
to the debris left on the loop.)

Use another sterile loop, get another loopful of purulent part of the specimen and inoculate
BAP, GBA, BCA and MAC with similar size and quality of sample onto the center of the first
quadrant.

Make an isolation streak onto each agar plate.

Incubate: BAP, GBA and BCA at 35 to 37 degree Celcius with CO2 5 – 10% incubator or a candle
jar for 18 – 24 hours; MAC at 35 to 37 degree Celcius in ambient air for 18 – 24 hours.

VIII. URETHRAL/VAGINAL/PENILE/ANORECTAL DISCHARGE

2 WOUND SWABS

1ST SWAB 2ND SWAB

Roll the sides and tip of the swab onto Make a smear and perform Gram stain

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the upper corner of the 1st quadrant of

BAP, CAP, MAC, MTM

Perform isolation streak technique onto

each agar plate

Incubate: BAP, CAP, MTM at 35- 37 degree

Celcius with 5 – 10% CO2 for 18 – 24 hours;
MAC at 35- 37 degree Celcius in ambient air
for 18 – 24 hours

IX. TISSUE

Get a piece of tissue form its container aseptically and roll over on top of the 1 st quadrant of
each plate. (BAP, CAP, MAC)

Make a smear for Gram stain

Then submerged he tissue in Thioglycholate Broth (If there is no other examination to be done
on the sample)

Perform isolation streak on each agar plate.

Incubate: BAP and CAP at 35- 37 degree Celcius with 5 – 10% CO2 for 18 – 24 hours; MAC at 35-
37 degree Celcius in ambient air

X. OTHER BODY FLUIDS

Perform gross examination of other body fluids and record

About 1 ml is preferred (if large volume is submitted, centrifuge is recommended and use the
sediment) for the bacteriological investigation. If volume is less than 1 ml, no centrifugation is
required

With sterile pasteur pipette, place 1 drop of body fluid onto the first quadrant of each plate
(BAP, MAC, CAP), 3 – 5 drops in BHIB, one drop for gram stain and one drop for india ink

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Prepare smear for Gram stain

Perform isolation streak onto each agar plate

Incubate: BAP and CAP, in 5 – 10% CO2 at 35 – 37 degree Celcius; MAC at 35 – 37 degree
Celcius, ambient air for 18 – 24 hours

Subculture from BHIB after 24 hours if no growth until 3 rd day (Final Result)
PREPARATION OF CULTURE MEDIA
Preparation of culture media is to make a synthetic media that contains nutrients for the isolation
of certain bacteria in the laboratory. It may be enriched, selective or differential media. If using
a dehydrated culture media, it is important to read carefully the manufacturer’s instructions: the
concentrations and how to use before the preparation.

General Materials Used in the Preparation of Culture Media:

Spatula Water bath

Erlenmeyer flasks (300 – 500ml capacity) Autoclave
Weighing balance Bunsen burner
Weighing paper Sterile serological pipette or 5 ml Syringe
Magnetic stirrer and stir bars Sterile Petri dish 100 x 15mm/ 150 x 15mm
Aluminum foil Sterile 25 ml test tube

I. BLOOD AGAR PLATE

This is the general-purpose medium for the best isolation of a wide variety of microorganism
including bacterial and fungal species.

Culture Media, Reagents and other Materials:

Trypticase Soy Agar/Columbia Agar Magnetic stirrer and stir bars

Distilled water Sterile Petri dish 100 x 15mm/ 150 x 15mm

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Sterile defibrinated Sheep/ Horse blood Sterile 25 ml test tube

Erlenmeyer flasks (300 – 500ml capacity)

Procedure:

1.1 Weigh the amount of TSA powder according to the manufacturer’s instruction and place in
an Erlenmeyer flask.
1.2 Add 250 ml distilled water and mix thoroughly
1.3 Cover with aluminum foil and label with the name of the medium and volume
1.4 Place in a boiling water bath, heat with frequent agitation and boil for 1 minute to completely
dissolve the powder.
1.5 Autoclave at 121 degree Celcius for 15 muntes.
1.6 Cool to 45 – 50 degree Celcius
1.7 Aseptically add 5-10% sterile defibrinated sheep or horse blood

Note:
1.7.1 Do not use citrated blood (citrated blood is inhibitory to some organisms)
1.7.2 Allow the blood to equilibrate at room temperature before adding o the medium.
1.7.3 Measure the blood in a sterile test tube o use a sterile serological pipette or a graduated
cylinder.

1.8 Mix by gentle swirling to avoid formation of bubbles.

1.9 Dispense or Pour about 20 ml into sterile petri dishes.

Note:

1.9.1 Depth of agar layer must be 4mm.

1.9.2 Frequent mixing or swirling at an interval of 10 plates poured
1.9.3 If bubble form, do a slight quick flame at the top of the molten agar with a Bunsen burner
1.10 Allow the medium to solidify.
1.11 Get the first and the last plate dispensed for sterility testing and another plate for culture
response test.
1.12 Store the rest of the plates at 4 degree Celcius in an inverted position of for longer shelf
life, place in a sterile tight plastic bag. (for up to 2-3 months)

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II. GENTAMICIN BLOOD AGAR

This is use as a selective medium for the isolation of Sterptococcus pneumoniae. The
incorporation of gentamicin inhibits the normal flora of the upper respiratory tract.

Culture Media, Reagents and other Materials:

Trypticase Soy Agar/Columbia Agar Magnetic stirrer and stir bars

Distilled water Sterile Petri dish 100 x 15mm/ 150 x 15mm
Sterile defibrinated Sheep/ Horse blood Sterile 25 ml test tube
Gentamicin, 80mg Water bath

Procedure:

2.1 Weigh the amount of TSA powder according to the manufacturer’s instruction and place in
an Erlenmeyer flask.
2.2 Add 250 ml distilled water and mix thoroughly
2.3 Cover with aluminum foil and label with the name of the medium and volume
2.4 Place in a boiling water bath, heat with frequent agitation and boil for 1 minute to completely
dissolve the powder.
2.5 Autoclave at 121 degree Celcius for 15 muntes.
2.6 Cool to 45 – 50 degree Celcius. Add 0.5ml of Gentamicin sulfate
2.7 Aseptically add 5-10% sterile defibrinated sheep or horse blood

Note:
2.7.1 Do not use citrated blood (citrated blood is inhibitory to some organisms)
2.7.2 Allow the blood to equilibrate at room temperature before adding o the medium.
2.7.3 Measure the blood in a sterile test tube o use a sterile serological pipette or a graduated
cylinder.

2.8 Mix by gentle swirling to avoid formation of bubbles.

2.9 Dispense or Pour about 20 ml into sterile petri dishes.

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Note:

2.9.1 Depth of agar layer must be 4mm

2.9.2 Frequent mixing or swirling at an interval of 10 plates poured
2.9.3 If bubble form, do a slight quick flame at the top of the molten agar with a Bunsen burner

2.10 Allow the medium to solidify.

2.11 Get the first and the last plate dispensed for sterility testing and another plate for culture
response test.
2.12 Store the rest of the plates at 4 degree Celcius in an inverted position of for longer shelf
life, place in a sterile tight plastic bag. (for up to 2-3 months)

III. CHOCOLATE AGAR PLATE

This medium is used in the isolation of a variety of non – fastidious and fastidious microorganisms
especially Neisseria spp., Haemophilus ssp. and Streptococcus-related spp.

Culture Media, Reagents and other Materials:

GC Agar Base Magnetic stirrer and stir bars

Hemoglobin powder Sterile Petri dish 100 x 15mm/ 150 x 15mm
Distilled water Sterile 25 ml test tube
Erlenmeyer flasks 2pcs. (300 – 500ml capacity) Isovitalex supplement or equivalent

Procedure:

3.1 Prepare Suspension A in an Erlenmeyer flask (100 ml Hemoglobin preparation)

3.1.1 Weigh 2 gms of Hemoglobin powder

3.1.2 Dissolve in 100 ml distilled water by poring a small amount of distilled water and mix by
swirling then pour the remaining water until a homogenous suspension is obtained. Or
use a magnetic mixer with a stir bar to mix. (Do not heat to dissolve)
3.1.3 Cover with aluminum foil and label.

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3.2 Prepare Suspension B in a separate Erlenmeyer flask (100 ml GC Agar Base Preparation).

3.2.1 Weigh 7.2 grams of GC Agar base and place the powder in an Erlenmeyer flask.
3.2.2 Dissolve to powder to 100 ml distilled water to a double strength base
3.2.3 Cover with aluminum foil and label.
3.2.4 Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.

3.3 Autoclave Suspensions A and B at 121 degree Celcius for 15 minutes.

3.4 Let suspensions A and B to cool to 45 – 50 degree Celcius.
3.5 Mix suspension A and B.
3.6 Aseptically add 2 ml(1%) Isovitalex enrichment to the mixture.
3.7 Mix by gentle swirling to avoid formation of bubbles.
3.8 Dispense/ Pour about 20 ml into sterile dishes.

Note:

3.8.1 Depth of agar layer must be 4mm

3.8.2 Frequent mixing or swirling at an interval of 10 plates poured
3.8.3 If bubble form, do a slight quick flame at the top of the molten agar with a Bunsen burner

3.9 Allow the medium to solidify.

3.10 Get the first and the last plate dispensed for sterility testing and another plate for culture
response test.
3.11 Store the rest of the plates at 4 degree Celcius in an inverted position of for longer shelf
life, place in a sterile tight plastic bag. (for up to 2-3 months)

IV. BACITRACIN CHOCOLATE AGAR

This is use as a selective medium for the isolation of Haemophilus spp. in respiratory specimens.
Bacitracin is incorporated to TSA with horse blood to inhibit the normal flora of the upper
respiratory tract. (GC Agar base-hemoglobin plus bacitracin can be used as an alternative
medium.)

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Culture Media, Reagents and other Materials:

Trypticase Soy Agar/Columbia Agar Magnetic stirrer and stir bars

Distilled water Sterile Petri dish 100 x 15mm/ 150 x 15mm
Sterile defibrinated Horse blood Sterile serological pipette or 5ml Syringe
Bacitracin, 300 ug/ml, 0.5 ml aliquot Water bath, 80 degree Celcius

Procedure:
4.1 Weigh the amount of TSA powder according to the manufacturer’s instruction and place in
an Erlenmeyer flask.
4.2 Add 250 ml distilled water and mix thoroughly
4.3 Cover with aluminum foil and label with the name of the medium and volume.
4.4 Place in a boiling water bath, heat with frequent agitation and boil for 1 minute to completely
dissolve the powder.
4.5 Autoclave at 121 degree Celcius for 15 muntes.
4.6 Cool at around 80 degree Celcius
4.7 Aseptically add 5-10% sterile defibrinated horse blood and mix gently by swirling.

Note:

4.7.1 Do not use citrated blood (citrated blood is inhibitory to some organisms)
4.7.2 Allow the blood to equilibrate at room temperature before adding o the medium.
4.7.3 Measure the blood in a sterile test tube o use a sterile serological pipette or a graduated
cylinder.

4.8 Place in an 80degree Celcius water bath for 15 – 20 minutes with frequent agitation.

Note:
4.8.1 Do not start the time if the blood is still reddish-brown. Timing of 15 – 20 minutes starts
when the blood is fully lysed or brown
4.9 Cool to 45 – 50 degree Celcius.
4.10 Add 0.5 ml Bacitracin suspension to the medium.
4.11 Mix by gentle swirling to avoid fprmation of bubbles.
4.12 Dispense/Pour about 20 ml into sterile petri dishes.

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Note:
4.12.1 Depth of agar layer must be 4mm
4.12.2 Frequent mixing or swirling at an interval of 10 plates poured
4.12.3 If bubble form, do a slight quick flame at the top of the molten agar with a Bunsen burner

4.13 Allow the medium to solidify.

4.14 Get the first and the last plate dispensed for sterility testing and another plate for culture
response test.
4.15 Store the rest of the plates at 4 degree Celcius in an inverted position of for longer shelf
life, place in a sterile tight plastic bag. (for up to 2-3 months)

V. MUELLER HINTON AGAR

This is the recommended medium in performing antimicrobial susceptibility testing of significant
non-fastidious bacterial pathogens.

Culture Media, Reagents and other Materials:

Mueller Hinton Agar (MHA) 100 ml Graduated cylinder

Distilled water Sterile Petri dish 100 x 15mm/ 150 x 15mm
Erlenmeyer flask Sterile 20 – 25ml serological pipette

Procedure:
5.1. Weigh the amount of MHA powder according to the manufacturer’s instruction and place in
an Erlenmeyer flask.
5.2 Add 500 ml distilled water and mix thoroughly.
5.3 Cover with aluminum foil and label, “MH” 500.
5.4 Place in a boiling water bath, heat with frequent agitation and boil for 1 minutes to
completely dissolve the powder.
5.5 Autoclave at 121 degree Celcius for 15 minutes.

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5.6 Cool to 45 – 50 degree Celcius.

5.7 Mix by gentle swirling to avoid formation of bubles.
5.8 By using a sterile serological pipette or graduated sylinder, dispense:
5.8.1 90-100 x 15 mm = 25 – 30 ml
5.8.2 150 x 15 mm = 60 – 70 ml
5.9 Frequent mixing/swirling is done at an interval of every 5 big plates poured (for small plates:
about 10 plates)
5.10 If bubbles form, a light quick passing of flame on top of the molten agar with a Bunsen
burner, If unable to remove the bubble, swirl the plate gently so that the bubble will go to the
edge of the plate. Discard if the bubbles remain.
5.11 Allow the medium to solidify.
5.12 Get the first and the last plate dispensed for sterility testing and another plate for culture
response test.
5.13 Store the rest of the plates at 4 degree Celcius in an inverted position of for longer shelf life,
place in a sterile tight plastic bag. (for up to 2-3 months)
VI. MUELLER HINTON AGAR WITH 5-1-% DEFIBRINATED SHEEP/HORSE BLOOD
This is recommended medium in performing antimicrobial susceptibility testing of Streptococcus
spp, Neisseria meningitidis, Beta – hemolytic Streptococci and other related species.
Culture Media, Reagents and other Materials:

Trypticase Soy Agar/Columbia Agar Magnetic stirrer and stir bars

Distilled water Sterile Petri dish 100 x 15mm/ 150 x 15mm
Sterile defibrinated Horse blood Sterile serological pipette or 5ml Syringe
Bacitracin, 300 ug/ml, 0.5 ml aliquot Water bath, 80 degree Celcius

Procedure:
6.1 Weigh the amount of MHA powder according to the manufacturer’s instruction and place in
an Erlenmeyer flask.

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6.2 Add 500 ml distilled water and mix thoroughly

6.3 Cover with aluminum foil and label with the name of the medium and volume
6.4 Place in a boiling water bath, heat with frequent agitation and boil for 1 minute to completely
dissolve the powder.
6.5 Autoclave at 121 degree Celcius for 15 muntes.
6.6 Cool to 45 – 50 degree Celcius
6.7 Aseptically add 5-10% sterile defibrinated sheep or horse blood
Note:
6.7.1 Do not use citrated blood (citrated blood is inhibitory to some organisms)
6.7.2 Allow the blood to equilibrate at room temperature before adding o the medium.
6.7.3 Measure the blood in a sterile test tube o use a sterile serological pipette or a graduated
cylinder.
6.8 Mix by gentle swirling to avoid formation of bubbles.
Note:

6.8.1 90-100 x 15 mm = 25 – 30 ml
6.8.2 150 x 15 mm = 60 – 70 ml

6.9 Allow the medium to solidify.

6.10 Get the first and the last plate dispensed for sterility testing and another plate for culture
response test.
6.11 Store the rest of the plates at 4 degree Celcius in an inverted position of for longer shelf
life, place in a sterile tight plastic bag. (for up to 2-3 months)

VII. MacCONKEY AGAR

This medium is use as a selective and differential medium for the isolation of Enterobacteriaceae
and mon of Non – Enterobacteriaceae. Crystal violet and ile salts in the medium inhibit the gram-
positive organisms
Culture Media, Reagents and other Materials:

MacCONKEY Agar (MAC) Erlenmeyer flask (500 ml – 1L capacity)

Distilled water Sterile Petri dish 90 - 100 x 15mm

Procedure:

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7.1 Weigh the amount of MAC powder according to the manufacturer’s instruction and place in
an Erlenmeyer flask.
7.2 Add 250 ml distilled water and mix thoroughly
7.3 Cover with aluminum foil and label with the name of the medium and volume
7.4 Place in a boiling water bath, heat with frequent agitation and boil for 1 minute to completely
dissolve the powder.
7.5 Autoclave at 121 degree Celcius for 15 muntes.
7.6 Cool to 45 – 50 degree Celcius
7.7 Aseptically add 5-10% sterile defibrinated sheep or horse blood
7.8 Mix by gentle swirling to avoid formation of bubbles.
7.9 Dispense or Pour about 20 ml into sterile petri dishes.
Note:
7.9.1 Depth of agar layer must be 4mm
7.9.2 Frequent mixing or swirling at an interval of 10 plates poured
7.9.3 If bubble form, do a slight quick flame at the top of the molten agar with a Bunsen burner

7.10 Allow the medium to solidify.

7.11 Get the first and the last plate dispensed for sterility testing and another plate for culture
response test.
7.12 Store the rest of the plates at 4 degree Celcius in an inverted position of for longer shelf
life, place in a sterile tight plastic bag. (for up to 2-3 months)

VIII. HAEMOPHILUS TEST MEDIUM

This is the recommended medium in performing antimicrobial susceptibility testing of
Haemophilus spp.
Preparation of reagents:

Nicotinamide
Mueller-Hinton adenine Distilled
Haematin Yeast extract
Agar dinucleotide Water
(NAD)
See procedure as 7. 5 ml 0.75 ml 1.25 g 250 ml
per 15 ml 1.5 ml 2.5 g 500 ml
22.5 ml 2.25 ml 3.75 g 750 ml

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manufacturer’s 30 ml 3.0 ml 5.0 g 1000 ml

instruction

Haematin Preparation:
Haematin – 5 g

0.01 M naOH - 100 ml

- Mix haematin and 0.01M NaOH in the water bath to dissolve completely.
- Aliquot 7.5 ml (for 250 ml preparation) and store at – 20 degree Celcius until ready for
use.
-
0.01 M NaOH Preparation
NaOH pellet – 0.04 f
Distilled water – 100 ml

- Dissolve the NaOH pellet in distilled water.

NAD Preparation (Nicotinamide adenine dinucleotide)

NAD – 0.5 g
Distilled Water – 100 ml
- Dissolve 0.5 g NAD in distilled water. Filter using a sterile syringe filter disc (0.22-0.25 um)
and aliquot 0.75 ml (for 250 ml preparation) and store at 20 degree Celcius until ready for
use.
Procedure:
8.1 Weigh MH for 250 ml preparation and add 1.25g of yeast extract.
8.2 Dissolve MH and yeast extract in distilled water and add haematin.
8.3 Autoclave at 121 degree Celcius for 15 minutes.
8.4 Cool at 45 – 50 degree Celcius. Add 0.75 ml NAD. Mix gently

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8.5 Dispense: 90- 100 x 15 mm petri dish = 25 ml; 150 x 15 mm = 60 ml

8.6 Allow the medium to solidify
8.7 Get the first and last plate dispensed or sterility testing and another culture response test
8.8 Store the rest of the plates at 4 degree Celcius in an inverted position of for longer shelf life,
place in a sterile tight plastic bag. (for up to 2-3 months)

IX. SORBITOL MAC CONKEY AGAR (SMAC)

This is the selectie medium for diarrheagenic Escherichia coli.
Procedure:
9.1 Weigh amount of SMAC powder according to manufacturer’s instruction and place in an
erlenmeyer flask
9.2 Add 250 ml distilled water and mix thoroughly.
9.3 Cover with aluminum foil and label with the name of the medium and volume
9.4 Boil until completely dissolved.
9.5 Autoclave at 121 degree celcius for 15 minutes.
9.6 Cool at 45 – 50 degree Celcius.
9.7 Mix by gentle swirling to avoid formation of bubbles.
9.8 Dispense about 20 ml in sterile petri dishes.
9.9 Allow the medium to solidify.
9.10 Get the first and the last for sterility testing and another for culture response test.
9.11 Store the rest of the plates at 4 degree Celcius in an inverted position or for longer shelf life
(for up to 2-3 months), place in a tight plastic bag.

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X. THOISULFATE CITRATE BILE SALT SUCROSE AGAR (TCBS)

This is the selective medium for Vibrio spp.
Procedure:
10.1 Weigh amount of TCBS powder according to manufacturer’s instruction and place in an
Erlenmeyer flask
10.2 Add 500 ml distilled water and mix thoroughly.
10.3 Cover with aluminum foil and label with the name of the medium and volume
10.4 Boil until completely dissolved.
10.5 Cool at 45 – 50 degree Celcius.
10.6 Mix by gentle swirling to avoid formation of bubbles.
10.7 Dispense about 20 ml in sterile petri dishes.
10.8 Allow the medium to solidify.
10.9 Get the first and the last for sterility testing and another for culture response test.
10.10 Store the rest of the plates at 4 degree Celcius in an inverted position or for longer shelf
life (for up to 2-3 months), place in a tight plastic bag.

XI. SALMONELLA SHIGELLA AGAR (SSA)

Procedure:
11.1Weigh amount of SSA powder according to manufacturer’s instruction and place in an
Erlenmeyer flask
11.2 Add 500 ml distilled water and mix thoroughly.
11.3 Cover with aluminum foil and label with the name of the medium and volume
11.4 Boil until completely dissolved.
11.5 Cool at 45 – 50 degree Celcius.
11.6 Mix by gentle swirling to avoid formation of bubbles.
11.7 Dispense about 20 ml in sterile petri dishes.
11.8 Allow the medium to solidify.

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11.9 Get the first and the last for sterility testing and another for culture response test.
11.10 Store the rest of the plates at 4 degree Celcius in an inverted position or for longer shelf
life (for up to 2-3 months), place in a tight plastic bag.

XII. CEFOPERAZONE CHARCOAL DEOXYCHOLATE AGAR (CCDA)

This is the medium specified for use as a selective medium for Campylobacter spp.
Procedure:
12.1 Weigh amount of SSA powder according to manufacturer’s instruction and place in an
Erlenmeyer flask
12.2 Add 500 ml distilled water and mix thoroughly.
12.3 Cover with aluminum foil and label with the name of the medium and volume
12.4 Boil until completely dissolved.
12.5 Cool at 45 – 50 degree Celcius.
12.6 Mix by gentle swirling to avoid formation of bubbles.
12.7 Dispense about 20 ml in sterile petri dishes.
12.8 Allow the medium to solidify.
12.9 Get the first and the last for sterility testing and another for culture response test.
12.10 Store the rest of the plates at 4 degree Celcius in an inverted position or for longer shelf
life (for up to 2-3 months), place in a tight plastic bag.

XIII. ALKALINE PEPTONE WATER (APW)

Procedure:
13.1 Weigh amount of APW powder according to manufacturer’s instruction for quantities and
volumes required.
13.2 Distribute the solution into sterile glass screw capped container (5 ml each container)
13.3 Autoclave at 121 degree Celcius for 15 minutes.
13.4 Prepared medium may be used immediately or can be stored at room temperature.

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XIV. SELENITE F (SF)

Procedure:
14.1 Weigh 23 g of sf powder in 1 L distilled water/deionized/purified water according to
manufacturer’s instruction and place in an Erlenmeyer flask.
14.2 Heat to boiling. Note: avoid over heating, DO NOT AUTOCLAVE.
14.3 Distribute the solution into sterile glass screw capped container (5 ml each container).
14.4 Prepared medium may be used immediately or can be stored at 4 degree Celcius.

MICROSCOPICAL EXAMINATION OF SPECIMENS

I. GRAM STAIN

Principle
The differences in cell wall composition of Gram positive and Gram negative bacteria accounts
for the Gram staining differences. Gram positive cell wall contain thick layer
of peptidoglycan with numerous teichoic acid cross linking which resists the decolorization.

The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of
the membranes of both gram-positive and gram negative bacteria.
The outer membrane of the Gram-negative cell (lipopolysaccharide layer) is lost from the cell,
leaving the peptidoglycan layer exposed. Gram-negative cells have thin layers of peptidoglycan,
one to three layers deep with a slightly different structure than the peptidoglycan of gram-
positive cells. With ethanol treatment, gram-negative cell walls become leaky and allow the large
CV-I complexes to be washed from the cell.

After decolorization, the gram-positive cell remains purple in color, whereas the gram-negative
cell loses the purple color and is only revealed when the counterstain, the positively charged
dye safranin, is added. Source: http://microbeonline.com/gram-staining-principle-procedure-
results/

Procedure
1. Air dry and heat fix by passing the slide 2-3 times over a flame and allow to cool.

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2. Flood the smear with crystal violet and let it stand for 1 minute.
3. Wash gently with water. Pour off excess water.
4. Flood the smear with Grams iodine and let it stand for 1 minute.
5. Wash gently with water. Pour off excess water.
6. Decolorize with acetone-alcohol mixture 5-1seconds until the alcohol runs almost clear. Be
careful not to over-decolorize.
7. Immediately wash with water. Air dry.
8. Examine under low power objective and estimate number of squamous epithelial cells and
leukocytes.
9. Examine under oil immersion objective to determine predominant and other organisms
present.

QUALITY CONTROL FOR STAINING A SMEAR

It is recommended:
 To clean the slide and heating or passing the slide under a flame 2-3 times.
 That controls be run concurrently with unknows or at least run on a daily basis using known
smears containing Gram-positive and Gram-negative bacteria.
 The standard color depend on the manufacturer, as long as the gram-positive is recognizable
from gram-negative.

II. POTASSIUM HYDROXIDE MOUNT (KOH)

Principle

KOH mounting is the most rapid method of determining fungal ethiology. A 10% KOH
concentration is used in skin and other samples, while 20-25% KOH concentration is used for
nails, tissues and other thick samples.

KOH dissolves keratin and cellular material. The fungal structures such as hyphae, large yeast
(Blatomyces), spherules and sphorangia may be distinguished.
REAGENTS AND MATERIALS
10% KOH Forceps
Microscope glass slides Rubber or Pencil eraser
Cover slips Bunsen burner

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Moist chamber Microscope

Types of Specimen
All types of specimen can be tested for KOH mount (including keratinized tissues like hair, skin
and nails).

Collection and Transport

1. Disinfect nails or skin 70% alcohol
2. Cleanse painful lesion with NSS or distilled water before collection.
3. Hair should be epilated with sterile forceps and place on a clean envelope and fold.
4. Place the skin scraping of infected lesions on a clean envelope or a sterile glass slide and
cover with another sterile slide.
5. Nail should be cut into smaller pieces with sterile nail cutter or nail scrapings and place on a
clean envelope or sterile dry vial.
6. Collect other types of specimen the way they are collected for bacteriological culture.

Procedure
1. Place 1 to 2 drops of 10% KOH on a clean glass slide. (In case of nail clippings, dissolve it first
in 20% KOH in a small test tube and stand for 20-30 mins. Before placing it on a slide.)
2. Place small amount of specimen then mix.
3. Pass the slide 2 to 3 times over a low flame in a Bunsen burner. DO NOT OVERHEAT.
Overheating may cause crystals to form.
4. Cover with a clean cover slip gently using a pencil eraser or forceps to disperse the specimen.
5. Place in a moist chamber to prevent drying
6. Stand for 5 minutes to allow clearing
7. Examine under the microscope with reduced light thru LPO.
8. Shift to HPO to confirm.
9. Report the result.

INTERPRETATION
Observe for the following structures :
Yeast cells (budding or single cell)
Pseudohyphae
Hyphal elements:

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May be HYALINE (light or no pigment) or DEMATIACEOUS (dark brown)

REPORTING OF RESULT
POSITIVE : Report as :POSITIVE FOR (FUNGAL ELEMENTS OBSERVED)”
Example: Yeast or hyphal elements

NEGATIVE : Report as “NO FUNGAL ELEMENTS FOUND.”

NOTE : Other specimens may have other structures to be observed.

QUALITY CONTROL
It is recommended:
1. To sterilize the glass slide by heating or passing the slide on flame 2 to 3 times.
2. Check if the reagent or glass slides are contaminated with yeasts or molds by examining
under the microscope.
3. Check if the KOH reagent has crystallized.

III. INDIA INK

Principle
Cryptococcus neoformans affects immunocompromised patients. The capsule of Cryptococcus
spp. excludes the ink particles, giving a clear halo around the organism in a semi-opaque
environment.

Note :
C. neoformans var. Neoformans and var. grubii are associated with pigeon droppings and soil
contaminated with those droppings.

C. gatii was originally identified in a eucalyptus tree but it has been suggested that soil is its main
reservoir.

Procedure:
1. Place a drop of India Ink on a clean glass slide.
2. Add a drop of sediment to the ink by using a sterile Pasteur pipette

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3. Slowly place the coverslip by touching the left edge of the coverslip to the left edge of the
mixture then leave to cover the entire smear.
4. Stand for 1 minute.
5. Examine under the microscope with a reduced light source at power field (slightly closing the
condenser) and look for cell that appears brilliant white against the dark background. (Some
RBC and WBC may look positive under LPO)
6. Report result.

INTERPRETATION
POSITIVE : Presence of encapsulated or clear halo around the yeast cells

NEGATIVE : Absence of encapsulated yeast cells

REPORTING OF RESULT:
POSITIVE : Report as “POSITIVE FOR ENCAPSULATED YEAST CELLS” or “ENCAPSULATED YEAST
CELLS FOUND”

NEGATIVE : Report as “NO ENCAPSULATED YEAST CELLS FOUND”

Note: Treated patients’ result may show small capsule or unencapsulated yeast cells.

- If unencapsulated, check if the India ink is contaminated with yeast cells, if it isn’t report the
result as “UNENCAPSULATED YEAST CELLS”
-
- If small capsules are seen, report as “ENCAPSULATED YEAST CELLS”

QUALITY CONTROL
It is recommended:
- To clean the slide by passing thru flame 2-3 times or by heating
- Make sure that the slides and reagents aren’t contaminated with yeasts, molds ( slides) or
Cryptococcus spp.(in the ink) by examining under the microscope.
- Check if the India ink has thickened.

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IV. ZIEHL-NEELSEN and KINYOUN STAINING TECHNIQUE

Principle

The Ziehl-Neelsen (Zn) and Kinyoun technique is used to stain Mycobacterium spp. including M.
tuberculosis, M. ulcerans, and M. leprae. Mycobacteria, unlike most other bacteria, do not stain
well by the Gram technique. Some Actinomycetes, Corynebacteia and bacterial endospores also
acid fast.

Acid fast-bacilli are difficult to stain but once stained it difficult to decolorized.

Differences between Ziehl-Neelsen and Kinyoun staining methods.

Ziehl-Neelsen Tecnique: Also called the Hot Method, this requires heat-fixation which shortens
the length of staining and it depends on the manufacturer instructions in commercially prepared
stain.

Kinyoun Technique: Also called the Cold Method, this does not requires heating of the primary
stain but with longer length of staining.

Procedure:
The preparation of sputum smears for the detection of M. tuberculosis is described on page no.,
urine smears on page no., and cerebrospinal fluid preparation on page no..
1. Alcohol-fix the dried smear by covering it with one or two drops of 70% or absolute
methanol for 2-3 minutes.
2. Cover the smear with filtered carbol fuchsin stain (primary stain).
3. For Ziehl-Neelsen technique: Heat the stain until vapour just begins to rise. Do not over
heat.

Allow the heated stain to remain on the slide for 5 minutes

For Kinyoun technique: Do not heat and stand it for 10 minutes
4. Wash off the stain with clean water.
5. Cover the smear with Acid alcohol until the smear is sufficiently decolorized. i.e., pale pink

Caution: Acid alcohol is flammable, therefore use with care well away from an open flame
6. Wash well with clean water.

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7. Cover the smear with methylene blue stain for 1 minute, using the longer time if the
smear is thin.
8. Wash off the stain with clean water.
9. Wipe the back of the slide, and place in a draining rack for the smear o air-dry (do not blot
dry).
10. Examine the smear microscopically first with the 40x objective to see the distribution of
material and then systematically with the oil immersion objective to look for AFB.

INTERPRETATION OF RESULT:

Acid fast Bacilli (AFB) – Red, straight or slightly curved rods, occurring singly or in small groups
Cells – Blue
Background material – Blue

Reporting of sputum smears

If any definite red bacilli are seen, report the smear as “AFB positive”, and give an indication
of the number of bacteria present as follows:

Finding of very few AFB: If only one or two AFB are seen, request a further specimen from
the patient. Tap water sometimes contains AFB that resemble tubercle bacilli and
occasionally stained scratches on a slide can be mistaken for AFB.

If no red bacilli are seen after examining the area of the smear, report the smear as “No AFB
seen”. Do not report “Negative” because organisms may be present but not seen in those
fields examined.

QUALITY CONTROL

At regular intervals and always when a new batch of stain is started, two sputum smears of
known high and low AFB positivity should be stained with the routine smears to check that
the carbol fuchsin dye, staining technique, and the microscopical examination of smears are
satisfactory.

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BIOCHEMICAL TESTING OF MICROORGANISMS

I. Gram positive cocci

1.1 Staphylococcus spp.

A. CATALASE TEST

Principle
This test is done to detect the presence of the enzyme Catalase which hydrolyzes hydrogen
peroxide to produce water and oxygen. This test will differentiate Catalase positive
(Staphylococci) from Catalase negative (Streptococci).

Reagents and Materials:

3% Hydrogen peroxide Bunsen burner / loop incinerator
Sterile glass slides Glass pipettes
Sterile applicator sticks / inoculating loop Bunsen burner
Rubber bulb Positive control : Staphylococcus spp.
Negative control : Streptococcus spp.

Procedure:
1. From an 18-20 hour culture of test organism, pick a colony.
2. Make a smear on a sterile glass slide and perform a positive and negative control
3. Place 1 drop of 3% hydrogen peroxide over the organism. Do not mix.
4. Observe for effervescence or bubbling.

INTERPRETATION:
POSITIVE : Immediate bubbling or effervescence
NEGATIVE : No bubbling

B. COAGULASE TEST

Prinicple
This test is done to detect the presence of enzyme coagulase which clots plasma. This is the most
reliable test to identify Staphylococcus aureus and differentiate it from other species of
Staphylococci.

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Coagulase plasma is lyophilized plasma specifically designed and pre-tested for direct
performance of coagulase test tube on Staphylococci. After reconstitution, coagulase plasma
will clot in the presence of the enzyme coagulase.

Reagents and Materials:

Procedure:
SLIDE TEST
Bound coagulase also know as “clumping factor” is attached to the bacterial cell wall.
Bacteria clump in fibrinogen-righ strands are formed between bacterial cell walls.

Reconstituted coagulase plasma Inoculating loop

Sterile NSS Glass slides
Sterile applicator stick Bunsen burner / loop incinerator
Rubber bulb 35 +/- 2 deg C incubator, ambient air
Test isolate Glass pipettes
NEGATIVE CONTROL : any coagulase- POSITIVE CONTROL : S.aureus
negative Staphylococcus spp. (CONS)
1. Divide a glass slide into 4 portions.
2. Mark the boundaries to separate each portion.
3. Add a drop of NSS to each portion.
4. Add a drop of coagulase plasma to the 2nd upto the last portion
5. Emulsify a colony of CONS on the 2nd drop of NSS.
6. Mix the bacterial suspension with the drop of coagulase plasma. It will serve as the NEGATIVE
CONTROL
7. Emulsify a colony of known S. aureus culture on the 3rd drop of NSS.
8. Mix the bacterial suspension with the drop of coagulase plasma.I t will serve as the POSITIVE
CONTROL
9. Emulsify a colony of the test isolate on the 4th drop of NSS.
10. Mix the bacterial suspension with the drop of coagulase plama
11. Observe and record results.

TUBE TEST

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Free coagulase is a thrombin-like extracellular enzyme which when incubated with plasma,
causes a formation of clot. Upon incubation with plasma, the organism produces free coagulase
which reacts with the serum substance CRF or coagulase-reacting factor to form a complex. This
complex reacts with fibrinogen to produce fibrin clot.

1. Emulsify a loopful of colony in a tube containing 0.5 mL of coagulase plasma.

2. Incubate the tube at 35 +/- 2 deg C for 2-4 hours and observe for a clot formation by gently
tilting the tube.
3. If no clot is formed, re-incubate the tube at 35 +/- 2 deg, ambient air and read after 18-24
hours.
4. Observe and record results.

INTERPRETATION

SLIDE TEST

POSITIVE : Presence of white precipitate or agglutination within 10-15 seconds

NEGATIVE : Smooth and milky/homogenous mixture

TUBE TEST

POSITIVE : Any degree of clotting

NEGATIVE : No clot formation

C. VOUGES-PROSKAUER TEST

Principle
This test is used to determine the ability of the organism to produce acetoin or acetyl-methyl
carbinol as the chief end product of glucose fermentation. The addition of 40% potassium
hydroxide in atmospheric oxygen converts acetoin to diacetyl with alpha-naphthol as the reagent
to produce a visible red production.
Materials and Reagents:
2 mL MRVP Broth Bunsen burner / loop incinerator
5% alpha-naphthol 35 +/- 2 deg C incubator
40% Potassium hydroxide Inoculating loop
Droppers Test isolate
POSITIVE CONTROL : Enterobacter cloacae NEGATIVE CONTROL : Eschericia coli

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Procedure:
1. Use a sterile inoculating loop to inoculate several colonies of the test isolate on the MRVP
broth.
2. Incubate the tube for 24 hours at 35 +/- 2 deg C incubator, ambient air.
3. After incubation, add 1.2 mL (12 drops) of 5% alpha-naphthol.
4. Followed by the addition or 0.4 Ml (4 drops) of 40% KOH.
5. Shake the tube gently and expose to atmospheric oxygen.
6. After 30 minutes, read and record results.

INTERPRETATION
POSITIVE : Red color
NEGATIVE : No color change or brownish color

D. DNAse TEST

Principle
This medium is used to differentiate microorganisms based on the production of DNAse. This
enzyme hydrolyzes the DNA in the medium, producing a clearing or clear halo around the colony.
DNAse-producing organisms require acidic conditions as a signal to produce this enzyme.

Reagents and Materials:

DNAse agar 35 +/- 2 deg C incubator, ambient air
Test isolate Glass pipettes
Rubber bulb Table lamp
1N HCl Bunsen burner / loop incinerator
Inoculating loop NEGATIVE CONTROL : Eschericia coli
POSITIVE CONTROL : Staphylococcus aureus

Procedure:
1. Divide the plate into three equal portions.
2. Label the portions as negative control, positive control and test isolate.

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3. With sterile inoculating loop, inoculate by making a straight line of about 1.5-2 cm for each of
the negative control, positive control and test isolate.
4. Incubate the plate at 35+/- 2 deg C incubator for 18-24 hours.
5. After incubation, flood with 1N HCl along the line of growth.
6. Stand for a few minutes.
7. After a few minutes, read under reflected light against a dark background.
8. Observe and record the results.

INTERPRETATION
POSITIVE : Distinct clear zone surrounding spot inoculate
NEGATIVE : No clear zone, the medium will turn hazy white

E. NOVOBIOCIN SUSCEPTIBILITY TEST

Principle

This confirmatory test is used to determine the effect of a small amount of Novobiocin in an
organism. This used to differentiate Coagulase Negative Staphylococci (CONS) which are S.
saprophyticus and S. epidermidis.

Reagents and Materials:

Novobiocin Inoculating loop
BAP Forceps
35 +/- degree celcius, 5-10% CO2 incubator Test isolate
Loop incinirator

Procedure:
1. Create a tree layered lawn on half of the BAP with a sterile loop containing a few colonies
of the test isolate. Make sure that the lawn is formed by tight side by side streaking to
ensure confluent growth.
2. Extend the streak to the other half of the BAP to isolate individual colonies
3. With sterile forceps, place novobiocin disk on the lawn. Take into account the ease of
reading the zones around each disk as they are set.
4. Gently tamp down the disks so that they adhere to the agar surface.
b. Incubate the plate for 18-24 hours at 35 +/- 2 degree Celsius, 5% CO2.
c. read and record results according to the interpretation below.

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INTERPRETATION:
Susceptible > 16 mm zone of inhibition
Resistant < 16 mm zone of inhibition

1.2 Streptococcus spp.

1.2.1 Alpha hemolytic

A. CATALASE TEST

(See page no. 34)

B. OPTOCHIN DISK TEST on Alpha-hemolytic colonies

Principle:

To test the organism’s susceptibility to the chemical ethyl hydrocuprein hydrochloride(optochin).

This is used to test the fragility of the bacterial cell membrane.

Reagents and Materials:

Optochin disk, 5 ug Inoculating loop
BAP Bunsen burner/ loop incinerator
Forceps 35 +/- 2 deg C incubator, 5-10% CO2
Caliper / ruler Test isolate

Procedure:
1. Create a three-layered lawn on half of the BAP with a sterile loop containing one colony of the
Alpha-hemolytic test isolate. Make sure that the lawn is formed by tight side by side streaking to
ensure confluent growth.
2. Extend the streak to the other half of the BAP to isolate individual colonies.
3. With sterile forceps, place an optochin disk on the lawn.
4. Gently tamp down the disk so that it adheres to the agar surface.
5. Incubate the plate for 18-24 hours at 35 +/- 2 deg C incubator, 5-10% CO2.
6. Read and record results.

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INTERPRETATION
ZONE SIZE INTERPRETATION / OTHER WORP-UPS
> or equal to 14 mm S.pneumoniae
9-13 mm Do bile solubility test
< 9 mm Other Streptococcus

C. BILE SOLUBILITY TEST

Principle
Another test used to confirm the identification of S.pneumoniae determines the tendency of the
bacterial cells to lyse in the presence of bile salts within a specific time and temperature.

Reagents and Materials:

10% sodium deoxycholate Bunsen burner / loop incinerator
NSS 35 +/- deg C incubator, 5-10% CO2
Sterile tubes Test isolate
Inoculating loop

Procedure:

BILE SOLUBILITY PLATE TEST

1. Place a drop of 10% sodium deoxycholate on well-isolated test colonies on BAP.

2. Incubate the plate for 30 minutes at 35 +/- 2 deg C incubator, 5-10% CO2. Do not invert the
plate.

BILE SOLUBILITY BROTH TEST

1. Prepare a heavy suspension of a pure culture in 1.0mL OF 0.85% saline.

2. Divide the suspension between two tubes (one test and one control)
3. Add 0.5 mL of 10% sodium deoxycholate to the test suspension and 0.5 mL of 0.85% saline to
the control
4. Gently mix both suspensions and incubate at 37 deg C for up to 15 minutes.
5. Examine for evidence of clearing of turbidity in the tube marked test compared with the saline
control.

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INTERPRETATION:

BILE SOLUBILITY PLATE TEST

BILE SOLUBLE : Disintegration of the colony and/or the appearance of a haemolytic zone in the
medium where the colony was located.
BILE INSOLUBLE : No change in appearance

BILE SOLUBILITY BROTH TEST

BILE SOLUBLE : Suspension clears in tube labelled test and remains turbid in control tube
BILE INSOLUBLE : Suspension remains turbid in broth tubes

Note : Partial clearing (partial solubility) is not considered positive for S. pneumonia
identification.
1.2.2 Beta-hemolytic and Variable Hemolytic

A. CATALASE TEST

(See page no. 34)

B. CAMP (CHRISTIE, ATKINS, MUNCH-PETERSEN) TEST

Principle
This test is based on the synergistic hemolysis between Beta hemolysin produced by most S.
aureus strains and an extracellular protein called CAMP factor produced by group B Sterptococci.
This phenomenon is seen with both haemolytic and non-hemolytic isolates of group B
Streptococci.

Reagents and Materials:

BAP Test isolate
Inoculating loop S. aureus ATCC 25923
Bunsen burner / loop incinerator POSITIVE CONTROL : Steproccus agalactiae
35 +/- 2 deg C incubator, 5-10% CO2 NEGATIVE CONTROL : Streptococcus pyogenes

Procedure
1. Make a vertical line using a sterile loop with a few colonies of S. aureus ATCC 25923 on BAP.
2. Draw a perpendicular line of the test isolate and positive and negative controls about 4-6m
away from S. aureus ATCC 25923 line.

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3. Label horizontal lines accordingly.

4. Incubate the plate for 18-24 hours at 35 +/2 deg C incubator, 5-10% CO2.
5. Read and record results.

INTERPRETATION:
SUSCEPTIBLE : Arrow-shaped zone of hemolysis
RESISTANT : No arrow-shaped hemolysis formed

C. PYR TEST

Principle
This test is used to detect the production of pyrrolidonyl amino peptidase (pyrrolidonil
arylamidase or PYRase) by the test isolate. Filter paper disks are impregnated with the substrate
L-pyrrolidonyl-Beta-naphthylamide. PYRase hydrolyzes the substrate resulting in the formation
of Beta-naphthylamine which appear as a bright pink to red substance in the presence p-
dimethylaminocinnamaldehyde (DMACA).

Reagents and Materials:

PYR Disk : L-pyrrolidonyl-Beta-naphthylamide Applicator Sticks
Color developer : DMACA (0.01% p- Distilled Water
dimethylaminocinnamaldehyde)
Glass slide Test isolate
Forceps

Procedure:
1. Using forceps, place PYR disk on a clean glass slide
2. Moisten the disk with a drop or with 5 – 10 ul of distilled water. Do not oversaturate the
disk with water. Alternatively, the disk may be placed in the surface of the agar medium
for rehydration.
3. Rub several colonies o the test isolate on the damp PYR disk
4. After 2 minutes, add a drop of the color developer on the disk
Caution: The color developer is toxic and may cause harm by inhalation, contact with skin
or eyes or if swallowed, may impair fertility or cause harm to unborn child. Refer to
Material Safety Data Sheet (MSDS)
5. Observe for color change 2 minutes after the addition of DMACA and record results
according to the interpretation below.

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INTERPRETATION:
Positive Bright pink to red colonies
Negative Cream, yellow or no colony color change

D. HIPPURATE HYDROLYSIS

Principle
This test determines the ability of bacteria to hydrolyze hippurate. One of the end products –
glycine – is detected after the addition of ninhydrin reagent.

Reagents and Materials:

Sodium hippurate disk Bunsen burner / loop incinerator
Ninhydrin reagent 35 +/- 2 deg C incubator, 5-10% CO2
Sterile tube Test isolate
Distilled water
Forceps POSITIVE CONTROL : Streptococcus agalactiae
Inoculating loop NEGATIVE CONTROL : Streptococcus pyogenes

Procedure:
1. Heavily suspend 1-3 colonies of the test isolate in a tube with 4 drops of distilled water.
2. Add a hippurate disk using your forceps to the bacterial suspension.
3. Incubate the bacterial suspension at 35 +/- 2 deg C incubator, ambient air for 2 hours.
4. After incubation, overlay the suspension with 2 drops of the ninhydrin reagent.
5. After incubation for another 30 minutes, observe and record results.

INTERPRETATION:

POSITIVE : Deep purple-blue color

NEGATIVE : Yellow or no color change

E. BILE ESCULIN TEST

Principle
Bile Esculin positive bacteria will grow in the presence of bile salts and hydrolyze esculin. Esculin
hydrolysis forms dextrose and esculetin. Esculetin is visualized in the presence of ferric ions as a
diffusible black to reddish brown complex.

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Reagents and Materials:

Bile esculin slant 35+/- 2 deg C incubator, 5-10% CO2
Inoculating needle Test isolate
Bunsen burner / loop incinerator

Procedure
1. Inoculate the bile esculin slant with 2-3 colonies of the test isolate with the inoculating needle.
2. Incubate the slant at 35+/- 2 deg C incubator, ambient air for 24-48 hours.
3. Observe and record results.

INTERPRETATION

POSITIVE : Diffuse blackening of more than half of the slant within 24-48 hours.
NEGATIVE : No blackening

F. SALT TOLERANCE TEST

Principle
This test is used to determine the ability of the organism to grow in high salt concentration. The
test separates Enterococcus spp. and other related catalase-negative cocci from group D
Streptococcus spp.

Reagents and Materials:

6.5 % NaCl broth 35 +/- 2 deg C, ambient air
Inoculating loop Test isolate
Bunsen burner / loop incinerator

Procedure
1. Emulsify 2-3 colonies of the test isolate into 6.5% NaCl broth.
2. Incubate the broth at 35 +/- 2 deg C incubator, ambient air overnight.
3. Observe and record results
4. Perform Gram stain from the turbid broth for the confirmation of cell morphology and to check
if the broth is not mixed with contaminants.

INTERPRETATION

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POSITIVE : Turbid or presence of obvious bacterial growth in the medium (Gram stain : gram-
positive cocci in pairs/chain )
NEGATIVE : Clear to no growth

II. Gram negative cocci

2.1. Neisseria spp .

A. Gram stain

(See page no. 27)

B. Check for the growth on Modified Thayer Martin

C. OXIDASE TEST

Principle
To determine the presence of bacterial cytochrome oxidase using the oxidation of the substrate
tetramethyl-p-phenylene dihydrochloride to indophenol, a dark purple-colored end product.

Reagents and Materials:

Oxidase strip / disk Test isolate
Sterile applicator stick Bunsen burner
NEGATIVECONTROL : Escherichia coli Glass slide
POSITIVE CONTROL : P. aeruginosa Forceps

Procedure
1. Sterilize the glass by passing through the flame. Allow to cool
2. Place the reagent disk / strip on a slide and moisten with sterile distilled water.
3. By using a sterile applicator stick, pick by touching the top of the colony from CA (Careful not
to touch the agar) and rub on the moistened disk.
4. Observe for the immediate development of a purple color

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NOTE : Never perform oxidase test on organism grown on stained media (ex: selective media,
differential media)

INTERPRETATION
POSITIVE : PURPLE
NEGATIVE : NO COLOR DEVELOPMENT

D. CARBOHYDRATE UTILIZATION TEST (CTA-METHOD)

Principle
Neisseria species produce acid from carbohydrates by oxidation not fermentation. Production of
acid is indicated by a color change of the phenol red indicator (red to yellow).

Reagents and Materials:

Test isolate BUNSEN BURNER
5 tubed CTA based media: INOCULATING NEEDLE
CTA w/o sugar, glucose, maltose, lactose and INCUBATOR : 35 +/- 2 deg C, ambient air
sucrose
Blood Agar Plate (BAP)

Procedure
1. Using a sterile needle, pick 5-10 colonies of the test organism from CA or BAP and stab 5-8
times into the following tube media : CTA only, CTA with glucose, maltose, lactose and sucrose.
Use different inoculating needle for each sugar test.
2. Incubate at 35 +/- 2 deg C, in ambient air for 18-24 hours.
3. Read after 24-48 hours.

INTERPRETATION:
POSITIVE : YELLOW
NEGATIVE : NO COLOR CHANGE OF ORANGE COLOR

E. CARBOHYDRATE UTILIZATION TEST (RAPID)

Reagents and Materials:

TEST ISOLATE
PHOSPHATE BALANCED SALT SOLUTION (BSS) BUNSEN BURNER

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STOCKED CARBOHYDRATE SOLUTIONS, 20% EACH: INOCULATING LOOP

GLUCOSE (G)
MALTOSE (M)
LACTOSE (L)
SUCROSE (S)
STERILE PASTEUR PIPETTES STERILE MICROTITER PLATE OR 5 PIECES
2-3mL STERILE TEST TUBES
TSI MEDIUM INCUBATOR : 35 +/- 2 deg C, ambient air
Procedure
1. Prepare about 0.5 mL of BSS in a sterile test tube.
2. Using a sterilized inoculating loop, make a heavy suspension of the test isolate from CA or BAP.
3. Prepare 5 sterile test tube or use the 5 wells in a sterile microtiter plate and label: “G” for
glucose, “M” Maltose, “L” Lactose, “S” for sucrose and “NC” for negative control.
4. By using a sterile Pasteur pipette, place 2 drops of BSS into each tube.
5. With another sterile Pasteur pipette, place a single drop of each appropriate carbohydrate.
Mix carefully.
6. Incubate at 35 +/2 deg C, in ambient air or in water bath for 1-2 hours.

INTERPRETATION
POSITIVE : YELLOW
NEGATIVE : NO CHANGE IN RED COLOR OR RED-ORANGE
Inoculate Test isolate to TSI to serve as negative control.

EXPECTED RESULT : NO GROWTH

If positive for growth, check if the growth is caused by a contaminant by performing gram stain.

2.2 Moraxella catarrhalis

A. Gram stain (See page no. 92 )

B. Carbohydrate utilization (see page no. 114)

C. HEMOLYSIS TEST
This is used to determine the haemolytic property of the organism.
a. REAGENTS AND MATERIALS

Test isolate NAD

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Staphylococcus aurues isolate Inoculating loop

Blood Hematin NAD Medium (BHN) Bunsen burner
Hematin Incubator : 5-10% CO2, 35 +/- 2 deg C

b. PROCEDURE
1. Prepare hemolysis test media. Use blood agar base or TSA instead of MHA. Add 5% sheep blood
after step no 8.4 (SEE THE PROCEDURE ON HOW TO MAKE HEMOLYSIS TEST MEDIUM ON
PAGE____)
2. By using a sterile loop, make one streak and make a separate stab on the agar just below the
streak.
3. Incubate in 5-10% CO2 incubator for 18-24 hours at 35 +/- 2 deg C.
d. INTERPRETATION
Alpha-hemolytic : partial lysis of red blood cells from the medium
Beta-hemolytic : complete hemolysis
Non-hemolytic : no hemolysis
D. SATELLITISM TEST
1. REAGENTS AND MATERIALS

Tryptic soy broth (TSB) Inoculating loop

NSS Sterile test tube (small)
BHI broth Incubator : 35 +/- 2 deg C, 5-10%CO2
BAP Bunsen burner
S.aureus isolate TSI Medium

2. PROCEDURE
1. Using the same suspension for the X and V growth requirement test, get a loopful and streak
in a close zigzag motion on BAP. Allow to dry.
2. Streak a vertical line of Staphylococcus aureus or middle of the zigzag pattern and incubate in
a 5-10% CO2 incubator for 18-24 hours at 35 +/- 2 deg C.

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3. Incubate TSI medium with Test isolate to serve as negative control.

III Gram negative bacilli

A. Enterobacteriaceae and Non - Enterobacteriaceae
1. Biochemical Tests
a. TSI
b. LIA
c. SIM
d. Citrate Utilization
e. Urease utilization
f. Oxidase Test
g. LOA (deamination and decarboxylation)
h. Carbohydrate Utilization

Additional Tests:
I.. OXIDASE TEST
1. PRINCIPLE
To determine the presence of bacterial cytochrome oxidase using the oxidation of the substrate
tetramethyl-p-phenylene dihydrochloride to indophenol, a dark purple-colored end product.
2. REAGENTS AND MATERIALS

Oxidase strip / disk Test isolate

Sterile applicator stick Bunsen burner
NEGATIVECONTROL : Escherichia coli Glass slide
POSITIVE CONTROL : P. aeruginosa Forceps
3. PROCEDURE
1. Sterilize the glass by passing through the flame. Allow to cool.
2.Place the reagent disk / strip on a slide and moisten with sterile distilled water.
3. By using a sterile applicator stick, pick by touching the top of the colony from CA (Careful not
to touch the agar) and rub on the moistened disk.

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4. Observe for the immediate development of a purple color.

NOTE : Never perform oxidase test on organism grown on stained media (ex: selective media,
differential media)
4. INTERPRETATION
POSITIVE : PURPLE
NEGATIVE : NO COLOR DEVELOPMENT

II. BETA-LACTAMASE TEST

1. PRINCIPLE
Routine testing is indicated to detect ampicillin and penicillin resistance. In some cases, there are
beta-lactamase negative but ampicillin resistance. Therefore, there is a need to do Ampicillin MIC
to check if it is truky resistant.
2. PROCEDURE
1. Sterilize the glass slide by passing through a flame.
2. Place the reagent disk on the slide. Moisten the disk with sterile distilled water.
3. Pick 1-2 colonies using an applicator stick and blot on the disk.
4. Observe for color change.

3. INTERPRETATION
POSITIVE : PINK COLOR WITHIN 30 SECONDS
NEGATIVE : NO COLOR CHANGE

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III. CARBOHYDRATE UTILIZATION TEST (CTA-METHOD)

1. PRINCIPLE
Neisseria species produce acid from carbohydrates by oxidation not fermentation. Production of
acid is indicated by a color change of the phenol red indicator (red to yellow).
2. REAGENTS AND MATERIALS

Test isolate BUNSEN BURNER

5 tubed CTA based media: INOCULATING NEEDLE
CTA w/o sugar, glucose, maltose, lactose and INCUBATOR : 35 +/- 2 deg C, ambient air
sucrose
Blood Agar Plate (BAP)

3. PROCEDURE
1. Using a sterile needle, pick 5-10 colonies of the test organism from CA or BAP and stab 5-8
times into the following tube media : CTA only, CTA with glucose, maltose, lactose and sucrose.
Use different inoculating needle for each sugar test.
2. Incubate at 35 +/- 2 deg C, in ambient air for 18-24 hours.
3. Read after 24-48 hours.
4. INTERPRETATION
POSITIVE : YELLOW
NEGATIVE : NO COLOR CHANGE OF ORANGE COLOR

IV. DECARBOXYLATION OF ORNITHINE

A. PRINCIPLE
To measure the enzymatic ability of an organism to decarboxylate the amino acid ornithine to
form an amine with resulting alkalinity.
B. REAGENTS AND MATERIALS

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Test isolate Inoculating loop

Sterile mineral oil Bunsen burner
Moeller decarboxylase base medium w/ 35 +/- 2 deg C, ambient air
ornithine
NEGATIVE CONTROL : Moeller decarboxylase base medium w/o amino acid
C. PROCEDURE
1. Using an inoculating loop, pick 3-5 colonies and emulsify some of the inoculum into a negative
control and slightly tap the excess fluid.
2. Without changing the loop, continue inoculating the rest of the inoculum into ornithine
3. Overlay with 5-8 drops of sterile mineral oil.
4. Incubate at 35 +/-2 deg C for 24-48 hours.
D. INTERPRETATION
POSITIVE : PURPLE TO VIOLET
NEGATIVE : YELLOW

V. PYOCYANIN PRODUCTION
1. PRINCIPLE
The agar medium is used for the enhancement of pyocyanin produced by some organisms.
2. REAGENTS AND MATERIALS

Test isolate Inoculating loop

POSITIVE CONTROL : Pseudomonas Bunsen burner
aeruginosa
NEGATIVE CONTROL : Burkholderia cepacia or 35 +/- 2 deg incubator, ambient air
Escherichia coli
KING A (pyocyanin) plate / tube slant

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3. PROCEDURE
1. Streak to plate or tube slant.
2. Incubate at 35 +/- 2 deg C for 18-24 hours. (Reincubate if there is no pigment production or
little growth at 25-30 deg C for 1-2 days.)
3. Observe for pigment production.
NOTE : SEE MANUFACTURER’S RECCOMENDATION
4. INTERPRETATION
POSITIVE : PRODUCES BLUISH-GREEN OR DARK GREEN PIGMENT
NEGATIVE : PRODUCES NO PIGMENT

VI. FLUORESCEIN PRODUCTION

1. PRINCIPLE
This agar is used for the enhancement of fluorescein produced by Pseudomonas aeruginosa. This
is used to differentiate Pseudomonas aeruginosa and Pseudomonas flourescens from other
Pseudomonas spp.
2. REAGENTS AND MATERIALS

Test isolate Inoculating loop

POSITIVE CONTROL : Pseudomonas Bunsen burner
aeruginosa
NEGATIVE CONTROL : Burkholderia cepacia or 35 +/- 2 deg incubator, ambient air
Escherichia coli
KING B (pyocyanin) plate / tube slant

3. PROCEDURE
1. Streak to plate of tube slant.
2. Incubate at 20-25 deg C (not more than 35 deg C) for 1-3 days. (Reincubate at 25-30 deg C for
102 days if there is no pigment production and place under UV light.)

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3. Observe for pigment production and place under UV light.

NOTE : SEE MANUFACTURER’S RECOMMENDATION.
4. INTERPRETATION
POSITIVE : COLONIES SURROUNDED BY A YELLOW TO GREENISH-YELLOW ZONE WHICH
FLUORESCE UNDER UV LIGHT.
NEGATIVE : NO PIGMENT PRODUCTION

VII. GROWTH 42 deg C

1. PRINCIPLE
This agar medium is used to differentiate Pseudomonas aeruginosa from Pseudomonas
flourescens and other fluorescent group of Pseudomonas spp.
2. REAGENTS AND MATERIALS

Test isolate Inoculating loop

TSB Bunsen burner
Test tube, 16 mm, 10 mL (sterile) 42 +/- 1 deg C incubator, ambient air
POSITIVE CONTROL : Pseudomonas NEGATIVE CONTROL : Pseudomonas
aeruginosa fluorescens or Pseudomonas putida

3. PROCEDURE
1. Put 1.2 – 2 mL of TSB in a 16mm tube with a maximal air surface.
2. Add 1 drop of a small inoculum in broth tube ( as for swabbing susceptibility plates ) taking
care that the broth is still completely clear after inoculation.
3. Use a positive control tube by a general loss of visibility.
4. INTERPRETATION
POSITIVE : PRESENCE OF GROWTH/TURBIDITY
NEGATIVE : ABSENCE OF GROWTH/CLEAR

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READING OF PLATES AND INTERPRETATION

I. Growth on BAP only or BAP and CAP

1.1 Describe the colony morphology: the size, shape, consistency: dry or moist; pigment;
transparency/opacity; hemolysis: alpha, Beta or non-hemolytic.
1.1.1 Perform Gram stain on colonies. Biochemical testing will depend on these
morphologies:
1.1.2 If gram-positive cocci in pairs and in chains, do further work-up for Beta-hemolytic
Streptococci and other catalase-negative cocci related species.
1.1.3 If small gram-positive rods: X,V, palisiding or Chinese characters fo further
coryneform organisms( Bacillus spp., Corynebacterium spp. and other coryneform
related spp)
1.1.4 If small gram-positive coccoid to tiny rods: do further work-up for Listeria spp. or
other related species.
1.1.5 If big gram-positive bacilli: do further work-up for Bacillus spp.
1.1.6 If gram-positive cocci in clusters, do further work-up for Staphylococcus spp. or other
related catalase-positive species.
1.1.7 If gram-negative diplococci, coccobacilli, do further work-up for gram-negative
organisms: Enterobacteriaceae, Non-Enterobacteriaceae, Neisseria spp. and for
Haemophilus spp. that require hematin (X factor) for growth.
1.1.8 If gram-negative bacilli/coccobacilli, do further work-up for gram-negative organisms
(Enterobacteriaceae/Non-Enterobacteriaceae/ other Haemophilus/ Aggregatibacter
spp.)
1.1.9 Fungal colonies:

1.1.9.1 If the organism reveals yeast cells/pseudo hyphae, do further work-up for the
identification of medically important yeast cells.
1.1.9.2 If the organism reveals yeast cells and hyphae or hyphae only, refer to Mycology
to do further work-up for the identification of medically important molds.
II. Growth on BAP, CAP AND, MAC

2.1. Compare, BAP, CAP and, MAC. Check if the colonies are the same and presence of
pigment on each medium. ( If other colonies are not the same, look also for other possible
pathogens specific for CAP or specific for BAP)
2.2. Describe the colonies on MAC:
2.2.1. LF: Lactose fermenter = Pink or Red

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2.2.2. NLF: Non-Lactose Fermenter colonies;

2.2.2.1 LLF: Late Lactose Fermenter colonies = Colorless to faint pink.
2.2.2.2 Describe if necessary the consistency as mucoid or dry
2.2.2.3 If mixed(different colony morphology)NLF or LF, describe also size, shape,
consistency: dry or moist; pigment.
2.2.3 Perform work – up for Enterobactericeae/ Non-Enterobacteriaceae
III. Growth on CAP only

3.1 Describe the colony morphology, the size, shape, consistency: dry or moist;
color/pigment, transparency/opacity, hemolysis (alpha, Beta or non-hemolytic).
3.2 Perform work – up for Haemophilus spp.
3.3 Perform work-up for gram-negative organisms. (Enterobactericeae/Non-
Enterobacteriaceae/ Neisseria spp.)

IV. Growth on BCA

4.1 Look for the possible morphology of Haemophilus spp. Disregard other colonies that is
obviously not Haemophilus spp. Describe the colony morphology, the size, sape,
consistency: ry or moist/mucoid and transparency.
4.2 Perform work – up for Haemophilus spp.

V. Growth on BAP and GBA

5.1 Look for suspicious Streptococcus pneumoniae and describe the colony morphology, the
size, shape, consistency and alpha hemolysis on BAP and GBA.
5.2 Perform work-up for Streptococcus pneumoniae

QUANTITATION OF COLONIES ON CULTURE MEDIA IN DIFFERENT SEPCIMENS

1. URINE
1.1 After incubation examine the plates for growth on BAP and MAC or BAP only.
1.2 Quantitation is done on colonies from BAP:
1.2.1 If 1 ul loop was used = 0.001 ml or 1 colony x 1000

(Example: 100 colonies x 1000 = 100,000 colonies per ml of urine)

1.2.2 If 10 ul loop was used = 0.01 or 1 colony x 100

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(Example: 100 colonies x 100 = 100,00 colonies per ml of urine)

2. STOOL/ RECTAL SWAB AND STERILE SPECIMENS

No quantitation is done; indicate “POSITIVE”.

2.1 Any organism that grow on sterile samples must be considered significant. A thorough
skin disinfection of skin surface eliminates the possibility of potential contaminants.
2.2 Significant enteropathogens are not normally present in stool specimen.

3. EXUDATES/ RESPIRATORY SECRETIONS (sputum, eye, ear discharge)

3.1 After incubation examine the plates for growth on BAP and MAC or BAP only.
3.2 Semi quantitation of colonies on BAP:
3.2.1 Not significant:
3.2.1.1 Very Few colonies
3.2.1.2 Few colonies
3.2.1.3 Light growth
3.2.2 Significant
3.2.2.1 Moderate growth
3.2.2.2 Moderately heave growth
3.2.2.3 Heavy growth
3.2.2.4 Confluent growth

4. Nasotracheal Aspirate NTA/Endotracheal aspirate (ETA)/Tracheal aspirate (TA)

4.1 Any quantitation is significant of significant pathogen in respiratory samples: Refer to C.
Exudates for quantitation.
4.2 If there is no significant pathogen observed, consider the colonies present

5. THROAT SWAB

Clinical impression: Tonsillopharyngitis

5.1 Semi-quantify the Beta-heamolytic pyogenic Streptococcus spp. Regardless of the
number of colonies as long as it is present in the sample it is considered significant.

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AND LABORATORY MEDICINE

5.2 There is no need look for C. diphtheriae.

6. THROAT SWAB/PSEUDOMEMBRANE

Clinical Impression: T/C or R/O diphtheria

6.1 No quantitation is done. The presence of Corynebaterium diphtheriae is significant.
6.2 Semi-quantify the Beta-hemolytic pyogenic Streptococcus spp. Still look for this organism
even if the diagnosis is diphteria.
7. NASAL SWAB/ THROAT SWAB
7.1 Clinical impression: MRSA infection/tracings of carriers
7.2 Any quantitation is significant. Refer to Exudates for quantitation.

REFERENCE: Training Course on the Laboratory Diagnosis of Medically Important Bacterial

Pathogens by Microbiology Department Research Institute for Tropical Medicine.

MICROBIOLOGY WORK INSTRUCTIONS