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Policy & Procedure Manual
Policy # MI\TECH\42\06\v01  Page 2 of 2
Technical Manual   

5.   Apply coverslip to wetted slide and examine with the fluorescent microscope using the
designated filter.  If there is a delay, add distilled water to the coverslip just prior to
examination.  Use a fresh tube of water daily.

6.   Optional for thicker smears .  Add few drops of the counter stain Fungi-Fluor solution B.
Rinse gently with tap water and th en proceed as in step 5 above.

Quality Control

Stain a smear of  Candida albicans daily.

Interpretation

Use 20x or 40x objective.

Fungal elements will appear yellow-green agains t a red-orange background  when counterstain is
used.  Observe for characteristic morphology.

References

1.   Manufacturers' Instructions (Data Sheet #316).  Fungi-Fluor  kit - Polysciences, Inc.,
July 1995

2.   Clin. Micro. Newsletter 9:33-36, March 1, 1987.
K.L. McGowna.  "Practical Approaches  to Diagnosing Fungal Infections in
Immunocompromised Patients".

3.   J. Clin. Micro. 28:393-394, Feb. 1990.  V.S. Baselski et al.  "Rapid Detection of
Pneumocystis carinii in Bronchoalveolar Lavage Sample s by Using Cellofluor Staining".


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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\07\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Gram Stain
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

GRAM STAIN

Principle

Bacteria can be recognized as gram positive (blue-black/purple) if they retain the primary dye
complex of crystal violet and iodine in the face of attempted decolourization, or as gram negative
(pink) if decolourization occurs as shown by the cell accepting the counterstain safranin.

Generally the mechanism of the Gram stain is:  The fixed bacteria are stained with the
triphenylmethane dye, crystal violet.  Next the smear is flooded with Grams solution which
oxidatively forms an insoluble complex with the crystal violet.  The smear is then flooded with
the organic solvent, acetone-alcohol.  Dependi ng on cell permeability the  crystal violet-iodine
complex will be washed from Gram negative bact eria in solvent but not from Gram positive
bacteria.  Upon counterstaining with safranin,  organisms which had been  discolorized by the
ethanol (Gram negative) will stain pink.  Gram positive organisms which retained the crystal
violet will appear blue-black/purple microscopically.

Materials

Crystal violet solution
Grams Iodine solution
Acetone alcohol
Safranin solution

Procedure

1.   Prepare the film on the slide and allow to air dry.
DO NOT HEAT TO DRY FILM.

2.   When film is dry, place slide on heating bloc k for several minutes.  Slide should be just
warm to your hand.
DO NOT OVERHEAT.

3.   Allow slide to cool - this will happen quickly  - in just a few seconds.
DO NOT ADD STAIN TO HOT SLIDE.

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Policy # MI\TECH\42\07\v01  Page 2 of 2
Technical Manual   

4.   Flood slide with crystal violet - leave 1 minute.

5.   Wash gently with water.

6.   Flood slide with Grams Iodine - leave 1 minute.

7.   Wash iodine from slide with acetone-alcohol mixture.  Add a few more drops of acetone-alcohol until no more colour comes from film - usually 30 seconds.

8.   Wash gently with water.

9.   Flood slide with safranin - leave 1 minute.

10.  Wash gently with water.  Clean back of slide with tissue and  place slide in tray.  

Precaution

1.   At no time should the film (smear) be exposed to too much heat.  When the specimen is
still wet, heat causes coagulation of the  protein resulting in heavy overstaining which
cannot be removed by the decolourizer.  A thic k smear will also show more tendency to
"lift off" during staining.

2.   Rinsing the Grams Iodine off with the decolorizer gives more stability to the CV-GI
complex and false over decolorizing will not take place.

3.   Flooding a hot slide with crystal violet will cause the stain to  precipitate and make
decolourizing much more difficult.

Quality Control

It is recommended that controls  be run concurrently with unknowns or at least run on a daily
basis using known smears containing Gram positive and Gram negative bacteria.


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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\43\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Staphaurex Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

STAPHAUREX TEST

Principle

A rapid slide latex test for the detection of clumping factor and prot ein A produced by most
strains of  S. aureus.

Reagents and Materials

Staphaureux latex suspensi on (store refrigerated)
Disposable reaction cards
Culture loop or wooden applicator stick

Procedure

1.  Confirm the identification of a suspect  Staphylococcus by Gram stain and catalase test.
2.  Allow the latex reagent to warm to room temperature before use.
3.  Shake the reagent so that all of the particles are resuspended.
4.  Dispense one drop of latex  reagent onto the reaction card.
5.  Add 1-3 colonies to the drop, mix well  with a loop or wooden applicator stick.
6.  Rock the slide for 20 seconds and look for clumping.
7.  Discard the slide into a discard container.

Interpretation

Positive test:  Clumping within 20 seconds  with the sensitized latex particles.
Negative test:  No clumping

Precautions

1.  False positive results may occur after 20 seconds.
2.  False positive agglutination can occur with   E. coli and 
  C.  albicans

PROCEDURE MANUAL
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Policy & Procedure Manual
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Quality Control

Test known positive and negative controls daily:

 Positive: S. aureus  (ATCC 25923)
 Negative: S. epidermidis  (ATCC 12228)

References

1.  Staphaurex Package insert, July 1992.


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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\44\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Streptococcal Grouping
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

STREPTOCOCCAL GROUPING
Principle

This test is used to determine the Lancefield gro up of an isolate.  Latex particles labelled with
specific group antisera will agglutin ate in the presence of the corre sponding antigen after extraction
with nitrous acid.

Reagents

Pro-lab Streptococcal grouping Latex kit.

Other Materials

Droppers
Disposable slides
Wooden stirring sticks
13x100 mm test tubes

Procedure

1.  Label one test tube for each isolate.
2.  Add one drop of Extracti on Reagent 1 to each tube.
3.  Suspend 4 beta-haemolytic colonies in the Extraction Reagent 1.
4.  Add 1 drop of Extraction Reagent 2 to each tube.
5.  Shake the tube and inc ubate for 2 minutes at RT.
6.  Add 7 drops of Extraction Re agent 3 to each tube.  Mix th e reaction by vortexing the tube
for 10 - 15 seconds.
7.  Dispense one drop of each la tex suspension to be  tested onto separate  circles on the test
card.
8.  Using a pasteur pipette, add one drop of extract to th e latex suspension.
9.  Mix the latex and extract wi th the wooden stick using the co mplete area of the circle.
10.  Gently rock the card for 2 minutes and lo ok for agglutination.

PROCEDURE MANUAL
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Policy & Procedure Manual
Policy # MI\TECH\44\v01  Page 2 of 2
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Interpretation

Positive: Strong vi sible agglutination  within 2 minutes.

Negative:  Milky appearance without visible agglutination.

Precautions

1.  False positive reactions have been known to occur with organisms from unrelated genera eg.
E. coli,  Klebsiella  sp.,  Pseudomonas sp.

Quality Control

Test reagents are checked weekly.

Each test should be tested with at least one extra grouping latex su spension as a negative control.

Reference

1. Pro-lab Streptococcal  Grouping package insert.

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Policy & Procedure Manual
Policy # MI\TECH\45\v01  Page 1 of  1
Section: Technical Manual  Subject Title: Tetrazolium Reduction
 Test (TTC)
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

TETRAZOLIUM REDUCTION TEST (TTC)

Principle

To differentiate between E. faecalis and  E. faecium.

E. faecalis reduces the colorless compound (Tetrazolium- chloride) to an insoluble substance -
formozan which is red.

Material

BHI broth/Tetrazolium-chloride (TTC)

Procedure

Inoculate a loopful of an overn ight plate culture to 1 ml of  TTC broth.  Incubate at 35
0
C and
observe the reaction at 2 hours.  If negative, reincubate  up to 8 hours / overnight.

Interpretation

Positive - deep magenta
Negative - colourless or faint pink

Quality Control

Positive:  E. faecalis   (ATCC 29212)
Negative:  E. faecium   (ATCC 19434)

References

1.   J. Gen. Microbiology (1965), 38, 279-287.
 

PROCEDURE MANUAL
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Book on Biological Laboratory penalty 14



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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\01\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Acid Fast Stain for
 Mycobacteria (Kinyoun)
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

ACID FAST STAIN FOR MYCOBACTERIA (KINYOUN)

Principle

To stain Mycobacteria present in specimens  and cultures.

Mycobacteria are different to stai n with common aniline dyes.  However, they will stain with basic
fuschsin.  Once stained, they retain the dye de spite treatment wi th mineral acid s i.e.  HCl H
2SO4
.
This property of acid fastness  may be due to a lipd fraction called mycolic acid.  Mycobacteria also
exhibit degrees of resistance to  decolourization with alcohol.

Materials

Kinyoun Carbol fuchsin
3% HCl in 95% ethanol
Brilliant green

Procedure

1.   Prepare smear over an  area of 2-3 sq. cm.

2.   Heat fix smear on heating block (56
0
C/1 hr).  Then hold to  incinerator for 10 secs.

3.   Place slide on stain rack and allow to cool.  Flood with Kinyoun Carbol fuschsin for 5 min.

4.   Rinse off stain with water.

5.   Decolourize with 3% acid alcohol for 3 mins.

6.   Rinse with water.

7.   Repeat decolourization for 1-2  mins. or until no red appears.

8.   Rinse with water.

9.   Flood slide 3-4 mins. with Brilliant green.

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10.  Rinse with water.

11.  Air dry.  DO NOT BLOT.

Microscopy

Place a drop of oil between the specimen and coverslip and oil again on top.  Smears are examined
with oil immersion lens.  The coverslip prevents cr oss contamination.  An average of 15 mins. is
spent on each slide.  The total area of the spec imen must be examined.

References

1.   Baker, F.J., Breach, M.R.   1980.  Medical Microb iological Technique, p. 15


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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\02\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Acid fast stain for Nocardia
(Modifed Kinyoun)
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

ACID FAST STAIN FOR NOCARDIA (MODIFED KINYOUN)

Principle

Nocardia  species possess the unique characteristic of resisting d ecolorization with acid alcohol.

Reagents

1.   Carbol-fuchsin
Basic fuchsin solution   (3 g basic fu chsin in 100 mL 95% ethyl alcohol)
10 mL
Phenol 5% aqueous    90 mL

2.   Decolourizer (1% sulfuric acid)
H2 SO4
 (concentrated)   1 mL
Distilled water   99 mL

3.   Methylene blue
Methylene blue    0.3 g
Distilled water   100 mL

Staining Procedure

1.   Fix the smear by gentle heating.
2.   Flood the smear with Carbol fuchsin solution.
3.   Allow the slide to stand for 5 minutes.
4.   Wash the smear with tap water.
5.   Decolorize the smear with 1% sulfuric acid until no more colour appears in the washing
(approx. 1 min.).
6.   Rinse with tap water.
7.   Counterstain with methylene blue about 1 minute.
8.   Rinse with tap water and air dry.

PROCEDURE MANUAL
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Policy & Procedure Manual
Policy # MI\TECH\42\02\v01  Page 2 of 2
Technical Manual   

Interpretation

The filaments of  Nocardia  species and Rhodococcus appear red-stained against a blue
background.

Quality Control

A positive control slide of Nocardia species is stained simultaneously with the clinical
specimens.

References

1.  Murray, PA. et al.  Manual of Clinical Microbiology, 7
th
 edition, 1999 ASM,
Washington, D.C.

PROCEDURE MANUAL
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\03\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Acridine Orange Stain
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

ACRIDINE ORANGE STAIN
Principle

Acridine orange is a fluorescent dye which will bi nd to the nucleic acid of  bacteria and other cells.
It is recommended for use  for the detection of microorganisms in direct smears.  It is useful for the
rapid screening of specimens from  normally sterile site s (eg. CSF) and blood smears, or smears
containing proteinaceous material where differentiation of organisms from background material
may be difficult.   

Reagents

Acridine Orange spot test dropper.  Stored at room temperature.
Absolute Methanol

Procedure

1.  Prepare a smear of the  specimen to be stained.

2.  Allow to air dry.

3.  Fix with methanol for 1 to 2 minutes.

4.  Hold the dropper upright and squeeze gently to crush the glass ampoule inside the dispenser.

5.  Flood the slide with the acridi ne orange and stain for 2 minutes.

6.  Rinse thoroughly with tap water and allow to air dry.

7.  Examine with a fluorescent microscope using low and oil immersion objectives.

Interpretation

Bacteria and fungus stain bright orange.  The background appears black to yellow green.
Leukocytes will stain yellow, orange and red.

PROCEDURE MANUAL
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Policy & Procedure Manual
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Quality Control

Stain a smear of Streptococcus pneumoniae (ATCC 6303)  each time the  test is performed.   

References

1.  Difco Spot Test Acridine Orange Stain package insert, 1984.

PROCEDURE MANUAL
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\04\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Bacto 3-Step Gram Stain
 Procedure
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

BACTO 3-STEP GRAM STAIN PROCEDURE

Principle

To be used for problem smears to determine the Gram reaction of organisms.

Materials

3-Step Stabilized Iodine Technique

Bacto Gram Crystal Violet
Bacto Stabilized Gram Iodine
Bacto 3-Step Gram Safranin-S

3-Step Technical Iodine Technique

Bacto Gram Crystal Violet
Bacto Gram Iodine
Bacto 3-Step Gram Safranin-T

Microscope slides
Bunsen burner or methanol
Bacteriological loop
Swabs
Blotting paper
Microscope with oil immersion lens
Bactrol  Gram Slide
Bactrol  Disks

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Policy # MI\TECH\42\04\v01  Page 2 of 2
Technical Manual   

Procedure

1.   Flood the fixed smear with primary stain (B acto Gram Crystal Violet) and stain for 1
minute.
2.   Remove the primary stain by gently washing with cold tap water.
3.   Flood the slide with mordant (Bacto Stabilized Gram Iodine or Bacto Gram Iodine
(traditional formulation) and retain on the  slide for 1 minute.  (Refer to LIMITATIONS
OF THE PROCEDURE, #5)
4.   Wash off the mordant with decolourizer / counterstain (Bacto 3-Step Gram Safranin-S or
Bacto 3-Step Gram Safranin-T).  ( NOTE : Do not wash off iodine with water).  Add more
decolourizer / counterstain solution to the slide and stain 20-50 seconds.
5.   Remove the decolourizer / counterstain solution by gently washing the slide with cold tap
water.
6.   Blot with blotting paper or pa per towel or allow to air dry.
7.   Examine the smear under an oil immersion lens.

Interpretation

REACTION   3-STEP TECHNIQUE
using either Bacto Gram Safranin-S or Bacto
Gram Safranin-T

                       Gram-positive

                       Gram-negative
   Purple-black
to purple cells

Red-pink to
fuchsia cells

Quality Control

Run controls daily using 18-24 hour cultures of known gram-positive and gram-negative
microorganisms. 

PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 

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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\42\05\v01  Page 1 of  1
Section: Technical Manual  Subject Title: Eosinophil Stain
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

EOSINOPHIL STAIN
Principle

A stain for the detection of eosinophils in clinical specimens.

Reagents

AJP Scientific Eosinophil stain:
  Solution I - Eosin Y
  Solution II - Buffer Ph 6.5
  Solution III - Methylene Blue
  Stored at room temperature

Procedure

1.  Make a thin smear and spread evenly.

2.  Fix slide by air drying  or with gentle heat.

3.  Cover slide with solution I and leave for 30 seconds.

4.  Add solution II to cover s lide.  Mix gently  and allow to stain for 3 to 5 minutes.

5. Wash off with  tap water and drain.

6.  Cover slide with solution III and immediately  wash off with tap wate r.  Drain and air dry.

Interpretation

Eosinophils stain with red cytoplasm and bright red granules.

Reference

1. A.J.P. Scientific INC package insert.

PROCEDURE MANUAL
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Policy & Procedure Manual
Policy #MI\TECH\42\06\v01  Page 1 of  2
Section: Technical Manual
Subject Title:  Fungi-fluor   Stain
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

FUNGI-FLUOR STAIN

Principle

The Fungi-Fluor  stain is used for the rapid identification of various fungal infections in fresh or
frozen clinical specimens.

The active, fluorescing dye in the staining solution is Cellufluor which is the disodium salt of
4,4'-bis[4-anilino-6-bis-(2-hydroxyethel) amino-s-tr iazin-2-ylamino]-2,2'-stilbenedisulfonic acid.
Fungi-Fluor  staining solution is a 0.05% solution of this  dye in deionized water with potassium
hydroxide added as a clearing agent.  The Fungi-Fluor  counter staining solution B is an
aqueous solution of Evans Blue dye used to re duce background fluorescence.  Cellufluor binds
nonspecifically to beta-linked polysaccharides found in the cell walls of various organisms such
as chitin and cellulose.

Materials

Staining Solution A
Counterstaining Solution B
Absolute alcohol
Water
Fluorescent Microscope (250-400 nm filter)

Precautions

1.  Store in a dark or opaque bottle, tightly sealed, at room temperature.

2.  Avoid eye or skin contact: use gloves and protective glasses.

Procedure

1.   Prepare smear of specimen and allow to air dry.

2.   Fix on the rack with absolute alcohol for 5 mins. until dry.  Fixed smears can be held
indefinitely until ready to stain and examine.

3.   Add few drops of Fungi-Fluor solution A (Cellufluor) for 1 minute.

4.   Rinse gently with tapwater.

PROCEDURE MANUAL
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\37\v01  Page 1 of  1
Section: Technical Manual  Subject Title: RapID ANA II System
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

RapID ANA II SYSTEM

Principle

The RapID ANA II System is a qualitative micromethod employing conventional and
chromogenic substrate for the identification of  medically important anaerobic bacteria of human
origin.

The tests used in it are based upon the microbial degradation of specifi c substrate detected by
various indicator systems.  The reactions are a combination of conventional tests and single-substrate chromogenic tests.

Materials

1.   RapID ANA II panels
2.   Suspension fluid
3.   Kovacs spot indole reagent
4.   RapID ANA II reagent
5.   RapID ANA ID forms

Procedure

Make an equivalent McFarland #3 turbidity suspension of 18-24 hours AnO
2
 culture (not more
than 72 hours) in the supplied suspension fluid.  Mix it thoroughly - can be used up to 15
minutes.  Inoculate an agar (BA FAA) plate for purity and incubate for 24 hours anaerobically.
Peel the lid off the panel marked "peel to inoculate".  Using th e Pasteur pipette, transfer the
entire contents into the right uppe r corner of the panel. Seal the panel. Level the contents in the
panel and slowly tilt the panel so that every chamber receives  an equal amount of suspension.
Incubate the panel at least four hour s (not more than six hours) in non-CO
2
 incubator at 35-37
0
C.
After the incubation period, read the panel prior to adding the r eagents and write results on the
ID form.  Add the reagents as per instructions.  Allow 30 seconds  but not more than two minutes.
Read it and score on the form.

Interpretation and Identification

Please follow the guidelines from the manufact urer and see the RapID ANA II ID Code Book.

See RapID ANA II System Insert #iii08-1/94 brochure which follows.

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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\38\v01  Page 1 of  2
Section: Technical Manual  Subject Title: RapID MGP Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

RapID MGP TEST

Principle

Rapid MGP Medium is a 5 hour test for the differentiation of  Enterococcus faecium  and  E.
faecalis  from  Enterococcus gallinarum and  E.  casseliflavus based on the ability to acidify the
carbohydrate methyl-glucopyranoside (MGP).

Reagents

Rapid MGP Medium (Hardy Diagnostics)
Bacteriology loop

Procedure

1.   Using a sweep of colonies from an 18-24 hour  pure culture of the organism to be tested,
stab the MGP media with the loop.  There should be a visible cell paste on the loop as the
media is inoculated.

2.   Incubate aerobically at 35
0
C for 5 hours.

3.   Observe for the development of a yellow colour along the stab line  indicating a positive
test.

4.   Reincubate weak reactions for 24 hours.

Interpretation

Positive:  yellow colour along stab line
Negative:  colour remains blue

E. casseliflavus +
E. gallinarum   +
E. faecalis   -
E. faecium   -

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Technical Manual   

Quality Control

Positive and negative controls are  run each time the test is set up.

Positive:  E. casseliflavus   (ATCC 12755)

Negative:  E. faecalis   (ATCC 19966) 

Reference

1.  Hardy Diagnostics package insert 1999.

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Policy & Procedure Manual
Policy # MI\TECH\39\v01  Page 1 of  1
Section: Technical Manual  Subject Title: RapID VP Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

RapID VP TEST

Purpose

To aid in the identification of  S. milleri.

Media

MR - VP broth

Procedure

1.   Transfer approximately 0.2 ml of VP broth into a sterile 13 x 100 test tube.

2.   Using a sterile inoculating wi re inoculate the test organi sm heavily into the broth.

3.   Incubate the tube at 35
0
C for 5 hours.

4.   After incubation add 1 drop of alpha-naphthol and 1 drop of 40% KOH.

5.   Shake the tube gently for one minute to expose the medium to air.  Allow 10-15 minutes
for reaction to develop.

Interpretation

 Positive - Red colour
  Negative  -  No colour change within 10-15 minutes

Precautions

The order of adding reagents is important; alpha-naphthol followed by 40% KOH.

Reference

Ruoff, K.L., Ferraro, M.J. 1986.  Presumptive identification of  S. milleri  in 5h J Clin Microbiol
24:495-497.


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Policy #MI\TECH\40\v01  Page 1 of  2
Section: Technical Manual  Subject Title: RapID Yeast Plus Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

RapID YEAST PLUS TEST
Purpose

Used for the identification of  yeast and yeast like organisms.

Materials

Rapid Yeast Plus Panel
Rapid Yeast Plus Reagent A and Reagent B
Rapid ID Inoculating fluid 2ml
Pasteur Pipettes
Cotton swabs

Procedure

1.   Use a cotton swab suspend sufficient growth of the yeast in the Inoculating fluid to give a
suspension heavy enough to obliterate the black lines on the inoculation card.

2.   Peel back the panel lid over the inoculation port by pulling the tab marked "Peel to
inoculate".

3.   Using a Pasteur pipette transfer the entire contents  of the inoculation fluid into the upper
right hand corner of the panel and then reseal the panel.

4.   Tilt the panel back away from the biochemical wells at approx. a 45% angle.

5.   While tilting back gently rock the panel from side to side to evenly distribute the
inoculum along the rear baffles.

6.   Slowly tilt the panel forward toward the reaction cavities until th e inoculum flows along
the baffles into the biochemical wells.

7.   Incubate panel at 30
0
C for 4 hours.

8.   After incubation peel the label lid from over the reaction cavities.

9.   Add 1 drop of reagent A to cavities 7 to 14 inclusive.

10.  Add 1 drop of reagent B to cavities 16-18 inclusive.

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11.  Read results after 30 seconds but no more th an 1 minute after addi ng reagent.  Record
results onto supplied report form, and look results up in the Rapid ID Yeast Plus code
book for interpretation.

Interpretation

Well # Positive Negative

1 to 5

Yellow  Blue to green
6

Yellow  Red, pink, orange, gold
7 to 14

Any yellow  Clear or cream
15

Red or dark red-orange  Yellow, yellow-orange or orange
16-18

Purple, red or dark pink  Clear straw, orange, pale to medium pink

Quality Control

Control strains are set up for each new lot number of panels.

References

1.  Rapid ID yeast plus package insert issue #7/98.

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Policy #MI\TECH\41\v01  Page 1 of  2
Section: Technical Manual  Subject Title: SIM (Sulfide-Indole-Motility)
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

SIM (SULFIDE-INDOLE-MOTILITY)
Principle

1.  To determine the ability of an organism to liberate  hydrogen sulfide (H
2
S) from sulphur-bearing amino acids pr oducing a visible, black colour reaction.
2.  To determine the ability of  an organism to split indole from the tryptophan molecule.
3.   To determine if the organism is motile or  non-motile.

This test is used, in conjunction with others, for the  identification of  Enterobacteriaceae  when
unable to identify usin g VITEK or API system.

Reagents

Kovac's Reagent

Other Materials

SIM Medium.
Inoculating wire or sterile glass pasteur pipette.

Procedure

1.  With a pasteur pipette, draw up a smal l amount of previously inoculated TSB.
2.  Stab vertically into the medium to within 1/4 to 1/2 inch fr om bottom: withdraw inoculating
needle following lin e of inoculation.
3. Incubate O
2, 35
o
C X 18-24 hours.
4.  Add a few drops of Kovac's reagent and  observe for development of a red colour.

Interpretation

H2
S production
  (a)  Positive:  any blackeni ng of the medium      
  (b)  Negative:  no blackening


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Motility
 (a) Positive: motile organisms migrate from the stab line  and diffuse into the
medium causing  turbidity. They may exhibit fuzzy streaks of
growth.  (Compare with  an uninoculated tube.)
 (b) Negative: bacterial growth accentuated along stab  line; surrounding medium
remains clear.
Summary:

  Results are recorded as follows.  Remember that H
2
S is first, then  indole and finally
motility.
  -/-/- no H2
S, indole neg, non motile
  -/-/+ no H2
S, indole neg, motile
    -/+/-     no H
2
S, indole pos, non motile
    -/+/+     no H
2
S, indole pos, motile
  +/-/-    H2
S, indole neg, non motile
  +/-/+    H2
S, indole neg, motile
  +/+/-    H2
S, indole pos, non motile
  +/+/+   H2
S, indole pos, motile

  Refer to Manual of Clinical Microbio logy for specific organism reactions.

Precautions

1. An H
2
S-producing organism may exhibit blackening on SIM medium, but none on TSI
medium.
2. Some H
2
S inhibition occurs when  the temperature exceeds 34
o
C.
3.  Many bacteria are motile  at one temperature and non -motile when at another.
4.  If a motility test is difficult to interpret,  compare with an uninoculated motility tube.  If still
in doubt, perform a wet prep or hanging drop pr eparation using a heavy loopful of an 18-24
hr culture.

Quality Control

Quality control must be performed on each  new lot of SIM before bein g put into general use.
  K. pneumoniae (ATCC 13883):   -/-/-
  P. vulgaris (ATCC 13315):       +/+/+

References

1. MacFaddin JF, Bioche mical Tests for identifi cation of Medical Bacteria, 2nd ed., Williams
and Wilkins, Baltimore MD, 1980, p162-173, 173- 183, 214-218.

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Book on Biological Laboratory penalty 12

Book on Biological Laboratory penalty 12

 

 

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Policy #MI\TECH\32\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Pastorex Staph Plus Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

PASTOREX STAPH PLUS TEST 

Principle

A rapid slide latex agglutination test for the detection of clumping factor, capsular
polysaccharide and protein A produced by most strains of S. aureus .

Reagents and Materials

Pastorex test latex suspension (store refrigerated)
Disposable reaction cards
Plastic stick

Procedure

1.   Confirm the identification of a suspect Sta phylococcus by Gram stain and catalase test.
2.   Allow the latex reagent to warm to  room temperature before use.
3.   Shake the reagent so that all of the particles are resuspended.
4.   Dispense one drop of latex test reagent in one of the circles on the reaction card.
5.   Dispense one drop of negative control r eagent in another ci rcle on the card.
6.   Emulsify 1 to 3 colonies into the test latex with a loop for 10 seconds.
7.   Repeat step 6 for the negative control reagent.
8.   Gently rock the card for 30 seconds and look for clumping.
9.   Discard the card into a discard container.

Interpretation

Positive test:  Clumping within 20 seconds  with the test latex particles only.

Negative test:  No clumping in either latex.

Uninterpretable test:  Clumping in the negative control.

Precautions

1.   False positive results may occur after 40 seconds.
2.   False positive agglutination can occur with organisms other than staphylococci.

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Quality Control

Test known positive and negative controls daily.

 Positive: S. aureus   (ATCC 29213)
 Negative: S. epidermidis   (ATCC 12228)

References

1.  Pastorex Staph Plus package insert Feb. 1999.

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Policy & Procedure Manual
Policy #MI\TECH\33\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Plate Streaking Methods
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

PLATE STREAKING METHODS

Blood Agar and MacConkey Agar for Urine Cultures

1 uL disposable loop
Inoculate in one continuous streak down the middle of  the plate.  With the sa me loop, streak out the
entire plate at 90
o
 to the initial inocul um.  Streak a minimum of 15 lines.

          1o
 inoculum

         


Martin-Lewis Agar

Inoculate plate with specimen swab in a "Z" pattern across the plate (with continuous rotation of the
swab while inoculating).  Streak out the entire plate with a sterile loop at 90
o
 to the initial inoculum.
Streak a minimum of 15 lines.


          1o
 inoculum



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Isoplater Streaking

Manual Streaking

Inoculate specimen with swab or loop onto the entire first quadrant of the agar plate.  Use a sterile
loop and streak out the se cond, third and fourth quadrants as per diagram:
1  2


     Use a sterile loop




              3                    4
Growth Quantitation:

  + / -
  1 + 
  2 +
  3 +

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Section: Technical Manual  Subject Title: Pro-Amp Glu-Amp Tests
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

PRO-AMP GLU-AMP TESTS
Principle

Rapid chromogenic tests for the identi fication of pathogenic Neisseria.

Reagents

Pro-Amp tablets
Glu-Amp tablets
Fast Blue BB solution
Sterile Saline

Other Materials

Sterile Tubes (13 x 100mm)

Procedure

1.  Suspend the growth from Choc  media in 2 tubes of 0.25 ml sa line to achieve the turbidity >
#2 McFarland standard.
2.  Add 1 tablet to the respective tube.
3.  Incubate at 36
o
C x 4 hours.
4.  After incubation add 3 drops of Fast Blue BB solution to each tube and read results after 10
minutes.

Interpretation

 Positive: Orange/salmon colour
 Negative: Yellow colour

Organism     Glu-Amp    Pro-Amp

 N. gonorrhoeae  - +
 N. meningitidis  +           v


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Quality Control

Test with control organisms when test is run:

 N. gonorrhoeae (ATCC 43069)
 N. menigitidis (ATCC 13090)

Reference

1.  Pro lab package insert, February 1985.

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Policy #MI\TECH\35\v01  Page 1 of 2
Section: Technical Manual  Subject Title: PYR Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

PYR TEST
Principle

PYR (L-pyrrolidonyl- β-naphthylamide) impregnated disks serve as a substrate for the detection
of pyrrolidonyl peptidase.  Following the hydrolysis of the substrate by the enzyme the resulting
β -naphthylamine produces a red co lour upon the addition of cinnamald ehyde reagent.  This test
is used, in conjunction with others, for the identi fication of catalase negative, gram positive cocci
including Enterococci and Group A Streptococci.

Reagents

PYR discs
Cinnamaldehyde reagent (0.01% p-dimethylamino-cinnamaldehyde)
(disks and reagents are both in PYR kit)
Glass slide
Innoculating loop
Forceps
Sterile distilled water

Procedure

1.  Place a PYR disk onto a glass slide and moisten it with one drop of st erile distilled water.
2.  Rub a loopful of the culture onto the moistened disk holding it in place with sterile
forceps.
3.  Leave at room temperature for 2 minutes.
4.  After 2 minutes, add 1 drop of cinnamaldehyde reagent.

Interpretation

Positive:  Pink or cherry red colour within one minute

Negative:  No colour change or slight yellow colour

Quality Control

Test knows positive and negative controls each time an unknown is run.

  Positive:  Group A streptococcus  (ATCC 19615)
  Negative:  Group B strept ococcus  (ATCC 13813)

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Reference

1.  Carr-Scarborough Microbiologicals package insert 1990.

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Section: Technical Manual  Subject Title: Quantitation of Organisms &
Cells on Smears & Culture
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

QUANTITATION OF ORGANISMS & CELLS ON SMEARS & CULTURE

Microscopic:
          Report as:
   ±  --- <1 per oil immersion field            ±
   +  --- 1 - 5 per oil immersion field             +
  ++  --- 5 - 10 per oil immersion field          ++
 +++  --- >10 per oil immersion field               +++

Culture:
          Report as:
   ± --- few colonies in primary inoculum          scant growth
   + --- confluent growth in prim ary inoculum    light growth
  ++ --- growth up to 2nd quadrant            moderate growth
 +++ --- growth in or >3rd quadrant            heavy growth

Note:  Quantitation precedes identification.

Size  of colonies
  lg    -  large
  med   - medium
  sm    -  small
  tiny  -  tiny
  ppt   -  pinpoint

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Precautions

1.  E. coli O157:H7 and non-motile stra ins which produce verotoxi n are MUG test negative.

Quality Control

The following controls  are tested weekly:
       MUG   INDOLE

  E. coli (ATCC 25922)      +       +

  P. mirabilis (ATCC 12453)     -       -

Reference

1.  Prolab package insert

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Policy #MI\TECH\26\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Neisseria Identification
  Sugars
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

NEISSERIA IDENTIFICATION SUGARS
Principle

The test determines the ability of bacteria to produ ce acid products from carb ohydrates.  Used as a
method to identify Neisseria species and other fastidious organisms.

Materials

Cystine Proteose Peptone  Agar (CPPA) media: - glucose, maltose, lactose, sucrose, control (no
sugar).
Inoculating loop.

Procedure

1.  For each tube, scrape a full 3 mm loopful of growth from the surface of a 24 hour chocolate
agar subculture plate.
2. Deposit this inoculum  a few millimetres below the surface of the medium.
3.  Incubate at 35
o
C.
4.  Examine tubes after 1,  4 and 24 hours incubation.

Interpretation

 Positive: Yellow colour at top of tube
  Negative:  Red (alkaline) to orange (neutral) colour.

 Organism      Glu Mal Lac Suc Cont

N. gonorrhoeae     +   -   -   -   -
  N. meningitides     +   +   -   -   -
  M. catarrhalis       -   -   -   -   -

Precautions

1. Inoculum must be heavy.
2. False positive results may occur if tubes  are incubated in CO
2
.
3.  Tubes that appear bright  yellow should be gram stained to check for contamination.

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Quality Control

The following controls are run each time the test is performed:

 N. gonorrhoeae (ATCC 43069)
 N. meningitidis (ATCC 13090)
 M. catarrhalis (ATCC 8176)

Reference

1.  Murray PA, et al.  Manual of Clinical  Microbiology, 7th ed ., 1999; pp. 592-598.

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Policy #MI\TECH\27\v01  Page 1 of  2
Section: Technical Manual  Subject Title: ONPG Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

ONPG TEST
Principle

This test is used to demonstrat e the presence or absence of the  enzyme B-galactosidase using the
substrate ortho-nitrophenyl-D-galactopyranoside in order to differ entiate lactose-fermenting from
non lactose-fermenting organism s and in the identification of B. cepacia.

Reagents

ONPG disks (Store refrigerated)
Sterile saline

Other materials

Sterile tube (13 x 100 mm)
Bacteriology loop
Sterile graduated Pasteur pipette

Procedure

1.  Place an ONPG disk into a sterile tube and add  0.2 mL saline.
2.  Heavily inoculate the tube with a loopful of the test isolate.
3.  Incubate at 35
o
C for up to 4 hours.

Interpretation

Positive:  yellow colour within 4 hours

Negative:  colourless at 4 hours

Precautions

1.  A heavy inoculum is necessary to obtain a high concentration of enzyme.

Quality Control

Test with known positive and negative controls  each time the test is performed.
 Positive: E. coli    (ATCC 25922) 
 Negative: P. vulgaris (ATCC 13315)

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References

1.   MacFaddin JF, Biochemical Tests for Identification of Medi cal Bacteria, 2nd ed., Williams
and Wilkins, Baltimore MD., 1980, p120-128.

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Policy #MI\TECH\28\v01  Page 1 of  2
Section: Technical Manual  Subject Title: ONPG-Phenylalanine-
 Motility Medium (ONPG-PAM)
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

ONPG-PHENYLALANINE-MOTILITY MEDIUM (ONPG-PAM)
Principle

This test is used to determine an organism's motility, its ability to ferm ent lactose and produce
phenylalanine deaminase. The medium is primarily  used as a screening procedure for the detection
of enteric pathogens.

Reagents

ONPG-PAM tube
10% Ferric chloride

Other materials

Culture wire

Procedure

1.  Inoculate the ONPG-PAM tube by stabbing the centre of it to the bottom of the tube.
2.  Incubate the tube in O
2
 at 35
o
C X 18 hours.
3.  Read the tube for  ONPG, motility and indole.
4.  Add 2 drops of 10% ferric chloride so lution and read the ph enylalanine result.

Interpretation

 ONPG:    positive: yellow
     negative: no colour change

 Motility:   positive:  diffuse growth from  line of inoculum
     negative: growth does not spread from line of inoculum

 Phenylalanine (PPA):  positive: green
     negative: yellow/brown



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Quality Control

Test with control organisms each time  a new batch of meda is prepared.

 ONPG Motility PPA  K. pneumoniae  (ATCC 13883)  +  -  -
P. vulgaris (ATCC 13315)  -  +  +


References

1.   Murray, PA, et al.  1999.  Manual of Clinical Microbiology, 7
th
 ed., American Society for
 Microbiology, Washington, D.C.

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Section: Technical Manual  Subject Title: Optochin Sensitivity Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

OPTOCHIN SENSITIVITY TEST
Principle

This test is used to determine an organism's susceptibilit y to the chemic al optochin
(ethylhydrocupreine hydrochloride) fo r the presumptive identification of  S. pneumoniae .

Reagents

Bacto Optochin Disks (5 µg  disk) Store refrigerated
Mueller Hinton Sheep Blood Agar (MHSBA)

Other Materials

Culture loop
Forceps
Cotton swabs

Procedure

1.  Inoculate the suspected alpha haemolytic colony onto a MHSBA to ob tain confluent growth.

2.  Using aseptic technique place an optochin disk onto the surface of the inoculated agar.
Press down with forceps.

3.  Incubate at 35
o
C in CO2
 for 18-24 hours.

Interpretation

Susceptibile: Zone of inhi bition of at least 14 mm
Resistant: Zone of inhi bition less than 14 mm

Quality Control

Test with known susceptible and  resistant control  strains weekly:

 Susceptible: S. pneumoniae    (ATCC 6303)
  Resistant:  Viridans Strep.   (LPTP 8610)

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References

1.   MacFaddin JF, Biochemical Tests for Identification of Medi cal Bacteria, 2nd ed., Williams
 and Wilkins, Baltimore MD., 1980, p245-249.

2.  Difco package insert, July 1983.

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Section: Technical Manual  Subject Title: Oxidase (API Strip)
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

OXIDASE (API STRIP)
Principle

This test determines whether an isolate produces oxidase enzymes.  This test is mainly used, in
conjunction with other tests,  for the identification of gram negative organisms and Bacillus species.

Reagents

API Oxidase Reagent
1.  0.2% Aqueous ascorbic acid: Reconstitute ascorbic  acid with 25 ml ster ile distilled water.
This solution may be refrigerated for up to 28 days.  The expiry date mu st be written on the
bottle.

2. N,N,N,-Tetramethyl-p-phenylenediamine-dihydrochloride:
  Reconstitute with 5 ml of the  0.2% aqueous ascorbic acid.  It is recommended that this be
re-constituted 4-5 hours before use.  This solu tion may be refrigerated fo r up to 7 days at 2 -
8
o
C.  The expiry date must be written on the bottle.

Other Materials

API filter paper
API oxidase tray
Wooden applicator stick

Procedure

1.  Place a filter paper in the oxidase tray and moisten entire pape r with oxidase  reagent. Allow
to air dry. May be used for up  to 1 week.                          
2.  Transfer a portion of the  colony to the filter paper using a wooden applicator stick.
3.  Observe for 30 seconds.

Interpretation

Positive: Development of a purple colour within 30 seconds
Negative: No colour change

Precautions

Nichrome wire may cause false positive reactions.

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Quality Control

Test daily with known positive and  negative controls.

 Positive: P. aeruginosa      (ATCC 27853)
 Negative: K. pneumoniae   (ATCC 13883)

References

1.  API Oxidase package insert 3/80.

2.   MacFaddin JF, Biochemical Tests for Identification of Medi cal Bacteria, 2nd ed., Williams
 and Wilkins, Baltimore MD, 1980, p249-260.

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Section: Technical Manual  Subject Title: Oxidase
  (Spot Test Dropper)
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

OXIDASE (SPOT TEST DROPPER)
Principle

This test determines whether an isolate produces oxidase enzymes and is used for the identification
of Neisseria species isolated from primary plates.

Reagents

Spot Test dropper.  Store at room temperature.

Procedure

1.  Hold the dropper upright and squeeze gently  to crush the glass ampule inside the dispenser.

2.  Add 2 - 3 drops directly to the colonies to be tested  and observe for 30 seconds.

Interpretation

Positive:    Development of a purple colour within 30 seconds

Negative:   No colour change

Note:    Colonies which are positive must be  subcultured immediately since prolonged
exposure will result in death of the organisms.

Quality Control

Test daily with known positive and  negative controls.
  P. aeruginosa (ATCC 27853)  :  positive
  E. coli (ATCC 25922)   :  negative

References

1.  Difco Spot Text Oxidase  reagent package insert 1985.

2.   MacFaddin JF, Biochemical Tests for Identification of Medi cal Bacteria, 2nd ed., Williams
 and Wilkins, Baltimore MD, 1980, p249-260.

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SUMMARY OF RESULTS - 18- 24 HOUR PROCEDURE (cont'd)

TUBE INCUBATION POSITIVE NEGATIVE COMMENTS

After reading GLU reaction, add 2 drops 0.8% sulfanilic acid
and 2 drops 0.5% N. N-dimethyl-alpha-naphthylamine

GLU
NO2

N2
 gas
Red

Bubbles: Yellow
after reagents
and zinc
Yellow

Orange after
reagents and
zinc

(1)  Before addition of reagents, observe GLU
tube (positive or negative) for bubbles.
Bubbles are indicative of reduction of
nitrate to the nitrogenous (N
2
) state.
(2)  A positive reaction may take 2-3 minutes
for the red colour to appear.
(3)  Confirm a negative test by adding zinc
dust or 20 mesh granular zinc.  A pink-orange colour after 10 minutes confirms a
negative reaction.  A yellow colour
indicates reduction of nitrates to the
nitrogenous (N
2
) state.


After reading carbohydrate reaction, add 1 drop 1.5% H2 O2


MAN
INO
SOR
Catalase
 
Bubbles

No bubbles

(1)  Bubbles may take 1-2 minutes to appear. 
(2)  Best results will be obtained if the test is
run in tubes which have no gas from
fermentation.

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Section: Technical Manual  Subject Title: API Test Strips - API 20NE
Issued by:  LABORATORY MANAGER   Original Date: August 3, 2003
Approved by: Laboratory Director

Revision Date:


IDENTIFICATION OF NON-ENTERIC  GRAM-NEGATIVE RODS (API 20NE)

Principle

The API 20NE system facilitates the identificat ion of non-fastidious Gram-negative rods not
belonging to the  Enterobacteriaceae within 48 hours.

The API 20NE strip consists of microtubes contai ning dehydrated media and substrates. The media
microtubes containing conventional tests are in oculated with a bacterial suspension which
reconstitutes the media. After incubation, the metabolic end produc ts are detected by indicator
systems or the addition of reagents. The substr ate microtubes contain assimilation tests and are
inoculated with a minimal medium. If the bact eria are capable of utilizing the corresponding
substrate, then they will grow.

Materials

API 20NE strips - store at 2-8
0
C
0.85% sterile saline
Mineral oil
Zinc dust
AUX Medium                                  }
James Reagent                                 }
Nitrate 1 - store at 2-8
0
C                }                Store at 2-8
0
C
Nitrate 2 - store at 2-8
0
C                }
      Oxidase Reagent

Procedure
1.   Preparation of Inoculum
a)   Add 2 ml. of 0.85% saline  to a sterile test tube.
b)   Using a sterile inoculating loop, carefully touch the centre of a well isolated
colony (2-3 mm. Diameter) and thoroughly em ulsify in the saline. The suspension
turbidity should be equal to a 0.5 McFarland standard.

2.   Preparation of the Strip

a)   An incubation tray and lid are supplied for each strip.
b)   Dispense 5 ml of distilled water in to the tray.

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3.   Inoculation of the Strip

a)   Remove the cap from the tube containing the bacterial suspension and insert a
sterile pipette.
b)   Tilt the API 20NE incubation tray an d fill the TUBE section of the NO 3
 to PNPG
microtubes by placing the pipette tip  against the side of the cupule.
c)   Open an ampule of AUX Medium and add 200 uL of the bacterial suspension to
the ampule. Mix well with a pipette while  avoiding the formation of air bubbles.
d)   Using the AUX Medium bacterial suspension, fill both the TUBE and CUPULE
section of [GLU ] to [PAC ]. Do not overfill the cupules. Fi ll to a flat or slightly
convex meniscus.
e)   After inoculation, completely fill the CUPULE section of the 3 unde rlined tests,      
  GLU, ADH  and URE  tubes with mineral oil.
f)   Using the excess bacterial suspension, i noculate an agar slant or plate (non-selective media such as nutrient agar, blood ag ar or tryptic (tryp ticase) soy agar is
suggested) as a purity check and for  oxidase testing, and/or additional
biochemical testing.  Incubate the slant or plate with the API 20NE strip.

4.   Incubation of the Strip

a)   After inoculation, place the plastic lid on the tray and incubate the strip for 24
hours at 30
0
C in a non-CO 2
 incubator.

5.   Reading the Strip

a)   After 24 hours incubation, record all reactions not re quiring the addition of
reagents.
b)   Perform the oxidase test.

A portion of the growth from the agar slate or plate, inoculated from the 20NE
bacterial suspension, should be rubbed  onto filter paper to which a drop of
oxidase reagent (1% tetramethyl-p-phenylen ediamine dihydrochloride) has been
added.  The area where the growth has been  added will turn dark purple within 10
seconds if the reaction is positive and will be colourless or light purple if
negative.
  Note:  (a)  Nichrome wire loops should NOT be used in performing
the oxidase test.  Nichrome wire can cause a false  positivereaction.

(b)  The oxidase test should NOT  be performed using bacterial
growth from selective media  such as MacConkey, EMB, etc.


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c)   Assimilation tests are observed for bacterial growth. An opaque cupule indicates a
positive reaction.
d)   Protect the assimilation tests with the incubation tray lid during the reading of the
Nitrate
and TRP tests.
e)   Perform the Nitrate test.

i.   Add one drop of Nitrate 1 and one  drop of Nitrate 2 reagents to NO
3
cupule.
ii.   After 5 minutes a red color indicates a positive reaction.
iii.  A negative reaction may be due to th e production of nitrogen. Add Zinc
dust to the NO 3
cupule. After 5 minutes a colorless cupule indicates a
positive reaction. A pink-red cupule indicates a negative reaction.

f)   Perform the TRP test.

i.   Add one drop of JAMES Reagent.
ii.   The reaction takes place immediately, producing a pink color in the entire
cupule if the reaction is positive.

Interpretation

1.   Use the API 20NE analytical profile index.
 
2.   The tests are separated into groups of three.   The following numerical value is assigned to
each positive reaction recorded:

1 - positive reaction in the first test of the group
2 - positive reaction in the second test of the group
4 - positive reaction in the third test of the group

  By adding the values corresponding to posit ive reactions in each group, a seven digit
            number is obtained.

3.   The strip must be reincubated in the following cases:

i.   If the profile cannot be found in the Analytical Profile Index.
ii.   If the following note is indicated for the profile obtained:

IDENTIFICATION NOT VALID
BEFORE 48-HR INCUBATION
iii.  If the strip is to be reincubate d, remove the reagents from the NO3
and
TRP cupules and then cover these tests with mineral oil.


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iv.  Reincubate the strip for another 24 hours at 30
0
C in a non-CO 2
 incubator.
v.   Read all the tests again, except for NO3,
 TRP and GLU.

READING TABLE

TESTS SUBSTRATES REACTONS/ENZYMES NEGATIVE
RESULTS
POSITIVE
RESULTS
NITrate reduction to
nitrites
NIT 1 + NIT 2 / 5 min
        colourless                   pink-red
N03
 Potassium
nitrate
NITrates to nitrogen  Zn / 5 min
       pink                      colourless
TRP tryptophane  indole production  JAMES / immediate
       colourless /                    pink
pale green / yellow
GLU  glucose  Acidification  blue to green  yellow
ADH arginine  arginine dihydrolase yellow orange/pink/red
URE  urea  Urease  yellow  orange/pink/red
ESC esculin  hydrolysis ( β -glucosidase)  yellow grey/brown/black
GEL gelatine
(with India ink)
hydrolysis (protease)  no pigment
diffusion
diffusion of black
pigment
PNPG  p-nitrophenyl- β -D-galactopyranoside
β -galactosidase  colourless




MUG negative: Colourless


 Indole positive: Red colour after addition of Kovac's
  Indole negative:  Kovac's remains yellow

 MUG   INDOLE   INTERPRETATION / ACTOIN

  +     +   report as E. coli
  -     +   set up VITEK  Identification
  +     -   set up VITEK  Identification
  -     -   set up VITEK  Identification

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c)   If the GLU is positive (yellow):

i.   Perform the oxidase test.

A portion of the growth from the agar slate or plate, inoculated from the
20E bacterial suspension, should be  rubbed onto filter paper to which a
drop of oxidase reagent (1% tetramethyl-p-phenylenediamine
dihydrochloride) has been added.  Th e area where the growth has been
added will turn dark purple within 10  seconds if the reaction is positive
and will be colourless or light purple if negative.

Note:  (a)  Nichrome wire loops should NOT be used in performing
  the oxidase test.  Nichrome wire can cause a false positive
reaction.

  (b)  The oxidase test should NOT  be performed using bacterial
  growth from selective media such as MacConkey, EMB,
etc.

Note:  (a)  Before addition of reagents, observe GLU tube (positive or
  negative) for bubbles.

(b)  The nitrate reduction and indole tests must be performed
last since these reactions re lease gaseous products which
interfere with the interpretati on of other tests on the strip.
The plastic incubation lid shoul d not be replaced after the
addition of these reagents.

ii.   Add the reagents to TDA and VP tubes.  If positive, the TDA reactions
will be immediate, whereas the VP reaction may be delayed up to 10
minutes.

iii.  The Kovacs' reagent should then be added to the IND tube.  

iv.  The Nitrate Reduction test should be  performed on all oxidase positive
organisms.  The reagents should be added to the GLU tube after the
Kovacs Reagent has been added to the IND tube.

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Interpretation

a)   Use the API 20E analytical profile index.  (For  18-24 hour tests, use white pages.  For 36-48 hour tests, use blue pages.)

b)   The tests are separated into groups of three.   The following numerical value is assigned to
each reaction recorded:

1-   positive reaction in the fi rst test of the group
2-   positive reaction in the second test of the group
  4-  positive reaction in any test
0-   negative reaction in any test

Reference

1.  Murray P.A., et al.  Manual of Clinical Microbiology, 7
th
 ed., 1999.

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SUMMARY OF RESULTS - 18-24 HOUR PROCEDURE

TUBE INCUBATION POSITIVE  NEGATIVE  COMMENTS
ONPG  Yellow Colourless (1)   Any shade of yellow is a positive
reaction.
(2)  VP tube, before the addition of reagents,
can be used a negative control.

ADH 18-24 hr
36-48 hr
Red or Orange
Red
Yellow
Yellow or Orange
Orange reactions occurring at 36-48 hours
should be interpreted as negative.

LDC 18-24 hr
36-48 hr
Red or Orange
Red
Yellow
Yellow or Orange
Any shade of orange within 18-24 hours is a
positive reaction.
At 36-48 hours, ora nge decarboxylase
reactions should be interpreted as negative.

ODC 18-34 hr
36-48 hr
Red or Orange
Red
Yellow
Yellow or Orange
Orange reactions occurring at 36-48 hours
should be interpreted as negative.

CIT  Turquoise or
Dark Blue
Light Green
Or Yellow
(1)  Both the tube and cupule should be filled.
(2)  Reaction is read in the aerobic (cupule)
area.

H2
S    Black Deposit  No Black Deposit  (1)   H2
S production may range from a heavy
black deposit to a very thin black line
around the tube bottom.  Carefully
examine the bottom of the tube before
considering the reaction negative.
(2)  A "browning" of the medium is a
negative reaction unless a black deposit
is present.  "Browni ng" occurs with TDA
positive organisms.

URE 18-24 hr
36-48 hr
Red or Orange
Red
Yellow
Yellow or Orange
A method of lower sensitivity has been chosen .
Klebsiella,  Proteus and  Yersinia routinely give
positive reactions.

TDA  Add 1 drop 10% Ferric chloride.

Brown-Red

Yellow
(1)  Immediate reaction.
(2)  Indole positive organisms may produce a
golden orange colour due to indole
production.  This is a negative reaction.

IND  Add 1 drop Kovacs Reagent

Red Ring

Yellow
(1)  The reaction should read within 2
minutes after the addition of the Kovacs
reagents and the  results recorded.
(2)  After several minutes, the HCl present in
Kovacs reagent may react with the plastic
of the cupule resulting in a change from a
negative (yellow) colour to a brownish-red.  This is a negative reaction.


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SUMMARY OF RESULTS - 18-24 HOUR PROCEDURE (cont'd)

TUBE INCUBATION POSITIVE NEGATIVE COMMENTS
Add 1 drop of 40% Potassium Hydroxide, then 1 drop of alpha-napthol.
VP
 Red Colourless
(1)  Wait 10 minutes before considering the
reaction negative.
(2)  A pale pink colour which appears
immediately after the addition of reagents
but which turns dark `pink or red after 10
minutes should be interpreted as positive.
Motility may be observed by hanging drop or
wet mount preparation.

GEL    Diffusion of the
pigment
No diffusion  (1)  The solid gelatin particles may spread
throughout the tube after inoculation.
Unless diffusion occurs, the reaction is
negative.
(2)  Any degree of diffusion is a positive
reaction.



GLU





MAN
INO
SOR
RHA
SAC
MEL
AMY
ARA










Yellow
Or Gray





Yellow


Blue or
Blue-Green





Blue or
Blue-Green

COMMENTS FOR ALL CARBOHYDRATES

Fermentation
(Enterobacteriaceae, Aeromonas, Vibrio)
(1)  Fermentation of the carbohydrates begins
in the most anaerobic portion (bottom) of
the tube.  Therefore, these reactions should
be read from the bottom of the tube to the
top.
(2)  A yellow colour at the bottom of the tube
only indicates a weak or delayed positive
reaction.

Oxidation (Other Gram-negatives)
(1)  Oxidative utilization of the carbohydrates
begins in the most aerobic portion (top) of
the tube.  Therefore, these reactions should
be read from the top to the bottom of the
tube. 
(2)  A yellow colour in the upper portion of the
tube and blue in the bottom of the tube
indicate oxidative utilization of the sugar.
This reaction should be considered
positive only for non- Enterobacteriaceae
gram negative rods.  This is a negative
reaction for fermentative organisms such
as Enterobacteriaceae.


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Procedure

1.   Preparation of Inoculum

a)   Only pure cultures of a single organism should be used (heavily inoculated sheep
BAP x 3; incubate for 24 hours at 35
0
C in 5% CO 2
).

b)   Using a sterile swab, harvest all the culture from 3 BAP and inoculate into 3 ml.
    sterile saline to give a turb idity of at least McFarland #6.

2.   Preparation of the Strip

a)   An incubation tray is supplied for each strip.

b)   Dispense 5 ml of water into the wells of the tray.

3.   Inoculation of the Strip

a)   Inoculate tests 1  → 11 of the strip (NIT to GEL).

b)   Avoid bubbles by tilting the strip slightly forward while  placing the pipette tip on
the side of the cupule.

c)   Add 3 drops into each cupule for tests NIT to ES.

d)   For the URE test fill the tube portion only.

e)   For the GEL test, fill  both the tube and cupule.  Then: 

f)   For the last nine tests of the strip (O to GLYG  transfer the rest of the bacterial
suspension  to an ampoule of GP medium .  Mix well.

g)   Distribute the new suspension into the tubes only of tests O to GLYG

h)   Overlay cupules URE  and O  to GLYG with mineral oil, forming a slight convex
meniscus.

i)   Cover with incubation lid and incubate the strip for 24 hours at 35
0
C (non-CO2
).

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Interpretation
REACTIONS TABLE

TESTS REACTONS NEGATIVE
RESULTS
POSITIVE
RESULTS
NIT NITrate reduction  NIT A + NIT B  (10 mn)
   Colourless Very pale pink
Dark pink
Red
PYZ PYraZinamidase  PYZ  (10 mn)
   Colourless Very pale brown
Very pale orange
Brown
Orange
PyrA Pyrrolidonyl Arylamidase  ZYM A  +ZYM B
    PyrA  → B
NAG (10 mn)
   Colourless Pale orange
Orange
PAL Alkaline Phosphatase Colourless
Beige-pale purple
Pale orange

Purple
β GUR  Beta GlucURonidase  Colourless
Pale grey
Pale beige

Blue
β GAL  Beta GALactosidase  Colourless
Beige-pale purple
Purple

∝  GLU
Alpha GLUcosidase  Colourless
Beige-pale purple
Pale green

Purple
BNAG N-Acetyl-B Glucosaminidase Colourless
Beige-pale purple
Pale brown
Pale grey

Brown



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REACTIONS TABLE (Cont'd)

TESTS REACTONS NEGATIVE
RESULTS
POSITIVE
RESULTS
ESC  ESCulin ( β  Glucosidase)
Colourless
Grey
Black
URE  UREase  Yellow Orange
Red
Pink
[GEL]  GELatine (hydrolysis)  No diffusion
of  black pigment
Diffusion of
black pigment
O
GLU
RIB
XYL
MAN
MAL
LAC
SAC
GLYG
Control (Fermentation )
GLUcose }
RIBose }
XYLOSE }
MANnitol }  Fermentation
MALtose }
LACtose }
Sucrose }
GLYcoGen }



Red

Orange



Yellow

Yellow-orange
CAT  CATalase (ESC or GEL test)  H 2 O2 
3% 1 min
   No bubbles Bubbles 
References

1.   Coyle, Marie B., Benjamin Lipsky.  1990.  Cor yneform Bacteria in Infectious Diseases:
Clinical and Laboratory Aspects.  Clinical Microbiology Reviews.  3:227-246.

2.   Freney, J.M.T.  Duperron, C. Couturier, W. Hansen, F. Allard, J.M. Boueufgras, D.
Monget, J. Fleurette.  Evaluation of API Coryne in Comparison with Conventional
Methods for Identifying Coryneform Bacteria, Journal of Clinical  Microbiology, January
1991, Vol. 29, p. 38-41.

3.   Murray P.A., et al.  Manual of Clinical Microbiology, 7
th
 ed., 1999.


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Section: Technical Manual  Subject Title: API Test Strips - API 20E
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

IDENTIFICATION OF  ENTEROBACTERIACEAE  (API 20E)
Principle

The API 20E system facilitates the 24-hour identification of Enterobacteriaceae  as well as 24 or
48-hour identification of othe r Gram negative bacteria.

The API 20E strip consists of microtubes containing dehydrated substrates for the demonstration
of enzymatic activity and carbohydrate (CHO) fermen tation.  The substrates are reconstituted by
adding a bacterial suspension.  After incubation, the metabolic end products are detected by
indicator systems or the addition of reagents.  CHO fermentation is detected by colour change in
the pH indicator.

Materials

API 20E strips - store at 2-8
0
C
0.85% sterile saline
Nitrate A - store at 2-8
0
C
Nitrate B - store at 2-8
0
C
Mineral oil
Zinc dust
Kovacs Reagent      
Voges - Proskauer Reagents    
Ferric Chloride         Store at 2-8
0
C 
H2 O2   

Oxidase Reagent  

OF Dextrose        ID of non-
Motility Medium         Enterobacteriaceae

Procedure
1.   Preparation of Inoculum
a)   Add 5 ml. of 0.85% saline  to a sterile test tube.
b)   Using a sterile inoculating loop, carefully touch the centre of a well isolated
colony (2-3 mm. Diameter) and thoroughly emulsify in the saline.

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2.   Preparation of the Strip

a)   An incubation tray and lid is supplied for each strip.
b)   Dispense 5 ml of water in to the tray.

3.   Inoculation of the Strip

a)   Remove the cap from the tube containing  the bacterial suspension and insert a 5
ml.  Pasteur pipette.
b)   Tilt the API 20E incubation tray and fill the tube section of the microtubes by
placing the pipette tip against the side of the cupule.

Note:   The ADH , LDC , ODC , H2S , AND URE  reactions can be interpreted best
if these microtubes are slightly underfilled.
c)   Fill both the TUBE and CUPULE section of [CIT ], [VP ] and [GEL ] tubes.
d)   After inoculation, completely fill the cupule section of the ADH , LDC , ODC ,
  H2S and URE  tubes with mineral oil.
e)   Using the excess bacterial suspension, i noculate an agar slant or plate (non-selective media such as nutrient agar, blood ag ar or tryptic (tryp ticase) soy agar is
suggested) as a purity check and for oxida se testing, serology, and/or additional
biochemical testing.  Incubate the  slant or plate for 18-24 hours at 35
0
C.

4.   Incubation of the Strip

a)   After inoculation, place the plastic lid on the tray and incubate the strip for 18-24
hours at 35
0
C in a  non-CO2
 incubator.

b)   Weekend incubation:  The biochemical reactions of the API 20E should be read
after 18-24 hours incubation.  If the strips cannot be read after 24 hours
incubation at 35
0
C, the strips should be removed from the incubator and stored at
2-8
0
C (refrigerator) until the reactions can be read.

5.   Reading the Strip

a)   After 18 hours of incubation and before 24 hours incubation, record all reactions
not requiring the addition of reagents.

b)   If the GLU tube is negative  (blue or green), do not add reagents.  Reincubate a
further 18-24 hours.

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