Book on the seventh penalty Biological Laboratory

Book on the seventh penalty Biological Laboratory

 

 

Page 67

TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\22\v01  Page 1 of  2
Section: Technical Manual  Subject Title: KOH String Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

KOH STRING TEST
Principle

The formation of a string  (DNA) in 3% KOH indicates that the  isolate is a gram negative organism.

Reagents

3% KOH

Other Materials

Glass slides
Culture loop

Procedure

1.  Place a drop of 3%  KOH onto a  glass slide.

2.  Emulsify in KOH a loopf ul of the culture from a BA  incubated for 18-24 hr.

3.  Continue to mix th e suspension for 60 sec and by slowly  lifting the loop, observe for the
formation of a string.

Interpretation

Positive:  formation of a string within  60 seconds

Negative:  failure to form a string

Precautions

1.  False positive and false negative results may occur.

Quality Control

Known controls should be tested  each time the test is performed.
 Positive: P. aeruginosa (ATCC 27853)
 Negative: S. aureus       (ATCC 25923)


PROCEDURE MANUAL
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Policy & Procedure Manual
Policy # MI\TECH\22\v01  Page 2 of  2
Technical Manual   

References

1.  Murray PA et al.  Manual  of Clinical Microbiolog y, 7th ed., 1999; p. 1671.

PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 

Page 69

TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\23\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Lap Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

LAP TEST
Principle

LAP (Leucine-β -naphthylamide) impregnated disks serve  as a substrate for the detection of
Leucine aminopeptidase.  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 usually used, in conjunction wi th other tests, for the identifi cation of streptococci and other
catalase negative gram positive cocci.

Reagents

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

Procedure

1.  Place a LAP 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 5 minutes.
4.  After 5 minutes, add 1 drop of cinnamaldehyde reagent.

Interpretation

Positive:  red colour within one minute

Negative:  no colour change or slight yellow colour

Quality Control

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

 Positive: E. faecalis  (ATCC 29212)
 Negative: Leuconostoc (ATCC 8923)

PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 

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Policy & Procedure Manual
Policy # MI\TECH\23\v01  Page 2 of  2
Technical Manual   

Reference

1.  Carr-Scarborough Microbiologicals package insert 1991.

PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 

Page 71

TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\24\v01  Page 1 of  3
Section: Technical Manual  Subject Title: Motility Test Medium
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

MOTILITY TEST MEDIUM
Principle

Motility Test Medium is a semi-solid agar  designed to demonstr ate motility by diffusion.

Motility Test Medium is a modification of the formula of Tittsler and Sandhoizer.  The medium
contains small amounts of agar and gelatin, as well as  triphenyltetrazolium chlo ride (TTC).  TTC is
a soluble compound which is taken  up by the bacterial cells.  Once the substance has been absorbed
by the bacteria, it is reduced, releasing the acid formazan, a hi ghly pigmented red, insoluble
compound.

Organisms are stabbed into the medium with an inoculating  wire.  If the organisms are motile, they
will diffuse into the soft medium laterally from the  line of inoculation, resulting in a diffuse, pink
color throughout the medium.  Nonmotile organisms grow along the line of inoculation only,
producing a pinkish-red line with no diffusion.

Storage

Upon receipt store at 2-8
0
C away from direct light.  Media should not be used if there are signs of
contamination, deterioration (shrinking or discolor ation), or if the expira tion date has passed.

Limitations

Motility tests often show a false-negative reaction.  The organism may be  weakly motile, or the
flagella may be damaged  due to heating, shaking, or other trauma.  A hang ing drop motili ty may be
performed from an inoculated tryptone broth incubated for 2-4 hours to co nfirm motility  results.
Consult appropriate microbiolog ical texts for procedure.

TTC may be inhibitory to some fastidious bacteria.

Most motility of bacteria should be inte rpreted at 35
0
C: however, certain bacteria such as Yersinia
enterocolitica demonstrate the  best motility at 25
0
C. 

PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 

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Policy & Procedure Manual
Policy # MI\TECH\24\v01  Page 2 of  3
Technical Manual   

Organisms that require oxygen for growth, such as  Pseudomonas aeruginosa, will produce a
spreading film on the surface of the medium, and will not fan  out from the inoculation line where
oxygen is depleted.

Procedure

1. Tube Method

Prior to inoculation, the medium  should be brought to  room temperature.  Inoculate selected
colonies of a pure 18 to 24 ho ur culture, or from a turbid broth culture 4-8 hours old.  Using
a straight needle, stab  the center of the medium  about 1/4" from the to p.  Incubate the tubes
with the caps loose at 35
0
C (see "Limitations") for 18-24 hours.   Observe for motility.

2. Plate Method

If using a multipoint inoculation  system, make a pour plate form  the 18 ml tube by gently
melting the agar in a boiling water bath and dispensing the liquid medium  into a sterile petri
dish.  Prepare the inoculum by touching the top of one  or tw o well isolated colonies and
inoculating into a broth.  Stab the inoculum into the medium using th e modified pins of a
replicator or by using a straight needle.  Incubate aerobically at 35
0
C (see "Limitations") for
16-18 hours.  Examine fo r the presence of a pink diffusion fr om the point of inoculation.  

Interpretation

Positive: A diffuse  pink color occurring  throughout the medium.
Negative:  A pinkish red line at th e stab site with no diffusion.


PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 

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Policy & Procedure Manual
Policy # MI\TECH\24\v01  Page 3 of  3
Technical Manual   

Quality Control

Test the following positive and negative control organisms each time the test is performed:

Positive:  Escherichia coli  (ATCC 25922 )
Negative:  Klebsiella pneumoniae   (ATCC 13883)

References

1.   Finegold, S.M., and E. J. Baron, Bailey  and Scott's Diagnostic Microbiology, 7
th
 ed.,
C.V. Mosby, St. Louis, 1986.  Koneman, E.W., et al., Color Atlas and Textbook of
Diagnostic Microbiology, J.B. Lippincott, Philadelphia,  1979.  Lennette, E.H., et al.,
Manual of Clinical Microbiology, 4
th
 ed., American Society for Microbiology,
Washington, D.C., 1985.

2.   MacFaddin, J.F., Biochemical Tests for Identification of Medical Bacteria, Williams and
Wilkins, Baltimore, 1980.  Tittsler R.P., and L.A. Sandhoizer, J. Bacteriol., 31:575, 1936.


PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 

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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\25\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Mug Test (PGUA)
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

MUG TEST (PGUA)
Principle

If an organism produces the enzyme glucur onidase it will break down the substrate ortho-nitrophenyl-beta-glucuronide liberating the ortho-nitrophenyl producing a yellow colour.  This test
is used, in conjunction with others, for  the identification of  E. coli.

Reagents and Materials

 PGUA tablets
 13x100mm tubes
 Tryptone water
 Kovac's reagent

Procedure

1.  Prepare a dense suspension of the test organi sm (lactose-fermenter only) in 0.25 mL of the
tryptone water.
2.  Add 1 PGUA tablet to the tube.
3.  Incubate at 36
o
C for 4 hours.
4.  Examine the tube for development of a yellow colour.
5.  Add 1 drop of Kovac's Indo le reagent and observe for the  development of a red colour.

Interpretation

 MUG positive:  Yellow colour







PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 

Page 9
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\02\v01  Page 4 of 5
Technical Manual   


SPS Disk for Differentiation of Anaerobic Cocci

Principle

Sodium polyanethol sulfonate ( SPS), a commonly used anticoagulan t, inhibits certain bacteria
such as Peptostreptococcus anaerobius and the aerobe  Gardnerella vaginalis .  Paper disks
impregnated with 5% SPS can be used as a tool for differentiating  P. anaerobius from other
anaerobic cocci.

Materials

A.   Reagents

1.   SPS Disks

a.   Combine the following in a flask.
    SPS   5 g
    Distilled water  100 ml
b.   After dissolving SPS, sterilize the mixture by filtration (0.22 µ m pore size
   filter).
c.   Dispense 20 µl onto sterile 1/4-inch diameter filter paper disks that are
spread inside empty, sterile petri dishes.  Allow these to dry for 72 hours
at room temperature.
d.   Store the disks at room temperature, and label with an expiration date of 6
   months.

2.   SPS disks are also commercially availa ble (Anaerobe Systems, Difco, Oxoid,
Remel).  Store as indicated by the manufacturers. 
3.   Brucella or other anaerobic blood agar plate.

B.   Supplies

1.   Single-disk dispenser or forceps
2.   Ruler (divided into millimeters)

Procedure

1.   Allow the container with disks to reach room temperature before use.
2.   Subculture the isolates on a BAP.  To ensure  an even, heavy lawn of growth, streak the
first quadrant back and forth se veral times.  Streak the other  quadrants to yield isolated
colonies.

PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 

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Policy & Procedure Manual
Policy # MI\TECH\02\v01  Page 5 of 5
Technical Manual   

3.   Place the SPS disk on the first quadrant.
4.   If you have several organisms to test, first streak all the plates and  then add the disks to
them at the same time.  You can use one plate for up to four tests.
5.   Incubate the plate(s) anaer obically for 48-72 hours at 35-37
0
C.
6.   Examine for a zone of inhibition of growth around the disk. 

Interpretation

A.   Susceptible: Zone of inhibition of  ≥  12mm

P. anaerobius usually gives a very large zone of inhibition ( ≥  16 mm), whereas other
anaerobic cocci that appear susceptible to SPS give smaller zones.  To presumptively
identify  P. anaerobius , you must also consider the  Gram stain, typical colonial
morphology, and odor.  Some strains of P. micros may be susceptible to SPS.  Examine
the Gram stain for the small cell size of P. micros  and chaining characteristic of  P.
anaerobius.

B.   Resistant: Zone of inhibition of <12 mm.

Quality Control

A.   Test each lot upon receipt and monthly thereafter.
B.   Test  P. anaerobius ATCC 27337 and  Peptostreptococcus asaccharolyticus  ATCC 29745
as described below under Procedure.  The results should show the following:
1.  P. anaerobius:   susceptible  to  SPS
2.  P. asaccharolyticus : resistant to SPS
C.   Record the results on a QC log.

PROCEDURE MANUAL
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Policy & Procedure Manual
Policy #MI\TECH\03\v02  Page 1 of  2
Section: Technical Manual  Subject Title: Anaerobic/Campylobacter
  Jar Set Up
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

ANAEROBIC/CAMPYLOBACTER JAR SET UP

Anaerobic Jar

1.   Anaerobic plates are kept in the nitrogen holding box until there is enough for a jar or
until the end of the day.

2.   Place the inoculated plates (max 14), biologi cal indicators and anaerobic indicator strip
into an empty anaerobic jar.

3.   Tear open an AnaeroGen foil sachet at the tear-nick indicated and remove the Anaero
Gen paper sachet from within.

4.   Immediately place the paper sachet in  the jar down the side of the plates.

5.   Place the lid on the jar (no catalyst re quired) and seal with the clamp.
Note:  The time between opening the foil sachet and sealing the jar should not exceed
 one minute.
Note:  Jar and lid must be labelled with the same number.

6.   Label jar with date and place in walk in incubator.

Control Testing

An anaerobic indicator is added to each jar as it is set up to visually check that anaerobic
conditions have been achieved and maintained.  Check the jar after 2 hours incubation to make
sure the indicator does not indicate oxygen present.

Biological Indicator

Inoculate a quarter anaerobic plate w ith the following test organisms:
  Bacteroides fragilis  ATCC 25285: growth
  Clostridium perfringens ATCC 13124: growth
  Clostridium difficile ATCC 9089: growth
  Pseudomonas aeruginosa ATCC 27853: no growth

Record results on the anaerobic jar QC sheet.

PROCEDURE MANUAL
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Policy & Procedure Manual
Policy # MI\TECH\03\v02  Page 2 of 2
Technical Manual   

Campylobacter Jar

1.   Campylobacter plates  are kept in the CO
2
 incubator until there is enough for a jar or until
4 p.m. (Any late cultures will be set up at the end of the shift).

2.   Place a dampened paper towel into the bottom of an anaerobic jar.

3.   Place the inoculated plates (max 14) into the ja r.  Include a plate fr eshly inoculated with
the control organism.

4.   Tear open an CampyGen foil sachet at the te ar-nick indicated and remove the CampyGen
paper sachet from within.

5.   Immediately place the paper sachet in  the jar down the side of the plates.

6.   Place the lid on the jar (no catalyst re quired) and seal with the clamp.
Note:  The time between opening the foil sachet and sealing the jar should not exceed
 one minute.
Note:  Jar and lid must be labelled with the same number.

7.   Label jar with date and place in walk in 42
o
C incubator.

Biological Indicator

Inoculate a campylobacter agar plate  with the following test organism:

Campylobacter jejuni  ATCC 29428: growth

Record results on the anaerobic jar QC sheet.

Note:  The technologist on the enteric bench is re sponsible for the daily subculturing of the
  control organism (3 new plates).  One newl y subcultured plate will be incubated with the
  reincubate culture jar.  The old control  plate and the remaining 2 newly subcultured
  plates will be kept in the CO
2
 incubator until the end of the day.

  The 2 new subcultured plates are for setting up new jars.  If more are needed, the
  technicians will subculture new plates from the old control plate.

PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 

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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\04\01\v01  Page 1 of  4
Section: Technical Manual  Subject Title: API Test Strips - API 
 CORYNE
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

IDENTIFICATION OF  CORYNEBACTERIUM (API CORYNE)

Principle

The API CORYNE system facilitates the 24 hour identification of C. jeikeium (CDC Group JK),
other medically important Corynebacteria, Rhodococcus equi,  Listeria species,  Erysipelothrix
rhusiopathiae , Actinomyces pyogenes, Arcanobacterium haemolyticum,  Brevibacterium
species and  Gardnerella vaginalis .

The API CORYNE strip consists of 20 microtubes containing dehydrated  substrates for the
demonstration of enzymatic act ivity or the fermentation of carbohydrates (CHO).  The addition
of a dense test suspension of bacteria rehydrates the enzymatic substrates.  The metabolic end
products produced during incubati on are detected through spontaneous coloured reactions or by
the addition of reagents.

The fermentation tests are inoculated with an enrichment medium (containing pH indicator)
which reconstitutes the CHO substrates.  Fermentation of CHO is detected by colour change in
the pH indicator.

Materials

API Coryne strips - store at 2 - 8
0
C
GP medium - store at 2 - 8
0
C
McFarland Standard #6
Nitrate A - store at Room Temperature
Nitrate B - store at 2 - 8
0
C
Zym A - store at 2 - 8
0
C in the dark
Zym B - store at 2 - 8
0
C in the dark
PYZ - store at 2 - 8
0
C in the dark
H2 O2
 - store at 2 - 8
0
C
Mineral oil
Sterile saline 3 ml

PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 
DEPARTMENT 
 

A book about the sixth penalty Biological Laboratory

A book about the sixth penalty Biological Laboratory

 

 

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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\17\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Gonogen
 (GC Coagglutination) Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

GONOGEN (GC COAGGLUTINATION)  TEST
Principle

The Gonogen II test is a coagglutination test for the confirmato ry identification of  N. gonorrhoeae.

Reagents

 I:  Buffer
  II: Gonogen reagent (antibodies)
 Positive control reagent
 Negative control reagent

Other Materials

 Test tray: consists of wells with special
  matrix and absorbent material
  Glass tubes (12 x 75mm) (not provided)
  Glass dropper rod assembly
  Plastic transfer pipets

Procedure

1. Preparation of sample

  a)  In a 12x75 mm tube dispense 500  µL of reagent I (buffer) using the glass dropper
rod assembly provided  (demarcation line).

  b)  Using a swab, make a suspension of approximately 30 colonies to match a
McFarland 1 turbidity standard.

  c)  Press swab against side of tube to express as much liquid as possible.

  d)  Vortex reagent II an d add 1 drop to the tube.

  e)  Mix and set sit fo r at least 5 minutes.

PROCEDURE MANUAL
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2. Test

  a)  With a plastic transfer pi pet, add 2 drops of each test suspension into a well of the
test tray using a separa te well for each test.

  b)  using a clean plastic transfer pipet,  add 1 drop of reagent  I (buffer) to each
completed test well.
Interpretation

  Positive:  Pink to red do t in well of test tray.

  Negative:  White to pale pink dot in well of test tray.

  Note:  1.  A colour reaction more intense th an the negative control should be
interpreted as positive.

   2. If color reaction is questionable, reincubate tube at RT for 3 minutes
and repeat test.

   3. If specimen suspension is made too turbid a faint background colour
will occur.  This should NOT  be interpreted as  a positive result.

Quality Control

The positive and negative controls must be tested whenever a test is run.  The test is performed in
the same manner except 1  drop of the control reagent is added to 500  µL of buffer rather than a
suspension of the test organism.

References

1. Gonogen II package insert, October 1993.

PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 

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Policy & Procedure Manual
Policy # MI\TECH\18\v01  Page 1 of  2
Section: Technical Manual  Subject Title: High Level
  Aminoglycoside Testing
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

HIGH LEVEL AMINOGLYCOSIDE TESTING
Principle

Enterococcal species where an identification and  sensitivity has been performed must be tested
for resistance to vancomycin and high level gentamicin and streptomycin (HLAR).

Materials

Entero HLAR Bi-plates
Brain Heart Infusion Agar  (BHI) - Control Plates

Procedure

1.   Using the VITEK colorimeter, prepare a 0.5 McFarland suspension in sterile saline
(inoculum from VITEK can be used).

2.   Using a sterile swab, spot inoculate the suspension onto each half of the plates.  Three
organisms can be tested on each plate.

3.   After the inocula has dried, incubate the plate at 35
o
C for up to 48 hours.

Interpretation

Check the control plate for adequate growth.  Then check the drug pl ates for absence or presence of
growth; any growth is considered sign ificant.  Read plates at 24 hours  and record results.  If there is
no growth on the streptomycin  plate, reincubate  plate for an add itional 24 hours.

PROCEDURE MANUAL
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Quality Control

  Control strains are tested daily.

      Expected results
      C G  S   

 E. faecalis (ATCC 19966)    +  +   -  
 E. gallinarum (ATCC 35038)    +   -   -  
  E. casseliflavus (ATCC 12755)    +   -   +  

  C = Growth Control; G = Gent amicin; S = Streptomycin; + = growth; - = no growth 

Reporting Results

Blood cultures and sterile fluids -- - Report with canned comment (Ref er to Susceptibility Testing
Manual).

Urines and other sites  --- Do not report HLAR.

Reference

1.  PML Technical Manual data sheet No. 323, Nov. 1993.

PROCEDURE MANUAL
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Policy & Procedure Manual
Policy # MI\TECH\19\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Hippurate Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

HIPPURATE TEST
Principle

This test determines the  ability of bacteria to hydrolyse sodium hippurate. One of the end products,
glycine is detected by the addition of ninhydrin reagent.

Reagents

Hippurate disks (store refrigerated)
Ninhydrin reagent
Sterile distilled water

Other Materials

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

Procedure

1.  Place a Hippurate disk into a sterile  tube and add 0.4 mL sterile water.

2.  Heavily inoculate the tube with a loopful of the test organism.

3.  Incubate at 35
0
C for 2 hours.

4.  Following incubation add 5  drops of ninhydrin reagent to the tube and shake gently.

5.  Reincubate tube for 10 minutes and read reaction.

Interpretation

Positive:   Deep purple-blue colour

Negative:   No colour change or light purple

PROCEDURE MANUAL
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Precautions

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

2.  Do not incubate longer than 30 minutes after addition of ni nhydrin reagent because a false
positive reaction could result.

Quality Control

Test with known positive and negative controls  each time the test is preformed.

Positive:  Campylobac ter jejuni   (ATCC 29428)

Negative:  Campylobacter coli    (CPI B7080)

Reference

1. Difco package insert.

PROCEDURE MANUAL
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Policy & Procedure Manual
Policy # MI\TECH\20\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Indole Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

INDOLE TEST

Principle

Bacteria that produce the enzyme tryptophanase will deaminate tryptophan to indole, pyruvic
acid and ammonia in the presence of a co-enzyme pyridoxal phosphate.

Indole combines with Ehrlich's / Kovac's  reagent to form a red-coloured complex.

Materials

Test A:   Filter paper strips impregnated with Ehrlich's reagent.

Test B:   Kovac's reagent
    2% Tryptone broth (Difco, Oxoid)

Method

A:  Filter paper strips are suspended over tubes of ONPG / PAM media, and incubated at
35
0
C overnight.

Interpretation

Positive test - development of red colour on the strip.
Negative test - white-yellow colour.

B:  1.  Inoculate the tryptone  broth, and incubate at 35
0
C overnight.
2.   Add a few drops of Kovac's reagent to the broth.

Interpretation

Positive test - red colour in the upper layer.
Negative test - light-yellow colour in the upper layer.

PROCEDURE MANUAL
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Quality Control

Test the following positive and  negative controls weekly:

Positive:  Proteus vulgaris  (ATCC 13315)
Negative:  Klebsiella pneumoniae (ATCC 13883)

Reference

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

PROCEDURE MANUAL
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Policy & Procedure Manual
Policy # MI\TECH\21\v01  Page 1 of  1
Section: Technical Manual  Subject Title: Koehler Illumination
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

KOEHLER ILLUMINATION

The microscope should be set up using Koehler il lumination for all parasitology examinations.
This ensures that all the light from the lamp is being focused onto the specimen and that the field
to be examined is evenly illuminated.

Procedure

1.   Turn the lamp on.

2.   Bring the condenser up to the top position, with the top lens swung in.

3.   Open the condenser diaphragm.

4.   Place a specimen on the stage and focus with the 10x objective.

5.   Close the field diaphragm.

6.   Lower the condenser until the edge of the field diaphragm is in sharp focus.

7.   Center the field diaphragm image  with condenser centering screws.

8.   Open the field diaphragm until the edge just disappears from view.

9.   Remove one eyepiece.

10.  Looking down the eyepiece tube, close the c ondenser diaphragm until the illumination is
approximately 2/3 full.

11.  Replace the eyepiece.

12.  Repeat for each objective lens when changed.

Reference

1.   Baron E., Finegold S.M., Bailey & Scott's Diagnostic Microbiology, 8
th
 ed., The C.V.
Mosby Company, p64.

PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT  

Book on Biological Laboratory fifth penalty

Book on Biological Laboratory fifth penalty

 

 

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Section: Technical Manual  Subject Title: Cryptococcal Antigen 

Interpretation of results

  Negative:  Negative result in in itial screening tests against ACGR.

  Positive:  The titre is reporte d as the highest dilution showing a 2+ or greater reaction
with ACGR and negative with NGR.

 Nonspecific Interference: The titre with ACGR is at least  4-fold higher than the titre
with NGR.

 Uninterpretable test: The titre with ACGR is less than 4-fold greater than the titre with
NGR.
III. Reporting

  Telephone all positive reports.

 Negative Report: "Cryptococcal antigen not detected by latex agglutination."

 Positive Report: "Cryptococcal antigen detected at  a titre of          by latex
agglutination."

  Non-specific or Uninterpretable Report:
    "Cryptococcal antigen uninterpretable by latex agglutination."

IV. Precautions

  The ring slide must be thoroughly  cleaned after each use as follows:

 (a) Soak in hypochlorite overnight.
 (b) Scrub using detergent.
  (c)  Rinse well with tap water.
  (d)  Rinse 3 times with distilled water.
  (e)  Dry thoroughly using paper towels.
  (f)  Wipe clean with lint-free tissue.

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Section: Technical Manual  Subject Title: Cryptococcal Antigen 

V. Quality Control

  The pattern of reactions for the controls must be as follows.

      NGR    -    -    +
 ACGR  +  -
   CAC  NC  AGC

  Failure to obtain this pattern  indicates that the test must be repeated and the patient test
results cannot be reported.

VI. References

Product Insert, 1986.  Meridian  Diagnostics Inc., 3471 Rive r Hills Dr., Cinc innati, Ohio
45244.  (513)-271-3700.


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Policy & Procedure Manual
Policy # MI\TECH\12\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Crystal MRSA ID System
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

CRYSTAL MRSA IDENTIFICATION SYSTEM
Principle

Used as a screening test for the detec tion of intrinsic methicillin-resistant  Staphylococcus aureus
from isolated colonies.

Materials

BBL Crystal panel (lid and base)
MRSA Id both-3.2ml
transfer pipette
(all provided in kit)

Method

1. Suspend test S. aureus  in 2 ml of Vitek saline a nd adjust to a McFarland 0.5.
2.  Vortex and transfer 0.5mL to the tube of MRSA Id broth and Vortex.
3.  Remove lid from panel base without  touching lid prongs and discard desiccant.
4.  Place a drop of sterile saline in the first well (positive control).
5.  Using the same pipette, place 3 drops of the ID broth suspension into the same well.
6.  Place 4 drops of the broth suspension into th e next 2 wells of the panel (oxacillin and
negative control). Leave the fourth  well empty. Remove any bubbles.
7.  Cover the panel base with the lid. Gently pre ss lid onto panel base w ith the lid onto panel
base with a snap. Lid should no longer  be removed.  Do not invert panel.
8. Incubate at 35
o
C for at least 4 but not more than 5 hours.
9.  Expose panel to UV light and record which wells are fluorescing.

Interpretation

Bacteria Well#1 Well#2 Well#3
Methicillin Sensitive:  +  _  _
Methicillin Resistant:  +  +  -


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Quality Control

Results are uninterpretable if positive control well is negative or the negative control well is
positive.

Reference

1.  BBL crystal MRSA ID System package insert August 1993.

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Policy & Procedure Manual
Policy # MI\TECH\13\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Denka MRSA Screen
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

DENKA MRSA SCREEN
Principle

To be used as a screen ing test for the detectio n of Methicillin Resistant S. aureus  (MRSA) from
isolated colonies.

Reagents

MRSA screening kit
Microcentrifuge tubes
Boiling water bath
Wooden sticks
Loops
Micropipettes

Method

1.   Add 4 drops of extraction reagent #1 into a microcentrifuge tube.

2.   Take one heavy loopful of the  Staphylococcus aureus  colonies from a blood plate and
suspend the cells in the microcentrifuge tube.

3.   Place in a boiling water bath for 3 minutes.

4.   Remove microcentrifuge tube and let cool to room temperature.

5.   Add one drop of extraction reagent #2 to the tube  and mix well.

6.   Centrifuge at high speed for 5 minutes.

7.   For each specimen to be tested , allot and label one circle of  the test card for testing with
sensitized latex and one with control latex.

8.   Place 50 microliters of the specimen onto 2 of the test ci rcles and add one drop of the
sensitized cells to one circle and one drop of the latex control to the other.

9.   Mix the sample and latex together.

10.  Rotate the card by hand for 3 minut es and observe fo r agglutination.

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11.  If negative rotate for another 3 minutes.

Interpretation

Sensitized latex  Control latex  Result
+ - MRSA
- - Not MRSA
+ or -  +  Indeterminant

Quality Control

Positive and negative controls must be set up once per week.

Positive:  S. aureus   (ATCC 43330)
 Negative: S. aureus  (ATCC 29213)

Reference

1.  Denka Seiken Co., Ltd., Tokyo, Japan, Denka MRSA Screen package insert June 1998.

PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 

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Policy & Procedure Manual
Policy # MI\TECH\14\v01  Page 1 of  1
Section: Technical Manual  Subject Title: DNAse Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

DNAse TEST
Principle

This test determines the ability of an organism to  produce deoxyribonuclease  (DNAse).  This test
used, in conjunction with othe rs, for the identification of  S. aureus ,  M. catarrhalis and  Serratia
species.

Reagents

DNAse test agar with methyl green

Other Materials

Culture loop or wooden applicator stick

Procedure

1.  Spot inoculate an isolate using gr owth from an 18-24 hr pure culture.
2. Incubate in O
2 at 35
o
C X 18-24 hr.

Interpretation

The plate should be observed  against a white background.

Positive:  Distinct clear zone surrounding spot inoculum
Negative: No clear zone

Quality Control

Test each new batch of media  and when in use with positi ve and negative controls. 

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

Reference

1.   MacFaddin JF, Biochemical Tests for Identification of Medi cal Bacteria, 2nd ed., Williams
 and Wilkins, Baltimore MD., 1980, p94-113.

PROCEDURE MANUAL
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Policy & Procedure Manual
Policy # MI\TECH\15\v01  Page 1 of  3
Section: Technical Manual  Subject Title: E. coli  O157 Latex Test
 (Oxoid)
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

E. coli  O157 LATEX TEST (OXOID)

Principle

The Latex test will demonstrate by slide agglutination, E. coli strains possessing the O157
antigen.  Sorbitol MacConkey Agar (SMAC) should be used as the primary screen.  Non-sorbital
fermenting colonies (NSF) are test ed with the latex reag ents, to determine if  the isolate belongs
to the O157 serogroup, and is therefore a poten tial vero-cytotoxin (VT) producing strain.

Reagents

DR621 Test Latex -  consists of latex partic les sensitized with  specific rabbit antibody
reactive with the O157 antigen.

DR622 Control Latex -  consists of latex particles sensitized with pre-immune rabbit
globulins.

Storage

Do not freeze.  Store at 2
0
C - 8
0
C.  Do not use kit beyond the expiry date.

Procedure

NSF colonies may be taken from SMAC plates or  alternatively NSF isolates may be inoculated
onto non-selective agar media for testing.

It is necessary to test up to 10 separate NSF co lonies to ensure a high probability of detection
from mixed cultures.

1)   Bring the latex reagents to room temperature. Make sure the latex suspensions are mixed
by vigorous shaking.  Expel any latex from the dropper pipette for complete mixing.

2)   Dispense 1 drop of the Test latex onto a circle of the black slide.  Place it close to the
edge of the circle.

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3)   Add some loopfuls or a pasteur pipette drop of sa line to the circle.  Ensure that the latex
and saline do not mix at this stage.

4)   Using a loop, pick off a portion of the colony to be tested and carefully emulsify in the
saline drop.

5)   Mix the Test latex and suspension together and spread to cover most of the reaction area
using the loop.  Flame the loop.

6)   Rock the slide in a circular motion, observing for agglutination.  Do not rock the card for
more than 1 minute and do not use a magnifying glass.

7)   If no agglutination occurs, then proceed to te st other NSF colonies if these are present.

8)   If agglutination with the test reagent does o ccur, then it is necessary to test a further
portion of the colony with the control reagent to ensure that the isolate is not an
autoagglutinating strain.

9)   When finished, dispose of the reaction slide into disinfectant.

Interpretation

a)  Positive result -    Agglutination of th e Test latex occurs within 1 minute.
     No agglutination of the Control latex.  *Perform
biochemical tests to confirm that the organism is an  E. coli
strain.

b)  Negative result -    no agglutination of the Test latex.

c) Non-interpretable result -  clumping of the Control latex.

References

1.   Borczyk A., Lior H., Crebin B. 1987.  Int. J. Food. Microbiol. 4, 347-349.

2.   Konowalchuk J., Speirs J. and Stavric S.  1977.  Infect. Immune. 18, 775-779.

3.   Scotland S., Day N. and Rowe B.  19 80.  FEMS Microbiol. Lett. 7, 15-17.

4.   Centers for Disease Control.   1982.  Morbid Mortal Wkly. 31, 580-585.

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Technical Manual   

5.   Karmali M., Steel B., Petric M. and Lim C.  1983.  Lancet 8, 619-620.

6.   Johnson W., Lior H. and Bezanson.  1983.  Lancet 8, 76.

7.   March S. and Ratnam.  1986.  J. Clin. Microbiol.  23, 869-872.

8.   Krishnan C., Fitzgerald V., Dakin S. and Behme R.  1987.  J. Clin. Microbiol. 25, 1043-1047.


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Policy # MI\TECH\16\v01  Page1 of  2
Section: Technical Manual  Subject Title: Germ Tube Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

GERM TUBE TEST
Principle

This is a rapid test for the presumptive identification of C. albicans.

Reagents

Bovine serum
  A small volume to be used as  a working solution may be stored
  at 2 to 8
o
C.  Stock soluti on can be dispensed into small
  tubes and stored at -20
o
C.

Other Materials

Clean glass microscope slides
Glass coverslips
Vitek tubes (13 x 100 mm)
Pasteur pipettes

Procedure

1.  Put 3 drops of serum into a small Vitek tube.
2.  Using a Pasteur pipette, touch a colony of yeas t and gently emulsify it  in the serum.  The
pipette can be left in the tube.
3.  Incubate at 37
o
C for 2-4 hours but no longer.
4.  Transfer a drop of the serum to a slide for examination.
5.  Coverslip and examine microscopically using x 40 objective.

Interpretation

Germ tubes are appendages half the width and 3 to 4 times the length of the ye ast cell from which
they arise.  There is no co nstriction between the yeast cell and the germination tube.

Positive test:  presence of short  lateral filament s (germ tubes)

Negative test: yeast cells only

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Precaution

C. tropicalis  may form pseudohyphae which may be  falsely interpreted as germ tubes.

Quality Control

Set up known controls each time a test is run.

 Positive: C. albicans  (ATCC 10231)
 Negative: C. tropicalis  (ATCC 13803)

Reference

1.  Murray PA, et al.  Manual of Clinical Microbiology, 7
th
 ed., 1999; pp. 1189-1191.



PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 
 

A book about the biological laboratory fourth penalty

A book about the biological laboratory fourth penalty

 

 

References

1.  MacFaddin JF, Bioche mical Tests for Identification of Medical Bacter ia, 2nd ed., Williams
and Wilkins, Baltimore MD., 1980, p51-58.

2.  Murray PA, et al.  Manual of Clinical  Microbiology, 7th ed ., 1999; pp 426-427.

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Policy # MI\TECH\10\v01  Page 1 of  1
Section: Technical Manual  Subject Title: Cetrimide Pseudomonas
 Selective Agar
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

CETRIMIDE PSEUDOMONAS SELECTIVE AGAR

Principle

Cetrimide Selective Agar is used for the identification of Pseudomonas aeruginosa.   Cetrimide is a
compound that has germ icidal activity against most organisms except  Pseudomonas aeruginosa.
Also pigment production is enhanced on this media. 

Procedure

1.   Divide the plate into approximately 8 pie shaped divisions.
2.   Streak the test organism (pure culture) onto one of the pie  shaped divisions.
3.   Incubate at 35
0
C for 18 - 24 hours.

Interpretation

Pseudomonas aeruginosa will grow on this media  and will be pigmen ted a pale green to dark blue-green colour.  All other organisms will not grow or will be non-pigmented.

Quality Control

Test with positive and nega tive controls ea ch time the test is set up.

 Positive:  Pseudomonas aeruginosa    (ATCC 27853) 
 Negative: Escherichia coli   (ATCC 25922)   

Reference

1.  PML Technical Manual,  1990. 

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Policy # MI\TECH\11\v01  Page 1 of 6
Section: Technical Manual  Subject Title: Cryptococcal Antigen
Issued by:  LABORATORY MANAGER   Original Date: March 20, 2000
Approved by: Laboratory Director

Revision Date: July 26, 2000

CRYPTOCOCCAL ANTIGEN

Latex particles coated with anti-cryptococcal globulin (ACGR) reacts with cryptococcal
polysaccharide antigen (in CSF or serum) causing a visible agglutination.

I. Specimen Collection and Processing

  5 mL of blood is collected in  a serum separator tube and separated by centrifugation. The
serum is removed to a vial and refrigerated until te sting. Specimens  are stored a
 -70o
C after testing.

  Spinal fluid is collected in clean, sterile, cent rifuge tubes.  Specimens are stored refrigerated
after testing.
  Note:  Fungus culture sh ould also be set up.

II. Procedure

 Reagents

  Meridian CALAS (Cryptococcal Antigen Latex Agglutination System)
  1.  GBDA - Glycine buffe red diluent with albumin.
  2.  ACGR - Anti-cryptococcal globulin reagent.
  3.  NGR - Normal globulin reagent.
  4.  AGC - Antiglobulin contro l.  Rehydrate with 1.5 mL dH
2
O.
  5.  NC - Negative control.  Rehydrate with 2.5 mL dH
2
O and  inactivate the negative
at 56
o
C for 30 minutes .
  6.  CAC - Cryptococcal antigen control (Positive control).
  7.  Pronase - Rehydrate with 2.5 mL dH
2
O.

Note:  Ensure that all reconstituted vials are thoroughly dissolved before use

  All reagents are stored refri gerated.  Do not interchange reagents with a kit having a
different lot number.  Allow reag ents to warm to room temperature before use.  Mix gently
before use.

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Section: Technical Manual  Subject Title: Cryptococcal Antigen 

 Other Materials
 Boiling water bath
 56o
C heating block
  1.0 x 0.1 mL pipettes
 Rotator
  Small serologic test tubes
  Test tube rack
 Marking pen
 Applicator sticks

  The following are provided by Meridian:
 Capillary pipettes
 Rubber bulb
 Ring slide

 Method

 Specimen preparation:

  1.  Store refrigerated if testing is not done immediately.

    (a)  Inactivate serum by mixing 500 µL of serum and 500 µL of pronase solution
in a 12 x 75 mm tube and incubate at 56
o
C for 15 minutes.  Further
inactivate in a boiling water bath for 5 minutes.   This constitutes a 1:2
dilution.

  (b) Centrifuge CSF at 3500 rpm for 15 mins.  Inactivate the supernatant in a
boiling water bath for 5 minutes.


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Section: Technical Manual  Subject Title: Cryptococcal Antigen 

Performing the tests:

Note:  Controls must be run each time a patient specimen is tested.

  1.  Set up and label  the slide as follows:

 
         










  2.  Gently resuspend the la tex particles in the ACGR an d NGR reagents.  Rock each
reagent just prior to use.
    Place one drop of ACGR or NGR into the designated rings.

3.   Place 25  µL (one drop) of the cryptococcal  antigen control (CAC) into the
designated rings.  Repeat with the negativ e control (NC) and anti-globulin control
(AGC)

 4. Place 25 µL of specimen in the designated rings.

  5.  Using a separate applicator stick, mi x the contents of each ring thoroughly,
spreading the contents over the entire surface area.

N
G
R


A
C
G
R

 CAC   NC  AGC   TEST
 (POS   (NEG  (Anti-
 Control)  Control) globulin
      Control)

Not
Used

PROCEDURE MANUAL
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Section: Technical Manual  Subject Title: Cryptococcal Antigen 

  6.  Place the slide on the rotator and rotate at 125 rpm for 5 minutes.

  7.  Read the reactions immediately.

 8. Rate the agglutination as follows:
    Positive = any evidence of agglutination (granula tion or clumping)
    Negative = a homogenous suspension  of particles with no  visible clumping.

  9.  Patient specimens showi ng any agglutination in ACGR should  be titrated against
both ACGR and NGR reagents.

    (a)  Prepare two-fold serial dilutions of  the specimen using 200 µL of GBDA in
each of 8 test tubes labelled as follows:

  Tube    1     2       3      4     5         6       7       8

  Serum  1:4   1:8   1:16   1:32   1:64    1:128   1:256 1:512

  CSF    1:2   1:4     1:8   1:16   1:32    1:64    1:128 1:256

    (b)  Transfer one drop of each dilution into 2 rings.
    (c)  Add one drop of ACGR  to one ring of each dilution.
    (d)  Add one drop of NGR  to each of the other rings.
  (e) Mix using separate applicator sticks.
(f)   Place the slide on the rotator and rotate at  125 rpm for 5 minutes.
  (g) Read the results as follows:

      1+ = fine granulation against a milky background
      2+ = small but definite clumps  against a slightly  cloudy background
      3+ = large and small clumps against a clear background
   4+ = large clumps against a clear background

    (h)  If tube #8 gives an agglutination of 2+ or
   greater, the specimen must be further serially
   diluted until a titre may be obtained.

PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT 
 

A book about the biological laboratory third penalty

A book about the biological laboratory third penalty

 

 

Reference

QC organisms to be used:
  Neisseria gonorrhoea    ATCC 31426
  Haemophilus influenzae   ATCC 10211
  Branhamella catarrhalis    ATCC 23246
  Haemophilus paraphrophilus    ATCC 49917

Reference Package Insert - api NH system for the identification of  Neisseria  and  Haemophilus
bioMerieux Inc., Missouri USA.

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Policy # MI\TECH\04\04\v01  Page 1 of  1
Section: Technical Manual  Subject Title: API Voice Response
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

API VOICE RESPONSE


1.  Dial (800) 645-7056 from a touch tone phone.

2.  Enter product code access number (0-14).

3.  Press * symbol.

4.  Enter profile number as outlined below.

5.  Press  # symbol.

6.  Press 1# to end the call.
  Press 2# to repeat the identification.
  Press 3# for the next profile.
  Press 4# to speak to a technologist.     

ACCESS CODES

Product Code Access Number  Incubati on Time  Profile Number Format

0  API 20E      24 hours      Enter 7 digits
1  API 20E      48 hours    Enter 9 digits only
6  API 20C      48-72 hours    Enter 7 digits
7 Coryne    24 hours  Enter 7 digits
9  STAPH-IDENT    24 hours    Enter 7 digits
12  API NE (Rapid NE)     24/48 hours    Enter 7 digits
35  20 Strep      24 hours    Enter 7 digits

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

Revision Date: February 15, 2002

BACITRACIN DISK TEST
Principle

This is a screening test for the presumptive identifi cation of Group A Streptococci which are
susceptible to 0.04U baci tracin.  Other beta-haemolytic streptoc occi are usually resistant to this
concentration of bacitracin.

Reagents

Bacto Differentiation Disks Bacitracin (0.04U).  Store refrigerated.
Blood Agar (BA)

Other Materials

Culture loop
Cotton swabs
Forceps

Procedure

1.  Inoculate the surface of the BA with the suspect beta haemolytic  Streptococcus.  Streak for
confluent growth.
2.  Using aseptic technique,  place a bacitracin disk onto the inoculated surface. 
3. Incubate in O
2 at 35
o
C X
18-24 hr.

Interpretation

Susceptible:  any zone of inhibition around the disk (Presumptive  Group A Stre ptococcus).
Resistant:    growth up  to the edge of the disk

Precautions

1.  Other beta-haemolytic streptococci may be susc eptible to bacitracin.  Therefore this test can
be used only for the presumptive  identification of Gp. A Strep.

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Quality Control

Test with known susceptible and  resistant control strains weekly.
 Susceptible: Gp.A Strep. (ATCC 19615)
  Resistant:  Gp.B Strep. (ATCC 13813)

Reference
1.  Difco Differentiation Disks Bacitracin package insert.

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Policy & Procedure Manual
Policy # MI\TECH\06\v02  Page 1 of  2
Section: Technical Manual  Subject Title: Beta-Lactamase (Cefinase)
   Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: November 1, 2002

BETA-LACTAMASE (CEFINASE) TEST
Principle

Cefinase discs are intended for use in  rapid testing of isolated colonies of  Neisseria gonorrhoeae,
Staphylococcus species,  Enterococcus species,  Hameophilus influenzae and anaerobic bacteria for
the production of beta-lactamase.

The Cefinase disc is impregnated with the chromogenic cephalosporin, Nitrocefin.  This compound
exhibits a very rapid colour change from yellow to  red as the amide bond in  the beta lactam ring is
hydrolyzed by a beta-lactamase.   When a bacterium produces this enzyme in signifi cant quantities,
the yellow-colored disc turns red in  the area where the  isolate is smeared.

Although other penicillins and ce phalosporins may be used as substrates for specific enzymes,
Nitrocefin has the wide spectrum of susceptibility and sensitivity of the comm ercially available beta
lactams.  It is not  known to react with other microbial enzymes.

Each disc is used to test one bacterial strain for the presence of beta-lactamase.

Materials

Cefinase discs impregnated with Nitrocefin.

Procedure

1)   Using a single disk dispenser, dispense the required number of disks fr om the cartridge into
an empty petri dish or onto a microscope slide.

2)   Moisten each disc with 1 drop of Sterile di stilled water.

3)   With a sterilized loop or applicator stick remove several well-isolated similar colonies and
smear onto a disk surface.

4)   Observe disk for colour change.

5)   Alternate procedure: Using forceps moisten disk  with one drop of Purified Water and then
wipe across colony.

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Interpretation

A positive reaction will show a yellow  to red colour change on the  area where the culture was
applied.  Note: colour ch ange does not usually develop over the entire disk.  A negative  result will
show no colour change on the disc.

For most bacterial strains a positive result will develop within 5 minutes.  However, positive
reactions for some stap hylococci may take up to 1 hour to develop.


Organisms

Result
Approx.
Reaction Time

Interpretation
Staphylococcus aureus  Positive  1 hr  Resistant to penicillin,
ampicillin, carbenicillin.
Probably susceptible to
cephalothin, methicillin,
oxacillin, naficillin and
other penicillinase-resistant penicillins.

Enterococcus faecalis  Positive 5 min Resistant to penicillin
and ampicillin.

Hameophilus influenzae  Positive 1 min Resistant to ampicillin
Susceptible to
cephalosporins.

Neisseria gonorrhoeae  and
Branhamella catarrhalis

Positive

1 min

Resistant to penicillin.

Anaerobic bacteria  Positive  30 mins  Probable identification is
Bacteroides  species.
Probably resistant to
penicillin an d may be
resistant to
cephalosporins including
cefotaxime and rarely
cefoxitin.
Controls:  Staphylococcus aureus  (ATCC 29213):  Positive
 Haemophilus influenzae   (ATCC 10211):  Negative

Reference

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

PROCEDURE MANUAL
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Policy & Procedure Manual
Policy # MI\TECH\07\v01  Page 1 of  1
Section: Technical Manual  Subject Title: Bile Esculin Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

BILE ESCULIN TEST
Principle

This test determines the ability of an organism to  grow in the presence of bile and to hydrolyze the
glycoside esculin to escu letin and glucose.  The test is used to presum ptively identify Group D
Streptococci.

Materials

Bile esculin agar slant / plate
Culture loop

Procedure

1.  Heavily inoculate a bile esculin slant / plate with the suspect organism.
2. Incubate in O
2 at 35
o
C for 18-24 hr.

Interpretation

Positive: Presence of  a dark brown to black  colour on the slant.

Negative:  No blackening of the medium.  Growth may occur, but this does not indicate esculin
  splitting.

Quality Control

Each new lot of media should be te sted with known control strains.

 Positive: E. faecalis  (ATCC 29212)
 Negative: Gp.B Strep. (ATCC 13813)
  No Growth:  Gp.A Strep.  (ATCC 19615)

References

1.   MacFaddin JF, Biochemical Tests for Identification of Medi cal Bacteria, 2nd ed., Williams
 and Wilkins, Baltimore MD, 1980, p4-12.


PROCEDURE MANUAL
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Policy & Procedure Manual
Policy # MI\TECH\08\v01  Page 1 of  1
Section: Technical Manual  Subject Title: Bile Solubility Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

BILE SOLUBILITY TEST
Principle

Tests the ability of alpha ha emolytic streptococci to lyse in the pr esence of bile salts.  This test is
used for the identification of Streptococcus pneumoniae.

Reagents

BBL Spot Test dropper (10% sodium desoxycholate).

Procedure

1.  Hold the dropper upright and squeeze gently to crush the glass ampoule inside the dispenser.
2.  Place 1 drop of the reagent directly  on isolated colonies of suspected S. pneumoniae .
3.  Keep the plates very level to prevent the reagent from running and washing a non-
pneumucoccal colony away, producing a false positive result.
4.  Incubate at room te mperature on the bench for 15-30 minutes until the  reagent drys.  Do not
invert the plate; leave the lid ajar.
5.  Examine the colonies for lysis.

Interpretation

  Positive (bile soluble):    Lysis of the colonies.

  Negative (bile insoluble):    No lysis of colonies.

Quality Control

Test with known positive and negative cont rol strains weekly.

 Positive: S. pneumoniae     (ATCC 6303) 
  Negative:  Viridans Streptococcus (LPTP 8610)

References

1.  Murray PA, et al.  Manual of Clinical Microbiology, 7
th
 ed., 1999; p. 1665.

2.  BBL Desoxycholate Reagent Dr oppers package insert, April 1991.

PROCEDURE MANUAL
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\09\v01  Page 1 of  2
Section: Technical Manual  Subject Title: Catalase Test
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

CATALASE TEST
Principle

Detects the presence of the enzyme catalase which hydrolyzes H2O2 to produce H
2O and O2
.  This
test is used to differentiate Staphylococci (catal ase positive) from Streptococci (catalase negative).

Reagents

Hydrogen peroxide (H 2O2
), 3%
  Store in a dark bottle and av oid any undue exposure to light.
  Keep refrigerated at a ll times when  not in use.

Other Materials

Clean glass microscope slides
Plastic culture loop or  wooden applicator stick

Procedure

1.  Pick a colony from an 18-24 hr culture and pl ace it on a clean glass slide.  Avoid carry over
of blood agar which can  cause false positives.
2.  Put one drop of 3% H
2O2
 over the organism on the slide.   Do not reverse  the order of the
procedure as false pos itive results may oc cur.  Do not mix.
3.  Observe for immediate bubbling (gas  liberation) and record the result.
4.  Discard the slide into a discard container.

Interpretation

Positive test:  Immediate bubbling, easily observed (0
2
 formed)

Negative test:  No bubbling

Precautions

1.  Carry over of blood  agar must be avoided.
2.  Growth for testing must be from an 18-24 hr culture.
3. 3% H
2O2 is caustic - avoid exposure to skin.  If H
2O2
 does get on the  skin, immediately
flood the area with 70% ethyl alcohol, not water.

PROCEDURE MANUAL
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Policy & Procedure Manual
Policy # MI\TECH\09\v01  Page 2 of  2
Technical Manual   

4.  Aerosols may be released  by the bubbling of the O
2
.
5. H
2O2
 is unstable and breaks down easily on exposure to light.  The solution must be kept
refrigerated in the dark.

Quality Control

H2O2
 is very unstable and should be tested  daily or immediately prior to its use.

 Positive: S. aureus  (ATCC 25923)
  Negative:  Gp. A. Strep.  (ATCC 19615)
 

A book about the second penalty Biological Laboratory

A book about the second penalty Biological Laboratory

 

 

yellow
[GLU ] glucose  Assimilation  transparent  opaque
[ARA ] arabinose  Assimilation  transparent  opaque
[MNE ] mannose  Assimilation  transparent  opaque
[MAN ] mannitol  Assimilation  transparent  opaque
[NAG ] N-acetyl-glucosamine Assimilation  transparent  opaque
[MAL ] maltose  Assimilation  transparent  opaque
[GNT ] gluconate  Assimilation  transparent  opaque
[CAP] caprate  Assimilation  transparent  opaque
[ADI] adipate  Assimilation  transparent  opaque
[MLT ] malate  Assimilation  transparent  opaque
[CIT ] citrate  Assimilation  transparent  opaque
[PAC] phenyl-acetate  Assimilation  transparent  opaque
OX  see oxidase test  cytochrome oxidase  colorless/
light purple
dark purple


PROCEDURE MANUAL
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Policy & Procedure Manual
Policy # MI\TECH\04\04\v03  Page 1 of  4
Section: Technical Manual  Subject Title: API  Test Strips - API NH
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: August 3, 2003

SYSTEM FOR IDENTIFICATION OF NEISSERIA & HAEMOPHILUS (API  NH)

Principle

The API NH strip consists of 10 microtubes containing dehydrated substrat es, which enable the
performance of 12 identification te sts (enzymatic reactions or suga r fermentations), as well as the
detection of a penicillinase (particular interest in  Haemophilus influenzae, Haemophilus
parainfluenzae, Branhamella catarrhalis (Moraxella catarrhalis)  and  Neisseria gonorrhoeae).

The reactions produced during incubation result in spontaneous color changes or are revealed by
the addition of reagents.

After a 2-hour incubation period at a temperature of 35-37
o
C, the reading of the reactions is
performed visually and identification is obtained by consulting the profile list.

Reagents

API NH strips
NaCl 0.85% Medium (2 ml)
JAMES reagent
ZYM B reagent
Swab
Incubation box
Result sheet
1 package insert
McFarland Standard, point 4 on the scale
Mineral oil
Pipettes
Ampule rack
Ampule protector

Procedure

1.   Specimen Processing

The microorganisms to be identified must first be  isolated as separate colonies by streaking the
specimen onto Blood agar, Chocolate agar or Martin-Lewis agar according to standard

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microbial techniques.

2.   Preparation of Strip

Each strip is composed of 10 cupules.  Each  cupule has an open and closed area (cupule
and tube).  An incubation tray is supplied  for each strip.  It serves as a support and
individual chamber while both  protecting the strip from contaminants in the air and
assuring the humid atmosphere necessary  to avoid dehydration during incubation.

•   Remove the strip from its individual packaging
•   Place the strip in the incubation box
•   Discard the desiccant sachet

Record the specimen number on the flat portion of the tray (do not record the number on
the lid as it may be misplaced during handling).

3.   Preparation of the Inoculum

•   Open an ampule of NaCl 0.85% Medium  (2 ml) with the ampule protector.
  •   Using a swab, pick up a few well-isolated  colonies and prepare a suspension with a
      turbidity equivalent to 4 McFarland,  ensuring it is well mixed .
  •   The suspension should be used immediately after preparation.

4.   Inoculation of the Strip

•   Distribute the prepared bacterial suspension  into the cupules, avoiding the formation of
    bubbles (tilt the stri p slightly forwards and place the tip of the pipette or PSIpette
against
    the side of the cupule):
-   Only fill the tube part of the first 7 microtubes (PEN  to URE ): about 50  µl.
-   Fill tube and cupule of the last 3 microtubes LIP/ProA , PAL/GGT ,  β GAL/IND:
about 150 µl, avoiding the formation of a convex meniscus.
  •   Cover the first 7 tests (PEN  to URE ) with mineral oil (underlined tests).

NOTE:  The quality of the filling is very import ant: tubes which are insufficiently or
          excessively full may cause false positive or false negative results.
        •   Close the incubation box.
        •   Incubate for 2 hours at 35-37
o
C  in aerobic conditions.


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5.   Incubation

  Incubate for 2 hours at 35-37
o
C in aerobic conditions.

6.   Reading the Strip

  Refer to the Reactions Table for a desc ription of how to  read the reactions.

  Note all spontaneous reactions (PEN to β GAL) and record them as + or -.
  •  Add 1 drop of ZYM B reagent to  microtubes 8 and 9: LIP/ProA  and PAL/GGT.
  •  Add 1 drop of JAMES reagent to microtube 10:  β GAL/IND.
  •   Wait 2 minutes then read the reactions by referring to  the Reading Table in this package
    insert and record them on the result sheet.
-   If the LIP reaction is positive (blue pigment), interpret the ProA  reaction as
negative , whether the ZYM B reagent has been added or not.
-   If, after a 2-hour incubation period, severa l reactions (fermentation, penicillinase)
are doubtful, re-incubate the strip for anot her 2 hours and read the reactions again
(the enzymatic tests should not  be re-read in this case).

Reactions Table

RESULTS  TESTS REACTIONS  SUBSTRATES QTY
(mg)
NEGATIVE POSITIVE

1) PEN

PENicillinase

Penicillin G

1.36

Blue
(penicillinase absent)

Yellow
Yellow-green
Yellow-blue
(penicillinase present

2) GLU
3) FRU
4) MAL
5) SAC

GLUcose (Acidification)
FRUctose (Acidification)
MALtose (Acidification)
SACcharose/Sucrose
(Acidification)

Glucose
Fructose
Maltose
Sucrose

0.5
0.1
0.5

Red
Red-orange

Yellow
Orange


6) ODC

Ornithine DeCarboxylase

Ornithine

0.55

Yellow-green
Grey-green

Blue

7) URE

UREease

Urea

0.41

Yellow

Pink-violet

8a) LIP

LIPase

5-bromo-3-indoxyl-caprate

0.033

Colorless
Pale grey

Blue
(+precipitate)


9a) PAL

Alkaline Phosphatase

Para-Nitrophenyl-phosphate 2CHA

0.038

Colorless
Pale yellow

Yellow


10a) β GAL

Beta GALactosidaase

Para-Nitrophenyl-BD
galactopyranoside

0.04

Colorless

Yellow

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Reactions Table (Cont'd)

RESULTS  TESTS REACTIONS  SUBSTRATES QTY
(mg)
NEGATIVE POSITIVE

ZYM B / 3 min


8b) ProA


Proline Arylamidase
If LIP is +. ProA is always -


Proline-4-methoxy-
β  naphthylamide


0.056


Yellow
Pale orange
(brown if LIP +)

Orange

ZYM B / 3 min


9b) GGT


Gamma Glutamyl Transferase


Gamma glutamyl
4-methoxy-
β  naphthylamide


0.049

Yellow
Pale orange
(yellow-orange if PAL +)

Orange


JAMES / 3 min


10b) IND


INDole


Tryptophane


0.036

Colorless


Pink

Quality Control

To be performed on receipt of every new  lot of strip by the Q.C bench technologist.

A book about the first penalty Biological Laboratory

PROCEDURE MANUAL

A book about the first penalty Biological Laboratory

TORONTO MEDICAL LABORATORIES \ MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT  

Page 1
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\v07  Page 1 of  128
Section: Technical Manual  Subject Title: Table of Contents
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Direct or  Revision Date: April 20, 2005
  Review Date: April 20, 2005

TECHNICAL MANUAL
TABLE OF CONTENTS
ALA (RAPID PORPHYRIN TEST) ............................................................................................4
ANAEROBIC IDENTIFICATION USI NG SPECIAL POTENCY DISKS
Anaerobic Identification by Means of Special Potency Disks......................................................6
SPS Disk for Differentiation of Anaerobic Cocci.........................................................................9
ANAEROBIC/CAMPYLOBACTER JAR SET UP ..................................................................11
API TEST STRIPS
     CORYNEBACTERIUM (API CORYNE) ...........................................................................13
     ENTEROBACTERIACEAE (API 20E) ................................................................................17
     IDENTIFICATION OF NON-ENTERIC  GRAM-NEGATIVE RODS (API 20NE) ..……24
     NEISSERIA & HAEMOPHILUS (API  NH).......................................................................28
     API VOICE RESPONSE.......................................................................................................32
BACITRACIN DISK TEST.......................................................................................................32
BETA-LACTAMASE (CEFINASE) TEST...............................................................................35
BILE ESCULIN TEST...............................................................................................................37
BILE SOLUBILITY TEST .........................................................................................................38
CATALASE TEST .....................................................................................................................39
CETRIMIDE PSEUDOMONAS SELECTIVE AGAR .............................................................41
CRYPTOCOCCAL ANTIGEN...................................................................................................42
CRYSTAL MRSA IDENTIFICATION SYSTEM....................................................................48
DENKA MRSA SCREEN ..........................................................................................................50
DNAse TEST ..............................................................................................................................52
E. coli  O157 LATEX TEST (OXOID).......................................................................................53
GERM TUBE TEST...................................................................................................................56
GONOGEN (GC COAGGLUTINATION) TEST .....................................................................58

PROCEDURE MANUAL
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Policy # MI\TECH\v07  Page 2 of  128
Section: Technical Manual  Subject Title: Table of Contents (Cont'd)
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Direct or  Revision Date: April 20, 2005
  Review Date: April 20, 2005

HIGH LEVEL AMINOGLYCOSIDE TESTING ......................................................................60
HIPPURATE TEST....................................................................................................................62
INDOLE TEST ...........................................................................................................................64
KOEHLER ILLUMINATION....................................................................................................66
KOH STRING TEST ..................................................................................................................67
LAP TEST ............................................................................................................................... ...69
MOTILITY TEST MEDIUM .....................................................................................................71
MUG TEST (PGUA)..................................................................................................................74
NEISSERIA IDENTIFICATION SUGARS ..............................................................................76
ONPG TEST............................................................................................................................... 78
ONPG-PHENYLALANINE-MOTILITY MEDIUM (ONPG-PAM) ........................................80
OPTOCHIN SENSITIVITY TEST............................................................................................82
OXIDASE (API STRIP) .............................................................................................................84
OXIDASE (SPOT TEST DROPPER)........................................................................................86
PASTOREX STAPH PLUS TEST ............................................................................................87
PLATE STREAKING METHODS............................................................................................89
PRO-AMP GLU-AMP TESTS...................................................................................................92
PYR TEST ............................................................................................................................... ...93
QUANTITATION OF ORGANISMS & CELLS ON SMEARS & CULTURE .......................95
RapID ANA II SYSTEM............................................................................................................96
RapID MGP TEST  .....................................................................................................................97
RapID VP TEST .........................................................................................................................99
RapID YEAST PLUS TEST.....................................................................................................100
SIM (SULFIDE-INDOLE-MOTILITY)..................................................................................102
STAINING METHODS:
     Acid Fast Stain for Mycobacteria (Kinyoun) .......................................................................104     

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Policy # MI\TECH\v07  Page 3 of  128
Section: Technical Manual  Subject Title: Table of Contents (Cont'd)
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Direct or  Revision Date: April 20, 2005
  Review Date: April 20, 2005

Acid fast stain for Nocardia (Modifed Kinyoun).................................................................106
     Acridine Orange Stain ..........................................................................................................108
     Bacto 3-Step Gram Stain Procedure....................................................................................110
     Eosinophil Stain ...................................................................................................................112
     Fungi-fluor   Stain ..............................................................................................................113
     Gram Stain ...........................................................................................................................115
STAPHAUREX TEST..............................................................................................................117
STREPTOCOCCAL GROUPING ...........................................................................................119
TETRAZOLIUM REDUCTION TEST (TTC) ........................................................................121
THERMONUCLEASE TEST..................................................................................................122
TRIBUTYRIN TEST ................................................................................................................124
TSI (TRIPLE SUGAR IRON)..................................................................................................125
TUBE COAGULASE TEST ....................................................................................................128
UREA SLANT..........................................................................................................................130
X AND V DISKS FOR IDENTIFICATION OF HAEMOPHILUS ........................................132
XYLOSE FERMENTATION ...................................................................................................134


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Policy #MI\TECH\01\v01  Page 1 of 2
Section: Technical Manual  Subject Title: ALA (Rapid Porphyrin
   Test)
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

ALA (RAPID PORPHYRIN TEST) 

Principle 

This test is used for rapidly detecting porphyrin as a means of speciating Haemophilus species.
Enzymes which convert ALA (delta - aminolevulinic acid) to porp hyrins in the biosynthesis of
hemin (X factor) are produced by Haemophilus parainfluenzae but not by  H. influenzae.  The
production of porphyrins is detected by examination with an ultra-violet (UV) light.

Reagents 

BBL  TAXO  Differentiati on Disks ALA.  (Store refrigerated in the da rk.  Allow 10-15 minutes
for the container to reach room temperature before opening).  
Sterile distilled water

Other Materials

Petri dish
Inoculating loop
Gauze
Long-wave UV lamp
Forceps

Procedure

1.  Place one ALA disk  for each organism to be tested on  the inside of a  Petri dish using
forceps.
2.  Moisten each disk with one drop of sterile water.
3.  Rub a loopful of the test organism onto the  moistened disk holding it in place with sterile
forceps.
4. Saturate gauze with  water, squeeze out any excess and place it in the petri dish as far away
from the disks as possible.
5.  Incubate at 35
o
C.
6.  Examine at hourly intervals for 6 hours by re moving the top of the petri dish and exposing
the disks to UV light  in a darkened room.   NB:  Wear UV safety goggles when using the
UV light.

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Interpretation 

A.  Positive:  Orange-red fluorescence

B.  Negative:  No fluorescence observed

Precautions 

1.  Use for differentiating  Haemophilus spp. only.

2.  Best results are obtained wh en a heavy inoculum is used.

3.  ALA is light sensitive.  Disks must be protected from light.

Quality Control

Test the following positive and negative co ntrols each time an  unknown is tested:

 Positive: H. parainfluenza  (ATCC 7901)
 Negative: H. influenzae        (ATCC 35056)

Reference

BBL  TAXO  Differentiation Disks ALA package insert, 1999.  Becton Dickinson Microbiology
Systems.

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Policy #MI\TECH\02\v01  Page 1 of  5
Section: Technical Manual  Subject Title: Anaerobic Identification Using
Special-Potency Disks
Issued by:  LABORATORY MANAGER   Original Date: July 31, 2000
Approved by: Laboratory Director

Revision Date: February 15, 2002

ANAEROBE IDENTIFICATION US ING SPECIAL-POTENCY DISKS 

Principle 

Special potency antimicrobial disks of vancomycin (5  µg), kanamycin (1,000  µg) and colistin
(10  µg) are used as an aid in determining the Gram reaction of anaerobes as well as in
preliminary categorization of some anaerobic genera and species (Table 1).  In general, gram
positive organisms are resistant to colistin and  susceptible to vancomycin, while most gram
negative organisms are resistant to vancomycin.  Th is difference is especially useful with some
Clostridia that consistently stain gram negative.

Table 1 - Anaerobic Identification  by Means of Special Potency Disks

Response
1
 to Disk:  
Type of Organism
Vancomycin
(5 µ g)
Kanamycin
(1,000  µ g)
Colistin
(10  µ g)

Gram negative
Gram positive
B. fragilis group
B. ureolyticus group
Fusobacterium spp.
Porphyromonas spp.
Veillonella spp.
R
S
R
R
S
R
V
R
S
R
S
V
R
S
R
S

1
 R - resistant;  S - susceptible;  V - variable

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Materials

A.   Reagents

1.   Special potency antibiotic disks: 

 Vancomycin  5 µg
 Kanamycin  1,000 µg
 Colistin  10 µg

Store a small supply of disks (one carton each) in a tight container with desiccants
at 4
0
C.

2.   Brucella or other anaerobic blood agar plate.

B.   Supplies

1.   Single disk dispenser or forceps
2.   Ruler (divided into millimeters)

Procedure

1.   Allow the container with disks to reach room temperature before opening it.
2.   Subculture the isolate on a BAP.  To ensure an even, heavy lawn  of growth, streak the
first quadrant back and forth se veral times.  Streak the other  quadrants to yield isolated
colonies.
3.   Place the three antibiotic disks on the first quadrant will apart fr om each other.  
4.   If you have several organisms to test, first streak all the plates and  then add the disks to
them at the same time.
5.   Incubate the plate(s) anaer obically for 48-72 hours at 35-37
0
C.
6.   Examine for zones of inhibition of growth around the disks.

Interpretation

A.  Susceptible:  Zone of inhibition of ≥  10 mm
B.  Resistant:  Zone of inhibition of < 10 mm

PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT  

Page 8

TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\02\v01  Page 3 of  5
Technical Manual    

Quality Control 

A.   Test special potency antibiotic disks by lot when initially received and weekly thereafter.

B.   Test  Bacteroides fragilis  (ATCC 25285),  Clostridium perfringens (ATCC 13124), and
Fusobacterium necrophorum  (ATCC 25286) as described below under Procedure.  The
results should show the following:

1.   B. fragilis: resistant to all  three antibiotics
2.   F. necrophorum: resistant to vancomycin; susceptible to colistin and kanamycin 
3.   C. perfringens : susceptible to vancomycin and kanamycin and resistant to colistin

C.   Record the results on a QC log.

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