Book on the seventh penalty Biological Laboratory
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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)
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References
1. Murray PA et al. Manual of Clinical Microbiolog y, 7th ed., 1999; p. 1671.
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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)
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Reference
1. Carr-Scarborough Microbiologicals package insert 1991.
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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.
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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.
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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
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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
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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.
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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.
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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.
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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.
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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
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