Transcript Slide 1

In the name of God
Yasuj University of Medical Sciences
Department of Microbiology
By: Dr. S. S. Khoramrooz
Department of Microbiology, Faculty of Medicine,
Yasuj University of Medical Sciences, Yasuj, Iran

The bacitracin susceptibility test is used for the presumptive
identification of group A β-hemolytic streptococci.

The test is performed on a blood agar medium with a
bacitracin differential disk (e.g., Bacitracin Disk, 0.04
unit).

Any zone of inhibition around the disk is considered a
positive test

Although this test is simple, inexpensive, and fairly
accurate for presumptive identification of group A
streptococci, it is not highly specific.
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
Over 10% of group C and G streptococcal strains are
also susceptible to bacitracin, as are about 5% of
group B strains.

Consequently, this test is often performed along with
the sulfamethoxazoletrimethoprim (SXT)
susceptibility test because:
 Groups C and G streptococci are usually susceptible
to SXT
 Whereas groups A and B streptococci are resistant
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
Some workers have advocated the use of bacitracin disks
directly on primary, nonselective blood agar for rapid
detection and identification of group A streptococci in throat
cultures.

However, this method will identify only 50-60% of isolates.

Placement of bacitracin disks on primary plates containing
selective media is considerably more sensitive.

The laboratory report should reflect the use of a presumptive
method: β-hemolytic streptococci, presumptively group A by
bacitracin” or "β-hemolytic streptococci, presumptively not group
A by bacitracin."
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
The SXT susceptibility test presumptively
distinguishes groups A and B streptococci from other
β-hemolytic streptococci.

When used in conjunction with the bacitracin test,
the SXT susceptibility test helps screen out those nonA, non- B S streptococci that may be susceptible to
bacitracin because both group A and B strains are SXTresistant, whereas groups C, F, and G are SXTsusceptible.
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
The test is performed in the same way as the bacitracin
test, except that a commercial disk containing 1.25 µg
trimethoprim and 23.75 µg of sulfamethoxazole is
used.

Any zone of inhibition indicates susceptibility to
SXT
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
The bile (sodium deoxycholate) solubility test
distinguishes S. pneumoniae from all other alpha hemolytic streptococci.

S. pneumoniae is bile soluble whereas all other alphahemolytic streptococci are bile resistant.

Sodium deoxycholate (2% in water) will lyse the
Pneumococcal cell wall
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A.Preparation of 2% sodium deoxycholate (bile salt)
solution
1. Dissolve 2 g of sodium deoxycholate into 100 ml
sterile distilled water.
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B.Performing the bile solubility test
1. Grow the isolate (s) to be tested for 18-24 hours on a BAP
at 35- 37°Cwith ~5% CO2 (or in a candle-jar)
2. Add bacterial growth from the overnight BAP to 1.0 ml of
0.85% saline to achieve turbidity in the range of a 0.5-1.0
McFarland standard.
3. Divide the cell suspension equally into 2 tubes (0.5 ml per
tube).
4.Add 0.5 ml of 2% sodium deoxycholate (bile salts) to one
tube. Add 0.5 ml of 0.85% saline to the other tube. Mix
each tube well.
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5. Incubate the tubes at 35-37°C in CO2
6.Vortex the tubes.
7.Observe the tubes for any clearing of turbidity after 10
minutes.
Continue to incubate the tubes for up to 2 hours at 3537°C in CO2 if negative after 10 minutes. Observe
again for clearing.
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C. Reading the bile solubility test results

A clearing of the turbidity in the bile tube but not in the saline
control tube indicates a positive test.
D.Troubleshooting

Partial clearing (partial solubility) is not considered positive
for pneumococcal identification.

Partially soluble strains that have optochin zones of inhibition
of less than 14 mm are not considered pneumococci.
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Q uality control

Each new lot of sodium deoxycholate should be tested
with positive and negative QC strains.
S. pneumoniae strain ATCC 49619 can be used as a
positive control
 S. mitis strain ATCC 49456 can be used as a negative
control.

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
S. pneumoniae strains are sensitive to the chemical
optochin (ethylhydrocupreine hydrochloride).

Optochin sensitivity allows for the presumptive
identification of alpha-hemolytic streptococci as S.
pneumoniae, although some pneumococcal strains are
optochin-resistant.

Other alpha-hemolytic streptococcal species are optochinresistant.
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Performing the optochin test

Optochin (P) disks (6 mm, 5 μg) can be obtained from a
commercial vendor.

Optochin disks are often called “P disks” and many
commercial versions are labeled with a capital “P”.

If a commercial source of P disks is not available, a 1:4000
solution of ethylhydrocupreine hydrochloride can be applied
to sterile 6 mm filter paper disks.
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1. Grow the strain(s) to be tested for 18-24 hours on a
BAP at 35-37°C with ~5% CO2 (or in a candle-jar).
2. Use a disposable loop to remove an isolated colony
from the overnight culture on the BAP and streak
onto one half of a BAP.
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
Two different isolates can be tested on the same plate,
but care must be taken to ensure that the cultures do
not overlap.
3. Place a P disk within the streaked area of the plate and
incubate the BAP overnight at 35-37°C with ~5%
CO2 (or in a candle-jar).
4. Observe the growth on the BAP near the P disk and
measure the zone of inhibition, if applicable.
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B. Reading the optochin test results

Using a 6 mm, 5 μg disk, a zone of inhibition of 14 mm or greater
indicates sensitivity and allows for presumptive identification of
pneumococci.

Zones of inhibition should be measured from the top surface of the
plate with the top removed.

Use either calipers or a ruler with a handle attached for these
measurements.

Measure the diameter of the zone holding the ruler over the center
of the surface of the disk when measuring the zone of inhibition.

In the case of an isolate completely resistant to optochin, the
diameter of the disk (6 mm) should be recorded.
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C. Troubleshooting

A smaller zone of inhibition (< 14 mm) or no zone of inhibition
indicates that the bile solubility test is required.

It is important to remember that pneumococci are sometimes
optochin-resistant.
D. Quality control

Each new lot of optochin disks should be tested with positive
and negative controls.

The growth of S. pneumoniae strain ATCC 49619 is inhibited by
optochin and growth of S. mitis strain ATCC 49456 is not
inhibited by optochin.
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NOVOBIOCIN DISK TEST
I. Principle
Coagulase-negative staphylococci can be divided into
novobiocin-susceptible and novobiocin-resistant species.
Among the novobiocin-resistant species, S. saprophyticus is the
one commonly recovered from humans as a cause of urinary
tract infections.
II. Reagents I. Novobiocin disks. 5 µg (NB 5, BD Microbiology
Systems, Franklin Lakes, NJ)
2. Sheep blood agar plate
III. Quality Control
A known S. saprophyticus strain and a S. epidermidis strain
should be tested with each new lot of novobiocin disks or on a
weekly basis.
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IV. Procedure
1. Prepare a suspension of the organism to be identified
in sterile distilled water or broth.
2. The suspension should be equivalent in turbidity to a
0.5 McFarland standard.
3. With a sterile swab, spread some of the suspension
over half of a blood agar plate.
4. Aseptically place a novobiocin disk on the inoculated
area.
Susceptibility to furazolidone may be assessed on the
same plate by placing the disks about 4 cm apart on the
inoculated area.
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Gently tamp the disk(s) with sterile forceps to assure contact
with the agar surface.
5. Incubate the plate aerobically for 18 to 24 hours at 35°C.
V. Results
A. Interpretation
1. S. saprophyticus are novobiocin-resistant and will show
zones of inhibition of 6 mm (no zone) to 12 mm.
2. Other coagulase-negative staphylococci and S. aureus
are novobiocin-susceptible and will show zones of 16 mm
or larger.
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
Most strains of S. aureus have a bound coagulase or
"clumping factor" on the surface of the cell wall.

This factor reacts directly with fibrinogen in plasma,
causing rapid cell agglutination.

The test can be performed with growth from blood agar,
CNA agar, or other nonselective nutrient medium, but
should not be performed from media with a high salt
content (e.g., mannitol salts agar) since the high salt
content causes some strains of S. aureus to
autoagglutinate.
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
Any strain that is negative on the slide coagulase test must
be confirmed with a tube coagulase test, because strains
deficient in clumping factor will usually produce free
coagulase.

Some strains of the human coagulase-negative species S.
lugdunensis and S. schleiferi subsp schleiferi also produce
clumping factor and may be positive with the slide test
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II. Media and Reagents
Rabbit plasma with EDTA (commercially available in
lyophilized form). Reconstituted plasma should be
refrigerated.
III. Quality Control
Coagulability of plasma may be tested by adding one
drop of 5% calcium chloride to 0.5 mL of the
reconstituted plasma.
A clot should form within 10 to 15 seconds.
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
A known Staphylococcus aureus strain and a
Staphylococcus epidermidis strain serve as positive and
negative controls, respectively.

Each reconstituted vial of rabbit plasma with EDTA
should be tested with 18- to 24-hour cultures of the
control strains.
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IV. Procedure
1- Slide test (bound coagulase): Place two drops of sterile water or
saline in two circles drawn on a glass slide with a wax pencil.
Gently emulsify colony material from the organism to be identified
in liquid in each of the circles.
Place a drop of coagulase plasma in the suspension in one of the
circles and mix with a wooden applicator stick.
Place another drop of water or saline in the other circle as a
control.
Rock the slide back and forth, observing for agglutination of the
test suspension.
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2. Tube test (free coagulase): Emulsify a small amount of
the colony growth of the organism in a tube containing
0.5 mL of coagulase plasma.
Incubate the tube at 35°C for 4 hours and observe for
clot formation by gently tilting the tube.
If no clot is observed at that time, reincubate the tube
at room temperature and read again after 18 hours.
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V. Results
A. Interpretation
1. Slide test: A positive reaction will be detected within
10 to 15 seconds of mixing the plasma with the
suspension by the formation of a white precipitate and
agglutination of the organisms in the suspension.
The test is considered negative if no agglutination is
observed after 2 minutes.
The saline control should remain smooth and milky.
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
If the control suspension agglutinates as well, the test is
uninterpretable.

All strains that are coagulase-positive can be reported
as coagulase-positive Staphylococcus or, less precisely,
Staphylococcus aureus.

All strains producing negative slide tests must be tested
with the tube coagulase test.
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
The coagulase detected by this method is secreted
extracellularly and reacts with a substance in the plasma called
coagulase-reacting factor (CRF) to form a complex, which, in
turn, reacts with fibrinogen to form fibrin (clot formation)

Tests that are negative after 4 hours of incubation at 35°C
should be held at room temperature and read again after 18
to 24 hours,

Because some strains will produce fibrinolysin on prolonged
incubation at 35°C, causing dissolution of the clot during the
incubation period.

Rare S. aureus strains may be coagulase-negative, and some
animal isolates (S. intermedius, S. hyicus, S. delphini, and S.
schleiferi subsp. Coagulans) may be tube coagulase_positive.
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
As mentioned above, the recommended medium for both
the slide and the tube coagulase procedures is rabbit plasma
with EDTA.

Citrated plasma should not be used, because organisms
that are able to use citrate (e.g., Enterococcus species) will
yield positive results if they are inadvertently mistaken for
staphylococci.


This error can be avoided by always performing a
catalase test first.
Human plasma (e.g.,outdated material from blood banks)
contains variable amounts of CRF and antistaphylococcal
antibodies and should not be used to perform coagulase
tests.
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2. Tube test: The tube coagulase test is considered positive
if any degree of clotting is noted.
The tube should be gently tilted and not agitated,
because this may disrupt partially formed clotted
material.
Fibrinolysins produced by the organism may also dissolve
the clot soon after formation.
Tube tests that are negative after 4 hours should be
incubated at room temperature overnight and read
after 18 hours.
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I. Principle

The cytochromes are iron-containing hemoproteins that act
as the last link in the chain of aerobic respiration by
transferring electrons (hydrogen) to oxygen, with the
formation of water.

The cytochrome system is found in aerobic, or
microaerophilic, and facultatively anaerobic organisms,
so the oxidase test is important in identifying organisms that
either lack the enzyme or are obligate anaerobes.

The test is most helpful in screening colonies suspected of
being one of the Enterobacteriaceae (all negative) and in
identifying colonies suspected of belonging to other genera
such as Aeromonas, Pseudomonas, Neisseria,
Campylobacter, and Pasteurella (positive).
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
The cytochrome oxidase test uses certain reagent dyes,
such as p-phenylenediamine dihydrochloride, that
substitute for oxygen as artificial electron acceptors.

In the reduced state, the dye is colorless; however, in
the presence of cytochrome oxidase and atmospheric
oxygen, p-phenylenediamine is oxidized, forming
indophenol blue.
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II. Media and Reagents
A. Tetramethyl-p-phenylenediamine dihydrochloride, 1%
(Kovac's reagent)
B. Dimethyl-p-phenylenediamine dihydrochloride, 1%
(Gordon and McLeod's reagent)
III. Quality Control
Bacterial species showing positive and negative reactions
should be run as controls at frequent intervals.
The following are suggested:
A. Positive control: Pseudomonas aeruginosa
B. Negative control: Escherichia coli
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IV. Procedure
The test is commonly performed by one of two methods:
I) the direct plate technique, in which two to three drops of reagent
are added directly to isolated bacterial colonies growing on
plate medium;
2) the indirect paper strip procedure, in which either a few drops
of the reagent are added to a filter paper strip or commercial
disks or strips impregnated with dried reagent are used.
The tetramethyl derivative of p-phenylenediamine is
recommended because the reagent is more stable in storage and is
more sensitive to the detection of cytochrome oxidase and is
less toxic than the dimethyl derivative.
In either method, a loopful of suspected colony is smeared into the
reagent zone of the filter paper.
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V. Results
A. Interpretation
Bacterial colonies having cytochrome oxidase activity
develop a deep blue color at the inoculation site within 10
seconds.
Any organism producing a blue color in the 10- to 60-second
period must be further tested because it probably does not
belong to the Enterobacteriaceae.
Stainless steel or Nichrome inoculating loops or wires
should not be used for this test because surface oxidation
products formed when flame-sterilizing may result in falsepositive reactions.
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I. Principle
Rapid test for the presumptive identification of both group A
β-hemolytic streptococci and enterococci.
Whereas the original test was described as a 16- to 20-hour
agar test, subsequent PYR test formats included a 4-hour
broth assay and several rapid (10- to 15-minute) tests in
which the PYR reagent is impregnated in filter paper disks or
strips that are inoculated with the organism to be tested.
This chart describes the 4-hour broth PYR test.
Disks impregnated with the PYR substrate can also be used for
an even more rapid test.
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
The substrate used for the PYR test is L-naphthylamideβ-naphthylamide.

This compound is hydrolyzed by a specific bacterial
aminopeptidase enzyme.

Hydrolysis of the substrate by this enzyme releases free βnaphthylamide,which is detected by the addition of
N,N-dimethylaminocinnamaldehyde.

This detection reagent couples with the naphthylamide
to form a red Schiff base.
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II. Media and Reagents
I.
PYR broth (Todd-Hewitt broth with 0.01% Lpyrrolidonyl-ρ-naphthylamide) dispensed into sterile
tubes in 0.20-mL volumes.
2. PYR reagent (0.01% p-dimethylaminocinnamaldehyde)
III. Quality Control
A. Positive control: Enterococcus faecalis or Streptococcus
pyogenes
B. Negative control: Streptococcus agalactiae
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IV. Procedure
I. With a sterile bacteriologic loop, pick up the growth of
two to three morphologically similar colonies and
emulsify them in the small volume of PYR broth.
2. Incubate the tube at 35°C for 4 hours.
3. Add one drop of the PYR reagent and observe for
color change.
The reaction should be read and recorded 1 minute
after the addition of reagent.
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V. Results
A. Interpretation
I. Positive: the development of a deep cherry red color
within a minute of addition of the reagent
2. Negative: a yellow or orange color
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VI. Procedure Notes

It is essential that testing be performed before the
PYR test to determine that the organism is a
streptococcus (i.e., gram-positive cocci, catalasenegative).

Other organisms (e.g., some aerococci, staphylococci,
nutritionally variant streptococci, Arcanobacterium
haemolyticum) may also be PYR-positive.
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

I. PrincipleCatalase is an enzyme that decomposes hydrogen peroxide (H202)
into water and oxygen.

Chemically, catalase is a hemoprotein, similar in structure to
hemoglobin, except that the four iron atoms in the molecule are in
the oxidized (Fe3+), rather than the reduced (Fe2+), state.

Excluding the streptococci, most aerobic and facultative bacteria
possess catalase activity.

Hydrogen peroxide forms as one of the oxidative end products of
aerobic carbohydrate metabolism.

If allowed to accumulate, it is lethal to bacterial cells.
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
Catalase converts hydrogen peroxide into oxygen and
water as shown by the following reaction:

The catalase test is most commonly used to
differentiate members of the Micrococcaceae from
members of the Streptococcaceae.
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II. Reagents
 A. Hydrogen peroxide 3% stored in a brown bottle
under refrigeration
 B. An 18- to 24-hour culture of the organism to be
tested
 III. Quality Control
 The hydrogen peroxide reagent must be tested with
positive and negative control organisms each day or
immediately before unknown bacteria are tested.
 A. Positive control: Staphylococcus aureus
 B. Negative control: Streptococcus species

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IV. Procedure
 I. With an inoculating needle or a wooden applicator
stick, transfer growth from the center of a colony to the
surface of a glass slide.


2. Add one drop of 3% hydrogen peroxide and observe
for bubble formation.
V. Results
 A. Interpretation
 The rapid and sustained appearance of bubbles or
effervescence constitutes a positive test.

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
Because some bacteria possess enzymes other than
catalase that can decompose hydrogen peroxide, a few
tiny bubbles forming after 20 to 30 seconds is not
considered a positive test.

In addition, catalase is present in red cells, so care must
be taken to avoid carryover of red cells with the colony
material.
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THE END
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