SEROLOGIC TEST
Minimum Bactericidal Concentration (MBC) –
SEROLOGICAL TEST AND IMMUNODIAGNOSIS
 BACTERIAL SEROTYPING:
o LANCEFIELD CLASSIFICATION
 Classification according to
carbohydrates present in the cell wall
of Streptococcus
o KAUFFMAN-WHITE SCHEME
 Classification of serotypes of Salmonella
based on surface antigen
o E. coli 0157:H7
 DIAGNOSTIC TEST
o Schultz- Charlton
 Diagnostic test for Streptococcus
pyogenes
o Widal test
 Detects antibody agains Salmonella
 Antigen used:
 O antigen – somatic antigen, heat
stable, lipopolysaccharide
 H antigen- Flagellar antigen, heat
labile, protein
 K/Vi antigen – capsular antigen, heat
labile, polysaccharide
o Weil-felix
 Detects antibody against Rickettsia
 Antigen used:
 OX-2 and OX-19: derived from
Protesus vulgaris
 OX-K : Derives from Protesus
mirabilis
 SUSCEPTIBILITY TEST
o Mantoux, Mendel, Tuberculin, Vollmer’s
vonPirket –tuberculosis
o Schick’s test – diphtheria
o Dick’s test – scarlet fever
o Mallein test – Glander’s disease
o Ascoli test – anthrax
o Francis test – S. pneumoniae infection
 Principle of serologic test
o Latex agglutination
o Precipitation test
o Labelled immunoassays ( EIA, FIA, RIA)
ANTIBIOTIC SUSCEPTIBILITY TESTING
TERMINOLOGIES:
1. Minimum Inhibitory Concentration (MIC) – the
lowest concentration of the antimicrobial agent
which inhibits the bacterial growth. 1 st dilution
tube with out visible growth
2. Minimum Lethal Concentration (MLC) – the
lowest concentration of the antimicrobial agent
which kills the bacterial growth when
subcultured to a fresh medium.
3.
the lowest concentration of the antimicrobial
agent needed to kill the bacterial growth.
Lowest concentration to kill 99.9%; subcultur
tubes near MIC to find 1st plate with no growth
4. Breakpoint (cutoff) – refers to the
concentration of an antimicrobial agent that
coincides with a susceptible or intermediate
MIC breakpoint for a particular drug.
5. Trailing Growth – Involves heavy bacterial
growth at lower concentrations followed by
one or more wells that show greatly reduced
growth in the form of a small button or a light
haze.
6. Skipped wells – Involve growth at higher
concentrations and no growth at one or more
of the lower concentrations; it may indicate
contamination, improper incubation, improper
concentration of the antimicrobials and
presence of unsual resistant isolate.
A. BROTH DILUTION METHOD
B. AGAR DILUTION METHOD
C. DISK DIFFUSION METHOD (Kirby-Bauer Test)
D. E TEST
E. MINIMUM BACTERICIDAL CONCENTRATION TEST
F. TIME KILL ASSAY
G. SYNERGY TEST
H. SERUM BACTERICIDAL TEST
A. BROTH DILUTION METHOD
 It involves challenging the organism of interest
with antimicrobial agents in a broth
environment (Mueller-Hinton broth).
 A specific amount of antibiotic is prepared in a
decreasing concentration in broth by serial
dilution technique, and a standard amount of
the test organism is inoculated.
 Absence of turbidity of broth signifies inhibition
of bacterial growth by the antibiotics being
tested.
Principle: to determine the lowest concentration of
the antimicrobial drug (MIC) required to inhibit
bacterial growth.
a.Broth Macrodilution
 Susceptibility medium: MH broth (nonfastidious
bacteria)
 Standard Inoculum size: 5 x 105 CFU/mL
 Incubation time: 16-24 hrs (overnight) at 35⁰C
 Advantage: to test antimicrobials not included
in the routine test or for fastidious bacteria;
used when MBC endpoints need to be
subsequently determined.
  Disadvantage: impractical to use if there are
several antimicrobial agents or isolates to be
tested.
b. Broth Microdilution
 Susceptibility medium: MH broth (nonfastidious
bacteria)
 Standard inoculum size: : 5 x 105 CFU/0.1 mL
well
 Incubation time: 16-24 hours (overnight) at
35⁰C
 Advantage: to test different antimicrobials (10-
15) at the same time against a single isolate.
 Disadvantage: inability to produce a penicillin
MIC that is consistently within the resistant
rangefor staphylococci that are low-level ß-
lactamase producers.
B. AGAR DILUTION METHOD
 The antimicrobial solutions brought together on
a molten and cooled culture medium
 A series of plates containing varying
concentration of each antimicrobials and
control plates are prepared.
 One or more bacterial isolates (up to 32
different isolates) are tested per plate; MIC can
also be determined.
 Procedure: A standard inoculum of bacteria is
spot-inoculated onto 100- mm plate.
 Susceptibility medium: MHA (aerobes)
MHA + 5% SB (fastidious
bacteria)
 Standard inoculum size: 1 x 104 CFU/mL
C. DISK DIFFUSION METHOD (Kirby-Bauer Test)
 Is limited to aerobic and facultatively anaerobic
bacteria.
 It is a qualitative method which provides the
greatest flexibility and cost – effectiveness – it
allows the laboratory to test ant 12
antimicrobial agents on a 150-mm MHA plate.
 Principle: the larger the zone of inhibition, the
lower the MIC – the zone of inhibition is
inversely related to MIC.
 Susceptibility standard medium: Mueller Hinton
Agar (MHA)
 Standard inoculum size: 1.5 x 108 CFU/mL
 Procedure: filter paper disks impregnated with
various antimicrobial agents of specific
concentrations are carefully placed on an agar
plate previously inoculated with the bacteria
being tested
■ Mueller – hinton Agar
Depth 4mm
 pH 7.2-7.4
 Physiologic concentration of Mg and Ca
 35 0C ambient air
 108 organism ( mc Farland 0.5)
 Weekly and with each new lot of agar or disc
( E. coli, S. aureus, P aeruginosa)
TURBIDITY STANDARD:
 The use of a standard inoculum size is as
important as culture purity and is accomplished
by comparison of the turbidity of the organism
suspension with a turbidity standard.
 0.5% Mc Farland Turbidity Standard (Barium
Sulfate Suspension)
o 99.5 mL of 1% H2SO4 + 0.5 mL 1.175% of
Barium Chloride
o is equivalent to 1.5 x 108 colony
forming units (CFU)/mL
o it should be tightly sealed and stored in
the dark at room temperature.
 Pure cultures are grown or are directly
prepared from agar plates to match the
turbidity of the 0.5 McFarland standard.
Preparation of Pure Culture for Susceptibility Testing:
1. Pure inocula (cultures) are obtained by selecting
4-5 colonies of the same morphology, then
inoculate into broth medium and incubate for
3-5 hours to achieve a turbid suspension.
2. Alternatively, 4-5 colonies 16-24 hours of age
may be selected from an agar plate and
suspended into broth medium or 0.85% NSS to
achieve a turbid suspension.
■ Direct inoculums suspension is preferred for
fastidious bacteria.
3. Matching turbidity using the unaided eye is
facilitated by holding the bacterial suspension
and McFarland tubes side by side and viewing
them against a black-lined background –
inoculums suspension should be used within 15
minutes.
4. If the bacterial suspension initially does not
match the standard’s turbidity, the suspension
may be diluted, or supplemented with more
organisms, as needed.
During streaking and placement of discs:
1. Turn the plate 60 degrees between each
streaking.
2. The surface of the medium should be allowed
to dry 3-5 minutes but not longer than 15
minutes.
 3. Within 15 minutes of inoculation, the
antimicrobial agents are applied on the MHA.
4. The discs must be positioned no closer than 24
mm from disc center to disc center and no
closer than 10-15 mm from the edge of the
plate. The disks are pressed firmly to ensure
contact with the agar.
5. Within 15 minutes of disk placement, plates are
inverted and incubated at 35⁰C for 16-18
hours. Plates are inverted to avoid
contamination of moisture on the agar surface
that can interfere with interpretation of test
results.
Reading of Plates and Interpretation of Results:
1. The lawn of growth must be confluent or almost
confluent.
2. Provided growth is satisfactory, the diameter or
each inhibition zone is measured using a caliper
or ruler.
3. Plates are examined 2-3 inches above a black,
nonreflecting surface and the zones are
measured from the back side of the plate.
4. Transmitted light (plate held up to light source)
rather than reflected light will improve the
accuracy of tests with penicillinase- resistance-
penicillins.
5. For media containing blood, the plate is
examined with the lid removed.
6. The appearance of individual colonies is
unacceptable.
7. The zone size of a motile, swarming organism
such as Proteus should be ignored (thin film of
growth)
8. Discontinuous, poor growth or tiny colonies
near the end of the zone size are also ignored.
9. Colonies within a clear zone should not be
ignored – these colonies may occur as a result
of contamination or testing a mixed culture. The
original colony should be retested,
10. Zone measurement is compared with the
interpretive tables of CLSI and results are
interpreted as susceptible, intermediate,
resistant or nonsusceptible.
– SUSCEPTIBLE – the patient’s infecting
organism should respond to therapy with
that antimicrobial agent using the
recommended dosage for the site of
infection, indicated by the presence of
zone of inhibition around the antibiotic
disk.
– INTERMEDIATE – the microorganism falls
into a range of susceptibility in which the
MIC approaches or exceeds the level of
antimicrobial agent that can be achieved
and for which clinical response is likely to
be less than with a susceptible strain
– RESISTANT – the microorganism is
inhibited by the usually achievable
concentrations of the antimicrobial agent
based on the dosages normally used with
that drug.
– NONSUSCEPTIBLE – when there are no
intermediate or resistance interpretative
criteria, only a susceptible criteria, and the
MIC or disk diffusion zone size for
classifying the organism as susceptible is
not achieved; it commonly occurs with
newer antimicrobials. It does not mean
that the organism is resistant to the
antimicrobial agent.
11. Quality control plates should be read prior to
reading results of patient isolates o determine
whether the test was performed correctly
Causes of False Resistance
1. Use of unduly heavy inocula of cultures or
undiluted specimen materials.
2. Late examination of test plates after zones have
become overgrown (18-24 hours) incubation.
3. Use of disc with inadequate drug concentration
due to prolonged storage, failure to refrigerate
as from disc container opened frequently.
4. Use of wrong organism.
NOTES TO REMEMBER:
 MHA is composed of beef infusions, nucleic
acids, vitamins, casein hydrolysate, (casein
neutralizes fatty acid) and agar.
 MHA containing 5% sheep’s blood is used for
testing streptococci and other fastidious
organisms.
 The MIC test represents a semiquantitative
method and the concentration (microgram/mL)
of a drug required to inhibit the growth of
bacterial isolate is reported together with a
susceptible, intermediate or resistant
interpretation.
 The diameter of the zone of inhibition around
disk is measured in mm using a caliper; a wide
zone surrounding a disk signifies more
susceptibility of the organism to the antibiotic.
 Zone width is related to antibiotic
concentration, diffusion rate through agar and
solubility.
  For long term-storage, disks are stored at 20⁰C
or below in an non-frost-free freezer while
working supply disks at 2-8⁰C for atleast 1 week.
 Routine susceptibility testing on ß-hemolytic
streptococci is generally not necessary.
Factors Affecting Zone of Inhibitions:
1. The amount of inoculum or test organism.
– only pure cultures can be tested.
– If the inoculum is too light it would
result to false susceptibility; if the
inoculum is too heavy it would result to
false resistance.
– Use of old cultures may result to false
susceptibility.
2. Thickness of the susceptibility agar plate (4 mm)
– If the agar is too thick, zone size would
be smaller, if agar is too thin, zone
sizes would be larger
3. The growth rate of the test organism.
– Air at 35⁰C for most bacteria (16-18
hours); N.gonorrhea for 20-24 hours.
– Lower temperatures may lead to larger
zones of inhibition.
– Temperatures higher than 35⁰C may
lead to false detection of MRSA.
– Prolonged incubation may result in false
resistance interpretation.
– Some MRSA may go undetected if less
than 24 hours of incubation.
– 5-7% CO2 for N. meningitides and
S.pneumonia.
4. The pH of the medium (7.2-7.4)
Incubation in CO2 results in decreased pH –
leads to decreased activity of aminoglycosides,
erythromycins, and clindamycin but increased
activity of tetracyclines.
5. The number of disk per plate.
– 12 disks/150 mm plate
– Placement of more than 12 disks may
result in overlapping zones (erroneous
results)
6. The concentration of divalent cations (Calcium
and Magnesium)
– It can affect testing of aminoglycosides
and tetracyclines against P.
aeruginosa.
– Elevated concentrations od the divalent
cations may result to diminish activity
of the aminoglycosides against
P.aeruginosa and decreased activity of
tetracyclines against all bacteria, and
– vise versa.
D. E TEST
 (+) result: ellipse of growth inhibition
 The MIC is read at the point on the scale where
the ellipse intersects the strip.
 The strip can be placed on special enriched
media or incubation atmosphere – used as an
alternative susceptibility test for fastidious
bacteria (S. pneumoniae and H. influenzae) and
anaerobic bacteria.
E. MINIMUM BACTERICIDAL CONCENTRATION TEST
 An in vitro determination of the amount of
antimicrobial agent required to kill as well as
inhibit the bacteria.
 It is performed together with a broth
macrodilution or microdilution MIC test – a mL
aliquot of each clear MIC tube or well is
subcultured to an agar medium.
 Standard inoculums: 5 x 105 CFU/mL
 Final dilution: 1:1000 (0.01 mL from the original
tube in 10 mL)
 Volume spread per plate: 0.1 mL
 Dilution Factor: 1:10,000 (104)
PROCEDURE;
• An actual colony count must be performed on
the test inoculums at the time the MIC test is
inoculated.
• The number of colonies that grow on subculture
are compared with the actual number of organisms
inoculated into the MIC test tubes or wells to
determine the extent of bactericidal activity at each
antimicrobial concentration.
INTERPRETATION OF RESULTS:
• If the numbers of colonies on a subculture plate
total less than 0.1% of the initial inoculums
(indicating 99.9% or more killing), a bactericidal
effect has been achieved.
• A 99.9% killing (or 0.1% survival) would be
accomplished if 5 or fewer colonies grow on
subculture of each clear well or tube after reading
of the MIC test.
• Paradoxic (eagle) effect – is decreased
bactericidal activity at a higher drug concentration.
• Tolerance – it is defined as an MBC:MIC ration
of > 32.