BACTERIOLOGY LABORATORY

S.Y. ‘23 - ‘24 | BY HUEN MINGARINE EXERCISE 18

TEXTBOOK ● Advantage: to test different

antimicrobials (10-15
1.0 Antimicrobial Susceptibility Testing antimicrobials) at the same time
against a single isolate
● Disadvantage: inability to produce a
1.1 Broth Dilution Method penicillin MIC that is consistently
within the resistant range for
staphylococci that are low-level
● It involves challenging the organism of B-lactamase producers
interest with antimicrobial agents in a broth
environment (Mueller-Hinton broth). NOTE:
● A specific amount of antibiotic is prepared in During inoculation, care must be taken to ensure that
a decreasing concentration in broth by serial all bacteria in the test inoculums are deposited
dilution technique, and a standard amount of directly into the antimicrobial solution, otherwise
the test organism is inoculated. bacteria may adhere to the wall of the tube or well
● Absence of turbidity of broth signifies above the meniscus of the antimicrobial solution and
inhibition of bacterial growth by the may remain viable during incubation of the MIC
antibiotics being tested. portion of the test.
● Principle: to determine the lowest
concentration of the antimicrobial drug (MIC)
required to inhibit bacterial growth. 1.2 Terminology
● Preferred method: Serial twofold-dilution
concentrations (μg/mL) 1. Minimum Inhibitory Concentration (MIC)
● Standard inoculum size: 5 x 105 CFU/mL ● The lowest concentration of the
● Can be used to determine MIC and MLC antimicrobial agent which inhibits
concentrations. bacterial growth.
● MH broth with 2% NaCl – to improve 2. Minimum Lethal Concentration (MLC)
detection of oxacillin-resistant staphylococci ● The lowest concentration of the
● MH broth with 2%-5% lysed horse blood – for antimicrobial agent which kills the
streptococci bacterial growth when subcultured
to a fresh medium.
A. Broth Macrodilution 3. Minimum Bactericidal Concentration (MBC)
● Susceptibility medium: MH broth ● The lowest concentration of the
(nonfastidious bacteria) antimicrobial agent needed to kill
● Standard inoculum size: 5 x 105 the bacterial growth.
CFU/mL 4. Breakpoint (cutoff)
● Incubation time: 16-24 hours ● Refers to the concentration of an
(overnight) at 35°C antimicrobial agent that coincides
● Advantage: to test antimicrobials with a susceptible or intermediate
not included in the routine test or MIC breakpoint for a particular
for fastidious bacteria; used when drug.
MBC endpoints need to be 5. Trailing Growth
subsequently determined ● Involves heavy bacterial growth at
● Disadvantage: impractical to use if lower concentrations followed by
there are several antimicrobial one or more wells that show greatly
agents or isolates to be tested reduced growth in the form of a
small button or a light haze.
B. Broth Microdilution 6. Skipped wells
● Susceptibility medium: MH broth ● Involve growth at higher
(nonfastidious bacteria) concentrations and no growth at
● Standard inoculum size: 5 x 105 one or more of the lower
CFU/0.1 mL well concentrations; it may indicate
● Incubation time: 16-24 hours contamination, improper incubation,
(overnight) at 35° improper concentration of the

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BACTERIOLOGY LABORATORY
S.Y. ‘23 - ‘24 | BY HUEN MINGARINE EXERCISE 18

antimicrobials and presence of ● Susceptibility standard medium: Mueller

unusual resistant isolate. Hinton Agar (MHA)
● Standard inoculum size: 1.5 x 10 CFU/mL
● Procedure: filter paper disks impregnated
1.3 Agar Dilution Method with various antimicrobial agents of specific
concentrations are carefully placed on an
● It is the reference method for testing agar plate previously inoculated with the
anaerobes and N. gonorrhoeae - because N. bacteria being tested.
gonorrhoeae has a tendency to lyze in broth
media, resulting in false-susceptibility. 2.0 Turbidity Standard
● It is used only in research studies because
plate preparation is laborious.
● Media for anaerobes: Brucella-laked SBA ● The use of a standard Inoculum size is as
blood (Wadsworth method) incubated at important as culture purity and is
35°C for 48 hours accomplished by comparison of the turbidity
● Shelf life of agar dilution plate: one week (for of the organism suspension with a turbidity
most antimicrobial agents) standard.
● Susceptibility medium: ● 0.5% McFarland Turbidity Standard (Barium
- MHA (aerobes) Sulfate Suspension)
- MHA +5% SB (fastidious bacteria) - 99.5ml of 1% H2SO4 + 0.5ml of
● Standard inoculum size: 1 x 10 CFU/mL 1.175% of barium chloride
● Procedure: - is equivalent to 1.5 x 10 colony
- A standard inoculum of bacteria is forming units (CFU)/mL
spot-inoculated onto a 100-mm - it should be tightly sealed and
plate. stored in the dark at room
- A series of plates containing temperature.
varying concentrations of each ● Pure cultures are grown or are directly
antimicrobial and control plates are prepared from agar plates to match the
prepared. turbidity of the 0.5 McFarland standard.
- One or more bacterial isolates (up
to 32 different isolates) are tested
per plate; MIC can also be 3.0 Preparation of Pure Culture for
determined. Susceptibility Testing

1.4 Disk Dilution Method (Kirby-Bauer 1. Pure inocula (cultures) are obtained by
Test) selecting 4-5 colonies of the same
morphology, then inoculate into broth
medium and incubate for 3-5 hours to
● Is limited to aerobic and facultatively achieve a turbid suspension. (Alternatively,
anaerobic bacteria. 4-5 colonies 16-24 hours of age may be
● It is a qualitative method which provides the selected from an agar plate and suspended
greatest flexibility and cost- effectiveness - it into broth medium or 0.85% NSS to achieve a
allows the laboratory to test any 12 turbid suspension).
antimicrobial agents on a 150-mm MHA - Direct inoculums suspension is
plate. preferred for fastidious bacteria.
● It depends on the formation of a gradient of 2. Matching turbidity using the unaided eye is
antimicrobial concentrations as the facilitated by holding the bacterial
antimicrobial diffuses into the agar the drug suspension and McFarland tubes side by
concentration decreases at increasing side and viewing them against a black-lined
distances from the disk. background (inoculum suspension should be
● Principle: the larger the zone of inhibition, the used within 15 minutes).
lower the MIC - the zone of inhibition is 3. If the bacterial suspension initially does not
inversely related to MIC match the standard's turbidity, the

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BACTERIOLOGY LABORATORY
S.Y. ‘23 - ‘24 | BY HUEN MINGARINE EXERCISE 18

suspension may be diluted, or supplemented 7. The zone size of a motile, swarming

with more organisms, as needed. organism such as Proteus should be ignored
(thin film of growth).
8. Discontinuous, poor growth or tiny colonies
4.0 During streaking and placement of near the end of the zone size are also
discs ignored.
9. Colonies within a clear zone should not be
ignored - these colonies may occur as a
1. Turn the plate 60 degrees between each result of contamination or testing a mixed
streaking (overlapping streaking). culture. The original colony should be
2. The surface of the medium should be retested.
allowed to dry 3-5 minutes but not longer 10. Zone measurement is compared with the
than 15 minutes. interpretive tables of CLSI and results are
3. Within 15 minutes of inoculation, the interpreted as susceptible, intermediate,
antimicrobial agents are applied on the MHA. resistant or nonsusceptible.
- When disks containing known ● Susceptible – the patient's infecting
concentrations of antimicrobial organism should respond to therapy
agents are placed on the surface of with that antimicrobial agent using
a freshly Inoculated plate, the agent the recommended dosage for the
immediately begins to diffuse and site of infection; indicated by the
establish a concentration gradient presence of a zone of inhibition
around the paper disk. around the antibiotic disk.
4. The discs must be positioned no closer than ● Intermediate – the microorganism
24mm from disc center to disc center and no falls into a range of susceptibility in
closer than 10-15mm from the edge of the which the MIC approaches or
plate. The disks are pressed firmly to ensure exceeds the level of antimicrobial
contact with the agar. agent that can be achieved and for
5. Within 15 minutes of disk placement, plates which clinical response is likely to
are inverted and incubated at 35°C for 16- 18 be less than with a susceptible
hours. Plates are inverted to avoid strain
contamination of moisture on the agar ● Resistant – the microorganism is
surface that can interfere with interpretation inhibited by the usually achievable
of test results. concentrations of the antimicrobial
agent based on the dosages
5.0 normally used with that drug.
Reading of Plates and Interpretation 11. Quality control plates should be read prior to
of Results reading results of patient isolates to
determine whether the test was performed
1. The lawn of growth must be confluent or correctly.
almost confluent.
2. Provided growth is satisfactory, the diameter 6.0 Causes of False Resistance
of each inhibition zone is measured using a
caliper or ruler.
3. Plates are examined 2-3 inches above a 1. Use of unduly heavy inocula of cultures or
black, non reflecting surface and the zones undiluted specimen materials.
are measured from the back side of the 2. Late examination of test plates after zones
plate. have become overgrown.
4. Transmitted light (plate held up to light 3. Use of disc with inadequate drug
source) rather than reflected light will concentration due to prolonged storage,
improve the accuracy of tests with failure to refrigerate as from disc container
penicillinase-resistant-penicillins. opened frequently.
5. For media containing blood, the plate is 4. Use of wrong organisms.
examined with the lid removed.
6. The appearance of individual colonies is
unacceptable.

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BACTERIOLOGY LABORATORY
S.Y. ‘23 - ‘24 | BY HUEN MINGARINE EXERCISE 18

NOTES TO REMEMBER: - Some MRSA may go undetected if

● MHA is composed of beef infusions, nucleic less than 24 hours of incubation. •
acids, vitamins, casein hydrolysate, (casein 5%-7% CO2 for N. meningitidis and
neutralizes fatty acid) and agar. S. pneumoniae.
● MHA containing 5% sheep's blood is used for 4. The pH of the medium (7.2-7.4).
testing streptococci and other fastidious - incubation in CO2 results in
organisms. decreased pH leads to decreased
● The MIC test represents a semiquantitative activity of aminoglycosides,
method and the concentration (μg/mL) of a erythromycins, and clindamycin but
drug required to inhibit the growth of increased activity of tetracyclines.
bacterial isolate is reported together with a 5. The number of disks per plate.
susceptible, intermediate or resistant - 12 disks/150mm plate
interpretation. - placement of more than 12 disks
● The diameter of the zone of inhibition around may result in overlapping zones
the disk is measured in millimeters using a (erroneous results).
caliper; a wide zone surrounding a disk 6. The concentration of divalent cations
signifies more susceptibility of the organism (calcium and magnesium)
to the antibiotic. - It can affect testing of
● Zone width is related to antibiotic aminoglycosides and tetracyclines
concentration, diffusion rate through agar against P. aeruginosa.
and solubility. - elevated concentrations of the
● For long term-storage, disks are stored at divalent cations may result in
20°C or below in a non-frost-free freezer diminished activity of the
while working supply disks at 2°-8°C for at aminoglycosides against P.
least 1 week. aeruginosa and decreased activity
● Routine susceptibility testing on B-hemolytic of tetracyclines against all bacteria,
streptococci is generally not necessary. and vice versa.

7.0 Factors Affecting Zone of Inhibition 8.0 E TEST

1. The amount of inoculum or test organism. ● is a dilution test based on the diffusion of a
- only pure cultures can be tested. continuous concentration gradient of an
- if the inoculum is too light it would antimicrobial agent from a plastic strip into
result in false susceptibility; if the an agar medium.
inoculums is too heavy it would ● It uses thin plastic strips that are
result in false resistance. impregnated on the under surface with an
- use of old cultures may result in antimicrobial concentration gradient and are
false susceptibility. marked on the upper surface with a
2. Thickness of the susceptibility agar plate (4 continuous MIC concentration index or
mm). scale.
- if the agar is too thick, zone sizes ● The strip is placed on the surface of the
would be smaller; if agar is too thin, culture medium inoculated with the desired
zone sizes would be larger. organism and the antibiotic diffuses into the
3. The growth rate of the test organism. surrounding agar.
- Recommendation: air at 35°C for ● (+) result: ellipse of growth inhibition
most bacteria (16-18 hours); N. ● The MIC is read at the point on the scale
gonorrhoeae for 20-24 hours. where the ellipse intersects the strip.
- lower temperatures may lead to ● The strip can be placed on a special enriched
larger zones of inhibition. media or incubation atmosphere - used as an
- temperatures higher than 35°C may alternative susceptibility test for fastidious
lead to false detection of MRSA. bacteria (S. pneumoniae and H. influenzae)
- prolonged Incubation may result in and anaerobic bacteria.
false resistance interpretation.

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