PHMC 4123

MICROBIOLOGY
LABORATORY REPORT 3

TOPIC: ANTIBIOTIC SENSITIVITY TEST

LECTURE’S NAME: MUHAMMAD SYAFFUAN AHMAD NAJIB

FULL NAME MATRIX NUMBER

SANJANA KAUR A/P PRAMJIT SINGH 2042222017
SARMITHA A/P KANNAN 2042222018
SHARMILA A/P RAVICHANDRAN 2042222020
LAB REPORT 3: ANTIBIOTIC SENSITIVITY TEST
INTRODUCTION:

It is not enough to just identify your organism. You also need to know what antimicrobial agents
your organism is susceptible to. There are several methods to determine this. Dilution testing is
used to quantitatively determine the minimal concentration (in mg/ml) of antimicrobial agent to
inhibit or kill the bacteria. This is done by adding two-fold dilutions of the antimicrobial agent
directly to an agar pour, a broth tube, or a micro-broth panel. The lowest level that inhibits the
visible growth of the organism is considered the Minimum Inhibitory Concentration (MIC). The
agar pour method is considered the reference test procedure in Europe. The broth dilution method is
more widely accepted in North America. The E test (AB Biodisk) is a plastic strip with a gradient
concentration of antimicrobial agents impregnated in it. The strip is placed directly on the surface of
an inoculated plate. The MIC is read from the strip where the growth inhibition intercepts the disk.
These strips are relatively expensive.
Many physicians however do not need any know the exact MIC, but just which antibiotics the
pathogen is susceptible, intermediate, or resistant to. The Kirby-Bauer agar diffusion method is well
documented and is the standardized method for determining antimicrobial susceptibility. White
filter paper disks (6 mm in diameter) are impregnated with known amounts of antimicrobial agents.
Each disk is coded with the name and concentration of the agent. For example, 10 µg of Ampicillin
is indicated on the disk by AM-10. The code is listed on the Disk Zone Diffusion Diameter Chart.
The impregnated disks are placed on an inoculated Mueller Hinton Agar (MHA) plate. The drug
diffuses through the agar. The plates are incubated for 16-24 hours. The agar may be supplemented
with blood or you may use blood agar for fastidious organisms. The diameter of the visible zone of
inhibition is measured and compared to reference values. There should be sufficient bacteria to
form a visible lawn of growth where it is not inhibited by the drug. The results are interpreted
qualitatively as resistant, intermediate, or susceptible. The standard protocol must be followed
exactly for you, or any clinical lab, to interpret the results reliably. There may be some inhibition of
growth and the organism could still be considered resistant to that antimicrobial agent if the zone
diameter is smaller than the reference values listed on the chart. Also note that different
antimicrobial agents have different measurements for resistant, intermediate, and susceptible. A
zone of inhibition may be considered susceptible to one antimicrobial agent and not to another. For
example, for ampicillin (AM-10) to be an effective antimicrobial agent, the zone of inhibition for
enterics and most strips must be greater than 16 mm while for staphs it must be greater than 28 mm.
When determining which antimicrobial agents are best for treatment when multiple zones of
inhibition are present, be sure to look at the relative zone of inhibition for that particular
antimicrobial agent and compare your measurements to that. For example, let’s say that your enteric
organism has a zone of inhibition around the Polymixin B disk of 20 mm and a zone of inhibition
around the Tetracycline disk of 20 mm. Because these measurements are larger than the
susceptibility zones listed on the Disk Zone Diffusion Diameter Chart, both of these antibiotics
would be considered possibilities for treatment.
However, when we look more closely, we see that a 16 mm zone of intermediate for amoxicillin is
only 1 mm larger than what is required to be susceptible while a 20mm zone of inhibition for
Polymixin B is 32mm larger than the minimum susceptibility measurement needed. In this
particular case then, Polymixin B and Tetracycline would both be adequate for treatment, but
Polymixin B would be the best choice.

OBJECTIVES:
1. To practice performing the antibiotic susceptibility testing following a step-by-step procedure.
2. To observe the zones of inhibition and to measure the zones using metric rulers, calipers, or a
special transparent template.
3. To record the susceptibility of the organisms to each antibiotic disk.

MATERIALS:
Actively growing broth or streaked plate of a single organism (pure culture), Gram stain materials
(optional), 1 Mueller Hinton Agar (MHA) plate (use BHI plate for all strips), 1 jar sterile saline, 1
sterile test tube, 1 sterile 5 mL pipette (with pipette bulb), 1 sterile swab, 0.5 McFarland test
standard, McFarland reference card, Spectrophotometer (optional), 8 disk dispenser or individual
disk dispensers and Antibiotic disk cartridges with

o Ampicillin (AM-10)
o Chloramphenicol (C-30)
o Nalidixic Acid (NA-30)
o Penicillin G (P-10)
o Polymixin B (PB-300)
o Streptomycin (S-10)
o Tetracycline (Te-30)
o Trimethoprim (TMP-5)
METHODOLOGY:

1. Perform a Gram Stain to confirm culture purity from your subculture plate.
2. Using a sterile 5 mL pipette, add 5 mL of sterile saline to a sterile test tube.
1. Alternatively, a tube of sterile water or a tube of sterile tryptic soy broth (TSB) can
be used.
3. Using an inoculating loop or needle, select several colonies from your subculture plate and
transfer them to a tube of sterile saline.

1. Select several colonies so you don’t inadvertently pick a non-representative colony.

1. Dilute your organism to obtain a turbidity equivalent to the 0.5 McFarland test standard.

1. Hold your diluted tube and the 0.5 McFarland test standard against the black-lined
McFarland reference card to accurately rate the turbidity.
2. This could also be measured in a spectrophotometer (87% transmittance at 686nm).

1. Within 15 minutes of diluting your organism, dip a sterile swab into the properly adjusted
inoculum. Lift it slightly out of the suspension and firmly rotate the swab several times
against the upper inside wall of the tube to express excess fluid.

1. If your swab is too wet your agar surface will not dry correctly and the antimicrobial
agents in the disk will diffuse through the wet surface and not into the agar.

1. Streak the entire surface three times with the swab, turning the plate 60 degrees between
streakings (turn the swab too) to obtain an even inoculation.

1. Close the lid and let sit for 3-5 minutes before applying the drug-impregnated disks.

2. Apply the disks using a dispenser using an aseptic technique. Deposit disks so that the
centers are at least 24 mm apart; up to 12 disks may be placed on a 150 mm plate, and 5
disks on a 100 mm plate.

1. We usually apply more than the recommended disk number to conserve plates and
our recommendations are not used to treat any patients!

3. Lightly press the disk down with a sterile swab to make contact with the surface.

1. You don't want to smush it into the agar itself!

4. Place your plate agar side up (inverted) in a 37°C incubator.

1. Streptococcus organisms should be on BHI instead of MHA plates.

2. Streptococcus organisms should be incubated in an atmosphere enriched with 5-10%
CO2.

5. Examine the plate after 16-24 hours of incubation.

6. Measure (in mm) only zones showing complete inhibition by gross visual inspection.

1. Hold the measuring device (ruler or calipers) over the back of the inverted plate over
a black non-reflective surface and illuminate from above.

7. Compare the values you obtained with those on the Disk Diffusion Zone Diameter Chart to
determine the susceptibility level to the antibiotics used.
8. Report the values as:

1. Resistant - indicates that clinical efficacy has not been reliable in treatment studies.
2. Intermediate - implies clinical applicability in body sites where the drug is
physiologically concentrated or when a high dosage of the drug can be used.
3. Susceptible - implies that an infection due to the organism may be treated with the
concentration of antimicrobial agent used unless otherwise contraindicated.
DISCUSSION:

We concentrated on how the antibiotic makes inactive the bacteria. We doing a group so we both
make this experiment t.Firstly we took the agar plate and we spirit the gram stain with the swab in
the agar space. Then we arrange the antibiotic in the agar plate which we spirit the gram stain. we
divide the 8 antibiotics so each person took 4 antibiotics. After that, we leave it for 1 day to see the
results. Here are the results:

Antibiotic Disc code Resistant (< or Intermediate Susceptible(=

=mm) (mm) or > mm)
Amoxicillin AMAX30 16
Ampicillin AMP10 32
Chloramphenic C10 25
ol
Ciprofloxacin CIP10 35
Gentamycin GM 13
Vancomycin VA30 0
Erythromycin E15 0
Pencilin P10 0

CONCLUSION:

We are dependent on antibiotics for the treatment of infectious diseases and they are critical for the
success of advanced surgical procedures, such as organ and prosthetic transplants. Antibiotic-
resistance mechanisms create an enormous clinical and financial burden on healthcare systems
worldwide. Despite the problem of antibiotic resistance in infectious bacteria, little is known about
the diversity, distribution, and origins of resistance genes, especially for the unculturable majority
of environmental bacteria. There exists high-level resistance both to antibiotics that have for
decades served as gold standard treatments and to those only recently approved for human use. The
study of the environmental resistance reservoir using metagenomic approaches will provide an early
warning system for future clinically relevant antibiotic-resistance mechanisms. Knowledge of such
natural variations will complement studies on clinical isolates to guide the rational development of
next-generation antibiotics that will be active against resistant strains.
Functional and sequence-based metagenomics has been used for the discovery of novel resistance
determinants and the improved understanding of antibiotic resistance mechanisms. Metagenomic
sequence data can be used to generate sample-specific and temporal antibiotic resistance profiles to
facilitate an understanding of the ecology of the microbial communities in an environment as well
as the epidemiology of antibiotic resistance gene transport between and among environments.
Traditional approaches to antibiotic resistance typically concentrate on human pathogens. There is a
clear need to expand the focus to include nonpathogenic bacteria in antibiotic research. This may
allow researchers to predict resistance before it emerges clinically; develop diagnostic techniques,
and build new therapeutic strategies to counteract resistance before it emerges in human pathogens.
With respect to functional metagenomics, we need to develop and apply new approaches to
cultivate the previously uncultivated and rare members of the microbial communities to assign
functions to the vast number of unknown or hypothetical genes and to develop novel genetic
systems that allow screening of the vast array of microbes on earth to identify antibiotic-resistance
genes.

Sequence-based metagenomics allows the comparison of microbial communities from different

hosts to investigate differences in the response to antibiotics and to select the most favorable
antibiotic to reduce side effects for certain hosts. The transition of next-generation sequencing into
clinical diagnostics is in the early stages of development in large reference laboratories and is being
leveraged for applications that require large amounts of sequence information. Sequence-based
metagenomic data can combine antibiotic-resistance gene abundance data with community
composition and metabolic pathway information to provide a more complete profile of specific
samples. This information is of great importance for the implementation of rational administration
guidelines for antibiotic therapies. We may also use these techniques to learn how human-
associated microbes can be manipulated by antibiotics or probiotics to reduce our dependency on
antibiotics, provide alternatives to existing antibiotics, and extend the lifetime of current and new
antibiotics. Many questions remain: the roles of the environmental reservoirs in clinical resistance
development are still hypothetical; we have little or no evidence that any of the putative resistance
genes identified in the environmental studies have been mobilized into pathogenic bacteria and
expressed as resistance phenotypes; we do not know how to rapidly identify new resistance genes;
and functional approaches are still relatively low throughput.

REFERENCES:
1. Leekha S, Terrell CL, Edson RS (February 2011). "General principles of antimicrobial
therapy". Mayo Clinic Proceedings. 86 (2): 156–67. doi:10.4065/mcp.2010.0639. PMC
3031442. PMID 21282489. Once microbiology results have helped to identify the etiologic
pathogen and/or antimicrobial susceptibility data are available, every attempt should be
made to narrow the antibiotic spectrum. This is a critically important component of
antibiotic therapy because it can reduce cost and toxicity and prevent the emergence of
antimicrobial resistance in the community
2. ^ Jump up to:a b c d e Kang CI, Kim J, Park DW, Kim BN, Ha US, Lee SJ, et al. (March
2018). "Clinical Practice Guidelines for the Antibiotic Treatment of Community-Acquired
Urinary Tract Infections". Infection & Chemotherapy. 50 (1): 67–100.
doi:10.3947/ic.2018.50.1.67. PMC 5895837. PMID 29637759.