Laboratory 7

Antimicrobial Zone of Inhibition (mm)

Agents (ug/mL)
Escherichia coli Pseudomonas aeruginosa Staphylococcus aureus
Diameter Interpretation Diameter Interpretation Diameter Interpretation
Amoxicillin (AMC) 12 mm R 0 mm R 0 mm R
Cephalothin (CF) 19 mm S 0 mm R 0 mm R
Chloramphenicol 31 mm S 15 mm I 0 mm R
(C)
Ciphofloxacin 30 mm S 40 mm S 0 mm R
(CIP)
Clindamycin (CC) 0 mm R 0 mm R 0 mm R
Erythromycin (E) 0 mm R 0 mm R 0 mm R
Oxacillin (OX) 0 mm R 0 mm R 0 mm R
Penicillin G (P) 0 mm R 0 mm R 0 mm R
Streptomycin (S) 19 mm S 0 mm R 0 mm R
Tetracycline (TE) 18 mm I 0 mm R 0 mm R
Tobramycin (TM) 25 mm S 18 mm S 0 mm R
Trimethoprim 29 mm S 0 mm R 0 mm R
sulfa (SXT)
Vanomycin (VA) - - - - 17 mm S

1. How is the information from the disk diffusion test used for the recommendation of the
clinical use of an antimicrobial drug?

The diffusion and dilution processes, according to the Clinical Laboratory Standards Institute, are two
options in doing the testing. The Kirby-Bauer disk diffusion assay, a diffusion method was made use
for this exercise. The results of the disk diffusion test can be used to prescribe the use of an
antimicrobial agent by evaluating the sensitivity of microbial cultures to a certain antimicrobial
regimen and, more specifically, as a reference for selecting effective antimicrobial treatment for
patients with infections and the management and prevention of infectious diseases.

2. Describe how bacteria can become resistant and the mechanism that may be involved in the
process.

Bacterial cells can develop antibiotic resistance in one of two ways. One way is by mutations in the
cell's DNA that arise during replication. Bacterial resistance may also be acquired by horizontal gene
transfer. There are three ways this can happen because in any case, genetic material from
antibioticresistant bacteria is passed to other bacterial cells, rendering them antibiotic-resistant as
well. When bacteria develop tolerance, antibiotic treatment destroys non-resistant bacteria while
antibioticresistant bacteria proliferate. Antibiotics are thwarted by bacterial cells through a variety of
pathways. Because of an unexpectedly impermeable cell membrane or a lack of the target that the
antibiotic attacks, certain bacteria are naturally immune. Other bacteria can produce enzymes that
inactivate antibiotics as they come into contact with them. Bacteria may alter the antibiotic's target,
making it useless in some cases. Bacteria may also make efflux pumps, which carry antibiotics out of
the bacterial cell until they have a chance to work (“How Bacteria Build Resistance at the Cellular
Level - Resources,” 2017).

3. What is the difference between dilution and diffusion methods? Which do you think gives
more reproductive results? Explain.

Dilution and Diffusion methods significantly differ from each other as the Dilution method includes a
varied amount of antimicrobial substances incorporated into a liquid or solid media and is followed
by inoculation of test bacteria. The diffusion method on the other hand makes use of either the
following: a filter disc, a porous cup, or a bottomless cylinder, containing a measured quantity of
drugs on a solid medium that has been seeded with test bacteria. The Kirby-Bauer disk diffusion
assay helps determine the susceptibility of a microorganism to various antimicrobial drugs. However,
to assess sensitivity and tolerance, the zones of inhibition assessed must be correlated to established
criteria, and they do not include details on bactericidal versus bacteriostatic behavior or allow for a
clear comparison of drug potencies. For the dilution method, on the other hand, the minimum
inhibitory concentration (MIC) of an antimicrobial agent against a particular microbe can be
determined using a variety of laboratory methods. The minimum bactericidal concentration (MBC)
can also be determined, generally as a follow-up experiment to the tube dilution method's MIC
determination. Technically both methods have promising results but the dilution method’s quite
ahead of that of the diffusion method.

4. How is the 0.5 McFarland standard prepared? What is the role of this standard in performing
antimicrobial susceptibility tests?

It is used to adjust the turbidity of the inoculum for antimicrobial susceptibility tests. By combining
barium chloride and sulfuric acid, the 0.5 McFarland turbidity standard is created. Shake well before
each use to ensure that the barium sulfate is uniformly distributed throughout the solution. The 0.5
McFarland Standard is the most widely used standard in clinical microbiology laboratories for
antimicrobial susceptibility tests and culture media performance testing. The McFarland standard
can be prepared in a variety of concentrations ranging from 0.5 to4, and the cell count density differs
based on the concentration. In microbiological laboratories, however, the most widely used
concentration for antimicrobial susceptibility testing and culture media performance testing is the
0.5 McFarland standard. The steps in preparing are as follows

• Prepare a 1% solution of anhydrous barium chloride (BaCl2) and 1% solution of sulfuric acid
(H2SO4)
• Combine and completely mix the barium chloride and sulfuric acid solutions to form a turbid
suspension.

• Place the resulting mixture in a foil-covered screw-cap tube.

• Store the McFarland standard at room temperature (25 °C) when not in use. McFarland
standard density solution will precipitate and clump over time, and it needs vigorous
agitation (vortexing) or shaking before each use. Note: Mark the tube to indicate the level of
liquid, and check before use to be sure that evaporation has not occurred.

• In the presence of good lighting, visually compare the turbidity by holding the bacterial
sample and McFarland Standard tubes up against the black and white bars printed on the
enclosed card.

• In case of heavy turbidity, adjust the turbidity of the log growth of the bacterial suspension
with the addition of broth or saline via sterile pipette to match the turbidity to that of a
known McFarland Equivalence Standard.

• If the test suspension is too light, inoculate with additional organisms or incubate tubes until
turbidity matches that of the standard.

5. Why is the Mueller-Hinton medium considered as the most suitable medium for
antimicrobial susceptibility testing?

The Mueller Hinton Agar (MHA) was developed by Mueller and Hinton in 1941 to isolate pathogenic
Neisseria species. The Kirby-Bauer disk diffusion approach has become more commonly used for
non-fastidious microorganism susceptibility testing. It is a non-selective, non-differential medium.
Just about all species that are plated on this medium will increase. It's a starch-based substance.
Bacterial toxins are seen to be ingested by starch, prohibiting them from meddling with antibiotics.
This provides for stronger antibiotic diffusion than most other plates.

6. Think of possible antimicrobial chemicals at home. Can you recommend and design an
experiment on how you can utilize these chemicals and assess their antimicrobial activities against
microorganisms?

Sterilants, disinfectants, and fungicides are types of antimicrobial compounds (biocides). Biocides are
synthetic or semisynthetic molecules that, when used at specific concentrations and under particular
conditions, destroy living cells under a certain time frame. Sterilants kill all types of microbial life;
disinfectants kill contagious pathogenic bacteria; sanitizers eliminate microbial contaminants; and
fungicides destroy fungi that are pathogenic to animals and humans on surfaces. To kill bacteria, a
variety of antimicrobial agents could be used individually or in conjunction. Add 2 teaspoons of
bleach to 4 cups of water to obtain effective results.
Ref:

How Bacteria Build Resistance at the Cellular Level - Resources. (2017, August 28). Retrieved March
19, 2021, from https://onlinepublichealth.gwu.edu/resources/antibiotic -resistance-at-
cellularlevel/#:%7E:text=There%20are%20two%20main%20ways,is%20through%20horizontal
%20gene%20t ransfer.

Testing the Effectiveness of Antimicrobials | Microbiology. (n.d.). Retrieved March 19, 2021, from
https://courses.lumenlearning.com/microbiology/chapter/testing -the-effectiveness-
ofantimicrobials/

N.A (2020). McFarland Standards- Principle, Preparation, Uses, Limitations. Retrieved from
https://microbenotes.com/mcfarland-standards/

Testing the Effectiveness of Antimicrobials | Microbiology. (2014). Retrieved from:

https://courses.lumenlearning.com/microbiology/chapter/testing -the-effectiveness-
ofantimicrobials/

Mueller-Hinton Agar - an overview | ScienceDirect Topics. (2018). Retrieved from:

https://www.sciencedirect.com/topics/immunology -and-microbiology/mueller-hinton-agar