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LAVALLE, Jestin B.

BMLS Intern Microbiology

Culture and Sensitivity

1. What is the method used in antibiotic sensitivity test? Name & describe other culture & sensitivity test.

Methods varies according to action (Diffusion & Dilution) and type of media used (Broth & Agar)

a. Disk Diffusion Method


o Most common used
o A standard inoculum of the organism is swabbed onto the surface of a Mueller-Hinton Agar plate, and the filter paper
disks impregnated with antimicrobial agents are placed on the agar.
o Zone of inhibition is qualitatively report after overnight incubation @ 35˚C if microorganism is sensitive (S),
intermediate (I), or resistant (R).
 Make a sub-culture using suitable broth medium, pick 4-5 pure isolated colonies of organism
 Incubate @ 35˚C for 2-8 hrs, then compare the turbidity of a subculture to a McFarland 0.5 barium sulfate
standard (99.5 ml of 1% H2SO4 and 0.5 ml of 1.175% BaCl2)
 If turbidity is greater than that of the McFarland Nephelometer Standard – adjust the suspension by adding
NSS (dilute)
 If less than – adjust by incubating the subculture further or by adding some more colonies
 Streak in MHA (4-6 mm depth) evenly in three different planes
 Allow to dry 3-5 minutes
 Distance from each disk: SJ - ≥30 mm ; M - ≥24 mm
 Distance from the edge of plate: SJ ≥10 mm ; M - ≥15 mm
 For the 90 mm plate not more than 80 disks should be used and with the 150 mm plate 12-14 disks may be
used without crowding (8 outer ring, 4 in center)
 Invert and incubate 16 to 18 hours @ 35˚C

b. Well Diffusion
o Antibiotics will drop to holes in agar and see to it if it is resistant or sensitive

c. Dilution Method
o Determine the best titer that is more effective in killing bacteria
o Different concentration of antimicrobial agents are incorporated onto MHA plates
o These are inoculated with test organisms adjusted to match 0.5 McFarland Standard
o Plates are incubated @ 35˚C overnight and read by observing the lowest drug concentration that inhibits visible bacterial
growth
o Concentration is reported as the MIC

d. Broth Methods
o Various concentrations of an antimicrobial drug are inoculated with a standard suspension of the test bacteria, then
overnight incubation
o The MIC is determined by observing the lowest concentration of the drug that will inhibit visible growth of the test
bacteria
o This method can be done using the test tube dilutions (macrodilution) or the plastic microtiter plates (microdilution
containing serial dilution of antimicrobial agent)

e. Epsilometer (E-test)
o Utilizes a rectangular plastic strip device that contains predefined gradient of antibiotic concentration that correspond to
MIC dilutions
o Plastic test device has a reading and interpretative scale on the surface of the strip
o Strip is applied onto the surface of an inoculated MHA plate, drug immediately diffuses in the agar
o After 24 hours, an elliptical zone of inhibition of bacterial growth is seen around the test strip
o Inhibitory concentration of the drug that inhibits the microorganism = zone edge intersects the plastic strip at a specific
level
LAVALLE, Jestin B. BMLS Intern Microbiology

2. Are all antibiotics used the same for all specimens? Explain.

No.

3. What is the recommended culture media for antibiotic sensitivity testing? How thick should the media be? How far should the antibiotic discs
from one another and from the edge of the petri plates?
 MHA (Mueller-Hinton Agar)
o Protocol
o Support growth of non-fastidious microorganisms
o Low concentration of inhibitors which is beneficial

Thickness should be 4-6 mm

 Too deep – smaller zones of inhibition


 To shallow – larger zones of inhibition

Distance from each disk: SJ - ≥30 mm ; M - ≥24 mm

Distance from the edge of plate: SJ ≥10 mm ; M - ≥15 mm

4. Enumerate and explain the mechanisms of antimicrobial actions.

 Inhibition of Cell wall synthesis

Peptidoglycan is found only in bacterial cell walls. Human cells don’t have cell wall (peptidoglycan), penicillin & its derivative have very little toxicity for
human host cells

Penicillin & certain other antibiotics prevent the synthesis of intact peptidoglycan, consequently the cell wall is weakened & cell undergoes lysis.

 Major Class
 Beta Lactams
 Binds to PBPs (Penicillin Binding Proteins) in the cell membrane, cell wall is inhibited & autolytic enzyme are
released degrading the cell wall  bacterial death
o Penicillins – Penicillium chrysogenum
o Cephalosphorins – Cephalosporium mold
 Other Beta Lactam antibiotics
o Monobactams – excellent against gram (-) like Pseudomonas sp.
o Carpabenems – excellent against aerobic & anaerobic gram (+) and gram (-) bacteria
 Mechanism of resistance
o Failure of antibiotic to penetrate (mutation: P. aeruginosa & imipenems)
o Failure to bind target site
o Hydrolysis of antibiotic by B-lactamase (Pen G & Staphylococci)

 Monobactams
 Aztreonam

 Glycopeptide
 Vancomycin
o Blocks repeat unit in the growing peptidoglycan chain (NAG-NAM-PEP)
o Inactivates against gm – bacteria, but used against oxacillin resistant staphylococci, C. difficile &
bacteria resistant to B-lactam antibiotics
o Resistant occurs in bacteria w/ altered terminal side chains

 Lipopeptides
 Bacitracin
o Inhibits recycling of bactrophenol phosphate
LAVALLE, Jestin B. BMLS Intern Microbiology
o Used topically for skin caused by gm + bacteria
o Resistant occurs where antibiotic fails to penetrate the bacterial cell

 Phenems
 Cycloserine
o Inhibits enzymes D-alanyl-D-alanine synthase & alanine racemase
o Has broad spectrum of activity
o Resistant occurs either by reduced drug uptake into bacterial cell or alteration of bacterial cell sites

 Injury to the Plasma membrane


o Polypeptides
 Polymyxin B – from Bacillus polymixa
 Polymyxin E (Colistin) – from Paenibacillus polymyxa var. (colistinus)
 Gram neg

Permeability change resulting to metabolite loss.

For antifungal drugs: amphotericin B & ketoconazole combines w/ sterols thus disrupting the membrane. Toxic to human cell however human cell
membrane has cholesterol rather than ergosterol w/c fungi has, so little toxicity is observe on human cells.

Resistant to detergent activity which prevent from penetrating the outer and inner cytoplasmic membrane.

 Inhibition to Protein Synthesis

Amino glycosides, tetracycline, chloramphenicaol, macrolides, clindamycin

Targets bacterial ribosome (70S) which differs from eukaryotic ribosome (80S), such account for the selective toxicity w/c affect CHON synthesis.
However, since eukaryotic mitochondria has 70S ribosome can therefore have adverse effects on host’s cells.

 Aminoglycosides
 Streptomycin, kanamycin, gentamycin, tobramycin, amikacin, netilmicin
 Exert effect by passing thru cell wall & membrane to the cytoplasm where they inhibit bacterial protein synthesis by
irreversibly binding ribosomes
 Secondary effect, faulty translation & disruption of bacterial membranes
 Finally they change the 30S shape causing incorrect reading on the mRNA
 Resistance
o Mutation of ribosome binding site
o Decreased antibiotic uptake
o Enzymatic modification by acetylation or phosphorylation

 Tetracycline
 Reacts w/ 30S of 70S bacterial ribosome by blocking the attachment of tRNA to ribosome preventing addition of AA to
growing polypeptide chain
 Resistance
 Increased efflux from the cell

 Chloramphenicol
 Inhibits peptide bond formation w/c reacts with 50S of 70S bacterial ribosome
 Can produce dyscrasias such as aplastic anemia by disrupting protein synthesis in human bone marrow cells

 Macrolides
 Reacts also w/ bacterial 50S ribosome only of gm + bacteria

 Clindamycin
 Binds 50S, inhibits peptidyl transferase by interfering w/ binding of AA acyl-tRNA complex

 Inhibition of Nucleic Acid Synthesis


LAVALLE, Jestin B. BMLS Intern Microbiology
Some have little usefulness since some interfere w/ mammalian DNA/RNA synthesis

 Rifampin
o From Streptomyces mediterranai
o Binds to DNA dependent RNA polymerase & inhibit initiation of RNA synthesis

 Quinolones
o Inhibit bacterial gyrases & topoisomerase, required to supercoil strands of bacterial DNA

 Inhibition of Enzymatic activity / Antimetabolites

Inhibit bacterial enzymatic activities by antimetabolites w/c closely resembles the normal substrate in a bacterial enzymatic activity

 Sulfonamides & Dapsone


o Compete with PABA, preventing synthesis of folic acid required by some bacteria

 Trimethoprim
o Has high affinity for dihydrofolate reductase, which prevent conversion into tetrahydrofolate, blocking formation of
thymidine, purine, methionine & glycine

5. Explain briefly antimicrobial resistance, sensitivity, and slight.

Resistant – This implies that an infection due to an organism is unlikely to respond / will not reliably respond to the antimicrobial agents

Susceptible – This implies that an infection due to an organism can be appropriately treated with the recommended dosage of antimicrobial dosage of
antimicrobial agent for that type of infection and infecting organism

Slight – Less effectivity of drug, efficient and therapeutic success effect is uncertain, antimicrobial agent to a patient can be increased beyond standard or
recommended dosages without serious risk of adverse reaction to obtain higher levels in the infected site

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