Lab -9- Antimicrobial Agent

Assist. Lec. Wasan Hatem

Antibiotics: these are chemotherapeutic agents of low
molecular weight produced by certain microorganisms,
that inhibit (bacteriostatic) or kill (bacteriocidal) other
microorganisms.
Factors affecting antimicrobial activity
Among the many factors that affect antimicrobial activity
in vitro, the following must be considered, because they
significantly influence the results of tests.
1-pH of environment
2-Components of medium
3-Stability of drug
4- Size of inoculum
5- Length of incubation
6-Metabolic activity of microorganisms
Antimicrobial susceptibility tests:
A)-Broth dilution assay: determines the minimum inhibitory
concentration (MIC) of the antibiotic, that is, the lowest
concentration at which it prevents growth of a given
organism. To determine the MIC, the antibiotic is first
serially diluted in a broth. An equal amount of bacterial
suspension of the test organism is then added to each tube.
Following overnight incubation (37ºC).Watch the turbidity
of each tube. Determine the highest dilution of the antibiotic
in which there was no growth of the organism or the nutrient
broth was clear (no turbidity). The lowest concentration in
the series to show no microbial growth is the MIC.
  Determination of the MIC is helpful in administering the
optimal concentration of the chosen antibiotic without
producing a toxic overdose of the antibiotic.
Broth dilution MIC titrations have the advantage that
the minimum bactericidal concentration (MBC)
can additionally be estimated by subculture of
dilutions of the antibiotic above that in which
inhibition has occurred overnight. The MBC is
usually taken as the lowest concentration able to kill
microorganism.
Procedure:( ‫) ل((الطالع‬
1- Place nine sterile tubes in a rack and label them.
TubeNo.: 1       2      3     4     5    6     7             8                           9
Label :     64    32    16     8    4    2     1     growth control
     sterility control
2- With a 5-ml pipette add 0.5 ml of sterile broth (Nutrient broth
or Mueller- Hinton broth) to each tube.
3- Add 0.5 ml of the ampicillin broth (128 μg/ ml ) to the first
tube. Discard the pipette. The concentration of ampicillin in this
tube is 64 μg per ml.
4- Take a fresh pipette, introduce it into the first tube (64 μg/ml ),
mix the contents thoroughly, and transfer 0.5ml from this tube
into the second tube (32 μg/ml). Discard the pipette.
5- With a fresh pipette, mix the contents of the second tube and
transfer 0.5 ml to the third tube (16 μg/ ml ).
6- Continue the dilution process through tube number 7. The
eighth and ninth tubes receive no antibiotic.
7- After the contents of the seventh tube are mixed, discard 0.5ml
of broth so that the final volume in all tubes is 0.5ml.
8- From the plate culture of E.coli prepare a suspension of the
organism in
5 ml of saline equivalent to a McFarland 0.5 standard.
9- With a sterile 1-ml pipette, transfer 0.1 ml of the E.coli
suspension into a tube containing 9.9 ml of saline. Discard the
pipette.
10- With a fresh pipette, mix the contents of the tube well. Add
0.1 ml of this organism suspension to the antibiotic-containing
broth tubes 1 through 7 and to the growth control tube.
11- Shake the rack gently to mix the tube contents and place the
tubes in the incubator for 18 to 24 hours.
B)-Disk diffusion test: the Kirby-Bauer method is a
standardized system that take all variables into
consideration. Discs impregnated with known
concentrations of antibiotics are placed on an agar plate
that has been inoculated or seeded uniformly over the
entire plate with a culture of the bacterium to be tested.
The plate is then incubated for 18-24 h. at 37ºC. During
this period, the antimicrobial agent diffuses through the
agar, and may prevent the growth of the organism. For
example, the outer boundary of the inhibitory zone is the
line of minimal inhibitory concentration. In this case, as
the antibiotic progressively diffused out from the disk
into the agar medium, it reached a concentration which
was unable to inhibit the growth of the organism.
Effectiveness of susceptibility is proportional to the
diameter of the inhibition zone around the disc. If there is
no inhibition, growth extends up to the rim of the disks
on all sides and the organism is reported as resistant (R)
to the antimicrobial agent in that disk. If a zone of
inhibition surrounds the disk, the organism is not
automatically considered susceptible (S) to the drug
being tested. The diameter of the zone must first be
measured (in millimeters) and compared for size with
values listed in a standard chart.
      A Mueller Hinton agar plate was used for susceptibility
testing of rapidly growing aerobic organisms according to
the disc diffusion technique (Bauer- Kirby method in
1966), while blood agar was used for Streptococcus sp.
and Chocolate agar for Haemophilus sp. and Neisseria sp.
Procedure: ( ‫) ل((الطالع‬
1- Three to four similar colonies were transferred into about 5ml of
nutrient broth and the turbidity was adjusted to give a
concentration of 1x108 cell/ml using  a McFarland tube .
2- A sterile cotton swab was dipped into the bacterial suspension
and the excess fluid was removed by rotating the swab with a
firm pressure against the inside of the tube above the fluid level.
3-The swab was streaked at 3 different planes, by rotating the plate
60 degrees each time.
4- Commercially available antibiotic discs were then placed on the
inoculated plate using sterile forceps and pressed firmly to ensure
complete contact.
5- The plates was incubated overnight at 37 ºC, then the diameter
of inhibition zones was measured by placing the plate against a
ruler and the result was interpreted according to the chart of
National Committee for Clinical Laboratory NCCL