SBT 721: MICROBIOLOGY

LAB

Dr.Akhileshwar Namani Ph.D

 Antibiotic sensitivity test by disc and
well diffusion methods
Antibiotic sensitivity testing or antibiotic susceptibility testing is
the measurement of the susceptibility of bacteria to antibiotics.
Susceptible-Microbes can't grow if the drug is present.
It is used because bacteria may have resistance to some
antibiotics.  
Antimicrobial resistance is the ability of a pathogenic microbe to
develop a resistance (oppose/defend) to the effects of an
antimicrobial medication.
Antibiotic resistance happens when germs like bacteria and fungi
develop the ability to defeat the drugs designed to kill them.
Kirby-Bauer Disk Diffusion Susceptibility Test Protocol
 Kirby-Bauer Disk Diffusion
Susceptibility
History:
The publication on penicillin by Alexander Fleming in 1928 is a
milestone in the history of medicine. As more antimicrobial
compounds were discovered, it was predicted that infectious diseases
would be eliminated through the use of these antimicrobials.
 
Unfortunately, the development of bacterial resistance to these
antimicrobials quickly diminished this optimism and resulted in the
need for physicians to request the microbiology lab to test a patient’s
pathogen against various concentrations of a given antimicrobial to
determine susceptibility or resistance to that drug.
 Kirby-Bauer Disk Diffusion
Susceptibility
The original method of determining susceptibility
to antimicrobials was based on broth dilution methods, which
although still the gold standard today, are time consuming to
perform.

Many researchers published variations

  for the broth dilution
procedure resulting in multiple protocols that resulted in widespread
confusion.

In 1960s, Kirby and his colleague, A. W. Bauer, extensively

reviewed the susceptibility testing literature. They consolidated and
updated all the previous descriptions of the disk diffusion method
and published their findings
 Kirby-Bauer Disk Diffusion
Susceptibility
Purpose
The purpose of the Kirby-Bauer disk diffusion susceptibility test is
to determine the sensitivity or resistance of pathogenic aerobic and
facultative anaerobic bacteria to various antimicrobial compounds in
order to assist a physician in selecting treatment options for his or her
patients.  

The pathogenic organism is grown on Mueller-Hinton agar in

the presence of various antimicrobial impregnated filter paper disks.
The presence or absence of growth around the disks is an indirect
measure of the ability of that compound to inhibit that organism.
 Mueller-Hinton agar
Mueller-Hinton II Agar
Beef extract 2.0 g
Acid hydrolysate of casein 17.5 g
Starch 1.5 g
Agar 17.0 g
Distilled or deionized water 1.0 L
pH 7.2–7.4
  at 25°C

Mueller Hinton Agar medium is non-selective and non-differential.

It allows the growth of almost all types of organisms.
The starch absorbs toxins released by the bacteria. Hence, the
toxins won't be able to affect the actions of antibiotics.
It is a loose agar. This allows for better diffusion of the antibiotics
than most other plates. A better diffusion leads to a truer zone of
inhibition.
Mueller-Hinton agar

 
 Principle
When a 6-mm filter paper disk impregnated with a known
concentration of an antimicrobial compound is placed on a MH agar
plate, immediately water is absorbed into the disk from the agar.
 The antimicrobial begins to diffuse into the surrounding agar.
The rate of diffusion through the agar is not as rapid as the rate of
extraction of the antimicrobial out of the disk.
The rate of diffusion of the antimicrobial through the agar is
 
dependent on the diffusion and solubility properties of the drug in
MH agar and the molecular weight of the antimicrobial compound.
 Larger molecules will diffuse at a slower rate than lower
molecular weight compounds.
These factors, in combination, result in each antimicrobial having a
unique breakpoint zone size indicating susceptibility to that
antimicrobial compound.
 Materials

✦ Two Mueller-Hinton agar plates

✦ Antibiotic disks (penicillin, chloramphenicol etc.)
✦ Antibiotic disk dispenser or forceps for placement
of disks
✦ Small beaker of alcohol (for sterilizing forceps)
 
✦ Two sterile cotton swabs
✦ One metric ruler (Scale)
✦ Sterile transfer pipettes
✦ Fresh broth cultures of: Escherichia coli
(McFarland 0.5 standard)
 McFarland 0.5 standard
In microbiology, McFarland
standards are used as a reference to
adjust
the turbidity of bacterial suspensions so
that the number of bacteria will be within
a given range to standardize microbial
testing.
An example of such testing  
is antibiotic susceptibility testing by
measurement of minimum inhibitory
concentration which is routinely used in
McFarland standards. No. 0.5, 1
medical microbiology and research. and 2
If a suspension used is too heavy or too
dilute, an erroneous result (either falsely
resistant or falsely susceptible) for any
given antimicrobial agent could occur.
Original McFarland standards were made by mixing specified
amounts of barium chloride and sulfuric acid together. Mixing
the two compounds forms a barium sulfate precipitate, which
causes turbidity in the solution.
A 0.5 McFarland standard is prepared by mixing 0.05 mL of
1.175% barium chloride dihydrate (BaCl2•2H2O), with 9.95 mL
of 1% sulfuric acid (H2SO4).

 
 
 Procedure
All aspects of the Kirby-Bauer procedure are standardized to
ensure reliable results.
Therefore, care must be taken to adhere to these standards.
Mueller-Hinton agar, which has a pH between 7.2 and 7.4, is
poured to a depth of 4 mm in either 150 mm or 100 mm Petri dishes.
The depth is important because of its effect upon the diffusion.
Thick agar slows lateral diffusion and thus produces smaller zones
 
than plates held to the 4 mm standard.
 Inoculation is made with a broth culture diluted to match a 0.5
McFarland turbidity standard.
Gently mix the E. coli culture (McFarland standard) until they
reach their maximum turbidity.
Dilute the broth with sterile saline until it appears to have the same
level of turbidity as the standard. If it is not turbid enough, incubate
it until it reaches that level
 
 Wickerham Card
For visual turbidity assessment.
Laminated background comparision card with black and white
stripes for visual turbidity assessment.

 
 
 
 
Method-1
Kirby-Bauer disk diffusion susceptibility
test protocol, placement
of antibiotic disks using an automated
disk dispenser. Step 1, a. through
d. An automatic disk dispenser can be
used to place multiple disks
simultaneously on a MH agar plate.  
Set the dispenser over the plate.
(B) Place the palm of your hand on the
top of the handle. (C) Press down
firmly and completely to dispense the
disks. The spring loaded handle
will return to the original position when
pressure is removed
Method-2
Placement of antibiotic disks using forceps
to manually place the disks. Antibiotic disks
can be manually placed on the MH agar plate
if desired. (A) Place the Mueller-Hinton agar
plate over the disk template. (B) Remove one
disk from the cartridge using forceps that
have been sterilized. (C) Lift the lid of the
plate and place the disk over one of the
positioning marks. (D) Press the disk with 
the forceps to ensure complete contact with
the agar surface. Replace the lid of the plate
between disks to minimize exposure to air-
borne contaminants
 
Results-Expected

 
 
 
 
Results-Your part

 
Results-Compare with standard
chart

 
 Oligodynamic action of copper on
bacteria
 
The oligodynamic effect (from Greek oligos "few", and dynamis
"force") is a biocidal effect of metals, especially heavy metals, that
occurs even in low concentrations.
A biocide is defined in the European legislation as a chemical
substance or microorganism intended to destroy, deter, render
harmless, or exert a controlling effect
  on any harmful organism.
The US Environmental Protection Agency (EPA) uses a slightly
different definition for biocides as "a diverse group of poisonous
substances including preservatives, insecticides, disinfectants, and
pesticides used for the control of organisms that are harmful to
human or animal health or that cause damage to natural or
manufactured products". When compared, the two definitions
roughly imply the same, although the US EPA definition includes
plant protection products and some veterinary medicines
 
 Principle
 
The action of very small concentrations of positive ions (mainly of
metals, such as copper, silver, and gold) on living organisms.
chemical having antimicrobial activity in minuscule amounts.
Oligodynamic action was discovered by  Karl Wilhelm von Nägel
(findings published in 1893), who observed the retardation of growth
of algae upon immersion of pieces of copper or silver in the vessels in
 
which the algae were being cultivated.
In oligodynamic action, metal ions concentrate on the surface of the
living object (bacteria, algae), which causes blocking of the free
carbonyl and sulfhydryl groups of the surface structures.
The metals react with thiol (-SH) or amine (-NH(1,2,3)) groups of
proteins, a mode of action to which microorganisms may develop
resistance.