antimicrobial test.pptx

Mechanism of Antimicrobial
Resistance

2
Motivation
• Any technology
involving
features with
sizes between 1
and 1000
nanometers can
be considered
nanotechnology.

3
Motivation
• Any technology
involving
features with
sizes between 1
and 1000
nanometers can
be considered
nanotechnology.

4
Motivation
• Nanotechnology is an emerging industry
which is bringing us exciting new products
and promises to change the way we live and
work in the future.

5
Motivation
• Nanotechnology is an emerging industry
which is bringing us exciting new products
and promises to change the way we live and
work in the future.

6
Food Containers
• Blue Moon Goods storage containers
http://www.bluemoongoods.com/silver_nanoparticle_food_containers.htm

7
Baby Bottles
• Baby Dream Silver Nano Poly products
http://babydream.en.ec21.com

8
Toothpaste
• Nano toothpaste
http://www.gnsnanogist.com/

9
Toothbrush
• Toothbrush by Songsing Nanotechnology
http://www.ssnano.net/ehtml/detail1.php?productid=73

10
Cutting Board
• Antibacterial Effect by Nano Silver Clean
http://www.adox.info/?doc=shop/item.php&it_id=000123

11
Computer Mouse
• IOGEAR's Personal Security Mouse
www.iogear.com/main.php?loc=product&Item=GMEFP1W6

12
Antibacterial Athletic Socks
• Eliminate smelly feet with antibacterial and
antifungal socks.
http://www.sharperimagebest.com/zn021.html

13
Cotton Sheets
• AgActive sheet sets are not only comfortable,
but they kill bacteria too!
http://www.agactive.co.uk/index.cfm/fuseaction/product.display/Product_ID/8/.htm

14
Washing Machine
• Silver Nano washing machine comes with a
silver wash option.
http://www.samsung.com/ph/products/washingmachine/drum/c1235a.asp?page=Specifications

15
Today's Activities
• Make nanoparticles.
• Soak filter in nanoparticles.
• “Inoculate” plate.
• Check results.

16
How to Make Silver Nanoparticles
• Put 2 ml .5 mM AgNO3 in a test tube.
– Start with a compound that has silver in it.
– Our solution is silver nitrate.

17
• Heat in boiling water bath for 5 minutes.
– Heating it will speed the reaction.

18
• Add 5 drops of 1% sodium citrate.
– Carefully add the sodium citrate;
the solution is HOT!
– Allows gold to form
stable nanoparticles

19
• Continue heating — wait for silver
nanoparticles to form.
– Watch for a change in color to indicate the silver
has formed.
– Let it heat a few more minutes to be sure the
color change is complete.

20
• Continue heating — wait for silver
nanoparticles to form.

21
Growth of Bacteria
• Bacteria may grow as
a group = colony
• Bacteria may cover
surface of plate =
lawn

22
Bacterial Antibiotic Sensitivity
• Antibiotics may
inhibit the growth of
some bacteria.
• Evidence of this is a
“halo”.
• A halo indicates a
zone where bacteria
are not present.

23
Procedure
• Cut filter paper into
small squares.
• Place squares in a
petri dish and pour
silver nanoparticles
over them.
• Let the squares
soak for about 10
minutes.

24
Procedure
• Mark the bottom of an agar
plate with your initials,
divide the bottom into
sections, and label.
• Put 1 to 2 drops of bacterial
culture on the plate.
• Spread the drops.

25
Procedure
• Place your nanoparticle
soaked filter paper
squares and your
control(s) in the
designated areas.
• Incubate your plate for 24
hours at 37ºC.

Antimicrobial Susceptibility Testing
•Dilution method
•vary amount of antimicrobial substances incorporated
into liquid or solid media
•followed by inoculation of test bacteria
•Diffusion method
•Put a filter disc, or a porous cup/a bottomless cylinder
containing measured quantity of drugs on a solid
medium that has been seeded with test bacteria

PRINCIPLES OF SUSCEPTIBILITY TESTING

KIRBY-BAUER TEST IS A AGAR DISK DIFFUSION METHOD
Kirby – Bauer Agar disk diffusion
method provides
qualitative interpretive category
results of susceptible, intermediate,
and resistant bacterial isolates
If the bacteria are
susceptible to a particular antibiotic,
an area of clearing
surrounds the wafer where bacteria
are not capable of
growing (called a zone of inhibition).

•Principle – impregnated disc absorbs moisture from the
agar and antibiotic diffuses into the agar medium
•As distance from disc increases, antibiotic concentration
decreases
•Visible growth of bacteria occurs on the surface of agar
where the concentration of antibiotic falls below the
inhibitory level for the test strain.
•Concentration of diffused antibiotic at the interface of
growing and inhibited bacteria approximates to the
(minimal inhibitor concentration) MIC obtained in dilution
tests.
Kirby Bauer Method

Antibiotic discs –
• commercially prepared discs 6mm in diameter are used
• Can be prepared from Whatman filter paper no.1, sterilized
in hot air oven
• Antibiotics delivered with 20 gauge wire loop, diameter 2mm.
This delivers 0.005ml antibiotic to each disc
• Discs and disc dispensers should be stored in sealed
containers with a dessicant
• Bulk stock kept at -20o
C
• Working stock kept in sealed containers with dessicant , stored
at less than 8 o
C

•Turbidity standard for inoculum
preparation –
•To standardize the inoculum
density
•BaSO4 turbidity standard,
equivalent to 0.5 McFarland is
used
•Prepared by 0.5ml 0.048 mol/L
BaCl2 + 99.5ml 0.18 mol/L H2SO4
•Density verified by using
spectrophotometer
•Absorbance at 625 nm should
be 0.008 – 0.10 for 0.5
McFarland standard
•Densities verified monthly

• Prepare a pure culture (18-24 hrs) of the sample
on a nonselective medium
• Adjust turbidity until it is equivalent to the 0.5
McFarland Turbidity Standard

•Procedure (Modified Kirby-Bauer
method: National Committee for
Clinical Laboratory Standards. NCCLS)
•Prepare applx. 108 (colony
forming unit) CFU/ml
bacterial inoculum in a saline
or tryptic soy broth tube (TSB)
or Mueller-Hinton broth (5
ml)
• Pick 3-5 isolated colonies from plate
• Adjust the turbidity to the same as
the McFarland No. 0.5 standard.
•Streak the swab on the
surface of the Mueller-Hinton
agar (3 times in 3 quadrants)
•Leave 5-10 min to dry the
surface of agar
Kirby Bauer Method

Kirby Bauer Method
Procedure (cont.)
Place the appropriate drug-
impregnated disc on the
surface of the inoculated agar
plate
Invert the plates and incubate
them at 35 oC, o/n (18-24 h)
 Measure the diameters of
inhibition zone in mm
Bacterial growth

Select colonies
Prepare inoculum
suspension

Mix well
Standardize inoculum
suspension

Remove sample
Within 15 minutes after
adjusting the turbidity of
the inoculum suspension,
dip a sterile non-toxic
swab on an applicator
into the adjusted
suspension.
Rotate the swab several
times, pressing firmly on
the inside wall of the
tube above the fluid level
to remove excess
inoculum from the swab.

INOCULATE THE PLATE WITH UNIFORMITY
Swab plate
Inoculate the dried surface of a
Muller-Hinton agar plate by
streaking the swab over the
entire sterile agar surface.
Repeat this procedure two more
times, and rotate the plate 60°
each time to ensure an even
distribution of inoculum.
Replace the plate top and allow
3 to 5 minutes, but no longer
than 15 minutes, for any excess
surface moisture to be absorbed
before applying the antibiotic
disks.

There should be an almost
confluent lawn of growth
when done properly.
If only isolated colonies grow,
the inoculum was too light
and the test should be
repeated.
To avoid extremes in
inoculum density, never use
undiluted overnight broth
cultures for streaking plates
GETTING IDEAL RESULTS DEPEND ON RIGHT
INOCULUM

The agar medium should have pH
7.2 to 7.4 at room temperature.
The surface should be moist but
without droplet of moisture.
The antibiotic disks should be
maintained at 8°C or lower or
freeze at -14°C or below until
needed, according to the
manufacturer’s recommendations.
Allow the disks to warm to room
temperature before use.
Don’t use expired disks.
BE CAUTIOUS

•Qualitative results
•Susceptible
•Intermediate – may respond if infection is at body site
where drug concentrates (e.g. urine) or if higher than
normal dose can be safely given
•Resistant
Kirby Bauer Method

Factors Affecting Size of Zone of Inhibition
• Inoculum density Larger zones with light inoculum and vice versa
• Timing of disc application If after application of disc, the plate is
kept for longer time at room temperature, small zones may form
• Temperature of incubation Larger zones are seen with
temperatures < 35 oC
• Incubation time Ideal 16-18 hours; less time does not give reliable
results
• Potency of antibiotic discs Deterioration in contents leads to
reduced size
• Composition of medium Affects rate of growth, diffusion of
antibiotics and activity of antibiotics

Factors Affecting Size of Zone of Inhibition
 Acidic pH of medium Tetracycline, novobiocin, methicillin
zones are larger
 Alkaline pH of medium Aminoglycosides, erythromycin
zones are larger
 Reading of zones Subjective errors in determining the clear
edge
 Size of the plate Smaller plates accommodate less number
of discs
 Depth of the agar medium (4 mm) Thin media yield
excessively large inhibition zones and vice versa
 Proper spacing of the discs (2.5 cm) Avoids overlapping of
zones

Modify methods for fastidious bacteria
Media recommended
for test of fastidious
bacteria

How silver ions kill bacterial?

untreated Adhesion of AgNPs
at 1 h
Disruption of cell
membrane After 5 h
Complete Disruption of cell
membrane After 8 h
Cell lysis at 12 h
Penetration of AgNPs8 h

Poly(lactide-co-
glycolide) copper
oxide (PLGA/CuO
hybrid nanofiber
scaffold)

Schematic illustration for the fabrication of
ZnS-PVAc electrospun nanofibers

Bactericidal activity; pristine PVAc and ZnS-PVAc
electrospun nanofiber mat with E. coli and S.
aureus bacteria

Schematic illustration showing the degradation
of cell membrane of bacteria by biologically
reactive oxygen species

antimicrobial test.pptx

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