Lecture 7 SUSCEPTIBILITY TESTING

Question 1

Differentiating growth on BA and Mac

Answer 1

If G+ve, then catalase and coagulase test are done to differentiate between S.aureus (positive beta hemo) (and S.saprophyticus (S.aureus is catalase +ve, coagulase +ve). S.saprophyticus /Strep. is
cat -ve and coag -ve
If G-ve, Typical colony colors on EMB or MAC are observed.

Question 2

CATALASE TEST

Answer 2

The presence ofcatalaseenzyme in thetestisolate is detected using hydrogen peroxide. If the bacteria possesscatalase(i.e. , arecatalase-positive) , when a small amount of bacterial isolate is added to hydrogen peroxide, bubbles of oxygen are observed.
Staph aur always cata post

Question 3

COAGULASE TEST

Answer 3

Staphylococcus aureus is known to produce coagulase, which can clot plasma into gel in tube or agglutinate cocci in slide. Thistestis useful in differentiating S.aureus from othercoagulase-negative staphylococci. Most strains of S.aureus produce two types ofcoagulase, freecoagulaseand boundcoagulase.
Post – clotting plasma
Neg – free flowing plasma

Question 4

CLED Medium

Answer 4

CLED is : Cysteine lactose electrolyte deficient agar.
CLED Agar is a differential culture medium for use in enumerating bacteria in urine.

It supports the growth of urinary pathogens and contaminants but prevents undue swarming of Proteus species due to its lack of electrolytes.

Question 5

Principle: of CLED

Answer 5

Lactose is an energy source for organisms capable of utilizing it by a fermentative mechanism.
Cystine permits the growth of “dwarf colony” coliforms.
Bromthymol blue is used as a pH indicator to differentiate lactose fermenters from lactose-nonfermenters.
Organisms which ferment lactose will lower the pH and change the color of the medium from bluish- green to yellow
Electrolyte sources are reduced in order to restrict the swarming of Proteus species

Question 6

Kirby-Bauer Test

Answer 6

The Manual method of performing the susceptibility tests involve streaking (swabbing) a Muller-Hinton agar plate with a dilution of the culture to be tested .
Disks containing the antibiotics are placed on the surface of the agar.
After incubation the plate is observed for the zone of inhibition (ZOI).
The test results are valid only if the standard parameter are maintained
Once the organism is identified, the susceptibility of the isolate is assessed by exposing the organism to varying concentrations of the antibiotic.
Traditionally it is done manually by the: The Disk Diffusion Method or Kirby-Bauer test.
Semi automated methods like the MIC (minimum inhibitory concentrations) are also used

Question 7

Standard Parameters for the Kirby Bauer test

Answer 7

Media: Mueller-Hinton agar(MH agar):preferred because of its relative reproducibility.
Inoculum size-0.5 MacFarland std- approximate number good for gram neg .
Temp. of incubation -35 o C
Time -16-18 hrs of incubation
Depth of the medium-4 mm – disc diffusion
Disk in use to be maintained at 6-8 o C
Stock discs to be stored at -14 o C
Chloride and sulfuric acid are used
BaCl2 + h2SO4 – which precipitates to BaSO4 and HCl – MacFarland Std
The turbidity of Mf is equal to 3.0 x 10 9 cells/mls

Question 8

Procedure for the Kirby-Bauer Test

Answer 8

1.A sterile swab is immersed in the standardized suspension of the test org.
The turbidity of the broth is compared with a standard suspension, like the McFarland standards
2.A Mueller -Hinton plate is uniformly inoculated by spreading the swab so as to form a lawn of the test org.
3.The surface is allowed to dry for 3-5 minutes.
4. Antibiotic discs with known conc. of the antibiotic are placed on the agar plate at a specific distance.
The antibiotic disks are placed depending on the type of test organism.
GRAM +ve: AM,PEN,ERY,GEN,OXA,VA (antibiotics)
GRAM-VE: AM,KF,GEN,OXA,SXT,TET
5. The plates are allowed to incubate overnight preferably 16-18 hrs of incubation .
6. If the antibiotic is effective than ZOI forms around the disk and the diameter of the zone is measured in mm.
The zone of inhibition (diameter in mL) is seen against a standard graph

Question 9

Interpretation of the test

Answer 9

After overnight incubation the plates are read for inhibition of growth around the antibiotic disk
The zone diameter is compared to the standard table for that drug and the org is reported as sensitive, intermediate (if bet sensitive and resistant)or resistant.
The ZOI is measured using a ruler.
Reported in mm.
AS a general rule ,the larger the zone of inhibition, the more sensitive the microbe is to that antibiotic