ANTIMICROBIAL STUDY.pptx

VARIOUS METHODS USED FOR
ANTIMICROBIAL STUDY (AST)
DR KOMAL JADHAV
2nd YEAR PG SCHOLAR
DEPT OF AGADA TANTRA

CONTENTS
Objective
Introduction
Need
Methods used for antimicrobial study
Uses
Summary
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various methods used for testing antimicrobial activity
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OBJECTIVE
• To understand in brief about the significance of antimicrobial studies.
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INTRODUCTION
• Microbes are minute organisms.
• It includes bacteria, virus, fungi and protozoa.
• Antimicrobial susceptibility tests (AST) are the tests carried out to see
the susceptibility of particular organisms for specific antimicrobial
agents.
• AST is performed only for pathogenic bacteria isolated from the
specimen.( E coli from urine specimen, but not from the stool)
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NEED
• AS A GUIDE FOR TREATMENT
oSensitivity of a given micro organism to known concentration of drugs.
oIts concentration in body fluids or tissues.
• AS AN EPIDEMILOGICAL TOOL
oThe emergence of resistant strains of major pathogens(salmonella typhi)
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NEW TERMS
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MBC
MIC
AST
MACRO
DILUTION
MICRO
DILUTION

METHODS
PHENOTYPIC GENOTYPIC
• Vitek,
phoenix &
• microscan
system
• Broth and
agar
dilution
methods
• Kirby-
bauer’s
DD Test
DISK
DIFFUS
ION
DILUTI
ON
TESTS
EPSILOM
ETER OR
E- TEST
AUTOM
ATED
AST
Such as PCR
detecting drug –
resistance genes
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I. PHENOTYPIC
1)DISK DIFFUSION METHOD
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Suitable for rapidly growing organisms
A Mueller- Hinton agar (plate) is uniformly inoculated
with the study organism
Add study drug / antibiotic with specific
concentrations to the agar plate
Incubated at 37 degree Celsius for 16-18hrs
0bserve for zone of inhibition of bacterial growth
Measure and interpret the results

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INTERPRETATION
EX: Stoke’s method of disc diffusion
• Resistant- zone size 3mm or less.
• Intermediate > 3mm, but smaller than the control by more than 3mm.
• Sensitive more or equal 3mm, smaller than control.
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EXAMPLE
 Different concentrations ( of study drug) will be incorporated into an agar medium
in a petridish along with varying concentration of Ceftriaxone and Vancomycin as
standard.
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2)DILUTION TESTS
• Antimicrobial agent is serially diluted, each dilution is tested
with the test organism AST and MIC is calculated.
• There are 2 types of dilution agar and broth.
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A)BROTH DILUTION METHOD
•MACROBROTH DILUTION –performed in test
tubes
•MICROBROTH DILUTION – performed in
microliter plate
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1. MACROBROTH DILUTION
Serial dilutions of the antimicrobial agent in Mueller- Hinton broth are taken in
tubes and each tube is inoculated with a fixed amount of suspension of the test
organism.
Tubes are incubated at 37 degree Celsius for 18- 24 hrs.
MIC is noted (min conc of drug where there is no visual bacterial growth).
MBC can be calculated by subculturing from each tube(showing no growth)on to a
nutrient agar plate without any antimicrobial agent.
The tube containing the lowest concentration of the drug that fails to show growth ,
on subculture, is the MBC of the drug for that test strain.
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• MIC procedure:
1. 9 dilutions of each drug have to be done with BHI for MIC.
2. In the initial tube 20microliter of drug will be added into the 380microliter of BHI broth.
3. For dilutions 200microliter of BHI broth will be added into the next 9 tubes separately.
4. Then from the initial tube 200microliter will be transferred to the first tube containing 200microliter of BHI broth. This was
considered as 10-1 dilution.
5. From 10-1 diluted tube 200microliter will be transferred to second tube to make 10-2 dilution.
6. The serial dilution will be repeated up to 10-9 dilution for each drug.
7. From the maintained stock cultures of required organisms, 5 microliter will be taken and added into 2ml of BHI (brain heart
infusion) broth.
8. In each serially diluted tube 200microliter of above culture suspension will be added.
9. The tubes were incubated for 24 hours and observed for turbidity.
10. Same procedure will be carried out for all microorganisms. All test tube will be incubated at 37c for 24hrs, without shaking.
11. After 24 hrs of incubation all the test tubes will read for MIC, turbidity indicates that bacterial growth has not been inhibited
by the concentration of preparation contained in the medium.
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2. MICROBROTH DILUTION
A polystyrene tray containing 96 wells is filled with small volumes of serial two- fold
dilutions of different antibiotics.
The inoculum suspension and standardization is done according to McFarland standard.
The bacterial inoculum is then inoculated into the wells and incubated at 37 degree Celsius
overnight.
The lowest concentration of antibiotic that completely inhibits visual growth of
bacteria(no turbidity) is recorded as MIC.
Can be simultaneously performed with many tests organisms/specimens, less reagent
required.
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3)EPSILOMETER OR E-TEST
This is quantitative method of detecting MIC by using the principle of both
dilution and diffusion of antibiotic into the medium.
It uses an absorbent strip containing predefined gradient (serial dilution) of
antibiotic concentration immobilized along its length.
It is applied to a lawn inoculum of a bacterium.
Following incubation of the test organism, an elliptical zone of inhibition is
produced surrounding the strip.
The antibiotic concentration at which the ellipse edge intersects the strip, is
taken as MIC value.
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4)AUTOMATED ANTIMICROBIAL
SUSCEPTIBILITY TESTS
• Most systems are computer assisted and have sophisticated softwares to
analyze the growth rates and determine the antibiotic susceptibility
report.
• They work by the principle of broth dilution.
• They use commercially available panel that contain antibiotic solution in
serial dilutions.
• They provide more rapid results compared with traditional methods.
• Ex – VITEK 2, PHOENIX SYSTEM, MICRO SCAN WALK AWAY
SYSTEM,
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VITEK 2
• Most widely used automated AST system in India.
• Can perform AST for bacteria and yeasts.
• It works on the principle of microbroth dilution.
• It uses a reagent card containg 64 wells, which contain doubling dilution of antimicrobial
agents.
• The organism suspension is added to the well.
• The cards are incubated in the system at 35.5degree Celsius.
• The reading is taken once in every 15minutes by the optical system of the equipment.
• It measures the presence of any turbidity which indicates the organism has grown in that
antibiotic well.
• The MIC is determined as the highest dilution of the antimicrobial agent which inhibit the
growth of organism and there is no turbidity in the well.
• The results are available within 8-10hrs for gram –ve bacilli and 16-18hrs for gram positive
cocci.
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B) GENOTYPIC
1)POLYMERASE CHAIN REACTION
• PCR is a technology in molecular biology used to amplify a single or
few copies of a piece of DNA to generate millions of copies of DNA.
• PRINCIPLE OF PCR – involves 3 basic steps
1)DNA extraction from the organism
2)Amplification of extracted DNA
3)Gel electrophoresis of amplified product
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APPLICATION
• MORE SENSITIVE – it can amplify very few copies of a specified
DNA
• MORE SPECIFIC – PCR can also detect the organisms that are highly
noncultivable by conventional culture methods.
• PCR can be used to detect the genes in organism responsible for drug
resistance.
• Detects genetic disease, such as sickle cell anemia, muscular dystrophy
etc
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DISADVANTAGES OF PCR
• Qualitative not quantitative.
• False positive amplification – contamination from environment.
• False negative.
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INTERPRETATION OF AST
Expressed in 4 categories.
SUSCEPTIBLE – antibiotic is clinically effective when used in standard
therapeutic dose.
INTERMEDIATE - antibiotic is not clinically effective when used in
standard therapeutic dose, but may be active when used in increasing
dose.
SUSCEPTIBLE DOSE DEPENDENT – antibiotic will be clinically
active only if given in increasing dose.
RESISTANT – antibiotic is not clinically effective when used in either
standard dose or increased dose.
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PREVIOUS STUDY
Pharmaceutico analytical and antimicrobial study of haratalsatwa.
Modification, stability and antimicrobial study of vachadi kwath.
Pharmaceutical modification of panchavalkakchurna into vaginal
pessary and its analytical, antimicrobial study.
Evaluation of efficacy of murvadi agada in antibiotic resistant diarrhea
(E coli)- An experimental study
Evaluation of anti-microbial activity of murvadi agada against multi-
drug resistant bacteria from clinical isolates of food poisoning - an in-
vitro study.
Evaluation of anti microbial activity of dashanga agada in multidrug
resistant clinical isolates of bacteria - an in-vitro study.
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INCORPORATION OF OWN
RESEARCH VIEWS
Evaluation of antimicrobial activity of ayurvedic drugs like kashay, vati,
choorna can be tested by using various other methods
Evaluation of antimicrobial property for fungi, yeast and viruses etc
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REFERENCES
Text book of Essentials of MEDICAL MICROBIOLOGY by Apurba S
Sastry and Sandhya Bhat
A Short text book of Medical Microbiology by Satish gupte
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ANTIMICROBIAL STUDY.pptx

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