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Culture media& antibiotic
sensitivity
Non selective media General media Blood agar Chocolate agar
Differential media MacConkey agar for Enterobacteriacae CLED for urine enter pathogenic
SSA for salmonella and shigella Carbohydrate fermentation TSI to differentiate lactose fermented from non
fermented Urea agar or broth for bacteria have Urease enzyme
Mannitol salt agar to differentiate staphylococcus
species
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Types of culture media
Blood agar
MacConkey agarMannitol salt agar
Blood agarChocolate agar
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Culture media& antibiotic
sensitivity
To stimulate growth of certain bacteria Loefflers media for diphtheria Lowenstein-Jensen for Tuberculosis TCBS for Vibrio cholera Bordet Gengou agar for Bordetella Sabouraud dextrose agar (SDA) for fungi
Enrichment media (broth media)
Enhance growth of certain bacteria Selenite broth for salmonella and shigella Thioglycollate and cooked meat for anaerobic
bacteria
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Antibiotic susceptibility media
Not interfering with antibiotic
Enhance growth of bacteria
According to National Committee ofClinical Laboratory Standard(NCCLS)
Muller Hinton agar
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Antimicrobial susceptibility testing
Indication
Choice of test
1st
Disk diffusion (qualitative)2nd Micro dilution broth test
(quantitative)
Selection of antimicrobial agents
According to NCCLS Member of medical staff
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Standardization as described by
NCCLS
Growth media (Mueller Hinton) PH (7.2-7.4) Serum Cation concentration (Ca, Mg)
Atmosphere Temperature (37) Inoculation One from log phase (4-6hr) Or density adjusted to 108 CFU/ml Comparing turbidity to a McFarland 0.5 BaSO4
standard 0.5 ml of 0.048M BaCL2 (1.175% w/v BaCL2. H2O) to
99.5 of o.36N H2SO4 Stored in the dark at room temperature
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Antibiotics
Disks stored at -20 Antibiotics powdered at (-20 to -
70) Quality control
Interpretation of results (NCCLS)
Selection of antibiotics to be
reported Appropriate for infection (UTI,
Meningitis)
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Methods used for antibiotics
susceptibility
1st Macro dilution broth method
Reference method
Broth media Serial dilution of antibiotic are made
100g/ml to 0.4g/ml in test tubes
Final cell number of bacteria
105cell/ml Reading of results
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Macro dilution broth method
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2nd Agar dilution method
Second reference method
Similar o broth method
104
cells per spot (CFU/spot) Can test many isolates on the same
plate
Reading of result
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Disk diffusion method
Principle of test
Disk contain antibiotic potency
Contact with moist agar
Water absorption Antibiotic diffuses into media
Creator extraction rate of antibiotic
Increase of antibiotic concentration
Decrease in antibiotic as distance increase
Clear zone around disk or growth
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Disk diffusion technique (Bauer, Kirby
method)
Test procedure
Following standard steps by NCCLS
Inoculated plates with tested bacteria
Allowed drying for 3-5min
Impregnate antibiotic disks no closer than15m to edge plate
Not more than 4 to 5 disk on a 100mmplate or 12 to 13 on 150mm plate
Invert plates and incubate after 15min
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Reading and interpretation
After 16-18hr of incubation
Measure zone diameter by ruler
Record results as sensitive,intermediate, or resistant accordingto NCCLS table
Limitation of the test
Only for bacteria thoroughlyevaluated
Not for slow or anaerobic bacteria
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Disk diffusion technique
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Detection of B-Lactmase
Used method
1stChromogenic cephalosporin test
Nitrocefin filter paper disk or stick
Color change from yellow to red
Test read after 15min
2nd acidimetric test
Change in PH
Phenol red as indicator
Bacteria Change in color from red to yellow
Less expensive but less sensitive
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Detection and screening of Methicillin
resistant staphylococcus aureus (MRSA)
Mueller Hinton agar with 4%NaCl
Methicillin (10g/ml) or Oxacillin(10g/ml)
Inoculate plate with testing strain
Carried out as for disk diffusion
Incubate plate at 35 for 24hr Sensitive no growth Resistant, growth is positive
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Border line Oxacillin resistant
Hyper production strain
Hydrolyzes Methicilin
Oxacillin and Augmentin disk usedfor detection
Placed apart from each other
Observe growth between the twodisks
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Extended spectrum B- lactamases
Produced by many gram negative bacteria Plasmid or chromosomes in origin Enable bacteria to be resistant to many antibiotics Have became established in many hospitals
such as klebseilla E. coli Enterobacter
Detection of Extended B-lactamases 1st showing resistant to Ceftazidime disk
(klebseilla, E. coli)
2nd
sensitive to other third generationcephalosporin 3th Resistant to other antibiotics
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Methods for detection
Double disk synergy test
Ceftotaxime and Augmentin
Placed 30mm apart Three-dimensional test
Circular hole in agar filled with
bacteria Growth at the point of cut indicated
the presence of enzyme
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E-Test method
Strip contain graded antibioticconcs.
An expansion of disk diffusionmethod
MIC read from point of strip
For general use
Useful for monitoring resistanttherapy