Culture media& antibiotic sensitivity

Culture media& antibiotic sensitivity: 

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

Types of culture media: 

Types of culture media Blood agar MacConkey agar Mannitol salt agar Blood agar Chocolate agar

Culture media& antibiotic sensitivity: 

Culture media& antibiotic sensitivity To stimulate growth of certain bacteria Loeffler’s 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

Antibiotic susceptibility media: 

Antibiotic susceptibility media Not interfering with antibiotic Enhance growth of bacteria According to National Committee of Clinical Laboratory Standard (NCCLS) Muller Hinton agar

Antimicrobial susceptibility testing : 

Antimicrobial susceptibility testing Indication Choice of test 1 st Disk diffusion (qualitative) 2 nd Micro dilution broth test (quantitative) Selection of antimicrobial agents According to NCCLS Member of medical staff

Standardization as described by NCCLS : 

Standardization as described by NCCLS Growth media (Mueller Hinton) PH (7.2-7.4) Serum Cation concentration (Ca, Mg) Atmosphere Temperature (37Ć) Inoculation One from log phase (4-6hr) Or density adjusted to 10 8 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

Antibiotics: 

Antibiotics Disks stored at -20Ć Antibiotics powdered at (-20 to -70Ć) Quality control Interpretation of results (NCCLS) Selection of antibiotics to be reported Appropriate for infection (UTI, Meningitis)

Methods used for antibiotics susceptibility: 

Methods used for antibiotics susceptibility 1 st Macro dilution broth method Reference method Broth media Serial dilution of antibiotic are made 100 µ g/ml to 0.4 µ g/ml in test tubes Final cell number of bacteria 10 5 cell/ml Reading of results

Slide 9: 

Macro dilution broth method

2nd Agar dilution method: 

2 nd Agar dilution method Second reference method Similar o broth method 10 4 cells per spot (CFU/spot) Can test many isolates on the same plate Reading of result

Disk diffusion method: 

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

Disk diffusion technique (Bauer, Kirby method): 

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 than 15m to edge plate Not more than 4 to 5 disk on a 100mm plate or 12 to 13 on 150mm plate Invert plates and incubate after 15min

Reading and interpretation : 

Reading and interpretation After 16-18hr of incubation Measure zone diameter by ruler Record results as sensitive, intermediate, or resistant according to NCCLS table Limitation of the test Only for bacteria thoroughly evaluated Not for slow or anaerobic bacteria

Disk diffusion technique: 

Disk diffusion technique

Detection of B-Lactmase : 

Detection of B-Lactmase Used method 1 st Chromogenic cephalosporin test Nitrocefin filter paper disk or stick Color change from yellow to red Test read after 15min 2 nd acidimetric test Change in PH Phenol red as indicator Bacteria Change in color from red to yellow Less expensive but less sensitive

Detection and screening of Methicillin resistant staphylococcus aureus (MRSA) : 

Detection and screening of Methicillin resistant staphylococcus aureus (MRSA) Mueller Hinton agar with 4%NaCl Methicillin (10µg/ml) or Oxacillin (10µg/ml) Inoculate plate with testing strain Carried out as for disk diffusion Incubate plate at 35Ć for 24hr Sensitive no growth Resistant, growth is positive

Border line Oxacillin resistant : 

Border line Oxacillin resistant Hyper production strain Hydrolyzes Methicilin Oxacillin and Augmentin disk used for detection Placed apart from each other Observe growth between the two disks

Extended spectrum B- lactamases : 

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 1 st showing resistant to Ceftazidime disk (klebseilla, E. coli ) 2 nd sensitive to other third generation cephalosporin 3th Resistant to other antibiotics

Methods for detection: 

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

E-Test method: 

E-Test method Strip contain graded antibiotic concs. An expansion of disk diffusion method MIC read from point of strip For general use Useful for monitoring resistant therapy