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

Chocolate agar Blood agar
Blood agar

MacConkey agar

Mannitol salt agar

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

 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

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

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

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)

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

Macro dilution broth method

 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

 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)

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

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

 Detection of B-Lactmase

Used method 1st Chromogenic 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

 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

 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

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 generation cephalosporin 3th Resistant to other antibiotics

 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

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