lab 6 culture media& antibiotic sensitivity

7/29/2019 Lab 6 Culture Media& Antibiotic Sensitivity

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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

lab 6 culture media& antibiotic sensitivity

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