antibiotic assay in blood and other body fluids.

1. ANTIBIOTIC ASSAY IN BLOOD AND
OTHER BODY FLUIDS
BY
SENI MB
I st yr Msc.MLT
MICROBIOLOGY
MIMS COAH

2. ANTIBIOTIC ASSAY IN BLOOD AND OTHER BODY FLUIDS.
A drug must reach a concentration at the site of infection
above the pathogens MIC to be effective.
In such cases of severe life threatening diseases it often
necessary to monitor the concentration of drugs in blood
and other body fluids.
This may be achieved by microbiological, chemical,
immunological, enzymatic or chromatographic assays.

3. 2 methods
1. Microbiological assays ( Bioassays)
Relay on susceptible microorganism to indicate the
presence of an antimicrobial agent.
Methods- Disc diffusion & Microdilution method
Adv: simple, less costly .
2. Non microbiological method
Chemical method
 HPLC, Chromatography, radioimmunoassay,
 Adv: Detect multiple type of antibiotics.

4. Disc diffusion bioassay
In this method a sensitive indicator organism is used to
detect antimicrobial activity in body fluid specimens that
have been inoculated on a filterpaper disc and place on
growth medium.
Procedure
Inoculating small filter paper disc with the body fluid
specimen of interest.
Place disc on agar plate that has been inoculated with an
indicator organism
Plate incubate overnight at 35-37 oc .

5. A specimen of sufficient concentration of antimicrobials
will create a zone of bacterial inhibition as it diffuses
through the agar.
This indicates the presence of antimicrobial activity in
bld and other body fluids.
+ ve and –ve control are usually run in parallel with the
test specimen to assure that the assay is performing as
expected.

6. Inhibition zone corresponds to a relative concentration of
antimicrobial compound.
Std.curves can be created using known std to obtain a
quantitative estimate of concentration if the specific
compound known.
Choice of urine, serum, plasma
Urine, blood both commonly been test for antimicrobial
assay.
Urine is generally accepted bez it is more sensitive than
serum, or plasma, chemical compounds concentrate in urine

7. Filter paper disc- 6mm
Indicator organism- S.aureus, B.subtilus used to detect broad
range of organisms.
2. MICRODILUTION METHOD
Sample preparation
Serum inactivated prior heating(56oc for 30 mints)
Bodyfluids such as urine, CSF were thought to contain
microorganism so sterilized by membrane filtration.

8. Dilution of sample
1:2, 1:25, 1:3, 1:35, arithmetic dilution
Use a multiwelled polystyrene tray.
Four rows A,B,C,D
50, 75, 100, 125 microliters of inoculam were added to A,B,C,D
Incubate at 37oC.
INOCULAM
 Bacterial inoculam standardised to 1X10^5 CFU.
REFERANCE BACTERIAL STRAINS
E.coli-4883++ gentamycin
S.aureus2834 methicillin
Organism maintained in trypticase soy agar slants.

9. READING
Highest dilution of serum showing no detectable growth
was determined to the end point.
Growth was defined as
Confluent turbidity or single or multiple clusters of
growth>2mm diameter.

10. 1. HPLC method
Extraction of drug from the biological samples.
Separation by HPLC and detection by UV spectrophotometry of
flourimetry.
Adv: rapid specific, and accurate moitoring of antibiotic
concentration in body fluids.
Can detect variety of antibiotics.
2, RIA
Rapid sensitive and specific method
Can be performed with min. samples.
Other methods
 Immunochemical methods
a. Haemagglutination inhibition
b. Homogenous enzyme immunoassay
c. UV and IR spectrophotometry

11. Ref:
 Analysis of drugs in biological fluids- Kurt M Dubowkii
 Microdilution assay of antibiotics in body fluids- Richard C.Tilton PhD