This document describes developing a microbiological bioassay using Bacillus pumilus spores to detect tetracycline residues in milk at concentrations corresponding to regulatory maximum residue limits (MRLs). Several experiments were conducted to optimize the bioassay, determining the ideal spore concentration, addition of chloramphenicol to decrease the detection limit, and assessing specificity, detection limit, and cross-reactivity. The optimized bioassay used 1.6 × 109 B. pumilus spores/ml with 2500 μg/l chloramphenicol, detecting tetracycline, chlortetracycline and oxytetracycline at their MRLs within 5 hours through a color change in the redox indicator. The

1. MICROBIOLOGICAL BIOASSY USING BACILLUS
PUMILUS TO DETECT TETRACYCLINE IN MILK
MICROBIOLOGICAL BIOASSY USING BACILLUS
PUMILUS TO DETECT TETRACYCLINE IN MILK
ABSTRACT
 The Tetracyclines (TCs) are widely used in the treatment of
several diseases of cattle and their residues may be
present in milk. To control these residues it is necessary to
have available inexpensive screening methods, user-
friendly and capable of analyzing a high number of
samples. The purpose of this study was to design a
bioassay of microbiological inhibition in microtiter plates
with spores of Bacillus pumilus to detect TCs at
concentrations corresponding to the Maximum Residue
Limits (MRLs). Several complementary experiments were
performed to design the bioassay. In the first study, we
determined the concentration of spores that produce a
change in the bioassay's relative absorbance in a short
time period. Subsequently, we assessed the concentration
of chloramphenicol required to decrease the detection
limit (DL) of TCs at MRLs levels. Thereafter, specificity, DL
and cross-specificity of the bioassay were estimated. The

2. most appropriate microbiological inhibition assay had a B.
pumilus concentration of 1·6 × 10 9 spores/ml, fortified
with 2500 μg chloramphenicol/l (CAP) in Mueller Hinton
culture medium using brilliant black and toluidine blue as
redox indicator. This bioassay detected 117 μg
chlortetracycline/l, 142 μg oxytetracycline/l and 105 μg
tetracycline/l by means of a change in the indicator's
colour in a period of 5 h. The method showed good
specificity (97·9%) which decreased slightly (93·3%) in milk
samples with high somatic cell counts (>250 000 cells/ml).
Furthermore, other antimicrobials studied (except
neomycin) must be present in milk at high concentrations
(from >5 to >100 MRLs) to produce positive results in this
assay, indicating a low cross specificity.
(Groot and et. al. diversity and role of germinant
receptors. Food Microbiologycal 28 199–208)

3. INTRODUCTION
The Tetracycline antibiotics are bacteriostatic . The mechanism
of action is through the reversible binding of bacterial 30s
ribosome and alternation of bacterial cytoplasmic membrane
.TCs are normally used against the infections of the urinary
tract, respiratory tract, and the intestines (Aga, DS and et.al).
They are also used in the treatment of Chlamydia, for patients
who are allergic to β-lactams and macrolides. It is orally fed
medicine. There are several of side effects while giving this
medicine to cattle. But here we discuss the effects on milk after
giving dose of tetracycline to cattle. Intake of contaminated
milk can result in adverse human health effects including
allergic reaction and the risk of teratogenicity when
administrated during 1st
trimester of pregnancy. Moreover
primary and permanent teeth discoloration often occurs if such
milk is consumed by infants or children less than 12 years old.
(Charm, S & Zomer and et.al)
FAO/WHO and EU have recommended a maximum residue
limit(MRL) of 100 μg/kg for tetracycline in milk. While FDA has
set an upper legal level of 300 μg/kg for combined residues of
TET, OXT and CHT.
Monitoring antibiotic residues is very important in controlling
food safety. For these reasons, several control authorities such
as the European Union and Codex have recommended a

4. maximum residue level (MRL) l μg of 100−1
(Codex
Alimentarius 2009; Council Regulation 2009).
Bioassays are quantitative biological assays used to estimate
the potency of agents by observing their effects on living
animals (in vivo) or tissue/cell culture systems (in vitro)
Tetracycline has been only detected in Nepal while detecting
other substances. It has never been a primary issue and single
subject of detection. Bioassays on detection of Tetracycline in
milk are cheaper and effective process by using Bacillus
pumilus. (Dhakal, I.P. 1997 )
OBJECTIVES
→ to start tetracycline detection method as main work, as it
has never been done in Nepal and was detected as secondary
need only.
→ to replace the costy chemical experiments
→to check quality of cattle milk in Nepal

5. METHODOLOGY
Following methods are applied to complete this work on
detection of tetracycline in milk of cattle
a) Primary methods
b) Secondary methods
A) Primary Methods
It involves direct interaction with farmers and lab experiments
to detect the amount of Tetracycline in milk.
B) Secondary Methods
It involves reference with Google scholar , Text book on
Agricultural microbiologyand different review article on related
topics
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7. THANK YOU!