Neomycin
Neomycin
Neomycin
ABSTRACT
The microbiological assay of an antibiotic is based upon a comparison of
the inhibition of growth of micro-organisms by measured concentrations
of the antibiotics under examination with that produced by known
concentrations of a standard preparation of the antibiotic having a known
activity.
INTRODUCTION:
Two general methods are usually employed, the cylinder-plate (or cup-
plate) method and the turbidimetric (or tube assay) method.
The cylinder-plate method (Method A) depends upon diffusion of the
antibiotic from a vertical cylinder through a solidified agar layer in a Petri
dish or plate to an extent such that growth of the added micro-organism
is prevented entirely in a zone around the cylinder containing a solution
of the antibiotic. The turbidimetric method (Method B) depends upon the
inhibition of growth of a microbial culture in a uniform solution of the
antibiotic in a fluid medium that is favourable to its rapid growth in the
absence of the antibiotic.
MEDIA:
Prepare the media required for the preparation of test organism inocula
from the ingredients listed in Table 1. Minor modifications of the
individual ingredients may be made, or reconstituted dehydrated media
may be used provided the resulting media have equal or better growth-
promoting properties and give a similar standard curve response.
* Quantity in ml, to be added after boiling the media to dissolve the agar.
The Standard Preparations for India are certified by the laboratory of the
Indian Pharmacopoeia Commission or by any other notified
laboratory(ies) and are maintained and distributed by the agency(ies)
notified for the purpose.
Buffer solution
Prepare by dissolving the following quantities given in Table 2 of
dipotassiumhydrogen phosphate and potassium dihydrogen phosphatein
sufficient waterto produce 1000 ml after sterilisation, adjusting the pH
with 8 M phosphoric acidor 10 M potassium hydroxide.
Test organisms
The test organism for each antibiotic is listed in Table 4, together with its
identification number in the American Type Culture Collection (ATCC).
Maintain a culture on slants of the medium and under the incubation
conditions specified in Table 5, and transfer weekly to fresh slants.
The following culture media have been found to be suitable for the
test. Fluid thioglycollate medium is primarily intended for the culture of
anaerobic bacteria; however, it will also detect aerobic bacteria.
Soyabean-casein digest medium is suitable for the culture of both fungi
and aerobic bacteria.
Mix the ingredients other than the thioglycollate or thioglycollic acid and
the resazurin sodium solution, in the order given above, in a mortar, with
thorough grinding. Stir in some heated distilled water, transfer to a
suitable container, add the remainder of the distilled water, and complete
the solution by heating in a boiling water-bath. Dissolve the sodium
thioglycollate or thioglycollic acid in the solution and, if necessary,
add 1M sodium hydroxide so that, after sterilisation, the solution will
have a pH of 7.1 ± 0.2. If filtration is necessary, heat the solution again
without boiling and filter while hot through moistened filter paper. Add
the resazurin sodium solution, mix and distribute the medium into
suitable vessels that provide a ratio of surface to depth of medium such
that not more than the upper half of the medium has undergone a colour
change indicative of oxygen uptake at the end of the incubation period.
Sterilise in an autoclave at 121º for 20 minutes. If the medium is to be
stored, cool promptly to 25º and store at 2º to 30º, avoiding excess of
light. If more than the upper one-third of the medium has acquired a pink
colour, the medium may be restored once by reheating in a water-bath or
in free-flowing steam until the pink colour disappears, and cooling
rapidly, taking care to prevent the introduction of non-sterile air into the
container. When ready for use, not more than the upper one-tenth of the
medium should have a pink colour. Medium more than 4 weeks old
should not be used.
Use fluid thioglycollate medium by incubating it at 30º to 35º.
L-Cystine 0.5 g
(K2HPO4)
DETERMINATION OF INOCULUM
For Method B. Proceed as described for Method A and, using the several
inocula, carry out the procedure as described for the specific antibiotic
assay running only the high and low concentrations of the standard
response curve. After incubation, read the absorbances of the appropriate
tubes. Determine which inoculum produces the best response between
the low and high antibiotic concentrations and use this inoculum for the
assay.
APPARATUS
All equipment is to be thoroughly cleaned before and after each use.
Glassware for holding and transferring test organisms is sterilised by dry
heat or by steam.
TEMPERATURE CONTROL.
Thermostatic control is required at several stages of a microbial assay,
when culturing a microorganism and preparing its inoculum and during
incubation in a plate assay. Closer control of the temperature is
imperative during incubation in a tube assay which may be achieved by
either circulated air or water, the greater heat capacity of water lending it
some advantage over circulating air.
SPECTROPHOTOMETER.
Measuring transmittance within a fairly narrow frequency band requires a
suitable spectrophotometer in which the wavelength of the light source
can be varied or restricted by the use of a 580-nm filter for preparing
inocula of the required density or with a 530-nm filter for reading a
absorbance in a tube assay. For the latter purpose, the instrument may
be arranged to accept the tube in which incubation takes place, to accept
a modified cell fitted with a drain that facilitates rapid change of contents,
or preferably fixed with a flow-through cell for a continuous flow-through
analysis. Set the instrument at zero absorbance with clear, uninoculated
broth prepared as specified for the particular antibiotic, including the
same amount of test solution and formaldehyde as found in each sample.
ASSAY DESIGNS
Microbial assays gain markedly in precision by the segregation of
relatively large sources of potential error and bias through suitable
experimental designs. In a cylinder plate assay, the essential
comparisons are restricted to relationships between zone diameter
measurements within plates, exclusive of the variation between plates in
their preparation and subsequent handling. To conduct a turbidimetric
assay so that the difference in observed turbidity will reflect the
differences in the antibiotic concentration requires both greater uniformity
in the environment created for the tubes through closer thermostatic
control of the incubator and the avoidance of systematic bias by a
random placement of replicate tubes in separate tube racks, each rack
containing one complete set of treatments. The essential comparisons are
then restricted to relationships between the observed turbidities within
racks. Within these restrictions, two alternative designs are
recommended; i.e. a 3-level (or 2-level) factorial assay, or a 1- level
assay with a standard curve. For a factorial assay, prepare solutions of 3
or 2 corresponding test dilutions for both the standard and the unknowns
on the day of the assay, as described under Preparation of the Standard
and Preparation of the samples. For a 1-level assay with a standard
curve, prepare instead solutions of five test dilutions of the standard and
a solution of a single median test level of the unknown as described in
the same sections. Consider an assay as preliminary if its computed
potency with either design is less than 60 per cent or more than 150 per
cent of that assumed in preparing the stock solution of the unknown. In
such a case, adjust its assumed potency accordingly and repeat the
assay. Microbial determinations of potency are subject to inter-assay
variables as well as intra-assay variables, so that two or more
independent assays are required for a reliable estimate of the potency of
a given assay preparation or unknown. Starting with separately prepared
stock solutions and test dilutions of both the standard and unknown,
repeat the assay of a given unknown on a different day. If the estimated
potency of the second assay differs significantly, as indicated by the
calculated standard error, from that of the first, conduct one or more
additional assays. The combined result of a series of smaller, independent
assays spread over a number of days is a more reliable estimate of
potency than that from a single large assay with the same total number
of plates or tubes.
METHODS
Carry out the microbiological assay by Method A or Method B.
Method. For preparing the standard curve, use a total of 12 Petri dishes
or plates to accommodate 72 cylinders or cavities. A set of 3 plates (18
cylinders or cavities) is used for each dilution. On each of the three plates
of a set fill alternate cylinders or cavities with solution S3 (representing
the median concentration of the standard solution) and each of the
remaining 9 cylinders or cavities with one of the other 4 dilutions of the
standard solution. Repeat the process for the other 3 dilutions of the
standard solution. For each unknown preparation use a set of 3 plates
(18 cylinders or cavities) and fill alternate cylinders or cavities with the
sample solution and each of the remaining 9 cylinders of cavities with
solution S3. Incubate the plates for about 18 hours at the specified
temperature and measure the diameters or the zones of inhibition.
Estimation of potency.
Average the readings of solution S3 and the readings of the concentration
tested on each sets of three plates, and average also all 36 readings of
solution S3. The average of the 36 readings of solution S3 is the
correction point for the curve. Correct the average value obtained for
each concentration (S1, S2, S4 and S5) to the figure it would be if the
readings for solution S3 for that set of three plates were the same as the
correction point. Thus, in correcting the value obtained with any
concentration, say S1, if the average of 36 readings of S3 is, for example,
18.0 mm and the average of the S3 concentrations on one set of three
plates is 17.8 mm, the correction is + 0.2 mm. If the average reading of
S1 is 16.0 mm the corrected reading of S1 is 16.2 mm. Plot these
corrected values including the average of the 36 readings for solutions S3
on two-cycle semilog paper, using the concentrations in Units or μg per
ml (as the ordinate logarithmic scale) and the diameter of the zones of
inhibition as the abscissa. Draw the straight response line either through
these points by inspection or through the points plotted for highest and
lowest zone diameters obtained by means of the following expressions:
Average the zone diameters for the sample solution and for solutions S3
on the plates used for the sample solution. If sample gives a large
average zone size than the average of the standard (solution S3), add
the difference between them to the zone size of solution S3 of the
standard response line. If the average sample zone size is smaller than
the standard values, subtract the difference between them from the zone
size of solution S3 of the standard response line. From the response line
read the concentration corresponding to these corrected values of zone
sizes. From the dilution factors the potency of the sample may be
calculated.
U1 and U2 are the sums of the zone diameters with solutions of the
unknown of high and low levels.
S1 and S2 are the sums of the zone diameters with solutions of the
standard of high and low levels.
I = ratio of dilutions.
If the potency of the sample is lower than 60 per cent or greater that 150
per cent of the standard, the assay is invalid and should be repeated
using higher or lower dilutions of the same solution. The potency of the
sample may be calculated from the expression.
At the same time prepare three control tubes, one containing the
inoculated culture medium (culture control), another identical with it but
treated immediately with 0.5 ml of diluteformaldehyde solution (blank)
and a third containing uninoculated culture medium.
Plot the values obtained for L and H and connect the points. Average the
absorbances for the sample and read the antibiotic concentration from
the standard response line. Multiply the concentration by the appropriate
dilution factors to obtain the antibiotic content of the sample.
Introduction
By definition, an antibiotic is either a natural product of a micro-
organism, an identical synthetic product or a similar semi synthetic
product, that inhibits the growth of other microorganisms (bacteriostatic
effect) or destroys other microorganisms (bactericide effect).
In the field of food hygiene and food control we deal with analysis of
antibiotic residues in food of animal origin due to the potential of
unwanted consequences . Among them are sensitivity to antibiotics,
allergic reactions and imbalance of intestinal microflora in people, spread
of resistance to antibiotics in microorganisms and losses in the food
industry where antibiotics can influence starter cultures used in the
production of meat and milk products.
Basic media for preparation of test plates were antibiotic agar No. 1
(MerckTM) and antibiotic agar No. 2 (MerckTM). Antibiotic agar No. 1 was
prepared as follows: 1000 ml of distilled water was added to 30, 5 g of
the medium, left for 15 min and then heated to boiling point so that the
medium was completely dissolved. The medium was then autoclaved at
121 oC for 15 min. For antibiotic agar No. 2 1000 ml of distilled water
was added to 15, 5 g of medium and then the same procedure was
followed. After autoclaving, the pH of the media was set to desired
values: pH 8 for Er, I BGA, Kin and AC plates and pH6 for E plates.
Preparation of test plates
Test plates were marked according to the bacterial strain added to the
medium: AC plate – Micrococcus luteus ATCC 2341, ER plate -
Staphylococcus epidermidis ATCC 12228, I-BGA plate - Bacillus subtilis
BGA, Kin plate - E. coli ATCC 10536 and E plate – Bacillus cereus ATCC
11778. The pH of the medium was maintained at 8.0 for AC, E and ER
plates and at 6.0for I-BGA and Kin plates. We defined the tolerance for
the width of inhibition zone at (as) 8.5 mm – 0.5 mm wider than the
width of the metal cylinder containing the sample. Inhibition zones
between 8 mm and 8.5 mm wide were considered a non-specific reaction.
To prepare a test plate 0.45 ml of suspension of bacterial culture was
added to 40 ml of basic medium and heated to 40 0C. Kin plate was an
exception where 0.2 ml of suspension was added to 50 ml of medium.
The mixture of medium and bacterial culture was poured into a petri dish
(5 ml of mixture into each petri dish). At room temperature the petri
dishes with silified medium were enveloped in a parafilm and stored in a
fridge. The storage period of test plates was one week. Before application
of samples to test plates, plates were warmed at room temperature for
20 to 30 min.
Confirmation solutions
To confirm the presence of antibiotic groups or their individual
representatives we used confirmation solutions. These solutions inhibit
the action of certain antibiotics and can help to distinguish between
antibiotic groups which cause inhibition zones on the same test plates.
Magnesium sulphate (MgSO4) was used to neutralise the aminoglicosides
and cephalosporinase enzyme to neutralise the cephalosporines.
Results
We have confirmed sensitive and resistant bacterial strains for all
antibiotic groups tested in our study. Based on our results we chose to
use Bacillus cereus ATCC 11778 (E plate) as the sensitive
and Micrococcus luteus ATCC 9341 (AC plate) as the resistant strain for
tetracycline and chlortetracycline from the tetracyclines group. For
tylosine and erythromycine from the macrolides group Micrococcus
luteus ATCC 9341 (AC plate) was chosen as the sensitive and Escherichia
coli ATCC10536 (Kin plate) as the resistant strain. For gentamycine,
sterptomycine and neomycine from the aminoglicosides group Bacillus
subtilis BGA (I-BGA plate) was chosen as the susceptible
and Staphylococcus epidermidis ATCC 12228 (ER plate) the resistant
strain. For cephalexine, cephoperasone and cephasoline from the
sensitive group Micrococcus luteus ATCC 9341 (AC plate) was chosen as
the susceptible and Staphylococcus epidermidis ATCC 12228 (ER plate)
as the resistant strain.
Discussion
Microbial methods for detection of antibiotic residues in food of animal
origin are used as a screening method in the majority of laboratories in
Europe that deal with analyses of drug residues in food.They are always
the method of choice for screening purposes as they allow qualitative
detection of antibiotics in the sample and identification of antibiotic
groups. This facilitates subsequent confirmation of specific antibiotic
residues with chemical methods. Microbial methods are relatively
inexpensive, easy to use, do not require expensive equipment and can be
efficiently adopted by laboratory staff. Although minimal expenditure is a
significant factor of analyses, no test is valuable if it does not give reliable
results. We succeeded in developing a microbial method which is
sensitive and meets the legislative requirements – to detect
concentrations of antibiotics below the MRL. For some antibiotics the level
of detection was at half the MRL or lower.Microbial methods are semi
quantitative, therefore any positive or suspicious result should be
confirmed by chemical methods. In accordance with the EC 2002/657/EC
regulation results of microbial methods are not reported as negative and
positive, but as satisfactory or suspect when the MRL is exceeded.
Although the STAR five-plate test is the official method approved by the
Community Reference Laboratory, many variations of microbial methods
are used across the world and most laboratories apply a specific approach
with a different number and types of bacterial strains and therefore a
different number of test plates.
REFERENCES
1. Indian Pharmacopoeia 2007, Published By The Indian Pharmacopoeia
Commission,Government Of India Ministry Of Health & Family