Chapter 12
Food additives analysis
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Determination of Antibiotics
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Introduction
Antibiotics are substances of natural, semi-synthetic, or
synthetic origin that exhibit antibacterial activity. Their
presence in foods is essentially due either to therapeutic
treatments or to the antibiotic-supplemented food gived
to certain animals. The consequences of the presence of
antibiotic residues are as numerous to human health as
they are to certain processing operations. Where human
health is concerned, a number of dangers must be
avoided, such as allergic effects and possibilities of
microbial selections and of mutations that essentially
have two consequences:
1. Selection of resistant strains.
2. Disequilibrium of the normal flora of the digestive
tract.
Methods of determination
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In processing operations, the presence of residues with
antibiotic activity in milk or meat makes them unsuitable
for some uses.
For all these reasons, the problems related to the
presence of the antibiotic residues in foods have been
extensively studied. The detection and the determination
of these residues are therefore essential elements in the
study of antibiotic evolution and the protection of the
consumer. The methods of determination used at
present can be classified into three groups:
·microbiological methods;
·eletrophoretic methods;
·physicochemical methods.
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Microbiological Methods
The most frequently used detection methods
are those that exploit the sensitivity of
certain bacterial strains vis-à-vis one or
several antibiotics. The manifestation of this
inhibition is affected either in liquid (e.g.,
acidification method) or solid media (e.g.,
agar diffusion method).
Liquid Medium Methods
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Principle
This technique is widely used for the detection
of antibiotics in milk. After pasteurization, the
sample is cultured with a strain sensitive to
antibiotics (e.g., Bacillus, Strentococcus, etc.).
After incubation, the production of latic acid, which
results from the growth of test bacteria in the
absence of antibiotic residues, is detected either
by a pH indicator or by the coagulation of the milk.
The bacterial growth can also be measured by
nephtelometry.
Methodology
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Several methods based on this principle are currently being
used.
1. Reduction of Colored Indicators
·Methylene Blue test
2. Measurement of the Coagulation Time
The test germ is yogurt fermenting agent (e.g., Streptococcus
thermophilus and Lactobacillus bulgaricus). The presence of
inhibiting substances is detected through the absence of milk
coagulation after a fixed culture period
3. Measurement of Acidity
A technique that consists of adding a strain of Streptococcus
thermophilus to milk and titrating the lactic acid produced after
incubation.
4. Measurement of Medium Turbidity
Measure the growth of the test germ by recording the
variations in medium turbidity over time.
Sub-summary
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By using method studying antibiotics in
liquid media, results can be obtained
rapidly. They allow the analysis of large
series of milk samples and can act as a
primary selection method. All positive of
questions samples must be subjected to
a confirmation test by the agar diffusion
method, which is to be described next.
Agar Diffusion Methods
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These techniques have been employed in
antibiotic analyses in all food products.
Principle
When one or more antibiotics in a solution are
brought into contact with an agar medium, they
diffuse into it. The diffusion is proportional to the
logarithm of their concentrations. The growth of
a test germ cultured in agar after incubation
shows the presence of an inhibiting substance
through the appearance of a clear zone in the
antibiotic diffusion zone, while everywhere else
the growth of the microorganism is visible.
Methodology
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Agar Culture Media
The composition of the agar medium depends on the strain used and
the antibiotic studied. For example, one may use: Healtey’s agar;
Chabbert medium; Bacto Whey Agar medium. The pH must be
adjusted to 6.6 or 7.8, depending on the antibiotic studied.
Sample Preparation
1. In its original state (milk);
2. After mixing it aseptically in a small quantity of sterile physiological
serum (e.g., curdled milk, cheese, or antibiotic supplemented food);
3. After solvent extraction (e.g., muscular tissues). For extraction,
three solvents: pure methanol, methanol + pH 8 bicarbonate 1/1
buffer (V/V); or distilled water + pH 8 bicarbonate 3/7 buffer (V/V)
are recommended.
Test Microorganisms
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The following strains are used most often:
·Bacillus stearothermophilus var calidolactis strain
·Bacillus subtilis ATCC 6633,
·Sarcina lutea ATCC 9341:
·Staphylococcus aureus,
·Bacillus megaterium ATCC 9855:
·Micrococcus luteus and Bacillus cereus.
* Other sensitive bacteria can also be used;
including Micro flavus, Bacillus cereus, Sarcina
lutea, and Escherichia coli.
Procedure
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1. The agar medium, which is melted and then
cooled to 45℃, is cultured with a diluted
suspension of the test germ. It is homogenized
and the mixture is poured into Petri dishes left to
cool horizontally. After pasteurization, the sample
is aseptically placed in contact with the agar
according to two techniques:
2. A filter paper disk is saturated with a fraction of
the sample product or the extraction solution (in
this case, drying is very important), and then
deposited on the surface of the cultured agar;
3. The sample is placed into hollow cavities in the
agar or small stainless steel cylinders applied to
the agar surface.
Interpretation of Results
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Petri dishes prepared as discussed are incubated. A clear
zone around the filter papar disk or the cavity indicates
the presence of a substance in the sample with antibiotic
activity. Otherwise, colonies propagate through the entire
agar surface. A control must always be performed with a
preparation that does not contain antibiotics. It is
important that this test be conducted under conditions
that are rigorously identical to the sample biological
liquid. Most biological products (e.g., serum, liver, milk,
muscle, urine, etc.) have enzyme binding or destructive
properties thar are likely to distort the results. It is
possible to detect penicillin by performing an assay with a
disk saturated with penicillinase. If the sample contains
penicillin at the start, then no inhibition zone appears
around the disk saturated with penicillinase. If it contains
an antibiotic other than penicillin, then an inhibition zone
appears around the disk saturated with penicillinase.
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Sub-summary
The agar diffusion method is relatively rapid and does not require much
labortory equipment. However, just like the liquid medium methods, it
is only an “all or none” technique that permits neither the
determination nor the identification of the antibiotic (except for
penicillin). It is especially well adapted when the sample antibiotic is
known, as is the case with pharmacodynamic studies of a product in a
particular animal. For a “regulatory type” control test, however, there
are other problems. One may work either with a whole “bank” of
bacterial strains of varying sensitivity in such a way as to cover the
largest range of antibiotics used, making it a cumbersome detection
system, or else neglect the antibiotic and make do with a few sensitive
bacteria that will not produce inhibition rings with the sample examined.
Finally, it should always be kept in mind that there are natural
antibiotics in the plant kingdom, and that in the animal world,
substances like lactic acid, the lactoperoxidoses, the aglutinins, and
the lactoransferinses can produce inhibition rings. That is why it is so
important to interpret the results carefully. In order to partially remedy
these drawbacks, a modification was made in the agar diffiusion
method. It consists of performing a preliminary electrophoretic
separation of the sample or extract to be analyzed.
Other methods
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1. The principle of electrophoresis method:
The sample to be tested is deposited in hollow cavities in
the agar or in wells. Under the effect of an appropriate
electric current, the antibiotics separate with different
and specific speeds and directions of migration, which
simultaneously permits the elimination of interfering
substances and provides information on the nature of
the antibiotics present in the sample.
A second layer of agar cultured with one or more test
microorganisms detects the diffusion of the antibiotics
into the gel after incubation.
A positive detection is indicated, as before, by the
formaiton of clear zones. In a certain sensitivity range
the diameter of the inhibition zones is proportional to
the concentration of the antibiotic present, which can
be used in its determination.
(*Billon and Taos (1979) used this electrophoretic technique by associating it with the
microbiological method. )
Advantages of Electrophoresis
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1. Elimination of “false positives” due to the seperation of
interfering substances. Thus, the control sample, void of
antibiotic, is no longer necessary, offering a great benefit to
official regulatory bodies.
2. Indentification of the antibiotics, which enables the
recognition of those used the most in human therapeutics
(e.g., penicillin, tetramycin, chlortetracycline, etc.) and
which can potentially be the most dangerous (e.g.,
antibioticresistance) to be the consumer.
3. Determination of the antibiotics. For fresh milk, when the
content is particularly high, the determination can aid in
making seizure of the milk simpler so that the responsible
suppliers can be penalized according to regulations.
4. Noticeably lower detection limits. The detection limits are
lower by four to ten times depending on the antibiotic or
antibiotics
This technique is rarely used in rountine work. It is an excellent reference method
for situations requiring expertise.
Other methods
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2. Physicochemical Methods
These techniques of antibiotic detection have developed
considerably in the past few years. They are special applications of
the analytical principles already employed for the determination of
other types of molecules.
3. Radioenzymatic Determinations
These are based on a specific biochemical reation between the
antibiotic molecule and a cofactor labled with carbon 14. Acording to
Charm (1979), the determination is effected in 10-15 minutes, and
the lower sensitivity limit is on the order of 0.05 Ul/ml for penicillin
(technique: “Charm test”).
4. Radioimmunological Determinations
Several companies have commercialized a determination “kit” that
includes the specific antibody and the antibody labeled with iodine
125. Despite a high theoretical sensitivity, this technique cannot
reach detection limits lower than 1 pg/ml of milk (Flavigny, 1980)
Other methods
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5. Determinations by Fluorimetry
Hamann and Heeschen (1975) have desrcibed a determination
technique using immunofluorescence, the principle of which consists
of labeling the antibiotic with fluoresceine. The determination occurs
by competition in the presence of an unlabeled antibiotic and a
specific serum. The measurement is affected by fluorimetry in
polarized or nonpolarized light. This technique is used for the
detection of tetracylines, which form fluorescent compounds after
being heated in neutral or alkaline medium (detection limit: 2 µg/g).
6. Determinations by Bioluminescence
Adenosine triphosphate (ATP) produced by bacterial cells is
transformed into adenosine diphosphate (ADP) in the presence of
luciferin luciferace. Photon emission, which accompanies the
reaction, is measured by a photometer. This technique therefore
involves a bacteriological step.
7. Spectrometry
Used in the ultraviolet, visible, and infrared spectra, spectrometry
permits the identification and the determination of penicillin
(detection limit: 10 µg/g), streptomycin, tetracyclines, and the like.
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8. Chromatographic Techniques
8.1Thin Layer Chromatography (TLC)
Hamann et al. (1979) used TLC to identify antibiotic residues in
milk, with or without preliminary extraction. The antibiotics
are identified on the plates by their RF. Determination can
be affected by fluorescence. The detection limits are:
·tetracyline: 0.025µg/ml
·chloramphenicol: 1µg/ml
·neomycin: 15µg/ml
·streptomycin: 0.5µg/ml
Bossuyt and Renterghem (1979) have also described a TLC
determination method after extraction of the antibiotic residues
with
acetone. Identification was affected bye using test strains of
Bacillus
cereus, Bacillus subtilis, Micrococcus flavrs, and Sareina lutea.
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8.2 Gas Chromatography
Hamann et al. (1979) have described different protocols for
extractions, identifications, and determinations of antibiotics
in milk by gas liquid chromatography. The detection limits
are the following:
·tetracycline: 0.5-10µg/ml
·chloramphenicol: 0.01µg/ml
·penicillin: 0.005Ul/ml
8.3 HPLC
This technique is now widely used for the detection and
determination of most antibiotics. In a number of methods,
the extraction of antibiotics is followed by a purification (e.g.,
on Sephadex columns) before seperation by HPLC
(Martinez and Shimoda, 1988; Pochard, 1987). Detection is
often affected by UV spectrophotometry, but other methods
can also be considered (Martinez and Shimoda, 1988).
Summary
Research and testing laboratories have at their
disposal a number of methods for the detection,
identification, and determination of the antibiotic
residues in foods. However, all of these techniques
are still not sufficiently classfied. The
microbiological methods are most frequently used
by all testing laboratories. The association of
electrophoretic technique with microbiological
detection has sighificantly increased reliability as
well as the sensitivity of determination. The use of
physicochemical methods is linked to the type of
equipment used by the laboratories, and the
complexity of the problems to be resolved.
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Determination of Antiseptics
Introduction-1
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The presence of antiseptic residues in foods is due
essentially to:
1. the processing aimed at stablilizing certain foods
(e.g., anhydrous sulphur in wines, biphenyls for
citrus fruits, etc.) In this case, regulation indicate
maximal quantities that must not be exceeded;
2. the contact of the products with inadequately
rinsed surface after the use of disinfectant product
(e.g., sodium hypochlorite, ammonium fluoride,
formaldehyde, etc.);
3. the addition of antiseptics whose use is
prohibited by law, in order to limit microbial growth
harming the marketable quality of a product.
Introduction-2
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The presence of antiseptic substances in
food poses problems similar to those
concerning antibiotics:
1. The health of the consumer, due to
the toxicity of the antiseptics;
2. At the technological level, certain
industrial transformation operations (e.g.,
inhibition of lactic ferments in the dairy
industry).
Microbiological Methods
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These technique are based on the fact that the growth of certain microorganisms
in inhibited by the presence of antiseptic residues.
1. General Method: Fermentation Test
Principle
The food is introduced into a fermentation tube (eudiometer after Einhorn
[Diemair and Postel, 1970]) and mixed with a standard quanity of a very active
Saccharomnyces cerevisiae suspension (i.e., baking yeast). After incubation,
the volume of carbon dioxide gas produced leads to a conclusion on the
presence or absence of antiseptics.
Sample preparation.
Liquid nutrients do not require any special preparation, with the possible
exception of dilutions for products that are too concentrated. However,
alcoholic drinks have to be dealcoholized by evaporation in a low vacuum at a
low temperature (30-40℃). The initial volume is reestablished bye adding
distilled water. The sugar content of the samples is raised to about 10% with
glucose. For solid or semi-solid foods, two samples are prepared for analysis
(about 10 g). One is mixed with 100 ml of 0.5% tartaric acid solution,
wherease the other is mixed with 100 ml of 0.1% sodium hydroxide solution.
Let them react for 10 min. The two extracts are filtered and adjusted to a pH
between 3 and 4. The sugar content is raised to about 10% with glucose.
Pasteurize the two extracts by healting for 1 in at 80℃.
In all cases, if the product is low in nitrogenated substances, then it is
necessary to add the yeast extract to a concentration of 2.5%.
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Preparation of Yeast.
In 100 ml of a sterile culture medium (glucose: 25 g; asparagine:1 g;
KH2PO4: 0.5 g; MgSO4H2O: 0.5 g)(Diemair and Postel, 1970),use
sterile technique to add 1 g of fresh baking yeast (test microorganism)
taken from an unopened packet stored in the cold (+2℃). Agitate in
such a way to disperse the yeast. Incubate for 24 hours at 25℃. Affect
a second culture using the the same nutrient medium taking 10 ml
from the first culture. Incubate for 48 hours at 25℃.
Fermentation test.
Eliminate the carbon dioxide gas from the second culture by filtering it
on sterile hydrophilic cotton. In a sterile Erlenmeyer flask, aspetically
introduce 10 ml of the yeast suspension thus prepared, and 50 ml of
the sample or sample preparation. Mix carefully and decant the
sample thus cultured in a sterile Einhorn’s endiometer in such a way
that the bowl is one-third filled and the tube is filled completely.
If possible, prepare a control with a sample or an extract free from
antiseptics under the same conditions.
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Results
Incubate for 20-24 hours at 25℃. After this period, read the
volume of carbon dioxide gas produced on the graduated
scale.
Assay without a reference.
If the volume of carbon dioxide gas released after
incubation is zero or less than 5 ml, then it may be
presumed that the inhibition is due to an antiseptic
(Diemair and Postel, 1970).
Assay with a reference.
If the sample examined release less gas than the
reference test, then the test must be considered as
positive when the difference between the two volumes of
gas is greater than 50%. One must always be prudent in
the interprepation of results, and keep in mind that certain
natural substances can have inhibiting effects vis-à-vis the
test strain. Chemical characterization of the antiseptic
provides the confirmation of positive results.
Application to a Few Foods
1. Milk and Milk Products: Kluyver Method
The sample to be analyzed is enriched in glucose, and
then cultured with a dilution of Saccharomyces cerevisiae in
peptonated water. A reference free from antiseptic is
prepared under the same conditions. After incubation at 25℃
for 48 hours, if the sample examined has gaseous release of
lower than 50% of that of the reference, then one may
conclude that it contains antiseptics like hydrogen peroxide,
quaternary ammoniums, and by-products of monobromacetic
acid at concentrations that are likely to cause manufacturing
flaws. This method can detect the antiseptics mentioned
previously to minimal concentrations of 0.02.-0.05 and
0.001%, repectively (Serres et al.).
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2. Wines, Beers, Worts, and Other Alcoholic Drinks
2.1 Official French method for detection of antiseptics in wines.
Sulfur anhydride is first removed from the wine by adding a requisite quantity
of acetaldehyde (… the wine must contain less than 20 mg/L of free SO2).
The wine is brought back to 10% alcohol and the quantity of
glucose is enhanced (20-50 g/L). A nutrient solution is added and the wine
is cultured with a suspension of Saccharomyces oviformis accustomed to
alcohol (10²*10³ yeast/ml of wine). The intensity of the fermentation,
measured in a Stella-Garoglio (Lecoq, 1965) zygometer, enable one to drow
conclusions about the presence or absence of antiseptics.
It is necessary to be able to compare the sample’s rate of fermentation
with that of a wine with a composition as close as possible to that of the
sample that is free of antiseptics. In normal wines, the release of dioxiode
begins after 48 hours of incubation at 25℃. The presence of antiseptics can
be confirmed if th commencement of fermentation is delayed by 48 hours as
compared with that of the reference test; however, the interpretation of
results with always be difficult with wines made from rotted grape. A delay in
fermentation could be possible, which suggests the content of antiseptic
traces.
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2.2 Kluyver-Mossel method, modified by Darmon.
This method consists of culturing the sample enriched in nutrients with a
Saccharomyces cerevisiae suspension, the growth of which can be studied
by measuring the volume of carbon dioxide gas released. With a drink free
of antiseptics, it is found that the curve representing the volume of CO2 as
a function of time is S-shaped, which represents a latency phase, a
logarithmic growth phase, and a slowed growth antiseptics, the latency
phase is more significant than that of the reference. By plotting the different
curves of growth on a graph, it can be seen that the presence of an
antiseptic residue in the samples creates a shift of the growth curve toward
the right.
For the methods to produce good results, the authors recommend:
1. the use of very active yeasts with concentration of between 50 and 100
cells/µl of sample liquid;
2. the elimination of sulfurous anhydride and alcohol by a stream of water
vapor;
3. conducting a reference test with a sample free of antiseptic.
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2.3 De Clerck, Aubert, and Jerumanis method. This
method (De Clerck, 1962) is used for beers. It consists of
adding to the beer a determined quantity of yeast cells
freshly grown in wort, and measuring their rate of
development in the wort that is cultured with this beer after
intervals of a day or two. In beers that contain an
antiseptic, yeast gets weaker. This is expressed as a
slowing of growth.
2.4 Baetsle, Mossel, and Verheyden method. This is the
official method of the Benelux countries (Baetsle et al.,
1955) for the detectiong of antiseptics in beer. Its principle
is also based on the use of Saccharomyces cerevisiae,
and the magnitude of the carbon dioxide gas released is
measured in the Einhorn tube.
Methods of Agar Medium Diffusion
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This method consists of incorporating the
suspect sample in a medium of agar culture.
The presence of the antiseptic is indicated
by the total or partical inhibition of
inoculated germs on the surface of th
culture medium, as compared with a
reference cultured under the same
conditions.
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1. Drieux and Thierry Method
The technique consists of introducing 10 ml of the sample and 0.1 ml if 1%
triphenyltetrazolium chloride in 10 ml of double concentration nutrient agar.
Homogenize and pour this mixture in a Petri dish. Under the same conditions,
prepare a reference Petri dish using a sample free of antiseptics. After setting the
agar, divide each dish into four parts and inoculate by streaking with four species of
bacteria:
·Escherichia coli;
·Serratia maresceus;
·Staphylococcus;
·Bacillus subtilis.
Incubate at 30-37℃ for 24-28 hours. In the same manner, preparea second series
of dishes that use four species that belong to the group of yeasts and molds.
·Saccharomyces cerevisiae;
·Penicillium glaucum;
·Aspergillus niger;
·Oidium.
Incubate at 20-25℃ for 4-5 days. Compare the growth of the germs after incubation
of the dishes. The absence of culture or a partical inhibition in the dish that contains
the sample to be examined reflects bactericidal or fungicical effect vis-à-vis the
species considered.
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2. Mossel and Eijgelaar Method
This method (Serres et al.) can be used to detect
antiseptics in butter. This is performed on the aqueous
phase of butter that is enriched in glucose and yeast
extract. The mixture prepared in this manner is mixed with
nutrient agar and poured into a Petri dish. As in the
previous technique, affect a reference test and divide the
dishes into two parts. Each part is cultured, one with a
strain of Candida The ever-increasing number of chemical
substances with antiseptic action considered, it is not
possible to provide an exhaustive list accompanied by
their methods of detection. This chapter is therefore limited
to detection and determination techniques that concern:
a) principle antiseptic substances, used as such and
authorized by regulation for the treatment of certain foods;
b) products with antiseptic action that are often used in the
operations of cleaning and disinfeceting;
c) a few antiseptics that are sometimes used illicitly.
Sorbic Acid and Sorbates
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The colorimetric method:
which was published by Schmidt in 1960, is applicable to
all foods. First, ascorbic acid is distilled by vapor. It is
collected in the distillate and oxidized by an acid solution
of potassium bichromate. It particularly forms malonic
dialdehyde, which with thiobarbituric acid produces a red
complex with a maximum absorption at 532 mm. The
sorbic acid concentration of the sample is read on a
standard curve that is formed from a solution of potassium
sorbate. The other antiseptics do not produce interference.
The precision of this method is on the order of ±5%.
High performance liquid chromatography:
can be used for the determination of sorbic acid.
Benzoic Acid and Derivatives
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A number of authors have published papers
on the determination of benzoic acid and its
derivatives (e.g., orthochlorobenzoic,
parachlorobenzoic, salicylic, and
parahydroxybenzoic acids, and methyl,
propyl, and butyl esters of
parahydroxybenzoic acid). Parkinson
advocates a chromatographic technique
after extraction with chloroform or a
chloroform-ethanol mixture.
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Official Method
Benzoic acid is extracted by ethyl eher. The solvent is evaporated,
and the odor and the points of melting and sublimation are determined
with one part of the residue. The other part is redissolved in hot water.
The liquid is then neutralized and iron perchloride is added, which
produces a characteristic precipitate in the presence of benzoic acid.
Blarez Method
This method (Ribereau-Gayon and Peynaud, 1958) modifies the
preceding technique by effecting destruction between benzoic acid
and succinic acid using the characteristic aspect of their crystalline
deposits.
Guillaume and Mehl
The Guillaume and Mehl (1951) Method uses ethyl extraction and the
determination of the antiseptic by borometric titration.
Sulfurous Anhydride and Sulfites
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Sulfurous anhydride is introduced into food liquids in the from
of sulfurous gas, which combines with sugars, ethanol,
and coloring matter. A fraction of free SO2 still remains
and is responsible for the characteristic odor and the
antiseptic action.
After the sample is heated, and water and phosphoric
acid are added, sulfurous anhydride can be detected in
two ways (Diemair and Postel, 1970):
·by odor;
·chemically by using a narrow starched band of paper
saturated with potassium. In the presence of SO2, the
paper takes on a blue tint that disappears if the duration of
the test is prolonged.
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A number of determination techniques can be used:
Liquid Products: Wines, Beers, Ciders, Fruit Juices, and Meads
The method, which is employed most often (Brun, 1963; RibereauGayon and Peynaud, 1958), is based on the oxidation of sulfrous
anhydride by iodine. The free SO2 is determined diretely by iodine in
an acid medium. On the other hand, sulfurous anhydride is not
oxidized by cold iodine in an acid medium, and therefore, it is
necessary to displace it by distillation and titrate it with iodine.
Semi-Liquid and Solid Products: Mustard, Dry Fruits, and Fish
Monier-Williams and Shipton (1954) have described methods in
which sulfurous anhydride reacts with hydrogen peroxide to form
sulfuric acid, which is then determined with a titrated alkaline solution.
Other Methods
Many other methods have been described (Coeur et al., 1961;
Jaulmes and Hamelle, 1961) among which are colorimetric methods
(Brun, 1963), gravimentric techniques (Code de la Charcuterie, 1980),
or that of Stone and Laschiver (1957), which are based on the purple
color produced by rosaniline with SO2
Other antiseptics
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Biphenyl, Orthophenyl-Phenol
Biphenyl
Orthophenyl-Phenol
Halogenated Acetic Acids – Monobromacetic Acid
Boric Acid
Formaldehyde and Substances Producing Formaldehyde
Formic Acid
Fluorinated Compounds: Sodium Fluoride and
Hydrofluosilicic Acid
Quaternary Ammoniums
Hydrogen Peroxide
Hypochlorites and Chloramines
Conclusion
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A general diagnosis that affirms that a food product
contains antiseptic residues is difficult to make using
chemical methods. Microbiological techniques, although
nonspecific, offer the advantage of being able to make a
rapid distinction between products that can cause
microbial inhibition and those that do not affect the
growth of sensitive strains. Unfortunately, the results can
be distorted by the presence of certain natural
substances that are harmful to the development of the
test strain, always necessitating a reference sample free
of antiseptics. Chemical methods can be used to verify
the microbiological results. They are affected, before the
biological test, every time it is necessary to verify
whether the adition of a parmissible antiseptic conforms
to regulations, or when there is a strong presumption of
the presence of a particular antiseptic.
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