ASTM E2186-02a(2023)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E2186 − 02a (Reapproved 2023)
Standard Guide for
Determining DNA Single-Strand Damage in Eukaryotic Cells
Using the Comet Assay
This standard is issued under the fixed designation E2186; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope cellular DNA repair rates (10), detection of the presence of
photoactive DNA damaging compounds (14), or detection of
1.1 This guide covers the recommended criteria for per-
specific DNA lesions (3, 18).
forming a single-cell gel electrophoresis assay (SCG) or Comet
1.3 This standard does not purport to address all of the
assay for the measurement of DNA single-strand breaks in
safety concerns, if any, associated with its use. It is the
eukaryotic cells. The Comet assay is a very sensitive method
responsibility of the user of this standard to establish appro-
for detecting strand breaks in the DNA of individual cells. The
priate safety, health, and environmental practices and deter-
majority of studies utilizing the Comet assay have focused on
mine the applicability of regulatory limitations prior to use.
medical applications and have therefore examined DNA dam-
1.4 This guide is arranged as follows:
age in mammalian cells in vitro and in vivo (1-4). There is
increasing interest in applying this assay to DNA damage in
Section
freshwater and marine organisms to explore the environmental
Scope 1
implications of DNA damage.
Referenced Documents 2
1.1.1 The Comet assay has been used to screen the geno-
Terminology 3
Summary of Guide 4
toxicity of a variety of compounds on cells in vitro and in vivo
Significance and Use 5
(5-7), as well as to evaluate the dose-dependent anti-oxidant
Equipment and Reagents 6
(protective) properties of various compounds (3, 8-11). Using
Assay Procedures 7
Treatment of Data 8
this method, significantly elevated levels of DNA damage have
Reporting Data 9
been reported in cells collected from organisms at polluted
Keywords 10
sites compared to reference sites (12-15). Studies have also Annex Annex A1
References
found that increases in cellular DNA damage correspond with
1.5 This international standard was developed in accor-
higher order effects such as decreased growth, survival, and
dance with internationally recognized principles on standard-
development, and correlate with significant increases in con-
ization established in the Decision on Principles for the
taminant body burdens (13, 16).
Development of International Standards, Guides and Recom-
1.2 This guide presents protocols that facilitate the expres-
mendations issued by the World Trade Organization Technical
sion of DNA alkaline labile single-strand breaks and the
Barriers to Trade (TBT) Committee.
determination of their abundance relative to control or refer-
ence cells. The guide is a general one meant to familiarize lab
2. Referenced Documents
personnel with the basic requirements and considerations
2.1 ASTM Standards:
necessary to perform the Comet assay. It does not contain
E1706 Test Method for Measuring the Toxicity of Sediment-
procedures for available variants of this assay, which allow the
Associated Contaminants with Freshwater Invertebrates
determination of non-alkaline labile single-strand breaks or
E1847 Practice for Statistical Analysis of Toxicity Tests
double-stranded DNA strand breaks (8), distinction between 4
Conducted Under ASTM Guidelines (Withdrawn 2022)
different cell types (13), identification of cells undergoing
3. Terminology
apoptosis (programmed cell death, (1, 17)), measurement of
3.1 The words “must,” “should,” “may,” “can,” and “might”
have very specific meanings in this guide. “Must” is used to
This guide is under the jurisdiction of ASTM Committee E50 on Environmental
Assessment, Risk Management and Corrective Action and is the direct responsibil-
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved Dec. 1, 2023. Published December 2023. Originally contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
approved in 2002. Last previous edition approved 2016 as E2186–02a(2016). DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/E2186-02AR23. the ASTM website.
The boldface numbers in parentheses refer to the list of references at the end of The last approved version of this historical standard is referenced on
this standard. www.astm.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2186 − 02a (2023)
express the strongest possible recommendation, just short of an 3.2.10 eukaryotic cell, n—cell with a membrane-bound,
absolute requirement. “Must” is only used in connection with structurally discrete nucleus and other well-developed subcel-
factors that relate directly to the acceptability of the test. lular compartments. Eukaryotes include all organisms except
“Should” is used to state that the specific condition is recom- viruses, bacteria, and cyanobacteria (blue-green algae).
mended and ought to be met if possible. Although violation of
3.2.11 ocular micrometer, n—a graduated grid placed be-
on “should” is rarely a serious matter, the violation of several
tween the viewer’s eye and an object being observed under a
will often render the results questionable. Terms such as “is
microscope, to measure the object’s size.
desirable,” “is often desirable,” and “might be desirable” are
3.2.12 single-stranded DNA, n—linear polymers of DNA
used in connection with less important factors. “May” is used
resulting from the breaking of hydrogen bonds between
to mean “is (are) allowed to,” “can” is used to mean “is (are)
complementary base pairs in double-stranded DNA.
able to,” and “might” is used to mean “could possibly.” Thus
the classic distinction between “may” and “can” is preserved
3.3 Definitions of Terms Specific to This Standard:
and “might” is never used as a synonym for either “may” or
3.3.1 comet, n—name based on the appearance of individual
“can.”
stained nuclear DNA and associated relaxed or fragmented
3.2 Definitions:
DNA migrating out from the nuclear DNA observed under the
3.2.1 CCD camera, n—charge coupled device (CCD) cam- microscope following these assay procedures.
era is a light sensitive silicon solid state device composed of
3.3.2 DNA migration distance, tail length, comet tail length,
many small pixels. The light falling on a pixel is converted into
n—distance in microns between the leading edge of electro-
a charge pulse which is then measured by the CCD electronics
phoretically migrating DNA and the closest edge of the
and represented by a number. A digital image is the collection
associated nuclear DNA (head).
of such light intensity numbers for all of the pixels from the
3.3.3 head, comet head, n—portion of a comet comprised of
CCD. A computer can reconstruct the image by varying the
the intact/immobile nuclear DNA.
light intensity for each spot on the computer monitor in the
proper order. Such digital images can be stored on disk,
3.3.4 tail, comet tail, n—portion of a comet comprised of the
transmitted over a computer network, and analyzed using
DNA migrating away from the intact/immobile nuclear DNA.
image processing techniques.
3.3.5 tail moment, n—a calculated value used to express the
3.2.2 cell lysis, n—the process of breaking open a cell by
distribution of DNA migrating from the comet head. Image
disruption of the plasma membrane.
analysis software applies an algorithm to the digitized image of
stained DNA and associated migrating DNA tail, which in
3.2.3 DNA, n—acronym for deoxyribonucleic acid, the sub-
stance that is the carrier of genetic information found in the essence defines the limits of the comet, subtracts background,
and determines the boundaries and staining intensity of the
chromosomes of the nucleus of a cell.
nucleus and comet tail. The calculated product of the percent of
3.2.4 DNA denaturation, n—refers to breaking hydrogen
DNA in the tail and the tail length is defined as the tail moment.
bonds between base pairs in double-stranded nucleic acid
molecules to produce two single-stranded polynucleotide poly-
4. Summary of Guide
mers.
4.1 Cells collected from organisms under different levels or
3.2.5 DNA lesion, n—a portion of a DNA molecule which
types of stress are dispersed and immobilized in agarose gel on
has been structurally changed.
microscope slides. The slides are placed in a solution to lyse
3.2.6 DNA supercoiling, n—the condition of DNA coiling
and disperse cell components, leaving the cellular DNA
up on itself because its helix has been bent, overwound, or
immobilized in the agarose. The DNA is denatured for a
underwound.
specified period of minutes by immersing the slides in an
alkaline solution. Strand breaks in the denatured cellular DNA
3.2.7 DNA supercoil relaxation, n—upon denaturation,
results in higher degree of supercoil relaxation: the more
DNA strand breaks allow the supercoiled DNA to unwind or
breaks, the greater the degree of relaxation. Given a sufficient
relax.
degree of relaxation, the application of an electric field across
3.2.8 double-stranded DNA, n—a structural form of DNA
the slides creates a motive force by which the charged DNA
where two polynucleotide molecular chains are wound around
may migrate through the surrounding agarose, away from the
each other, with the joining between the two strands via
immobilized main bulk of cellular DNA. Following
hydrogen bonds between complementary bases.
electrophoresis, the alkaline conditions are neutralized by
3.2.9 electrophoresis, n—a method of separating large mol- rinsing the slides in a neutral pH buffer and fixation of slide and
ecules (such as DNA fragments or proteins) from a mixture of its contents in ethanol. The DNA in the fixed slides is stained
similar molecules. An electric current is passed through a with fluorescent DNA stain and visualized using a fluorescent
medium containing the mixture, and each kind of molecule microscope. Migration distance of DNA away from the
travels through the medium at a different rate, depending on its nucleus, comet tail length, can be measured by eye using an
electrical charge and size. Separation is based on these differ- ocular micrometer. Comet tail length, percent DNA in tail, tail
ences. Agarose and acrylamide gels are the media commonly moment, and other DNA migration values can be calculated
used for electrophoresis of proteins and nucleic acids. with the use of image analysis software.
E2186 − 02a (2023)
5. Significance and Use use of T4 endonuclease V (3). Modifications of this type vastly
expand the utility of this assay and are good examples of its
5.1 A common result of cellular stress is an increase in DNA
versatility.
damage. DNA damage may be manifest in the form of base
5.2 A sufficient knowledge of the biology of cells examined
alterations, adduct formation, strand breaks, and cross linkages
using this assay should be attained to understand factors
(19). Strand breaks may be introduced in many ways, directly
affecting DNA strand breakage and the distribution of this
by genotoxic compounds, through the induction of apoptosis or
damage within sampled cell populations. This includes, but is
necrosis, secondarily through the interaction with oxygen
not limited to, influences such as cell type heterogeneity, cell
radicals or other reactive intermediates, or as a consequence of
cycle, cell turnover frequency, culture or growth conditions,
excision repair enzymes (20-22). In addition to a linkage with
and other factors that may influence levels of DNA strand
cancer, studies have demonstrated that increases in cellular
damage. Different cell types may have vastly different back-
DNA damage precede or correspond with reduced growth,
ground levels of DNA single-strand breaks due to variations in
abnormal development, and reduced survival of adults,
excision repair activity, metabolic activity, anti-oxidant
embryos, and larvae (16, 23, 24).
concentrations, or other factors. It is recommended that cells
5.1.1 The Comet assay can be easily utilized for collecting
representing those to be studied using the SCG/Comet assay be
data on DNA strand breakage (9, 25, 26). It is a simple, rapid,
examined under the light or fluorescent microscope using
and sensitive method that allows the comparison of DNA
stains capable of differentially staining different cell types.
strand damage in different cell populations. As presented in this
Morphological differences, staining characteristics, and fre-
guide, the assay facilitates the detection of DNA single strand
quencies of the different cell types should be noted and
breaks and alkaline labile sites in individual cells, and can
compared to SCG/Comet damage profiles to identify any
determine their abundance relative to control or reference cells
possible cell type specific differences. In most cases, the use of
(9, 16, 26). The assay offers a number of advantages; damage
homogenous cell populations reduces inter-cell variability of
to the DNA in individual cells is measured, only extremely
SCG/Comet values. The procedures for this assay, using cells
small numbers of cells need to be sampled to perform the assay
from many different species and cell types, have been pub-
(<10 000), the assay can be performed on practically any
lished previously (1, 2, 3, 5, 8, 10, 13, 14, 17, 18, 32-38). These
eukaryotic cell type, and it has been shown in comparative
references and others should be consulted to obtain details on
studies to be a very sensitive method for detecting DNA
the collection, handling, storage, and preparation of specific
damage (2, 27).
cell types.
5.1.2 These are general guidelines. There are numerous
5.3 The experimental design should incorporate appropriate
procedural variants of this assay. The variation used is depen-
controls, reference samples, and replicates to delineate the
dent upon the type of cells being examined, the types of DNA
influence of the major sources of experimental variability.
damage of interest, and the imaging and analysis capabilities of
the lab conducting the assay. To visualize the DNA, it is stained
with a fluorescent dye, or for light microscope analysis the 6. Equipment and Reagents
DNA can be silver stained (28). Only fluorescent staining
6.1 Equipment:
methods will be described in this guide. The microscopic
6.1.1 Water Bath, set at a temperature of 35 °C to 40 °C to
determination of DNA migration can be made either by eye
keep slide coating agarose liquefied during the preparation of
using an ocular micrometer or with the use of image analysis
slides.
software. Scoring by eye can be performed using a calibrated
6.1.2 Centrifuge, capable of exerting a 600X g force and of
ocular micrometer or by categorizing cells into four to five
handling 1.5 mL microcentrifuge tubes. Lower g force will
classes based on the extent of migration (29, 30). Image
require longer centrifugation times and refrigeration to mini-
analysis systems are comprised of a CCD camera attached to a
mize stress to cells.
fluorescent microscope and software and hardware designed
6.1.3 Electrophoresis Chamber and Power Supply, a sub-
specifically to capture and analyze images of fluorescently
marine electrophoresis chamber, and a power supply able to
stained nuclei. Using such a system, it is possible to measure
deliver a constant current up to 300 mA and a voltage gradient
the fluorescence intensity and distribution of DNA in and away
of 0.4 to 1.3 V/cm.
from the nucleus (8). Using different procedural variants, the
6.1.4 Fluorescent Microscope, DNA is visualized by stain-
assay can be utilized to measure specific types of DNA
ing with one of a number of fluorescent DNA stains such as
alterations and DNA repair activity (1, 3, 8, 10, 13, 14, 17, 18).
ethidium bromide (EtBr), propidium iodine, or YOYO®. In
Alkaline lysis and electrophoresis conditions are used for the
this guide, EtBr is used with a fluorescent microscope with a
detection of single-stranded DNA damage, whereas neutral pH
510 to 560 nm excitation filter and 590 nm barrier emission
conditions facilitate the detection of double-strand breaks (31).
filter to view the EtBr-stained DNA.
Various sample treatments can be used to express specific types
of DNA damage, or as in one method, to preserve strand
damage at sites of DNA repair (10). Nuclease digestion steps
The sole source of supply of the apparatus known to the committee at this time
can be used to introduce strand breaks at specific lesion sites.
is Molecular Probes Inc., 4849 Pitchford Ave. Eugene, OR 97402-9165. If you are
Using this approach, oxidative base damage can be detected by
aware of alternative suppliers, please provide this information to ASTM Interna-
the use of endonuclease III (18), as well as DNA modifications
tional Headquarters. Your comments will receive careful consideration at a meeting
resulting from exposure to ultraviolet light (UV) through the of the responsible technical committee, which you may attend.
E2186 − 02a (2023)
6.1.5 Image Analysis System, an image analysis system nated items and waste properly.), dissolve 10 mg ethidium
comprised of a CCD camera attached to the microscope which bromide (EtBr) in 1 mL distilled water, store in light-protected
is connected to a computer loaded with the systems software. container at 4 °C.
6.2.6.1 Ethidium Bromide Working Solution, 10 μL EtBr
6.2 Reagents:
stock solution (6.2.6) in 5 mL distilled water. Store in a
6.2.1 Slide Coating Agarose, low-endo-osmotic agarose,
light-protected container at room temperature.
melting point of ~ 30 °C for slide base coat dissolved in TAE
6.2.7 95 % Ethanol, reagent grade 95 % ethanol, stored in a
buffer, to a final concentration of 0.5 to 1.0 %.
freezer at -20 °C.
6.2.1.1 Stock TAE Buffer (50X), Tris base 242 g, glacial
acetic acid 57.1 mL, 0.5 M EDTA pH 8.0 100 mL, mix in
7. Assay Procedures
distilled water to final volume of 1 L.
7.1 Slide Preparation:
6.2.1.2 TAE Working Buffer (1X), dilute 50X TAE (6.2.1.1)
7.1.1 Slides on which agarose cell suspensions may be
1:49, 10 mL 50X TAE in 490 mL distilled water.
applied are prepared as follows.
6.2.2 Cell Suspension Agarose (CSA), low melting point
agarose (LMA) for cell suspension and slide application,
NOTE 1—Gloves should be worn whenever handling slides and
melting point of 30 °C. LMA dissolved to 0.65 to 1.0 % in
slidecovers, skin oils reduce the adherence of agarose to the microscope
slides.
buffer solution suitable for the cells being studied. Kenny’s salt
solution (6.2.2.1) has been successfully used with marine
Prepare 100 mL of melted slide coating agarose (6.2.1).
invertebrate cells (13, 14, 17) and phosphate buffered saline for
Solid agarose preparations can be liquefied in a microwave
most vertebrate cells (8, 31).
oven with alternating short pulses of microwaves followed by
6.2.2.1 Kenny’s Salt Solution, to 900 mL of distilled water gentle swirling. Using 3 in. by l in. by 1 mm clear glass
add 23.5 g NaCl (0.4 M), 0.7 g KCl (9 mM), 0.1 g K HPO (0.7
microscope slides with a frosted label area, grasp the slide at
2 4
mM), and 0.2 g NaHCO (2 mM). Adjust pH to 7.5 with the label area, dip into the melted agarose, and leave immersed
NaOH, bring to a final volume of 1 L and filter through 0.45 for at least 10 s. Remove from the agarose and wipe excess
μm filter to sterilize. Store at 4 °C. agarose off the back of the slide, and then place on a level
6.2.2.2 Phosphate Buffered Saline Stock, 10X PBS, 1.361 g surface and allow to dry for 2 h at room temperature or for 30
KH PO (0.01 M), 14.2 g Na HPO (0.1 M), 80.1 g NACl, min in a drying oven at 37 °C. Store dry slides in a moisture-
2 4 2 4
(1.37 M), 20.1 g KCl (0.027M), adjust to pH 7.0, bring to a free slide storage box at room temperature. Slides prepared in
final volume of 1 L and filter through 0.45 μm filter to sterilize. this manner may be stored almost indefinitely and require no
Store at 4 °C. further preparation before sample application.
6.2.2.3 PBS Working Solution, dilute 10X PBS (6.2.2.2) 1:9,
7.2 Sample Preparation:
10 mL 10X PBS in 90 mL distilled water. After dilution pH
7.2.1 This assay requires very small quantities of cells.
should be 7.4.
Researchers should be familiar with maintenance media most
6.2.3 Stock Lysis Solution, 146.4 g NaCl (2.5 M), 37.2 g
likely to reduce stress on the cell types used. Cells suspended
EDTA (0.1 M), 1.2 g TrisHCl (0.01 M), combine chemicals in
in 50 μL to 1000 μL maintenance media are placed in a 1.5 mL
500 mL of distilled water; while stirring slowly, add 8 to12 g
centrifuge tube and the cells are pelleted (for example, at 600
NaOH to facilitate the dissolution of EDTA and bring the pH to
× g for 2 min). Supernatant is carefully drawn off and
10. Adjust volume to 1 L, filter through 0.45 μm filter to
discarded. In most cases, a cell pellet ~1 to 2 mm in diameter
sterilize and store at 4 °C.
is more than sufficient to yield cell densities of between 5 to 20
6.2.3.1 Working Lysis Solution, 89 mL Lysis stock solution
cells per microscopic field of view at 200×, when resuspended
(6.2.3), 10 mL DMSO, 1.0 mL Triton X-100, final volume 100
in 50 μL to 400 μL of cell suspension agarose (CSA, 6.2.2).
mL. Make fresh on the day of use. Refrigerate to 4 °C prior to
Experience estimating and adjusting cell densities will reduce
use.
the number of overlapping and superimposed nuclei encoun-
6.2.4 Electrophoresis/Unwinding Solution, to 968 mL dis-
tered during scoring. CSA is melted and placed in a 35 °C to
tilled water add 30 mL (10 N) NaOH and 2 mL (0.5 M) EDTA,
40 °C water bath and the temperature is allowed to stabilize.
pH 8.0, final volume 1 L. Make fresh on the day of use.
The pellet is gently resuspended in the appropriate volume of
6.2.5 Stock 1 M Tris Buffer, pH 7.5, dissolve 121.1 g Tris
CSA at 35 °C to 40°C, as determined by the researcher. Before
Base, in 800 mL distilled water, add 63 mL concentrated HCl,
the agarose solidifies, an aliquot of the cell suspension (for
adjust pH to 7.5 and bring to the final volume of 1L.
example, 50 μL to 75 μL) is transferred to an agarose-coated
6.2.5.1 Working 0.4 M Tris Neutralization Buffer, pH 7.5,
slide (7.1). The applied sample is then uniformly spread over
mix 200 mL (1 M) Tris pH 7.5 (6.2.5) with 300 mL distilled
the slide by placing a clean glass coverslip on top of the still
water for 500 mL final volume. Store at 4 °C.
liquefied sample. The slide is then placed on a level ice-cold
6.2.6 Ethidium Bromide Stock Solution, (Warning—Use
metal or glass surface until the agarose has solidified. Once
extreme caution when handling. Ethidium bromide is a muta-
solidified, a top-coat of 50 μL to 75 μL CSA is added by
gen. Wear appropriate safety protection. Dispose of contami-
slipping the coverslip off the slide, applying agarose and
replacing the coverslip on top. The agarose is allowed to
solidify as before. After the agarose has solidified, the coverslip
is slipped off and the slide placed in a glass slide jar filled with
Information on many of the available systems can be found at
www.geocities.com/cometassay/index.html. ice-cold working Lysis solution (6.2.3.1), so that the solution
E2186 − 02a (2023)
completely immerses the area of the slide on which the sample length light on to the sample, with barrier filters allowing the
was applied. The minimum lysis period is usually one hour, passage of emission wavelength light to be viewed through the
with no apparent maximum. However, gels can become more
microscope should be used. Slides viewed in this way reveal
fragile with extended lysis times.
stained comets as brightly fluorescent orange balls 10 μm to 40
μm in diameter. Scoring may be conducted in several ways: (1)
7.3 Unwinding and Electrophoresis Conditions:
by eye, using a calibrated ocular micrometer (7.5.2) or by
7.3.1 Slides processed through the lysis step, 7.2, should be
categorizing comets into four or five classes based on the
rinsed in distilled water or neutralizing buffer to remove the
extent of migration (29, 30), or (2) using an image analysis
lysis salts and detergents. This can be accomplished by
system (7.5.3), comprised of a CCD camera attached to an
immersing the slides for 2 min and replacing with fresh
epifluorescent microscope and software and hardware designed
distilled water/neutralizing buffer three times. The slides are
specifically to capture and analyze images of fluorescently
placed in the electrophoresis chamber and the chamber filled
stained nuclei. With the use of an image analysis system, it is
with electrophoresis/unwinding solution (6.2.4) to a depth of 3
possible to measure the fluorescence intensity and distribution
to 4 mm above the slides. The slides should be set close
of DNA throughout the comet (32). In this way, the percentage
together; multiple rows may be formed on the chamber
of DNA in the comet tail, the length of the tail, tail moment
platform. Optimum unwinding and electrophoresis times can
(which is the product of the fraction of DNA in the tail and tail
be determined by comparing the extent of migration in
length), as well as numerous other measures can be deter-
untreated control target cells and target cells exposed to a DNA
mined.
damaging agent (for example, gamma-radiation, hydrogen
peroxide, methlymethane sulphonate). In order for historical
7.5.1.1 Regardless of the method used for collecting data on
data to be useful, the negative control cells should exhibit some
DNA migration, the number of comets measured per slide must
level of DNA migration. The slides should be left undisturbed
be determined by the researcher to yield adequate statistical
in the electrophoresis/unwinding solution for from 15 to 60
power. Generally, the recommended number of cells to score
min to unwind or denature the nuclear DNA. The power supply
per sample is from 50 to 100 (39, 40). Fewer cells would
is set to run for from 5 min to 60 min at 300 mA constant
eliminate the ability to identify the presence of subpopulations
current, applying a voltage gradient that may range from 0.4
of cells with altered migration among a larger population of
V/cm to 1.3 V/cm. After electrophoresis, the power is turned
cells with migration patterns not different from the control
off, the slides are removed from the chamber, and the alkali
sample. Because of inherent variability within and across
neutralized by three rinses of 2 min duration in 0.4 M Tris, pH
electrophoresis runs, it is recommended that two slides be
7.5 (6.2.5.1). The DNA in the agarose gel is then fixed by
scored per sample, with 50 % of the data obtained from each
soaking for 5 min in ice cold 95 % ethanol (6.2.7). Fixed slides
replicate slide. Comets are scored in different sectors of the
can then be dried at room temperature or in a 37 °C oven. The
slide. It is important to avoid scoring comets near the edge of
dried slides can be stored in slide boxes until stained and
the slide and not to score slides with high background levels of
scored.
staining. Once the field of view is randomly moved to a sector,
7.4 Staining:
a systematic method of scoring is used, such as scoring comets
7.4.1 There are many commercially available fluorescent
from left to right starting
...