Inoculation Methods

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Inoculum, Genes, Wills

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Microbiological challenge testing of

foods
E. Komitopoulou, in Food and Beverage Stability and Shelf Life, 2011

16.2.4 Choosing the right method of inoculation

The choice of the inoculation method is another crucial parameter in the design of
challenge trials. A challenge test can be considered successful if inoculation does not
affect any of the product’s intrinsic or extrinsic properties. The methodology em-
ployed also needs to be reproducible and properly validated, one that also includes
pre- and post-inoculation analysis of the critical characteristics of the product’s
formulation (e.g., moisture content, water activity and pH), to ensure that these have
remained unaffected by the inoculation process.

The choice and volume of the liquid inoculum carrier used in the inoculation
methodology are crucial parameters affecting the success of the trials. Independent
of the type of carrier used, the aim should always be to use the minimum possible
volume of carrier to also ensure the least possible change in the product character-
istics. In liquid products the inoculum can be suspended in a sample of the product
matrix itself, creating a stock which is then used to inoculate different samples of
the same product. In cases where maintaining the moisture level is important, the
inoculum carrier can be the same diluent used to adjust the moisture content of the
product formulation in the first case.
The successful inoculation methodology needs to ensure even distribution of the
inoculum within the product matrix to minimize errors in the subsequent sampling
and enumeration of the challenge organism, whilst at the same time maximizing
microbial exposure to the product’s environment. This can prove particularly prob-
lematic when inoculation is carried out using a syringe through the packaging wall
containing a rubber septum. When adequate mixing cannot be achieved, samples
are first inoculated and then re-packed making sure that packaging after inoculation
matches the normal packaging conditions of the product.

Mixing of inoculum within the product matrix can easily be done in products with
water activities higher than aw = 0.96 (e.g., sauces) using the minimum volume of
the inoculum carrier possible. Spraying has also been suggested and employed for
inoculation of product surfaces, although care should be taken to ensure that this is
carried out using the appropriate protective equipment to make sure that exposure
to pathogenic aerosols is minimized.

> Read full chapter

DNA Vaccines for Biodefense and

Emerging and Neglected Infectious
Diseases
Shan Lu, ... Shixia Wang, in Vaccines for Biodefense and Emerging and Neglected
Diseases, 2009

Prime and Boost Approach

Since the most successful DNA vaccine inoculation method (PMED) for eliciting
immune responses in nonhuman primates and in humans is not commercially
available for clinical use, it has become important to utilize other techniques and
methods to increase the immunogenicity of DNA vaccines that are currently in
development. One such method is the prime-boost approach. The rationale under-
lying this strategy is that gene-based vaccines, presented by a recombinant viral
vector or as DNA plasmids, elicit immune responses by producing antigens in vivo;
however, these immune responses have not been strong, particularly in humans. In
order to increase the levels of immune responses, in particular the cell-mediated
immune responses, strategies have been developed to prime the immune system
with the DNA vaccine and boost with a recombinant viral vector (Pancholi et al.,
2000; Ramshaw and Ramsay, 2000; McShane et al., 2001; Schneider et al., 2001;
Gonzalo et al., 2002; Mellquist-Riemenschneider et al., 2003; Woodland, 2004;
Seaman et al., 2005; Wu et al., 2005; Perkins et al., 2006), or an attenuated virus (Yuan
et al., 2005). This DNA–viral vector combination has proven effective in eliciting
high-level cell-mediated immune responses in nonhuman primate studies (Letvin
et al., 2004; Boyer et al., 2005) and even in humans (Mwau et al., 2004; Vuola et al.,
2005; Goonetilleke et al., 2006; Hanke et al., 2007).

Alternatively, recombinant protein can also be used to boost the immune system
that has been primed with DNA vaccines (Barnett et al., 1997; Letvin et al., 1997;
Richmond et al., 1998; Pal et al., 2005, 2006; Wang et al., 2005b, 2006b; Lu, 2006; Law
et al., 2007). The DNA prime-protein boost combination has been proven effective
in eliciting balanced humoral and cell-mediated immune responses even in human
studies (Wang et al., 2008a). The DNA prime or protein boost components when
used alone have been shown to be less effective in inducing immune responses
when compared to prime plus boost approach (Wang et al., 2005b, 2006b).

> Read full chapter

Techniques for Oral Microbiology

In Atlas of Oral Microbiology, 2015

2.2.3.2 Sample Inoculation

Spread method, drop method, and spiral inoculation method are used for oral
clinical bacteriology samples. Appropriate dilutions of the specimen solution are
quantitatively inoculated onto the agar plate.

1. Spread method: Ten microliters from appropriately diluted samples are taken
with a micropipettor and placed on the surface of the agar medium. The
sample is then evenly coated on the agar surface using a sterile glass spreader
(triangle rod and L-shaped rod). At the appropriate dilution, each bacterial cell
from the specimen should form a single colony after incubation (Figure 2.15(A)
and (B)).Figure 2.15. (A) Spread method protocol. (B) Colonies on a plate
using spread method (periodontal bag samples). (C) Drop method protocol.
(D) Colonies on a plate using drop method (saliva samples). (E) Colonies on a
plate using spiral plater (plaque samples).
2. Drop method: Twenty-five or 50 μl samples at the appropriate dilution are
dropped onto the surface of the agar using a micropipettor. Then, the plates
are placed directly into the dry incubator (Figure 2.15(C) and (D)).
3. Spiral plater method: The spiral plater is the most advanced method for
inoculating bacteria for the purpose of counting colony-forming units. The
sample liquid is automatically diluted and inoculated using a needle tip on
the surface of the agar plate by the instrument, and the bacteria colonies
grow uniformly along the spiral trajectory after incubation (Figure 2.15(E)). As
a result, sample counting and bacterial colony observation are more accurate
and reproducible. Details are included in Section 2.4 of this chapter.

> Read full chapter

Handbook of Modern Pharmaceutical

Analysis
Gregory A. Birrer, ... Judy Estrada, in Separation Science and Technology, 2001

A. Sterility Test Methods

The USP describes two general methods for conducting the test: the direct transfer,
or direct inoculation, method and the membrane filtration method. As the name
indicates, the direct inoculation method involves the aseptic transfer of a sample
of test product solution into the sterility test growth medium. To use this method,
it must first be demonstrated that the product solution itself does not inhibit the
growth of typical “indicator” microorganisms specified in the USP method. It should
be self-evident why it is important to perform testing to negate the chance of
product inhibition of possible microbial contaminants, as this is the purpose of
the sterility test. The direct inoculation method, while not theoretically complex,
requires the utmost technical precision and aseptic manipulation techniques for
proper execution. As a consequence of the repetitive motions involved, it is prone to
human error.

The membrane filtration method is specified by the USP to be used whenever

“the nature of the product permits.”3 The direct transfer method should be used
only when it is not possible to properly perform the membrane filtration method.
Therefore, membrane filtration is the most widely used method in the industry. It
involves a pressure or vacuum filtration of the test product solution through a sterile
filtration apparatus fitted with a filter membrane (thus capturing any microbial
contaminants on the membrane) then either a wash step with a sterile diluent and/or
subsequent plating of the membrane filter onto the surface of an agar plate. When
the membrane filtration method is used, it is imperative that a sterile diluting fluid
be used to (1) rinse away any microbial cells residing on the filtration apparatus
and ensure their impingement onto the filter surface and (2) dilute or otherwise
wash away any product residue that might inhibit subsequent microbial cell growth.
There are commercially available presterilized systems that are widely used. These
save time in preparation and setup, serve to expedite the testing, and help reduce
the chance of laboratory contamination.
> Read full chapter

DNA Vaccine: Does it Target the Double

Stranded-DNA Sensing Pathway?*
Cevayir Coban, ... Ken J. Ishii, in Biological DNA Sensor, 2014

Ideal vaccines are expected to give lifetime protection from infectious diseases, and if
possible, from allergic diseases, autoimmune diseases and cancer. DNA vaccination
was introduced two decades ago as a simple plasmid inoculation method with a
capability of inducing both cellular and humoral immune responses. Recent studies
have provided insights into the molecular mechanisms by which the double-strand-
ed structure of DNA vaccine induces the activation of type-I interferon (IFN)-me-
diated innate immune responses via STING/TBK1 complex, similar to cytosolic
double stranded DNA (dsDNA) recognition of immune cells. In this chapter, DNA
vaccines and the current knowledge on their mechanism of action will be introduced.
The possibilities of using this knowledge for improving immunogenicity of DNA
vaccines in humans will then be discussed.

> Read full chapter

Disease Resistance in Sorghum

I.K. Das, P. Rajendrakumar, in Biotic Stress Resistance in Millets, 2016

2.3.1.9 Viral diseases

A repeatable and efficient screening technique not only helps to identify stable
resistant sources but also is essential to identify susceptible stages of the host,
maintenance of virus inoculum, mass multiplication of insect vectors, and devel-
oping disease assessment scales. Field screening as well as greenhouse screening
techniques have been reported for sorghum viruses. Group seedling inoculation
method for greenhouse screening for resistant to MStV is a simple and efficient
technique that can develop 98% virus infection. The young nymphs of the insect
vector (Peregrines maidis) are allowed to feed on virus-infected sorghum leaves for 4
days and allowed to incubate for a week to get viruliferous. The viruliferous adults are
used for inoculation of sorghum plants both in greenhouse and in field conditions.

In a field screening technique, a sorghum genotype highly susceptible to MStV is

planted in the field as an infector row after every five blank rows. Each seedling at 3–4
leaf stages are infested with two viruliferous vectors per plant in the leaf-whorl. The
test materials are planted in the following day in five consecutive rows in-between
two infector rows. The viruliferous insects inoculate the virus to plants in infector
rows and also multiply and the subsequent generations of insects from infected
plants move to young seedlings to feed and at the same time inoculate the virus
to fresh young seedlings. The observations on number of plants showing disease
symptoms over total plants are expressed as percent disease.

> Read full chapter

Sunflower
Yalcin Kaya, in Breeding Oilseed Crops for Sustainable Production, 2016

Stem Canker (Phomopsis/Diaporthe helianthi)

Phomopsis is a severe disease in almost all sunflower-growing countries of Europe
as well as in the United States and Canada. The first sunflower-resistant genotypes
were determined by Škorić in the 1980s derived from interspecific hybrids (cultivated
sunflower × H. tuberosus), a cross between H. argophyllus × Armavirski 9345 and
the restorer line SNRF-69 after a long breeding process in Serbia (Škorić, 2012).
Sunflower resistance to Phomopsis is horizontal and is positively correlated with the
stay-green trait in stems at ripening, with Macrophomina and Phoma resistance,
and with drought tolerance (Masirevic, 2000).

As inoculation methods to evaluate breeding materials, leaf and petiole infec-

tion were found to be successful methods. In vitro screening of sunflower calli
to determine the reaction to a filtrate of Phomopsis is possible too (Quaglia and
Zazzerini, 2007). It is advised to eliminate susceptible plants from early breeding
generations before observations are made about F3 or F4 families, because of the
difficulty of distinguishing intermediate and resistant genotypes at the mycelium.
A rich germplasm has been developed for Phomopsis resistance, especially from wild
Helianthus species, to increase genetic variability and to develop resistant hybrids.
Based on recent results, leaf resistance was found to be controlled by one major gene
and at least two complex factors were found to influence resistance in petiole and
stem tissues (Masirevic, 2000). The recent use of molecular markers in breeding has
led to recombinant inbred lines derived by crossing LR4-17 (resistant) with HA89
(susceptible) major QTLs for different leaf and stem resistance components being
determined and pyramided (Langar et al., 2004, Liu and Jan, 2012; Qi et al., 2012;
Škorić, 2012).
> Read full chapter

The Sterility Test

Tim Sandle, in Sterility, Sterilisation and Sterility Assurance for Pharmaceuticals,
2013

18.3 Pharmacopeia Sterility Test

There are two principle methods of Sterility Testing, as defined in the pharma-
copoeiae [3], membrane filtration and direct inoculation. Of these methods, mem-
brane filtration is the method of choice, because a larger size can be tested. The
pharmacopoeia require that the entire contents of a small volume product are
filtered and at least half the contents of a large volume product pass through a
membrane filter. With the direct inoculation method, only a few millilitres of a
liquid product are transferred into the test media. Furthermore, any microorganisms
present are far more likely to be separated from potentially inhibitory substances in
the product through the act of filtration or can be eliminated by rinsing the filter. It is
also common for membrane filtration systems to be enclosed, such as the Steritest™
system (introduced in 1975), which minimises risk of contamination by reducing
transfer steps [4].

Membrane filtration is the appropriate method for all aqueous, alcoholic, oily and
solvent products that can pass through a sterile filter with a porosity of 0.45 μm
(Figure 18.1). The standard filter is manufactured from cellulose esters or other
similar plastics. The filter acts to separate the product from any microorganisms,
so that the product passes through the filter and any microorganisms present in
the product are trapped within the filter matrix. A rinse solution (i.e. phosphate
buffered saline, saline or Ringer’s solution) is used to remove any product residues.
This washing process is normally performed three or four times and the filter should
remain wet throughout.
Figure 18.1. Inspecting a membrane filtration test chamber

(image courtesy of Tim Sandle)

The filter is divided into two portions, or more than one filter is used, as in the
widely used Steritest™ polycarbonate filtration system. To each filter, culture media
is added, so that any microorganisms trapped in the filter membrane, following
incubation at a suitable temperature, will replicate. Two culture media are used.
The pharmacopoeias recommend fluid thioglycollate medium (FTM), incubated at
30–35°C to isolate bacteria (aerobic and anaerobic) and soya bean casein digest
medium (tryptone soya broth, TSB), incubated at 20–25°C to isolate aerobic bacteria
and fungi. FTM has resazurin, an oxidation-reduction indicator, added to create a
chemical layer (indicated by a pink colouration) to allow the growth of both aerobic
and anaerobic microorganisms.

However, many products will not readily filter (i.e. protein-based products that
will block the filter pores), as they are so inherently anti-microbial that the direct
inoculation method is used. Direct inoculation may also be preferred over mem-
brane filtration if the membrane filtration method simply cannot be validated. When
the direct inoculation method is selected, the laboratory should be able to justify
why it has selected this method over the membrane filtration technique. The direct
inoculation technique involves the addition of a portion of the product to two
different culture media, FTM and TSB, as per the membrane filtration technique.
This is half the contents of the product vial to each culture medium – for product
between 50 mg and 300 mg – or the entire contents for product of less than 50 mg.
For large volumes of product, a concentrate of the culture medium is sometimes
added to the product.
For the direct inoculation technique, products which have antimicrobial activity must
be neutralised before a portion of the product is added to the culture medium. This
is performed either by the addition of a neutraliser or by dilution of the product.

Each Sterility Testing session should have a negative control consisting of the test
media and test consumables. Such a control is designed to indicate if the culture
media, or some aspect of the test environment, could result or increase the risk of a
false positive developing in the Sterility Test. All test consumables should be recorded
for each test.

The incubation time for both test methods is 14 days. The previous incubation, which
stood for 50 years, was 7 days. This was increased to 14 days in 1997. This was
because it was estimated that 30% of Sterility Test failures occurred between 7 and 14
days, due to the time taken for sub-lethally damaged or stressed microorganisms to
grow [5]. Microorganisms isolated from a Sterility Test are more likely to be stressed
due to the transfer from their environment into a more hostile environment (the
product) and then into a completely different nutrient-rich environment (the culture
media). These microorganisms are also likely to be low in number, as little as one
cell. These factors contribute to a relatively long lag phase at the start of the microbial
cell growth cycle in the culture medium (Figure 18.2) [6].

Figure 18.2. A technician preparing direct inoculation test bottles

(image courtesy of Tim Sandle)

For products which produce suspension, flocculation, turbidity or deposits, so that

the presence or absence of microbial growth cannot be readily seen, a subculture
step is required. To subculture a suitable portion of the culture, media is transferred
to a container of the same media type and incubated for a further time period (as
discussed below).

The items incubated must be clearly labelled with the identity of the product, the
medium used, the temperature of incubation and the date of testing. Throughout
the incubation of the Sterility Test, the articles must be examined regularly for
growth, which is often every day or every other day. When inspecting the items being
tested, care must be taken to prevent undue agitation, especially of the thioglycollate
medium. If anaerobic conditions are not maintained, this will be indicated by
the resazurin indicator. At the end of the incubation period, the articles must be
inspected, by gentle swirling, for visible turbidity against an artificial light source.

If turbidity is seen, an investigation must be performed (this is examined in Chapter

19). For the test to be valid, certain conditions must be met:

The culture media used is sterile, often shown by incubating articles of culture
media alongside the Sterility Test.
The culture media can support microbial growth, from growth promotion
testing.
The product does not have a microstatic or microbicidal effect, or can be
eliminated – as indicated by Sterility Test validation.
Contamination is not introduced into the test by external sources.

> Read full chapter

Plant Cell and Hairy Root Cultures –

Process Characteristics, Products, and
Applications
Wei Wen Su, Kung-Ta Lee, in Bioprocessing for Value-Added Products from Renew-
able Resources, 2007

3.2.2 Upstream processing

The physical structure of the roots poses challenges to inoculation and homo-
geneous root distribution in a liquid culture. As a result, reduced productivities
have often been noted upon culture scale-up [102, 103]. Some attempts had been
made to solve the inoculation problem. Ramakrishnan et al. [104] reported an
inoculation method that consisted of briefly homogenizing the bulk root cultures
of Hyoscyamus muticus, Beta vulgarus, and Solanum tuberosum, then aseptically
transferring the slurry to the reactor. The effects of specific excision on root cultures
of related species were examined by Falk and Doran [105] and Woo et al. [106].
The effects of the cut treatment on root growth, morphology, and alkaloid content
were further investigated in flask cultures. The data showed that hairy roots of A.
belladonna with a suitable length (longer than 1 cm) retained the ability to grow and
produce tropane alkaloids after a cut treatment [107].

After inducing hairy roots and selecting high-producing cell lines, it is necessary
to optimize medium components and culture conditions before the culture can be
successfully scaled up. Hairy roots can be cultivated without the addition of exoge-
nous hormones, because the t-DNA from A. rhizogenes codes for auxin synthesis
[108]. However, growth regulators may still affect hairy root growth, organogenesis,
and the formation of both primary and secondary metabolites. The accumulation
of hyoscyamine and scopolamine could be significantly enhanced in hairy root
cultures of Hyoscyamus muticus by adding the auxins IAA or NAA [109].

Elicitors are generally defined as molecules that can stimulate the defense responses
of plants, including the formation of phytoalexins. The effects of elicitor on plant
secondary metabolite production by hairy roots and suspension cells are summa-
rized in Table 5. Biotic elicitors, such as the cell wall components of filamentous
fungi, yeast, and microalgae, have been shown to stimulate the production of
antimicrobial compounds in plants. Abiotic elicitors, such as jasmonate (JA) and its
methyl ester (MeJA), and salicylic acid, are generally considered to be secondary
signals, thus modulating many physiological events in higher plants, including
defense responses, flowering, and senescence. They are regarded as a new class of
phytohormone. Some secondary metabolites may also be stimulated by heavy metals
and synthetic substances [114]. It has been reported that exogenously applying MeJA
induced the biosynthesis of terpenoids [122]. MeJA was also reported to stimulate
saponin production in cultured ginseng cells [123] and Bupleurum falcatum root
fragments [124], but the detailed mechanisms responsible for these stimulatory
effects remain unevaluated. The elicitation of plant cells and tissues can lead to
increased yields, and hence the use of biotic and abiotic elicitors has been considered
a viable strategy for improving the yield of plant secondary products. The application
of elicitors to plant cell cultures is not only useful for enhancing the biotechnological
productivity of valuable secondary metabolites in fermentation systems but also for
the study of plant-microbe interactions. An added biotechnological benefit of their
use is that they may also promote the liberation of metabolites into the medium
[125].

Table 5. Stimulation of plant secondary metabolite production by elicitation

Plant Elicitor Function Reference

Hairy root cultures
Ammi majus Higher accumulation [110]
of coumarins
 Benzo(1,2,3)-thiadia-
zole-7-carbothionic
acid S-methyl ester

Artemisia annua (22S, 23S)-homo- Enhancement of [111]

brassinolide; fungal artemisinin production
elicitor
Beta vulgaris Micro algal Enhancement of be- [112]
talines production
Catharanthus roseus CdCl2 Increase in indole alka- [113]
loid production
Cichorium intybus Fungal elicitor Production of volatile [114]
compounds
Ocimum basilicum Fungal cell wall Enhancement of ros- [115]
marinic acid produc-
tion
Panax ginseng Methyl jasmonate Improving ginsenoside [116]
yield
Salvia miltiorrhiza Yeast; Ag+ Enhancement of tan- [117]
shinones production
Solanum tuberosum Fungal elicitor Production of phy- [118]
toalexins
Tagetes patula Micro algal elicitor Enhancement of thio- [112]
phenes production
Suspension cultures

Glycyrrhiza glabra Methyl jasmonate Stimulation of soyas- [119]

aponin biosynthesis
Taxus chinensis 2-hydroxyethyl Increase in taxuyunna- [120]
jasmonate/trifluo- nine C production
roethyl jasmonate
Taxus canadensis Methyl jasmonate Increase in taxoid pro- [121]
duction

> Read full chapter

Control of Plant Virus Diseases

Marina Barba, ... Graziella Pasquini, in Advances in Virus Research, 2015

4.6 Selection of tolerant and/or resistant crop cultivars

Apart from the control of the virus vector and the use of virus-free material, the
development of virus-resistant varieties appears to be the most effective approach
to achieve control of plant viruses, especially for perennial crops that can become
infected during their long life span. The use of resistant or tolerant cultivars and/or
rootstocks could be the most important aspect of virus disease management, es-
pecially in areas in which virus infections are endemic. The conventional breeding
for virus-tolerant or -resistant fruit tree cultivars using available germplasm is a
long-term strategy, and the development and production of these cultivars may take
decades, if successful. In particular, the selection is slow and difficult due to the
transfer of undesirable characteristics and other constraints typical of fruit trees such
as long biological cycle with extended juvenile phase and high level of heterozygosity
(Decroocq, Badenes, & Neumüller, 2011; Garcia et al., 2013).

Many studies have been performed on fruit tree cultivars for evaluation of their
susceptibility to different viruses, based on field observations under natural infection
pressure or involving models obtained by grafting or chip budding or by viruliferous
vector transmission in the field or under greenhouse conditions. Obtained data in
most cases were not comparable as they were influenced by virus isolates, virus
inoculation methods, classification of plant response to viruses (Cooper & Jones,
1983), and types of diagnostic tests with different sensitivity used for evaluation of
germplasm infection.

In these studies, only limited information is available on tolerance or resistance

sources for the majority of these pathogens. With the exception of PPV in stone
fruits (Decroocq et al., 2011) and possibly Cherry leaf roll virus in walnut, there are
currently no conventional breeding efforts for tolerance or resistance to viruses of
fruit trees.

Many efforts have been made for the evaluation of tolerance and resistance
germplasm for PPV, the most detrimental for stone fruits cultivation, but with very
few natural sources of resistance identified in Prunus species. Resistant apricot
germplasm, originated from North America, has been used in breeding programs
and used in crosses with local cultivars in several European countries (Audergon,
1997; Badenes & Llácer, 2006; Bassi, 2006; Karayiannis et al., 2006). PPV resis-
tance seems to be controlled in apricot by at least two genes (Karayiannis, Tho-
midis, & Tsaftaris, 2008; Martínez-Gómez & Dicenta, 2000; Moustafa, Badenes,
Martínez-Calvo, & Llácer, 2001).

No peach germplasm was resistant to PPV, but resistance has been identified in
wild species Prunus davidiana. This genotype was included in breeding programs for
introducing PPV resistance in peach cultivars (Decroocq et al., 2005). Quantitative
trait locus analysis performed on F1 progeny allowed the identification of several
genomic regions involved in the resistance (Decroocq et al., 2005; Marandel, Salava,
Abbott, Candresse, & Decroocq, 2009; Pilařová et al., 2010).

Also, interspecific peach rootstocks and almond cultivars seem to be a source of

resistance to PPV (Pascal, Pfeiffer, & Kervella, 2002; Rubio, Martínez-Gómez, &
Dicenta, 2003).

The hexaploid genome of plum has made it difficult to study PPV resistance in this
species. No data on molecular markers are available for any trait in plum (Decroocq et
al., 2011). The hypersensitive response (Kegler, Grüntzig, Fuchs, Rankovic, & Ehrig,
2001), characterized by a localized cell death, is an effective resistance mechanism to
 PPV under natural and artificial inoculation (Garcia et al., 2013) and has been used
in plum breeding programs (Hartmann, 1998).

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