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From Mainberg@aol.com Fri Oct 11 13:36:50 1996
To: warren@gas.sci.monash.edu.au
cc: ahicks@nmt.edu, MElliott@pri.pulmonary.ubc.ca, asj@iastate.edu
Subject: Re: Seed sterilization paper(Part I)
A REVIEW OF THE DISINFECTION OF ORCHID SEED
by Fred J. Bergman
During the routine disinfection of Phalaenopsis seed in
1991, it was observed that 56 % more crosses germinated
when seed was disinfected using a solution prepared from
old calcium hypochlorite powder. Analysis of the calcium
hypochlorite powder and a series of experimental sowings
confirmed that the improved germination resulted from a
decreased hypochlorite concentration. The old calcium
hypochlorite powder had lost 75% of its strength so that a
standard 7.1% solution (10g/140ml) was now equivalent to a
1.8% solution (F. J. Bergman, unpublished).
In an effort to understand the relationship between
lower concentrations and an increase in the number of
crosses that germinate, a decision was made to review the
early literature. When Knudson sowed the first orchid seed
under aseptic conditions on December 7, 1918 (Knudson,
1922), he disinfected the seed using calcium hypochlorite
(Wilson, 1915). Wilson had studied the use of calcium
hypochlorite as a seed disinfectant while a graduate
student of Knudson's at Cornell (Knudson, 1948). Although
the procedure most cited for orchid seed disinfection is
Wilson's (1915), a fresh review of his paper uncovered
information frequently overlooked. This information
includes the following: (1) orchid seed was not one of the
22 varieties of seed Wilson investigated; (2) the
procedure stated that "dilutions from the known strength
may be used as well as full strength"; (3) the exposure
time effect study used a 1:1 dilution of the stock
solution, equivalent to 5g/140ml; (4) the names chloride
of lime, bleaching powder and calcium hypochlorite were
all used to describe a single reagent that contained 28%
available chlorine; and (5) the stock solution was
described both in terms of wt % available chlorine and wt
% reagent.
Noting that current commercial calcium hypochlorite
contains 65-75 wt % available chlorine in contrast to the
hypochlorite containing 28 wt % available chlorine used by
Wilson, it appeared worthwhile to review seed disinfectant
procedures recommended over the last several decades in
the literature. This paper provides overview and comments
on the literature describing disinfectants used with
orchid and other seed. A search of English publications
[Literature search extended from Wilson's paper (1915) up
to and including papers published in 1933]. located one
hundred and seventy-three papers describing orchid seed
disinfection. A review of 147 orchid books provided an
additional 14. Knudson's publication (1922), was followed
by 93 descriptions recommending calcium hypochlorite
concentrations from 2 to 12 wt %. Forty-eight papers
recommended 10g/140ml (7.1 wt %), while 37 papers used
minor modifications using essentially the same 7.1 wt %
concentration. Five papers used the 10g/100ml (10 wt %)
first recommended by Thompson (1977). Only Ryerson (1952)
and Redlinger (1961) used concentrations significantly
lower. Ryerson used 2.4 wt % for "clean seed" while
Redlinger used 2 wt %. One paper (Hamilton, 1988)
recommended "7g/l of water"; believed to be an error since
the author cited Arditti (1982) who recommends a saturated
calcium hypochlorite solution of 7g/100 ml.
A review of literature on general uses of chlorine
disinfectants provided information helpful to this study.
Wilson (1915) described his chloride of lime as "titrating
28% chlorine", a term derived from the method of analyzing
activity. It is the concentration of the hypochlorite ion
referred to as available chlorine, that determines the
germicidal strength of a solution (Rubbo and Gardner,
1965; Sykes, 1965; Segall, 1968; Dychdala, 1983).
Available chlorine is defined as the measure of oxidizing
capacity, not the chlorine content, and is equivalent to
two chlorine atoms for each hypochlorite ion (Snell and
Hilton, 1971; Dychdala, 1983). Chlorine "available" in
calcium hypochlorite and bleaching powder is traditionally
determined by titration using either arsenious oxide
(AOAC, 1990) or iodometrially using standard sodium
thiosulfate (Snell and Hilton, 1971). The two methods are
equivalent if acetic acid is used to acidify the sample
when using the iodometric procedure (Mellor, 1956).
Current literature refers to the product containing
28-35 wt % available chlorine as bleaching powder and/or
chloride of lime and the product containing 70-75 wt %
available chlorine as calcium hypochlorite (Mellor, 1956;
Snell and Hilton, 1971; Wink and Henkels, 1978; Dychdala,
1983). Products containing concentrations greater than 35
wt % available chlorine first became available in the U.
S. in the early thirties. According to Mellor (1956), it
is now known that the product called chloride of lime or
bleaching powder consists of a mixture of partially
hydrated Ca(OCl)2:Ca(OH)2 and Ca(OCl)2:2Ca(OH)2 while
calcium hypochlorite consists of Ca(OCl)2:3H2O. Because of
the larger amount of available chlorine in calcium
hypochlorite compared to chloride of lime or bleaching
powder (from 28 wt % to the present 70-75 wt %), the
standard practice of defining concentration using wt %
becomes unacceptably inaccurate.
The error created by using wt % to indicate
disinfectant concentration is compounded by the poor
stability of powdered calcium hypochlorite. Several
authors describe powdered calcium hypochlorite as
unstable. Mellor (1956) states "it is well known that
bleaching powder deteriorates in storage. Impurities and
storage conditions are significant factors, but because
decomposition rates are so variable decomposition rates
are seldom published". Mellor (1956) does provide one
example of a loss rate of 1.44 wt % per month in the
summer and 0.61 wt % per month in the winter, an average
of 12.3 wt % per year. Knudson (1948) acknowledged that
calcium hypochlorite was unstable, recommending storage in
a tightly closed bottle under refrigeration and
replacement at regular intervals. Liddell (1946) reported
finding a "fairly fresh sample" that contained a
negligible amount of chlorine. The implications are that
the freshness of powdered calcium hypochlorite can affect
a solution's final strength. However, only one article in
the orchid literature (Knudson, 1948) recognized the
stability of powdered calcium hypochlorite as a potential
problem.
CALCIUM HYPOCHLORITE/CONCENTRATION-Because authors most
often use wt % to define concentration, the actual
strength (hypochlorite concentration) used for orchid seed
was available from only four reports. In addition to the
1-2 wt % available chlorine used by Wilson (1915), Hegarty
(1955) reported using 7.5 wt % of chloride of lime (24 wt
% available chlorine) for a solution strength of 1.8 wt %
available chlorine. Castle and Nickell (1942) and Fennell
(1956) used 3.6 wt % of calcium hypochlorite (70 wt %
available chlorine) for a solution strength of 2.5 wt %
available chlorine. Sweet and Bolton (1979) obtained
optimum disinfection using a concentration of 0.5 wt %
available chlorine, but with non-orchid seed.
An attempt was made to determine the actual
concentrations used on orchid seed when disinfectant
strength was only reported in wt %. Data was arranged
using the strength of calcium hypochlorite powder
available at the time. The first group had used calcium
hypochlorite (bleaching powder) that contained 28-30 wt %
available chlorine. This group extended from the first
publication by Knudson (1922) to the early thirties. The
second group extended from the thirties to the fifties,
using calcium hypochlorite that contained anywhere from 28
to 70 wt % available chlorine. The third group extends
from the fifties to the present and uses calcium
hypochlorite that contains about 70 wt % available
chlorine. Knudson apparently used bleaching powder
containing 28 wt % available chlorine in his studies
(Knudson, 1948). Knudson reported germinating Cattleya and
Epidendrium seed (1922) and Cymbidium, Phalaenopsis,
Dendrobium, Ophrys, and an Odontoglossum cross (1924).
Northen (1953) reported that Phalaenopsis seed frequently
failed to germinate when disinfected using calcium
hypochlorite at 10g/140 ml. Northen was reporting at a
time when calcium hypochlorite contained 28 to 75 wt %
available chlorine. Kano (1971) reported that there was
"no doubt" that calcium hypochlorite often kills seed of a
number of genera, including Angraecum, Ascocenda,
Phalaenopsis and Vanda. Kano's observations covered a time
period when calcium hypochlorite contained 70-75 wt %
available chlorine. Ryerson (1952) was successful in
germinating Phalaenopsis seed using calcium hypochlorite
at concentrations equivalent to 1.4 wt %, while Garrard
(1966) successfully used a 2.5 wt % solution. Both Ryerson
and Garrard used calcium hypochlorite that could have
contained anywhere from 28 to 75 wt % available chlorine.
The evaluation of concentration effects was simplified
by converting wt % to wt % available chlorine [Wt % is
converted to wt % available chlorine by multiplying the
weight of hypochlorite by the % available chlorine, then
dividing by the liquid volume and multiplying by 100. For
example, Knudson's solution (1922) equals 10 g x 0.28
divided by 140ml x 100 = 2 wt % available chlorine]. The
conversion shows that Knudson (1922-1948) used 2 wt %
available chlorine, and Garrard (1966) used 0.7 or 1.9 wt
% available chlorine, Ryerson (1952) used 0.4 or 1.0 wt %
available chlorine. Knudson, Ryerson, and Garrard, all
using 2 wt % available chlorine or less, reported success
in germinating Phalaenopsis seed. Northen (1953),
describing disinfection using calcium hypochlorite at 2 to
5 wt % available chlorine reported that Phalaenopsis seed
generally failed to germinate. Kano (1971), reporting on
work that used calcium hypochlorite at 5 wt % available
chlorine observed that Phalaenopsis seed always failed to
germinate. This indicates that using available chlorine
concentrations of 2 wt % or less increases the number of
crosses that can be successfully germinated.
The hypochlorite ion [OCl-] is the active agent in the
disinfection process (Sweet and Bolton, 1979; Rubbo and
Gardner, 1965; Sykes, 1965; Segal, 1968; and Dychdala,
1983). This fact suggests that calcium hypochlorite should
be effective at the same concentration of available
chlorine as that established for sodium hypochlorite.
After sodium hypochlorite was suggested as a substitute
for calcium hypochlorite, Liddell (1947), Northen and
Northen (1947, 1948) and Knudson (1948) conducted
comparison studies. Both Northen and Northen and Knudson,
using Cattleya seed, compared calcium hypochlorite at a
concentration of 10g/140ml to various concentrations of
sodium hypochlorite. For comparison purposes, the sodium
hypochlorite concentrations expressed as dilution ratios
were converted to wt % available chlorine. Northen and
Northen (1947, 1948) reported that sodium hypochlorite was
effective at concentrations equivalent to 0.01-0.5 wt %
available chlorine while Knudson (1948) reported that
sodium hypochlorite was effective at concentrations
equivalent to 0.125-0.5 wt % available chlorine. Liddell
(1948) found sodium hypochlorite effective at
concentrations equivalent as low as 0.093 wt % available
chlorine. This indicates that calcium hypochlorite should
also be effective at a concentration of 0.1 to 0.5 wt %
available chlorine, significantly less than the 2-5 wt %
available chlorine conventionally used for seed
disinfection.
SODIUM HYPOCHLORITE/CONCENTRATION-Household bleaches are
aqueous solutions usually containing around 5 wt % sodium
hypochlorite and sold under various trade names that
include Clorox, Purex and Domestos. A 5.25 wt % solution
of Clorox contains 5 wt % available chlorine. Liddell's
paper (1946) was the first of 50 papers to recommend
Clorox as an orchid seed disinfectant. Liddell recommended
using dilution ratios of 1:10-1:32 (1946), stating that he
preferred 1:20. Liddell changed his dilution ratio to 1:54
(1947), and reported success germinating Cattleya,
Cymbidium, Vanda, and some Dendrobium. Liddell (1948)
reported dilution ratios as low as 1:120 were effective
and that Clorox was safe for Cattleya and probably other
genera but toxic to Phalaenopsis at 1:10. For Phalaenopsis
he recommended using 1:54 having found that concentration
adequate as a disinfectant. Northen and Northen (1948)
reported success using Clorox at a low dilution of 1:500.
Dilutions of 1:10-1:20 are frequently reported (Harvais
and Hadley,1967; Syoichi, 1989; Yam and Weatherhead, 1988).
Confusing directions in the literature frequently make
it necessary to assume which concentrations were actually
employed. The use of available chlorine would eliminate
this confusion. Liddell (1946) reported that a 1:32
dilution contained 6000 ppm available chlorine (0.6 wt %).
Liddell was the only author working with orchid seed to
provide a sodium hypochlorite concentration in terms of
available chlorine.
CONTACT TIME/CALCIUM HYPOCHLORITE-Contact time has been
examined as a potential variable in the germination of
orchid seed. Knudson (1948) treated Cattleya seed at 5,
20, and 30 min and found a 5 minute treatment adequate,
although stating he normally used 15 min. Northen and
Northen (1947) reported that 66 % of viable Cattleya seed
still germinated after contact with calcium hypochlorite
for 48 hr. Sweet and Bolton (1979), germinating non-orchid
seed, reported no improvement with contact times greater
than 10 minutes. Extending the contact time appears to
have little effect on seed germination when disinfecting
with calcium hypochlorite. Overall the literature
indicates that a contact time of 5-10 min should be
sufficient for disinfection.
CONTACT TIME/SODIUM HYPOCHLORITE-Information on the effect
of a longer contact time on germination was provided by
Northen and Northen (1947, 1948) and Knudson (1948).
Northen and Northen (1947, 1948) used an eye dropper,
transferring 2-4 drops of seed containing disinfectant to
each flask. They found that with Clorox, extending the
contact time caused a significant decrease in germination.
In addition, they reported that duplicate treatments
produced highly variable results. Knudson (1948) proposed
the variable germination rates were related to the
quantity of Clorox added to the flask along with the seed.
La Garde (1929) was the first to suggest that orchid
seed sowing would benefit from disinfectant removal prior
to seed sowing. Current practice follows La Garde's
suggestion to remove disinfectant from the seed and rinse
with sterile distilled water before sowing. Rinsing
effectively stops the seed/disinfectant contact.
Disinfectant contact times reported for investigators
removing Clorox and rinsing vary from 5 min to 120 min
(Syoichi, 1989; Harvais and Hadley, 1967).
STABILITY/CALCIUM HYPOCHLORITE-Frequently, authors
indicate calcium hypochlorite solutions are unstable.
Castle and Nickell (1942) wrote that the solution must be
fresh; Wright (1948) Arditti (1982) wrote that solutions
should be prepared fresh daily; and Scott and Arditti
(1959) recommended preparing a fresh solution every 24 hr.
Redlinger (1961) prepared a fresh solution every 4 hr and
Northen (1970) recommended preparing a fresh solution for
each sowing. In contrast, Dychdala (1983) describes
calcium hypochlorite solutions as fairly stable. The use
of distilled or deionized water free from metallic ions
significantly improves the stability of hypochlorite
solutions (Dychdala, 1983). Knudson (1948) stated that in
their laboratory they prepared 300 ml of calcium
hypochlorite solution and stored it in a glass stoppered
bottle in a refrigerator for use over a period of time.
Sweet and Bolton (1979) stored a 5 wt % available chlorine
solution of calcium hypochlorite under refrigeration and
diluted it to working strength prior to use. Stock
solution activity was verified monthly by analysis.
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