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From Mainberg@aol.com Fri Oct 11 13:37:06 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 II)
Part II
SATURATED SOLUTION/CALCIUM HYPOCHLORITE-A solution
prepared by dissolving 10 g of powdered calcium
hypochlorite in 140 ml of water (Wilson, 1915), equivalent
to 7.1 wt %, is frequently referred to as saturated. Smith
(1932) was first to refer to Wilson's solution as
saturated. Ryerson (1952), Hey and Hey (1966) and Arditti
(1982) also describe a 7 wt % solution as saturated. In
contrast, Linke (1958) gives the solubility of calcium
hypochlorite as 21.8 wt % and Mellor (1956) gives the
solubility as 22.5 wt % at 18 C. A review of the original
papers, reveals a discrepancy in data quality that tends
to support the solubility provided by Linke and Mellor.
Therefore, a 7% solution should not be described as
saturated since this implies that the correct strength can
be obtained without weighing.
EFFECT OF pH-Segall (1968) listed factors influencing the
effectiveness of a disinfection procedure (in decreasing
order of importance) as: concentration, contact time, pH,
and temperature. Using pure cultures, spore kill was found
to be the greatest between 5 and 15 min with a significant
improvement obtained when the pH was lowered below 7.5.
Dychdala (1983) reported that the disinfecting efficiency
of hypochlorus acid (HOCl) is greater than the
hypochlorite ion (OCl-) by a factor of about 80. He found
that 100 ppm (0.01 wt %) available chlorine, at a pH of
8.2, was equivalent to 1000 ppm (0.1 wt %) available
chlorine at a pH of 11.3. Dychdala suggested that mold
spores are very resistant to chlorine disinfectants and
inactivation within a few minutes would require 135 to 500
ppm (0.0135-0.05 wt %) available chlorine. Sykes (1965)
reported that 1.9 ppm (0.00019 wt %) available chlorine at
a pH of 6.2 was equivalent in killing power to 450 ppm
(0.0450 wt %) at a pH of 10.5. Sweet and Bolton (1979), in
their studies of non-orchid seed, concluded that
hypochlorite was most effective at a pH of 6. For
convenience they adjusted the pH with a phosphate buffer.
From this evidence, pH appears to be a frequently
uncontrolled variable that might be responsible for some
of the variable germination results reported in the
literature.
EFFECT OF TEMPERATURE-Dychdala (1983) reported that with
hypochlorite at 25 ppm (0.0025 wt %) and a pH of 10
disinfection required 121 min at 20 C and 38.7 min at 35
C. This equals a 60% improvement for a 10 C rise. Segall
(1968) reported that as exposure time increases, the
temperature effect decreases. The orchid literature did
not discuss potential temperature related disinfection
effects.
CONTACT DECOMPOSITION OF HYPOCHLORITES-Frequently found in
the literature is the statement that rapid decomposition
occurs when hypochlorites contact germination media. Rapid
decomposition is cited as the reason seed can be
successfully sown by injecting a combination of seed and
disinfectant into the flasks. No data were found to
support this claim. It would appear that if rapid
decomposition does not occur, contact time would be
effectively increased in an uncontrolled manner. The
increased contact time would be especially detrimental
when disinfecting using Clorox which is very time-
sensitive.
SURFACTANTS-Twelve papers recommended the use of
surfactants, first proposed by Withner (1942), to enhance
orchid seed disinfection. Sweet and Bolton (1979) cited a
study showing that surfactants increase the effectiveness
of hypochlorite solutions. Only three of the twelve orchid
papers (Butcher and Marlow, 1986; Syoichi, 1989; Light,
1994) reported using surfactants with Clorox. Ernst,
Arditti and Healey (1971) investigated wetting agents by
incorporating various surfactants in nutrient media. They
found, that even when added directly to the media at
concentrations below 100 ppm (0.01 wt %), effects on
germination were minor; some wetting agents even improved
germination. Considering the reported non-toxicity of low
levels of surfactants and the improved disinfection
obtained using surfactants, it would appear safe to accept
the use of surfactants at 100 ppm (0.01 wt %) or below as
beneficial.
ALTERNATE DISINFECTANTS-Several papers investigated
alternate disinfectants. The results were mostly negative,
with many of the alternates having already been evaluated
and rejected by Wilson (1915). The exception was hydrogen
peroxide. Twenty papers described using 3% hydrogen
peroxide, first used with orchid seed by Smith (1932).
Five authors, including Smith, reported poor results.
Thomasen (1964) reported improving results by soaking the
seed in either 4 % bouillon or honey for 4 hr at 37 C
prior to disinfection. Most authors have tried hydrogen
peroxide in an effort to overcome the lack of germination
obtained using hypochlorites. The arguments supporting
hydrogen peroxide disinfection were not persuasive.
OTHER CONSIDERATIONS-Knudson (1948) believed that calcium
hypochlorite was superior to sodium hypochlorite, stating
that sodium from the decomposition of sodium hypochlorite
was toxic to seed. The superiority of calcium hypochlorite
was supported by Northen and Northen (1947,1948) and Sweet
and Bolton (1979). Sweet and Bolton found that slash pine
seedlings treated with sodium hypochlorite had a 15%
germination rate and abnormal cotyledon development while
those exposed to calcium hypochlorite at the same
available chlorine concentration developed normally and
had a 77% germination rate. No mention of sodium
hypochlorite's potential to adversely affect orchid
seedling development was found in the orchid literature.
Contact volume was first described by Wilson(1915) who
stated that "the solution employed should be about five
times or more the volume of the seed". There were no
papers that demonstrated an optimum ratio.
DISCUSSION
There can be no question that using weight percent does
not adequately describe the strength of a calcium
hypochlorite solution. Since 1915, the available chlorine
content of powdered calcium hypochlorite has increased
from 28 to 75 wt %. The use of wt % with today's stronger
calcium hypochlorite produces solutions that contain 5 wt
% available chlorine, significantly stronger than the 2 wt
% available chlorine used by Knudson (1922). The solution
used by Wilson (1915) containing 1 wt % available
chlorine, when prepared using today's calcium hypochlorite
containing 70 wt % available chlorine, requires only 1.45g
of calcium hypochlorite per 100 ml of solution not the 7.1
g specified in most literature.
It is recommended that future reports use wt %
available chlorine to describe hypochlorite concentration.
This term was used by Wilson (1915) and is almost
universally used to describe the strength of
chlorine-containing disinfectants. For critical work,
calcium hypochlorite solutions should be analyzed just
prior to use. For routine work, as a minimum, reports
should provide the powdered reagent weight, the powdered
reagent's strength as available chlorine, and confirmation
that the powdered reagent was fresh.
Most investigators working with orchid seed apparently
assume that effective disinfection using calcium
hypochlorite requires a 7.1 wt % solution. The literature
review does not support this belief. The authors working
with orchids who reported using lower concentrations
reported improved germination, especially with
Phalaenopsis (Knudson, 1922-1948; Garrard, 1966; Ryerson,
(1952). Sweet and Bolton (1979) described the optimum
concentration for non-orchid seed as 0.5 wt % available
chlorine, equivalent to a 0.73 wt % solution using today's
calcium hypochlorite.
Disinfectant concentration in the range of 2-5 wt %
appears to be especially critical when sowing Phalaenopsis
seed. Successful germination was reported when using a
calcium hypochlorite concentration of 2 wt % or lower
available chlorine while seed disinfected using higher
concentrations frequently failed to germinate.
Literature on sterilization and disinfection describes
the hypochlorite ion as the disinfecting agent. This
suggests that a concentration of 0.1-0.5 wt % available
chlorine, established by Liddell (1948) using sodium
hypochlorite, will be equally effective using calcium
hypochlorite. A solution containing 0.2 wt % available
chlorine, prepared using today's calcium hypochlorite
containing 70 wt % available chlorine, requires only 0.29g
of calcium hypochlorite per 100 ml of solution.
The widely held belief that solutions of calcium
hypochlorite are unstable, thereby requiring frequent
preparation, has discouraged the use of calcium
hypochlorite. The belief that calcium hypochlorite is
unstable was not validated by this literature review.
Indeed, successful disinfection was reported using
solutions stored for extended time periods (Knudson, 1948;
Sweet and Bolton, 1979). The stability of a dilute calcium
hypochlorite solution is enhanced by using distilled or
deionized water (free from destabilizing ions) and
protecting the solution from light and air (Knudson, 1948;
Mellor, 1956). It was suggested that refrigeration would
further extend the useful life of hypochlorite solutions
(Knudson, 1948).
The recent trend of removing disinfectant from the seed
by rinsing with sterile water prior to sowing is supported
by the literature. In addition, disinfectant removal
permits precise control of contact time with the
disinfectant, a critical factor for some seed. The
addition of a surface-active agent to increase the
effectiveness of disinfectants is also supported by the
literature.
Segal (1968) states that the effectiveness of the
disinfection process is dependent upon concentration,
contact time, pH, and temperature. By the manipulation of
these variables, it should be possible to tailor a
disinfecting procedure to maximize germination. The fact
that seed survives long contact times with calcium
hypochlorite solutions (Northen and Northen, 1948)
indicates that the preferred approach, when working with
seed showing heavy or resistant strains of contamination,
would be to increase contact time keeping concentration as
low as possible. Seed from genera other than Phalaenopsis
may also be sensitive to hypochlorites. Using lower
concentrations of calcium hypochlorite may improve
germination with seed that in the past has proven
difficult or impossible to germinate. This suggests that
previous studies with seed that appeared viable, but
failed to germinate when disinfected using standard
strength solutions, should be repeated using calcium
hypochlorite at a concentration of 0.2 wt % available
chlorine. In difficult cases, consideration should be
given to using a concentration lower than 0.2 wt %
available chlorine, accepting a slightly increased
contamination rate in an effort to obtain a higher
germination rate.
The orchid literature did not describe the effects of
pH or temperature on orchid seed disinfection. It is
uncertain if there is any advantage in disinfecting using
a lower concentration of available chlorine at a lower pH
or a higher temperature. If damage to the embryo results
from the hypochlorous acid concentration rather the
hypochlorous ion itself, then altering the pH and
temperature will produce little gain. However, the orchid
grower should remain aware of the potential effect of
altering pH and/or temperature.
The literature clearly demonstrates that an extended
contact time is less damaging using calcium hypochlorite
than using Clorox (Northen and Northen, 1947-1948;
Knudson, 1948). Better germination was obtained with
Cattleya seed disinfected using calcium hypochlorite at 2
wt % available chlorine than with sodium hypochlorite at
0.125 wt % available chlorine (Knudson, 1948). This
clearly demonstrates the superiority of calcium
hypochlorite. Considering its relative mildness and that
no critical time frame is necessary, calcium hypochlorite
is clearly the preferred disinfectant.
The volume of solution used to disinfect a given
volume of seed was not delineated in the literature but a
ratio of 5 or more was occasionally specified. The use of
lower concentrations of disinfectant suggests that a
substantial excess should be employed to provide a
hypochlorite reserve during disinfection.
The use of Clorox may be justified for the routine
sowing of less sensitive seed. Most recently published
procedures have used either a (1:10) or (1:20)
concentration (0.5 or 0.25 wt % available chlorine).
Liddell (1948) reported that a (1:54) or approximately a
0.1 wt % available chlorine concentration was optimum.
This would suggest that germination might be improved by
those using Clorox by lowering the concentration.
In summary, the literature indicates that optimum
disinfection of orchid seed will be obtained using calcium
hypochlorite at a concentration of 0.1-0.5 wt % available
chlorine, adding surfactants at 100 mg/l, using a large
excess of solution and contact time of 10-15 min, and
removing disinfectant by filtration followed by several
sterile water rinses. Unused disinfectant stock, protected
from light, air, and excessive temperatures, may be stored
for future use.
LITERATURE CITED
AOAC, 1990. Official Methods of Analysis. Method 935.09.
Chlorine (available) in calcium hypochlorite.
Association of Official Analytical Chemists.
Arlington, Va 22209.
Adritti, J. 1982. Orchid seed germination and seedling
culture - a manual. Pages 243-370 in J. Arditti [ed.],
Orchid biology: reviews and perspectives, vol. II.
Cornell University Press, Ithaca, New York.
Butcher, D., and S. A. Marlow, 1986. Asymbiotic
germination of epiphytic and terrestrial orchids.
Pages 31-38 in H. W. Pritchard [ed.], Modern methods
in orchid conservation: the role of physiology,
ecology and management. Cambridge University Press,
New York, 1989.
Castle, H., and L. G. Nickell. 1942. The
aerosol-hypochlorite technique for the sterilization
of orchid seed. Amer. Orchid Soc. Bull. 11: 200-201.
Dychdala, G. R. 1983. Chlorine and chlorine compounds.
Chapter 19 in S. S. Block [ed.], Disinfection,
sterilization and preservation. 3rd edition, Lea and
Febiger, Philadelphia, Penn.
Ernst, R., J. Arditti and P. L. Healey. 1971. Biological
effects of surfactants. New Phytol. 70:457-475.
Fennell, T. A. Jr. 1956. Orchids for home and garden.
Pages 104-105. Rinehart & Co. Inc., New York.
Garrard, J. 1966. Growing orchids for pleasure. A. S.
Barnes & Co., New York.
Hamilton, R. M. 1988. The new orchid doctor. Robert M
Hamilton, Richmond, B. C.
Harvais, G., and G. Hadley. 1967. The development of
Orchis purpurella in asymbiotic and inoculated
cultures. New Phytol. 66: 217-230.
Hegarty, C. P. 1955. Observations on the germination of
orchid seed. Amer. Orchid Soc. Bull. 24: 457-464.
Hey, G. L., and M. G. Hey. 1966. Raising rare orchids from
seed. Pages 35-38 in L. R. De Garmo [ed.], Proc. 5th
World Orchid Conf.
Kano, K. 1971. Seed germination of oriental cymnbidium and
their shoot tip culture. Page 141 in M. J. G. Corrigan
[ed.], Proc. 6th World Orchid Conf.
Knudson, L. 1922. Nonsymbiotic germination of orchid
seeds. Bot. Gaz. 73: 1-25.
.1924. Further observations on nonsymbiotic
germination of orchid seeds. Bot. Gaz. 77:212-226.
.1948. Clorox and calcium hypochlorite as
disinfectants for orchid seed. Amer. Orchid Soc. Bull.
17: 347-353.
La Garde, R. V. 1929. The growing of orchids from seed.
Ann. Missouri Bot. Gard. 18 (2): 29-36.
Liddell, R. W. 1946. A simplified technique for
germinating orchid seed. Amer. Orchid Soc. Bull. 15:
9-15 and 69-73.
.1947. On sterilizing seed with clorox. Amer.
Orchid Soc. Bull. 16:538.
.1948. Further experiments with clorox in the
germination of Cattleya seed. Amer. Orchid Soc.
Bull. 17:354-357.
Light, M. H. S. 1994. Flasking problems and solutions. The
Orchid Rev. 102:104-108.
Linke, W. F. 1958. Solubilities - inorganic and metal-
organic compounds. D. Van Nostrand and Co. Inc.,
Princeton, N. J.
Mellor, J. W., [ed.]. 1956. Inorganic and theoretical
chemistry, Supplement II, Part I, Chlorine oxides and
oxyacids, pages 556-569, and the determination of
hypochlorite and the analysis of bleach liquors, pages
666-668. Longmans, Green and Co., New York.
Northen, H. T., and R. T. Northen. 1947. Effect of
sodium hypochlorite (Clorox) on the germination of
Cattleya seed. Amer. Orchid Soc. Bull. 16: 498-499.
.1948. Further studies of the effects of sodium
hypochlorite (Clorox) on the germination of Cattleya
luegeae seed. Amer. Orchid Soc. Bull. 17: 358-360.
Northen, R. T. 1953. Phalaenopsis seed sowing. Amer.
Orchid Soc. Bull. 22: 122-123.
.1970. Home orchid growing. Page 103. Van
Nostrand Reinhold Co., New York.
Redlinger, J. R. 1961. Sterilizing agents for orchid seed
flasking. Amer. Orchid Soc. Bull. 30: 800-801.
Rubbo, S. D., and J. F. Gardner. 1965. Sterilization and
disinfection, page 140. Lloyd-Luke Ltd., London.
Ryerson, D. 1952. Ryerson expansible orchid folder. Page
B. Daniel Ryerson Pub., Homestead, Florida.
Scott, R. J., and J. Arditti. 1959. Cymbidiums from pod to
pot. Amer. Orchid Soc. Bull. 28: 823-829.
Segal, R. H. 1968. Fungal effectiveness of chlorine as
influenced by concentration, temperature, pH, and
spore exposure time. Phytopathology. 58: 1412-1414.
Smith, F. E. V. 1932. Raising orchid seedlings
asymbotically under tropical conditions. Gardners
Chronical. 91(2349): 9-11.
Snell, F. D., and C. L. Hilton, [eds.]. 1971. Bleaching
agents. Vol. 7. Pages 221-227, and Hydrogen Peroxide.
Vol. 14. Pages 432-433. Encyclopedia of industrial
chemical analysis. Interscience Publishers, New York.
Sweet, H. C., and W. E. Bolton. 1979. The surface
decontamination of seeds to produce axenic seedlings.
Amer. J. Bot. 66: 692-698.
Sykes, G. 1965. Disinfection and sterilization. Chapter
15, The halogens. pages 381-395. E. & F. N. Spon LTD,
London.
Syoichi, I. 1989. Seed germination of Ponerorchis
graminifolia. Lindleyana. 4: 161-163.
Thomasen, H. B. 1964. New technique in the sterilization
of orchid seed. Amer. Orchid Soc. Bull. 33: 111.
Thompson, P. A. 1977. Orchids from seed. Royal Botanic
Gardens, Kew, Wakehurst Place: HMSO.
Wilson, J. K. 1915. Calcium hypochlorite as a seed
sterilizer. Amer. J. Bot. 2: 420-427.
Wink, R. J., and P. L. Henkels. 1978. Calcium
hypochlorite. Page 423 in Kirk-Othmer [ed.],
Encyclopedia of chemical technology. 4th Ed. John
Wiley & Sons Inc., New York.
Withner, C. L. 1942. Nutrition experiments with orchid
seedlings. Amer. Orchid Soc. Bull. 11: 112-114.
Wright, H. D. 1948. Orchid seed germination for the
beginner. Amer. Orchid Soc. Bull. 17: 27-32.
Yam, T. W., and M. A. Weatherhead. 1988. Germination
and seedling development of some Hong Kong orchids.
Lindleyana. 5: 156-160.
Date: Mon, 14 Oct 1996 14:59:41 -0400
From: Mainberg@aol.com
Subject: [10711] Source of Calcium hyplchlorite
I have received several requests for sources of calcium
hypochlorite. In the US it is available from most chemical supply
companies with the exception of Sigma for about $40 a pound. For those
without the credentials to make such purchases, it can be obtained
from G & B Laboratory, 2426 Cherimoya Dr. Vista, CA 92084. For those
outside the US, a very good source for economical, high purity calcium
hypochlorite are companies that carrying swimming pool supplies. In
the US it is called Super Sock It. A 1 lb supply in a sealed plastic
bag cost about $3. It is labled as containing 75% available chlorine,
the number you will use in calculating how much to use. I have
analyzed Super Sock It and found it to be essentially pure calcium
hypochlorite.
Unfortunately it is labled "for swimming pool use only" so in the US
you cannot be legally use it to disinfect orchid seed. You must buy it
from a laboratory supply. I suppose you could treat your swimming pool
and then use your swimming pool water to disinfect your orchid
seed. Isn't it fun, you can swim in it but you can't use it on orchid
seed. Fred Bergman
Date: Thu, 24 Oct 1996 17:45:54 -0400
From: Mainberg@aol.com
Subject: [10854] Re: Request for info on Ca(OCl)2
To Ed & Others; Yes you have figured it right. 1g/433 ml of calcium
hypochlorite (65% AC) will give you a soluition containing 1500 ppm
(mg/l) AC. A note of caution, this is a concentration that is
tentative in nature. As far as I know I am the only one who has used
it extensively. I don't ordinarly sow seed for others. But reciently a
new young member of the local society asked me to sow a cross for
him. I had to go to 3000 mg/l to get a clean culture. I believe there
are benifits in using the lowest effective concentration posible. It
therefore makes sense to harvest the seed as clean as possible to
permit the use of low concentrations. I watch a capsule and when its
color starts to change, and as soon as it starts to splits I remove
the capsule. The capsule is placed on a clean sheet of paper and
opened. I haven't found it necessary to disinfect the outside of the
capsule. The capsule and seed are allowed to air dry for 24 hrs, the
seed shaken out and folded into a small unglazed paper packet. The
packet is labled with the cross number and placed in a small jar over
anhydrous silica gel. The jar then goes into the vegetable crisper of
the refrigerator. I stir a wetting agent into the disinfectant just
before adding the seed (100 mg/l ,about 1/12 of a ml). I shake the
seed-solution very hard when the seed is first added and then about
once every 3-4 min. thereafter. Frequency is not important since the
initial shaking thoroughly wets the seed. To answer your question on
weighing the calcium hypochlorite, I would say your scale should be
fine. If you were publishing you should try for more accuracy, but
for everyday practicle work this is not necessary. At this time I
would expect that even with Phal seed you will not see an unacceptable
difference between 5.0 g/l and 1.5 g/l AC. The real problem occurs
when you use the 50 g/l that most recomend. And because we are all on
a learning curve, me included, might I suggest you make a double or
triple sowing at different concentrations. Let OLD now how it comes
out. Fred Bergman
Date: Thu, 07 Nov 1996 23:37:14 -0500
From: Mainberg@aol.com
Subject: [11097] Re: Comments on mud, Fert,, clorox
-Response to Bob Hamilton's comments. Bob; there are a couple of
points that I would like to make is response to your comments, to my
comments, to your comments. Is everyone lost? Like you I am lazy. So
if I were to disinfect my seed using clorox and germination was poor
or nonexistent, I would resow using calcium hypochlorite. So the easy
way to go is to use calcium hypochlorite in the first place and save
the second sowing. Most of my experience has been with Phals. but I
have also tried a few other genera. A Ctna I had made earlier failed
to germinate using normal procedures (1 in 500 germinated). The cross
was repeated using calcium hypochlorite at 2000 mg/l available
chlorine and near 100 % germination was obtained. Some Phal crosses
germinate using stronger disinfectants, but crosses that deliquesce in
less that 7 mo. fail to germinate unless calcium hypochlorite at a low
concentration is used. Formally I examined all of my seed using a
microscope and the tetrazolium test. Frequently, seed that tested
viable failed to germinate. I no longer test my seed because I have
found that if the dry pod, when shaken over a sheet of paper produces
even a small quantity of dust like seed, I have viable seed which will
germinate. I agree that clorox will work for many genera, especially
Cats. However it seems unlikly to me that Phals would be the only
genera that is sensitive to sodium hypochlorite or calcium
hypochlorite at higher concetrations. My contention all along is that
bleach will work for many genera but in those cases, where apparently
viable seed fails to germinate, a repeat sowing should be made using a
low concentration of calcium hypochlorite. My work has only touched
the surface of what needs to be done and I hope others will join in
the determinations of when the alternate disinfection procedure
produces improved results. As a final note for those of you who are
considering using bleach-no objection intended- note that Bob uses a
contact time of only 5 min. and washes the seed 4 times after
disinfection.Fred Bergman
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