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Sterile (Aseptic) Technique

Aseptic technique is absolutely necessary for the successful establishment and


maintenance of plant cell, tissue and organ cultures. The in vitro environment in which
the plant material is grown is also ideal for the proliferation of microorganisms. In most
cases the microorganisms outgrow the plant tissues, resulting in their death.
Contamination can also spread from culture to culture. The purpose of aseptic
technique is minimize the possibility that microorganisms remain in or enter the cultures.
The environmental control of air is also of concern because room air may be highly
contaminated. Eample! "neezing produces #$$,$$$ % &$$,$$$ aerosol droplets which
can then attach to dust particles. These contaminated particles may be present in the
air for wee's. ()ave you ever viewed the air around you when you open the curtains on
a sunny day*+. Air may also contain bacterial and fungal spores, as do we.
I. Contaminants
A. Bacteria, fungi, and insects
1. Bacteria
,acteria are the most frequent contaminants. They are usually introduced with the
eplant and may survive surface sterilization of the eplant because they are in
interior tissues. "o, bacterial contamination can first become apparent long after a
culture has been initiated (see below+. "ome bacterial spores can also survive the
sterilization procedure even if they are on the tissue surface. -any 'inds of bacteria
have been found in plant tissue cultures including Agrobacterium, Bacillus,
Corynebacterium, Enterobacter, Lactobacillus, Pseudomanas, Staphylococcus, and
Xanthomonas. ,acteria can be recognized by a characteristic .ooze./ the ooze can
be many colors including white, cream, pin', and yellow. There is also often a
distinctive odor.
2. Fungi
0ungi may enter cultures on eplants or spores may be airborne. 0ungi are
frequently present as plant pathogens and in soil. They may be recognized by their
.fuzzy. appearance, and occur in a multitude of colors.
3. Yeast
1east is a common contaminant of plant cultures. 1easts live on the eternal
surfaces of plants and are often present in the air.
4. iruses, etc.
2iruses, mycoplasma%li'e organisms, spiroplasmas, and ric'ettsias are etremely
small organisms that are not easily detected. Thus, plant culture is not necessarily
pathogen%free even if microorganisms are not detected, and this can influence
culture success. "pecial measures such as meristem culture are often necessary to
eradicate such contaminants.
!. "nsects
The insects that are most troublesome in plant cultures include ants, thrips, and
mites. Thrips often enter cultures as eggs present on the eplants. Ants and mites,
however, usually infest already established cultures. -ites feed on fungus and mite
infestations are often first detected by observing lines of fungal infection that lead
from the edge of the culture vessel to the plant tissue, having been introduced by the
insect. It is very difficult to eradicate insect infestations. Careful lab practices and
cleanliness should prevent most infestations.
B. "nitial #$nta%inants
-ost contamination is introduced with the eplant because of inadequate
sterilization or 3ust very dirty material. It can be fungal or bacterial. This 'ind of
contamination can be a very difficult problem when the plant eplant material is
harvested from the field or greenhouse. Initial contamination is obvious within a few
days after cultures are initiated. ,acteria produce 4ooze5 on solid medium and
turbidity in liquid cultures. 0ungi loo' 4furry5 on solid medium and often accumulate
in little balls in liquid medium.
#. &atent #$nta%inati$n
This 'ind of contamination is usually bacterial and is often observed long after
cultures are initiated. Apparently the bacteria are present endogenously in the initial
plant material and are not obviously pathogenic in situ. 6nce in vitro, however, they
increase in titer and overrun the cultures. 7atent contamination is particularly
dangerous because it can easily be transferred among cultures.
'. "ntr$duced #$nta%inati$n
Contamination can also occur as a result of poor sterile technique or dirty lab
conditions. This 'ind of contamination is largely preventable with proper care.
(. 'etecti$n $f #$nta%inants
Contamination is usually detected by the .eyeball. method in research labs.
)owever, indeing is possible, and is frequently done in commercial settings. This
involves ta'ing a part of the plant tissue and culturing it in media that are specific for
bacteria and fungi. -edia that have been used for this purpose include 89A (potato
detrose agar+ and :, broth (with salts, yeast etract and glucose+. This is the most
reliable method for detecting bacteria and fungi, but, as indicated above, there may
be infecting organisms that won;t be detected.
II. The Transfer Hood
7aminar airflow hoods are used in commercial and research tissue culture settings. A
horizontal laminar flow unit is designed to remove particles from the air. <oom air is
pulled into the unit and pushed through a )E8A ()igh Energy 8article Air+ filter with a
uniform velocity of =$ ft>min across the wor' surface. The air is filtered by a )E8A (high
efficiency particulate air+ filter so nothing larger than $.? micrometer, which includes
bacterial and fungal spores, can pass through. This renders the air sterile. The positive
pressure of the air flow from the unit also discourages any fungal spores or bacteria
from entering. 9epending on the design of the hood, the filters are located at the bac'
or in the top of the bo.
III. Sterilization and Use of Supplies and Equipment:
A. Sterili)ing t$$ls, %edia, *essels etc.
1. Aut$cla*ing
Autoclaving is the method most often used for sterilizing heat%resistant items and our
usual method for sterilizing items. In order to be sterilized, the item must be held at
#&#C, #@ psi, for at least #@ minutes. It is important that items reach this temperature
before timing begins. Therefore time in the autoclave will vary, depending on volume in
individual vessels and number of vessels in the autoclave. -ost autoclaves
automatically ad3ust time when temperature and psi are set, and include time in the
cycle for a slow decrease in pressure. There are tape indicators that can be affied to
vessels, but they may not reflect the temperature of liquid within them. There are also
4test 'its5 of microorganisms that can be run through the autoclave cycle and then
cultured.
Empty vessels, bea'ers, graduated cylinders, etc., should be closed with a cap or
aluminum foil. Tools should also be wrapped in foil or paper or put in a covered
sterilization tray. It is critical that the steam penetrate the items in order for sterilization
to be successful.
2. Aut$cla*ing and Filter+sterili)ing ,edia and -ther &iquids
Two methods (autoclaving and membrane filtration under positive pressure+ are
commonly used to sterilize culture media. Culture media, distilled water, and other heat
stable mitures can be autoclaved in glass containers that are sealed with cotton plugs,
aluminum foil, or plastic closures. )owever, solutions that contain heat%labile
components must be filter%sterilized. 0or small volumes of liquids (#$$ ml or less+, the
time required for autoclaving is #@%&$ min, but for larger quantities (&%A liter+, ?$%A$ min
is required to complete the cycle. The pressure should not eceed &$ psi, as higher
pressures may lead to the decomposition of carbohydrates and other components of a
medium. Too high temperatures or too long cycles can also result in changes in
properties of the medium.
6rganic compounds such as some growth regulators, amino acids, and vitamins may
be degraded during autoclaving. These compounds require filter sterilization through a
$.&& Bm membrane. "everal manufacturers ma'e nitrocellulose membranes that can be
sterilized by autoclaving. They are placed between sections of a filter unit and sterilized
as one piece. 6ther filters (the 'ind we use+ come pre%sterilized. 7arger ones can be set
over a sterile flas' and a vacuum is applied to pull the compound dissolved in liquid
through the membrane and into the sterile flas'. "maller membranes fit on the end of a
sterile syringe and liquid is pushed through by depressing the top of the syringe. The
size of the filter selected depends on the volume of the solution to be sterilized and the
components of the solution.
:utrient media that contain thermo labile components are typically prepared in several
steps. A solution of the heat%stable components is sterilized in the usual way by
autoclaving and then cooled to ?@C%@$C C under sterile conditions. "olutions of the
thermo labile components are filter%sterilized. The sterilized solutions are then combined
under aseptic conditions to give the complete medium.
In spite of possible degradation, however, some compounds that are thought to be heat
labile are generally autoclaved if results are found to be reliable and reproducible.
These compounds include A,A, IAA, I,A, 'inetin, pyridoine, &%ip and thiamine.
3. (th.lene -/ide 0as
8lastic containers that cannot be heated are sterilized commercially by ethylene oide
gas. These items are sold already sterile and cannot be resterilized. Eamples of such
items are plastic petri dishes, plastic centrifuge tubes etc.
4. 1 2adiati$n
It is possible to use germicidal lamps to sterilize items in the transfer hood when no one
is wor'ing there. De do not do this. E2 lamps should not be used when people are
present because the light is damaging to eyes and s'in. 8lants left under E2 lamps will
die.
!. ,icr$3a*e
It is also possible to sterilize items in the microwave/ we do not do this.
4. ,$re #$%%ents
Fnow which of your implements, flas's, etc. are sterile and which are not. "terile
things will have been autoclaved and should be wrapped with some 'ind of
protective covering, e.g. foil, for transport from the autoclave to the hood.
6ur usual autoclave time of &$ minutes is intended for relatively small volumes.
7arge flas's of media, water, etc. may require longer autoclaving periods. It is
preferable to put no more than one liter of liquid in a container to be autoclaved.
Also, be sure to leave enough room in the container so that the liquid does not boil
over.
"terilized items should be used within a short time (a few days at most+.
Items that come pac'aged sterile, e.g. plastic petri plates, should be eamined
carefully for damage before use. If part of a pac'age is used, seal up the remainder
and date and label. Ese up these items unless there is some question about their
sterility/ they are epensive.
De use :algene vessels for some purposes in the lab. These will melt if eposed to
high heat.
IV. Working in the Transfer Hood:
The hoods in our lab run continuously. If for some reason one has been turned off,
turn it on and let it run for at least #@ minutes before using.
-a'e sure that everything needed for the wor' is in the hood and all unnecessary
things are removed. As few things as possible should be stored in the hood.
Chec' the bottom of the hood to ma'e sure there is no paper or other debris
bloc'ing air inta'e.
<emove watches, etc., roll up long sleeves, and wash hands thoroughly with soap
(preferably bactericidal+ and water.
"pray or wipe the inside of the transfer hood (bottom and sides, not directly on the
filters+ with G$H Et6). 6thers use disinfectants such as 7ysolI. Dipe the wor' area
and let the spray dry.
Dipe hands and lower arms with G$H Et6). It is not necessary to flame them (This
is a 3o'e.+.
"pray everything going into the sterile area with G$H ethanol. 0or eample, spray
bags of petri dishes with G$ H alcohol before you open them and place the desired
number of unopened dishes in the sterile area.
Dor' well bac' in the transfer hood (behind the line+. Especially 'eep all flas's as far
bac' to the bac' of the hood as possible. -ovements in the hood should be
contained to small areas. A line drawn across the distance behind which one should
wor' is a useful reminder.
-a'e sure that materials in use are to the side of your wor' area, so that airflow
from the hood is not bloc'ed.
9on;t touch any surface that is supposed to remain sterile with your hands. Ese
forceps, etc.
Instruments (scalpels, forceps+ can be sterilized by flaming % dipping them in =@H
Et6) and then immediately placing them in the flame of an alcohol lamp or gas
burner. This can 5e danger$us if the *essel h$lding the alc$h$l tips $*er and
an alc$h$l fire results. A fairly deep container, li'e a coplin%staining 3ar, should be
used to hold the ethanol. Ese enough ethanol to submerge the business ends of the
instruments but not so much that you burn your hands. "ome people wear gloves in
the hood for certain procedures. If you do this, be very careful not to get them near
the flame. 6ther methods of sterilization that do not require alcohol are with a
bacticinerator or glass bead sterilizer. There is not as much ris' from fire with these,
but the instruments can still get etremely hot, causing burns.
Arrange tools and other items in the hood so that your hands do not have to cross
over each other while wor'ing. 0or a right%handed person, it is best that sterilizing
implements and tools be placed on the right. The plant material should be placed to
the left. All other items in the hood should be arranged so that your wor' area is
directly in front of you, and between J and #$ inches in from the front edge. :o
materials should be placed between the actual wor' area and the filter. Feep as little
in the hood as possible.
8lant material should be placed on a sterile surface when manipulating it in the
hood. "terile petri dishes (epensive+, sterile paper towels, or sterile paper plates
wor' fine. 8re%sterilized plastic dishes have two sterile surfaces%the inside top and
inside bottom.
"terilize your instruments often, especially in between individual petri plates, flas's,
etc. The tools should be placed on a holder in the hood to cool or should be cooled
by dipping in sterile water or medium before handling plant tissues.
Dipe up any spills quic'ly/ use G$H Et6) for cleaning. Clean hood surface
periodically while wor'ing.
Ese of glass or plastic pipettes! Klass pipettes are put into containers or wrapped
and then autoclaved. 8lastic pipettes are purchased presterilized in individual
wrappers. To use a pipette, remove it from its wrapper or container by the end
opposite the tip. 9o not touch the lower two%thirds of the pipette. 9o not allow the
pipette to touch any laboratory surface. Insert only the untouched lower portion of
the pipette into a sterile container.
"terilize culture tubes with lids or caps on. Dhen you open a sterile tube, touch only
the outside of the cap, and do not set the cap on any laboratory surface. Instead,
hold the cap with one or two fingers while you complete the operation, and then
replace it on the tube. This technique usually requires some practice, especially if
you are simultaneously opening tubes and operating a sterile pipette. After you
remove the cap from the test tube, pass the mouth of the tube through a flame. If
possible, hold the open tube at an angle. 8ut only sterile ob3ects into the tube.
Complete the operation as quic'ly as you reasonably can, and then flame the mouth
of the tube again. <eplace the lid.
Inoculating loops and needles are the primary tools for transferring microbial
cultures. De use plastic ones that come sterile. If you are moving organisms from an
agar plate, touch an isolated colony with the transfer loop. <eplace the plate lid.
6pen and flame the culture tube, and inoculate the medium in it by stirring the end of
the transfer tool in the medium. If you are removing cells from a liquid culture, insert
the loop into the culture. Even if you cannot see any liquid in the loop, there will be
enough cells there to inoculate a plate or a new liquid culture.
If you donLt have to be careful about the volume you transfer, a pure culture or sterile
solution can be transferred to a sterile container or new sterile medium by pouring.
0or eample, we do not measure a specific volume of medium when we pour culture
plates, although after you have done it for a while, you become pretty consistent.
<emove the cap or lid from the solution to be transferred. Thoroughly flame the
mouth of the container, holding it at an angle as you do so. <emove the lid from the
target container. )old the container at an angle. Muic'ly and neatly pour the
contents from the first container into the second. <eplace the lid.
If you must transfer an eact volume of liquid, use a sterile pipette or a sterile
graduated cylinder. Dhen using a sterile graduated cylinder, complete the transfer
as quic'ly as you reasonably can to minimize the time the sterile liquid is eposed to
the air.
<emove items from the hood as soon as they are no longer needed. All cultures
must be sealed before leaving the hood.
Dhen transferring plant cultures, do contaminated cultures last. "ituate the cultures
so that the contaminated part is closest to the front of the hood (so contaminants
blow outward away from the interior of the hood.
8lace waste in the proper containers! Empty (e.g. after transfer+ or old petri plates
used in transformation eperiments go in the big bag to be autoclaved, as do other
disposables that were in contact with recombinant bacterial or plant material. All
needles go in the sharps bo, needles used with bacteria get autoclaved. "mall bags
used in the hood for waste go in the big bag to be autoclaved/ do not overfill the
small bags or leave full bags in or on the hood for someone else to dispose of.
Klassware that comes in contact with bacteria is placed in a separate pan to be
autoclaved.
Dhen finished in the hood, clean up after yourself. <emove all unnecessary
materials and wipe the hood down with G$H Et6).
It is pointless to practice good sterile technique in a dirty lab. "pecial problems are
contaminated cultures, dirty dishes and solutions where microorganisms can grow.
"tore cultures in a sequestered area. De will discuss this area later. Chec' cultures
every ?%@ days for contamination.
V. Surfae!sterilizing "lant #aterial
1. 6reparati$n $f St$c7 6lants
8rior good care of stoc' plants may lessen the amount of contamination that is present
on eplants. 8lants grown in the field are typically more 4dirty5 than those grown in a
greenhouse or growth chamber, particularly in humid areas li'e 0lorida. 6verhead
watering increases contamination of initial eplants. 7i'ewise, splashing soil on the plant
during watering will increase initial contamination. Treatment of stoc' plants with
fungicides and>or bacteriocides is sometimes helpful. It is sometimes possible to harvest
shoots and force buds from them in clean conditions. The forced shoots may then be
free of contaminants when surface%sterilized in a normal manner. "eeds may be
sterilized and germinated in vitro to provide clean material. Covering growing shoots for
several days or wee's prior to harvesting tissue for culture may supply cleaner material.
Eplants or material from which material will be cut can be washed in soapy water and
then placed under running water for # to & hours.
2. (than$l ($r "s$pr$p.l Alc$h$l)
Ethanol is a powerful sterilizing agent but also etremely phytotoic. Therefore, plant
material is typically eposed to it for only seconds or minutes. The more tender the
tissue, the more it will be damaged by alcohol. Tissues such as dormant buds, seeds, or
unopened flower buds can be treated for longer periods of time since the tissue that will
be eplanted or that will develop is actually within the structure that is being surface%
sterilized. Kenerally G$H ethanol is used prior to treatment with other compounds.
3. S$diu% 8.p$chl$rite
"odium hypochlorite, usually purchased as laundry bleach, is the most frequent choice
for surface sterilization. It is readily available and can be diluted to proper
concentrations. Commercial laundry bleach is @.&@H sodium hypochlorite. It is usually
diluted to #$H % &$H of the original concentration, resulting in a final concentration of
$.@ % #.$H sodium hypchlorite. 8lant material is usually immersed in this solution for #$ %
&$ minutes. A balance between concentration and time must be determined empirically
for each type of eplant, because of phytotoicity.
4. #alciu% 8.p$chl$rite
Calcium hypochlorite is used more in Europe than in the E.". It is obtained as a powder
and must be dissolved in water. The concentration that is generally used is ?.&@ H. The
solution must be filtered prior to use since not all of the compound goes into solution.
Calcium hypochlorite may be less in3urious to plant tissues than sodium hypochlorite.
!. ,ercuric #hl$ride
-ercuric chloride is used only as a last resort in the E.". It is etremely toic to both
plants and humans and must be disposed of with care. "ince mercury is so phytotoic,
it is critical that many rinses be used to remove all traces of the mineral from the plant
material.
4. 8.dr$gen 6er$/ide
The concentration of hydrogen peroide used for surface sterilization of plant material is
?$H, ten times stronger than that obtained in a pharmacy. "ome researchers have
found that hydrogen peroide is useful for surface%sterilizing material while in the field.
9. (nhancing (ffecti*eness $f Sterili)ati$n 6r$cedure
"urfactant (e.g.Tween &$+ is frequently added to the sodium hypochlorite.
A mild vacuum may be used during the procedure.
The solutions that the eplants are in are often sha'en or continuously stirred.
:. 2insing
After plant material is sterilized with one of the above compounds, it must be rinsed
thoroughly with sterile water. Typically three to four separate rinses are done.
;. 1se $f Anti5i$tics and Fungicides in itr$
De have found that the use of antibiotics and fungicides in vitro is not very effective in
eliminating microorganisms and these compounds are often quite phytotoic. ,ut there
are compounds that could be further evaluated.
1<. 6lant 6reser*ati*e ,i/ture
88-N is a proprietary broad%spectrum biocide, which can be used to control
contamination in plant cell cultures, either during the sterilization procedure, or as a
medium component. 88-N comes in an acidic liquid solution (p) ?.J+. The
recommended dose is $.@O&.$ m7 of 88-N per liter of medium. )igher doses are
required to treat endogenous contamination and for Agrobacterium.
Its ma'ers say that 88-N has several advantages over antibiotics! It is effective
against fungi as well as bacteria, thus it can be substituted for a coc'tail of antibiotics
and fungicides. 88-N is less epensive than antibiotics, which ma'es it affordable for
wide and routine use. The formation of resistant mutants toward 88-N is very unli'ely
because it targets and inhibits multiple enzymes. -any antibiotics adversely affect plant
materials. If used as recommended, 88-N does not adversely affect in vitro seed
germination, callous proliferation, or callous regeneration. "eeds and eplants with
endogenous contamination can be sterilized at doses of @%&$ m7>7 of 88-N. This is
useful when routine surface sterilization is insufficient.