PlantCell Reports(1986) 5:292-294

PlantCell
Reports
© Springer-Verlag 1986

Stability of tissue culture medium pH as a function of autoclaving,

time, and cultured plant material
Robert M. Skirvin, Mel C. Chu, Mary L. Mann, Heather Young, Joseph Sullivan, and Thomas Fermanian

University of Illinois, Department of Horticulture, 1707 South Orchard Street, Urbana, IL 61801, USA

Received September 18, 1985 / Revised version received May 21, 1986 - Communicated by A. R. Gould

ABSTRACT pH of 6.0 during a 4-week dark incubation. The drift

was slow in control media without callus and acceler-
Autoclaving is a standard procedure for steril- ated in media with callus.
izing nutrient media for plant tissue cultures. Most Dougall (5) has reviewed the literature associ-
tissue cultures are grown at pH 5.2 to 5.8 with pH ated with in vitro pN changes, and he believes that
adjustments being made prior to autoclaving. This the cause o--f-such pH changes is best explained in
paper reports that there are significant differences terms of ammonium (NH4+) and nitrate (NO3-) uptake
between initial pH levels and pH levels following from the medium. Dougall also presents evidence that
autoclaving, particularly in the pH range of 5.7 to initial medium pH can influence the ultimate pH of the
8.5. This effect is noted with and'without agar. medium by influencing the uptake rate of nitrate
In addition, we report that with time the pH of the and/or ammonium.
medium drifts into the acid range. When Cucumis In spite of all that is known about in vitro pH
callus was added to the medium, the pH was changed stability, most tissue culturists are unaware of the
significantly within 48 hours. The amount and situation in their own medium. This study was initi-
direction (increase or decrease of pH) was signifi- ated to examine the extent to which initial pH of a
cantly correlated with the original pH. This sug- modified Murashige and Skoog (MS) (i0) nutrient medi-
gests that researchers should be wary of the true pH um would be altered after autoclaving and to investi-
situation in their medium. In addition, in publica- gate pH stability with time with and without plant
tions authors should specify whether their medium pH material.
value was determined before or after autoclaving.
MATERIALS AND METHODS
INTRODUCTION
The medium used in this investigation was a modi-
Plant tissue cultures are known to tolerate a fied Murashige and Skoog (MS) medium (13). Six batch-
wide range of pH's; a value between 5.2 and 5.8 is es of media were made. Each batch was adjusted to a
most often provided (7). The standard procedure for different pH (5.0, 5.7, 6.4, 7.1, 7.8, and 8.5) prior
pH adjustment in tissue culture media is to correct to autoclaving with 0.5N KOH and HCI. Agar (DifCo
the pH of the nutrient medium with acid and/or base Bacto, 6 g/liter) was added to half of the samples,
prior to autoclaving. Although some researchers heated, and then dispensed into 25 X 150 mm culture
have measured pH after autoclaving and reported this tubes at i0 mls of medium per tube. The samples with-
"post-autoclave" pH value, the determination of this out agar were dispensed directly into tubes. The
value has not been routine laboratory practice. tubes were covered with plastic caps. After auto-
There are reports that the high temperatures of auto- claving for 15 min at 15 psi, the tubes were cooled
claving may cause the pH to drift (2,4,7,8,12). A to room temperature. Post-autoclave pH was determined
recent study suggested that changes in pH after auto- immediately after cooling and at weekly intervals for
claving are less pronounced with increasing agar an additional 6 weeks with a C o m i n g 125 pH meter
levels (12). Behagel (2) discussed the dependence using an Orion 91-35 combination electrode. The pH
of post-autoclave pH on the course of temperature meter was standardized using 2 buffers (pH 7.0 and
during autoclaving. He concluded that differences pH 4.01). The electrode was pushed into the medium
in post-autoclave pH were unavoidable due to the many and pH was recorded once the machine had equilibrated.
chemical reactions that occur during media steriliza- For each treatment at each date, a sample of i0 differ-
tion which are both temperature and pH dependent. ent tubes was sacrificed to estimate pH changes over
Other factors which can influence media pH include time.
the type of autoclave, position within the autoclave, In another experiment, MS media without agar were
quality of mineral nutrients, the quality of water determined to have post-autoclave p~S of 3.33, 5.11,
used in the medium, and the duration of autoclaving. 6.63, and 7.98. About i00 mg of Cucumis melo callus
In addition to physical factors which influence (14) was added to the media, pH changes were monitor-
pH changes i n vitro, it is also known that the pres- ed by harvesting 5 culture tubes of each pH every 2
ence of plant tissue affects the medium's ultimate pH. hours for a 48 hour period. Some tubes were maintain-
For instance, Pelet et al. (ii) reported time related ed without callus (control) to estimate the amount
pH changes when Populus deltoides and Ulmus americana of drift that occurred during this period.
callus were grown at various post-autoclave pH's, Comparisons of correlations between agar and liq-
ranging from 3.5 to 8.0. All media drifted towards a uid media at each pH were performed. The time studies
were analyzed by regression analysis.

Offprint requests to: R. M. Skirvin

 293

RESULTS same at all pre-autoclave pH values. With time, the

Following autoclaving, pH changes were observed, medium pH drifted to a more acid condition. As ob-
particularly in the pre-autoclave range of 5.7 to 7.8 served by Singha (12), agar slightly reduced media
(Table i). Post-autoclave pH readings were lower acidification. The amount of reduction depended upon
than the pre-autoclave pH readings. For example, pH the pre-autoclave pH. Of particular importance is the
7.1 pre-autoclave medium changed to about 5.7 after fact that autoclave-induced pH changes occur prominent-
autoclaving. ly within the pH range used by most tissue culturists,
5.2 to 5.8. It may be that for a particular species
Table i. pH changes after autoclaving on modified the combination of pH drift and autoclav-induced pH
Murashige and Skoog medium adjusted to various pH's. changes may be drastic enough to result in non-
optimal growing conditions.
The addition of Cucumis callus to the media ac-
celerated the rate of pH change regardless of original
pH. The statistical equation that described the
Number of weeks Pre-autoclave pH values -r-"
change in pH was different for each original pH (Fig-
after autoclaving 5.0 5.7 6.4 7.1 7.8 8.5
ure i). For instance, the post-autoclave pH of 3.33
increased to a final average pH of 4.87 while the 7.98
pH was reduced to 4.71 in the same 48 hour period
Liquid medium (-Agar)
(Table 2). These results correspond to those of
Pelet et al. (ii) who reported that the addition of
0 4.2 ~/ 4.6 5.1 5.8 6.7 8.1
poplar or elm callus to media of various pH's result-
1 4.1 4.4 5.0 6.0 6.8 7.4
ed in a drift towards a pH of 6.0.
2 4.1 4.3 5.0 5.9 6.8 7.4
Ion exchange, as a function of type of nitrogen
3 4.1 4.2 4.9 5.8 6.7 7.2
in the medium, is a probable source of the extra
4 4.0 4.2 4.8 5.6 6.4 7.1
hydrogen or hydroxide ions required to explain the pH
5 4.0 4.3 4.8 5.6 6.4 7.0
changes (5). When the medium was only slightly acid
6 4.0 4.1 4.7 5.2 6.3 6.8
to basic (pH 5.11 to 7.98), the addition of Cucumis
Semi-solid (+Agar) callus resulted in more acidity (Table 2, Figure i).
0 4.4 4.6 5.0 5.7 6.7 8.0 It has been shown that as plant cells absorb ammonium,
a hydrogen ion is exchanged. These H+ ions probably
1 4.4 4.6 5.0 5.8 6.7 7.4
contributed to the pH decrease observed on post-
2 4.3 4.6 4.9 5.7 6.6 7.3
autoclave pH 5.11, 6.63, and 7.98 media (Table 2). The
3 4.4 4.5 4.7 5.6 6.5 7.1
pH changes also might have been due partially to in-
4 4.2 4.4 4.7 5.4 6.4 7.0
trusion of H+ ions into cell walls to create a pH
5 4.2 4.5 4.8 5.5 6.3 6.9
optimum for cell wall loosening enzymes (6).
6 4.2 4.4 4.7 5.3 6.2 6.8
When CucUmis callus was added to the Very acid
medium, pH = 3.37, the medium became less acid (pH =
~!pH adjusted with 0.5N KOH or HCI before autoclaving 4.87) (Table 2, Figure i). Acidity favors the uptake
~!Each value is the average of i0 readings of nitrate (NO3-)ions. As nitrate is absorbed, bicar-
bonate (HCO3-) is extruded to the medium. The bicar-
The pH of each test medium drifted to a more acid bonate ion, in turn, joins with a proton (H+) (from
condition over the 6-week test period (Table i). ionized water) to yield carbonic acid (H2CO3). The
When Cucumis callus was added to the media, the decrease of H+ ions (and concomitant increase in OH-
pH changed rapidly (Figure i). For all media with concentration), results in pH change to a more basic
condition.
pH 5.11 or greater, there was a gradual acidifi-
cation over the 48 hour test period. The very acid The prediction curve for the pH 3.33 medium was
medium (pH = 3.33) became less acid in a linear fash- best fit by a linear, not quadratic, equation. On
ion throughout the 48 hour period. After 48 hours, the basis of this equation, the predicted change in
there were no significant differences among the pH's pH with increasing time would result in very basic
for any of the media (Table 2). Without plants, the medium. We have not found this to be the case (data
pH drift encountered during the 48 hour period was not presented). On the contrary, with time the pH
negligible (Table 2). tends to stabilize near 4.5 to 4.8. Therefore, our
prediction curve probably cannot be extrapolated past
Table 2. Changes in pH after 48 hours on tissue the first 48 hours. The curvilinear prediction equa-
culture medium with and without Cucumis tions for the other pH's are probably valid over a
callus. longer period of time.
Because the pH of all four media were so similar
after 48 hours (Table 2), it is tempting to suggest
that the plant material has an active role in estab-
Original pH Observed pH (48 hours) Predicted pH lishing an optimum pH environment. It also might be
+ plants - plan~s that the direction and extent of the pH change might
be influenced by the parent plant's in vivo pH opti-
mum. Such a hypothesis can only be verified by com-
3.33 4.87 3.27 4.82 -+ 0.12 ~ paring species with known in vivo requirements. For
5.11 4.55 4.99 4.52 -+ 0.15 instance, if this is true, then members of the
6.63 4.58 6.68 4.60 ± 0.40 Ericaceae (Heath family), which require acid soils,
7.98 4.71 8.00 4.42 -+ 0.36 sh0uld acidify the medium more than a species that
prefers basic soils. We leave these investigations
to others.
~IPredicted! pH ± 95% confidence intervals The pH changes associated with our modified MS
should not be assumed to be identical for all media.
DISCUSSION It is suggested that similar experiments be conducted
in every tissue culture laboratory under the particu-
Changes in pH do occur after autoclaving and lar growing conditions available in that laboratory.
these changes may be expected with or without t h e In addition, we suggest that all tissue culture re-
addition of agar. The extent of change was not the searchers should take care to measure the pH of
294
5.5
5.4

• UPPER 957- CL @" " •UPPER 957. CL

~etOBSERVED PH 5.2 * •g , * ;, *ORSER,EO P.
• iLOHER 957- CL • A ALOHER 957. CL
e'"ePREOICTEO PH IT=3.4O + .OSX} .~ AID I e ' " e P R E D t C T E R PH (T=5. 40 - . O5X + . 0006 (%
r2 = O. 7 0 4 2 ~ j . - " ..... " 5
• "®.,. r2 = 0.6345
4.5 • s.v
ptt 4.8
* "e. •
lk "es. • v
_4.[-. - 4. S tr" ~"

• . "tP-o..~. . ........ •
• .|-'• " ilHtial 4.4
• ~..~"~" • p H : 3,33 t
3.5 • tlr'a initial
it" A
4.2 pH=5.1i

4
I f I 1 I l I I I J I I 1 I I ] I l I I I I I J
4 8 L2 16 20 24 28 32 36 40 44 48 4 8 12 16 20 24 28 32 36 40 44 48

TIME (HOURS) TIME (HOURS]

6
.'=,.
",~r
5. 75
• •UPPER 95Z CL "UPPER 95~ CL
1~ ~rOBSERVEO PH ~¢OBSERVEB PH
• •LOHER 95% CL • ALOHER 95% CL
5.5 e'"OPREDI~TEO PH (T=S. 58 - . I 2 X + . 002) ~ ' " ¢ P R E D I C T E n PH (T=R.44 - . IEX + . 0 0 1 6

5. 25 a ", r2 = 0 . 5 7 8 9 ~,,t" r2 = 0.8321

pH
5 %{., e,,
4.75 A'e * • •
• "lit, ,e
initial m'e,•
4.5 ~'e, •
initial pH -- 7.98 A'e,,
PH = 6.(;3
492E • A • ..... ¢

4 I I I I I I l I I I I I I I L I i I l I I I I
4 S 12 IE 20 24 28 32 36 40 44 48 4 8 12 L8 20 24 28 32 38 40 44 48

TIME IMOUOS] TIME (HOURS]

Figure I. pH changes which have been induced in vitro by the addition of Cucumis callus to Murashige and Skoog
media of various initial pH's.

nutrient medium both before and after autoclaving as 4. Cousson A, Tran Thanh Van K (1981) Physiol Plant
well as at various times during culture. 51:77-84
5. Dougall DK (1980) Nutrition and metabolism. In:
ACKNOWLEDGEMENTS Staba EJ (ed) Plant tissue culture as a source of
biochemicals, Chemical Rubber Company Press, Boca
Funds for this research were provided by the Raton, Florida, pp 21-58
Illinois Agricultural Experiment Station. Thanks 6. Hansen J, Triuavas (1982) New Phytologist 90:1-18
to Ms. Holly Kerns, Dr. Mohamed Kouider, and Dr. Ed 7. Kruse, PF, Patterson MK (1973) Tissue culture:
Hellman for assistance with this project. Special methods and applications, Academic Press
thanks to Dr. Mary Ann Lila Smith for help with 8. Mann ML, Skirvin RM, Chu MC (1982) HortScience
editing. 17:534
9. Martin SM (1980) Environmental factors. B. Tem-
REFERENCES perature, aeration, and pH. In: Staba EJ (ed)
Plant tissue culture as a source of biochemicals,
i. Anderson WC (1975) Proc Int Plant Prop Soc 25:129 Chemical Rubber Company Press, Boca Raton, Florida
2. Behagel HA (1971) The pH and sterilization. In: pp 143-148
Van Bragt J, Mossel DAA, Pierik RLM, Veldstra H i0. Murashige T, Skoog F (1962) Physiol Plant 15:473-
(eds) Effects of sterilization on components in 479
nutr:ent media. Veenman H and Zonen N.V. Nether- ii. Pelet F, Hildebrandt AC, Riker AJ, Skoog F (1960)
lands, pp 117-120 Amer J Bot 47:186-195
3. Bridson EY (1978) Diets, culture media, and food 12. Singha S (1982) J Amer Soc Hort Sci 107:657-660
supplements. In: Rechcigl M Jr (ed) Handbook 13. Skirvin RM, Chu MC (1979) HortScience 14:608-610
series in nutrition and food, vol III, section G, 14. Young H, Skirvin RM, Juvik J (1983) Cucurbit
Chemical Rubber Company Press, Boca Raton, Genetics Coop Rept 6:56-57
Florida, pp 148-149