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British Phycological Journal

ISSN: 0007-1617 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tejp19

The use of antibiotics to obtain axenic cultures of


algae

A.K. Jones , Muriel E. Rhodes & Susan C. Evans

To cite this article: A.K. Jones , Muriel E. Rhodes & Susan C. Evans (1973) The use of
antibiotics to obtain axenic cultures of algae, British Phycological Journal, 8:2, 185-196, DOI:
10.1080/00071617300650211

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Br. phycol. Z8:185-196
30 June 1973

T H E U S E O F A N T I B I O T I C S TO O B T A I N
A X E N I C C U L T U R E S OF A L G A E
By A. K. JONES, MURIEL E. RHODES and SUSAN C. EVANS
Department of Botany and Microbiology, University College of Wales,
Penglais, Aberystwyth SY23 3DA

Attempts to obtain undamaged axenic cultures of six algae by means of short-term exposure
to various antibiotic mixtures were successful in four cases. Apparently pure cultures of the
two diatoms, Nitzschia capitellata Hust. and Amphora coffeaeformis var. perpusilla (Grun.)
Cleve were obtained after short exposure to penicillin + Ceporin + aureomycin, followed by
mechanical separation. No changes in cytology or pigmentation and growth characteristics
could be detected. Axenic Pediastrum boryanum (Turp.) Menegh. var. boryanum resulted after
short exposure to penicillin + tetracycline + chloramphenicol + aureomycin; further addi-
tion of neomycin, Ceporin or streptomycin here effected no advantage, and 20-0p.g/ml neomy-
cin was algicidal. For Chlorella vulgaris Beij. var. vulgaris, the addition of neomycin at 1.25-
20-0/xg/ml to the 4-antibiotic mixture above was necessary to eliminate a yeast. The Tribonema
viride Pasch. and Oscillatoria animalis J. Ag. cultures remained contaminated with Penicillium
and bacteria; the antibiotic sensitivity patterns of the residual bacterial flora are given, and
suggestions made for their elimination. The practical difficulties involved when trying to prove
the absence of all bacteria are briefly discussed.

Algae coated with mucilages are notoriously difficult to obtain in axenic cul-
ture; the associated micro-organisms may be many and various, with bacteria
especially abundant. Such associations have been shown by Lange (1971) to be
beneficial or even essential to the growth of the alga, and explicable in terms of
bacterial production of growth factors or CO2 subsequently incorporated by the
alga. Additional evidence suggesting a possible obligatory relationship was also
obtained during the attempted culture of pelagic diatoms by Droop & Elson
(1966); successful diatom culture was not achieved in the absence of all asso-
ciated bacteria. Thus, to elucidate such relationships, pure 'axenic' or mono-
bacterial cultures are obligatory, though not easy to achieve or maintain. A
typical large-scale continuous culture of Chlorella sorokiniana Shihira et Krauss
for example, was found by Litchfield, Colwell & Prescott (1969) to be heavily
infected with Pseudomonas, Acinetobacter, Flavobacterium and Bacillus bacteria,
even when grown at the selective temperature of 39 ° C.
Three major isolation methods, used singly or in combination, are applicable
to algae (see Droop, 1969): nutritional enrichment methods, mechanical manip-
ulations, and techniques involving the use of antibiotics. Fortunately, many
plant and animal eukaryotes are different in their resistance to antibiotics com-
pared with bacteria, and so algae may be purified if the algal material is clonal,
although antibiotics are advocated only when other methods fail, because of
possible damage to the algal cell. For this reason also, minimal antibiotic
exposure times and concentrations are desirable, and such have proved effective
for the purification of some physically-delicate and easily-damaged gelatinous
algae.
The antibiotics most widely used are penicillin, streptomycin and chloram-
phenicol. The wide range of bacteria found in algal culture renders a single
185
186 A.K. JONES, MURIEL E. RHODES AND SUSAN C. EVANS

antibiotic less efficaceous than mixtures, the components of which are now often
known to exert various modes of action, such as inhibition of bacterial cell wall
synthesis (penicillin) or impairment of substrate oxidation and inhibition of
RNA and protein synthesis on 70S ribosomes (neomycin, tetracyclines and
chloramphenicol). Thus knowledge of the identity of the associated bacteria
should be helpful in the formulation of effective antibiotic mixtures, bearing in
mind that possible damage or alteration of the algae must be, ideally, non-
existent. Berland & Maestrini (1969), for example, found that aureomycin,
terramycin, polymyxin B and chloramphenicol were very efficient for purifying
their diatom cultures, but algal damage resulted. Their paper contains an excel-
lent review of much literature pertinent to this problem, and further extensive
experiments concerning the detailed action of 144 antibiotics on Euglena were
carried out by Ebringer (1972) and fully reported.
Preliminary attempts here to obtain bacteria-free cultures of two diatom
cultures, Nitzschia capitellata Hust. and Amphora coffeaeformis var. perpusilla
(Grun.) Cleve, by standard plating and micromanipulative techniques proved
unsuccessful. Adherent mucilage was demonstrable by negative staining of cell
suspensions with nigrosin, and confirmed by positive staining with 50~ alco-
holic Gentian Violet. McDaniel, Middlebrook & Bowman (1962) attempted to
overcome the same problem by exposure to detergent plus phenol solutions, but
when their technique was applied here the phenol proved lethal for the algae, yet
insufficient to eliminate bacteria. The use of ultraviolet light was also investi-
gated. Some of the bacterial contaminants of the Nitzschia culture were grown
on Tryptone Soy Agar and exposed for varying periods to a U.V. source emitting
at 235.7 nm. A 5 min exposure was sufficient to prevent any visible bacterial
growth, but even a 10 min exposure of a thin layer of the contaminated Nitzschia
culture in a liquid medium did not result in bacteria-free cultures. Furthermore,
the longer exposure time resulted in morphologically-aberrant diatoms. Thus
both methods resulted in damaged algae still contaminated with viable bacteria.
Antibiotic methods were therefore necessarily but cautiously investigated:
Droop's (1967) successful techniques with a wide range of algae and inverte-
brates depended on exposure to high concentrations of antibiotics for a short
time. He claimed that there was 'seldom any difficultyin purifying the organisms;
the hard part is continuing to grow them afterwards'. His diatom mixture V was
therefore applied to cultures of Nitzschia capitellata and Amphora coffeaeformis,
but even a 24 h exposure resulted in bleached chloroplasts in both diatoms.
Similar findings were reported by Ebringer, Mego, Ju?a~ek & Kada (1969) for
Euglena gracilis, with 100~o bleaching after 24 h exposure to 500 ~g/ml
streptomycin. Krauss (1962) also presented data showing that streptomycin
was inhibitory to a wide range of algae between 1-40 /zg/ml, and so the
800-2000 ~g/ml streptomycin in Droop's mixture was deduced to be the prob-
able bleaching agent. Droop's diatom mixture was therefore significantly modi-
fied to contain only 2000 /zg/ml benzyl penicillin sulphate, plus 1600 t~g/ml
Ceporin (cephaloridine) plus 200 tzg/ml aureomycin, a mixture without strepto-
mycin and deliberately designed as least likely to affect eukaryotes. A short
exposure to this mixture yielded axenic cultures of both diatoms after subsequent
plating. Immediate dilution and streak-plating after a short exposure to the
antibiotics was essential because of the possibility of a few residual bacteria.
Purification of algal cultures 187

Thus bacteria-free algal cells were finally s e p a r a t e d m e c h a n i c a l l y on conven-


t i o n a l a g a r streak-plates. N o detectable changes o c c u r r e d in the algae, a n d they
grew well subsequently. T h e r e was no i n d i c a t i o n t h a t either d i a t o m culture was
altered b y the antibiotic t r e a t m e n t , in so far as t h e g r o w t h rates a n d m i c r o -
scopical a p p e a r a n c e s were n o r m a l , a n d the p i g m e n t s o f Amphora coffeaeformis
were unchanged, as j u d g e d b y their a b s o r p t i o n s p e c t r a a n d o n e - d i m e n s i o n a l
thin-layer c h r o m a t o g r a p h i c b e h a v i o u r . N u t r i t i o n a l studies also failed t o reveal
any a b n o r m a l i t i e s ; the o r g a n i s m s grew as before in the same m e d i u m (a m o d i -
fication o f D r o o p ' s $36 medium).
This success p r o v o k e d f u r t h e r a t t e m p t s to purify f o u r l a b o r a t o r y cultures o f
representatives o f different classes o f algae, viz. Oscillatoria animalis J. Ag,
C C A P No. B1459/6, Chlorella vulgaris Beij. var. vulgaris C C A P No. 211/1 l b .
Pediastrum boryanum (Turp.) Menegh. var. boryanum C C A P No. LB261/2, a n d
Tribonema viride Pasch. C C A P N o . LB 880/3, all originally o b t a i n e d f r o m the
Culture Collection o f A l g a e a n d P r o t o z o a m a i n t a i n e d at 36 S t o r e y ' s W a y ,
Cambridge, England.
M o d i f i c a t i o n s o f the p r o c e d u r e devised b y D r o o p (1967) were e m p l o y e d ;
various c o m b i n a t i o n s o f b r o a d - s p e c t r u m antibiotics such as c h l o r a m p h e n i c o l ,
tetracycline, s t r e p t o m y c i n a n d n e o m y c i n were used for s h o r t exposure p e r i o d s at
various low concentrations, t o g e t h e r with benzyl penicillin which is effective
only against G r a m - p o s i t i v e bacteria. C e p o r i n (cephaloridine), a specific bacteri-
cide with a structural nucleus similar to the penicillins, was also used because o f
its activity t o w a r d s some G r a m - n e g a t i v e as well as G r a m - p o s i t i v e p r o k a r y o t e s .
The short exposure p e r i o d was f a v o u r e d as less likely to e n c o u r a g e the develop-
m e n t o f antibiotic-resistant algal o r bacterial v a r i a n t s ; the l a t t e r were c o m -
m o n l y f o u n d b y Soli (1963) after 10-11 d a y exposures t o streptomycin, n e o m y -
cin, etc. S o m e o f the b a c t e r i a a s s o c i a t e d with the algae b o t h before a n d after the
a n t i b i o t i c t r e a t m e n t were isolated, purified a n d identified as far as possible, to
p r o v i d e m o r e detailed d a t a o n the extent a n d nature o f a n y a n t i b i o t i c resistance.
Such i n f o r m a t i o n s h o u l d e n a b l e m o r e r a t i o n a l modifications o f a n t i b i o t i c mix-
tures to increase their effectiveness, c o n c o m i t a n t with absence o f d a m a g e to the
host, as is p r a c t i s e d in clinical situations.

METHODS
BACTERIOLOGICAL STUDIES
The four stock cultures, Oscillatoria, Chlorella, Pediastrum and Tribonerna were grown in
Bold's borate-buffered, EDTA-chelated salts plus NaNO 3 basal medium (Deason & Bold,
1960). Loopfuts of each culture were streaked on to duplicated dried plates of glucose nutrient
agar, pH 7.2, and King's B glycerolpeptone agar, pH 7"2 (King, Ward & Raney, 1954). One
set of plates was incubated at 25 ° C and the other at 37° C for at least 7 days.
A variety of colonies was evident on the 25 ° C plates, and a random selection of 12 isolates
was made for purification and identification, using an incubation temperature of 25 ° C and
standard bacteriological procedures such as Gram-staining, flagella-staining, the determination
of colonial characters on various nutrient agar media, and the biochemical reactions of the
isolates towards gelatin, casein, nitrate, glucose, sucrose and lactose. Some of the relevant
experimental details are given in Table II, and Cowan & Steel (1965) may be consulted for full
details of standard bacteriological media and reagents. Spot tests for catalase and oxidase were
carried out on suitable young cultures, and thus the isolates were identified as far as possible
with reference to the descriptions given in the Bergey Manual (Breed, Murray & Smith, 1957)
and various later taxonomic papers. Information concerning the antibiotic sensitivity of the
isolates was necessary for identification purposes, as well as to supply data for future experi-
ments. Therefore Oxoid Multidisks (Oxoid Ltd., Southwark Bridge Road, London, S.E.1)
188 A. K. JONES, M U R I E L E. RHODES A N D SUSAN C. EVANS

and Evans Sentest antibiotic tablets (Evans Medical Ltd., Speke, Liverpool) were used on pour-
plates of all isolates, and zones of inhibition recorded. For details of the concentrations of the
antibodies used, see Table III.
After the various antibiotic treatments of the algal cultures (detailed below), 1 ml volumes of
all the treated cultures were tested for sterility by the pour-plate method using molten cooled
glucose nutrient agar, and incubating the set plates aerobically at 25 ° C for at least 5 days. Any
resultant growth was purified and characterised using the above procedures.

ANTIBIOTIC EXPERIMENTS
10 ml volumes of each of the four algal cultures in Bold's basal medium (Deason & Bold,
1960) were inoculated aseptically into 100 ml fresh sterile medium in cotton-wool-plugged
Erlenmeyer flasks. These test cultures were incubated for 2 weeks at room temperature (c.
1 6 ° C ) o n a west-facing window-sill.

(a) Experiments with C h l o r e l l a , P e d i a s t r u m and T r i b o n e m a


A sterile mixture of seven antibiotics was applied at six concentrations to 4.0 ml aliquots in
test-tubes of the above young test cultures, using a simple serial dilution method, and sterile
medium as the diluent. The final concentrations of the antibiotics in contact with each of the
three cultures is given in Table I. For convenience the antibiotics were weighed out, thoroughly
mixed, and stored at 4 ° C as 'dry mixes' ; they were dissolved only immediately prior to use, due
to the instability of some aqueous solutions of antibiotics. The dry antibiotic mixtures were
dissolved in 48"0 ml of Bold's basal medium plus 2.0 ml N/1 NaOH to effect solution. The
final solutions were filter-sterilised using Oxoid filter membranes of A.P.D. 0'45/~m.
Further cultures were similarly prepared for all three algae using a streptomycin-free anti-
biotic mixture of the remaining six antibiotics at various concentrations (see Table I for details).
Mixtures minus both streptomycin and neomycin, and minus streptomycin, neomycin and
Ceporin (see Table I) were also employed. All four series of the three algae were incubated on
the west-facing window-sill for 24 h.
After the above exposure to the various antibiotics, 2.0 ml volumes of each treated culture
were inoculated into 8.0 ml sterile Bold's basal medium. These cultures were incubated a
further 2 weeks to allow growth of the algae, plus or minus bacteria. Pour-plate sterility tests
were then carried out on 1-0 ml volumes from each tube, as described above. Algal viability was
checked by microscopic examination of all cultures for green, normal-looking cells, and asso-
ciated bacteria. Further tests for algal viability were made by streaking a loopful of culture
from tubes A and B (highest antibiotic concentrations) on to proteose agar slopes (Difco
Proteose Peptone, 0"1; KNO3, 0.02; K~HPO4, 0.002; MgSO4.7H20 , 0.002; Difco agar 1"0; all
~/o w/v in distilled water, pH 7"0-7"2), which were incubated on the window-sill.

(b) Experiments with O s c i l l a t o r i a


As a prokaryote, Oscillatoriamore closely resembles bacteria and this similarity may include
antibiotic sensitivity. Therefore an 8-tube serial dilution experiment using much lower
concentrations of antibiotics than heretofore (for details, see Table I), was carried out using the
same basic technique.
In all experiments the original algal cultures were also further incubated in the absence of
antibiotics for control purposes.

RESULTS
BACTERIOLOGICAL STUDIES
W h e n t h e f o u r o r i g i n a l s t o c k c u l t u r e s w e r e s t r e a k e d o n to g l u c o s e n u t r i e n t
agar (GNA) and King's B glycerol agar, no bacterial growth developed on any
p l a t e i n c u b a t e d at 37 ° C. A b l u e - g r e e n Penicillium sp. g r e w o n t h e G N A p l a t e
i n o c u l a t e d f r o m the Tribonema c u l t u r e , b u t all t h e rest w e r e sterile. A t 25 ° C a
v a r i e t y o f b a c t e r i a , as j u d g e d b y c o l o n i a l a p p e a r a n c e , g r e w o n all p l a t e s f r o m all
f o u r c u l t u r e series. F r o m t h e 25 ° C G N A plates, 12 c o l o n i e s w e r e r a n d o m l y
selected, c h a r a c t e r i s e d a n d i d e n t i f i e d as far as p o s s i b l e ; t h e s e d a t a ( f o r 11 iso-
lates, b e c a u s e o n e s u b s e q u e n t l y died) a r e p r e s e n t e d in T a b l e s I I a n d I I I . T h e
results c o n c e r n i n g the p r o p e r t i e s o f t h e m i c r o - o r g a n i s m s identified f r o m t h e
Purification of algal cultures 189

sterility-test plates (which h a d been i n o c u l a t e d with 1-0 ml volumes o f the anti-


b i o t i c - t r e a t e d cultures) are given in Table IV. I t is evident t h a t a l t o g e t h e r a
variety o f m i c r o - o r g a n i s m s , yeasts, m o u l d s , G r a m - p o s i t i v e a n d G r a m - n e g a t i v e
bacteria, r e m a i n e d in the cultures, b u t a p p a r e n t sterility was achieved for s o m e
o f the Pediastrurn a n d Chlorella cultures when certain c o n c e n t r a t i o n s o f anti-
biotics were employed. A t lower concentrations, g r o w t h f r o m the Pediastrum
cultures consisted o f a m i x t u r e o f G r a m - p o s i t i v e a n d G r a m - n e g a t i v e r o d s a n d
yeasts. The Chlorella cultures yielded an a b u n d a n c e o f b o t h yeasts a n d o r a n g e
colonies o f isolate T y p e N o . 1, it was strange, however, t h a t only f r o m the m o s t
c o m p l e x a n t i b i o t i c mixture were bacterial colonies visible. F r o m the Tribonema
cultures, a heavy g r o w t h o f a blue-green Penicillium sp. o b s c u r e d bacterial
g r o w t h on all plates, a n d it was f o u n d that, when tested in p u r e cultures, this
Penicillium inhibited the g r o w t h o f the associated b a c t e r i a (Types 8 a n d 9, a n
Achromobacter sp.).

TABLE I. Final concentrations of antibiotics (/~g/ml) present in cultures of


(a) Chlorella, Pediastrum and Tribonema; (b) Oscillatoria

Series (a) 1. Dilution tube


t
Antibiotic A B C D E F

Benzyl-penicillin-SO4 2000 1000 500 250 125 62-5


Tetracycline 500 250 125 62.5 31.25 15.6
Chloramphenicol 60 30 15 7-5 3.75 1-9
Aureomycin 50 25 12-5 6.25 3"12 1-55
Ceporin 400 200 100 50 25 12-5
Neomycin-SO4 10 5 2-5 1.25 0"6 0"3
Streptomycin-SO4 10 5 2'5 1-25 0.6 0"3
Series (a) 2. No Streptomycin

Benzyl-penicillin-SO4 4000 2000 1000 500 250 125


Tetracycline 1000 500 250 125 62-5 31.25
Chloramphenicol 120 60 30 15 7'5 3.75
Aureomycin 100 50 25 12-5 6.25 3-I
Ceporin 800 400 200 100 50 25
Neomycin-SO4 20 10 5 2-5 1"25 0.6
Series (a) 3. No Streptomycin or Neomycin

Details as for Series (a) 2 except for omission of Neomycin-SO4


Series (a) No 4. Streptomycin, Neomycin or Ceporin

Details as for Series (a) 2 except for omission of Neomycin-SO4 and


Ceporin
Series (b), for Oscillatoria only

An 8-tube (A to H) twofold dilution series as above containing Benzyl-


penicillin SO4 from 1"0 to 0-007, plus Chloramphenicol from 40-0 to 0"313,
plus Neomycin from 16"0 to 0"125 (all/zg/ml)
TABLE II. The properties and identification of the bacteria isolated f r o m the four stock cultures o f algae.
A n incubation temperature o f 25 ° C was used t h r o u g h o u t

Isolate n u m b e r s
Property 1 &2 3 8&9 4&ll 7 13 10 12

Source 1. Ped; 2, Chlor Ose Trib Osc Ose Trib Osc OSC
Gram- G m + rods G m - - rods G m - - rods G m - - rods G m - - rods G m - - rods Gm + Gm+&--
staining 1'5-3.0 x 0 ' 4 ~ m 1'5-2.5 x 1"5-3"0 × 1.0-1.5 x 1.5-2-0 x & filaments filaments cocci in
N o branching in 0.5 p,m 1.0/.Lm 0'4/~m 0.5/zm common 0-8 F,m wide, tetrads,
y o u n g cultures Capsules 1"5-20'0 x branched 1.0 x 1-5/~m,
also pairs
Acid-fastness _ _ 0.8 p,m
(to 5 % & 15%
H~SO4)
G N A colonies Pale orange Cream S Cream S Cream S Cream S Rust-brown Dull white Sulphur
at 3 days S. 1.0 m m 1 "0 m m 1 "0 m m 1"0 m m pin-point S; adhesive powdery yellow S
pin-point 1"0 m m 2.0 m m
Anaerobic . . . . . . .
growth
G r o w t h at 41 ° C . . . . . . + +
Catalase + + + + + + + +
Oxidase -- + + + + + + +
Motility -- ? + + + + _
Flagellation -- -- Peri Polar Peri Peri --
(capsules) 1-5 1-4 1-5 3-8
G r o w t h with + . . . . . . +
5 % NaC1
H u g h & Leifson
Glucose Ox A~ Ox At4 -- Ox A 1 -- Ox A 7 --
Lactose -- -- -- Ox Aa0 -- -- --
Sugar broths
Glucose A14 -- -- A2 -- -- Ala --
Lactose A14 . . . . . . .
Sucrose A14 . . . . . A14 --
NO~ to NOz + -- -- + -- (+) + --
NO 3 to N 2 -- -- -- + . . . .
Litmus milk ALK R N pellicle ALK N ALK ALK R ALK N
Orange pellicle White
pellicle powdery
pellicle
V.P. & M.R. -- . . . . . . .
G r o w t h in +14 -- ? +1 +5 -- -- --
Koser's citrate
H~S from cysteine -- + -- -- + -- -- --
Gelatin hydrolysis . . . . . . . +
Starch hydrolysis . . . . . + -- +
Arginine dihydrolase
Aerobic -- + + + + + -- --
Anaerobic -- -- -- + . . . .
Sensitivity to
Streptomycin + + -- + + -- + +
Terramycin + + + + 4- + + +
Penicillin (+) . . . . . . +
0/129 vibriostat* . . . . . . . +
Identification Coryne- Achromo- Achromo- Pseudomonas Achromo- Flavo- Strepto- Sarcina
bacterium bacter s p . bacter s p . putida* bacter sp. bacterium myces s p . lutea
sp. (I) (II) (III) sp

KEY: Ped = Pediastrum, Osc : Oscillatoria, Trib = Tribonema, Chlor : Chlorella. * : v i b r i o s t a t 2, 4, - d i a m i n o - 6 , 7-di-iso-propyl p t e r i d i n e
f r o m A l l e n & H a n b u r y , W a r e , E n g l a n d . t = G r e e n - f l u o r e s c e n t p i g m e n t t y p i c a l o f Pseudomonas p r o d u c e d o n K i n g ' s B a g a r . U n d e r
colonies, S = shining, smooth surface; measurement refers to colony diameter. Peri : peritrichate flagella, Ox ~ oxidative attack on
sugar, A = acid reaction, and the subscripts indicate the number of days incubation for a positive reaction. Under litmus milk, ALK :
alkaline reaction, R = reduction, N = neutral reaction.
q- = p o s i t i v e r e a c t i o n ; ( + ) = w e a k l y p o s i t i v e r e a c t i o n ; - - : n e g a t i v e r e a c t i o n .
A l l c u l t u r e s w e r e i n c u b a t e d u p t o 14 d a y s , e x c e p t t h a t n i t r a t e d e t e r m i n a t i o n s , g e l a t i n h y d r o l y s i s a n d V . P . t e s t s w e r e d e t e r m i n e d a f t e r
both 3 and 7 days also. Gram-stained preparations from 24 h nutrient agar cultures. Flagella-stained preparations from 24 h cultures on
n u t r i e n t a g a r + w a t e r s l o p e s ( R h o d e s , 1958).
192 A.K. JONES, MURIEL E. R H O D E S AND SUSAN C. E V A N S

TABLE III. The antibiotic sensitivity of the bacteria isolated from the
four stock cultures of algae

Isolate number
l&2 3 8&9 4&ll 7 13 10 12

Using Oxoid
Multidisks with/zg
Ampicillin 5 (+) . . . . . (6-) +
25 + 6- -- _ 6- + + +
Cepl~aloridine 5 6- . . . . . . 6-
Chloramphenicol 10 ÷ 6- -- _ _ 6- 6-
50 + + 6- + 6- 6- 6- +
Cloxacilli'n 5 (+) (+) . . . . (+) +
Colistin 50 (+) 6- -- 6- 6- -- 6- 6-
200 + 6- -- + + 6- 6- +
Erythromycin + 6- + -- _ 6- 6- 6-
Fusidic acid 10 6- + . . . . 5 6- 6-
Kanamycin 5 6- + (+) + + + 6- 6-
30 6- 6- + + + + + 6-
Lincomycin 2 6- 6- . . . . 6- 6-
Methicillin 10
Nitrofurantoin 200 6- + (6-) -- (+) + + +
Neomycin 10 6- + 6- 6- 6- 6- + 6-
Novobiocin 5 6- + + -- _ 6- + 6-
Penicillin G 1"5 U . -- . . . . . . 6-
S t r e p t o m y c i n I0 + 6- -- + 6- -- 6-
25 + + -- 6- 6- -- 6- +
Sulfaf'urazole 100 + + (+) -- _ 6- -- +
500 + + (4-) -- (+) + -- 6-
Tetracycline 10 + + + + 6- + + (+)
,, 50 6- 6- 6- + + 6- + 6-

Using Evans
Sentest tablets
Streptomycin (low) 6- + -- + 6- -- + +
Streptomycin (high) + 6- -- + 6- -- 6- +
Terramycin (low) + 6- 6- + 6- 6- 6- 6-
Penicillin (high) (6-) . . . . . . 6-

KEY: 6- = isolate sensitive to the antibiotic;


( + ) = slightly sensitive;
- - = c o m p l e t e resistance s h o w n .

ANTIBIOTIC EXPERIMENTS
The results of the sterility tests after the exposure of the four algae to various
concentrations of various mixtures of antibiotics are summarised in Table IV.
They have already been discussed in the previous section.
V i a b i l i t y o f t h e a l g a e w a s c h e c k e d i n t w o w a y s , m i c r o s c o p i c a l l y a n d cul-
t u r a l l y . T h e r e s u l t s o f t h e m i c r o s c o p i c a l e x a m i n a t i o n o f all s u b - c u l t u r e s f o l l o w -
i n g a n t i b i o t i c t r e a t m e n t a r e s u m m a r i s e d i n T a b l e IV. I n all c a s e s s o m e b r o w n i s h
cells w i t h d e g r a d e d c h l o r o p h y l l , t o g e t h e r w i t h cells o f n o r m a l h e a l t h y a p p e a r a n c e ,
w e r e p r e s e n t a t all t h e h i g h e r a n t i b i o t i c c o n c e n t r a t i o n s . O n l y i n t h e Pediastrum
c u l t u r e (a) 2 A w e r e n o v i a b l e a l g a l cells f o u n d ; t h e y w e r e p r e s u m a b l y d e s t r o y e d
b y t h e 20.0 t~g/ml n e o m y c i n , b e c a u s e , w h e n t h i s w a s o m i t t e d , h e a l t h y a l g a e w e r e
present at the corresponding concentrations for other antibiotics.
A f t e r 14 d a y s i n c u b a t i o n o f t h e s t r e a k e d p r o t e o s e a g a r s l o p e s , Chlorella c u l -
t u r e s f r o m all f o u r e x p e r i m e n t s g r e w well. N o g r o w t h o f t h e Pediastrum w a s
Purification of algal cultures 193

TABLE IV. Antibiotic treatments of four algae. The results of the sterility tests from 1.0 ml
volumes of the antibiotic-treated cultures plated with glucose nutrient agar and incubated
for 5 days at 25 ° C, together with the microscopical appearances of the algae

Culture
Antibiotic Dilution r
mixture tube Pediastrum Chlorella Tribonema Oscillatoria
Bact. Algae Bact. Algae Bact. Algae Bact. Algae
No. (a) 1 A • + Ys + + F,W ++
(7 components) B • ++ Ys, O +++ F,W +++
C-F O +++ Ys, O +++ F,W +++
No. (a) 2 A • -- • + F,W +
(6 components, B • + • ++ F,W ++
no streptomycin) C • +++ • +++ F,W +++
D • +++ • +++ F,W +++
E,F Y,Q,W +++ • +++ F,W +++
No. (a) 3 A • ++ Ys + + F,W +
(5 components, B • ++ Ys +++ F,W ++
no streptomycin C • +++ Ys +++ F,W +++
or neomycin) D • +++ Ys +++ F, W +++
E, F Y, O +++ Ys +++ F,W +++
No. (a) 4 A • + Ys + + F,W +
(4 components, B • ++ Ys +++ F,W ++
no streptomycin, C • +++ Ys +++ F,W +++
neomycin or D • +++ Ys +++ F,W +++
Ceporin) E, F O +++ Ys +++ F,W +++
No. (b)
(Oscillatoria A-H W +++
only, 3
components)
Original stock Y,O,W +++ Ys, O +++ F,W,Y +++ W,Y +++
cultures

KEY" • signifies no growth; O = orange bacteria, Corynebacterium type (see Table II);
Ys = yeasts; W = white bacteria, Achromobacter spp. ; Y = yellow bacteria, Flavo-
bacterium sp. in the case of Tribonema and Sarcina lutea in the Oscillatoria culture;
F = Fungus, a blue-green Penicillium sp. with septate hyphae 3"3 p.m wide and green
conidia 3"0/~m diameter.
- - signifies that no healthy green algal cells were present; + = some, + + = large
numbers, and + + + = normal bright green cells as in the controls.
For details of the antibiotic mixtures used, see Table L

o b t a i n e d , a l t h o u g h t h e c o n t r o l g r e w well. T h e Tribonema slopes showed a dense


o v e r g r o w t h by t h e b l u e - g r e e n Penicillium sp. w h i c h w a s also p r e s e n t in t h e
o r i g i n a l s t o c k c u l t u r e . A l l t h e Oscillatoria c u l t u r e s g r e w well. No cytological
a b e r r a n c e s w e r e d e t e c t e d in all t h e t u b e s C t o F o f all f o u r a l g a e when they were
compared with the original cultures.

DISCUSSION
T h e s e a t t e m p t s to e l i m i n a t e t h e a s s o c i a t e d m i c r o - o r g a n i s m s f r o m algal cul-
tures by m e a n s o f a n t i b i o t i c m i x t u r e s w e r e successful f o r f o u r o u t o f t h e six cul-
tures tested. T h e Tribonema r e m a i n e d h e a v i l y i n f e c t e d w i t h t h e b l u e - g r e e n
Penicillium sp., a n d a h e a v y i n c i d e n c e o f residual yeasts was r e c o r d e d f r o m m o s t
194 A.K. JONES, MURIEL E. RHODES AND SUSAN C. EVANS

of the Chlorella cultures. This suggests that the inclusion of a specific fungicide
such as Actidione, or a polyene antibiotic such as nystatin or amphotericin B,
might be advantageous. Actidione is certainly actively antifungal (2-3 /~g/ml
inhibits Penicillium according to Booth, 1971) but it is unpromising here because
Berland & Maestrini (1969) reported a complete cell mortality for 5 species
of diatoms at an Actidione concentration of only 5.0/~g/ml, and Krauss (1962)
lists growth inhibition of representatives of the Chlorophyceae, Xanthophyceae
and Bacillariophyceae even at 1-2/zg/ml. More promising is nystatin, and other
polyene antibiotics such as amphotericin B, which are known to bind specifically
to sterols located on fungal cell membranes, leading to membrane damage and
cell lysis (Franklin & Snow, 1971). Digitonin also complexes with sterols and
instantly lysed fungal protoplasts (Villanueva & Acha, 1971). Booth (1971)
reviewed the literature on antifungal antibiotics and recommended nystatin,
pimaricin, trichlorodinitobenzene, Endomycin, GS-388 (a mercapto-pyridine
derivative) or HRS 1950. A search of the mycological Volume 4 of Methods in
Microbiology (1971) resulted in finding no other successful fungicides, other than
the satisfactory use of propionate for the selective inhibition of mould growth in
yeast cultures (Beech & Davenport, 1971). Franklin & Snow (1971) mention
that griseofulvin and oligomycin show a useful, but very limited range of anti-
fungal activity. Currently, the new antimycin is most promisingly selective, and
inhibits the growth of many yeasts and moulds by inhibiting cytochrome-
mediated respiration in some way which leaves bacterial pathways unaffected. If
however this antibiotic blocks mitochondrial enzyme complexes, as has been
suggested, its use with algae is likely to be damaging.
Yeast-free healthy cultures of Chlorella were obtained with the streptomycin-
free antibiotic mixture No. 2. It was surprising that the richer mixture, No. 1, did
not eliminate the orange coryneform bacteria; it may have been due to anta-
gonistic effects in the antibiotic mixtures. A comparative scrutiny of Table IV
shows that the neomycin in mixture No. 2 apparently inhibited the yeast growth,
and so its inclusion at concentrations of 1.25-20.0/zg/ml was vital in producing
bacteria- and yeast-free cultures of Chlorella, and its wide antibacterial activity
is evident in Table III.
For Pediastrum, the antibiotic mixture No. 1 was again unexpectedly less
effective than the rest for obtaining bacteria-free cultures; 20.0/zg/ml neomycin
destroyed the Pediastrum. Axenic cultures were however obtained even from the
limited antibiotic mixture No. 4 containing only penicillin + tetracycline +
chloramphenicol + aureomycin; thus the addition of Ceporin, neomycin or
streptomycin effected no advantages in this case.
Antibiotic treatment of Oscillatoria successfully eliminated bacteria except
for the green-fluorescent pseudomonads, common aquatic organisms and
notoriously antibiotic-resistant. All Pseudomonas-infected cultures of Oscilla-
toria remained healthy and viable, and might well tolerate higher concentrations
of antibiotics than those used here, but polymyxin B or one of the new peni-
cillins such as Carbenicillin (specifically developed to combat human Pseu-
domonas infections), Kanamycin or Colistin (see Table III) would probably
eliminate the residual fluorescent pseudomonads. Kanamycin, however, causes
chloroplast bleaching and may induce the growth of chloroplast-deficient
strains, as reported for Euglena by Ebringer (1972). Polymyxin B has been used
Purification of algal cultures 195

in algal cultures: Soli (1963) for example, found that it was effective and rela-
tively non-toxic when tested on his three diatom cultures; 50/~g/ml caused no
visible damage, and bacterial growth was reduced but not eliminated, because
polymyxin-resistant mutants were recorded in three out of his five Chaetoceros
and Asterionella cultures. His 10-11 day exposure, however, would enhance the
establishment of such antibiotic-resistant bacteria and permanent algal damage,
so Droop's one-day exposure time is preferable, with the inclusion of polymyxin
(up to 50.0/~g/ml). However, as Berland & Maestrini (1969) emphasise, it is not
possible to forecast accurately the response of an alga towards any antibiotic,
because detailed conditions such as medium constituents, pH, Eh, presence of
glucose, density of algal cells and mucilages, are such modifying influences. Thus
the data in Table III cannot be accepted categorically. Krauss (1962) indeed
cites the finding that polymyxin B was much more toxic to Chlorella pyrenoidosa
than to C. vulgaris, and so, unfortunately, each species must be individually
tested.
It is preferable to employ antibiotics for culture purification only when
mechanical procedures fail, and then only at minimal concentrations and for
minimal periods. Hesitancy in their use, because of possible mutagenic action
towards the algae, is prudent, but, in fact, three of the natural associants found
in these algal cultures, i.e. the Penicillium sp., the Streptomyces sp. (isolate No.
10, see Table II) and the Pseudomonasputida (isolates Nos. 4 and 11), themselves
produced antibiotics, whose activity was revealed when tested against the
Achromobacter sp. (isolates 8 and 9). Pseudomonas antibiotics are well known
and potent, and probably account for the absence of other bacteria in the
Oscillatoria cultures here (see Table IV). The action of antibiotics is now better
understood (see Franklin & Snow, 1971), and so they may be more carefully and
rationally selected, and the most likely sites of damage particularly scrutinised,
as was done here with respect to the detailed pigment characteristics of Amphora
coffeaeformis before and after purification with the simple penicillin + Ceporin
+ aureomycin mixture. It is noteworthy that axenic cultures were obtained in the
absence of streptomycin, a well-documented chloroplast bleaching agent.
It is worth emphasising that in these experiments, and indeed in most of those
published, although there was no cytological evidence (from Gram-stained
preparations and examination of wet mounts) that any bacteria remained, there
was no absolute proof that any culture was finally strictly bacteria-free. Test
conditions were such that only the relatively common aerobic bacteriological
'weeds' would be revealed by the sterility tests employed. Anaerobic and nutri-
tionally-fastidious heterotrophs, and photosynthetic or chemoautotrophic
bacteria (although not seen microscopically) might remain associated with the
algae in a dormant state. Considerable amenities and bacteriological expertise
would be necessary to demonstrate their presence. Paasche (1971) for example,
claimed the separation of red photosynthetic bacteria from Euglena cultures by a
simple pipette method, but his 'sterility tests' were not adequate for the demon-
stration of growth from any such residual anaerobic photosynthetic bacteria. In
practice, it is impossible to prove the absence of all bacteria by growth tests;
there are many aquatic organisms which we are still unable to grow on labora-
tory media. To check for the presence of all known types of bacteria in the lunar
soil samples, the basic test protocol involved the use of at least 166 cultures
196 A . K . JONES, MURIEL E. RHODES AND SUSAN C. EVANS

(excluding controls a n d replicates), representing several m e d i u m c o m b i n a t i o n s ,


adjusted to various p H values, a n d i n c u b a t e d at different temperatures in b o t h
the light a n d the dark, in a wide range o f gaseous e n v i r o n m e n t s , see Taylor,
F e r g u s o n & T r u b y (1970) for details. Even so, n o n e of those conditions w o u l d
have allowed the growth of, for example, Leucothrix, one o f the most c o m m o n
bacteria epiphytic o n algae. Such bacteria, u n a b l e to grow on the usual 'sterility
test' media, should however n o t be overlooked as possible i m p o r t a n t symbionts,
perhaps providing growth factors, antibiotics, or acting as physico-chemical
buffering or detoxifying agents in these m o s t interesting algal associations which
are p r o v i n g so deceptively difficult to investigate.

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