The Effect of Carbon Dioxide on Spore Germination in Some Clostridia

HOLLAND, DIANA. BARKER, A. N. AND WOLF, J. (1970). J . appl. Bad. 33. 274-284.

The Effect of Carbon Dioxide on Spore Germination in Some Clostridia

DIANA HOLLAND, A. N. BARKER AND J. WOLF

Division of Food and Agricultural Bacteriology, Department of Bacteriology, The University of Leeds, England

(Received 13 July 1969)

SUMMARY. The rate and extent of spore germination in 3 cultures of clostridia have been investigated under controlled gas environment conditions. Germination in each of them was markedly stimuleted by carbon dioxide in a variety of media. Quantitative memure- ments on 2 of the cultures indicated that while Cloatrddium bifermentana gave rapid and complete germination with as little &s 3 m?d bicarbonate, CZ. sporogenea PA 3879/S, required higher concentrations for maximal response.

A CAREFUL SEARCH of the literature concerning the effect of CO, or bicarbonate on the germination of spores of clostridia indicates the lack of a consistent behaviour pattern. In fact, for certain specific strains the reported findings would appear to be contradictory.

Evidence for an essential or distinctly stimulatory role would seem to have been confirmed for Clostridium botulinum 62A by several workers (Wynne & Foster, 1948; Andersen, 1951, 1952; Treadwell, Jann & Salle, 1958). In the following group of organisms an apparently satisfactory rate of germination has been recorded in the absence of additional CO,: Cl. chuvoei, Cl. histolyticum. Cl. welchii (Wynne & Poster, 1948), Cl. bifermentans (Gibbs, 1964, 1967), C1. botulinum 33A (Kuritza-Olejnikow & Grecz, 1965) and Cl. botulinum type E (Ward & Carroll, 1966). A good example of contradictory findings by independent workers is represented by Cl. sporogenes PA 3679/h. Thus, while Riemann (1963) and Uehara & Frank (1965) successfully dispensed with the addition of CO,, Lund (1957) did not obtain satisfactory germination without it.

Our own preliminary observations with Cl. sporogenes PA 367915, (Wolf, unpublished) clearly indicated the potent stimulatory effect of CO, on germination. It was thus of some interest to study the conditions affecting the operation of thia factor and to compare this organism with others in which a specific CO, effect had not been clearly manifested.

Materials and Methods Origin of cultures

The organisms used in these investigations were: C1. sporogenes strain PA 3679/S,, supplied by Dr T. A. Roberts, Meat Research Institute, Bristol; C1. sporogenes strain PA 3679/h, supplied by Dr H. A. Frank, Honolulu, Hawaii, and Cl. bqermentuns strain CN 1617, supplied by Dr P. A. Gibbs, Ministry of Agriculture, Elmwood Avenue Belfast.

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Effect of carbon dioxide on spore germination in clostridia 275

P r e p r u t h of spore swpensions Solid sporulation media were used for the production of spore c r o p because they gavc cleaner suspensions than did liquid media. An 18 h broth culture was used to inoculate the surface of the agar which was sloped in 4 oz flat-sided bottles and kept horizontal during anaerobic incubation a t 37". After 5 days the spore crops were harvested and washed 10-15 times with successive 20 ml quantities of water. The resulting suspension of spores and vegetative cells was stored a t 4" until autolysis of the latter was complete. Vegetative debris was then removed by rewashing the spores 3 times. The final suspension contained c. loe spores/ml and < 1 % of vegetative cells.

Culture P A 3679/S, Spores were produced on RCM Agar (Oxoid) supplemented with nickel sulphate,

cobalt sulphate, managanese sulphate, and calcium chloride, each at a final concentration of 10 p/m.

Culture P A 3679/h The sporulation medium consisted of the supernatant fluid from meat broth

(Southern Group Hospital Laboratory, Hither Green, London, S.E.13.) solidified with 1.5% agar.

Clostridium bifermentans Spores were produced on RCM agar.

Heat shock treatments Spores were heat shocked before each experiment aa follows: (a) PA 367915, a t SOo/10 min in water; (b) PA 3679/h a t SOo/l0 min in 400 mM sodium phosphate buffer, pH 7.4; (c) CZ. bifermentans a t 8So/l0 min in 500 mM phosphate buffer, pH 7.4.

Germination media Each of thc 3 organisms was tested in 2 types of germination medium. The first, based on extracts of natural organic substances, is referred to as the complex medium; the second, based on chemically defined ingredients, as the synthetic medium.

Complex medium E'or the 2 strains of PA 3679 the complex medium, freshly prepared for each experi-

ment, contained (yo w/v) : Brain Heart Infusion (Difco dehydrated) 5.0; yeast extract (Oxoid) 0.4; in 50 mM sodium phosphate buffer, pH 7.4. For C1. &fermentam the casein digest medium (CMB) of Gibbs (1964) was supplemented with 2% of yeast extract (Oxoid) and 100 mM phosphate buffer, pH 7.4.

Synthetic medium The medium for PA 3679/S, contained; 50 rrml L - a l h e (B.D.H.); 10 mIu lactate

(20% solution of lactic acid, Koch Light Laboratories); 60mM sodium phosphate

276 Diane Holland, A. N. Barker and J. Wolf

buffer, pH 7.0. For PA 3679/h it contained: 100 mna L-alanine; 20 m~ lactate ; 100 mM phosphate buffer, pH 7.4. For CI. bifermentuns the phenylalanine-alanine-lactate medium of Gibbs (1964) was used.

Neutralized solutions of the lactic and amino acids were stored a t -20". Stored solutions were de-aerated by immersion in boiling water before use.

Sodium bicarbonate solutions These solutions (usually at 1.2 M) were freshly prepared for each experiment.

Determination of the rate of germination The extent of germination was determined both nephelometrically, as changes in O.D., and microscopically, as changes in phase brightness. Nephelometric measure- ments were made with a Model 9 Nepho-Colorimeter (Coleman Instruments Inc., Maywood, Ill., US. A.) .

Tube experiments Tubes (20 x 155 mm) were used and germination was generally assessed nephelometrically. The total volume of germinants and spores was 10 ml/tube. Apart from the use of freshly autoclaved or preheated media, no special precautions were taken to exclude oxygen or carbon dioxide. (To facilitate presentation of results, experiments of this type are illustrated in the figures by means of unbroken lines whilst broken lines signify the controlled gas environment experiments described below.)

Controlled gas environment experiments These investigations were carried out in a specially designed 'H' shaped glass vessel (Fig. 1). The longer arm of the vessel consisted of a Coleman nephelometer tube and the shorter arm housed the pH and Eh electrodes. The electrode system consisted of a combined glasslreference electrode and a separate platinum electrode. All readings were taken by means of a Pye pH/millivolt meter, model 78.

Both arms of the vessel were fitted with gas inlet tubes long enough to discharge into the medium; only the longer arm possessed a gas outlet. Introduction of spores and sampling for phase contrast measurement was effected by means of a Pasteur pipette inserted through the gas outlet. For nephelometric readings the medium was tipped into the longer arm.

In these experLents anaerobic conditions were obtained by the passage of oxygen- free nitrogen (White Spot quality, British Oxygen Company, London). The gas was passed through a series of Drechsel bottles containing alkaline pyrogallol, concentrated sulphuric acid, barium hydroxide and distilled water. The gas flow system had provision for the introduction of CO, which was passed through water alone. Nitrogen was passed a t c. 500 ml/min and CO, a t a rate which maintained a stable pH and compensated for the loss of CO, from the bicarbonate medium. The germination medium (12 ml) was placed in the shorte rarm of the vessel and gas was passed for 2-3 h, a period required to achieve a steady Eh and pH. The spore suspension (3 ml) was then introduced into the longer arm. After a further 15 min of equlibration the experiment was started by tipping the spore suspension into the germination medium.


,-..-Gas outlet and sampling orifice

I

Combined glass/ reference electrode

Fig. 1. The spore germination tube.

Time (mid

Fig. 2. Static tube experiments. The effect of bicarbonate on the germinstion of spores of PA 3679/S, at 26' in the oomplex medium.

Closed triangles, 60,90,120,180,240 and 300 mM bicarbonate; closed ciroles, 30 m x bicarbonate; closed squares, 600 RIM bicarbonate; open triangles, bicarbonate omitted.

278 Diana Holland, A. N. Barker and J. Wolf

Results PA 367915,

Earlier observations on this strain had shown that addition of CO, or bicarbonate to both synthetic and complex media was essential for rapid and complete germination (Wolf, unpublished) and these findings have been confirmed.

In static tube experiments with the complex medium, in the absence of bicarbonate (Fig. 2) germination was relatively slow (30% in 30 min). Addition of 30 mM bicarbonate proved distinctly stimulatory, while 60 mM increased the rate even further. Higher concentrations (up to 600 mM) gave no additional stimulation.

In similar static tube experiments with the synthetic medium the need for bicarbonate was more obvious, and in its absence germination was extremely low (Fig. 3). Additions of 30-240 mM bicarbonate resulted in progressively increasing germination rates, but concentrations > 240 mM were suboptimal.

The CO, effect became more evident under controlled gas environment conditions (Figs 4 and 5), and in both types of media, for rapid and complete germination, there was an absolute CO, requirement. This is well illustrated by the stop-go experiment in the synthetic medium (Fig. 5).

PA 3679/h Uehara & Frank (1964) had not reported a CO, or bicarbonate requirement for the

‘h’ variant of PA3679, but in our experiments, using both complex and synthetic media, bicarbonate, with or without added CO,, substantially stimulated germination.

Time (rnin)

Fig. 3. Statio tube experiments. The effeot of bicarbonate on the germinetion of spores of PA 3679/S, at 26’ in the synthetic medium.

Closed triangles, 240 mx bioarbomte; squares, 120, 180 300 and 600 mM bicarbonate; oiroles, 60 and 90 mx bioarbonete; o m , 30 mM bicarbonate; open triangles, bicarbonate omitted.


4

I I 0 20 40 60 80

Time ( m i d

Fig. 4. Germination of spores of PA 3679/S, at 25" in the complex medium. Solidline, static tube experiment ; brokenlines, gas flow experiments; closedtriangles,

240 mM bicarbonate ; open triangles, bicarbonate omitted.

100 -

-

80 -

- - $ 60- - c 0

0 - ._ .- .-

E 8 4 0 -

t Y

- \

I I I I J 0 20 40 60 80

Time ( m i d

Fig. 6. Germination of S P O ~ S of PA 3679/S, at 25" in the synthetic medium. Solid line, static tube experiment ; brokenlines, gmflow experiments. Closed triangles,

240 mM bicarbonate ; open triangles, bicarbonate omitted.

280 Diana Holland, A. N. Barker and J. Wolf

Thus, in the controlled gas experiments germination in the complex medium was virtually CO, dependent and even in the static tube experiments the stimulatory effect of CO, is clearly apparent (Fig. 6).

Results in the synthetic medium were similar to those shown by the S2 variant (Fig. 6).

Clostridium bifermentans These experiments were extended to spores of C1. bijermentans, an organism for

which no CO, requirement had been reported and whose germination requirements had been investigated (Gibbs, 1964). It also had the advantage of differing biochemi- cally from the previous 2 proteolytic strains.

Time (min)

Fig. 6. Germinstion of spores of PA 3679/h at 37" in complex medium. Solid lines, static tube experimente; broken lines, ges flow experiments. Closed

triangles, 120 mM bicarbonate; open triangles, bicarbonate omitted.

In static tube experiments using the synthetic medium, a comparison of the germination rates of the 3 organisms, in the absence of added bicarbonate, shows that Cl. bifermenhna germinates faster than the 2 proteolytic strains and is even responsive to low concentrations of bicarbonate (Figs 3, 6, 7). The stop-go experiment under controlled gas conditions convincingly illustrates the powerful stimulatory effect of CO, (Fig. 8). These rates of germination were virtually reproduced in similar experiments using the complex medium.

Additional experiments in synthetic mediu It has been shown that a combination of alanine, phenylalanine, lactate and bicarbonate represents a highly effective germination medium for Cl. biferwntarm and that the omission of bicarbonate results in a marked fall in both rate and extent of germina-


tion (Figs 7, 9E, G). The absence of lactate from this system resulted in a similar level of reduction, but the simultaneous exclusion of both lactate and bicarbonate virtually suppressed germination (Fig. 9B, D).

In comparison with the significant role of lactate in this system, that of phenylalanine proved of secondary importance (Fig. 9C, F). It is clearly evident that with this strain of C1. bijerermentans, L-alanine alone is a most ineffective germinant even when augmented by bicarbonate (Fig. 9A).

I 1 I I 1 0 20 40 60 80

Time(mln)

Pig. 7. Static tube experiments. The effect of bicarbonate on the germination of spores

Closed triangles, 6, 30, 60, 90, 120 and 180 mM bicarbonate; squeres, 1.2 mY of C1. b?@mwtane at 26' in synthetic medium.

bicarbonate; circles, 0.6 mar bicarbonate; open triangles, bicarbonate omitted.

In contrast to the above organism, spores of PA 3679/S, responded moderately well to the combination of L-alanine + bicarbonate, although L-alanine alone was completely ineffective (Fig. 10A, C). The important role of bicarbonate is even more clearly evident when tested in the presence of lactate (Fig. 10B, D), where its addition converts a virtually negligible response into one which is both rapid and complete (90% in 10 min).

Discussion The distinct stimulation of germination by CO, shown by esoh of the 3 clostridia was more obvious in controlled gas experiments than in open tubes. In the latter, 2 factors could interfere with the full manifestation of its effect : (i) the presence of Co,

282

ia

8

- E s E d 4

c 0

0 .- c

._

2

Diana Holland, A. N. Barker and J. Wolf

A -4

I 40 60 80 20

Time (min)

Fig. 8. Germination of spores of C1. bifermentane at 26' in synthetio medium. Solid line, static tube experiment; broken lines, gas flow experiments. Closed

triangles, 60 mM bicarbonate ; open triangles. bicarbonate omitted.

C

0 4 J GO 80

Tlmc (min)

Fig. 9. Germination of spores of Cl. bifementana in various synthetio media. Bicarbonate at 60 mM, other ingredients m Gibbs (1964). Crosses alenine+

bioarbonate ; closed squares, alanine +phenylalanine + bicarbonate ; closed triangles, alanine + phenylalanine + lactate + bicarbonate ; closed circles, alanine +lactate+ bicarbonate ; open squares, alanine + phenylalanine ; open circles, Janine +lactate ; open triangles, alanine + phenylalanine + lactate.


Time ( m i n )

Fig. 10. Static tube experiments. Germination of spores of PA 3679/S, in various synthetic media.

Crosses, 6 0 m ~ Janine + 240mnr bicarbonate; closed circles, 6OmMdenine + 240mnr bicarbonate, 4- 10 my hotate; plus signs, 60 mar ahnine; open circles, 50 mM alenine + 10 m M lactate.

derived from the atmosphere; and (ii) the presence of dissolved oxygen. This latter factor, whilst of little importance with spores which germinate readily in the presence of oxygen, might materially influence the germination of spores sensitive to it. We have shown elsewhere (Holland, Barker & Wolf, 1969) that spores of PA3679/h germinate more slowly under aerobic than anaerobic conditions and this could account for the extremely poor response noted in tube experiments using synthetic media devoid of bicarbonate. When oxygen was removed by the continuous passage of nitrogen, a higher rate of germination resulted. Since no oxygen effect was observed with Cl. bijermentum (Holland el al., 1969) its marked germination in tube experiments with no added bicarbonate could be attributed to atmospheric CO, (Fig. SB), a feature strongly suggested by the very low rate of germination in the total absence of CO, (Fig. 8A).

It would appear that spores of Cl. bqermentans have a lower CO, requirement than those of PA 3679/S,. The former gave maximal germination with as little as 3 mM of added bicarbonate whilst the latter required 30 mM (complex medium) or 240 m~ (synthetic medium). To what extent such differences could be attributable to the presence and activation of spore decarboxylases remains to be shown.

Quantitative differences in the CO, requirements in the 2 types of media might be associated with the presence of CO, replacement factors (CRF) in the complex media. Such factors have been shown to operate in the nutrition of the vegetative cells of some clostridia (Kindler, Mager & Grossowicz, 1966). With C1. bifermentans, the

Diana Holland, A. N. Barker and J. Wolf

similarity in response produced by bicarbonate or lactate (in the presence of L-alanine +phenylalanine, Fig. 9B, D, E) need not be regarded as indicative of the function of lactate as a CRF-the 2 compounds are basically complementary and their combined presence is essential for maximal germination. Similar evidence for the complementary nature of these compounds was also obtained from the studies on PA3679/S2 (Fig. 10).

CO, could exert its effects in the gaseous state or in solution as bicarbonate or even carbonate. No attempt has been made to distinguish these different forms and their respective contributions remain to be determined.

A specific stimulatory role in spore germination has been ascribed to the Na+ ion (Gibbs, 1967; Shoesmith & Holland, 1968). The stimulatory effect of sodium bicarbonate in our experiments was found to be due to the bicarbonate ion rather than the Naf ion.

It is relevant to comment on the ccological significance of the CO, requirements in the germination of clostridial spores. Their germination and therefore ultimate survival may be influenced by both CO, and oxygen (Holland et at., 1969). Thus even where spores are insensitive to the latter, their germination may still be subject to the presence of an adequate CO, concentration and hence, in nature, such organisms (via their spores) would have a distinct survival advantage because they would germinate only in the elevated CO, concentrations characteristic of anaerobic conditions.

References ANDERSEN, A. A. (1951). A rapid plate method of counting spores of Cb8tTidium botuliwm.

J . Bad. 62, 425. ANDERSEN, A. A. (1952). Effect of subtilin on spores of Clostridium botulinum. J . Bact. 64, 145. GIBES, P. A. (1964). Factors affecting the germination of spores of Clostridium bifemntam.

J . gen. Microbiol. 37, 41. GIBES, P. A. (1967). The activation of spores of Clostridium bifementans. J . gen. Microbbl.

46,285. HOLLAND, D., B ~ K E R , A. N. & WOLF, J. (1069). Factors affecting germination of clostridia.

In spores I V . Ed. L. L. Campbell. Washington, D.C.: Am SOC. Microbiol. KINDLER, A. H., hlaoER, J. & GROSSOWICZ, N. (1956). Nutritional studies with the ClOatridiUm

botulinum group. J . gen. Microbiol. 15, 386. KURITZA-OLEJNIKOW, H. & GRECZ, N. (1965). Studies on germination of spores of Clostridium

botulinum. Bact. Proc. p. 7. LUND, A. J. (1957). Investigation of factors influencing food sterilization and preservation:

Sporulation in the genus Cloatridium. Ann. Rep. Horml Innet., Minneapolis. Minn. RIEMANN, H. (1963). Germination of bacterial spores with chelators. Thesis, University of

Copenhagen. SHOESMITH, J. G. & HOLLAND, K. T. (1968). The germination requirements of spores of Clostridium

tetani. Biochem. J . 106,38P. TREADWELL, P. E., JA”, G. J. & SALLE, A. J. (1958). Studies on faotors affecting the rapid

germination of spores of Clostridium botulinum. J . Bact. 76, 549. UEHARA, M. & FRANK, H. A. (1965). Factors affecting alanine-induced germination of clostridial

spores. InSporea I I I . Eds L.L. Campbell & H. 0. Halvorson. Ann Arbor, Michigan, U.S.A.: Am. SOC. Microbiol.

WARD, B. Q. & CARROLL, B. J. (1966). Spore germination and vegetative growth of Clostridium botulinum type E in synthetic media. Can. J . Microbiol. 12, 1145.

W m , E. S. & FOSTER, J. W. (1948). Physiological studies on spore germination with special referonce to Clostridium botulinum, 111. J . Bact. 55, 331.

The Effect of Carbon Dioxide on Spore Germination in Some Clostridia

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