J. appl. Bacf. 1975, 38 245-253

Nutritional Requirements for Growth and Sporulation of Clostridiurn perfringens

S. I. MUHAMMED*, S. M. MORRISON A N D W. L. BOYD Department of Microbiology, Colorado State University, Fort Collins, Col. 8052 I ,

U.S.A. Received 1 October 1974 and accepted 5 March 1975

Two strains of Clostridium perfringens grew in a chemically defined medium consisting of L-tryptophan, L-arginine, L-glutamic acid, L-histidine HC1, L-leucine, DL-threonine, DLphenylalanine, DL-tryrosine, DL-valine, L-cystine, ascorbic acid, Ca d-pantothenate, pyridoxine, biotin, adenine HCI, glucose, salts and mercaptoacetic acid. Alanine, aspartic acid and methionine were highly stimulatory but not essential for growth. Growth did not occur in the absence of glucose, but other fermentable carbohydrates were not tested. Acetone, isopropyl alcohol, succinic acid, acetic acid, butanol, butyric acid, lactic acid, pyruvic acid, oxaloacetic acid or acetaldehyde did not eliminate the requirement for glucose. Methionine was required for sporulation ; one strain also required riboflavin, isoleucine, serine and lysine. Butanol increased the degree of sporulation in a complex thioglycolate medium. Failure of CI. perfringens to sporulate in inadequately buffered media containing glucose was shown to be caused by the high H-ion concentration developing in the culture medium. In addition, some possible end-products of glucose metabolism such as lactic acid, oxaloacetic acid and acetaldehyde, reduced sporulation in one strain appreciably.

THEELEMENTAL REQUIREMENTS and some of the functions of metallic ions in Clostridium perfringens metabolism have been investigated (Webb, 1948; Shankar & Bard, 1952, 1955a, b). Pappenheimer & Shaskan (1944) found that iron was necessary for the acetic-butyric acid type of fermentation and, when deficient, a shift to a lactic acid fermentation occurred. Ferrous iron also stimulated haemotoxin production by Cl. perfringens (Locke & Main, 1931). As with other organisms, sulphur was essential for the growth of C1. perfringens and this could be supplied by cysteine or cystine, replaceable by glutathione, but not by inorganic sources (Fuchs & Bonde, 19576). Amino acid and vitamin requirements of CI. perfringens were studied by Boyd, Logan & Tytell (1948) and Fuchs & Bonde (1957~).Requirements varied between strains and some amino acids, though not essential, were found to stimulate growth. Several media have been developed empirically for the production of spores by CZ. perfringens (Ellner, 1956; Angelotti, Hall, Foter & Lewis, 1962; Kim, Cheney & Woodburn, 1967; Ting & Fung, 1972). This study was designed to find some of the factors required for growth and sporulation of CI.perfringens.

* Present address: Department of Veterinary Pathology and Microbiology, University of Nairobi, P.O. Box 29053, Kabete, Kenya.

t2451

246

S. I. MUHAMMED, S. M. MORRISON AND W. L. BOYD

Materials and Methods Organisms and preparation of inocula Two strains of Cl. perfringens isolated from water were used in this study. These are designated as CI.perfringens 6 D 0 and colo. The organisms were identified following the procedure of Cowan & Steel (1965). The organisms also produced an opalescence in egg yolk medium which was neutralized by CI. perfringens type A antitoxin (Burroughs Wellcome & Co., Beckenham, Kent). Standard suspensions were prepared by growing the organisms in Bacto Fluid Thioglycollate Medium (Difco) with glucose (FTM+glucose) for 16 h at 37". The cultures were centrifuged at 3670 g for 30 min and washed twice with 0 . 3 mM-phosphate-buffered saline, pH 7.2 (PBS). The final pellets were resuspended to original volumes in PBS and used as inocula. Estimation of the number of viable cells in the suspensions was carried out by the drop method of Miles & Misra (1938). Known volumes of the suspensions were diluted in peptone water [O.1 % peptone (Oxoid) + 0 . 5 % NaCl] and various dilutions were inoculated on blood agar plates [Tryptose Blood Agar Base (0xoid)i- 10% bovine blood] previously dried at 37" for 2 h. The inoculated plates were dried in an atmosphere of nitrogen for 2 h at 37 O, inverted and incubated at 37" for 24 h. Colony counts were made using a dissection stereoscope at a magnification of x 10.

Chemically defined media The composition of the chemically defined media used in the study of growth and sporulation requirements are given in Tables 1 and 2. The media were prepared as TABLE1 Composition of chemically defined Basic Medium 1 used in growth and sporulation studies of Clostridium perfringens ~~

Nutrients KzHPO4 KH2P04 MgS04 FeSO4 MnSO4 NaCl Ca d-pantothenate Pyridoxine Biotin Adenine HCI Glucose Mercaptoaceticacid

8.3 g 1.6g 200.0 mg 10.0 mg 10.0 mg 2.5 g 1.0 rng 800.0 pg 5.0 pg

10.0 mg 10.0 g 0 . 3 ml

Nutrient

Amount (rng/l)

Ascorbic acid L-Tryptophan L-Arginine L-Glutamic acid L-Histidine HCI L-Leucine DL-Threonine DL-Phenylalanine DL-Tyrosine DL-Valine L-Cystine

250.0 250.0 250.0 750.0 250.0 250.0 250.0 250.0 250.0 375.0 120.0

follows. A salt solution was made by dissolving magnesium sulphate, sodium chloride and glucose in a small amount of water and then adding solutions of ferrous sulphate and manganese sulphate. Amino acids and ascorbic acid were dissolved in boiling water, and cystine in N-HCI; adenine, vitamins and salts solutions were added to the

247

NUTRITION OF CL. PERFRINGENS

cooled amino acids solution. The pH value of the medium was adjusted to 7.2 with N-NaOH before phosphates in solution were added. The final volume was made up to 1100 ml. This preparation constituted Basic Medium 1 (BM 1). The medium was distributed into bottles marked 1 to 11 in 100 ml portions and additional nutrients added to bottles 2 to 11 to give the media listed in Table 2. Water, double distilled in glass, was used in the preparation of all defined media. TABLE2 Nutrients added to Basic Medium I to make media 2 to I1 Medium No. Nutrient

Amount (m€!/l)

-

Glycine L-Lysine DL-Serine DL-Methionine L-Aspartic acid DL-Alanine ~~-1solucine Riboflavin Uracil

500 500 750 250 500 500 250 0-50

0.0125

2

3

4

5

6

7

8

+ + + + + + + + +

+ + + + + + + + -

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

9 l O l i

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ , nutrient included in the medium; -, nutrient omitted from the medium. Partially defined medium The partially defined medium contained (/l): K2HP04,8.3 g; KH2P04,1-6 g; MgS04, 200 mg; FeS04, 10.0 mg; MnS04, 10.0 mg; Ca d-pantothenate, 1 .O mg; pyridoxine, 800 pg; biotin, 5 pg; adenine HCl, 10 mg; L-tryptophan, 50 mg; Bacto vitamin free casamino acids, 15 g; mercaptoacetic acid, 0.3 ml. All media were dispensed into screwcapped test tubes and autoclaved at 121" for 15 min. Glassware was washed with detergents, rinsed in tap water and subsequently in distilled water before being dried and sterilized in a hot air oven at 160"for 2 h. Preparation of a standard curi'e Total cell counts in growth and sporulation experiments were estimated using a standard curve. Clostridium perfringens 6D0 was grown in FTM glucose for 40 h at 37". Cells were harvested by centrifuging, washed in PBS and the final pellet resuspended in PBS. Various dilutions of the suspension were made in PBS and optical density ( o n ) read in a Spectronic 20 (Bausch & Lomb) spectrophotometer using a wave length of 620 nm. One-tenth ml of the standard suspension inoculated into 10 ml of PBS was used as a blank. The number of cells /ml of each diluted suspension was estimated with a haemocytometer. The O.D. values were then plotted against cell numbers to obtain the standard curve.

+

Growth and sporulation of Cl. perfringens in chemically defined media One-tenth ml of standard suspensions of Cl. perfringens 6D0 and colo containing ca. 1-2-2 x 108 cells were inoculated into 10 ml amounts of chemically defined media

248

S. I. MUHAMMED, S. M. MORRISON AND W. L. BOYD

1 to 11 (Table 2) and the cultures incubated for 40 h at 37 '. Cultures were then centrifuged for 1 h at 3670 g and the pellets resuspended to original volume in PBS. The suspensions were read for turbidity with a Spectronic 20 spectrophotometer at 620 nm using a suspension of 0.1 ml of the same strain of Cl. perfringens in PBS as control. The total cell count was then estimated using the standard curve. The organisms were also inoculated into the chemically defined media containing 0.3 % of CaC03. These cultures were incubated for 72 h before they were tested for sporulation. Growth and sporulation of C1. perfringens with various carbon compounds One-tenth ml of standard suspensions of Cl. perfringens 6 D 0 and colo were inoculated into tubes of FTM or partially defined media each having 27.7 mM of one of the compounds listed in Table 5. Control media did not contain any of the additional substances. The organisms were also inoculated into FTM at pH 4-5. The initial pH value of all other media was 7-2. Cultures in partially defined media were checked for growth after 72 h at 37 O, while cultures in FTM were checked for growth and sporulation after 48 h. The total cell count in FTM with the various carbon compounds was estimated using the standard curve as described above. Testing for sporulation In sporulation experiments cultures were incubated at 37" for 48 h or 72 h. One ml quantities of the spore cultures were pasteurized at 80" for 10 min, then inoculated into FTM+glucose and incubated at 37" for 24 h to test for the presence of viable spores. When the effect of various carbon compounds on sporulation was investigated the number of spores in samples of pasteurized cultures was estimated by the method of Miles & Misra (1938), using 0.1 % peptone water as diluent and blood agar as plating medium.

Growth requirements of C1. perfringens Mean O.D. values and total cell counts of Cl.perfringens strains 6 D 0 and colo growing in 11 defined media are presented in Table 3. Three experiments were conducted and in each, triplicate cultures were set up for each strain and medium. The 2 strains grew in all media. For both strains, growth in BM 1 was minimal but was, however, comparable to growth in medium 6 (without alanine). Strain 6 D 0 also grew poorly in medium 8 (without methionine) and strain colo grew poorly in medium 7 (without aspartic acid). When the ability of CI. perfringens 6 D 0 and colo to grow in media containing different carbon compounds as energy sources was tested, both strains grew in partially defined medium with glucose but not in media with the other compounds listed in Table 5. No growth was observed in the control medium in the absence of these compounds,

249

NUTRITION OF CL. PERFRINGENS

Sporulation requirements of C1. perfringens Clostridium perfringens 6 D 0 and colo growing in chemically defined media 1 to I 1 containing glucose but no CaC03 did not form spores. The results for sporulation obtained when 0 . 3 of CaC03 was incorporated in the media are presented in Table 4.

TABLE3 Mean* and standard error of optical density, and estimated total cell count of Clostridium perfringens (600 and colo) cultures grown in chemically de$ned media CI. perfringens 6D0 7 O.D.3 TCC _____0.1650.02 6 . 8 106 ~ 0.3650.11 1 . 4 5 lo9 ~ 0.46f0.10 1 . 8 5 ~ 1 0 9 0.2850.08 1 . 1 5 109 ~ 0.22k0.02 9 . 0 108 ~ 0.16f0.01 6 . 8 lo* ~ 0.3650.06 1 . 4 5 109 ~ 0.19+0.06 7 . 5 108 ~ 0.32k0.03 1 . 3 109 ~ o.5050.00 2 . 0 109 ~ 0.28+0*05 1 . 1 5 ~ 1 0 ~

Medium? 1 2 3 4 5 6

7 8 9 10 11

CI. perfringens colo

TCC

O.D.

6.8 x lo8 1.65x109 1 . 5 5 lo9 ~ 1 . 9 5 lo9 ~ 1*35x109 3 . 5 lo8 ~ 8 . 0 10s ~ 8.5x10" 1 . 1 5 lo9 ~ 1.25~10~ 1 * 9 x109

0*16+0.01 0.4150.17 0.39+0.09 0.48f0.14 0.33k0.02 0.08+0*01 0.20+0.01 0.21+0.01 0.28k0.03 0.3150.02 0.47+0-06

* Mean of 3 experiments. t See text formulation of each medium. 3 Abbreviations used:

o.D., optical density (at 620 nm); TCC, total cell countlml. Incubation was at 37" for 40 h.

TABLE 4 Sporulation" of Clostridium perfringens 6 0 0 and colo grown in chemically defiried media containing 0.3 of CaCO3 Defined medium No. A

Strain

__

6D0

cola

__ -

{ {

Expt

1

2

3

4

5

I

6

7

8

9 1 0 1 1

_____~~.___

1 2 3 1 2 3

+ - - + - - - - - + - - - + - - - - + + - - + + - - - -

+

- + + - + + + - + - - + + - + + + - - + - + + + - + + - + + +

* + , sporuiation occurred; -, sporulation did not occur. The 2 strains did not sporulate in BM 1 or in medium without methionine. Both strains sporulated in complete medium 2 and in medium 6 (without alanine). The 2 strains varied in their ability to sporulate in the other media. In some media spores were not detected consistently in any of the 3 experiments.

250

S. I. MUHAMMED, S. M. MORRISON A N D W. L. BOYD

Different carbon compounds were incorporated into FTM to observe whether they would inhibit the sporulation of CI.perfringens 6D0 and colo in this medium. Table 5 shows that glucose prevented the sporulation of both strains; but the sporuIation of CI. perfringens 6 D 0 was also affected adversely by lactic acid, oxaloacetic acid and acetaldehyde. On the other hand butanol increased the sporulation of both strains. Table 5 also presents the final pH values of the cultures. Sporulation did not occur in FTM when the initial pH value of the medium was 4.5.

TABLE 5 Total cell count and spore count of Clostridium perfringens 6 0 0 and colo cultures in thioglycollate base medium (FTM) with added carbon compounds, andfinal p H values of cultures Cl. perfringens colo

Cl. perfringens 6DO L

I

Carbon compound

TCC*

SP

-

L

1

PH value

TCC*

SP

>

PH value

~~

Glucose Acetone Isopropyl alcohol Succinic acid Acetic acid Butanol Butyric acid Lactic acid Pyruvic acid Oxaloacetic acid Acetaldehyde FTM pH 7.2 FTMpH4.5

2.3 x 109 NS 6.7 x 102 8 . 0 lo8 ~ ~ 8 . 0 ~ 1 0 ~3 . 2 103 8.0~ 108 2.1 x 102 1.3~104 8.Ox 108 9.ox 108 1.1x 105 8 . 0 lo8 ~ 4.5 x 103 1 5 9.0 x 108 9 * o x108 5.6 x lo3 4 . 0 lo8 ~