Chia sẻ tài liệu về phân lập vi sinh vật
- Thread starter gvthang
- Start date
tài liệu phân lập VSV ở VN có rất nhiều, bạn tìm bộ Một số Phương pháp nghiên cứu VSV do Nguyễn Lân Dũng chủ biên, 3 tập; tập 1 chỉ tất cả pp phân lập VSV.
Bạn cứ nói nhóm vi sinh vật mà bạn đang làm, định làm, mọi người sẽ giúp. Chú ý lần sau gõ tiếng Việt có dấu.
cảm ơn anh nhiều !
Phải nói là em đang rất cần những tài liệu về phân lập vi sinh vật nói chung va phân lập chủng có hoạt tính xellulo cao nói riêng .
Em đang làm nghiên cứu và đang bế tắc...
Anh giúp em cái gì có liên quan đến những vấn đề đó em cung rất quí .
Email của em : Gvthang01sh@yahoo.com
Phải nói là em đang rất cần những tài liệu về phân lập vi sinh vật nói chung va phân lập chủng có hoạt tính xellulo cao nói riêng .
Em đang làm nghiên cứu và đang bế tắc...
Anh giúp em cái gì có liên quan đến những vấn đề đó em cung rất quí .
Email của em : Gvthang01sh@yahoo.com
hay quá anh SHMT ơi !
Em đang học năm 5 rồi cũng đang cần 3 quyển sách của thầy Lân Dũng, em đã đi lục lọi tất cả các quầy sách củ nhưng "bất lực", anh có cách nào giúp em với nhưng em ở tận ĐÀ NẴNG luôn
Lê đức Dũng. lớp 01SH - ĐẠI HỌC BÁCH KHOA ĐÀ NẴNG
EMAIL: leducdungbksh@yahoo.com
Em đang học năm 5 rồi cũng đang cần 3 quyển sách của thầy Lân Dũng, em đã đi lục lọi tất cả các quầy sách củ nhưng "bất lực", anh có cách nào giúp em với nhưng em ở tận ĐÀ NẴNG luôn
Lê đức Dũng. lớp 01SH - ĐẠI HỌC BÁCH KHOA ĐÀ NẴNG
EMAIL: leducdungbksh@yahoo.com
Vũ Thành Lâm
Senior Member
chuyến này căng đây
cho anh xin với SHMT ơi
mấy hôm trước thầy Dũng còn lên bảo các chị phòng anh cho mượn lại mấy ?cuốn nhưng thầy thấy nát bèm cả rùi (mấy cụ biện luận với thầy rằng Nát chứng tỏ nhiều người dùng chỉ sợ mãi mà vẫn mới thôi ?hehhe)
chú có thể gửi qua bưu điện hoặc mail cho anh hoặc nhiệt tình mang cho anh luôn 8)
đa tạ luôn
cho anh xin với SHMT ơi
mấy hôm trước thầy Dũng còn lên bảo các chị phòng anh cho mượn lại mấy ?cuốn nhưng thầy thấy nát bèm cả rùi (mấy cụ biện luận với thầy rằng Nát chứng tỏ nhiều người dùng chỉ sợ mãi mà vẫn mới thôi ?hehhe)
chú có thể gửi qua bưu điện hoặc mail cho anh hoặc nhiệt tình mang cho anh luôn 8)
đa tạ luôn
Cảm ơn anh nhiều !
Em hiện giờ đang làm ở trường nhưng địa chỉ liên lạc theo trường giờ chắc chưa được...
Em rất cần nó.
Anh hứa giúp em thì giúp đến cùng nha !
Em sẽ để địa chỉ cho anh và anh cũng cho em địa chỉ và giá cả 2 cuốn sách tập 1và 2 .
Địa chỉ em : Giáp văn thắng
lớp 01sh -khoa hoá -DH bách khoa đà nẵng .
Em sẽ chờ hồi âm của anh .
Em hiện giờ đang làm ở trường nhưng địa chỉ liên lạc theo trường giờ chắc chưa được...
Em rất cần nó.
Anh hứa giúp em thì giúp đến cùng nha !
Em sẽ để địa chỉ cho anh và anh cũng cho em địa chỉ và giá cả 2 cuốn sách tập 1và 2 .
Địa chỉ em : Giáp văn thắng
lớp 01sh -khoa hoá -DH bách khoa đà nẵng .
Em sẽ chờ hồi âm của anh .
Hiện tại mình đang giữ cuốn tập 2 thôi, còn hai tập nữa thì phân tán đi đâu rồi í.
Nhưng điều này không quan trọng, cơ bản là chúng ta cần phải thỏa thuận lại về tiền photo tài liệu và gửi sách?????. Nếu gvthang ở HN thì đơn giản thôi.
to lamvt
em nghĩ chỗ anh không thiếu tài liệu này mà.
Nếu thật sự cần thì ở trường có đấy anh à.
Nhưng điều này không quan trọng, cơ bản là chúng ta cần phải thỏa thuận lại về tiền photo tài liệu và gửi sách?????. Nếu gvthang ở HN thì đơn giản thôi.
to lamvt
em nghĩ chỗ anh không thiếu tài liệu này mà.
Nếu thật sự cần thì ở trường có đấy anh à.
Phạm Ngọc Dương
Senior Member
hiện nay mình dang gặp khó khăn trong việc phân lập chủng vi khuẩn Clostridium một chủng vi khuẩn gây hương có trong chượp chín của nước mắm, ai có tài liệu gì về vấ đề này giúp minh với nhé !
General Media for Enrichment and Isolation
A number of complex media have been widely used for the enrichment and isolation of saccharolytic and proteolytic clostridia: reinforced clostridial medium (RCM) (Gibbs and Hirsch, 1956; Hirsch and Grinstedt, 1954); differential reinforced clostridial medium (DRCM) (Gibbs and Freame, 1965); cooked meat media (CM and CMC) (Holdeman et al., 1977; Robertson, 1915–1916); peptone-yeast extract medium (PY) (Holdeman et al., 1977); Viande-Levure medium (VL) (Beerens and Fievez, 1971; Willis, 1977). RCM and DRCM contain glucose and starch; the others can be supplemented with further ingredients, such as carbohydrates or certain amino acids, to make them more selective. DRCM is recommended especially for the detection and enumeration of clostridial spores in pasteurized samples of food (Freame and Fitzpatrick, 1971; Gibbs and Freame, 1965).
Any medium that is rich in carbohydrates and that contains some peptone and yeast extract as well as reducing agents is suitable for the enrichment of the common saccharolytic clostridia. A selective medium containing antimicrobial agents for the isolation of C. butyricum and a few other butyric acid-producing, saccharolytic clostridia from human feces has been described by Popoff (1984).
Potato mash medium, as described by Ruschmann (1928), has been used by several investigators in the past to study the clostridia in retting flax, in manure, in silage, and in milk (Dührsen, 1937; Glathe, 1934; Ritter, 1932; Ruschmann, 1928; Ruschmann and Bavendamm, 1925; Ruschmann and Harder, 1931). Its starch and pectin content favors the development of starch- and/or pectin-fermenting clostridia. Maize mash medium (Weizmann, 1919) and maize liver medium (McClung and McCoy, 1934) have been used for enrichment and isolation of the butanol-acetone-producing clostridia (Beesch, 1953; Weizmann, 1919; Weyer and Rettger, 1927), the pigment-producing strains (Hellinger, 1947; McClung, 1943), and other saccharolytic species (Gilliland and Vaughn, 1943; McClung and McCoy, 1934; Weizmann and Hellinger, 1940). Milk, without or with supplementations, has been found convenient for the enrichment of several clostridia from soil, wounds, and plant material or of the clostridia commonly present in the milk itself (Meyn, 1933; van Beynum and Pette, 1940; Weinzirl and Veldee, 1915; Weizmann and Hellinger, 1940; Winkler, 1961).
Potato tubers, stabbed and immersed in water, provide a simple method for the enrichment of common saccharolytic clostridia. The following procedure has been described by Veldkamp (1965).
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PROCEDURE: Enrichment of Saccharolytic Clostridia with Potato Tubers (Veldkamp, 1965)
A potato is washed under the tap and subsequently stabbed once or twice with a knife. It is then placed in a beaker, and enough water is added just to cover the potato; the beaker is covered with a watch-glass and incubated at 37°C. The oxygen that might be introduced into the tissue is consumed by its cells. In the anaerobic environment Clostridium rapidly starts to decompose the pectin in between the plant cells. The tissue is thus macerated. When the tuber floats, due to profuse gas formation, the water is poured out of the glass; the potato is washed and dissected. Microscopic examination of the tuber contents invariably shows the typical pleomorphic clostridial cells; among these, spore-bearing spindle-shaped cells are often encountered.
Isolation can easily be achieved as follows. A sample of the macerated tissue is inoculated into yeast extract-glucose broth and after pasteurization (10 min at 80°C in a water bath) the culture is incubated at 37°C in N2 atmosphere. A pure culture can be obtained by streaking a sample on yeast extract-glucose agar and incubating under N2.
A double-layered polypectate agar medium has been used to isolate pectolytic Clostridium spp. from tubers and carrots and from suspensions of field and rhizosphere soils (Lund, 1972; Lund et al., 1981; Perry, 1982; Perry, 1985).
Media given by various authors for enrichment and growth of saccharolytic clostridia frequently show variations that are not essential for growth of these organisms. The following medium is recommended for the growth of many species.
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PROCEDURE: Medium for Growth of Saccharolytic Clostridia
The medium contains:
1 M potassium phosphate, pH 7.5 30 ml
1 M MgSO4 1 ml
Solution M (see below) 0.5 ml
0.2M FeSO4 in 0.1 M H2SO4 0.2 ml
Trypticase 10 g
Yeast extract 6 g
Sodium thioglycolate 0.5 g
Energy source (sucrose or glucose) 10–20 g
Distilled water 1 liter
The pH is adjusted to 7.0–7.2. The medium is autoclaved for 20 min at 121°C.
Solution M contains:
MnCl2 10 mM
CaCl2 30 mM
CoCl2 5 mM
Na2MoO4 5 mM
Since stock solutions of the salts can be stored in the laboratory, this medium can be prepared rather quickly. It is best to autoclave the sugar solutions separately.
The fixation of molecular nitrogen is a property shared by many representatives of the saccharolytic clostridia (Rosenblum and Wilson, 1949). Media that contain glucose or sucrose but lack a source of nitrogen have been used for the isolation of C. pasteurianum and other species. The comprehensive literature was summarized by Skinner (1971). Based on the method of Augier (1957), the following liquid medium for counting nitrogen-fixing clostridia in soil by the most probable number method (MPN) and for subsequent isolation has been recommended by Skinner (1971).
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PROCEDURE: Isolation of Nitrogen-Fixing Saccharolytic Clostridia (Skinner, 1971)
The medium contains:
K2HPO4 0.8 g
KH2PO4 0.2 g
MgSO4·7H2O 0.2 g
NaCl 0.2 g
FeSO4·7H2O 10 mg
MnSO4·4H2O 10 mg
CaCl2 10 mg
Na2MoO4·2H2O 25 mg
Yeast extract 10 mg
Trace element solution (see below) 1 ml
Soil extract (see below) 10 ml
Glucose or sucrose 10.0 g
Sodium thioglycolate 1.0 g
Distilled water 1 liter
The pH is adjusted to 7.2.
The trace element solution contains:
Na2B4O7·10H2O 50 mg
CoNO3·6H2O 50 mg
CdSO4·2H2O 50 mg
CuSO4·5H2O 50 mg
ZnSO4·7H2O 50 mg
MnSO4·H2O 50 mg
Distilled water 1 liter
The stock solution should be saturated with CO2.
The soil extract is prepared as follows (Augier, 1956): Equal weights of a neutral garden soil and water are combined and heated at 130°C for 1 h. After cooling, it is filtered through paper, bottled, autoclaved, and stored until use.
Sodium thioglycolate is added just before the medium is distributed in 10-ml portions into narrow test tubes fitted with Durham tubes, capped or plugged, and autoclaved at 121°C for 15 min. After inoculation with 1 ml of decimal dilutions of pasteurized soil, the tubes are incubated in anaerobic jars under a nitrogen atmosphere at 30°C for 2 weeks. Positive tubes show abundant gas formation, turbidity with or without a viscoid, whitish deposit or surface pellicle, and odor of butyric acid.
Isolations are made by streaking small inocula from positive tubes on plates prepared from the above medium plus 2% agar. The plates are incubated in anaerobic jars under an atmosphere of nitrogen. Colonies can be picked within 7 days at 30°C and purified by repeated streaking on nitrogen-free agar. Pasteurization can be applied at any stage, provided that spores are present.
The above enrichment procedure can be made rather specific for C. pasteurianum if the concentration of sucrose is increased to 15% (Spiegelberg, 1944; Witz et al., 1967). Inocula from such enrichments are again pasteurized and streaked on the above agar containing 2% glucose.
Clostridia, such as C. butyricum and C. tyrobutyricum, which ferment lactate plus acetate to butyrate, CO2, and H2, have been frequently found as the dominating anaerobic sporeformers in certain silages (Bryant and Burkey, 1956; Gibson, 1965; Gibson et al., 1958; Rosenberger, 1951, 1956) and milk products (Goudkov and Sharp, 1965). C. tyrobutyricum is responsible for spoilage of certain types of cheese by formation of gas (“late blowing” of hard cheese) or rancid odor (Goudkov and Sharp, 1966; Kutzner, 1963, 1966).
A medium originally developed by Bhat and Barker (1947) has been used for the detection and the isolation of lactate-utilizing sporeformers in silage (Rosenberger, 1951, 1956), in cheese (Kutzner, 1963, 1966), and in milk (Halligan and Fryer, 1976). Both C. butyricum and C. tyrobutyricum will grow in this medium; a distinction between these species is easily possible because of the striking differences in the utilization of carbohydrates: C. butyricum grows on maltose, raffinose, lactose, and starch, which are not utilized by C. tyrobutyricum.
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PROCEDURE: Isolation of Clostridia That Ferment Lactate plus Acetate (Bhat and Barker, 1947)
The medium contains:
K2HPO4 0.5 g
(NH4)2SO4 0.5 g
MgSO4·7H2O 0.1 g
FeSO4·7H2O 20 mg
Yeast extract 0.5 g
Biotin 0.1 μg
p-Aminobenzoate 100 μg
Sodium L-lactate 10.0 g
Sodium acetate 8.0 g
Sodium thioglycolate 0.5 g
Distilled water 1 liter
Sodium thioglycolate is added immediately before autoclaving. The pH is adjusted to 6.0–7.0, and the medium is autoclaved for 20 min at 121°C.
A slightly acidic pH value of the medium favors growth of C. butyricum and C. tyrobutyricum. The latter has been shown to start growing in media with a pH value of as low as 5.3 (Kutzner, 1963). When a sodium lactate solution is used for preparing the medium, the pH should be checked after autoclaving (it tends to become more acidic).
Enrichment cultures are set up in glass-stoppered bottles (filled to the neck). They are inoculated with pasteurized garden soil or other material and incubated at 37°C. Turbidity and gas formation are observed after a few days, accompanied by an increase of the pH. Two transfers into fresh medium (5–10% inoculum) are made before isolation is performed by streaking material on agar medium of the same composition as above plus 2% agar. The plates are incubated in anaerobic jars under a nitrogen atmosphere.
Using a slightly modified medium, C. butyricum was found to be the dominant lactate-fermenting sporeformer in the ensiling process of perennial ryegrass (Gibson et al., 1958). Clostridia other than C. butyricum and C. tyrobutyricum that are found occasionally in silage (C. paraputrificum, C. tetanomorphum, C. perfringens, and C. sphenoides) require more complex media or are not able to use lactate plus acetate as energy source.
Procedures for the detection of the very low numbers of spores of C. tyrobutyricum in milk used in making cheese have been described (Fryer and Halligan, 1976; Halligan and Fryer, 1976). First, an enrichment is done using a complex medium that contains calcium lactate. Then the presence of C. tyrobutyricum is confirmed by subculturing positive enrichments in a slightly modified medium as given above.
There have been many reports on the enrichment and isolation of cellulose-decomposing clostridia, but the cultures obtained were often not pure and subcultures are not available any more. In addition to the two classical cellulose-decomposing species C. cellobioparum (Hungate, 1944) and C. thermocellum (McBee, 1948, 1950; Viljoen et al., 1926), several new mesophilic and thermophilic species have been validly described (Van Gylswyk, 1980; Madden et al., 1982; Madden, 1983; Petitdemange et al., 1984; Sleat et al., 1984; LeRuyet et al., 1985; Sleat and Mah, 1985; Murray et al., 1986; Sukhumavasi et al., 1988; Palop et al., 1989).
Samples for the isolation of cellulolytic clostridia can be taken from soil, feces of herbivorous animals (horse manure has long been recognized as an excellent source for thermophilic strains also), compost, decayed plants, and from anaerobic sewage or mud. Except for the different incubation temperature, the procedures for enrichment and isolation of both mesophilic and thermophilic cellulolytic species are about the same.
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PROCEDURE: Enrichment and Isolation of Cellulolytic Clostridia (Omelianski, 1902; Skinner, 1960, 1971)
For enrichment, the medium contains:
K2HPO4 1.0 g
(NH4)2SO4 1.0 g
MgSO4·7H2O 0.5 g
CaCO3 2.0 g
NaCl 0.5 g
Resazurin 1 mg
Cellulose (chopped filter paper) 20 g
Distilled water 1 liter
The final pH is adjusted to 7.1.
Enrichments are set up in completely filled, glass-stoppered bottles (50- or 100-ml), inoculated with 0.1 g of pasteurized sample. The sample material provides enough growth factors for the primary enrichment, and reducing conditions are established by the contaminating microflora. Incubations are made at 30–37°C. As soon as digestion of the filter paper becomes visible (1 or 2 weeks), isolation of the cellulose-degrading clostridia is performed by repeatedly streaking on cellulose agar medium.
The cellulose agar medium contains:
K2HPO4 7.5 g
KH2PO4 3.5 g
(NH4)2SO4 0.5 g
NaCl 1.0 g
MgSO4·7H2O 50 mg
CaCl2 50 mg
Resazurin 1 mg
Yeast extract 1.0 g
Cellulose 10.0 g
Agar 15.0 g
L-Cysteine hydrochloride 0.5 g
Distilled water 1 liter
The pH is adjusted to 7.1.
A suitable substrate for these enrichments is powdered cellulose (e.g., MN 300, Machery and Nagel; CF-11, Whatman) or a cellulose preparation made by wet-grinding of finely divided filter paper (e.g., Whatman No. 1 ashless cellulose paper) in a pebble mill (Hungate, 1950). To keep the cellulose particles suspended, Skinner (1960) recommended the addition of about 0.1% sodium carboxymethylcellulose (substitution range, 0.65–0.85) to the medium.
The cysteine hydrochloride is added to the medium shortly before autoclaving. The medium is autoclaved for 20 min at 121°C and distributed to petri dishes, which are kept in anaerobic jars under an atmosphere of 90% N2 + 10% CO2. Alternatively, screw-capped bottles (Skinner, 1971) or roll tubes (Hungate, 1950) may be used. Digestion of cellulose is visible after incubation at 30–37°C for 1 week or longer, depending on the activity of the strains. Colonies are often very small, and material for transfers should be picked carefully under a dissecting microscope. Colonies that contain spores are suspended in cellulose-free basal medium and pasteurized (10 min at 75°C) before streaking again.
For purification, an alternate streaking on cellulose agar or on agar that contains 0.5% cellobiose instead of cellulose can be advantageous. Cellobiose is readily fermented by all the cellulolytic isolates described. However, spore production on cellobiose agar is not as pronounced as on cellulose agar. Therefore, colonies must be examined carefully before being pasteurized.
The “double-layer” method may be used at an early stage to detect cellulose-decomposing colonies by formation of clear halos. Petri dishes are filled with 20 ml of cellulose-free agar medium and subsequently overlayed with 5 ml of cellulose-containing agar medium.
The selective isolation of C. sphenoides from mud samples using citrate as the energy source for growth has been described (Walther et al., 1977). It has not been demonstrated whether this method is also applicable to the isolation of this organism from silage or human infections in which C. sphenoides has been reported to occur (Gibson, 1965; Smith, 1949). C. sphenoides is a representative of the relatively small group of saccharolytic bacteria that do not form butyrate but ferment carbohydrates to ethanol, acetate, CO2, and H2.
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PROCEDURE: Direct Isolation of Clostridium sphenoides (Walther et al., 1977)
The medium contains:
K2HPO4 2.0 g
KH2PO4 3.4 g
MgSO4·7H2O 0.2 g
(NH4)2SO4 0.3 g
NaCl 0.6 g
CaCl2·2H2O 60 mg
Yeast extract 4.0 g
Peptone 2.0 g
Trisodium citrate dihydrate 14.7 g
Resazurin 1 mg
Agar 15.0 g
Distilled water 1 liter
Adjust the pH to 6.7–7.0. 0.3 g of L-cysteine hydrochloride is added after the agar has been dissolved.
Applying the Hungate technique, 5-ml portions are added to 15-ml tubes. After autoclaving (20 min at 121°C), the tubes are kept in a water bath at 50°C. Then 0.3 ml of a pasteurized mud sample is used for the preparation of decimal serial dilutions. Roll tubes are prepared which are incubated at 37°C. After 48–72 h, single colonies that show an increased size as compared to the background growth of contaminating organisms are picked, inoculated into 10 ml of liquid citrate medium, and incubated at 37°C for 24 h. For further purification, serial dilutions in roll tubes containing citrate agar are repeated at least three times.
During isolation, the selected colonies are checked for the presence of wedge-shaped cells and of spherical, nearly terminally located spores, both of which are typical for C. sphenoides.
A number of single amino acids can be used by certain clostridial species as sources of energy, carbon, and nitrogen for growth (Andreesen et al., 1989). Media that contain a certain amino acid, small amounts of yeast extract, and minerals have been used for enrichment and isolation of some clostridial species. The use of L-alanine led to the isolation of C. propionicum (Cardon and Barker, 1946, 1947) and of γ-aminobutyrate and δ-aminovalerate to “C. aminobutyricum” and C. aminovalericum, respectively (Hardman and Stadtman, 1960a, 1960b). C. tetanomorphum has been isolated by Kornberg using L-histidine as the principal substrate (see Wachsman and Barker, 1955a), and the use of L-glutamate led to the isolation of C. cochlearium as well as of C. tetanomorphum (Barker, 1937, 1939). Lysine was used to enrich and isolate Clostridium SB4 (Costilow et al., 1966), which subsequently was identified as C. subterminale (H. A. Barker, personal communication). It appears that the possible advantage of using single amino acids as selective substrates for the isolation of certain clostridia has never been studied systematically. Investigations by Mead (1971), Elsden et al. 1976, and Elsden and Hilton (1978, 1979) have shown that a considerable number of clostridia are able to ferment single amino acids.
In the following procedure, the enrichment and isolation of C. cochlearium or C. tetanomorphum is described, using L-glutamate as the specific substrate.
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PROCEDURE: Enrichment and Isolation of Glutamate-Fermenting Clostridia (Barker, 1937, 1939)
The medium contains:
K2HPO4 0.2 g
MgSO4·7H2O 0.1 g
Yeast extract 0.5 g
Sodium L-glutamate 10.0 g
Sodium thioglycolate 0.5 g
Tap water 1 liter
The final pH is ajusted to 7.6. Enrichments are set up in completely filled glass-stoppered bottles with pasteurized soil as inoculum (0.5 g soil per 100 ml). Within 1–2 days at 37°C, an abundant growth of sporeforming anaerobes is observed accompanied by a moderate production of gas. After transferring a volume of 2 ml once or twice to 100-ml bottles containing medium of the above composition, isolations are made by repeatedly streaking on L-glutamate agar plates or by diluting in agar roll tubes.
Laanbroek et al. (1979) described the enrichment of C. cochlearium from anaerobic sludge in an L-glutamate-limited anaerobic chemostat.
For mass culture of C. cochlearium or C. tetanomorphum, the “Medium for Growth of Saccharolytic Clostridia” (this chapter) may be used. Instead of sugars, sodium L-glutamate (17 g per 1 liter of medium) is added as energy source and Trypticase is omitted.
A selective isolation medium for C. sporogenes and related clostridia that are capable of growth by means of coupled oxidation-reduction reactions between appropriate pairs of amino acids has been developed by Fryer and Mead (1979). The semidefined medium contains alanine and proline as the principal energy sources and polymyxin. When samples from soil or mud were directly plated on agar medium, a high percentage of the isolates were strains of the C. sporogenes/C. botulinum group.
The anaerobic purine fermenters, C. acidiurici and C. cylindrosporum, were originally isolated by Barker and Beck (1942) from enrichment cultures with uric acid as the sole source of carbon, nitrogen, and energy. Several more strains of both species were obtained by Champion and Rabinowitz (1977) using the same method. Uric acid agar supplemented with tryptone, meat extract, liver extract, and chicken fecal extract was used by Barnes and Impey (1974) for the isolation of uric acid-decomposing anaerobes from avian cecum. The isolated strains of anaerobic sporeformers were designated C. malenominatum. Another purinolytic species, C. purinolyticum, was isolated using adenine as the selective substrate (Dürre et al., 1981).
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PROCEDURE: Isolation of Purine-Fermenting Clostridia (Champion and Rabinowitz, 1977; Barker and Beck, 1942)
The medium contains:
Uric acid 2.0 g
10 N KOH 3.0 ml
K2HPO4·3H2O (70% solution) 1.5 ml
MgSO4·7H2O 50 mg
CaCl2·2H2O 5 mg
FeSO4·7H2O 2 mg
Yeast extract 1.2 g
Resazurin 1 mg
Mercaptoacetic acid 1.5 ml
Distilled water (see below) 1 liter
The medium is prepared as follows: 500 ml distilled water plus KOH plus the K2HPO4 solution are brought to a boil. Then uric acid is slowly added. It goes into solution instantaneously. The solution is cooled down and the other ingredients of the medium are added. Mercaptoacetic acid is added shortly before autoclaving, and the pH is adjusted to 7.2 using a sterile, 60% K2CO3 solution. Other reducing agents may be used instead of mercaptoacetic acid: sodium thioglycolate (750 mg/liter) or cysteine hydrochloride (1 g/liter) plus dithiothreitol (0.1 g/liter).
After autoclaving (20 min at 121°C), the medium is immediately used to completely fill 50-ml glass-stoppered bottles. After inoculation with 0.5 g of pasteurized soil or chicken feces, the bottles are incubated at 37°C. Growth occurs within 24–48 h and is accompanied by an increase of alkalinity. Utilization of uric acid can be monitored by the decrease in absorbancy at 290 nm of a 1 to 100 dilution of the growth medium. After one or two more transfers (0.5% inoculum) to 50-ml bottles containing the medium above, isolation is made under strict anaerobiosis by repeatedly streaking on uric acid agar plates incubated in anaerobic jars or on roll tubes.
The purinolytic clostridia require strictly anaerobic conditions for growth, and the application of stringent anaerobic culture techniques is necessary. The three accepted species, C. acidiurici, C. cylindrosporum, and C. purinolyticum, are clearly differentiated on the basis of DNA homology, whereas the form and position of their spores differ (Schiefer-Ullrich et al., 1984). C. cylindrosporum was named after its cylindrical spores that are variably situated and do not cause swelling of the mother cell (Barker and Beck, 1942; Andreesen et al., 1985).
The isolation of adenine-fermenting clostridia requires a medium of slightly different composition. Instead of uric acid, it contains 0.2% adenine, 0.2% sodium bicarbonate, and 10–7 M each of thiamine, sodium selenite, sodium molybdate, and sodium tungstate to replace yeast extract (Dürre et al., 1981).
C. kluyveri has specialized on the conversion of ethanol and acetate to butyrate, caproate, and H2 (Barker and Taha, 1942; Bornstein and Barker, 1948a). It is unable to utilize carbohydrates or amino acids for growth and requires only biotin and p-aminobenzoate as growth factors. Therefore, the medium used for enrichment is very selective.
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PROCEDURE: Isolation of Clostridium kluyveri (Bornstein and Barker, 1948a; Stadtman and Burton, 1955)
The medium contains:
K2HPO4 0.30 g
KH2PO4 0.20 g
NH4Cl 0.25 g
MgSO4·7H2O 0.20 g
CaCl2·2H2O 10 mg
FeSO4·7H2O 5 mg
MnSO4·4H2O 2 mg
Na2MoO4·2H2O 2 mg
Biotin 10 μg
p-Aminobenzoate 200 μg
Ethanol 20 ml
Potassium acetate 5.0 g
Glacial acetic acid 2.5 ml
Resazurin 1 mg
Sodium thioglycolate 500 mg
Distilled water 1 liter
Thioglycolate is added shortly before autoclaving (20 min at 121°C). After autoclaving, the medium is rapidly cooled, and the pH is adjusted to 7.0 using a 60% K2CO3 solution autoclaved separately (about 8–10 ml are required).
The enrichment is made at 35°C in 100-ml glass-stoppered bottles completely filled with the anaerobically prepared sterile medium and inoculated with pasteurized mud from ensiled leaves of sugar beets (turnips), other decaying plant material, fresh water, or sewage digester. After 1 or 2 weeks, enrichment cultures that show gas production and that smell of butyric and caproic acids are used for inoculating fresh liquid medium (10% inoculum). After two to three transfers, serial dilutions are prepared in agar roll tubes using the above medium supplemented with 2% agar. The colonies of C. kluyveri that develop after several days are generally small (1–3 mm), fluffy, spherical or compact, and lens-shaped. The colonies should contain large cells, about 1 × 10 μm in size. Typical colonies are picked and transferred to a new series of roll tubes. If cells of selected colonies contain spores (oval, terminal), a pasteurization step can be applied after suspending material of a colony in a small volume of growth medium.
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A number of complex media have been widely used for the enrichment and isolation of saccharolytic and proteolytic clostridia: reinforced clostridial medium (RCM) (Gibbs and Hirsch, 1956; Hirsch and Grinstedt, 1954); differential reinforced clostridial medium (DRCM) (Gibbs and Freame, 1965); cooked meat media (CM and CMC) (Holdeman et al., 1977; Robertson, 1915–1916); peptone-yeast extract medium (PY) (Holdeman et al., 1977); Viande-Levure medium (VL) (Beerens and Fievez, 1971; Willis, 1977). RCM and DRCM contain glucose and starch; the others can be supplemented with further ingredients, such as carbohydrates or certain amino acids, to make them more selective. DRCM is recommended especially for the detection and enumeration of clostridial spores in pasteurized samples of food (Freame and Fitzpatrick, 1971; Gibbs and Freame, 1965).
Any medium that is rich in carbohydrates and that contains some peptone and yeast extract as well as reducing agents is suitable for the enrichment of the common saccharolytic clostridia. A selective medium containing antimicrobial agents for the isolation of C. butyricum and a few other butyric acid-producing, saccharolytic clostridia from human feces has been described by Popoff (1984).
Potato mash medium, as described by Ruschmann (1928), has been used by several investigators in the past to study the clostridia in retting flax, in manure, in silage, and in milk (Dührsen, 1937; Glathe, 1934; Ritter, 1932; Ruschmann, 1928; Ruschmann and Bavendamm, 1925; Ruschmann and Harder, 1931). Its starch and pectin content favors the development of starch- and/or pectin-fermenting clostridia. Maize mash medium (Weizmann, 1919) and maize liver medium (McClung and McCoy, 1934) have been used for enrichment and isolation of the butanol-acetone-producing clostridia (Beesch, 1953; Weizmann, 1919; Weyer and Rettger, 1927), the pigment-producing strains (Hellinger, 1947; McClung, 1943), and other saccharolytic species (Gilliland and Vaughn, 1943; McClung and McCoy, 1934; Weizmann and Hellinger, 1940). Milk, without or with supplementations, has been found convenient for the enrichment of several clostridia from soil, wounds, and plant material or of the clostridia commonly present in the milk itself (Meyn, 1933; van Beynum and Pette, 1940; Weinzirl and Veldee, 1915; Weizmann and Hellinger, 1940; Winkler, 1961).
Potato tubers, stabbed and immersed in water, provide a simple method for the enrichment of common saccharolytic clostridia. The following procedure has been described by Veldkamp (1965).
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PROCEDURE: Enrichment of Saccharolytic Clostridia with Potato Tubers (Veldkamp, 1965)
A potato is washed under the tap and subsequently stabbed once or twice with a knife. It is then placed in a beaker, and enough water is added just to cover the potato; the beaker is covered with a watch-glass and incubated at 37°C. The oxygen that might be introduced into the tissue is consumed by its cells. In the anaerobic environment Clostridium rapidly starts to decompose the pectin in between the plant cells. The tissue is thus macerated. When the tuber floats, due to profuse gas formation, the water is poured out of the glass; the potato is washed and dissected. Microscopic examination of the tuber contents invariably shows the typical pleomorphic clostridial cells; among these, spore-bearing spindle-shaped cells are often encountered.
Isolation can easily be achieved as follows. A sample of the macerated tissue is inoculated into yeast extract-glucose broth and after pasteurization (10 min at 80°C in a water bath) the culture is incubated at 37°C in N2 atmosphere. A pure culture can be obtained by streaking a sample on yeast extract-glucose agar and incubating under N2.
A double-layered polypectate agar medium has been used to isolate pectolytic Clostridium spp. from tubers and carrots and from suspensions of field and rhizosphere soils (Lund, 1972; Lund et al., 1981; Perry, 1982; Perry, 1985).
Media given by various authors for enrichment and growth of saccharolytic clostridia frequently show variations that are not essential for growth of these organisms. The following medium is recommended for the growth of many species.
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PROCEDURE: Medium for Growth of Saccharolytic Clostridia
The medium contains:
1 M potassium phosphate, pH 7.5 30 ml
1 M MgSO4 1 ml
Solution M (see below) 0.5 ml
0.2M FeSO4 in 0.1 M H2SO4 0.2 ml
Trypticase 10 g
Yeast extract 6 g
Sodium thioglycolate 0.5 g
Energy source (sucrose or glucose) 10–20 g
Distilled water 1 liter
The pH is adjusted to 7.0–7.2. The medium is autoclaved for 20 min at 121°C.
Solution M contains:
MnCl2 10 mM
CaCl2 30 mM
CoCl2 5 mM
Na2MoO4 5 mM
Since stock solutions of the salts can be stored in the laboratory, this medium can be prepared rather quickly. It is best to autoclave the sugar solutions separately.
The fixation of molecular nitrogen is a property shared by many representatives of the saccharolytic clostridia (Rosenblum and Wilson, 1949). Media that contain glucose or sucrose but lack a source of nitrogen have been used for the isolation of C. pasteurianum and other species. The comprehensive literature was summarized by Skinner (1971). Based on the method of Augier (1957), the following liquid medium for counting nitrogen-fixing clostridia in soil by the most probable number method (MPN) and for subsequent isolation has been recommended by Skinner (1971).
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PROCEDURE: Isolation of Nitrogen-Fixing Saccharolytic Clostridia (Skinner, 1971)
The medium contains:
K2HPO4 0.8 g
KH2PO4 0.2 g
MgSO4·7H2O 0.2 g
NaCl 0.2 g
FeSO4·7H2O 10 mg
MnSO4·4H2O 10 mg
CaCl2 10 mg
Na2MoO4·2H2O 25 mg
Yeast extract 10 mg
Trace element solution (see below) 1 ml
Soil extract (see below) 10 ml
Glucose or sucrose 10.0 g
Sodium thioglycolate 1.0 g
Distilled water 1 liter
The pH is adjusted to 7.2.
The trace element solution contains:
Na2B4O7·10H2O 50 mg
CoNO3·6H2O 50 mg
CdSO4·2H2O 50 mg
CuSO4·5H2O 50 mg
ZnSO4·7H2O 50 mg
MnSO4·H2O 50 mg
Distilled water 1 liter
The stock solution should be saturated with CO2.
The soil extract is prepared as follows (Augier, 1956): Equal weights of a neutral garden soil and water are combined and heated at 130°C for 1 h. After cooling, it is filtered through paper, bottled, autoclaved, and stored until use.
Sodium thioglycolate is added just before the medium is distributed in 10-ml portions into narrow test tubes fitted with Durham tubes, capped or plugged, and autoclaved at 121°C for 15 min. After inoculation with 1 ml of decimal dilutions of pasteurized soil, the tubes are incubated in anaerobic jars under a nitrogen atmosphere at 30°C for 2 weeks. Positive tubes show abundant gas formation, turbidity with or without a viscoid, whitish deposit or surface pellicle, and odor of butyric acid.
Isolations are made by streaking small inocula from positive tubes on plates prepared from the above medium plus 2% agar. The plates are incubated in anaerobic jars under an atmosphere of nitrogen. Colonies can be picked within 7 days at 30°C and purified by repeated streaking on nitrogen-free agar. Pasteurization can be applied at any stage, provided that spores are present.
The above enrichment procedure can be made rather specific for C. pasteurianum if the concentration of sucrose is increased to 15% (Spiegelberg, 1944; Witz et al., 1967). Inocula from such enrichments are again pasteurized and streaked on the above agar containing 2% glucose.
Clostridia, such as C. butyricum and C. tyrobutyricum, which ferment lactate plus acetate to butyrate, CO2, and H2, have been frequently found as the dominating anaerobic sporeformers in certain silages (Bryant and Burkey, 1956; Gibson, 1965; Gibson et al., 1958; Rosenberger, 1951, 1956) and milk products (Goudkov and Sharp, 1965). C. tyrobutyricum is responsible for spoilage of certain types of cheese by formation of gas (“late blowing” of hard cheese) or rancid odor (Goudkov and Sharp, 1966; Kutzner, 1963, 1966).
A medium originally developed by Bhat and Barker (1947) has been used for the detection and the isolation of lactate-utilizing sporeformers in silage (Rosenberger, 1951, 1956), in cheese (Kutzner, 1963, 1966), and in milk (Halligan and Fryer, 1976). Both C. butyricum and C. tyrobutyricum will grow in this medium; a distinction between these species is easily possible because of the striking differences in the utilization of carbohydrates: C. butyricum grows on maltose, raffinose, lactose, and starch, which are not utilized by C. tyrobutyricum.
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PROCEDURE: Isolation of Clostridia That Ferment Lactate plus Acetate (Bhat and Barker, 1947)
The medium contains:
K2HPO4 0.5 g
(NH4)2SO4 0.5 g
MgSO4·7H2O 0.1 g
FeSO4·7H2O 20 mg
Yeast extract 0.5 g
Biotin 0.1 μg
p-Aminobenzoate 100 μg
Sodium L-lactate 10.0 g
Sodium acetate 8.0 g
Sodium thioglycolate 0.5 g
Distilled water 1 liter
Sodium thioglycolate is added immediately before autoclaving. The pH is adjusted to 6.0–7.0, and the medium is autoclaved for 20 min at 121°C.
A slightly acidic pH value of the medium favors growth of C. butyricum and C. tyrobutyricum. The latter has been shown to start growing in media with a pH value of as low as 5.3 (Kutzner, 1963). When a sodium lactate solution is used for preparing the medium, the pH should be checked after autoclaving (it tends to become more acidic).
Enrichment cultures are set up in glass-stoppered bottles (filled to the neck). They are inoculated with pasteurized garden soil or other material and incubated at 37°C. Turbidity and gas formation are observed after a few days, accompanied by an increase of the pH. Two transfers into fresh medium (5–10% inoculum) are made before isolation is performed by streaking material on agar medium of the same composition as above plus 2% agar. The plates are incubated in anaerobic jars under a nitrogen atmosphere.
Using a slightly modified medium, C. butyricum was found to be the dominant lactate-fermenting sporeformer in the ensiling process of perennial ryegrass (Gibson et al., 1958). Clostridia other than C. butyricum and C. tyrobutyricum that are found occasionally in silage (C. paraputrificum, C. tetanomorphum, C. perfringens, and C. sphenoides) require more complex media or are not able to use lactate plus acetate as energy source.
Procedures for the detection of the very low numbers of spores of C. tyrobutyricum in milk used in making cheese have been described (Fryer and Halligan, 1976; Halligan and Fryer, 1976). First, an enrichment is done using a complex medium that contains calcium lactate. Then the presence of C. tyrobutyricum is confirmed by subculturing positive enrichments in a slightly modified medium as given above.
There have been many reports on the enrichment and isolation of cellulose-decomposing clostridia, but the cultures obtained were often not pure and subcultures are not available any more. In addition to the two classical cellulose-decomposing species C. cellobioparum (Hungate, 1944) and C. thermocellum (McBee, 1948, 1950; Viljoen et al., 1926), several new mesophilic and thermophilic species have been validly described (Van Gylswyk, 1980; Madden et al., 1982; Madden, 1983; Petitdemange et al., 1984; Sleat et al., 1984; LeRuyet et al., 1985; Sleat and Mah, 1985; Murray et al., 1986; Sukhumavasi et al., 1988; Palop et al., 1989).
Samples for the isolation of cellulolytic clostridia can be taken from soil, feces of herbivorous animals (horse manure has long been recognized as an excellent source for thermophilic strains also), compost, decayed plants, and from anaerobic sewage or mud. Except for the different incubation temperature, the procedures for enrichment and isolation of both mesophilic and thermophilic cellulolytic species are about the same.
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PROCEDURE: Enrichment and Isolation of Cellulolytic Clostridia (Omelianski, 1902; Skinner, 1960, 1971)
For enrichment, the medium contains:
K2HPO4 1.0 g
(NH4)2SO4 1.0 g
MgSO4·7H2O 0.5 g
CaCO3 2.0 g
NaCl 0.5 g
Resazurin 1 mg
Cellulose (chopped filter paper) 20 g
Distilled water 1 liter
The final pH is adjusted to 7.1.
Enrichments are set up in completely filled, glass-stoppered bottles (50- or 100-ml), inoculated with 0.1 g of pasteurized sample. The sample material provides enough growth factors for the primary enrichment, and reducing conditions are established by the contaminating microflora. Incubations are made at 30–37°C. As soon as digestion of the filter paper becomes visible (1 or 2 weeks), isolation of the cellulose-degrading clostridia is performed by repeatedly streaking on cellulose agar medium.
The cellulose agar medium contains:
K2HPO4 7.5 g
KH2PO4 3.5 g
(NH4)2SO4 0.5 g
NaCl 1.0 g
MgSO4·7H2O 50 mg
CaCl2 50 mg
Resazurin 1 mg
Yeast extract 1.0 g
Cellulose 10.0 g
Agar 15.0 g
L-Cysteine hydrochloride 0.5 g
Distilled water 1 liter
The pH is adjusted to 7.1.
A suitable substrate for these enrichments is powdered cellulose (e.g., MN 300, Machery and Nagel; CF-11, Whatman) or a cellulose preparation made by wet-grinding of finely divided filter paper (e.g., Whatman No. 1 ashless cellulose paper) in a pebble mill (Hungate, 1950). To keep the cellulose particles suspended, Skinner (1960) recommended the addition of about 0.1% sodium carboxymethylcellulose (substitution range, 0.65–0.85) to the medium.
The cysteine hydrochloride is added to the medium shortly before autoclaving. The medium is autoclaved for 20 min at 121°C and distributed to petri dishes, which are kept in anaerobic jars under an atmosphere of 90% N2 + 10% CO2. Alternatively, screw-capped bottles (Skinner, 1971) or roll tubes (Hungate, 1950) may be used. Digestion of cellulose is visible after incubation at 30–37°C for 1 week or longer, depending on the activity of the strains. Colonies are often very small, and material for transfers should be picked carefully under a dissecting microscope. Colonies that contain spores are suspended in cellulose-free basal medium and pasteurized (10 min at 75°C) before streaking again.
For purification, an alternate streaking on cellulose agar or on agar that contains 0.5% cellobiose instead of cellulose can be advantageous. Cellobiose is readily fermented by all the cellulolytic isolates described. However, spore production on cellobiose agar is not as pronounced as on cellulose agar. Therefore, colonies must be examined carefully before being pasteurized.
The “double-layer” method may be used at an early stage to detect cellulose-decomposing colonies by formation of clear halos. Petri dishes are filled with 20 ml of cellulose-free agar medium and subsequently overlayed with 5 ml of cellulose-containing agar medium.
The selective isolation of C. sphenoides from mud samples using citrate as the energy source for growth has been described (Walther et al., 1977). It has not been demonstrated whether this method is also applicable to the isolation of this organism from silage or human infections in which C. sphenoides has been reported to occur (Gibson, 1965; Smith, 1949). C. sphenoides is a representative of the relatively small group of saccharolytic bacteria that do not form butyrate but ferment carbohydrates to ethanol, acetate, CO2, and H2.
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PROCEDURE: Direct Isolation of Clostridium sphenoides (Walther et al., 1977)
The medium contains:
K2HPO4 2.0 g
KH2PO4 3.4 g
MgSO4·7H2O 0.2 g
(NH4)2SO4 0.3 g
NaCl 0.6 g
CaCl2·2H2O 60 mg
Yeast extract 4.0 g
Peptone 2.0 g
Trisodium citrate dihydrate 14.7 g
Resazurin 1 mg
Agar 15.0 g
Distilled water 1 liter
Adjust the pH to 6.7–7.0. 0.3 g of L-cysteine hydrochloride is added after the agar has been dissolved.
Applying the Hungate technique, 5-ml portions are added to 15-ml tubes. After autoclaving (20 min at 121°C), the tubes are kept in a water bath at 50°C. Then 0.3 ml of a pasteurized mud sample is used for the preparation of decimal serial dilutions. Roll tubes are prepared which are incubated at 37°C. After 48–72 h, single colonies that show an increased size as compared to the background growth of contaminating organisms are picked, inoculated into 10 ml of liquid citrate medium, and incubated at 37°C for 24 h. For further purification, serial dilutions in roll tubes containing citrate agar are repeated at least three times.
During isolation, the selected colonies are checked for the presence of wedge-shaped cells and of spherical, nearly terminally located spores, both of which are typical for C. sphenoides.
A number of single amino acids can be used by certain clostridial species as sources of energy, carbon, and nitrogen for growth (Andreesen et al., 1989). Media that contain a certain amino acid, small amounts of yeast extract, and minerals have been used for enrichment and isolation of some clostridial species. The use of L-alanine led to the isolation of C. propionicum (Cardon and Barker, 1946, 1947) and of γ-aminobutyrate and δ-aminovalerate to “C. aminobutyricum” and C. aminovalericum, respectively (Hardman and Stadtman, 1960a, 1960b). C. tetanomorphum has been isolated by Kornberg using L-histidine as the principal substrate (see Wachsman and Barker, 1955a), and the use of L-glutamate led to the isolation of C. cochlearium as well as of C. tetanomorphum (Barker, 1937, 1939). Lysine was used to enrich and isolate Clostridium SB4 (Costilow et al., 1966), which subsequently was identified as C. subterminale (H. A. Barker, personal communication). It appears that the possible advantage of using single amino acids as selective substrates for the isolation of certain clostridia has never been studied systematically. Investigations by Mead (1971), Elsden et al. 1976, and Elsden and Hilton (1978, 1979) have shown that a considerable number of clostridia are able to ferment single amino acids.
In the following procedure, the enrichment and isolation of C. cochlearium or C. tetanomorphum is described, using L-glutamate as the specific substrate.
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PROCEDURE: Enrichment and Isolation of Glutamate-Fermenting Clostridia (Barker, 1937, 1939)
The medium contains:
K2HPO4 0.2 g
MgSO4·7H2O 0.1 g
Yeast extract 0.5 g
Sodium L-glutamate 10.0 g
Sodium thioglycolate 0.5 g
Tap water 1 liter
The final pH is ajusted to 7.6. Enrichments are set up in completely filled glass-stoppered bottles with pasteurized soil as inoculum (0.5 g soil per 100 ml). Within 1–2 days at 37°C, an abundant growth of sporeforming anaerobes is observed accompanied by a moderate production of gas. After transferring a volume of 2 ml once or twice to 100-ml bottles containing medium of the above composition, isolations are made by repeatedly streaking on L-glutamate agar plates or by diluting in agar roll tubes.
Laanbroek et al. (1979) described the enrichment of C. cochlearium from anaerobic sludge in an L-glutamate-limited anaerobic chemostat.
For mass culture of C. cochlearium or C. tetanomorphum, the “Medium for Growth of Saccharolytic Clostridia” (this chapter) may be used. Instead of sugars, sodium L-glutamate (17 g per 1 liter of medium) is added as energy source and Trypticase is omitted.
A selective isolation medium for C. sporogenes and related clostridia that are capable of growth by means of coupled oxidation-reduction reactions between appropriate pairs of amino acids has been developed by Fryer and Mead (1979). The semidefined medium contains alanine and proline as the principal energy sources and polymyxin. When samples from soil or mud were directly plated on agar medium, a high percentage of the isolates were strains of the C. sporogenes/C. botulinum group.
The anaerobic purine fermenters, C. acidiurici and C. cylindrosporum, were originally isolated by Barker and Beck (1942) from enrichment cultures with uric acid as the sole source of carbon, nitrogen, and energy. Several more strains of both species were obtained by Champion and Rabinowitz (1977) using the same method. Uric acid agar supplemented with tryptone, meat extract, liver extract, and chicken fecal extract was used by Barnes and Impey (1974) for the isolation of uric acid-decomposing anaerobes from avian cecum. The isolated strains of anaerobic sporeformers were designated C. malenominatum. Another purinolytic species, C. purinolyticum, was isolated using adenine as the selective substrate (Dürre et al., 1981).
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PROCEDURE: Isolation of Purine-Fermenting Clostridia (Champion and Rabinowitz, 1977; Barker and Beck, 1942)
The medium contains:
Uric acid 2.0 g
10 N KOH 3.0 ml
K2HPO4·3H2O (70% solution) 1.5 ml
MgSO4·7H2O 50 mg
CaCl2·2H2O 5 mg
FeSO4·7H2O 2 mg
Yeast extract 1.2 g
Resazurin 1 mg
Mercaptoacetic acid 1.5 ml
Distilled water (see below) 1 liter
The medium is prepared as follows: 500 ml distilled water plus KOH plus the K2HPO4 solution are brought to a boil. Then uric acid is slowly added. It goes into solution instantaneously. The solution is cooled down and the other ingredients of the medium are added. Mercaptoacetic acid is added shortly before autoclaving, and the pH is adjusted to 7.2 using a sterile, 60% K2CO3 solution. Other reducing agents may be used instead of mercaptoacetic acid: sodium thioglycolate (750 mg/liter) or cysteine hydrochloride (1 g/liter) plus dithiothreitol (0.1 g/liter).
After autoclaving (20 min at 121°C), the medium is immediately used to completely fill 50-ml glass-stoppered bottles. After inoculation with 0.5 g of pasteurized soil or chicken feces, the bottles are incubated at 37°C. Growth occurs within 24–48 h and is accompanied by an increase of alkalinity. Utilization of uric acid can be monitored by the decrease in absorbancy at 290 nm of a 1 to 100 dilution of the growth medium. After one or two more transfers (0.5% inoculum) to 50-ml bottles containing the medium above, isolation is made under strict anaerobiosis by repeatedly streaking on uric acid agar plates incubated in anaerobic jars or on roll tubes.
The purinolytic clostridia require strictly anaerobic conditions for growth, and the application of stringent anaerobic culture techniques is necessary. The three accepted species, C. acidiurici, C. cylindrosporum, and C. purinolyticum, are clearly differentiated on the basis of DNA homology, whereas the form and position of their spores differ (Schiefer-Ullrich et al., 1984). C. cylindrosporum was named after its cylindrical spores that are variably situated and do not cause swelling of the mother cell (Barker and Beck, 1942; Andreesen et al., 1985).
The isolation of adenine-fermenting clostridia requires a medium of slightly different composition. Instead of uric acid, it contains 0.2% adenine, 0.2% sodium bicarbonate, and 10–7 M each of thiamine, sodium selenite, sodium molybdate, and sodium tungstate to replace yeast extract (Dürre et al., 1981).
C. kluyveri has specialized on the conversion of ethanol and acetate to butyrate, caproate, and H2 (Barker and Taha, 1942; Bornstein and Barker, 1948a). It is unable to utilize carbohydrates or amino acids for growth and requires only biotin and p-aminobenzoate as growth factors. Therefore, the medium used for enrichment is very selective.
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PROCEDURE: Isolation of Clostridium kluyveri (Bornstein and Barker, 1948a; Stadtman and Burton, 1955)
The medium contains:
K2HPO4 0.30 g
KH2PO4 0.20 g
NH4Cl 0.25 g
MgSO4·7H2O 0.20 g
CaCl2·2H2O 10 mg
FeSO4·7H2O 5 mg
MnSO4·4H2O 2 mg
Na2MoO4·2H2O 2 mg
Biotin 10 μg
p-Aminobenzoate 200 μg
Ethanol 20 ml
Potassium acetate 5.0 g
Glacial acetic acid 2.5 ml
Resazurin 1 mg
Sodium thioglycolate 500 mg
Distilled water 1 liter
Thioglycolate is added shortly before autoclaving (20 min at 121°C). After autoclaving, the medium is rapidly cooled, and the pH is adjusted to 7.0 using a 60% K2CO3 solution autoclaved separately (about 8–10 ml are required).
The enrichment is made at 35°C in 100-ml glass-stoppered bottles completely filled with the anaerobically prepared sterile medium and inoculated with pasteurized mud from ensiled leaves of sugar beets (turnips), other decaying plant material, fresh water, or sewage digester. After 1 or 2 weeks, enrichment cultures that show gas production and that smell of butyric and caproic acids are used for inoculating fresh liquid medium (10% inoculum). After two to three transfers, serial dilutions are prepared in agar roll tubes using the above medium supplemented with 2% agar. The colonies of C. kluyveri that develop after several days are generally small (1–3 mm), fluffy, spherical or compact, and lens-shaped. The colonies should contain large cells, about 1 × 10 μm in size. Typical colonies are picked and transferred to a new series of roll tubes. If cells of selected colonies contain spores (oval, terminal), a pasteurization step can be applied after suspending material of a colony in a small volume of growth medium.
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hay quá , có sách cậu pót cả lên cho anh em tham khảo được chứ
Đây không phải là sách mà là tài liệu online tôi truy cập được qua cổng của một trường đại học. Ý tôi nói phần tham khảo cụ thể ở đây là tế tác giả, năm xuất bản, tên bài báo, tên tạp chí mà cái bài trên trích dẫn.
À ! anh SHMT ơi, nếu anh photo sách thì anh photo dùm cho em một bản luôn anh hấy! Vì em và gvthang học chung một lớp. Còn cái khoản kia thì...nếu anh có tài khoản thì bọn em sẽ chuyển vào cho anh còn nếu không thì bọn em sẽ chuyển theo địa chỉ của anh.
địa chỉ của bọn em: Lê Đức Dũng. Lớp 01SH- Công nghệ sinh học- Trường Đại Học Bách Khoa -ĐN
địa chỉ của bọn em: Lê Đức Dũng. Lớp 01SH- Công nghệ sinh học- Trường Đại Học Bách Khoa -ĐN
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