Chapter 12: Clostridium botulinum

Updated: 8/3/00

Contents

Potential Food Safety Hazard

The following information on Clostridium botulinum is from Solomon and Lilly (1998).

C. botulinum is an anaerobic, rod-shaped sporeformer that produces a protein with characteristic neurotoxicity. Botulism, a severe food poisoning, results from ingestion of food containing botulinal toxin produced during the growth of these organisms in food. Although this food poisoning is rare, the mortality rate is high; the 962 recorded botulism outbreaks in the United States from 1899 to 1990 (CDC, 1979) involved 2320 cases and 1036 deaths. In outbreaks in which the toxin type was determined, 384 were caused by type A, 106 by type B, 105 by type E, and 3 by type F.

In two outbreaks, the foods implicated contained both types A and B toxins. The limited number of reports of C and D toxins as the causative agent of human botulism have not been generally accepted. However, all types except F and G, which have not been as thoroughly studied, are important causes of animal botulism.

Antigenic types of C. botulinum are identified by complete neutralization of their toxins by the homologous antitoxin; cross-neutralization by heterologous antitoxins does not occur or is minimal. There are seven recognized antigenic types: A, B, C, D, E, F, and G. Cultures of five of these types apparently produce only one type of toxin but all are given type designations corresponding to their toxin production. Types C and D cross-react with antitoxins to each other because they each produce more than one toxin and have at least one common toxin component. Type C produces predominantly C1 toxin with lesser amounts of D and C2, or only C2, and type D produces predominantly type D toxin along with smaller amounts of C1 and C2. Mixed toxin production by a single strain of C. botulinum may be more common than previously realized. There is a slight reciprocal cross-neutralization with types E and F, and recently a strain of C. botulinum was shown to produce a mixture of predominantly type A toxin, with a small amount of type F.

Aside from toxin type, C. botulinum can be differentiated into general groups on the basis of cultural, biochemical, and physiological characteristics. Cultures producing types C and D toxins are not proteolytic on coagulated egg white or meat and have a common metabolic pattern which sets them apart from the others. All cultures that produce type A toxin and some that produce B and F toxins are proteolytic. All type E strains and the remaining B and F strains are nonproteolytic, with carbohydrate metabolic patterns differing from the C and D nonproteolytic groups. Strains that produce type G toxin have not been studied in sufficient detail for effective and satisfactory characterization.

C. botulinum is widely distributed in soils and in sediments of oceans and lakes. The finding of type E in aquatic environments by many investigators correlates with cases of type E botulism that were traced to contaminated fish or other seafoods. Types A and B are most commonly encountered in foods subjected to soil contamination. In the United States, home-canned vegetables are most commonly contaminated with types A and B, but in Europe, meat products have also been important vehicles of food-borne illness caused by these types.

Optimum temperature for growth and toxin production of proteolytic strains is close to 35ºC; for nonproteolytic strains, it is 26-28ºC. Nonproteolytic types B, E, and F can produce toxin at refrigeration temperatures (3-4ºC). Toxins of the nonproteolytics do not manifest maximum potential toxicity until they are activated with trypsin; toxins of the proteolytics generally occur in fully (or close to fully) activated form. These and other differences can be important in epidemiological and laboratory considerations of botulism outbreaks. Clinical diagnosis of botulism is most effectively confirmed by identifying botulinal toxin in the blood, feces, or vomitus of the patient. Specimens must be collected before botulinal antitoxin is administered to the patient. Identifying the causative food is most important in preventing additional cases of botulism. See Examination of Canned Foods, Chapter 5.

Botulism in infants 6 weeks to 1 year of age was first recognized as a distinct clinical entity in 1976. This form of botulism results from growth and toxin production by C. botulinum within the intestinal tract of infants rather than from ingestion of preformed toxin. It is usually caused by C. botulinum types A or B, but a few cases have been caused by other types. Infant botulism has been diagnosed in most U.S. states and in every populated continent except Africa (Arnon, 1987).

Constipation almost always occurs in infant botulism and usually precedes characteristic signs of neuromuscular paralysis by a few days or weeks. There is a broad range of severity of illness. Some infants show only mild weakness, lethargy, and reduced feeding and do not require hospitalization. Many have shown more severe symptoms such as weakened suck, swallowing, and cry; generalized muscle weakness; and diminished gag reflex with a pooling of oral secretions. Generalized muscle weakness and loss of head control in some infants reaches such a degree of severity that the patient appears "floppy." In some hospitalized cases, respiratory arrest has occurred, but most were successfully resuscitated, and with intense supportive care have ultimately recovered. As a result, the case-fatality rate (2%) for this form of botulism is low. Recovery usually requires at least several weeks of hospitalization (Arnon, 1987).

Honey, a known source of C. botulinum spores, has been implicated in some cases of infant botulism. In studies of honey, up to 13% of the test samples contained low numbers of C. botulinum spores (Hauschild et al., 1988). For this reason, the FDA, the Centers for Disease Control and Prevention (CDC), and the American Academy of Pediatrics recommend not feeding honey to infants under the age of 1 year.

Contents

Control Measures

Measures to prevent botulism include reduction of the microbial contamination level, acidification, reduction of moisture level, and whenever possible, destruction of all botulinal spores in the food. Heat processing is the most common method of destruction. Properly processed canned foods will not contain viable C. botulinum. Home-canned foods are more often a source of botulism than are commercially canned foods, which probably reflects the commercial canners' great awareness and better control of the required heat treatment.

A food may contain viable C. botulinum and still not be capable of causing botulism. If the organisms do not grow, no toxin is produced. Although many foods satisfy the nutritional requirements for the growth of C. botulinum, not all of them provide the necessary anaerobic conditions. Both nutritional and anaerobic requirements are supplied by many canned foods and by various meat and fish products. Growth in otherwise suitable foods can be prevented if the product, naturally or by design, is acidic (of low pH), has low water activity, a high concentration of NaCl, an inhibitory concentration of NaNO2 or other preservative, or two or more of these conditions in combination. Refrigeration will not prevent growth and toxin formation by nonproteolytic strains unless the temperature is precisely controlled and kept below 3ºC. Foods processed to prevent spoilage but not usually refrigerated are the most common vehicles of botulism (Solomon and Lilly, 1998).

Contents

FDA Guidelines

Table 12-1. FDA Guidelines for C. botulinum in fish and fishery products.
Product 
Guideline
Reference 
All fish  Presence of viable spores or vegetative cells in products that will support their growth; or, presence of toxin.  FDA, 1998b 
Salt-cured, air dried uneviscerated fish  Illegal product (potential for C. botulinum toxin). Note: Small fish, less than 5 inches (12.7 cm) in length (e.g. anchovies and herring sprats), that are processed in a manner that prevents toxin formation, and that reach a water phase salt content of 10%, a water activity of below 0.85 (Note: this value is based on the minimum water activity for growth of S. aureus), or a pH of 4.6 or less are exempt from the evisceration requirement.  FDA, 1998b 
FDA, 1998a 

Contents

Growth

Table 12-2. Limiting conditions for C. botulinum type A and proteolytic B and F growth.
Parameter 
Values Reported 
Reference 
Min. aw 
0.935
 FDA, 1998c
Min. pH  4.6 Hauschild, 1989 
Max. pH  9.0  Hauschild, 1989 
Max. %NaCl  10  Hauschild, 1989 
Min. temp. 
10ºC (50ºF) 
 Hauschild, 1989
Max. temp. 
48ºC (118.4ºF)
 FDA, 1998c

Table 12-3. Limiting conditions for C. botulinum type E and nonproteolytic B and F growth.
Parameter 
Values Reported 
Reference 
Min. a w 
0.97 
 Hauschild, 1989 
Min. pH 
5.0 
 Hauschild, 1989 
Max. pH 
9.0 
 Hauschild, 1989 
Max. %NaCl
5 
 Hauschild, 1989 
Min. temp. 
3.3ºC (37.9ºF)
 FDA, 1998c 
Max. temp. 
45ºC (113ºF) 
 Hauschild, 1989 

Contents

Heat Resistance

Table 12-4. Spore heat resistance of C. botulinum nonproteolytic type E.
Temp. 
D-Value 
Strain 
Medium 
Reference 
(ºC) 
(ºF) 
(min.) 
      
73.89
165
12.97 
 Beluga  Crabmeat Lynt et al., 1977 
73.89
165 
10.39 
 Alaska  Crabmeat  Lynt et al., 1977 
73.89
165 
6.80 
 G21-5  Crabmeat  Lynt et al., 1977 
73.9 
165 
8.66 
 Mixed  Surimi  Rhodehamel et al., 1991
76.67
170 
4.07 
 Beluga  Crabmeat  Lynt et al., 1977 
76.67
170 
3.04 
 Alaska  Crabmeat  Lynt et al., 1977 
76.67
170 
2.38 
 G21-5  Crabmeat  Lynt et al., 1977 
76.7 
170 
3.49 
 Mixed  Surimi  Rhodehamel et al., 1991 
79.4 
175 
2.15 
 Mixed  Surimi  Rhodehamel et al., 1991 
79.44
175 
1.65 
 Beluga  Crabmeat  Lynt et al., 1977 
79.44
175 
1.35 
 Alaska  Crabmeat  Lynt et al., 1977 
79.44
175 
1.10 
 G21-5  Crabmeat  Lynt et al., 1977 
82.2 
180 
1.22 
 Mixed  Surimi  Rhodehamel et al., 1991 
82.22
180 
0.74 
 Beluga  Crabmeat  Lynt et al., 1977 
82.22
180 
0.51 
 Alaska  Crabmeat  Lynt et al., 1977 
82.22
180 
0.63 
 G21-5  Crabmeat  Lynt et al., 1977 
82.22
180 
0.62 
 25V-1  Crabmeat  Lynt et al., 1977 
82.22
180 
0.49 
 25V-2  Crabmeat  Lynt et al., 1977 
85.00
185 
0.29 
 Beluga  Crabmeat  Lynt et al., 1977 

Table 12-5. Spore heat resistance of C. botulinum type B.
Temp. 
D-Value
Strain 
Medium 
Reference 
(ºC)
(ºF) 
(min.) 
      
88.9
192
 12.9 
N/S 
Crabmeat 
 Peterson et al., 1997 
89.5
193
 11.1 
N/S 
Crabmeat 
 Peterson et al., 1997 
90.0
194
 9.5 
N/S 
Crabmeat 
 Peterson et al., 1997 
90.6
195
 8.2 
N/S 
Crabmeat 
 Peterson et al., 1997 
91.1
196
 7.1 
N/S 
Crabmeat 
 Peterson et al., 1997 
91.7
197 
 6.1 
N/S 
Crabmeat 
 Peterson et al., 1997 
92.2
198 
 5.3 
N/S 
Crabmeat 
 Peterson et al., 1997 
92.8
199 
 4.5 
N/S 
Crabmeat 
 Peterson et al., 1997 
93.4
200 
 3.9 
N/S 
Crabmeat 
 Peterson et al., 1997 
93.9
201 
 3.4 
N/S 
Crabmeat 
 Peterson et al., 1997 
94.5
202 
 2.9 
N/S 
Crabmeat 
 Peterson et al., 1997 
 N/S = Not specified

Contents

Incidence in Fish and Fishery Products

Dungeness crab

Table 12-6. Occurrence of C. botulinum in the intestinal tract, gills, and shell of Dungeness crab (Cancer magister) crab from the Pacific Coast of the United States (Eklund and Poysky, 1967).
Area No. crab % crab positive C. botulinum type
Ketchikan, AK 21 57 E
Bellingham, WA 18 61 B, E
6 66 E
32 75 E
Gray's Harbor, WA 40 75 A, E
Columbia River 20 87 E
Eureka, CA 50 14 E
Fort Bragg, CA 43 30 A, B, E
San Francisco, CA 50 12 B, C, E

Great Britain fishing grounds

Table 12-7. Distribution of C. botulinum type E in the marine environment of Great Britain (Cann et al., 1967).
Source Sample No. tested No. containing C. botulinum type E
Shops in Britain Vacuum packed fish 646 5
Skagerrak White fish intestines 130 0
Skagerrak Whole white fish 130 0
North Sea White fish intestines 96 0
North Sea Herring intestines 200 0
Norwegian Sea Herring intestines 22 3
Norwegian Sea Whole herring 44 24
England & Wales Shellfish 106 0

Gulf of Maine fish

Table 12-8. Total numbers of composite fish intestine (10-1/2 intestines per sample) assayed and the number containing C. botulinum toxin (Nickerson et al., 1967).
Species
Number of composite intestine samples
Number of samples containing type E toxin
Haddock
116
6
Cod
41
1
Flounder, black back
45
4
Flounder, dab
31
11
Pollock
9
0
1Non-specific

Table 12-9. Toxin tests on Most Probable Number (MPN) cultures of frozen intestines corresponding to samples previously found to contain type E C. botulinum (Nickerson et al., 1967).
Sample Sample number MPN Type E cells per 100g
Haddock
1
10
2
<4
3
4
4
4
5
<4
6
<4
Cod
1
<4
Black back
1
<4
2
4
3
<4
4
<4

Menhaden surimi

Seven of 565 (1.2%) test portions (250g) of menhaden surimi without added cryoprotectants contained C. botulinum type E spores (Rhodehamel et al., 1991).

Seafood cocktails

C. botulinum type E was not detected in 35 samples of commercial seafood cocktails, ranging in pH from 4.10 to 4.85 (Lerke, 1973).

Smoked eel

Two of 10 (20%) different samples of smoked eel tested positive for C. botulinum type E (Abrahamsson, 1967).

Smoked salmon

119 samples of smoked salmon in Denmark were examined for C. botulinum spores. C. botulinum type B was isolated from 2 samples (1.68%). A strong inhibitory effect of formaldehyde against C. botulinum in smoked salmon was noted (Nielsen and Pedersen, 1967).

U.S. Pacific coast fish

Table 12-10. C. botulinum toxicity screening results from fish gills and viscera (Craig and Pilcher, 1967).
Fish
No. of samples tested
No. of samples toxic
% toxic
No. of samples typed as type E
Sockeye salmon
56
12
21.4
4
Chinook salmon
91
15
16.5
4
Silver salmon
210
21
10.0
10
Sturgeon
27
3
17.0
3
Steelhead
36
7
19.4
5, 2B
Bottom fish
113
19
16.8
13, 1A

Table 12-11. Comparative incidence of toxic specimens in ocean-caught and river caught salmon (Craig and Pilcher, 1967).
Species/location
Samples tested
No. toxic samples
% toxic samples
Silver salmon      
Port Angeles
65
1
1.5
La Push
50
3
6.0
Westport
43
4
9.3
Columbia River
28
9
32.3
Newport-Depot Bay
34
1
2.9
Chinook salmon      
Columbia River
74
15
20.3
Westport Newport
75
5
6.7

Table 12-12. Comparative incidence of toxic specimens in bottom fish caught near Columbia River and in a distant coastal location (Craig and Pilcher, 1967).
Location
No. tested
No. toxic
% toxic
Coos Bay 54 5 9.2
Astoria 59 14 23.7

Table 12-13. Comparative incidence of toxic specimens in miscellaneous samples tested (Craig and Pilcher, 1967).
Sample No. tested No. toxic % toxic
Clams 8 1 12.5
Oysters 3 0 0
Smelt 2 0 0
Smoked fish 15 2 7.5

U.S. Retail fish

Table 12-14. Occurrence of C. botulinum in random samplesa of fish and other seafoods obtained from retail markets, wholesale distributors/processors, a fish farm, and fish hatchery, between 1984-1985 (Baker et al., 1990).
Common name Origin No. pos./ No. tested % Types
Butterfish California Coast
0/5
 0 NA
Cat fish, farm raised Modesto, CA
3/4
 75.0 3A, 1F
Indianola, MS
1/5
 20.2 1A
Halibut Pacific Coast
3/11
 27.3 3A
King fish Unknown
0/5
 0 NA
Ling cod Pacific Coast
1/5
 20.0 1A
Mackerel Unknown
0/5
 0 NA
Ocean perch Pacific Coast
2/7
 28.6 2A
Pacific oyster Pacific Coast
0/5
 0 NA
Prawns Unknown
0/5
 0 NA
Rockfish, G.I. tract
3/10
 30.0 3A
Rockfish, compositea
6/6
 100.0 3A
Rockfish, retail market fillets
0/8
 0 NA
Rex sole California coast
2/9
 22.0 1A, 1B
King salmon gills Nimbus River, CA
2/18
 11/1 2A
King salmon G.I. tract Nimbus River, CA
3/8
 37.5 3A
King salmon compositeb Nimbus River, CA
3/3
 100.0 3A
King salmon retail market fillet Alaska
2/14
 14.3 2A, 1B
Sardines Canada
2/5
 40.0 1A, 1B
Scallops East Coast
1/6
 16.6 1A
Shark Unknown
0/1
 0 NA
Shrimp Unknown
0/5
 0 NA
Smelt Unknown
0/5
 0 NA
Squid Unknown
0/5
 0 NA
Trout Unknown
2/6
 33.3 1A, 1B
aSample units were 70g of muscle homogenate
bComposite sample: Homogenized flesh from 4-9 fish.
NA = Not applicable.

Contents

Analytical Procedures

Contents

C. botulinum (Solomon and Lilly, 1998)

  1. Equipment and materials
    1. Refrigerator
    2. Clean dry towels
    3. Bunsen burner
    4. Sterile can opener (bacteriological or puncture type)
    5. Sterile mortar and pestle
    6. Sterile forceps
    7. Sterile cotton-plugged pipets
    8. Mechanical pipetting device (never pipet by mouth)
    9. Sterile culture tubes (at least a few should be screw-cap tubes)
    10. Anaerobic jars (gaspak or case-nitrogen replacement)
    11. Transfer loops
    12. Incubators, 35 and 28ºc
    13. Sterile, reserve sample jars
    14. Culture tube racks
    15. Microscope slides
    16. Microscope, phase-contrast or bright-field
    17. Sterile petri dishes, 100 mm
    18. Centrifuge tubes
    19. Centrifuge, refrigerated, high-speed
    20. Trypsin (1:250; difco laboratories, Detroit, MI)
    21. Syringes, 1 and 3 ml, sterile, with 25 gauge, 5/8 inch (1.6 cm) needles for injecting mice
    22. Mice, 16-24 g (for routine work, up to 34 g)
    23. Mouse cages, feed, water bottles, etc.
    24. Millipore filters: 0.45 µm pore size

  2. Media and reagents
    1. Alcoholic solution of iodine (4% iodine in 70% ethanol) (R18)
    2. Chopped liver broth (M38) or cooked meat medium (M42)
    3. Trypticase-peptone-glucose-yeast extract (TPGY) (M151) broth or with trypsin (TPGYT) (M151a)
    4. Liver-veal-egg yolk agar (M84) or anaerobic egg yolk agar (M12)
    5. Sterile, gel-phosphate buffer, pH 6.2 (R29)
    6. Absolute ethanol
    7. Gram stain reagents (R32), crystal violet (R16), or methylene blue (R45) solutions
    8. Sterile physiological saline solution (R63)
    9. Monovalent antitoxin preparations, types A-F (obtain from CDC)
    10. Trypsin solution (prepared from Difco 1:250)
    11. 1 N Sodium hydroxide solution (R73)
    12. 1 N Hydrochloric acid solution (R36)

  3. Sample preparation

    4. Preliminary examination. Refrigerate samples until testing, except unopened canned foods, which need not be refrigerated unless badly swollen and in danger of bursting. Before testing, record product designation, manufacturer's name or home canner, source of sample, type of container and size, labeling, manufacturer's batch, lot or production code, and condition of container. Clean and mark container with laboratory identification codes.

    Solid and liquid foods. Aseptically transfer foods with little or no free liquid to sterile mortar. Add equal amount of gel-phosphate buffer solution and grind with sterile pestle before inoculation. Alternatively, inoculate small pieces of product directly into enrichment broth with sterile forceps. Inoculate liquid foods directly into enrichment broth with sterile pipets. Reserve sample; after culturing, aseptically remove reserve portion to sterile sample jar for tests which may be needed later. Refrigerate reserve sample.

    Opening of canned foods (see Chapter 5)

    Examine product for appearance and odor. Note any evidence of decomposition. DO NOT TASTE the product under any circumstances. Record the findings.

  4. Detection of viable C. botulinum

    1. Enrichment. Remove dissolved oxygen from enrichment media by steaming 10-15 min and cooling quickly without agitation before inoculation.

      2. Inoculate 2 tubes of cooked meat medium with 1-2 g solid or 1-2 ml liquid food per 15 ml enrichment broth. Incubate at 35ºC.

      Inoculate 2 tubes of TPGY broth as above. Incubate at 28ºC. Use TPGYT as alternative only when organism involved is strongly suspected of being a nonproteolytic strain of types B, E, or F.

      Introduce inoculum slowly beneath surface of broth to bottom of tube. After 5 d of incubation, examine enrichment cultures. Check for turbidity, gas production, and digestion of meat particles. Note the odor.

      Examine cultures microscopically by wet mount under high-power phase contrast, or a smear stained by Gram reagent, crystal violet, or methylene blue under bright-field illumination. Observe morphology of organisms and note existence of typical clostridial cells, occurrence and relative extent of sporulation, and location of spores within cells. A typical clostridial cell resembles a tennis racket. At this time test each enrichment culture for toxin, and if present, determine toxin type according to procedure in 6, below. Usually, a 5 d incubation is the period of active growth giving the highest concentration of botulinal toxin. If enrichment culture shows no growth at 5 d, incubate an additional 10 d to detect possible delayed germination of injured spores before discarding sample as sterile. For pure culture isolation save enrichment culture at peak sporulation and keep under refrigeration.

    2. Isolation of pure cultures. C. botulinum is more readily isolated from mixed flora of enrichment culture or original specimen if sporulation has been good.

      3. Pre-treatment of specimens for streaking. Add equal volume of filter-sterilized absolute alcohol to 1 or 2 ml of enrichment culture in sterile screw-cap tube. Mix well and incubate 1 h at room temperature. To isolate from sample, take 1 or 2 ml of retained portion, and add an equal volume of filter-sterilized absolute alcohol in sterile screw-cap tube. Mix well and incubate 1 h at room temperature. Alternatively, heat 1 or 2 ml of enrichment culture or sample to destroy vegetative cells (80ºC for 10-15 min). DO NOT use heat treatment for nonproteolytic types of C. botulinum.

      Plating of treated cultures. With inoculating loop, streak 1 or 2 loopfuls of ethanol or heat-treated cultures to either liver- veal-egg yolk agar or anaerobic egg yolk agar (or both) to obtain isolated colonies. If necessary, dilute culture to obtain well-separated colonies. Dry agar plates well before use to prevent spreading of colonies. Incubate streaked plates at 35ºC for about 48 h under anaerobic conditions. A Case anaerobic jar or the GasPak system is adequate to obtain anaerobiosis; however, other systems may be used.

  5. Selection of typical C. botulinum colonies

    6. Selection. Select about 10 well-separated typical colonies, which may be raised or flat, smooth or rough. Colonies commonly show some spreading and have an irregular edge. On egg yolk medium, they usually exhibit surface iridescence when examined by oblique light. This luster zone, often referred to as a pearly layer, usually extends beyond and follows the irregular contour of the colony. Besides the pearly zone, colonies of C. botulinum types C, D, and E are ordinarily surrounded by a wide zone (2-4 mm) of yellow precipitate. Colonies of types A and B generally show a smaller zone of precipitation. Considerable difficulty may be experienced in picking toxic colonies since certain other members of the genus Clostridium produce colonies with similar morphological characteristics but do not produce toxins.

    Inoculation. Use sterile transfer loop to inoculate each selected colony into tube of sterile broth. Inoculate C. botulinum type E into TPGY broth. Inoculate other toxin types of C. botulinum into chopped liver broth or cooked meat medium. Incubate as described in 4-a, above, for 5 d. Test for toxin production as described in 6, below. To determine toxin type, see 6-c, below.

    Isolation of pure culture. Restreak toxic culture in duplicate on egg yolk agar medium. Incubate one plate anaerobically at 35ºC. Incubate second plate aerobically at 35ºC. If colonies typical of C. botulinum are found only on anaerobic plate (no growth on aerobic plate), the culture may be pure. Failure to isolate C. botulinum from at least one of the selected colonies means that its population in relation to the mixed flora is probably low. Repeated serial transfer through additional enrichment steps may increase the numbers sufficiently to permit isolation. Store pure culture in sporulated state either under refrigeration, on glass beads, or lyophilized.

  6. Detection and identification of botulinal toxin

    1. Preparation of food sample. Culture one portion of sample for detection of viable C. botulinum; remove another portion for toxicity testing, and store remainder in refrigerator. Centrifuge samples containing suspended solids under refrigeration and use supernatant fluid for toxin assay. Extract solid foods with equal volume of gel-phosphate buffer, pH 6.2, by macerating food and buffer with pre-chilled mortar and pestle. Centrifuge macerated sample under refrigeration and use supernatant fluid for toxin assay. Rinse empty containers suspected of having held toxic foods with a few milliliters of gel-phosphate buffer. Use as little buffer as possible to avoid diluting toxin beyond detection. To avoid or minimize nonspecific death of mice, filter supernatant fluid through a 0.45µm millipore filter before injecting mice. For non-proteolytic samples or cultures, trypsinize after filtration.

    2. Determination of toxicity in food samples or cultures

      3. Trypsin treatment. Toxins of nonproteolytic types, if present, may need trypsin activation to be detected. Therefore, treat a portion of food supernatant fluid, liquid food, or TPGY culture with trypsin before testing for toxin. Do not treat TPGYT culture with trypsin since this medium already contains trypsin and further treatment may degrade any fully activated toxin that is present. Adjust portion of supernatant fluid, if necessary, to pH 6.2 with 1 N NaOH or HCl. Add 0.2 ml aqueous trypsin solution to 1.8 ml of each supernatant fluid to be tested for toxicity. (To prepare trypsin solution, place 0.5 g of Difco 1:250 trypsin in clean culture tube and add 10 ml distilled water, shake, and warm to dissolve. Analysts who are allergic to trypsin should weigh it in a hood or wear a face mask.) Incubate trypsin- treated preparation at 35-37ºC for 1 h with occasional gentle agitation.

      Toxicity testing. Conduct parallel tests with trypsin-treated materials and untreated duplicates. Dilute a portion of untreated sample fluid or culture to 1:5, 1:10, and 1:100 in gel-phosphate buffer. Make the same dilutions of each trypsinized sample fluid or culture. Inject each of separate pairs of mice intraperitoneally (i.p.) with 0.5 ml untreated undiluted fluid and 0.5 ml of each dilution of untreated test sample, using a 1 or 3 ml syringe with 5/8 inch (1.6 cm), 25 gauge needle. Repeat this procedure with trypsin-treated duplicate samples. Heat 1.5 ml of untreated supernatant fluid or culture for 10 min at 100ºC. Cool heated sample and inject each of a pair of mice with 0.5 ml undiluted fluid. These mice should not die, because botulinal toxin, if present, will be inactivated by heating.

      Observe all mice periodically for 48 h for symptoms of botulism. Record symptoms and deaths. Typical botulism signs in mice begin usually in the first 24 h with ruffling of fur, followed in sequence by labored breathing, weakness of limbs, and finally total paralysis with gasping for breath, followed by death due to respiratory failure. Death of mice without clinical symptoms of botulism is not sufficient evidence that injected material contained botulinal toxin. On occasion, death occurs from other chemicals present in injected fluid, or from trauma.

      If after 48 h of observation, all mice except those receiving the heated preparation have died, repeat the toxicity test, using higher dilutions of supernatant fluids or cultures. It is necessary to have dilutions that kill and dilutions that do not kill in order to establish an endpoint or the minimum lethal dose (MLD) as an estimate of the amount of toxin present. The MLD is contained in the highest dilution killing both mice (or all mice inoculated). From these data, the number of MLD/ml can be calculated.

    3. Typing of toxin. Rehydrate antitoxins with sterile physiological saline. Do not use glycerin water. Dilute monovalent antitoxins to types A, B, E, and F in physiological saline to contain 1 international unit (IU) per 0.5 ml. Prepare enough of these antitoxin solutions to inject 0.5 ml of antitoxin into each of 2 mice for each dilution of toxic preparation to be tested. Use the toxic preparation that gave the higher MLD, either untreated or trypsinized. Prepare dilutions of the toxic sample to cover at least 10, 100, and 1000 MLD below the previously determined endpoint of toxicity if possible (see b, above). The untreated toxic preparation can be the same as that used for testing toxicity. If a trypsinized preparation was the most lethal, it will be necessary to prepare a freshly trypsinized fluid. The continued action of trypsin may destroy the toxin.

      4. Inject the mice with the monovalent antitoxins, as described above, 30 min to 1 h before challenging them with i.p. injection of the toxic preparations. Inject pairs of mice (protected by specific monovalent antitoxin injection) i.p. with each dilution of the toxic preparation. Also inject a pair of unprotected mice (no injection of antitoxin) with each toxic dilution as a control. The use of 4 monovalent antitoxins (types A, B, E, and F) for the unknown toxic sample prepared at 3 dilutions requires a total of 30 mice--6 mice for each antitoxin (24 mice) plus 2 unprotected mice for each of the 3 dilutions (6 mice) as controls. Observe mice for 48 h for symptoms of botulism and record deaths. If test results indicate that toxin was not neutralized, repeat test, using monovalent antitoxins to types C and D, plus polyvalent antitoxin pool of types A through F.

Contents

Screening Procedure for Clostridium botulinum Type E Spores in Smoked Fish

  1. Equipment and materials

    1. 12 mice (16-24 g, or up to 34 g) per subsample (24 or more required for positives)
    2. Types A, B, E antisera
    3. Saline, sterile, 0.85% NaCl (R63)
    4. Trypsin (Difco); 1:250, 5% solution
    5. Syringes, 1 and 3 ml, 25 gauge, 5/8 inch (1.6 cm) needle
    6. Incubator 28ºC
    7. TPGY medium (M151)
    8. Water bath, 37ºC
    9. Gel-phosphate diluent (R29)
    10. Centrifuge, refrigerated
    11. Plastic bags, strong and water-tight

  2. Procedure

    3. Incubation. Place each smoked fish subsample (which may consist of 1 or more fish, depending on size, and may be either vacuum-packed or bulk-smoked fish) in a strong water-tight plastic bag. Add freshly steamed and cooled TPGY broth to subsample. NOTE: Add enough TPGY broth to completely cover fish. Squeeze bag to expel as much air as possible and seal it with hot-iron bag sealer or other airtight closure device. Incubate at 28ºC for 5 d. Precautions should be taken during incubation period since bag may swell and split from gas formation.

    Cultures. At end of incubation period, centrifuge 20 ml of TPGY culture from each subsample at 7500 x g rpm for 20 min. Use refrigerated centrifuge. Determine pH of TPGY. If above 6.5, adjust to 6.0-6.2 with HCl. Refrigerate for overnight storage.

    Trypsinization. To 3.6 ml of culture, adjusted to pH 6.0-6.2, add 0.4 ml of 5% solution of trypsin. Incubate at 35-37ºC for 1 h. Remove culture and let cool to room temperature before injecting mice.

    Toxicity screening. Dilute trypsinized and nontrypsinized broth cultures to 1:5, 1:10, and 1:100 in gel-phosphate diluent. (NOTE: Do not store trypsinized material overnight.) Inject mice i.p. with 0.5 ml of each dilution. Inject 2 mice per dilution, i.e., trypsinized and nontrypsinized (total 12 mice per subsample). Observe mice for botulism symptoms and record condition of mice at frequent intervals for 48 h. If no deaths occur, no further tests are indicated. Deaths are presumptive evidence of toxin and should be confirmed.

    Confirmation with protected mice. Dilute new portion of nontrypsinized or trypsinized culture (whichever showed the highest titer) to 1:5, 1:10, and 1:100 in gel-phosphate diluent. Inject 6 mice i.p. with 0.5 ml of 1:5 saline dilution of type E antiserum. These will be compared to 6 mice without this protection (controls). After 30 min, inject 0.5 ml of each dilution into 2 mice protected with antiserum and into 2 mice not so protected. Record their condition at intervals up to 48 h. If unprotected mice die and protected mice live, the presence of type E toxin is indicated. If all protected mice die, repeat confirmation with higher dilutions of toxic culture in type E-protected mice and with mice protected against C. botulinum types A and/or B antiserum. If all antiserum-protected mice die, send toxic culture media on dry ice to Division of Microbiological Studies (HFS-516), FDA, 200 C Street, S.W., Washington, DC 20204, for further tests. Isolate and identify cultures from samples containing toxin of type E, if possible.

    Obtain C. botulinum antisera from Centers for Disease Control and Prevention, Atlanta, GA 30333, USA. Reconstitute lyophilized antisera with sterile saline. Dilute sera 1:5 with sterile saline for mouse injection.

    If you have questions about the method, contact Haim Solomon, FDA. Telephone (202) 205-4469; FAX (202) 401-7740.  

Contents

General Hints Regarding C. botulinum Toxin Analysis

  1. The first 24 h are the most important time regarding symptoms and death of mice: 98-99% of animals die within 24 h. Typical symptoms of botulism and death may occur within 4 to 6 h.
  2. If deaths occur after 24 h, be very suspicious, unless typical botulism symptoms are clearly evident.
  3. If deaths occur in mice injected with the 1:2 or 1:5 dilution but not with any higher dilution, be very suspicious. Deaths may have been from nonspecific causes.
  4. Mice can be marked on tails with dye to represent various dilutions. Dye does not come off easily.
  5. Mice injected with botulinal toxin may become hyperactive before symptoms occur.
  6. Food and water may be given to the mice right away; it will not interfere with the test.
  7. Rehydrated antitoxin may be kept up to 6 months under refrigeration, and may be frozen indefinitely.
  8. TPGY medium is relatively stable and can be kept 2-3 weeks under refrigeration.
  9. With cooked meat medium, vortex tubes completely; toxin may adhere to meat particles.
  10. Trypsin is not filtered. Use 0.5 g in 10 ml of distilled water. It can be kept up to 1 week under refrigeration.  

Contents

Interpretation of Data

(NOTE: Laboratory tests are designed to identify botulinal toxin and/or organisms in foods)

  1. Toxin in a food means that the product, if consumed without thorough heating, could cause botulism.
  2. Viable C. botulinum but no toxin in foods is not proof that the food in question caused botulism.
  3. The presence of toxin in food is required for an outbreak of botulism to occur.
  4. Ingested organisms may be found in the alimentary tract, but are considered to be unable to multiply and produce toxin in vivo, except in infants.
  5. Presence of botulinal toxin and/or organisms in low-acid (i.e., above pH 4.6) canned foods means that the items were underprocessed or were contaminated through post-processing leakage.
  1. The protection of mice from botulism and death with one of the monovalent botulinal antitoxins confirms the presence of botulinal toxin and determines the serological type of toxin in a sample.
  2. The following reasons may explain why deaths occur in mice that are protected by one of the monovalent antitoxins:

- There may be too much toxin in the sample.

- More than one kind of toxin may be present.

- Deaths may be due to some other cause.

Retesting at higher dilutions of toxic fluids is required, and mixtures of antitoxins must be used in place of monovalent antiserum. Some other toxic material, which is not heat-labile, could be responsible if both heated and unheated fluids cause death. The heat-stable toxic substance could possibly mask botulinal toxin.  

Contents

Safety Precautions for the Clostridium botulinum Laboratory

  1. Place biohazard signs on doors to restrict entrance and keep the number of people in the laboratory to a minimum.
  2. All workers in the laboratory should wear laboratory coats and safety glasses.
  3. Use 1% hypochlorite solution to wipe laboratory table tops before and after work.
  4. NEVER PIPETTE ANYTHING BY MOUTH. USE MECHANICAL PIPETTORS.
  5. Use a biohazard hood for transfer of toxic material, if possible.
  6. Centrifuge toxic materials in a hermetically closed centrifuge with safety cups.
  7. Personally take all toxic material to the autoclave and see that it is sterilized immediately.
  8. Do not work alone in the laboratory or animal rooms after hours or on weekends.
  9. Have an eye wash fountain and foot-pedaled faucet available for hand washing.
  10. No eating and drinking in the laboratory when someone works with toxins.
  11. In a very visible location, list phone numbers where therapeutic antitoxin can be obtained in case of emergency. THIS IS VERY IMPORTANT!
  12. Reduce clutter in the laboratory to a minimum and place equipment and other materials in their proper place after use.  

Contents

Other analytical procedures

Contents

Commercial Test Products

Table 12-15. Commercial test products for C. botulinum.

Test Kit

Analytical Technique

Approx. Total Test Time1

Supplier

Probelia PCR System

Polymerase chain reaction

30 h

BioControl Systems, Inc.
Contact: Robin Forgey
12822 SE 32nd St.
Bellevue, WA 98005
Phone: 425/603-1123
E-mail: info@rapidmethods.com
Web: www.rapidmethods.com
1Includes enrichment

Contents

References

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AOAC. 1995. Clostridium botulinum and its toxins in foods: Microbiological method. Sec. 17.7.01, Method 977.26. In Official Methods of Analysis of AOAC International, 16th ed., P.A. Cunniff (Ed.), p. 46-48. AOAC International, Gaithersburg, MD.

Arnon, S.S. 1987. Infant botulism. In Pediatrics, 18th ed., A.M. Rudolph and J.I.E. Hoffman (Eds.), p. 490-492. Appleton & Lange, Norwalk, CT.

Baker, D.A., Genigeorgis, C., and Garcia, G. 1990. Prevalence of Clostridium botulinum in seafood and significance of multiple incubation temperatures for determination of its presence and type in fresh retail fish. J. Food Protect. 53(8):668-673.

Cann, D.C., Wilson, B.B., and Shewan, J.M. 1967. C. botulinum in the marine environment of Great Britain. In Botulism 1966: Proceedings of the Fifth International Symposium on Food Microbiology, M. Ingram and T.A Roberts (Eds.), p. 62-65. Chapman and Hall Ltd., London.

Centers for Disease Control. 1979. Botulism in the United States, 1899-1977. Handbook for epidemiologists, clinicians, and laboratory workers. DHEW Publ. No. (CDC) 74-8279, Washington, DC, plus additional reports by CDC at annual meetings of the Interagency Botulism Research Coordinating Committee (IBRCC).

Craig, J.M. and Pilcher, K.S. 1967. The natural distribution of C. botulinum type E in the Pacific coast areas of the United States. In Botulism 1966: Proceedings of the Fifth International Symposium on Food Microbiology, M. Ingram and T.A Roberts (Eds.), p. 56-61. Chapman and Hall Ltd., London.

Eklund, M.W. and Poysky, F. 1967. Incidence of C. botulinum type E from the Pacific Coast of the United States. In Botulism 1966: Proceedings of the Fifth International Symposium on Food Microbiology, M. Ingram and T.A Roberts (Eds.), p. 49-53. Chapman and Hall Ltd., London.

FDA. 1998a. Clostridium botulinum toxin formation. Ch. 13. In Fish and Fishery Products Hazards and Controls Guide, 2nd ed., p. 151-174. Department of Health and Human Services, Public Health Service, Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Seafood, Washington, DC.

FDA. 1998b. FDA & EPA guidance levels. Appendix 5. In Fish and Fishery Products Hazards and Controls Guide, 2nd ed., p. 245-248. Department of Health and Human Services, Public Health Service, Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Seafood, Washington, DC.

FDA. 1998c. Bacterial pathogen growth. Appendix 4. In Fish and Fishery Products Hazards and Controls Guide, 2nd ed., p. 241-244. Department of Health and Human Services, Public Health Service, Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Seafood, Washington, DC.

Hauschild, A.H.W. 1989. Clostridium botulinum. Ch. 4. In Foodborne Bacterial Pathogens, M.P. Doyle (Ed.), p. 111-189. Marcel Dekker, Inc., New York.

Hauschild, A.H.W., R. Hilsheimer, K.F. Weiss, and R.B. Burke. 1988. Clostridium botulinum in honey, syrups, and dry infant cereals. J. Food Prot. 51:892-894.

Lerke, P. 1973. Evaluation of potential risk of botulism from seafood cocktails. Appl. Microbiol. 25(5):807-810.

Lynt, R.K., Solomon, H.M., Lilly, T, and Kautter, D.A. 1977. Thermal death time of Clostridium botulinum Type E in meat of the blue crab. J. Food Sci. 42(4):1022-1025.

Nickerson, J.T.R., Goldblith, S.A., DiGioia, G., and Bishop, W.W. 1967. The presence of C. botulinum, type E in fish and mud taken from the Gulf of Maine. In Botulism 1966: Proceedings of the Fifth International Symposium on Food Microbiology, M. Ingram and T.A Roberts (Eds.), p. 25-33. Chapman and Hall Ltd., London.

Nielsen, S.F. and Pedersen, H.O. 1967. Studies on the occurrence and germination of C. botulinum in smoked salmon. In Botulism 1966: Proceedings of the Fifth International Symposium on Food Microbiology, M. Ingram and T.A Roberts (Eds.), p. 66-72. Chapman and Hall Ltd., London.

Peterson, M.E., Pelroy, G.A., Poysky, F.T., Paranjpye, R.N., Dong, F.M., Pigott, G.M., and Eklund, M.W. 1997. Heat-pasteurization process for inactivation of nonproteolytic types of Clostridium botulinum in picked Dungeness crabmeat. J. Food Protect. 60(8):928-934.

Rhodehamel, E.J., Solomon, H.M., Lilly, T. Jr., Kautter, D.A. and Peter, J.T. 1991. Incidence and heat resistance of Clostridium botulinum type E spores in menhaden surimi. J. Food Sci. 56(6):1562-3, 1592.

Solomon, H.M. and Lilly, T.,Jr. 1998. Clostridium botulinum. Ch. 17. In Food and Drug Administration Bacteriological Analytical Manual, 8th ed. (revision A), (CD-ROM version). R.L. Merker (Ed.). AOAC International, Gaithersburg, MD.