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Bacillus cereus foodborne disease
Clinica, Microbiology lewsletter 13 July 1r, 1981
yright © 1981 by G. K. Hall & Co. ISSN 0196-4399 :illus cereus
F o o d b o r n e Disease
I fin S. Bergdoll, Ph.D. . d Research Institute r. .,ersity o f Wisconsin r.'~5 Willow Drive I~ lison, Wisconsin 53706
Foodborne disease in the United States has been associated with a plumber of different microorganisms. One of these, Bacillus cereus, has been implicated in several food poisoning outbreaks, although more have been attributed to the staphylococci, salmonellae, and Clostridium p e r f r i n g e n s . Six outbreaks attributed to B. cereus were reported to the Centers for Disease Control in 1978. In addition, a number of outbreaks in which the patients had symptoms similar to those of B. cereus food poisoning were reported but were not identified with any particular organism.* It is possible that some of these were caused by B. cereus but not identified as such, because attempts to isolate these 'organisms were not always made.
Clinical Disease Two types of illness result from the ingestion of foods containing B. cereus: a) symptoms of abdominal 9ain, watery diarrhea, and moderate aausea that develop 10-12 hours after :he ingestion of food containing the srganisms; and b) symptoms of musea and vomiting that develop in ]-5 hours with occasional diarrhea I sometime later. In the first type, :linical recovery occurs about 12 ,ours after the onset of symptoms md in the second type in 6-24 hours. IFhe symptoms of the second type are imilar to those of staphylococcal ood poisoning, but in several cases ~'Foodborne Disease Surveillance, Annual ISummary 1978, Centers for Disease Control.
I
the only organism that could be found was B. cereus. That B. cereus could cause this latter type of illness was shown when rice in which these organisms had grown (administered intragastrically) produced emesis in monkeys (7). There are a number of foods that have been implicated in the first type of food poisoning, such as cooked meat and poultry, meat and vegetable soups, and various desserts. A typical outbreak occurred in South Dakota in 1978 from the consumption of mashed potatoes that had been held under inadequate refrigeration for one day after preparation. The potato flakes used to prepare the mashed potatoes were contaminated with B. cereus organisms, and the heating during preparation was inadequate to destroy the heat-resistant spores. If the potatoes had been served on the day of preparation no problems would have arisen, because relatively large numbers ( > lOS/g) of these organisms are needed to cause illness. Proper refrigeration is the best control for this type of foodborne disease, since it is impossible to eliminate B. cereus organisms from the environment. The second type of B. cereus illness has been associated with rice prepared in Chinese restaurants, primarily in England. Usually it is caused by fried rice made from boiled rice that is prepared in large quantity and inadequately cooled, thus giving the B. cereus organisms an opportunity to grow and produce toxin. Frequently the frying is done quickly, providing inadequate heating to destroy any toxin that might be present. Also, the fried rice may be prepared in advance and kept at
room temperature for " f l a s h " frying before serving. Spores of B. cereus can survive the boiling and frying, and vegetative cells can grow very rapidly in cooked rice at room temperature (10). The addition of beef, chicken, or egg can enhance the growth of these organisms. It appears that the clinically different types of B. cereus food poisoning are related to two different toxins. The first type of disease is caused by a toxin produced by the organisms growing in the digestive tract after the ingestion of contaminated food; this explains the longer incubation period. The second type is caused by another toxin produced by the organisms when they grow in rice; upon ingestion it can cause vomiting, thus the designation emetic toxin. This accounts for the faster development of the symptoms of this type.
Laboratory Diagnosis B. c e r e u s i s a large gram-positive, rod-shaped, aerobic, spore-forming organism capable of growing under anaerobic conditions. The organism grows in the temperature range of 10°-48°C with optimum growth occurring at 28°-35°C. The spores are quite heat-resistant, and some will survive when heated at temperatures i
In This Issue Bacillus cereus
Food Poisoning . . .
85
Blood Culture Survey Response (Part 2) . . .
87
E r y s i p e l o t h r i x Infection . . .
89
Letters . . .
90
Positions...
90
m
...1
up to 100°C. These organisms are common in soil and on vegetation and have been isolated from a wide variety of foods. Their widespread distribution renders it impossible to eliminate them from our environment. Association of a particular foodborne illness with B. c e r e u s requires the isolation of large numbers of these organisms from the implicated food, especially if the disease is the diarrheal type. In this type, relatively large numbers must be present in the ingested food to result in illness. It is possible that in the case of the emetic toxicosis the organisms could be present in fewer numbers and still cause illness because the responsible toxin is heat-stable and would remain even though the organisms were destroyed. Various methods have been used for the isolation of the B . c e r e u s . Hauge (5, 6), in his initial work with this type o f food poisoning, used blood agar incubated anaerobically at 37°C for 18 hours and then aerobically for 18-24 hours. Colonies surrounded by a clear zone of hemolysis were picked for lecithinase activity. Another method is the most probable number method of Nygren (1 I). Usually the numbers of organisms present in the foods involved are sufficiently high that enrichment is not necessary. Gilbert and Taylor (2) used horse blood agar incubated at 35°-37°C for 24 hours for the isolation and enumeration of these organisms. This method was particularly useful for colony counts on clinical specimens and contaminated food stuffs. They subcultured any suspect colonies onto Kendall's B.C. medium for further identification. Details of their procedures can be obtained from their paper. Taylor and Gilbert have proposed a scheme for serotyping the B . c e r e u s isolates from food poisoning outbreaks (12). They identified 23 different serotypes and found the serotyping useful in the epidemiologic investigation of food poisoning outbreaks. A great majority o f the strains involved in the emetic type of illness belonged to serotype 1, while
86
only two of those involved in the diarrheal type were of this serotype; however, strains cannot be identified specifically by this method as either emetic or diarrheal. Serotyping is useful in identifying the source of the organisms involved in any particular outbreak. Toxin Studies
Several attempts have been made to identify the mechanism(s) responsible for the diarrheal type o f food poisoning, but to date no one has succeeded in isolating the toxin or in identifying it with a specific antibody. Currently, work is under way in the Food Research Institute at the University of Wisconsin (funded by the Food and Drug Administration) to isolate the toxin, and good progress is being made. The major problem with the isolation studies is the lack of a suitable assay for the toxic substance. The ileal-loop test in rabbits appears to be the most reliable method but is a very difficult one to perform. Results vary from rabbit to rabbit, and thus a large number of animals must be used. A skin assay with rabbits has been proposed (3) and is used by some investigators (13), but there appears to be more than one agent that gives a positive reaction. This test can be helpful in following the purification of the toxic agent, but it is not specific, and other tests must be used. Studies in monkeys have been done in an attempt to identify fractions that contain the diarrheal toxin (4), but this method is limited because of the costs involved. An attempt has also been made to purify the agent responsible for the emetic action in food poisoning victims, but no conclusive results have been achieved (8, 9). Emesis in rhesus monkeys has been used to follow the purification of the toxin, but because of both the interference of reagents employed in the purification and the lack of monkeys, this work has been temporarily discontinued (Melling, personal communication). It appears that the emetic toxin has a relatively low molecular weight and is nonantigenic, because the animals could be used
repeatedly without development of resistance to the toxin (8). Summary
Although B . c e r e u s foodborne disease is not a serious illness and may occur infrequently in the United States, more attention should be given to those illnesses that are similar to C. p e r f r i n g e n s and staphylococcal food poisoning but in which C. p e r f r i n g e n s and staphylococci cannot be isolated. The chances are good that B . c e r e u s organisms may be responsible for many of these outbreaks.
References
I. Gilbert, R. J. 1979. Bacillus cereus gastroenteritis, pp. 495-518. In H. Riemann and F. L. Bryan (eds.), Food-borne infections and intoxications. Academic Press Inc., New York. 2. Gilbert, R. J., and A. J. Taylor.
1976. Bacillus cereus food poisoning, pp. 197-213. In F. A. Skinner and J. G. Carr (eds.), Microbiology in agriculture, fisheries and food. Society for Applied Bacteriology symposium series no. 4 (London). Academic Press Inc., New York. 3. Glatz, B. A., W. M. Splra, and
4.
5.
6. 7.
8.
9.
J. M. Goepfert. 1974. Alteration of vascular permeability in rabbits by culture filtrates of Bacillus cereus and related species. Infect. Immun. 10:229-303. Goepfert, J. M. 1974. Monkey feeding trials in the investigation of the nature of Bacillus cereus food poisoning. Proc. IV Int. Cong. Food Sci. Technol. I11:178-181. Hauge, S. 1950. Matforgiftninger fremkalt av Bacillus cereus. Nordisk. Hyg. Tidskr. 31:18%206. Hauge, S. 1955. Food poisoning caused by aerobic spore-forming bacilli. J. Appl. Bacteriol. 18:591-595. Melling, J., et al. 1976. Identification of a novel enterotoxigenic activity associated with Bacillus cereus. J. Clin. Pathol. 29:938-940. Meiling, J., et ai. 1978. Identification and characterization of Bacillus cereus emetic toxin. J. Appl. Bacteriol. 45:xxv. Meiling, J., and B. J. Capel. 1978. Characteristics of Bacillus cereus toxin. FEBS Lett. 4:133-135.
10. Morita, T. N., and M. J. Woodburn.
1977. Stimulation of Bacillus cereus growth in cooked rice combinations. J. Food Sci. 42:1232-1235. 11. Nygren, B. 1962. Phospholipase C-
producing bacteria and food poisoning. Acta Pathol. Microbiol. Scand. [Suppl.] 160:1-89. 12. Taylor, A. J., and R. J. Gilbert.
Properties and production characteristics of vomiting, diarrheal, and necrotizing toxins of Bacillus cereus. Am. J. Clin. Nutr. 32:219-228
1975. Bacillus cereus food poisoning: A provisional serotyping scheme. J. Med. Microbiol. 8:543-550. 13. Turnbull, P. C. B., et al. 1979.
Results of the Survey of Blood Culture Methods (Part 2) Here are the rest o f the blood culture survey results, covering methods o f handling positive blood cultures. More than half the laboratories attempt to identify some organisms rapidly, depending on the Gram stain. The most commonly used tests are the API and Micro-ID for gram-negative rods, and the coagulase test for gram-positive cocci in clusters. The number and types o f media used for routine subculture o f positive blood cultures demonstrate again the creativity o f clinical microbiologists. Almost all laboratories perform direct antibiotic susceptibility tests from positive blood cultures. The disc diffusion was used by the majority o f laboratories. We thank you for your responses and look forward to your participation in future surveys. 30. Do you attempt to rapidly identify bacteria recovered from blood cultures, i.e., on the same day? 202 Yes 126 No If yes, what method(s) do you employ for identifying: Gram-negative rods: API (102); Micro-ID (88); AMS (9); MS-2 (5); Minitek (1); Microscan (1); Autobac (1); Oxidase (15); Indole (5); Urease (1); Serology (5); Enterotube (1); Motility (1). Gram-positive cocci in clusters: Coagulase (121); Catalase (26); Mannitol (6); DNAse (4); Lysostaphin (2); A disc (2); P disc (3); Bile-esculin (1); C A M P (1); fl-lactamase (1). Gram-positive cocci in chains: A disc (54); P disc (50); Serology (54); Bile solubility (14); Catalase (16); NaC1 (16); C A M P (15); PSE (8); API (7); Hippurate (3); Quad plate (1). Gram-negative cocci: Oxidase (18); Sugars (25); O N P G (1); Serology (16). Possible anaerobes: FA (13); Minitek (9); API (8); PRAS sugars (1); Catalase (3); Bile (1); EYA (1); Quad plate (i). Yeast: Germ tube (1). 31. Assume that you examine a Gram stain o f a turbid blood culture bottle. Beside the Gram stain observations listed below, indicate the medium and incubation environment that would be used for subculture. Do not include other tests done. Number of Laboratories Reporting No bacteria seen
Gram-pos. cocci
Gram-pos. rods
Gram-neg. cocci
Gram-neg. rods
Mixed*
Yeast
Mediumt
Aer. Anaer. Aer. Anaer. Aer. Anaer. Aer. Anaer. Aer. Anaer. Aer. Anaer. Aer. Anaer.
Blood agar Chocolate MacConkey's EMB CNA (or PNBA) PEA LKV or KV Schaedler's agar Brucella agar Thioglycolate TM or MTM Sabouraud's
115 242 15 3 5 2 0 I 0 11 0 0
162 43 0 0 1 3 5 21 14 12 0 0
256 156 18 2 22 4 0 1 0 11 0 0
200 31 0 2 12 8 7 24 6 11 0 0
236 134 21 3 11 4 0 1 0 11 1 0
197 30 4 0 9 6 l0 24 4 9 0 0
170 246 25 6 8 4 0 I 0 8 34 0
154 48 1 0 5 3 13 20 4 7 1 0
213 196 181 55 13 5 2 1 0 9 0 0
186 29 5 8 6 7 65 27 4 7 0 0
224 174 184 53 86 67 1 1 0 8 3 0
170 26 5 3 44 50 85 21 2 4 0 0
187 71 4 10 6 3 0 0 0 3 1 154
61 12 0 0 2 0 0 4 1 0 0 12
*Mixture of gram-positive and gram-negative organisms tlncubated aerobically or anaerobically, as indicated Many laboratories reported using multiple media for subculture. The following media were reported by less than 10 laboratories: Mannitol salt; Staph 110; Chapman; Bile-esculin; Mueller-Hinton; Columbia; Birdseed; Mycosel; Mycobiotic; Tergitol-7; TSB; Babdex; A7; Mycoplasma; Tellurite; Hektoen; TSA; Youseff's; PSE; NaC1; Azide; IM; EYA; Neomycin; GVH; CM; CMG; Pre-reduced; Peptic digest; Sabhi; Cornmeal; HB; Endo; BBK; BMB; SE; KBE; BHCC; NYC; EBA; Lecithinase-lactose; Trypan blue; Blood with staph streak.
87
yright © 1981 by G. K. Hall & Co. ISSN 0196-4399 :illus cereus
F o o d b o r n e Disease
I fin S. Bergdoll, Ph.D. . d Research Institute r. .,ersity o f Wisconsin r.'~5 Willow Drive I~ lison, Wisconsin 53706
Foodborne disease in the United States has been associated with a plumber of different microorganisms. One of these, Bacillus cereus, has been implicated in several food poisoning outbreaks, although more have been attributed to the staphylococci, salmonellae, and Clostridium p e r f r i n g e n s . Six outbreaks attributed to B. cereus were reported to the Centers for Disease Control in 1978. In addition, a number of outbreaks in which the patients had symptoms similar to those of B. cereus food poisoning were reported but were not identified with any particular organism.* It is possible that some of these were caused by B. cereus but not identified as such, because attempts to isolate these 'organisms were not always made.
Clinical Disease Two types of illness result from the ingestion of foods containing B. cereus: a) symptoms of abdominal 9ain, watery diarrhea, and moderate aausea that develop 10-12 hours after :he ingestion of food containing the srganisms; and b) symptoms of musea and vomiting that develop in ]-5 hours with occasional diarrhea I sometime later. In the first type, :linical recovery occurs about 12 ,ours after the onset of symptoms md in the second type in 6-24 hours. IFhe symptoms of the second type are imilar to those of staphylococcal ood poisoning, but in several cases ~'Foodborne Disease Surveillance, Annual ISummary 1978, Centers for Disease Control.
I
the only organism that could be found was B. cereus. That B. cereus could cause this latter type of illness was shown when rice in which these organisms had grown (administered intragastrically) produced emesis in monkeys (7). There are a number of foods that have been implicated in the first type of food poisoning, such as cooked meat and poultry, meat and vegetable soups, and various desserts. A typical outbreak occurred in South Dakota in 1978 from the consumption of mashed potatoes that had been held under inadequate refrigeration for one day after preparation. The potato flakes used to prepare the mashed potatoes were contaminated with B. cereus organisms, and the heating during preparation was inadequate to destroy the heat-resistant spores. If the potatoes had been served on the day of preparation no problems would have arisen, because relatively large numbers ( > lOS/g) of these organisms are needed to cause illness. Proper refrigeration is the best control for this type of foodborne disease, since it is impossible to eliminate B. cereus organisms from the environment. The second type of B. cereus illness has been associated with rice prepared in Chinese restaurants, primarily in England. Usually it is caused by fried rice made from boiled rice that is prepared in large quantity and inadequately cooled, thus giving the B. cereus organisms an opportunity to grow and produce toxin. Frequently the frying is done quickly, providing inadequate heating to destroy any toxin that might be present. Also, the fried rice may be prepared in advance and kept at
room temperature for " f l a s h " frying before serving. Spores of B. cereus can survive the boiling and frying, and vegetative cells can grow very rapidly in cooked rice at room temperature (10). The addition of beef, chicken, or egg can enhance the growth of these organisms. It appears that the clinically different types of B. cereus food poisoning are related to two different toxins. The first type of disease is caused by a toxin produced by the organisms growing in the digestive tract after the ingestion of contaminated food; this explains the longer incubation period. The second type is caused by another toxin produced by the organisms when they grow in rice; upon ingestion it can cause vomiting, thus the designation emetic toxin. This accounts for the faster development of the symptoms of this type.
Laboratory Diagnosis B. c e r e u s i s a large gram-positive, rod-shaped, aerobic, spore-forming organism capable of growing under anaerobic conditions. The organism grows in the temperature range of 10°-48°C with optimum growth occurring at 28°-35°C. The spores are quite heat-resistant, and some will survive when heated at temperatures i
In This Issue Bacillus cereus
Food Poisoning . . .
85
Blood Culture Survey Response (Part 2) . . .
87
E r y s i p e l o t h r i x Infection . . .
89
Letters . . .
90
Positions...
90
m
...1
up to 100°C. These organisms are common in soil and on vegetation and have been isolated from a wide variety of foods. Their widespread distribution renders it impossible to eliminate them from our environment. Association of a particular foodborne illness with B. c e r e u s requires the isolation of large numbers of these organisms from the implicated food, especially if the disease is the diarrheal type. In this type, relatively large numbers must be present in the ingested food to result in illness. It is possible that in the case of the emetic toxicosis the organisms could be present in fewer numbers and still cause illness because the responsible toxin is heat-stable and would remain even though the organisms were destroyed. Various methods have been used for the isolation of the B . c e r e u s . Hauge (5, 6), in his initial work with this type o f food poisoning, used blood agar incubated anaerobically at 37°C for 18 hours and then aerobically for 18-24 hours. Colonies surrounded by a clear zone of hemolysis were picked for lecithinase activity. Another method is the most probable number method of Nygren (1 I). Usually the numbers of organisms present in the foods involved are sufficiently high that enrichment is not necessary. Gilbert and Taylor (2) used horse blood agar incubated at 35°-37°C for 24 hours for the isolation and enumeration of these organisms. This method was particularly useful for colony counts on clinical specimens and contaminated food stuffs. They subcultured any suspect colonies onto Kendall's B.C. medium for further identification. Details of their procedures can be obtained from their paper. Taylor and Gilbert have proposed a scheme for serotyping the B . c e r e u s isolates from food poisoning outbreaks (12). They identified 23 different serotypes and found the serotyping useful in the epidemiologic investigation of food poisoning outbreaks. A great majority o f the strains involved in the emetic type of illness belonged to serotype 1, while
86
only two of those involved in the diarrheal type were of this serotype; however, strains cannot be identified specifically by this method as either emetic or diarrheal. Serotyping is useful in identifying the source of the organisms involved in any particular outbreak. Toxin Studies
Several attempts have been made to identify the mechanism(s) responsible for the diarrheal type o f food poisoning, but to date no one has succeeded in isolating the toxin or in identifying it with a specific antibody. Currently, work is under way in the Food Research Institute at the University of Wisconsin (funded by the Food and Drug Administration) to isolate the toxin, and good progress is being made. The major problem with the isolation studies is the lack of a suitable assay for the toxic substance. The ileal-loop test in rabbits appears to be the most reliable method but is a very difficult one to perform. Results vary from rabbit to rabbit, and thus a large number of animals must be used. A skin assay with rabbits has been proposed (3) and is used by some investigators (13), but there appears to be more than one agent that gives a positive reaction. This test can be helpful in following the purification of the toxic agent, but it is not specific, and other tests must be used. Studies in monkeys have been done in an attempt to identify fractions that contain the diarrheal toxin (4), but this method is limited because of the costs involved. An attempt has also been made to purify the agent responsible for the emetic action in food poisoning victims, but no conclusive results have been achieved (8, 9). Emesis in rhesus monkeys has been used to follow the purification of the toxin, but because of both the interference of reagents employed in the purification and the lack of monkeys, this work has been temporarily discontinued (Melling, personal communication). It appears that the emetic toxin has a relatively low molecular weight and is nonantigenic, because the animals could be used
repeatedly without development of resistance to the toxin (8). Summary
Although B . c e r e u s foodborne disease is not a serious illness and may occur infrequently in the United States, more attention should be given to those illnesses that are similar to C. p e r f r i n g e n s and staphylococcal food poisoning but in which C. p e r f r i n g e n s and staphylococci cannot be isolated. The chances are good that B . c e r e u s organisms may be responsible for many of these outbreaks.
References
I. Gilbert, R. J. 1979. Bacillus cereus gastroenteritis, pp. 495-518. In H. Riemann and F. L. Bryan (eds.), Food-borne infections and intoxications. Academic Press Inc., New York. 2. Gilbert, R. J., and A. J. Taylor.
1976. Bacillus cereus food poisoning, pp. 197-213. In F. A. Skinner and J. G. Carr (eds.), Microbiology in agriculture, fisheries and food. Society for Applied Bacteriology symposium series no. 4 (London). Academic Press Inc., New York. 3. Glatz, B. A., W. M. Splra, and
4.
5.
6. 7.
8.
9.
J. M. Goepfert. 1974. Alteration of vascular permeability in rabbits by culture filtrates of Bacillus cereus and related species. Infect. Immun. 10:229-303. Goepfert, J. M. 1974. Monkey feeding trials in the investigation of the nature of Bacillus cereus food poisoning. Proc. IV Int. Cong. Food Sci. Technol. I11:178-181. Hauge, S. 1950. Matforgiftninger fremkalt av Bacillus cereus. Nordisk. Hyg. Tidskr. 31:18%206. Hauge, S. 1955. Food poisoning caused by aerobic spore-forming bacilli. J. Appl. Bacteriol. 18:591-595. Melling, J., et al. 1976. Identification of a novel enterotoxigenic activity associated with Bacillus cereus. J. Clin. Pathol. 29:938-940. Meiling, J., et ai. 1978. Identification and characterization of Bacillus cereus emetic toxin. J. Appl. Bacteriol. 45:xxv. Meiling, J., and B. J. Capel. 1978. Characteristics of Bacillus cereus toxin. FEBS Lett. 4:133-135.
10. Morita, T. N., and M. J. Woodburn.
1977. Stimulation of Bacillus cereus growth in cooked rice combinations. J. Food Sci. 42:1232-1235. 11. Nygren, B. 1962. Phospholipase C-
producing bacteria and food poisoning. Acta Pathol. Microbiol. Scand. [Suppl.] 160:1-89. 12. Taylor, A. J., and R. J. Gilbert.
Properties and production characteristics of vomiting, diarrheal, and necrotizing toxins of Bacillus cereus. Am. J. Clin. Nutr. 32:219-228
1975. Bacillus cereus food poisoning: A provisional serotyping scheme. J. Med. Microbiol. 8:543-550. 13. Turnbull, P. C. B., et al. 1979.
Results of the Survey of Blood Culture Methods (Part 2) Here are the rest o f the blood culture survey results, covering methods o f handling positive blood cultures. More than half the laboratories attempt to identify some organisms rapidly, depending on the Gram stain. The most commonly used tests are the API and Micro-ID for gram-negative rods, and the coagulase test for gram-positive cocci in clusters. The number and types o f media used for routine subculture o f positive blood cultures demonstrate again the creativity o f clinical microbiologists. Almost all laboratories perform direct antibiotic susceptibility tests from positive blood cultures. The disc diffusion was used by the majority o f laboratories. We thank you for your responses and look forward to your participation in future surveys. 30. Do you attempt to rapidly identify bacteria recovered from blood cultures, i.e., on the same day? 202 Yes 126 No If yes, what method(s) do you employ for identifying: Gram-negative rods: API (102); Micro-ID (88); AMS (9); MS-2 (5); Minitek (1); Microscan (1); Autobac (1); Oxidase (15); Indole (5); Urease (1); Serology (5); Enterotube (1); Motility (1). Gram-positive cocci in clusters: Coagulase (121); Catalase (26); Mannitol (6); DNAse (4); Lysostaphin (2); A disc (2); P disc (3); Bile-esculin (1); C A M P (1); fl-lactamase (1). Gram-positive cocci in chains: A disc (54); P disc (50); Serology (54); Bile solubility (14); Catalase (16); NaC1 (16); C A M P (15); PSE (8); API (7); Hippurate (3); Quad plate (1). Gram-negative cocci: Oxidase (18); Sugars (25); O N P G (1); Serology (16). Possible anaerobes: FA (13); Minitek (9); API (8); PRAS sugars (1); Catalase (3); Bile (1); EYA (1); Quad plate (i). Yeast: Germ tube (1). 31. Assume that you examine a Gram stain o f a turbid blood culture bottle. Beside the Gram stain observations listed below, indicate the medium and incubation environment that would be used for subculture. Do not include other tests done. Number of Laboratories Reporting No bacteria seen
Gram-pos. cocci
Gram-pos. rods
Gram-neg. cocci
Gram-neg. rods
Mixed*
Yeast
Mediumt
Aer. Anaer. Aer. Anaer. Aer. Anaer. Aer. Anaer. Aer. Anaer. Aer. Anaer. Aer. Anaer.
Blood agar Chocolate MacConkey's EMB CNA (or PNBA) PEA LKV or KV Schaedler's agar Brucella agar Thioglycolate TM or MTM Sabouraud's
115 242 15 3 5 2 0 I 0 11 0 0
162 43 0 0 1 3 5 21 14 12 0 0
256 156 18 2 22 4 0 1 0 11 0 0
200 31 0 2 12 8 7 24 6 11 0 0
236 134 21 3 11 4 0 1 0 11 1 0
197 30 4 0 9 6 l0 24 4 9 0 0
170 246 25 6 8 4 0 I 0 8 34 0
154 48 1 0 5 3 13 20 4 7 1 0
213 196 181 55 13 5 2 1 0 9 0 0
186 29 5 8 6 7 65 27 4 7 0 0
224 174 184 53 86 67 1 1 0 8 3 0
170 26 5 3 44 50 85 21 2 4 0 0
187 71 4 10 6 3 0 0 0 3 1 154
61 12 0 0 2 0 0 4 1 0 0 12
*Mixture of gram-positive and gram-negative organisms tlncubated aerobically or anaerobically, as indicated Many laboratories reported using multiple media for subculture. The following media were reported by less than 10 laboratories: Mannitol salt; Staph 110; Chapman; Bile-esculin; Mueller-Hinton; Columbia; Birdseed; Mycosel; Mycobiotic; Tergitol-7; TSB; Babdex; A7; Mycoplasma; Tellurite; Hektoen; TSA; Youseff's; PSE; NaC1; Azide; IM; EYA; Neomycin; GVH; CM; CMG; Pre-reduced; Peptic digest; Sabhi; Cornmeal; HB; Endo; BBK; BMB; SE; KBE; BHCC; NYC; EBA; Lecithinase-lactose; Trypan blue; Blood with staph streak.
87