Microbiological Quality and Safety in Dairy Industry

Module 5. Microbiological quality and safety evaluation of milk and milk products

Lesson 28
ENUMERATION OF BACILLUS CEREUS, CLOSTRIDIUM BOTULINUM AND CLOSTRIDIUM PERFRINGENS

28.1 Introduction

Bacillus cereus rods are aerobic, spore forming Gram–positive with square ends in short to long chains. The spores are ellipsoidal, central to sub terminal, thin-walled and do not swell. B. cereus is responsible for a minority of food borne illnesses causing severe nausea, vomiting and diarrhoea. It produces pinkish color colonies on MYPA medium. Food poisoning caused by B. cereus may occur when foods are prepared and held without adequate refrigeration for several hours before serving. B. cereus is commonly found in soil, on vegetables, and in many raw and processed foods. Consumption of foods that contain >106 B. cereus/ g may result in food poisoning. Foods incriminated in past outbreaks include cooked meat and vegetables, boiled or fried rice, vanilla sauce, custards, soups, and raw vegetable sprouts. Two types of illness have been attributed to the consumption of foods contaminated with B. cereus. The first and better known type is characterized by abdominal pain and diarrhoea; it has an incubation period of 4-16 hours and symptoms that last for 12-24 hours. The second, which is characterized by an acute attack of nausea and vomiting, occurs within 1-5 h after consumption of contaminated food; diarrhoea is not a common feature in this type of illness. Twenty, mostly European laboratories took part in a collaborative study to validate the general 1993 ISO 7932 standard for the enumeration of B. cereus in foods. The precision of the test method appeared to be unaffected by the type of food or the concentration of B. cereus present in the test sample.

28.2 Enumeration Principle of B. cereus

• B. cereus is mannitol-negative. The mannitol content of the medium thus allows differentiation of the accompanying mannitol-positive microbial flora which is identified by a change in colour of the indicator phenol red to yellow.
• B. cereus is not affected by concentrations of polymyxin which inhibit the common accompanying microbial flora. Addition of polymyxin is necessary, however, if the sample material is suspected to contain high numbers of accompanying microorganisms.
• B. cereus produces lecithinase. The insoluble degradation products of egg-yolk lecithin accumulate around the cereus colonies to form a white precipitate.
• A lecithinase reaction occurs very early in many strains, B. cereus colonies can, therefore, often be rapidly identified before accompanying polymyxin-resistant microorganisms have had a chance to fully develop.
• B. cereus selective supplement contains polymyxine-B in lyophilized form.
• It suppresses the growth of the accompanying bacterial flora during the culture of B. cereus.

28.3 Mannitol-Egg-Yolk-Polymyxine-Agar (MYP Agar)

28.3.1 Typical composition (g/litre)

Peptone from casein 10.0; meat extract 1.0; D (-) mannitol 10.0; sodium chloride 10.0; phenol red 0.025; agar-agar 12.0 Also to be added (per litre of medium); egg-yolk emulsion 100 ml; polymyxin B sulfate 100,000 IU B. cereus Selective Supplement.

28.3.2 Preparation

Suspend 21.5 g in 450 ml de-mineralized water, autoclave (15 min at121°C). Cool to about 45 to 50 °C add 50 ml (this volume can be varied depending on the degree of turbidity desired) of sterile egg-yolk emulsion and the contents of one vial B. cereus Selective Supplement, mix. pH: 7.2 ± 0.2 at 25 °C. Pour plates. The plates (including egg-yolk) are evenly turbid and slightly orange (red without egg-yolk).

28.3.3 Typical colony characteristics

B. cereus appears as rough, dry colonies with a pink to purple base which are surrounded by a ring of dense precipitate (Fig. 28.1). Colonies surrounded by yellow or a clear zone are not B. cereus. Further tests should be performed to confirm the identity of B. cereus (anaerobic degradation of D (+) glucose, degradation of gelatin, positive nitrate reduction)

Fig. 28.1 B. cereus colonies on mannitol-egg-yolk-polymyxine-agar (MYP Agar)

28.4 Detection of B. cereus

Enumeration, isolation and identification of B. cereus has been described in three basic steps

1. Direct plating on (selective) media
2. Direct (selective) enrichment
3. Pre-enrichment, followed by a selective enrichment

Depending on the number of cells expected in a sample and/or the standards described, one or more of these procedures may be used for the detection or enumeration of bacteria. Often an enrichment procedure is required for the detection of pathogens, whereas, due to higher expected numbers, direct plating is possible for the detection of indicator and spoilage organisms. The success of the basic protocols depends on: 1) the number and the state of the micro-organisms in the sample; 2) the selectivity of the media (a balance between inhibition of competitors and inhibition of the target organism); 3) conditions of incubation (time, temperature, presence of oxygen) and 4) the selectivity of the isolation medium (the ease of distinguishing the target organism from competitive micro flora). Another improvement on the traditional method is the development of chromogenic isolation and enumeration media, which make it easy to distinguish the target organisms.

28.4.1 Media and reagent

Mannitol egg-yolk polymyxin agar (MYPA) base plates, polymyxin B solution for MYPA (0.1%), Voges-Proskauer medium & reagent, Nutrient agar for B. cereus & Butterfield’s Phosphate-buffered dilution water.

28.4.2 Sample preparation

Using aseptic technique, weigh 25 g of sample into sterile blender jar or macerate with sterile mortar pastel. Add 225 ml Butterfield's phosphate-buffered dilution water (1:10 dilution) and mixed well for 2 min. Using the 1:10 dilution, make serial dilutions of sample for enumeration of B. cereus.

28.4.3 Plate count of B. cereus

Prepare serial dilutions from 10-2 to 10-6 by transferring 10 ml homogenized sample (1:10 dilution) to 90 ml dilution blank, mixing well with vigorous shaking, and continuing until 10-6 dilution is reached. Inoculate duplicate MYPA plates with each dilution of sample (including 1:10) by spreading 0.1 ml evenly onto surface of each plate with sterile glass spreading rod. Incubate plates 24 hours at 30°C and observe for colonies surrounded by precipitate zone, which indicates that lecithinase is produced. B. cereus colonies are usually a pink color which becomes more intense after additional incubation.

28.4.4 Confirmation of B. cereus

Pick 5 or more eosin pink, lecithinase-positive colonies from MYPA plates and transfer to nutrient agar slants. Incubate slants 24 hours at 30°C. Prepare Gram-stained smears from slants and examine microscopically. B. cereus will appear as large Gram-positive bacilli in short-to-long chains; spores are ellipsoidal, central to sub terminal.

28.5 Clostridium Perfringens

Food poisoning caused by Clostridium perfringens may occur when foods such as meat or poultry are cooked and held without maintaining adequate heating or refrigeration before serving. The presence of small numbers of C. perfringens is not uncommon in raw meats, poultry, dehydrated soups and sauces, raw vegetables, and spices. Because the spores of some strains are resistant to temperatures as high as 100°C for more than one hour, their presence in foods may be unavoidable. Furthermore, the oxygen level may be sufficiently reduced during cooking to permit growth of the Clostridia. Spores that survive cooking may germinate and grow rapidly in foods that are inadequately refrigerated after cooking. Thus, when clinical and epidemiological evidence suggests that C. perfringens is the cause of a food poisoning outbreak, the presence of hundreds of thousands or more of these organisms per gram of food substantiates the diagnosis.

Illness typically occurs 8-15 hours after ingestion of the contaminated food. The symptoms, which include intense abdominal cramps, gas, and diarrhea (nausea and vomiting are rare), have been attributed to a protein enterotoxin produced during sporulation of the organism in the intestine. The enterotoxin can be detected in sporulating cultures, and a method for this purpose is included. A high correlation has been established between the ability of C. perfringens strains to produce enterotoxin and their ability to cause food poisoning. However, it is difficult to obtain consistent sporulation with some strains.

C. perfringens cells lose their viability when foods are frozen or held under prolonged refrigeration unless special precautions are taken. Such losses may make it difficult to establish C. perfringens as the specific cause of a food poisoning outbreak. It is recommended that samples which cannot be examined immediately be treated with buffered glycerin-salt solution and stored or shipped frozen to the laboratory as described below.

28.5.1 Principle

Conventional methods for the detection of clostridia have traditionally incorporated heat killing of vegetative cells of clostridia and contaminating bacteria (to identify the presence or quantify the clostridial spores present). This is followed by the use of a nutritionally rich base medium, e.g. meat broth or blood agars, to promote spore germination. The addition of starch in many media is to facilitate germination and in some methods gentle heating of the sample prior to inoculation is recommended.

Reinforced Clostridial Medium (RCM) is based on non-selective growth of contaminating bacteria so some media now contain inhibitors and other selective agents. Sulphide and an iron source are usually used as indicators. The clostridia reduce the sulphide to sulphite which gives a black precipitate with the iron present in the medium. Sulphite reducing clostridia are then enumerated as black colonies if solid media is used.

Sodium sulphite and ferric citrate may be added to RCM to become differential RCM (DRCM) which was recommended for the detection of sulphite reducing clostridia in drinking water and is specified in the ISO standard 6461-1(1986) liquid enrichment method for water.

Tryptose sulphite cycloserine agar (TSC) was a medium for detection of vegetative and spore forms of C. perfringens in foodstuffs and clinical specimens, which contains cycloserine as an inhibitor of accompanying bacterial flora and causes the colonies which develop to remain smaller. In addition to incorporation of sulphite and iron, this medium utilizes sulphadiazine, oleandomycin phosphate and polymyxin B sulphate to give a high degree of selectivity and specificity for C. perfringens. Other Clostridium species, e.g. C. bifermentans and C. butyricum, are inhibited.

28.5.2 Plate count of viable C. perfringens.

Using aseptic technique, place 25 g food sample in sterile blender jar. Add 225 ml peptone dilution fluid (1:10 dilution). Homogenize 1-2 min at low speed. Obtain uniform homogenate with as little aeration as possible. Using 1:10 dilution prepared above, make serial dilutions from 10-1 to 10-6 by transferring 10-90 ml peptone dilution fluid blanks. Mix each dilution thoroughly by gently shaking before each transfer. Pour 6-7 ml TSC agar without egg yolk into each of ten 100 × 15 mm petri dishes and spread evenly on bottom by rapidly rotating dish. When agar has solidified, label plates, and aseptically transfer one ml of each dilution of homogenate to the center of duplicate agar plates. Pour additional 15 ml TSC agar without egg yolk into dish and mix with inoculum by gently rotating dish.

28.5.2.1 TSC agar containing egg yolk emulsion

An alternative plating method preferred for foods containing other types of sulphite-reducing organisms is to spread 0.1 ml of each dilution with sterile glass rod spreader over previously poured plates of TSC agar containing egg yolk emulsion. After inoculum has been absorbed (about 5 min), overlay plates with 10 ml TSC agar without egg yolk emulsion. When agar has solidified, place plates in upright position in anaerobic jar. Establish anaerobic conditions and place jar in 35°C incubator for 20-24 hours. (TSC agar containing egg yolk is incubated 24 hours). After incubation, remove plates from anaerobic jar and select those containing 20-200 black colonies for counting. C. perfringens colonies in egg yolk medium are black with a 2-4 mm opaque white zone surrounding the colony as a result of lecithinase activity.

28.5.3 Presumptive confirmation test

Select 10 typical C. perfringens colonies from TSC or TSC-egg yolk agars plates and inoculate each into a tube of freshly deaerated and cooled fluid thioglycollate broth. Incubate in standard incubator 18-24 hours at 35°C. Examine each culture by Gram stain and check for purity. C. perfringens is a short, thick, Gram-positive bacillus. If there is evidence of contamination, streak contaminated culture on TSC agar containing egg yolk and incubate in anaerobic jar 24 hours at 35°C. Surface colonies of C. perfringens are yellowish gray with 2-4 mm opaque zones caused by lecithinase activity. This procedure is also used for isolating C. perfringens from chopped liver broth whenever the organism is not detected by direct plating on TSC agar.

28.5.4 Completed confirmation test

28.5.4.1 Gelatin liquefaction

Stab-inoculate motility-nitrate (buffered) and lactose-gelatin media with 2 mm loopfuls of pure fluid thioglycollate medium culture or portion of isolated colony from TSC agar plate. Stab lactose-gelatin repeatedly to ensure adequate inoculation, and then rinse loop in beaker of warm water before flaming to avoid splattering. Incubate inoculated media 24 hours at 35°C. Examine lactose-gelatin medium cultures for gas production and color change from red to yellow, which indicates acid production. Chill tubes for one hour at 5°C and examine for gelatin liquefaction. If medium gels, incubate an additional 24 hours at 35°C and examine for gelatin liquefaction.

28.5.4.2 Gram’s staining

Inoculate sporulation broth with one ml fluid thioglycollate medium culture and incubate 24 hours at 35°C. Prepare Gram stain of sporulation broth and examine microscopically for spores. Store sporulated cultures.

28.5.4.3 Motility

C. perfringens is non-motile. Examine tubes of motility-nitrate medium for type of growth along stab line. Non-motile organisms produce growth only in and along stab. Motile organisms usually produce diffuse growth out into the medium, away from the stab.

28.5.4.4 Nitrate reduction

C. perfringens reduces nitrates to nitrites. To test for nitrate reduction, add 0.5 ml reagent A and 0.2 ml reagent B (R48) to culture in buffered motility-nitrate medium. Violet colour which develops within 5 min indicates presence of nitrites. If no color develops, add a few grains of powdered zinc metal and let stand a few minutes. A negative test (no violet color) after zinc dust is added indicates that nitrates were completely reduced. A positive test after addition of zinc dust indicates that the organism is incapable of reducing nitrates.

28.6 Clostridium Botulinum

Clostridium botulinum is an anaerobic, rod-shaped spore forming bacterium that produces a protein with characteristic neurotoxicity. Under certain conditions, these organisms may grow in foods producing toxin(s). Botulism, a severe form of food poisoning results when the toxin-containing foods are ingested. Although this food illness is rare, its mortality rate is high; the 962 recorded botulism outbreaks in the United States from 1899 to 1990 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. Due to a limited number of reports, type C and D toxins have been questioned as the causative agent of human botulism. It is suspected that these toxins are not readily absorbed in the human intestine. However, all types except F and G, which have not been as studied thoroughly, are important causes of animal botulism.

There are seven recognized antigenic types: A through 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 each antigenic type produces 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.

28.6.1 Pathogenicity

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 non-proteolytic, with carbohydrate metabolic patterns differing from the C and D non-proteolytic groups. Strains those 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 sea-foods. Types A and B are most commonly encountered in foods associated with 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. 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.

28.6.2 Growth characteristics

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 (aw), 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 non-proteolytic strains unless the temperature is precisely controlled and kept below 3°C. Foods processed to prevent spoilage but not usually refrigerated are the most common vehicles of botulism.

Optimum temperature for growth and toxin production of proteolytic strains is close to 35°C; for non-proteolytic strains it is 26-28°C. Non-proteolytic types B, E, and F can produce toxin at refrigeration temperatures (3-4°C). Toxins of the non-proteolytics 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.

28.6.3 Enumeration and detection

28.6.3.1 Sample preparation

1. 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.
2. 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 pipettes. Reserve sample; after culturing, aseptically remove reserve portion to sterile sample jar for tests which may be needed later. Refrigerate reserve sample.
3. Opening of canned foods. Examine product for appearance and odour. Note any evidence of decomposition. Do Not Taste the product under any circumstances. Record the findings.

28.6.3.2 Enrichment

Remove dissolved oxygen from enrichment media by steaming 10-15 min and cooling quickly without agitation before inoculation. 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 35°C. Inoculate two tubes of TPGY broth as above. Incubate at 28°C. Use TPGYT (Trypticase-peptone-glucose-yeast extract) as alternative only when organism involved is strongly suspected of being a non-proteolytic strain of types B, E, or F. Introduce the inoculum slowly beneath surface of broth to bottom of tube. After 5 days of incubation, examine enrichment cultures. Check for turbidity, gas production, and digestion of meat particles. Note the odour.

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 clostridia cells, occurrence and relative extent of sporulation, and location of spores within cells. A typical clostridia cell resembles a tennis racket. At this time test each enrichment culture for toxin, and if present, determine toxin type according to procedure in F, below. Usually, a 5-day incubation is the period of active growth giving the highest concentration of botulinal toxin. If enrichment culture shows no growth at 5 days, incubate an additional 10 days 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.

28.6.3.3 Isolation of pure cultures

C. botulinum is more readily isolated from the mixed flora of an enrichment culture or original specimen if sporulation has been good.

28.6.3.4 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 hour 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 hour at room temperature. Alternatively, heat (80°C for 10-15 min) 1 or 2 ml of enrichment culture or sample to destroy vegetative cells.

28.6.3.5 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 the agar plates well before use to prevent spreading of colonies. Incubate streaked plates at 35°C for about 48 hours under anaerobic conditions. A Case anaerobic jar or the GasPak system is adequate to obtain anaerobiosis; however, other systems may be used.

28.6.3.5.1 Selection of typical C. botulinum colonies

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.

28.6.3.5.2 Isolation of pure culture

Re-streak toxic culture in duplicate on egg yolk agar medium. Incubate one plate anaerobically at 35°C. Incubate second plate aerobically at 35°C. The culture may be pure if colonies typical of C. botulinum are found only on anaerobic plate (no growth on aerobic plate). 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.