Iron Milk Medium Research Articles

Comparison of methods for the enumeration of Clostridium perfringens spores in water

Four methods for enumerating Clostridium perfringens spores in water were evaluated: (1) the IMM (Iron Milk Medium) method (MPN); (2) the LS (Lactose Sulfite Broth) method (MPN); (3) the m-CP (membrane filtration Clostridium perfringens Agar) method (membrane filtration); and (4) the TSC (Tryptose Sulfite Cycloserine Agar) method (membrane filtration). The performance of these methods was compared with that of the DRCM (Differential Reinforced Clostridium Medium) method (MPN) as adopted by CETESB (Brazil's Environmental Sanitation Technology Company) for the analysis of C. perfringens spores in water. Statistical analysis was performed according to ISO 17994:2004 (Water Quality - Criteria for Establishing Equivalence between Microbiological Methods). The LS, m-CP, and TSC methods were considered not equivalent to the DRCM method, as they gave significantly lower results. The IMM showed inconclusive results and, according to ISO 17994:2004, analysis of a greater number of samples is needed to draw definitive conclusions comparing IMM and DRCM.

Occurrence of Clostridium perfringens in the Broiler Chicken Processing Plant as Determined by Recovery in Iron Milk Medium

Over 30 years ago, Clostridium perfringens was reported as a contaminant of the processing plant and processed carcasses of broiler chickens. Poultry processing procedures and methods for detecting C. perfringens have changed since that time. Therefore, a study was conducted to determine the incidence and numbers of C. perfringens in the water of the scald tank, the water of the chill tank, and the rinse water of the processed carcasses from modern broiler chicken processing plants. In trial 1, collected samples were inoculated into iron milk medium (IMM) and incubated at 46°C for 18 h (the traditional method) or at 37°C for 3 h followed by incubation at 46°C for 15 h (an injury recovery method). Each of three preselected broiler chicken flocks from two integrators were the first processed for that processing shift. The overall incidence of confirmed C. perfringens in samples associated with the three flocks was 40% of postprocessing scald water samples, 13% of preprocessing chill water samples, 13% of postprocessing chill water samples, and 19% of carcass rinses. The incidence of C. perfringens in samples incubated in IMM using the injury recovery procedure was significantly higher than in samples incubated in IMM by the traditional method, but only when all samples associated with the three flocks were pooled. In trial 2, water samples from each tank of a three-tank counterflow scalder, water samples from the prechill and chill tank, and samples of carcass rinses were collected in the middle of a processing shift during multiple visits to a processing plant. Samples were inoculated into IMM with neomycin and polymyxin B sulfate (IMMA) and incubated using the traditional and injury recovery procedures. The incidence of C. perfringens in water samples was 100% from scald tank 1, 100% from scald tank 2, 100% from scald tank 3, 88% from the prechill tank, and 63% from the chill tank. The incidence in carcass rinse samples was 67%. The mean most probably number (MPN) of C. perfringens for contaminated samples decreased from log10 5.07/100 ml of water in scald tank 1 to log10 1.26/100 ml of water in the chill tank. The mean MPN in carcass rinse samples was log10 1.20 C. perfringens per 100 ml. The incidence and mean MPN of C. perfringens in these samples after heat shock at 75°C for 20 min was somewhat less, but high enough to indicate that much of the contamination arises from heat-resistant spores of this organism. In trial 2, there were no differences in incidence and MPN of C. perfringens in samples incubated in IMMA with the traditional method or the injury recovery method.

Iron Milk Medium Method for Recovering Clostridium perfringens from Shellfish: Collaborative Study

Eleven laboratories participated in a collaborative study analyzing shellfish (oysters, Crassostrea gigas) for the detection and enumeration of Clostridium perfringens by the iron milk medium (IMM) method. The IMM method was compared to AOAC Official Method 976.30. Shellfish were artificially inoculated with C. perfringens cells (vegetative and spores) at low (1 x 10(3) colony forming units [cfu]/g), medium (1 x 10(4) cfu/g), and high (1 x 10(6) cfu/g) levels. Negative controls (zero level) were analyzed by each laboratory. C. perfringens FD-1, the strain involved in a foodborne illness, was used. Blind duplicates of each inoculum level were analyzed, giving a total of 16 samples per laboratory. The selectivity of IMM relies solely on the rapid growth of C. perfringens at 45 degrees C indicated by stormy fermentation reaction within 18 h. C. perfringens is detected and enumerated using the most probable number technique. A statistical evaluation of the data found no significant differences between the estimates from the 2 methods. The IMM method for detection of C. perfringens from shellfish has been adopted first action by AOAC INTERNATIONAL.

Incidence of Clostridium perfringens in Fresh Sausages in Argentina

A simple iron-milk medium was used for the isolation and enumeration (MPN) if Clostridium perfringens from fresh sausages. Samples were diluted (log 10) in 0.1% peptone water, aliquots seeded and incubated at 45°C for 16–18 h, and cultures with clots and stormy fermentation were considered positive. Confirmation of C. perfringens was made by subculturing in tryptose-sulfite-neomycin agar under anaerobic conditions. Suspect colonies were identified by standard procedures, including nitrate reduction, gelatin liquefaction, lipase and lecithinase production, starch hydrolysis, hemolysis and reverse CAMP test. From 136 samples studied, 110 were positive. Twenty-six, 48, 25, 7, 3, and 1 samples contained, respectively 10, 102, 103, 104, 105, and 109C. perfringens per g of food. Three per cent of samples contained enough bacteria to produce food poisoning. The frequency of distribution approximately follows the Poisson equation with a μ=1.793.

Comparison of Media for Enumeration of Clostridium perfringens in Foods

ABSTRACTIron Milk Medium (IMM), Tryptose Sulfite Cycloserine (TSC) and Shadihi‐Ferguson‐perfringens (SFP) agar were compared for enumeration of Clostridium perfringens in four typical foods. The overall recovery in 160 samples tested at two inoculum range levels (102–103 and 104−105 per mL of sample homogenate) indicated that any of the three media could be used for most routine analyses. There were no significant differences in recovery abilities of the three media used for clams, oysters and turkey meats. In 15 naturally contaminated samples, recoveries of C. perfringens were not different in IMM compared to TSC. Therefore, the iron milk medium which is inexpensive, rapid and simple to prepare can be used as an alternative method for routine analyses for C. perfringens in foods such as shellfish and lean meats.

Differentiation of Clostridium perfringens from Related Clostridia in Iron Milk Medium

The stormy fermentation reaction of Clostridium perfringens in iron milk medium was compared to that of several C. perfringens-like strains. These clostridia, C. barati, C. perenne, C. absonum, and C. paraperfringens are very similar to C. perfringens on the basis of certain biochemical reactions and, consequently, are often difficult to distinguish from C. perfringens. Furthermore, these related clostridia may also be present in foods. Results of this study demonstrate that after 18 h of incubation at 45°C, only C. perfringens gave a positive reaction in iron milk with inocula as low as 22 cells/g. Some of the other strains began to show only gas production at 18 h. After 24 to 42 h some strains gave positive results and after 72 h all were positive. Enumeration of C. perfringens from food samples in iron milk medium by a 3-tube most probable number (MPN) technique gave similar results to enumeration by plate count using Shahidi-Ferguson Perfringens (SFP) agar. Furthermore, a rapid positive response occurred after only 2 and 3 h incubation of iron milk inoculated with 108 and 107 cells/ml, respectively. The high selectivity, ease of identification and rapid growth of C. perfringens in iron milk make the iron milk MPN procedure a valuable assay for accurate enumeration and differentiation of C. perfringens from related Clostridia in food products.

Comparison of Iron Milk and Official AOAC Methods for Enumeration of Clostridium perfringens from Fresh Seafoods

Fresh seafoods collected from Seattle area markets were evaluated for the presence and numbers of Clostridium perfringens. In a comparison of the official AOAC Shahidi-Ferguson perfringens and the simple iron milk methods, 29 of 287 samples of fresh fish and shellfish were positive for C. perfringens. All 29 positive samples were detected by the iron milk medium; however, only 7 of the 29 were detected by the AOAC methods. A statistical evaluation of the data showed a significant difference between the 2 methods.

Use of Iron Milk Medium for Enumeration of Clostridium perfringens

Abstract A simple iron milk medium was used for isolation and enumeration of Clostridium perfringens from soil, sludge, and water samples. The whole milk contained only iron powder as a reducing agent; no other inhibitors were added. The iron milk most probable number (MPN) procedure was compared with 4 plating media: sulfite-polymyxin-sulfadiazine, Shahidi-Ferguson perfringens, tryptose-sulfite- cycloserine (both with and without egg yolk), and tryptone-sulfite-neomycin. The selectivity of the iron milk relies solely on the rapid growth of C. perfringens at 45°C and the stormy fermentation reaction within 18 h. Isolates were confirmed as C. perfringens by standard biochemical tests. The iron milk MPN procedure compared very well with the 4 plating media tested. Selectivity of incubation temperature, short incubation time, and ease of identification by the characteristic stormy fermentation make this method ideal for enumerating C. perfringens from large numbers of samples.

Studies of the genus Clostridium. 3. The taxonomic study of the genus Clostridium with special reference to the organism isolated from the digestive tract and the soil

A total of 436 strains were subjected to a taxonomical survey. All of them wereof spore-forming, obligatory anaerobic, Gram-positive or negative catalase-negative rods.Of them, 418 strains had been isolated from the feces or intestinal contents of black Neo-pards, leopards, tigers, cheetahs, pumas, jaguar, chickens, horses, cattle, pigs, dogs, cats>rabbits, ferrets, guinea pigs, mice and seal, lesions in horse and cattle, the tonsils of dogs, the uterus of cattle, soil, and silage, the remaining 18 strains were named stock cultures.The results obtained were as follows.l. The genus Clostridium was divided into four sub-genera, sixteen groups, on thebasis of four characters ; namely, the shape of the spore, rnotiNity, liquefaction of gelatin, and the growth type in iron-milk medium.2. Further studies are necessary to achieve the classification of this genus by thebiological and serological features, pathogenicity, and source of origin.