Meat Medium Research Articles

Dependence of Clostridium botulinum gas and protease production on culture conditions.

Reports that Clostridium botulinum toxin can sometimes be detected in the absence of indicators of overt spoilage led to a systematic study of this phenomenon in a model system. Media with various combinations of pH (5.0 to 7.0) and glucose (0.0 to 1.0%) were inoculated with vegetative cells of C. botulinum 62A and incubated anaerobically at 35 degrees C. Although growth and toxin production occurred at all pH and glucose combinations, accumulation of gas was delayed or absent in media with low pH, low glucose levels, or both. Other proteolytic C. botulinum strains gave similar results. Trypsin activation was required to detect toxin in some low pH cultures. The trypsinization requirement correlated with low proteolytic activity in the cultures. Proteolytic activity of the strains examined was 5- to 500-fold lower in botulinal assay medium than in cooked meat medium. The results indicate that the absence of gas accumulation does not preclude the presence of botulinal toxin and that proteolytic cultures grown under adverse conditions may require trypsinization for the detection of toxin.

The occurrence ofClostridium botulinumat aquatic sites in and around Auckland and other urban areas of the North Island

Samples of bottom muds from lakes and waterways in the Auckland area were inoculated into bottles of cooked meat medium. After incubation, the media were tested for production of Clostridium botulinum toxin which was detected with samples from 11 of the 20 sites examined. Although toxins produced from all positive samples were neutralized by specific antisera of both type C and D toxins, it is likely that C. botulinum type Calpha is the only type present in the Auckland area. All samples from other urban areas of the North Island gave negative results.

Transport Media forcampylobacter Fetus Venerealis

Abstract Sir. — Cultural confirmation of venereal campylobacteriosis in cattle can only be achieved if samples arc cultured on the day of collection or a suitable transport medium is used. Clark et al.(3) have developed a selective enrichment medium for Campylobacter fetus venerealis which is also an effective transport medium (8). However, this transport medium is very cumbersome to prepare in that it requires a special gas mixture and large quantities of fresh bovine serum. This letter reports on the evaluation of Clark’s (8), cooked meat medium with antibiotics (CMMA), Weybridge (4), modified EMJH (5), Stuart’s (Oxford) and Cairy Blair (Oxoid) media, as transport media for C. fetus veneralis.

Further Studies on the Antibotulinal Effectiveness of Nisin in Acidic Media

ABSTRACTThe antibotulinal effectiveness of nisin in TPYG broth was increased somewhat by lowering the pH to pH 5.5. The ability of nisin to inhibit the outgrowth of strain 56A spores was markedly increase at pH 5.5 by comparison to its effectiveness at higher pHs observed in previous studies. The increased effectiveness of nisin at pH 5.5 was less notable for the strain 69A, 113B, and 213B spores. The nisin sensitivity of the type E spores was essentially unchanged from that observed in earlier studies at higher pHs. At pH 6, nisin levels of 5000 I.U./ml were insufficient to prevent spore outgrowth by C. botulinum in cooked meat medium. Comparatively, much lower levels of nisin were effective in preventing botulinal outgrowth in TPYG broth at pH 6. The decreased effectiveness of nisin in cooked meat medium may be due to the binding of nisin to meat particles, and this binding is apparently not affected by lowering the pH to pH 6.0.

Effect of Nisin on the Outgrowth of Clostridium botulinum Spores

ABSTRACTNisin, an antibiotic produced by certain strains of Streptococcus lactis, is effective in preventing the outgrowth of Clostridium botulinum spores. Type A C. botulinum spores were the most resistant to the inhibitory action of nisin requiring 1000‐2000 I.U. of nisin/ml for a 50% inhibition of outgrowth on TPYG agar plates. Type E spores were more sensitive requiring only 50‐100 I.U./ml for 50% inhibition of outgrowth on TPYG agar plates. Type B spores displayed an intermediate level of sensitivity requiring 500‐1000 I.U. of nisin/ml for 50% inhibition of outgrowth on TPYG agar plates. Similar levels of nisin were necessary to prevent spore outgrowth in TPYG broth and BHI broth over a 7‐day incubation period. With prolonged incubation periods of up to 65 days in TPYG broth, spore outgrowth was observed sporadically at higher nisin levels with the type A and B spores which may indicate some decomposition of nisin with storage. Nisin levels of 5000 I.U./ml for the type A spores and 2000 I.U./ml for the type B spores and the Minnesota E spores were insufficient to prevent spore outgrowth by C. botulinurn in cooked meat medium. For the Beluga E spores, a nisin level of 2000 I.U./ml was necessary to prevent spore outgrowth in cooked meat medium. The need for higher levels of nisin in cooked medium to prevent spore outgrowth may be due to the binding of the nisin by meat particles.

A cooked meat blood culture medium; shelf-life and clinical evaluation compared with other systems

The recovery of anaerobic bacteria from small inocula was examined in an in vitro simulated blood culture system. A freshly prepared cooked meat medium gave the best results while Difco Thiol medium and a commercially available cooked meat medium gave good recovery; Brewer's thioglycollate medium was unsatisfactory. Those media tested performed less well after storage. However, stored Difco Thiol medium and laboratory prepared cooked meat media could be revitalised by steaming, and restored to full efficacy. In a clinical trial of blood culture media, freshly prepared cooked meat medium was compared with an established two-bottle system of Brewer's thioglycollate and Brain Heart Infusion media. The inclusion of freshly prepared cooked meat medium increased the percentage of positive blood cultures from 10·1 to 11·8 per cent. Eighty six per cent of positive cultures could have been detected by our previous technique; 98 per cent could be isolated by our adoption of a freshly prepared cooked meat medium with Brain Heart Infusion Broth.

Common antigens of stable L-forms of group A streptococcus and human myocardial muscle fibers

Common antigenic features of stable streptococcal L-forms and of the cytoplasmic membrane of muscle fibers of the human myocardium were demonstrated by an immunofluorescence method. The common antigen is a component of the surface membrane of the muscle cell, adjacent to the sarcolemmal sheath, and of the membranes of the transverse tubules of the macroplasmic reticulum, which pass through the sarcomeres of the muscle fiber in the zone of the L-disks. The reaction was completely prevented by exhaustion of the antiserum against antigen of L-forms by means of a human myocardial tissue homogenate or a suspension of cultures of L-forms grown on a meat or casein medium. Exhaustion with a tissue homogenate of other organs (liver) or with concentrated nutrient medium had virtually no effect on the intensity of the reaction. In the authors' view, the presence of common antigens in cultures of stable L-forms of group A streptococcus and the myocardium may be one cause of the long persistence of L-forms in the human and animal body.

Behaviour of proteolytic Clostridium botulinum types A and B near the lower temperature limits of growth

The behaviour of spores of Clostridium botulinum type A and proteolytic C. botulinum type B has been studied in cooked meat medium at 10°C, 12°C, 15°C, and 20°C, using mixed cultures (9 groups of in total 41 strains) and pure cultures (41 strains).

Changes in the Population of Clostridium perfringens Type A Frozen in a Meat Medium

Survival of eight strains of Clostridium perfringens type A frozen in a meat medium at −23 C and held under aerobic conditions for 1,7, and 14 days was 17, 12, and 6.2%, respectively. Holding under anaerobic conditions for 1, 7, and 14 days gave survival percentages of 51, 26, and 10%, respectively. Survival was not affected by cooling rates of 0.5, 1.5, and 20 C per min. Addition of 10% sucrose to the meat medium did not affect survival. The meat medium with glycerol concentrations of 1.0 and 2.0M gave survival percentages of 13, 63, 144, and 58, respectively, when frozen at −23 C and held for 22 to 24 h.

An Improved Cooked Meat Medium for the Detection of Clostridium botulinum

A new medium composed of cooked meat and fluid thioglycolate broth was tested with 20 proteolytic and 11 saccharolytic strains of Clostridium botulinum. All of the proteolytic strains produced a black ring at the surface of the broth, presumably due to hydrogen sulfide production, while the saccharolytic strains produced white opaque rings. Since the black ring is formed rapidly and is easily seen, the new medium may be useful for the rapid identification of proteolysis in unknown isolates. Growth and toxin production were better in the new medium than in the usual cooked meat medium or in cooked meat medium plus 0.5% glucose. These results suggest that the new medium will be useful in facilitating identification of C. botulinum.

Collaborative Study of a Method for the Detection of Clostridium botulinum and Its Toxins in Foods

The mouse toxicity and protection technique for the detection and identification of Clostridium botulinum and its toxins in foods was collaboratively studied by 11 laboratories. Each laboratory received 4 samples of cream of mushroom soup; 2 contained spores and toxin of C. botulinum type A, 1 contained spores and toxin of C. botulinum type E, and 1 contained spores of C. sporogenes. The media used were cooked meat medium (beef heart or chopped liver broth) and trypticase peptone glucose yeast extract broth with trypsin. The results indicate that this method has a high degree of repeatability and reproducibility. All 11 laboratories correctly identified the toxins and the nontoxic sample in the food and detected and identified the viable spores in the samples by means of the subsequent cultures. This method has been adopted as official first action.

Phage conversion to hemagglutinin production in Clostridium botulinum types C and D.

Five toxigenic strains of Clostridium botulinum types C and D were incubated at 37 degrees C for 7 days in 15 ml of the following media: LYG medium, cooked-meat medium, egg meat medium, and N-Z-amine medium. The supernatants of these cultures were tested for hemagglutinin production with 1% erythrocytes obtained from mice, guinea pigs, chickens, sheep, monkeys, and humans. Four toxigenic strains produced hemagglutinin. The highest hemagglutinin titer was obtained with a combination of human erythrocytes and cultures incubated in LYG medium. When the same experiment was carried out with many nontoxigenic strains, hemagglutination was observed in only one strain, C-N71. Strains producing hemagglutinin also produced phages. The phages obtained from toxin- and hemagglutinin-producing strains converted nontoxigenic indicator strains to produce both toxin and hemagglutinin. The phage obtained from a toxin-positive hemagglutinin-negative strain could only induce cultures to produce toxin, and the phage from a toxin-negative hemagglutinin-positive strain could only induce production of hemagglutinin. These studies suggest that the production of hemagglutinin by C. botulinum types C and D is governed by bacteriophages and that hemagglutinin production can be transmitted separately or concomitantly with toxin production.

Initial studies on "six months disease" in sheep

Enterotoxaemia in sheep due to Clostridium welchii type D was indicated by field and laboratory investigations in Nepal. Morphological, cultural, biochemical, biological and toxin-producing characteristics observed were used to type the isolates. In anaerobic meat medium, all isolates produced pinkish discoloration of meat. All the strains fermented lactose, maltose, dextrose and sucrose whereas, salicine was fermented only by 17 strains. All but five strains were MR negative. Out of 200 isolated, 166 produced both alpha and epsilon toxins and the remaining 34 non-toxogenic strains are likely to be variants which have lost their toxogenicity. Epidemiologically the local name "Six months disease" and enterotoxaemia are considered to be identical diseases.

The stability of toxigenicity in Clostridium botulinum types C and D.

Several type C and D strains of Clostridium botulinum, which had been converted to the toxgenic state by phages, were serially transferred through cooked meat medium with and without specific anti-phage serum. Most of the converted strains lost their toxigenicity even during transfer without antiserum, and the non-toxigenic variants that appeared were resistant to lysis and conversion by the original phage. However, in some combinations of phage and host bacteria toxigenicity was stable after ten transfers, though it showed a transient decrease, and the non-toxigenic variants that arose remained sensitive to lysis and conversion. When converted strains were transferred in medium containing anti-phage serum, toxigenicity was lost more rapidly than in the absence of serum and the non-toxigenic variants that appeared remained sensitive to lysis and conversion by the parent phage. Filtrates of the supernatants of culture fluids of strains transferred without anti-phase serum converted non-toxigenic strains to toxigenicity at varying rates.

Efficiency of microbe filtration of air filter for rearing germfree animals (author's transl)

Filtration efficiency of 3 types of air filter frame and cylinder (Fig. 1) was examined by culture of the air obtained from the operating isolators at intervals of 3 to 21 days. The air of isolator was bubled in 30 ml of nutrient broth for 30 or 120 minutes (4approximately15 l/min.) and each of 1 ml of the broth was inoculated into thioglycollate medium (TGC), cooked meat medium (CMM), heart infustion broth (HIB) and glucose peptone water (GPW). The media were incubated for 7 to 14 days at 30 or 25 degrees C. As seen in Table 1, microbes could be effectively removed through all types of filter for over 10 months, though there were a very few positive cases suggesting technically acidental contamination at sampling.

Inhibition of Clostridium botulinum by Strains of Clostridium perfringens Isolated from Soil

Thirty-one soil samples were examined for the presence of organisms capable of inhibiting growth and toxin production of strains of Clostridium botulinum type A. Such organisms were found in eight samples of soil. Inhibiting strains of C. perfringens were found in five samples, of C. sporogenes in three and of Bacillus cereus in three. Three of the C. perfringens strains produced an inhibitor effective on all 11 strains of C. botulinum type A against which they were tested, seven of eight proteolytic type B strains, one nonproteolytic type B strain, five of nine type E strains and all seven type F strains, whether proteolytic or nonproteolytic. They did not inhibit any of 26 type C strains, 6 type D strains, 4 type E strains, or 24 C. sporogenes strains. In mixed culture, an inhibitor strain of C. perfringens repressed growth and toxin production by a C. botulinum type A strain even though it was outnumbered by the latter about 40 times. It also repressed growth and toxin production of C. botulinum in mixed culture of soils in which this latter organism naturally occurred when cooked meat medium but not when trypticase medium was used.

Inhibition of Clostridium botulinum by Strains of Clostridium perfringens Isolated from Soil

Thirty-one soil samples were examined for the presence of organisms capable of inhibiting growth and toxin production of strains of Clostridium botulinum type A. Such organisms were found in eight samples of soil. Inhibiting strains of C. perfringens were found in five samples, of C. sporogenes in three and of Bacillus cereus in three. Three of the C. perfringens strains produced an inhibitor effective on all 11 strains of C. botulinum type A against which they were tested, seven of eight proteolytic type B strains, one nonproteolytic type B strain, five of nine type E strains and all seven type F strains, whether proteolytic or nonproteolytic. They did not inhibit any of 26 type C strains, 6 type D strains, 4 type E strains, or 24 C. sporogenes strains. In mixed culture, an inhibitor strain of C. perfringens repressed growth and toxin production by a C. botulinum type A strain even though it was outnumbered by the latter about 40 times. It also repressed growth and toxin production of C. botulinum in mixed culture of soils in which this latter organism naturally occurred when cooked meat medium but not when trypticase medium was used.

Low-temperature-growing clostridia from marine sediments

Marine sediments inoculated into cooked meat medium were enriched for 5 months at 0–5 °C. Organisms isolated and purified were tested for growth at low temperatures, and those growing only anaerobically at 5 °C and below were retained for further study. The growth–temperature range was determined by growing 18 isolates on a temperature-gradient incubator over a temperature range of 0 to 34 °C. On the bases of minimum, optimum, and maximum growth temperatures, the 18 isolates were separated into obligate and facultative psychrophilic and psychrotrophic clostridia. The DNA was extracted from cells grown at 12 °C and the percentage of G + C for all the isolates fell within the range characteristic of Clostridium species. The volatile alcohols and acids produced on a glucose medium were detected by gas chromatography. Ten of the isolates produced no alcohols and only acetic and butyric acids. One isolate produced butanol and acetic and butyric acids. The remaining seven isolates produced one or two alcohols, ethanol and butanol, and up to six acids. The 18 isolates fell into distinct groups with a close relationship between growth–temperature range and volatile alcoholic and acidic products. Biochemical tests showed that the isolates resembled Clostridium species described in the literature; however, they were not like any known species and were not named.

INHIBITION OF CLOSTRIDIUM PERFRINGENS BY STREPTOCOCCUS FAECALIS IN A MEDIUM CONTAINING CURING SALTS

Inhibition of two strains of Clostridium perfringens by Streptococcus faecalis in Cooked Meat Medium containing curing salts was investigated. Inhibitory effects were evaluated by growth-curve studies and by measurement of inhibition zones on agar. Both strains of C. perfringens were inhibited by S. faecalis in a medium containing glucose, although to different degrees. Addition of nitrite to the medium containing glucose increased the inhibitory effect of S. faecalis towards C. perfringens. Production of lactic acid by S. faecalis appears to play an important role in this inhibition of C. perfringens.

A comparison of media used in vitro to isolate non-sporing Gram-negative anaerobes from blood

Five anaerobic media were compared in a model blood culture system for their ability to recover small inocula of Gram-negative non-sporing anaerobes. Dehydrated cooked meat medium was the least effective; USP thioglycollate medium was the most effective isolation medium and is recommended for routine use. Freshly prepared cooked meat medium has the advantage of allowing prolonged survival of strains.