Peptone water

As illustrated in Table-1, 500 samples were randomly collected from 100 apparently healthy ducks (50 alive and 50 freshly slaughtered ducks) and 100 diseased ducks (50 alive and 50 freshly died and emergency slaughtered ducks) from commercial farms and traditional slaughterhouses at Ismailia Governorate, Egypt. Tracheal swabs and internal organs from freshly died and slaughtered ducks were collected. Samples were collected in peptone water (Oxoid, USA) under the complete aseptic conditions and rapidly transported to the lab for bacteriological examination.

Microbial survivors were measured by the corresponding plating of aliquots of wine samples diluted in peptone water (Oxoid, Basingtok, Hampshire, UK) and plated onto the appropriate agar medium. For yeast enumeration, Potato Dextroxe Agar (Oxoid) was used, and plates were incubated at 25 °C for 48 h. Lactic acid bacteria (LAB) survivors were enumerated in Mann Rogosa Sharpe (MRS) Agar (Oxoid) and the plates were incubated in anaerobic conditions (<1% O₂) at 30 °C for 24 to 72 h. After the plate incubation, the number of counted colonies corresponds with the number of viable microorganisms expressed as a colony form unit per milliliter (CFU/mL) or its decimal logarithm (Log₁₀ CFU/mL). The survival fraction was calculated by dividing the number of microorganisms that survived the treatment (N_t) by the initial number of viable cells (N₀).

Samples for total aerobic mesophilic and lactic acid bacteria (LAB) were taken on the 1st and 30th days of ripening. For microbiological analysis, an internal procedure based on ISO 15214:1998 [36 ] was used. A 10 g sample of sausage was aseptically weighed in a sterile plastic bag. Subsequently, samples were homogenized with 90 mL of a sterile solution of 0.1% (w/v) peptone water (Oxoid, Unipath, Basingtoke, UK) for 2 min at 20–25 °C in a Masticator blender (IUL Instruments, Barcelona, Spain), thus making a 1/10 dilution. Serial 10-fold dilutions were prepared by mixing 1 mL of the previous dilution with 9 mL of 0.1% (w/v) sterile peptone water. Total aerobic mesophilic bacteria were enumerated following standard ISO 4833, 2003 (colony-count technique at 30 °C), by aseptically spread plating 1 mL of each of the serial 10-fold dilutions on Plate Count Agar (PCA). After incubation at 30 °C for 72 h, the CFU per gram of sample was determined. For lactic acid bacteria enumeration, 100 mL samples of similar 10-fold dilutions were plated on Man, Rogosa, and Sharpe (MRS) agar. After incubation at 35 °C for 3 days or 30 °C for 5 days in an aerobic atmosphere supplemented with 5% carbon dioxide, the cfu per gram of sample was determined.

Bacteriophage MS2 (ATCC 15597-B1) was propagated using the double agar layer method. The bottom layer (of the tryptone soya agar (TSA) Escherichiacoli agar plates) consisted of TSA (Oxoid, Basingstoke, Hampshire, UK) and the top layer consisted of 4.5 mL of soft TSA mixed with 500 µL of overnight E. coli (ATCC 700891) culture (which had been incubated overnight at 37 °C in typtone soya broth (Oxoid)) and 200 µL of freeze thawed MS2 bacteriophage solution. The plates were then incubated overnight at 37 °C. The plaques were harvested in peptone water (Oxoid) and purified by centrifugation at 3000 rpm for 15 min to separate the host cell debris and the bacteriophage. The supernatant was filtered through a 0.22 μm Millex-GP Syringe Filter Unit (Millipore, catalog number SLGP033RS, Tullagreen, Cork Ireland) and used as a stock solution. This stock was serially diluted in sterile water and the concentration was determined by plating and counting plaques using the double agar layer method described above.

The breasts of the carcasses were used as sampling points, and only regions which were not wounded by lead shot were used. Each sample was prepared by excising a piece of skin 10 cm² in area with a sterile knife blade and a template. The samples were placed in a sterile stomacher bag containing 90 mL of sterile 0.1% (w/v) peptone water (Oxoid Ltd., Hampshire, UK) and homogenized (Masticator IUL, Barcelona, Spain) for two minutes. Decimal dilutions in sterile 0.1% (w/v) peptone water were prepared from this homogenate. Table 1 shows the culture media, incubation conditions and references used for each of the microbial groups studied. All inoculations were carried out in duplicate. Plates with between 25 and 250 colonies (spread plate technique) or between 30 and 300 colonies (pour plate technique) were counted, and average counts were calculated.

The isolation and identification of C. perfringens from pork and chicken meat samples were carried out according to the previously described method of the American Public Health Association (APHA) [25 ]. Briefly, a sample (25 g) was added to a bag (Seward Ltd, Worthing, UK) containing 225 mL of 0.1% Peptone Water (Oxoid Ltd, Hants, UK), homogenized by a Seward stomacher 400 circulator (Seward Ltd, Worthing, UK), and incubated for 24 h under anaerobic conditions at 37 °C. After incubation, the homogenate was serially diluted and 1 mL of an appropriate dilution was mixed with soft tryptose sulfite cycloserine agar (Oxoid Ltd, Hants, UK), supplemented with 5% egg yolk and perfringens selective supplement, and then poured on a Petri dish. After solidification, the plate was overlayed with a second TSC layer and incubated anaerobically for 24 h at 37 °C. Colonies with a black color and white halo were considered presumptive C. perfringens. Well-separated colonies were picked up for biochemical testing using an API 20A kit (Biome’rieux, Marcy I’ Etoile, France). For further confirmation, up to five single colonies per each plate were selected to detect the species-specific 16S-rRNA gene using the method previously described by Tonooka et al. (2005) [26 (link)]. All C. perfringens strains were stored at −86 ˚C for further experiments.

10 g of lettuce, soil, and manure were weighed into well-labelled sterile zip lock bags and 90 ml of peptone water (Oxoid, UK) was added and pulsified using a pulsifier (PUL 100E; Stuart Scientific Co. Ltd., UK) for 30 s. One millimeter (1 ml) of the resultant stock solution was inoculated in 5 ml of MacConkey broth (Oxoid, UK) and incubated for 24 ± 2 hours at 44°C. 1 ml of the overnight positive culture was transferred into test tubes containing 5 ml of Tryptone Soy Broth (Oxoid, UK) and incubated for 24± 2 hours at 44°C. A few drops of Kovac's Indole Reagent (LOBA CHEMIE PVT. Ltd., India) were added to each positive tube. All tubes that retained the red color ring following simple agitation were selected, and a loop full streaked on Eosin Methylene Blue Agar (EMBA) (Oxoid, UK) and incubated for 24± 2 hours at 37°C. Colonies with a shiny green metallic appearance were confirmed as E. coli and stored in Brain Heart Infusion (Oxoid, UK) with 20% glycerol.