Comparison of monolayer and bilayer plates used in antibiotic assay.

Standard curves of 5 antibiotics were determined in an antibiotic assay using bilayer and monolayer agar plates and AOAC-specified test organisms and agar media. Micrococcus luteus ATCC 9341a and antibiotic medium No. 2 were used to prepare the penicillin G standard curve. The same organism and antibiotic medium No. 11 were used to prepare the erythromycin standard curve. Standard curves for streptomycin, tetracycline, and gentamicin were prepared, respectively, with antibiotic medium No. 5 and Bacillus subtilis ATCC 6633, antibiotic medium No. 8 and B. cereus ATCC 11778, and antibiotic medium No. 11 and Staphylococcus epidermidis ATCC 12228. Assays of inhibition by meat fortified with penicillin, streptomycin, gentamicin, tetracycline, erythromycin also were performed on monolayer and bilayer plates. Differences in standard curves and inhibitory responses obtained with monolayer and bilayer plates were < 10%. Thus, monolayer plates are acceptable for use in analyses of meat and poultry for antibiotics residues, with savings in laboratory resources and time.

Biological and physicochemical characteristics of four serotypes of Salmonella enteritidis.

Four serotypes of Salmonella enteritidis, Anatum ATCC 9270, Newbrunswick ATCC 1608, Oranienburg 200 E, and Pullorum RM, were studied to determine biological, chemical, or physical differences which might explain variations in Salmonella virulence as previously reported by McCullough and Eisele (J. Infect. Dis. 88:278-289, 1951; 89:259-265, 1951). These investigators found that serotype Pullorum was significantly less virulent than serotypes Newport, Derby, Barielly, Meleagridis and Anatum when fed to healthy humans. Results of our own experiments showed that serotype Pullorum RM had a generation time approximately twice that of serotype Anatum 9270. The volume of serotype Pullorum was approximately one-half the volume of the other serotypes used (Anatum 9270, Newbrunswick 1608, Oranienburg 200 E, Cubana 12007, and Meleagridis DR). The number of cells required to yield 1 g dry weight was substantially higher for serotype Pullorum RM than for serotypes Anatum 9270, Newbrunswick 1608, and Oranienburg 200 E. The yield of endotoxin per gram dry weight for serotype Pullorum RM averaged 22 mg/g, whereas yields of endotoxin for serotypes Anatum 9270, Newbrunswick 1608, and Oranienburg 200 E averaged 32 to 35 mg/g. The relative abundance of the four major fatty acids (measured by gas chromatography) also showed distinct differences among the serotypes. Pullorum RM contained less lauric and 3-hydroxymyristic acids and more myristic and palmitic acids than the other three serotypes. The identity of 3-hydroxymyristate was confirmed by mass spectroscopy. Serotype Pullorum RM required 10 times more lipopolysaccharides (endotoxin) to obtain a 50% lethal dose in mice than the other three serotypes. When the lipid part was separated from the polysaccharide and solubilized with bovine serum, the 50% lethal dose of serotype Pullorum RM was equal to that of the other three.

Twenty-four-hour immunofluorescence technique for the detection of salmonellae in nonfat dry milk.

A detection procedure was developed in which a newly devised lysine-iron medium was used as a one-step selective and enrichment medium for detection of salmonellae by the fluorescent-antibody technique. Incubation was conducted in two steps: initially at 30 C for 5 hr to resuscitate sublethally stressed cells, followed by incubation at 39 C for 17 hr. Twenty-seven strains of salmonellae from groups A-I were utilized in the development of this procedure which was sensitive enough to detect one Salmonella bacterium in 100 g of nonfat dry milk.

Increased sensitivity of immunofluorescent assay for Salmonella in nonfat dry milk.

The necessity of developing a quick, sensitive, and reliable test for Salmonella in nonfat dry milk (NDM) is evident from the recent tracing of Salmonella outbreaks to this product. Normally, coagulation of casein occurs when assaying NDM under regular cultural conditions, raising the possibility of trapped bacteria. After 20 hr of incubation of NDM in preenrichment lactose broth, enrichment was achieved by using Selenite-Cystine Broth. Smears from the enrichment broth were examined by the fluorescent-antibody technique (FAT) with a commercially available polyvalent O globulin conjugated with fluorescein. Standard cultural methods (SCM) were performed for comparison with FAT. Sensitivity of FAT was definitely improved by the use of trypsin. Casein coagulation of NDM can be avoided by addition of trypsin to samples during initial preenrichment in lactose broth. Samples containing approximately one Salmonella per 10 g were easily detected by FAT with the use of trypsin-treated samples. The method required only 42 hr to complete. Additionally, the use of trypsin enhanced recovery of Salmonella by use of SCM, as evidenced by alteration in the observed coliform to Salmonella ratios.

Two loci controlling genetic cellular resistance to avian leukosis-sarcoma viruses.

Female chickens known to be heterozygous for resistance to subgroups A and B of the avian leukosis-sarcoma viruses were mated to males known to be homozygously resistant to both. The progeny were assayed both on the chorioallantoic membrane (CAM) and in tissue culture for resistance to representative viruses of the A, B, and tentatively defined C subgroups. Segregation ratios of resistance to A and B subgroup viruses agreed with the previously suggested hypothesis of single-autosomal-recessive genes controlling resistance to each subgroup. Mixed infection on the CAM and replicate plate infection in tissue culture with subgroup A and B viruses showed that resistance to the A and B subgroups was inherited independently. Assays with viruses tentatively classified as subgroup C indicated that they were largely composed of a mixture of subgroup A and B viruses or of particles possessing the host range specificity of both. However, virus stocks of the subgroup C category, as well as some stocks classified as subgroup B, produced small numbers of pocks or foci on individuals known to be resistant to subgroup A and B viruses. It is suggested that these Rous sarcoma virus stocks carry between 1 and 10% of a true subgroup C virus.