A stabilized L-form of Streptococcus pyogenes continues to synthesize glycerol teichoic acid. This polymer was obtained from S. pyogenes and its L-form, treated in identical fashion, and compared. Highly purified glycerol teichoic acid from only the L-form was found to be devoid of D-alanine and to have a shorter chain length. Otherwise, the glycerol teichoic acid from these two organisms was found to be a 1, 3-phosphodiester-linked glycerophosphate polymer substituted with D-glucose. Evidence is presented that most, if not all, of the glycerol teichoic acid in.this streptococcus lies between the wall and membrane. A possible need for the continued synthesis of a minute amount of glycerol teichoic acid by this L-form for survival is discussed in terms of the known function of teichoic acids in bacteria.MATERIALS AND METHODS Bacteria, media, and cell fractionation. S. pyogenes, type 12, and its stabilized L-form were used (7). S. pyogenes (5 to 8 liters) was incubated at 37 C for 16 h and harvested when in late stationary phase by centrifugation (17,000 x g) at 4 C. The streptococcus was cultured in brucella broth (Pfizer Diagnostics, Brooklyn, N.Y.) containing either bovine serum albumin, fraction V (8 g/liter; Armour Pharmaceutical Co., Chicago, Ill.) or supplemented with (in grams per liter): yeast extract, 8; sodium acetate, 10; glucose, 19; and KH2P04, 4.5, with the pH adjusted to 7.3. L-form cells were cultured in brucella broth containing bovine serum albumin with the addition of 3% NaCl (7) with and without penicillin G (1,000 U/ml). Where indicated, L-form and coccal media were supplemented, except that yeast extract was omitted. Supplemented medium readily caramelized when autoclaved. Thus, precautions had to be taken (i.e., rapid cooling after autoclaving) to prevent excessive caramelization. An alternative procedure, referred to as noncaramelized medium, denotes the addition of the supplement to the autoclaved basal medium after sterilization by membrane filtration (Millipore Corp.; 934 on July 31, 2020 by guest

Enzymic lipid peroxidation by light and dark muscle microsomes of herring (Clupeu harengus) required ATP or ADP, NADH and Fe. NADPH could not effectively replace NADH. Inhibition was observed at high concentrations of ADP and NADH but not Fe. The optimal pH for the reaction of both types of microsomes was between 6 and 7. The average peroxidation rate was 362 and 1143 nmoles MDA per mg protein per hr at 6'C for the light and dark muscle microsomes, respectively. The energy of activation for the light and dark muscle microsomes was similar. The light muscle microsomes lost activity faster than the dark muscle microsomes when exposed to 35°C. Ferrous ion stimulated enzymic lipid peroxidation of light and dark muscle microsomes over that observed with ferric ion.

Membrane lipoteichoic acid continues to be synthesized by an osmotically fragile, stabilized L-form of Streptococcus pyogenes. Chromatographic and electrophoretic comparisons indicate that the lipid componenent of lipoteichoic acid in this L-form and its parental streptococcus is glycerophosphoryldiglucosyl diglyceride and not phosphatidylkojibiosyl diglyceride. Based upon dry weight determinations, the yield of lipoteichoic acid from the L-form is 0.19%, as compared with 0.97% from the streptococcus. When grown with bacitracin the L-form contains the same amount of teichoic acid as when grown without this antibiotic; however, its lipoteichoic acid content is reduced by 85%. Similarly, the L-form grown with novobiocin for 10 h contains only 17% of the teichoic acid found in control cells.

The effect of preprocess holding conditions (3 and 12% brine at 7.2 and 0.6"C) on the quality of canned sardines (Clupea harengus) was examined. The sardines appeared to have a relatively short shelf life and had to be canned within 36 hr when lightly salted (3% brine) and held at 7.2"C. At a lower temperature (0.6"C) and a higher salt concentration (12% brine) the sardines could be held up to 3 days. Low viable bacterial counts detected during storage experiments indicated spoilage not to be primarily due to bacterial activity. Sensory evaluation of the raw sardines could not be used with any accuracy to predict quality of the canned product, because raw sardines, which were still considered acceptable before canning, resulted in an unacceptable product. Of the objective quality tests performed on raw sardines (TMA, TBA, tyrosine and pH) only TMA-N content appeared to possess high accuracy (P 4 0.001) in predicting quality of the canned products.

A microsomal fraction isolated from herring (Clupea harengus) muscle was shown to oxidize the lipids in an emulsion prepared from extracted lipids of herring muscle. The oxidation of the lipids of the emulsion was dependent on enzymic oxidation of the microsomal lipids. In the absence of either the microsomes or NADH (previously shown to be required for enzymic lipid peroxidation), no measurable oxidation occurred in either the lipid fraction of the microsomes or the lipids of the emulsion. Disappearance of eicosapentaenoic and docesahexaenoic acids from peroxidizing assay systems in presence and absence of lipid emulsion corresponded to the increase in TBA reactive substances.

Soluble fractions from the light and dark muscle tissue of herring (Clupea harengus) were tested for their effect on lipid peroxidation of the isolated mierosomal fractions from the respective muscle tissues. To determine the nature of the components involved, the supernatant fractions were heated and/or dialyzed before treatment of the mierosomal fraction. In addition, some mierosomal samples were pretreated for 60 min with the supernatant fractions prior to initiating lipid peroxidation, whereas in other cases the supernatant fractions were added on initiation of the lipid peroxidation reaction. Results indicated that light muscle cytosol contained factors inhibitory to mierosomal lipid peroxidation. Dark muscle cytosol did also, but in addition appeared, to contain activators. Preheating, dialysis, or preincubation indicated the nature of some of the compound's involved. High and low molecular weight compounds were involved as were thermostable and thermolabile substances.

Reproducibility of aerclbic plate count (APC), coliform and fecal coliform counts in frozen cod was examined by 14 laboratories (3 government, 4 universjty and 7 industry) using primarily FDA recommended procedures. In order to assure homogeneity of samples, the fish was comminuted and thoroughly mixed. The 35°C and the "room temperature" (26°C) APC on Sample A (a good quality product) were 1.3 x lO'/g and 2.6 x lO'/g, respectively, and for Sample B (a questionable quality product) the counts were 2.9 x lO'/g and 6.4 x lO'/g, respectively. Standard deviations among counts (logro values) were 0.26 and 0.32 for the 35'C counts and 0.24 and O.,tO for the 26°C counts of Sample A and B, respectively. The std. lev. were reduced to 0.19, 0.22, 0.17 and 0.25, respectively, when counts that did not show significant overlap (based on Dur.can's New Multiple Range Test) were excluded from the calculations. Coliform counts on Sample A (not inoculated) ranged fron <30 to 4.6 x 10s/lOOg. Coliform and fecal colif&n counts for Sample B (inoculated at estimated level of 1 x 104/100z) were 5.8 x 103/100g and 5.5 x 103/100a. with a std. dev. of 0.43 and C.37, respectiveiy. The std. dev: for

The behavior of two strains of Listeria monocytogenes (147 and ATCC 19111) was evaluated at different stages of salmon processing. At lower temperatures of 2, 7, and 11 degrees C, L. monocytogenes survived on dry wood surfaces for at least 3 days without added nutrients but was unrecoverable after 2 days at 22 degrees C. Moisture or minimal nutrients on the wood surface increased viability of L. monocytogenes at all incubation temperatures. When large amounts of nutrients were provided, the recoveries of L. monocytogenes at low temperatures (< or = 11 degrees C) were essentially unchanged over the 3-day holding period, and rapid growth was observed at room temperature. In the presence of natural microflora, L. monocytogenes died off rapidly in seawater within 36 h at room temperature. When held at < or = 11 degrees C, L. monocytogenes lost viability throughout storage but was still detectable after more than 6 days of incubation. In the absence of natural microflora, both strains of L. monocytogenes were static during the holding period at all temperatures. At 2, 7, and 11 degrees C, L. monocytogenes in nonsterile salmon blood-water remained viable even after 6 days of incubation, whereas in sterile blood-water, growth of L. monocytogenes was observed at 7 and 11 degrees C. In the absence of natural microflora, L. monocytogenes grew better than it did in the presence of natural microflora. L. monocytogenes 147 was more competitive with background organisms than was L. monocytogenes ATCC 19111. No L. monocytogenes could be detected in the digestive tract of salmon 3 days after its introduction. The survival pattern of L. monocytogenes in fish digestive tracts was similar, regardless of whether the fish were feeding or not. A noticeable decline in the pathogen was observed as early as 3 h after introduction.