Recovery of spores of Clostridium botulinum in yeast extract agar and pork infusion agar after heat treatment.

Yeast extract agar, pork infusion agar, and modifications of these media were used to recover heated Clostridium botulinum spores. The D- and z-values were determined. Two type A strains and one type B strain of C. botulinum were studied. In all cases the D-values were largest when the spores were recovered in yeast extract agar, compared to the D-values for spores recovered in pork infusion agar. The z-values for strains 62A and A16037 were largest when the spores were recovered in pork infusion agar. The addition of sodium bicarbonate and sodium thioglycolate to pork infusion agar resulted in D-values for C. botulinum 62A spores similar to those for the same spores recovered in yeast extract agar. The results suggest that sodium bicarbonate and sodium thioglycolate should be added to recovery media for heated C. botulinum spores to obtain maximum plate counts.     

Recovery of spores of Clostridium botulinum in yeast extract agar and pork infusion agar after heat treatment.

Yeast extract agar, pork infusion agar, and modifications of these media were used to recover heated Clostridium botulinum spores. The D- and z-values were determined. Two type A strains and one type B strain of C. botulinum were studied. In all cases the D-values were largest when the spores were recovered in yeast extract agar, compared to the D-values for spores recovered in pork infusion agar. The z-values for strains 62A and A16037 were largest when the spores were recovered in pork infusion agar. The addition of sodium bicarbonate and sodium thioglycolate to pork infusion agar resulted in D-values for C. botulinum 62A spores similar to those for the same spores recovered in yeast extract agar. The results suggest that sodium bicarbonate and sodium thioglycolate should be added to recovery media for heated C. botulinum spores to obtain maximum plate counts.     

Recovery of Heated Clostridium perfringens Type A Spores on Selective Media

The enumeration of Clostridium perfringens spores on sulfite-polymyxin-sulfadiazine agar (SPS), tryptone-sulfite-neomycin agar (TSN), Shahidi-Ferguson-perfringens agar (SFP), tryptone-sulfite-cycloserine agar (TSC), and TSN lacking antibiotics (BASE) was studied. The spores were heated at 105 to 120 C by the capillary-tube method. The media were about equally efficient for the enumeration of heat-activated spores. Efficiency of the media for the recovery of spores surviving heat treatments at ultrahigh temperatures varied as follows: TSC >/= SFP > BASE > SPS > TSN. Greater recovery when survivors were enumerated on TSC or SFP was attributed to germination of injured spores by the lysozyme present in the egg yolk emulsion used in these media. Low recovery of survivors on TSN and SPS was due to both the absence of lysozyme and inhibition of injured spores by the selective agents of these media. Recovery of heated spores was reduced greatly by polymyxin, neomycin, and kanamycin, and slightly by sulfadiazine and D-cycloserine. The addition of lysozyme to SPS or TSN did not improve the percentage of heat-injured spores recovered because the selective agents of these media interfered with the action of lysozyme. The suitability of the selective media for the enumeration of survivors was greatly affected by the presence of certain foods.     

The nature and site of biocide-induced sublethal injury in Bacillus subtilis spores

Spores of Bacillus subtilis NCTC 8236 exposed at 22 degrees C to test biocides (alkaline glutaraldehyde, an iodophor, Lugol's solution, sodium hypochlorite and sodium dichloroisocyanurate) demonstrated varying degrees of injury to stressing agents (sodium hydroxide, sodium lauryl sulphate, polymyxin B sulphate or cetylpyridinium chloride) incorporated into a recovery agar medium. This injury to stressing agents was expressed mainly during outgrowth.     

THE ADAPTATION OF FUNGI TO FUNGICIDES: ADAPTATION TO COPPER AND MERCURY SALTS

The susceptibility of Botrytis cinerea to copper sulphate in liquid media increased when the volume, and therefore the depth, of the medium in culture bottles exceeded certain values; when the volume was 40 ml. the maximum concentration allowing growth was 300 p.p.m.
By growing mycelium in media containing progressively higher concentrations of copper sulphate a strain was produced which grew at a concentration of 750 p.p.m.
In high concentrations of copper sulphate growth always started at the edge of the liquid, and inocula grew only if they were placed in this position.
In germination tests spores from the resistant strain were more resistant to copper sulphate than were spores of the parent strain.
The resistance of mycelium, and to a lesser extent of spores, was retained after growth of the resistant strain for six months in fungicide-free media.
Spore and mycelial inocula grew in much higher concentrations of copper sulphate when nutrient media were solidified with agar.
The strain resistant in liquid media was no more resistant than the parent strain on agar media.
The resistance of the fungus was not increased after growth for long periods on agar containing high concentrations of copper sulphate. The resistance of the strain resistant in liquid media was not lost after growth on agar media for 3 months.
Attempts to produce strains more resistant than the parent to mercuric chloride were unsuccessful.
The results obtained with phenyl-mercuric acetate were essentially similar to those obtained with copper sulphate, but relatively much more resistant strains were produced.     

Relationship Between the Increased Sensitivity of Heat Injured Clostridium perfringens Spores to Surface Active Antibiotics and to Sodium Chloride and Sodium Nitrite

Spores of Clostridium perfringens NCTC 8798 became increasingly sensitive to inhibition by sodium chloride, sodium nitrite and a mixture of polymyxin and neomycin when heated at 70–100°C. That the increased sensitivity to each agent was caused by the same cellular damage was suggested by: (1) the temperature coefficients for induction of cellular damage leading to increased sensitivity to all three agents were the same; (2) in the absence of the agents, the spores regained resistance to all three at about the same time and in each case in the presence of cell wall-, protein- and DNA-synthesis inhibitors; (3) combinations of the agents were generally no more inhibitory to the heated spores than were the individual agents.     

Recovery of Viability and Radiation Resistance by Heat-Injured Conidia of Penicillium expansum Lk. ex Thom

Spores heated in water at 54 C for up to 1 hr were plated on nutrient agar immediately or held for 3 days in aerated water at 23 C and then plated. Under these conditions, holding was optimal for recovery, increasing survival percentage up to 20-fold over values for immediate plating. Recovery was prevented partially or completely, however, when spores were held in any of the following solutions: glucose, potassium phosphate, ammonium or sodium acetate, sodium azide, or 2,4-dinitrophenol, or in the sodium or potassium salts of pyruvate, and tricarboxylic acid cycle acids. Both anaerobiosis and incubation at 0 C prevented recovery. Survivors of a heat treatment were more sensitive to gamma radiation than were unheated spores. Conditions which affected the recovery of viability had the same effect on restoration of radiation resistance. Thus, many of the processes for restoration of radiation resistance seem involved also in recovery of viability after heating. After a 99% inactivating treatment (about 30 min at 54 C), heated spores respired as fast as unheated spores, or faster. Malate, citrate, succinate, and acetate stimulated respiration in unheated spores and inhibited it in heated spores.     

Role of Curing Agents in the Preservation of Shelf-stable Canned Meat Products

Experiments were conducted to gain a better understanding of the mechanism by which sodium chloride, sodium nitrate, and sodium nitrite supplement the action of heat in preserving canned cured meat products. Heated spores of putrefactive anaerobe 3679h were less tolerant of all three curing agents in the outgrowth medium than were unheated spores. When the curing agents were added to the heating menstruum, but not to the outgrowth medium, sodium chloride and sodium nitrate tended to protect the spores against heat injury, but sodium nitrite did not. When the spores were both heated and cultured in the presence of the curing agents: (i) nitrate and salt increased the apparent heat resistance at low concentrations (0.5 to 1%) but decreased it at concentrations of 2 to 4%; (ii) nitrite was markedly inhibitory, especially at pH 6.0. At the normal pH of canned luncheon meats (approximately 6.0), nitrite appears to be the chief preservative agent against spoilage by putrefactive anaerobes.     

The activity of polymyxins against dense populations of Escherichia coli.

The activities of polymyxin B sulphate, colistin (polymyxin E) sulphate and their sulphomethyl derivatives were compared by continuous turbidimetric monitoring of dense cultures of an Escherichia coli strain exposed to these agents. Judged by the concentration of antibiotic which caused a rapid fall in opacity of the culture, polymyxin B sulphate and colistin sulphate had similar activities, but the sulphomethyl compounds differed considerably: sulphomyxin sodium induced lysis of the culture at a concentration four times that of the parent compound, whereas colistin sulphomethate sodium induced a delayed fall in opacity consistent with recruitment of activity as the inactive sulphomethyl derivative was broken down to the parent compound. Durign overnight incubation, regrowth of cultures which had initially succumbed to polymyxin action occurred, apparently due to the selection of phenotypically resistant variants from within the population. In this way cultures could easily be adapted to growth in concentrations of antibiotic well above the conventionally-determined minimum inhibitory concentration. The comparative ease of adaptation was in the order: colistin sulphomethate greater than sulphomyxin greater than colistin sulphate greater than polymyxin B sulphate.     

Studies on the Mechanism of the Sporicidal Action of Glutaraldehyde

S ummary . Low concentrations (0.025–0.125%) of glutaraldehyde inhibited or prevented colony formation by Escherichia coli, Bacillus subtilis and B. pumilis in agar, and inhibited germination of spores of the Bacillus spp. in L-alanine plus D-glucose. Higher concentrations (2%) of glutaraldehyde at pH 8.5 were sporicidal. Pre-treatment of spores with glutaraldehyde lessened release of dipicolinic acid when the spores were subsequently heated at 100°, but not at 121°. Spores treated with glutaraldehyde and then with 0.5 M thioglycollic acid in 6 M urea at 70° were less sensitive to lysis by hydrogen peroxide than spores which had not been exposed to glutaraldehyde. Glutaraldehyde was less effective in preventing peroxide induced lysis if added to spores which had been previously exposed to thioglycollic acid plus urea at 70°. The mechanism of the sporicidal activity of glutaraldehyde is discussed in relation to these findings.     

Spore membrane(s) as the site of damage within heated Clostridium perfringens spores.

Clostridium perfringens spores were injured by ultrahigh-temperature treatment at 105 C for 5 min. Injury was manifested as an increased sensitivity to polymyxin and neomycin. Since many of the survivors could not germinate normally the ultrahigh-temperature-treated spores were sensitized to and germinated by lysozyme. Polymyxin reportedly acts upon the cell membrane. Neomycin may inhibit protein synthesis and has surface-active properties. Injured spores were increasingly sensitive to known surface-active agents, sodium lauryl sulfate, sodium deoxycholate, and Roccal, a quaternary ammonium compound. Injured spores sensitive to polymyxin and neomycin also were osmotically fragile and died during outgrowth in a liquid medium unless the medium was supplemented with 20% sucrose, 10% dextran, or 10% polyvinylpyrrolidone. The results suggested that a spore structure destined to become cell membrane or cell wall was the site of injury. Repair of injury during outgrowth in the presence of protein, deoxyribonucleic acid, ribonucleic acid and cell wall synthesis inhibitors was consistent with this hypothesis.     

Quantitative Analysis of Polymyxin B Released from Polymyxin B-Treated Dormant Spores of Bacillus subtilis and Relationship between Its Permeability and Inhibitory Effect on Outgrowth

Polymyxin B, one of the cyclic polypeptide antibiotics, binds to the coat of Bacillus subtilis dormant spores and inhibits them from growing after germination. When about 2.8 × 10⁸ cells/ml of polymyxin B-treated dormant spores were incubated in heart infusion broth, 3.6 μg/ml of polymyxin B were released into the liquid medium during germination. Incubation of the same concentration of polymyxin B-treated ones in 100 mM CaCl₂ solution released 4.0 μg/ml of the antibiotic. The effect of various concentrations of polymyxin B on germination, outgrowth and vegetative growth of the dormant spores was investigated; the results showed that concentrations of 4.0 μg/ml and higher of the antibiotic inhibited their outgrowth and vegetative growth after germination. Young vegetative cells were less sensitive to the antibiotic than germinated spores. In addition to these results, immunoelectron microscopy with colloidal gold particles indicated that polymyxin B permeated into the core of the germinated spores and inhibited them from outgrowing.     

The Effect of Incubation Temperature on the Recovery of Spores of Bacillus subtilis 8057

S ummary . The recovery of Bacillus subtilis spores was studied after different heat treatments at 95° and incubation at different temperatures in roll tubes in a gradient temperature incubator. Plate count agar and brain–heart infusion agar were used in the roll tubes. Unheated spores showed similar recoveries at 16–48° whereas heated spores had an optimum recovery temperature of c. 30.9. The rate of germination of untreated spores was greatest at c. 41° and ceased at 50°. Heated spores germinated at 52°5°, suggesting that recovery of heat-treated spores is not limited by their ability to germinate. Outgrowth of spores at different incubation temperatures was similar for germinated and ungerminated spores. Accordingly it is outgrowth rather than germination which is sensitive to temperature.     

Culture media for the differentiation of isolates of Yersinia ruckeri, based on detection of a virulence factor

M.D. FURONES, M.L. GILPIN AND C.B. MUNN. 1993. Strains of the bacterial fish pathogen Yersinia ruckeri were identified with the API 20E system and distinguished on the basis of whole cell agglutination with antisera, sorbitol fermentation and polymyxin B sensitivity. Strains which were shown to possess the virulence-associated heat-sensitive factor (HSF) were shown to grow preferentially on culture media containing sodium dodecyl sulphate (SDS) and to produce a creamy deposit around the colonies. By contrast, strains lacking this factor (HSF^-) grew poorly and without forming a deposit. Enhancement of the differentiation between the two types was shown by the incorporation of Coomassie brilliant blue dye into agar containing 1% SDS, and the uptake of Coomassie blue and Congo red was shown to be temperature-dependent. Most strains tested were shown to belong to serotype I, and were sensitive to polymyxin and did not ferment sorbitol. With the medium developed most serotype I strains but not those of other serotypes were shown to possess HSF. It is suggested that the medium is used in epidemiological studies of Y. ruckeri.     

Comparison of coats and surface-dependent properties of Bacillus cereus T prepared in two sporulation environments

The surface or coat-associated properties of Bacillus cereus T spores produced from modified G medium (MGM) and fortified nutrient agar (FNA) were compared. The two populations appeared structurally similar by transmission electron microscopy. Spores prepared on FNA were more susceptible to ozone inactivation than MGM-prepared spores. When activated by heating for 15 min at 70–85°C, FNA-prepared spores were optimally activated at 85°C and did not become hydrophilic on heat activation while MGM spores were optimally activated at 70°C and became hydrophilic on activation. Susceptibility to removal of coat and outer membrane by chemical and enzymatic extraction treatments was measured by monitoring reduced ability to germinate in nutrients and acquired ability to germinate in the presence of lysozyme. Bacillus cereus T MGM-prepared spores germinated in lysozyme upon<1 h exposure to sodium dodecyl sulphate-dithiothreitol. FNA-prepared spores were lysozyme sensitive after > 2 h treatment. Thus, B. cereus T FNA spore coats and outer membranes were more resistant to these denaturing agents. Transmission electron micrographs revealed no change in appearance of extracted spores. Sporulation environment must be considered when laboratory-prepared spores are used to assess or predict the effect of control procedures on spores present in nature.     

THE ADAPTATION OF FUNGI TO FUNGICIDES: ADAPTATION TO THIRAM, ZIRAM, FERBAM, NABAM AND ZINEB

The concentrations of thiram preventing germination of spores of Botrytis cinerea in drops of a 1% solution of sucrose, and on the surface of a sucrose-nitrate agar have been determined. Thiram had much less effect on germination in the agar medium, even when a purified agar was used. There was no growth on sucrose-nitrate agar if the concentration of thiram exceeded 31 p.p.m. Attempts to obtain strains able to grow at higher concentrations were unsuccessful.
Similar results were obtained with ziram, nabam and zineb.
Ferbam also was more effective in preventing spore germination in spore drops than on agar media; this effect was obtained with ordinary and with purified agar.
On a sucrose-nitrate agar generally there was no growth if the concentration of ferbam exceeded 125 p.p.m., but in one of forty-eight plates containing 250 p.p.m. ferbam, five slowly growing colonies were produced, and from these colonies arose mycelium which grew and sporulated rapidly on 500 p.p.m. ferbam agar. Agar disk inocula were transferred from these cultures to agar containing higher concentrations of ferbam and in this way, and by repeating the process, a strain was obtained which grew slowly but continuously, and sporulated on agar containing 5000 p.p.m. ferbam. However, the poor solubility of this fungicide made it difficult to assess quantitatively the degree of adaptation.
A proportion of the spores from this strain germinated in drops containing about twice the concentration of ferbam which prevented germination of parent spores.
The resistance of the mycelium of the resistant strain was not lost after repeated subculture on fungicide-free agar. The resistant strain was as susceptible as the parent strain to thiram, ziram, nabam and zineb.
Attempts to obtain strains of Venturia inaequalis resistant to thiram, ferbam, ziram and zineb were unsuccessful.     

Growth from Spores of Clostridium perfringens in the Presence of Sodium Nitrite

The method by which sodium nitrite may act to prevent germination or outgrowth, or both, of heat-injured spores in canned cured meats was investigated by using Clostridium perfringens spores. Four possible mechanisms were tested: (i) prevention of germination of the heat-injured spores, (ii) prior combination with a component in a complex medium to prevent germination of heat-injured spores, (iii) inhibition of outgrowth of heat-injured spores, and (iv) induction of germination (which would render the spore susceptible to thermal inactivation). Only the third mechanism was effective with the entire spore population when levels of sodium nitrite commercially acceptable in canned cured meats were used. Concentrations of 0.02 and 0.01% prevented outgrowth of heat-sensitive and heat-resistant spores, respectively. Nitrite-induced germination occurred with higher sodium nitrite concentrations.     

Decreased particulate NADH oxidase activity in Bacillus subtilis spores after polymyxin B treatment

The activities of several enzymes of polymyxin B-treated dormant and germinated spores of Bacillus subtilis were examined. The particulate NADH oxidase of the antibiotic-treated spores showed considerably lower specific and total activities compared with those of untreated ones. The specific and total NADH oxidase activities of untreated spores increased 12- and 15-fold respectively during germination, whereas increases during germination of polymyxin B-treated spores were inhibited. The specific and total activities of particulate NADH cytochrome c reductase of dormant spores were decreased by polymyxin B treatment in almost the same proportion as those of the particulate NADH oxidase. The specific activity of NADH dehydrogenase of dormant spores remained unchanged after antibiotic treatment but the total activity fell considerably. The activities of other enzymes examined were similar for untreated dormant and germinated spores and antibiotic-treated spores. The respiration of polymyxin B-treated dormant spores was inhibited at the same time as the start of germination. Morphologically, polymyxin B-treated dormant spores lost a laminar structure of the cortex and details of the spore protoplast. The inhibitory mechanism of particulate NADH oxidase activity of polymyxin B-treated dormant spores is discussed.     

Revival of Bacillus subtilis spores from biocide-induced injury in the germination process

Spores of Bacillus subtilis NCTC 8236 were treated with glutaraldehyde, Lugol's iodine, polyvinylpyrrolidone-iodine (PVP-I), sodium hypochlorite or sodium dichloroisocyanurate (NaDCC). After exposure survivors were enumerated on nutrient agar containing potential revival agents (subtilisin, lysozyme, calcium dipicolinate, calcium lactate). Of these, only calcium lactate had any significant enhancing effect and then only with iodine-treated spores. Calcium lactate (9 mmol 1⁻¹) in nutrient broth enhanced the rate and extent of germination of iodine-treated spores but not of spores previously subjected to glutaraldehyde, hypochlorite or NaDCC.     

Interaction of Bacillus subtilis spores with sodium hypochlorite, sodium dichloroisocyanurate and chloramine-T

S.F. BLOOMFIELD AND M. ARTHUR. 1992. Solutions of chlorine-releasing agents (CRAs) show varying activity against Bacillus subtilis spores; sodium hypochlorite (NaOCl) shows higher activity than sodium dichloroisocyanurate (NaDCC) which is more active than chloramine-T. Investigations with coat- and cortex-extracted spores indicate that resistance to CRAs depends not only on the spore coat but also the cortex. Whereas extraction of alkali-soluble coat protein increased sensitivity to NaOCl and NaDCC, degradation of coat and cortex material was required to achieve significant activity with chloramine-T. NaOCl (in the presence and absence of NaOH) and NaDCC (in the presence of NaOH only) produced degradation of spore coat and cortes material which may be related to their rapid sporicidal action at low concentrations under these conditions. By contrast, chloramine-T produced no degradation of cortex peptidoglycan and was only effective against normal and alkali-treated spores at high concentrations, requiring extraction of peptidoglycan with urea/dithiothreitol/sodium lauryl sulphate (UDS) or UDS/lysozyme to achieve significant activity at low concentrations. Results suggest that the sporicidal action of CRAs is associated with spore coat and cortex degradation causing rehydration of the protoplast allowing diffusion to the site of action on the underlying protoplast.