Evaluation of the Spiral Plate Maker for the Enumeration of Micro-organisms in Foods

A statistically based comparison of five methods of enumerating bacteria in foods has been undertaken to assess the potential application of the Spiral Plate Maker (Gilchrist et al. 1973) which is claimed to reduce very significantly the costs involved in the quantitative estimation of viable micro-organisms in foods. The performance of the Spiral Plate Maker was compared with that of three conventional methods (pour plate, surface spread plate and drop count) for the examination of four types of food by four different operators. Analysis of variance showed that there were no differences between the methods at the 5% level although some isolated interactions occurred. Regression and correlation coefficients between the various methods were all highly significant and the results obtained by the spiral plate method were within the limits of error for traditional quantitative methods. The spiral plate method, in many cases, can replace advantageously any of the other methods for the quantitative estimation of viable microorganisms in foods. Labour requirements for the enumeration of micro-organisms by the spiral plate method were only 31% of that required for one conventional method. In addition considerable savings in materials were achieved.     

Enumeration of petroleum-degrading microorganisms.

A variety of factors, including concentration of oil, antibiotics, dyes, and inoculum washes, were examined to determine their effect on the total counts of microorganisms on oil-containing media. The media found to be best for enumerating petroleum-degrading microorganisms contained 0.5% (vol/vol) oil and 0.003% phenol red, with Fungizone added for isolating bacteria and streptomycin and tetracycline added for isolating yeasts and fungi. Washing the inoculum did not improve recovery of petroleum degraders. Specifically, silica gel-oil medium and a yeast medium are recommended for enumeration of petroleum-degrading bacteria and yeasts and fungi, respectively. It is suggested that counts of petroleum degraders be expressed as percentage of the total population rather than total numbers of petroleum degraders per se. Incubation temperature and presence of oil was found to influence the numbers of petroleum-degrading microorganisms at a given sampling site.     

Comparison of the Precision Obtained in Counting Viable Bacteria by the Spiral Plate Maker, the Droplette and the Miles & Misra Methods

The Spiral Plate Maker yielded counts of viable bacteria in pure suspensions that were at least as precise as those obtained under comparable conditions by the Droplette and Miles & Misra methods. A tendency of the Miles & Misra method to produce erroneous counts of Escherichia coli was demonstrated and the advantages of the mechanical method over established counting methods is discussed.     

Enumeration of sublethally heated staphylococci in some dried foods.

The effect of 45 substances to restore the salt tolerance of sublethally heat-injured Staphylococcus aureus was tested. Sodium pyruvate, yeast extract, L-histine, casitone (Difco), adenosine triphosphate, and acetylphosphate were effective. For enumeration a repair medium was first used, containing sodium pyruvate and penicillin in 1% skim milk. This step was followed by counting on Baird-Parker agar with penicillinase. This method was selective; fewer than 100 staphylococci/g food could be enumerated and it gave counts about 8 times higher than the method of Giolitti and Cantoni used as a five-tube most probable number technique. Heat injury sensitized S. aureus to polymyxin.     

Novel bioemulsifiers from microorganisms for use in foods

The main objective of this study was to test a range of microorganisms for production of extracellular, high molecular weight emulsifiers for potential use in foods. A standard emulsification assay developed specifically for assessing food emulsifiers was used to examine 24 extracellular microbial products from bacteria, yeasts and algae. Of the 24 products tested, nine had emulsification ability that was as good as and eight had emulsifying properties that were better than those of the commonly used food emulsifiers gum arabic and carboxymethylcellulose. The eight good producer organisms included the yeasts Candida utilis, Candida valida, Hansenula anomala, Rhodospiridium diobovatum and Rhodotorula graminis, the red alga Porphiridium cruentum, and the bacteria Klebsiella spp. and Acinetobacter calcoaceticus. Of these, C. utilis was selected for further study due to the excellent emulsification properties of its extracellular products and the food-grade status of the organism. Crude preparations of the bioemulsifier from C. utilis exhibited low viscosity and had a carbohydrate content of over 80%. Preliminary trials showed that the bioemulsifier from this organism had potential for use in salad cream.     

Plate screening methods for the detection of polysaccharase-producing microorganisms

Polysaccharide-degrading enzymes (polysaccharases) are widely applied in industry. One of the sources of these enzymes are polysaccharide-degrading microorganisms. To obtain such microorganisms from enrichment cultures, strain collections or gene libraries, efficient plate screening methods are required that discriminate between intact and degraded polysaccharide. This can be achieved by making use of specific physicochemical properties of the polysaccharide, such as complex formation with dyes and gelling capacity, or by the application of dye-labelled polysaccharides. This review presents a survey of plate methods based on these principles. Both theoretical and practical aspects of the methods are discussed.     

Agar Plate Method for Detection and Enumeration of Alkylbenzenesulfonate-Degrading Microorganisms

A simple method for detection and enumeration of alkylbenzenesulfonate (ABS)-degrading microorganisms by using agar plates was developed and used in microbiological studies of coastal marine and polluted river waters. The method depends upon the color responses of neutral red in alkaline medium. Neutral red changes from pink, when it enters into ABS micelles, to yellow, when the ABS is degraded, and does not form micelles. When neutral red-tris(hydroxymethyl)-aminomethane buffer solution and then cationic surfactant solution were sprayed onto the agar surface of ABS-nutrient agar cultures, transparent haloes appeared around the colonies of ABS-degrading microorganisms against a pink background. Viable counts of ABS-degrading bacteria isolated from both seawater and freshwater environments were considerably higher in polluted waters than in less polluted areas. Viable counts of ABS-degrading bacteria averaged 1.5 x 105/ml in samples from the surface water of polluted Tokyo Bay and 3.0 x 104/ml in samples from the surface water of polluted Tamagawa River but were fewer in number in samples from less polluted waters.     

Enumeration of petroleum-degrading marine and estuarine microorganisms by the most probable number method.

Several media designed for use in a most probable number (MPN) determination of petroleum-degrading microorganisms were compared. The best results, i.e., largest numbers, were obtained using a buffered (32 mM PO4=) liquid medium containing 1% hydrocarbon substrate. Of 104 presumptive oil degraders tested, 20 grew on oil agar medium but did not utilize oil or a mixture of pure paraffinic hydrocarbons (C10 to C16 n-alkanes) in liquid (MPN) medium. Visible turbidity in the liquid medium was correlated with hydrocarbon utilization. Counts of petroleum degraders obtained using liquid medium (MPN) were in most cases higher than those obtained on an oil-amended silica gel medium. Both procedures yield an estimation of oil degraders, and the oil-amended agar permits growth of organisms which do not degrade crude oil. All strains of oil-degrading microorganisms examined in this study were lipolytic, but the converse was not always true.     

Rapid enumeration of microorganisms in foods by the direct epifluorescent filter technique.

Filtration of "stomachered" food suspensions through nylon filters (pore size, 5 microns) removed most of the food debris without affecting the recovery of microorganisms. Two to ten milliliters of these prefiltered suspensions could be filtered in the direct epifluorescent filter technique (DEFT). The technique takes less than 30 min to complete and has a lower sensitivity of less than 60,000 microorganisms per g for all products examined. Vegetative bacterial cells, spores, fungal hyphae, and yeasts could be distinguished with the technique. For fresh meat and fish, the DEFT count of prefiltered suspensions agreed well with the plate count of unfiltered suspensions over the range of 10(4) to 10(10)/g (correlation coefficient of 0.91). For frozen meat and fish and frozen vegetables, the two counting methods had correlation coefficients of 0.87 and 0.66, respectively. The poor correlation for frozen vegetables was due to the inclusion in the DEFT count of nonviable bacteria killed by the blanching process used to inactivate enzymes. Good agreement was obtained between the prefiltered DEFT count and unfiltered plate count for cooked meats, cream doughnut, and whole peppers. Possible reasons for the poor agreement between the DEFT count and plate count for certain products are discussed.     

Enumeration and characterization of acidophilic microorganisms isolated from a pilot plant stirred-tank bioleaching operation

Microorganisms were enumerated and isolated on selective solid media from a pilot-scale stirred-tank bioleaching operation in which a polymetallic sulfide concentrate was subjected to biologically accelerated oxidation at 45 degrees C. Four distinct prokaryotes were isolated: three bacteria (an Acidithiobacillus caldus-like organism, a thermophilic Leptospirillum sp., and a Sulfobacillus sp.) and one archaeon (a Ferroplasma-like isolate). The relative numbers of these prokaryotes changed in the three reactors sampled, and the Ferroplasma isolate became increasingly dominant as mineral oxidation progressed, eventually accounting for >99% of plate isolates in the third of three in-line reactors. The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits (e.g., oxidation of iron and/or sulfur and autotrophy or heterotrophy) were examined. More detailed studies were carried out with the Leptospirillum and Ferroplasma isolates. The data presented here represent the first quantitative study of the microorganisms in a metal leaching situation and confirm that mixed cultures of iron- and sulfur-oxidizing prokaryotic acidophiles catalyze the accelerated dissolution of sulfidic minerals in industrial tank bioleaching operations. The results show that indigenous acidophilic microbial populations change as mineral dissolution becomes more extensive.     

Improved Stomacher 400 Bag Applicable to the Spiral Plate System for Counting Bacteria

A stomacher 400 bag was slightly modified, constructed, and tested in combination with the spiral plating technique. Use of the modified bag resulted in more rapid sample preparation and saved both labor and glassware because of the avoidance of large particles of solids and fatty foods blocking the hole of the dispensing stylus tube.     

Discrepancies in the Enumeration of Escherichia coli

Stationary-phase cells of Escherichia coli were enumerated by the pour plate method on Trypticase soy agar containing 0.3% yeast extract (TSYA), violet red-bile agar, and desoxycholate-lactose agar, and by the most-probable-number method in Brilliant Green-bile broth and lauryl sulfate broth. Maximum counts were assumed to be those on TSYA. In general, numbers detected were lower with the selective solid media and higher with the selective liquid media. Inhibitory effects, especially on selective solid media varied with the strains of E. coli. The lower detection on selective solid media was partly due to the stress induced in some cells by the temperature of the melted media used in the pour plate method. These cells apparently failed to repair and form colonies in the selective media. Improved detection on the selective solid media was achieved by using 1% nonfat milk solids, 1% peptone, or 1% MgSO(4).7H(2)O in the dilution blanks. Higher detection on selective agar media was effected by surface plating or by surface-overlay plating of the cells. The surface-overlay method appeared to be superior for the direct enumeration of E. coli in foods.     

Comparison of the Pour, Spread, and Drop Plate Methods for Enumeration of Rhizobium spp. in Inoculants Made from Presterilized Peat

Inoculants prepared with presterilized peat were enumerated by the pour, spread, and drop plate techniques. Results indicated that the three plating methods were interchangeable. The drop plate method was preferred because of its economy in materials and labor.     

Enumeration of antigen-presenting cells in mice infected with Sendai virus

Substantial progress has been made in understanding Ag presentation to T cells; however, relatively little is known about the location and frequency of cells presenting viral Ags during a viral infection. Here, we took advantage of a highly sensitive system using lacZ-inducible T cell hybridomas to enumerate APCs during the course of respiratory Sendai virus infection in mice. Using lacZ-inducible T cell hybridomas specific for the immunodominant hemagglutinin-neuraminidase HN421-436/I-Ab and nucleoprotein NP324-332/Kb epitopes, we detected APCs in draining mediastinal lymph nodes (MLNs), in cervical lymph nodes, and also in the spleen. HN421-436/I-Ab- and NP324-332/Kb-presenting cells were readily detectable between days 3 and 9 postinfection, with more APCs present in the MLN than in the cervical lymph nodes. Interestingly, no infectious virus was detected in lymphoid tissue beyond day 6, suggesting that a depot of noninfectious viral Ag survives, in some form, for 2-3 days after viral clearance. Fractionation of the MLN demonstrated that APC frequency was enriched in dendritic cells and macrophages but depleted in the B cell population, suggesting that B cells do not form a large population of APCs during the primary response to this virus.     

Enumeration of Bacillus cereus in Foods

For the enumeration of vegetative cells and spores of Bacillus cereus in foods, a mannitol-egg yolk-phenol red-agar has been developed which exploits the failure of B. cereus to dissimilate mannitol, and the ability of most strains to produce phospholipase C. When a high degree of selectivity was required, polymyxin B sulfate in a concentration of 10 ppm appeared to be the most effective selective additive. Useful characteristics for the identification of presumptive isolates of B. cereus were found to be: morphology, dissimilation of glucose mostly to acetyl methyl carbinol under anaerobic conditions, hydrolysis of starch and gelatin, reduction of nitrate, and growth on 0.25% chloral hydrate agar.     

Enumeration of Airborne Bacteria in Hospital

An investigation of the “normal” bacterial content of the air of a large general hospital is described. In many different places within five different areas 70 to 200 settle-plate or slit-sampler bacterial counts were carried out. Average counts were most often of the same order as or lower than other published results and were proportional to human activity. The use of the logarithms of the counts showed no advantage, and conventional statistics should be applied with caution in evaluating such studies. Slitsampler and settle-plate counts of all bacteria showed no correlation, whereas those of Staph. aureus were correlated. There is a lack of parallelism between hospital infection and air bacteria counted by current methods, which are, therefore, not suitable for routine use.     

Enumeration of Bacteria in Sea Water

The choice of methods to be used for the enumeration of bacteria in sea water must depend on the objectives of the investigators. Comparisons of data based on different methods may be invalid. An operational definition is given for a colony-generating unit (CGU) which, under some conditions, may be a single viable bacterium. Such units can conveniently be enumerated by a modification of the “minimum probable number” method currently employed in aquatic bacteriology. Counts of “viable bacteria” (CGU) in surface sea water samples from the region of the North Pacific Gyre (28° N, 155°W) were estimated to range from 10,000 to 200,000 per litre.