A simple and effective plating method to screen polycyclic aromatic hydrocarbon-degrading bacteria under various redox conditions

Agar plates with a polycyclic aromatic hydrocarbon (PAH) layer have been used to screen for microorganisms that degrade PAHs, leaving clear zones around colonies; however, there are several problems with previous methods such as undesired contamination in the fume hood and difficulty in controlling the amount of PAH on the plates. In this study, we developed a modified screening method to address the drawbacks encountered with previous screening methods. A uniform white layer of PAHs was generated by spreading PAHs dissolved in volatile solvents over a surface of solidified agar medium, followed by the evaporation of the solvents. An inoculation was then performed by spreading a molten agar medium containing microbial samples over the solidified agar medium with a PAH layer. Subsequently, the white PAH layer migrated to the surface of the molten agar medium. This essential modification enabled us not only to solve problems of the previous screening methods but also to prepare an agar plate with a PAH layer without a complicated experimental scheme in the anaerobic chamber. After solidification of the molten agar medium and incubation of the plates, clear zones were successfully detected around colonies with aerobic and anaerobic PAH-degrading microbial cultures.     

Development of a Method for Detection of Lactic Acid Bacteria Producing Exclusively the l-(+)- Isomer of Lactic Acid

A method was developed for the detection and isolation, within a population of lactic acid bacteria, of strains producing exclusively the l-(+)- isomer of lactic acid; the visual detection of colonies of these particular strains can be carried out directly on agar plates (50 to 70 colonies per plate). The method is based on an enzymatic stereospecific reaction involving d-(-)-lactate dehydrogenase and linked to a staining reaction; the diffusion area of the d-(-)- isomer stains red around the d-(-)- and the dl-lactic acid-producing colonies, while the colonies producing exclusively l-(+)-lactic acid are detected by the absence of the colored halo. The intensity of staining was increased when cellulose powder and Tween 20 were added to the agar medium.     

A simple agar plate assay for screening siderophore producer yeasts.

Yeasts produce hydroxamate-type siderophores (iron-binding compounds) in response to Fe-stress conditions. Because these siderophores are important to the biocontrol of postharvest diseases of apple and pears, a method for screening siderophore producer yeast was developed.The screening method was carried out in special Petri dishes with eight or nine wells (25-mm diameter). These wells were filled with siderophore production medium and seeded with yeasts isolated from epiphytic apple microflora. After yeasts grew (24-48 h), holes (2-mm diameter) were made in the agar of each well. Holes were filled with an acid solution of ferric perchlorate. After 10-15 min, reddish halos appeared in the bottom of the plate and their intensities were compared with standards. Standards were prepared in the same special dish with rhodotorulic acid solutions (concentrations between 0.05 and 1 g/l) plus 2% agar. When agar solidified into wells, holes were made and filled with ferric perchlorate solution. Color intensities of reddish halos were proportional to siderophore concentration and the detection limit was 0.1 g/l. It was possible to correlate the production of siderophore in solid medium with the results obtained in liquid medium. The methodology was also a useful tool for making a preliminary assessment of the influence of different factors on the siderophore production.     

Immunodiffusion method for detection of type A Clostridium botulinum

A simple gel immunodiffusion agar procedure was developed for detecting toxigenic strains of Clostridium botulinum type A. The method consisted of overlaying colonies grown on thin-layer tryptone-peptone-glucose-yeast extract agar with gel diffusion agar containing desired levels of C. botulinum type A antitoxin. Concentric precipitin zones formed around colonies of C. botulinum type A. Strains of C. botulinum type A were detected by this procedure. However, C. botulinum type B reacted to a lesser degree with this system. No reaction was noted with types E, F, Langeland, F8G, Clostridium perfringens, or with strains of nontoxigenic Clostridium sporogenes. Thickness of the plating medium, incubation time and temperature, environmental growth conditions, and levels of both agar an antitoxin were important factors affecting the efficiency of the procedure, whereas the age of the culture (used as inoculum) was not critical. Thin agar medium (5 ml per plate [15 by 100 mm]) containing 1.5% agar gave consistent results, but more agar limited diffusion, and lower levels encouraged spreaders. The optimal concentration of antitoxin incorporated in to the gel diffusion agar overlay was 1.2 IU/ml gel diffusion agar. Rabbit type A antitoxin prepared with purer immunizing agent gave similar reactions. The addition of type A antitoxin in tryptone-peptone-glucose-yeast extract agar medium before inoculation with type A C. botulinum showed promising results.     

Immunodiffusion method for detection of type A Clostridium botulinum.

A simple gel immunodiffusion agar procedure was developed for detecting toxigenic strains of Clostridium botulinum type A. The method consisted of overlaying colonies grown on thin-layer tryptone-peptone-glucose-yeast extract agar with gel diffusion agar containing desired levels of C. botulinum type A antitoxin. Concentric precipitin zones formed around colonies of C. botulinum type A. Strains of C. botulinum type A were detected by this procedure. However, C. botulinum type B reacted to a lesser degree with this system. No reaction was noted with types E, F, Langeland, F8G, Clostridium perfringens, or with strains of nontoxigenic Clostridium sporogenes. Thickness of the plating medium, incubation time and temperature, environmental growth conditions, and levels of both agar an antitoxin were important factors affecting the efficiency of the procedure, whereas the age of the culture (used as inoculum) was not critical. Thin agar medium (5 ml per plate [15 by 100 mm]) containing 1.5% agar gave consistent results, but more agar limited diffusion, and lower levels encouraged spreaders. The optimal concentration of antitoxin incorporated in to the gel diffusion agar overlay was 1.2 IU/ml gel diffusion agar. Rabbit type A antitoxin prepared with purer immunizing agent gave similar reactions. The addition of type A antitoxin in tryptone-peptone-glucose-yeast extract agar medium before inoculation with type A C. botulinum showed promising results.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp.

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

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.     

Culture medium for selective isolation and enumeration of Gram-negative bacteria from ground meats.

We developed a new medium, designated peptone bile amphotericin cycloheximide (PBAC) agar, which contains (per liter) 10 g of peptone, 300 mg of bile salts, 1 mg of amphotericin B, 1 g of cycloheximide, and 15 g of agar. When 21 samples of fresh ground beef were studied and plate count agar counts were used as references, we obtained a mean recovery of 28% of total counts with violet red bile agar overlay, whereas we obtained 48% recovery with PBAC agar. With 12 samples of frozen ground beef, recovery on violet red bile agar overlay was 29% of the recovery on plate count agar, whereas the corresponding value on PBAC agar was 45%. PBAC agar allowed the enumeration of 1.4 times as many gram-negative bacteria as violet red bile agar overlay. None of eight strains of gram-positive bacteria and none of eight strains of yeasts grew on PBAC agar. Of 158 colonies randomly selected from pour plates of eight fresh ground meat samples, 95% stained gram negative. In comparison, only 70% of 151 colonies selected from corresponding plate count agar plates were gram negative. The lack of background color, turbidity, and ease of use make PBAC agar easier to handle than other media used for gram-negative bacteria, such as violet red bile agar, violet red bile agar overlay, and crystal violet tetrazolium agar. In the preparation PBAC agar, all ingredients are autoclaved together except amphotericin B, which is filter sterilized and added before the plates are poured.     

Nitrogen-deficient Medium in the Differential Isolation of Klebsiella and Enterobacter from Feces

On a nitrogen-deficient agar medium, the tribe Klebsielleae formed large, glistening, mucoid colonies which were easily distinguished from other colony types. Of 113 Klebsielleae isolates from human feces which were characterized, Klebsiella accounted for 88% of the total; 75% were K. pneumoniae; K. ozaenae (13%) was isolated from one individual only. The remaining strains (12%) were identified as Enterobacter cloacae. Counts (for the tribe) ranged from 10(2) to 10(6), with a median of 10(4); 9 of 53 stool specimens were negative. K. pneumoniae was also isolated from 6 of 41 frozen foil-pack foods. Anaerobic studies at room temperature and 37 C revealed no appreciable differences from aerobic plates. The nitrogen-deficient medium appeared better than E M B for isolation of Klebsielleae when they were present in low numbers relative to other coliforms; slime production by Klebsielleae concomitant with minimal growth of other bacteria is involved.     

Accurate assessment of antibiotic susceptibility and screening resistant strains of a bacterial population by linear gradient plate

The dynamics of a bacterial population exposed to the minimum inhibitory concentration (MIC) of an antibiotic is an important issue in pharmacological research. Therefore, a novel antibiotic susceptibility test is urgently needed that can both precisely determine the MIC and accurately select antibiotic-resistant strains from clinical bacterial populations. For this purpose, we developed a method based on Fick's laws of diffusion using agar plates containing a linear gradient of antibiotic. The gradient plate contained two layers. The bottom layer consisted of 15 mL agar containing the appropriate concentration of enrofloxacin and allowed to harden in the form of a wedge with the plate slanted such that the entire bottom was just covered. The upper layer consisted of 15 mL plain nutrient agar added with the plate held in the horizontal position. After allowing vertical diffusion of the drug from the bottom agar layer for 12 h, the enrofloxacin concentration was diluted in proportion to the ratio of the agar layer thicknesses. The uniform linear concentration gradient was verified by measuring the enrofloxacin concentration on the agar surface. When heavy bacterial suspensions were spread on the agar surface and incubated for more than 12 h, only resistant cells were able to form colonies beyond the boundary of confluent growth of susceptible cells. In this way, the true MIC of enrofloxacin was determined. The MICs obtained using this linear gradient plate were consistent with those obtained using conventional antibiotic susceptibility tests. Discrete colonies were then spread onto a gradient plate with higher antibiotic concentrations; the boundary line increased significantly, and gene mutations conferring resistance were identified. This new method enables the rapid identification of resistant strains in the bacterial population. Use of the linear gradient plate can easily identify the precise MIC and reveal the dynamic differentiation of bacteria near the MIC. This method allows the study of genetic and physiological characteristics of individual strains, and may be useful for early warning of antibiotic resistance that may occur after use of certain antimicrobial agents, and guide clinical treatment.     

Development of Streptococcal L-Form Colonies

The development and architecture of L-form agar colonies produced from protoplasts and L-phase bodies were studied by both light and scanning electron microscopy. Agar blocks containing L-phase microcolonies of group A Streptococcus strains ADA and GL8 and group D Streptococcus strain F24 as well as longitudinal sections of mature colonies were used as samples. Initially, granules of about 0.5 mum in diameter were produced by multiple condensation and fragmentation of protoplasts and large bodies. Surface growth by granules ensued and infiltration into agar occurred only after 10 to 11 hr of incubation at 37 C. Club-shaped granules were noted and division seemed to take place by simple fission. The configuration of large bodies and granules in mature colonies suggested budding as another means of replication. Acellular spaces inside the colonies appeared to have been formed by lysis of large bodies or by the envelopment of space by the extending growth of minute granules. Whereas no significant strain variation was noted in colonies of less than 24 hr of incubation, fully mature colonies were differentiated on uniform media.     

Identification of carbapenems resistant genes on biofilm forming K. pneumoniae from urinary tract infection

The multi-drug resistant effect of the Gram negative bacteria K. pneumoniae was identified by disc diffusion method using specific UTI panel discs of Kleb 1 HX077 and Kleb 2 HX090 HEXA. Among the multi-drug resistant bacteria, the carbapenem resistant (CR) effect of the K. pneumoniae was screened by specific carbapenem detection antibiotics of HEXA HX066 and HX0103 HEXA by disc diffusion method. In addition, the effective antibiotics were further performed against K. pneumoniae by minimum inhibition concentration method. Further, the carbapenemase genes of VIM 1 and IMP 1 were detected from the isolated strains by multiplex PCR method. Furthermore, the biofilm forming ability of selected carbapenem resistant K. pneumoniae was initially identified by tissue culture plate method and confirmed by exopolysaccharide arrest ability of congo red agar assay. Finally, our result was proved that the identified K. pneumoniae is carbapenemase producing strain, and its virulence was extended with strong biofilm formation.     

Development of a differential medium for bile salt hydrolase-active Lactobacillus spp.

An agar plate assay was developed to detect bile salt hydrolase activity in lactobacilli. On Lactobacillus-selective MRS or Rogosa SL medium supplemented with taurodeoxycholic, taurocholic, or taurochenodeoxycholic acids, bile salt hydrolysis was manifested at two intensities: (i) the formation of precipitate halos around colonies or (ii) the formation of opaque granular white colonies. Sixty-six lactobacilli were tested for bile salt hydrolase activity by both the plate assay and a sensitive radiochemical assay. No false-positive or false-negative results were detected by the plate assay. Based on results of experiments with Eubacterium lentum and Bacteroides species, the plate assay was dependent on two factors: (i) the presence of bile salt hydrolytic activity and (ii) the ability of the organism to sufficiently acidify the medium to protonate free bile acids. The availability of a differential medium for determination of bile salt hydrolase activity will provide a rapid method for determining shifts in a specific functional activity of intestinal Lactobacillus species and provide a rapid screening capability for identifying bile salt hydrolase-deficient mutants. The latter application should allow bile salt hydrolase activity to be used as a marker enzyme in genetic experiments.     

Development of a differential medium for bile salt hydrolase-active Lactobacillus spp

An agar plate assay was developed to detect bile salt hydrolase activity in lactobacilli. On Lactobacillus-selective MRS or Rogosa SL medium supplemented with taurodeoxycholic, taurocholic, or taurochenodeoxycholic acids, bile salt hydrolysis was manifested at two intensities: (i) the formation of precipitate halos around colonies or (ii) the formation of opaque granular white colonies. Sixty-six lactobacilli were tested for bile salt hydrolase activity by both the plate assay and a sensitive radiochemical assay. No false-positive or false-negative results were detected by the plate assay. Based on results of experiments with Eubacterium lentum and Bacteroides species, the plate assay was dependent on two factors: (i) the presence of bile salt hydrolytic activity and (ii) the ability of the organism to sufficiently acidify the medium to protonate free bile acids. The availability of a differential medium for determination of bile salt hydrolase activity will provide a rapid method for determining shifts in a specific functional activity of intestinal Lactobacillus species and provide a rapid screening capability for identifying bile salt hydrolase-deficient mutants. The latter application should allow bile salt hydrolase activity to be used as a marker enzyme in genetic experiments.     

Plate ethanol-screening assay for selection of the Pichia stipitis and Hansenula polymorpha yeast mutants with altered capability for xylose alcoholic fermentation

A new method for the selection of Pichia stipitis and Hansenula polymorpha yeast mutants with altered capability to ferment xylose to ethanol was developed. The method is based on the ability of P. stipitis and H. polymorpha colonies to grow and produce ethanol on agar plates with xylose as the sole carbon and energy source. Secreted ethanol, in contrast to xylose, supports growth of cells of the indicator xylose-negative strains (the wild-type strain of Saccharomyces cerevisiae or Δxyl1 mutant of H. polymorpha) mixed with agar medium. The size of the tester culture-growth zone around xylose-grown colonies appeared to be dependent on the amount of secreted ethanol. Mutants with altered (decreased or elevated) ethanol production in xylose medium have been isolated using this method. The mutants exhibited pleiotropic alterations in enzymatic activities of the intermediary xylose metabolism.     

Isolation and Screening of Alkaline Lipase-producing Fungi from Brazilian Savanna Soil

Summary Fifty-nine lipase-producing fungal strains were isolated from Brazilian savanna soil by employing enrichment culture tecniques. An agar plate medium containing bile salts and olive oil emulsion was employed for isolating and growing fungi in primary screening assay. Twenty-one strains were selected by the ratio of the lipolytic halo radius and the colonies radius. Eleven strains were considered good producers under conditions of submerged liquid fermentation (shaken cultures) and solid-state fermentation. The most productive strain, identified as Colletotrichum gloesporioides, produced 27,700 U/l of lipase under optimized conditions and the crude lipase preparation was capable of hydrolysing a broad range of substrates including lard, natural oils and tributyrin.     

Isolation of bacteria and fungi from human sputum and faeces using carbon monoxide as source of energy and carbon

Seventy-five sputa and 10 faeces of human origin were examined for organisms capable of growing in carbon monoxide (CO). Specimens were plated onto a buffered agar medium containing trace vitamins, and incubated (2–4 weeks) in 60% v/v CO as the sole source of carbon and energy. All specimens yielded growth except for three sputa; many individual colonies were shown to comprise at least two different organisms. While no recognized carboxydotrophs were found, presumptive identification revealed that streptococci, staphyloccoci, coliform bacilli, yeasts and moulds were all represented. Using a specific enzyme test, evidence for CO dehydrogenase activity was found in a significant proportion of mixed and purified cultures. These studies indicate that CO-tolerant organisms which may be using CO as a source of carbon and energy are associated with man.     

The Processing of Mammalian Haemopoietic Cells in Thin Layer Agar Cultures for Electron Microscopy

Human and mouse haemopoietic cells cultured by the thin layer agar technique have been studied with the electron microscope. To process colonies of haemopoietic cells or individual cells which appeared in these colonies, a special technique had to be developed. The technique presented covers methods of selection, isolation, and sectioning that were devised for this purpose.

Haemopoietic cells are cultured in small plastic Petri dishes containing a culture system with 0.25% agar. Cell colonies and individual cells intended for light as well as for electron microscopic study are examined and selected microscopically with the aid of a numbered grid which is placed under the closed Petri dish.

Cells in the agar gel are fixed with glutaraldehyde which is pipetted directly onto the cultures. In order to facilitate their removal from the medium, the consistency of the agar solution is increased by evaporating liquid with controlled mild warming.

Pieces of agar containing colonies or single cells are cut out with a fine trephine and postfixed in osmium tetroxide. Agar pieces are embedded cell side up in a thin layer of Epon. After polymerization, the Epon-embedded pieces of agar are appropriately oriented at the head of flat embedding molds filled with fresh Epon. After another polymerization procedure, the top of the Epon blocks containing the cells are trimmed to a smooth surface with a glass knife.

The exact distance between the smooth surface of the blocks and the cells is measured by use of the vertical micrometer of a standard light microscope. The Epon layer around the specimen is trimmed away to expose selected cells for subsequent semi-thick and ultrathin sectioning. Sections are stained and examined microscopically.

With minor modifications the technique described also enables the processing of extremely small quantities of biological materials derived from other experiments for both light and electron microscopic observation.     

An improved method for screening alpha-acetolactate producing mutants.

Alpha-Acetolactate-deficient Lactococcus lactis ssp. lactis biovar. diacelylactis are utilised in several industrial processes for producing diacetyl and alpha-acetolactate. They can be selected by screening after random mutagenesis. We improved a previously described screening method [Monnet, C., Schmitt, P., Diviès, C., 1997. Appl. Environ. Microbiol. 63, 793-795], which makes it possible to screen up to 1000 colonies per agar plate, whereas the previous method allowed to screen only 60 colonies per agar plate. The new screening method facilitates selection of alpha-acetolactate-deficient mutants.