A chromogenic plating medium for the isolation and identification of Enterobacter sakazakii from foods, food ingredients, and environmental sources

A chromogenic agar, R&F Enterobacter sakazakii chromogenic plating medium (ESPM), was developed for isolating presumptive colonies of E. sakazakii from foods and environmental sources. ESPM contains two chromogenic substrates (5-bromo-4-chloro-3-indoxyl-alpha-D-glucopyranoside and 5-bromo-4-chloro-3-indoxyl-beta-D-cellobioside), three sugars (sorbitol, D-arabitol, and adonitol), a pH indicator, and inhibitors (bile salts, vancomycin, and cefsulodin), which all contribute to its selectivity and differential properties. On ESPM, 79 pure culture strains of E. sakazakii (10 clinical isolates and others from food and environmental sources) yielded blue-black (three strains were blue-gray) raised colonies, 1 to 2 mm in diameter with and without halos after 24 h at 35 degrees C. Other enteric organisms plus Pseudomonas aeruginosa yielded white, yellow, green, or clear colonies with and without clear halos. Of these genera, only Shigella sonnei and one Pantoea strain produced blue-black to blue-gray colonies. ESPM was used to isolate E. sakazakii from a variety of foods: corn, wheat, and rice flours; powdered infant formula; dairy products (dried milk, whey, and caseinates); cereals; and environmental sources. Most false-positive results on ESPM were eliminated by observing acid production on either sucrose or melibiose after 6 h at 35 degrees C on a R&F E. sakazakii screening medium (ESSM) biplate. In an analysis of 240 samples, the number of samples positive for E. sakazakii by the ESPM-ESSM method and the U.S. Food and Drug Administration protocols (violet red bile glucose agar and tryptic soy agar) were 27 and 16, respectively, with sensitivity and specificity values of 100.0 and 96.9% versus 59.3 and 43.7%, respectively. These data support the fact that E. sakazakii confirmation should be based on more than one confirmation system. Both the API 20E and Biolog Microlog3 4.20 systems should be used for confirmation of E. sakazakii isolates.     

A selective differential medium for Enterobacter sakazakii, a preliminary study

Enterobacter sakazakii can cause fatal invasive infection of neonates associated with the presence of this organism in powdered infant milk formula. A new chromogenic medium (Druggan-Forsythe-Iversen agar, DFI) is described for the selective detection of this emergent pathogen. The medium is based on the alpha-glucosidase reaction which is detected using 5-bromo-4-chloro-3-indolyl-alpha,D-glucopyranoside (XalphaGlc). Ent. sakazakii hydrolyses this substrate to an indigo pigment, producing blue-green colonies on this medium. DFI was compared with the current method of detection on violet red bile glucose agar (VRBGA) followed by pigment production on tryptone soy agar (TSA) after 48-72 h at 25 degrees C and subsequent biochemical profile determination using Biomerieux API20E. Ninety-five clinical and food strains of Ent. sakazakii were detected on the DFI chromogenic medium 2 days sooner than the alternative method. The characteristics of 148 strains representing 17 genera of non-Ent. sakazakii Enterobacteriaceae were compared using the two methods. Only 16/18 Escherichia vulneris strains, 2/3 strains of Pantoea spp. and 1/8 Citrobacter koseri strains gave false positive results on DFI agar. Eight alpha-glucosidase positive strains were identified as Pantoea using their API20E biochemical profile, but had higher percentage identification as Ent. sakazakii using ID32E. Therefore the DFI medium enables the detection of Ent. sakazakii within mixed cultures of Enterobacteriaceae, whereas the organism could be missed when using VRBGA since the latter is a general Enterobacteriaceae selective medium. In addition, the common use of API20E to check yellow pigmented colonies on TSA may lead to false negative results and consequently the acceptance of a batch of infant formula milk (IFM) that contains Ent. sakazakii.     

利用选择性培养基快速初步鉴定食品中的致病微生物

目的 建立快速区分鉴别某种(类)微生物的方法。方法 选择几种常用的培养基, 将不同的菌株接种在培养基上, 通过培养基成分对不同微生物的选择性、显色反应及不同菌株在选择性培养基上具有的特征性菌落形态, 初步并快速判定微生物, 尤其是致病类微生物。结果 大肠埃希氏菌会与其他致病菌混淆, 可以通过结晶紫中性红胆盐(crystal violet neutral red bile salt, VRBA)琼脂将其区分; 志贺氏菌在培养基上菌落基本均为半透明, 体现的均为培养基本身的颜色; 亚硫酸铋(sulfurous acid bismuth, BS)琼脂的选择性较强, 但鼠伤寒沙门氏菌在该培养基上长势良好。结论 该方法可有效提高鉴别致病菌的效率。     

Overlay technique for direct detection and identification of haemolytic Listeria on selective plating medium. Comparison of five media

An overlay technique is proposed for the identification and counting of haemolytic Listeria colonies directly on selective plating media. The technique was applied to different Listeria-selective plating media. In pure culture studies with collection strains, the overlay technique was more efficient and reliable for detection haemolytic Listeria species compared with the incorporation of blood into the agar. The efficacy of the overlay technique for the direct detection of haemolytic colonies of Listeria from raw milk samples was related to agar selectivity. The best results were obtained with Listeria-selective agar medium modified (LSAMM). Catalase assay, together with reactions for aesculin and tellurite, were useful and reliable criteria for the identification of Listeria. All colonies on LSAMM which were positive for catalase, tellurite and aesculin while those displaying typical haemolysis corresponded in most cases to L. monocytogenes.     

Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp

A selective and differential medium (PALCAM agar) was elaborated for the isolation and enumeration of Listeria monocytogenes. PALCAM is based on Columbia agar with 0.05% glucose made selective by the addition of 0.001% polymyxin B, 0.0005% acriflavin, 1.5% lithium chloride and 0.002% ceftazidime. The diagnostic traits were attained by the incorporation of (i) 0.08% aesculin and 0.05% ferric salt; and (ii) 1% mannitol plus 0.008% phenol red. PALCAM recovered test strains of L. monocytogenes and other Listeria spp quantitatively and suppressed most other bacteria of common occurrence in fresh food. L. monocytogenes colonies were approximately 2 mm grey-green with a black sunken centre and a black halo on a cherry-red background. The occasional Enterococcus or Staphylococcus strains developing on the medium gave rise to grey colonies with a brown-green halo or yellow colonies with a yellow halo. PALCAM was the preferred medium out of 13 tested Listeria selective agars in current use. A similar differential enrichment broth, L-PALCAMY was developed based on peptone yeast extract broth with 2.5% egg yolk emulsion. The diagnostic traits and inhibitors used in this medium were the same as in PALCAM agar, through in different concentrations. Growth rate and cellcrop of L. monocytogenes in L-PALCAMY were of the same order as in Columbia broth. The growth of the majority of other bacteria of common occurrence in fresh foods was inhibited. The medium recovered L. monocytogenes more effectively from severely contaminated food than other current enrichment media.     

A comparative study between overlay method and selective-differential media for recovery of stressed Enterobacter sakazakii cells from infant formula

This study compares the performance of different selective-differential media with the overlay method for recovery of stressed cells of Enterobacter sakazakii from infant formula milk (IFM). Five different selective-differential media were used in this study: OK medium, violet red bile agar (VRBA), Druggan-Forsythe-Iversen agar (DFI), Enterobacteriaceae enrichment (EE) agar, and fecal coliform agar (FCA). Tryptic soy agar supplemented with 0.1% sodium pyruvate (TSAP) was used as a control. The overlay method involved applying a thin layer (8ml) of each of the selective media onto TSAP after spreading a sample onto TSAP. Reconstituted IFM was inoculated by ca 1x10(7)CFU/ml of a mixture of four strains of E. sakazakii and subjected to different stress conditions: heat (55 degrees C for 10min), a freeze-thaw cycle (-20 degrees C for 24h, thawed at room temperature, frozen again at -20 degrees C, and thawed), acidic pH (pH 3.56 for 15min), alkaline pH (pH 11.04 for 15min), and desiccation (E. sakazakii was inoculated onto powdered IFM at a level of ca 1x10(6)CFU/g, held at 21 degrees C, water activity of the inoculated product was 0.29 and examined at 0, 15, and 30d). No major differences were noticed between the control (TSAP) and the overlay methods. However, the overlay method recovered significantly higher numbers of stressed E. sakazakii cells compared to selective-differential media. Also, the selective-differential media exhibited some variability in terms of their capabilities to recover stressed cells of E. sakazakii. Among all the examined selective-differential media, DFI performed better for recovering stressed E. sakazakii cells. This study suggests that the overlay method may serve as a potential alternative to direct selective plating for best recovery of E. sakazakii from IFM.     

Assessment of different selective agar media for enumeration and isolation of Listeria from dairy products

Different selective agar media were compared for the recovery and isolation of five species of Listeria from raw milk and cheese. The selective media examined were Beerens medium, MacBride medium and that described by Dominguez et al. (1984) with 6 mg/l acriflavine, listeria selective agar medium (LSAM), and LSAM with 12 mg/l acriflavine (LSAM X 2A); a non-selective yeast glucose Lemco agar was included for comparison. When the difference between listeria and the natural microflora of raw milk and cheese was 10(2) cfu/ml, listeria could be isolated by direct plating on all media tested. When it was lower than 10(3)-10(4) cfu/ml, listeria were isolated by direct plating only on LSAM and LSAM X 2A. When the difference was greater than 10(4) cfu/ml, a previous enrichment was necessary to isolate them. LSAM and LSAM X 2A media performed better than the other media tested for isolating listeria by direct plating and improved their isolation from dairy products. This superior performance was evaluated by the ability of these media to support colony formation of different species of Listeria tested, the easy recognition of these colonies from those formed by other microorganisms and by their capacity to inhibit the natural microflora of these foods.     

Rapid and specific enzyme immunoassay on hydrophobic grid membrane filter for detection and enumeration of thermophilic Campylobacter spp. from milk and chicken rinses

Six commercially available anti-Campylobacter antibodies were examined for their applicability in an enzyme immunoassay on hydrophobic grid membrane filters, both for the detection and enumeration of Campylobacter spp. When a panel of nine Campylobacter (seven Campylobacter jejuni and two Campylobacter coli) and eight non-Campylobacter strains were used in a dot-blot format enzyme immunoassay to test the specificity of these antibodies, only one polyclonal antibody (Biodesign) detected all Campylobacter strains. Escherichia coli O157:H7 produced weak nonspecific signals due to endogenous peroxidase activity. The specificity of this Biodesign antibody was further tested against 30 more Campylobacter strains and more than 600 non-Campylobacter strains on hydrophobic grid membrane filters grown on modified Campylobacter agar with charcoal and deoxycholate, a Campylobacter selective medium. All the Campylobacter strains were detected, whereas only two (Acinetobacter calcoaceticus, Salmonella Minnesota) of the approximately 130 non-Campylobacter strains, which grew on modified Campylobacter agar with charcoal and deoxycholate, gave false-positive signals. This simple, rapid, and specific enzyme immunoassay also detected Campylobacter spp. from inoculated milk and chicken rinses and naturally contaminated chicken rinses.     

Psychrotrophic, proteolytic and lipolytic properties of Enterobacteriaceae isolated from milk and dairy products

Most of the Enterobacteriaceae strains (73 out of 75) isolated in a previous study (Wessels et al., 1988) were psychrotrophic on agar plates, with the exception of Enterobacter cloacae strains. The Enterobacteriaceae strains were largely non-proteolytic on milk agar medium although limited numbers of E. cloacae, Serratia rubidaea and Klebsiella oxytoca strains were capable of proteolytic activity at 25 degrees C. The E. cloacae and K. oxytoca strains positive at 25 degrees C were also proteolytic at 7 degrees C. Most of the species tested were non-lipolytic on Victoria blue butterfat agar. The majority of Serratia marcescens and Klebsiella pneumoniae strains and a minority of E. cloacae and K. oxytoca strains, however, were lipolytic on this medium.     

Evaluation of various selective media for the detection of Pseudomonas species in pasteurized milk

Pseudomonas spp. are common gram-negative, post-pasteurization contaminants that contribute to spoilage of pasteurized dairy products. This study evaluated 5 common selective media for detecting Pseudomonas spp. in pasteurized milk. The performance of each selective medium for recovering 12 different Pseudomonas isolates (selected to represent a diversity of pasteurized milk isolates) was compared with that of standard plate count agar pour plates. Pseudomonas isolates showed varying abilities to produce colonies on different selective media. For 2 of 12 isolates, a 48-h incubation time was required for colony formation on any of the media tested. Violet red bile agar and coliform Petrifilm (3M, St. Paul, MN) were less effective than standard plate count agar pour plates at recovering Pseudomonas, regardless of incubation time, and MacConkey agar showed poor detection efficiency compared with SPCP after a 48-h incubation (R(2) = 0.26). Therefore, the use of violet red bile agar, MacConkey agar, or coliform Petrifilm may not be sufficient for detecting common Pseudomonas spp. in milk. The methods showing the highest detection efficiencies were crystal violet tetrazolium agar (CVTA) pour plates (R(2) = 0.95) and CVTA plates inoculated by spiral plating (R(2) = 0.89) incubated at 32 °C for 48 h. Overall, plating milk samples on CVTA followed by a 48-h incubation at 32 °C was the most effective selective method for recovering a diversity of Pseudomonas spp. from milk.     

Isolation of Enterobacter sakazakii and other Enterobacteriaceae from powdered infant formula milk and related products

The presence of Enterobacter sakazakii and other Enterobacteriaceae was surveyed in 82 powdered infant formula milk (IFM) and 404 other food products. The presence of Ent. sakazakii was detected using the conventional method (growth on violet red bile glucose agar plus yellow pigment production on TSA) and a new chromogenic medium (Druggan–Forsythe–Iversen agar, DFI) which enables results to be obtained 2 days earlier than the conventional method. Ent. sakazakii was isolated from 2/82 powdered IFM, 5/49 dried infant foods, 3/72 milk powder, 2/62 cheese products and various dry food ingredients, especially herbs and spices (40/122). Ent. sakazakii was isolated from 67 samples using the DFI medium, however only 19 of the samples were positive following the conventional method. The largest difference in isolation between the two methods was with dry food ingredients.Although Enterobacteriaceae were enumerated from one powdered IFM sample (Klebsiella ozaenae, 200 cfu/g), 7/82 had detectable Enterobacteriaceae after enrichment in EE broth. Using the ISO 6579 2002 method and immuno-magnetic separation technique no Salmonella serovars were isolated from powdered IFM, dried infant foods or milk samples. Therefore hygienic production of powdered IFM and milk production as monitored by control of Salmonella and enumeration of Enterobacteriaceae did not control Ent. sakazakii.     

食品中沙门氏菌分离、鉴别方法的比较研究

目的筛选用于分离、鉴别食物样本沙门氏菌的适宜方法。方法比较了3种沙门氏菌显色培养基与亚硫酸铋(BS)琼脂、HE琼脂和木糖赖氨酸脱氧胆盐(XLD)琼脂传统培养基的检测敏感性、特异性、准确性和食品样本的适用性。结果 3种沙门氏菌显色培养基对杂菌的抑制效果不同,但检测结果直观,检测灵敏度高;BS琼脂方法分离沙门氏菌特异性强;HE琼脂及XLD琼脂分离沙门氏菌,当样品污染菌数量多时,鉴别结果易受影响。结论建议使用传统培养基分离、鉴别沙门氏菌时,补充使用一种抑制杂菌效果好的沙门氏菌显色培养基,以提高沙门氏菌的检测效率。     

Evaluation of a rapid fluorogenic method for the detection of Escherichia coli in dairy products.

A rapid fluorogenic medium was evaluated for the detection of Escherichia coli in dairy products. The medium was capable of detecting Esch. coli after 7.5 h incubation at 41.5 degrees C. Samples of pasteurized milk (136), raw milk (63), soft cheese (60) and pasteurized cream (39) were examined with media based on 4-methylumbelliferyl-beta-D-glucuronide (MUG-7) and Violet red bile agar and there were no significant differences between the numbers of Esch. coli detected on the two media. MUG-7 medium had a specificity of 98.6% and the small number of organisms giving a false positive reaction were identified as Klebsiella pneumoniae. The incidence of false negative results was approximately 2%. MUG-7 medium was suitable for pour plate, spread plate and membrane filtration methods. Possible applications of the method are discussed.     

椰毒假单胞菌酵米面亚种选择性分离培养基的比较研究

目的比较4种椰毒假单胞酵米面亚种选择性分离培养基(马铃薯葡萄糖琼脂,PDA;改良马铃薯葡萄糖琼脂,mPDA;椰毒假单胞菌酵米面亚种分离琼脂,PCFA;银耳卵黄氯霉素琼脂,TYCA)的分离效果,为修订GB/T4789.29—2003《食品卫生微生物学检验椰毒假单胞菌酵米面亚种检验》提供技术支持。方法接种椰毒假单胞菌酵米面亚种于4种选择性分离培养基,观察各培养基上目标菌菌落形态,计算生长率;接种10种常见致病菌和环境中常见菌于4种选择性分离培养基,进行生长特异性试验;通过直接涂布和增菌划线的加标试验,验证这4种选择性分离培养基对粪便、食品和环境土壤等不同标本/样品中椰毒假单胞菌酵米面亚种的分离检出情况。结果 mPDA和PCFA能够分别抑制8种和6种致病菌的生长,且椰毒假单胞菌酵米面亚种的生长率都高于75%;在mPDA和PCFA上,目标菌与多数杂菌形态有明显区别,而在PDA和TYCA上,形态相近不易区分;在粪便、土壤和食品等不同标本/样品的直接涂布和增菌划线分离的加标试验中,mPDA和PCFA上的检出率均超过80%,明显高于PDA和TYCA。结论 mPDA和PCFA分离效果比原标准的PDA明显提高,建议在标准修订中增加此两种选择性分离培养基。     

Evaluation of five selective media for isolation of catalase-negative gram-positive cocci from bulk tank milk

Five selective media including Edwards modified medium, Edwards modified medium supplemented with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L), Streptococcus selective medium, Streptosel agar, and thallium-crystal violet-toxin-ferric citrate medium were evaluated for the isolation of streptococci and streptococci-like organisms from raw milk. The sensitivity and specificity of these selective media for streptococci and streptococci-like organisms were determined by using American Type Culture Collection reference strains. Under experimental conditions Edwards modified medium with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L) showed the highest sensitivity (100%) and specificity (100%) for streptococci and streptococci-like organisms followed by thallium-crystal violettoxin-ferric citrate medium, Edwards modified medium, Streptococcus selective medium, and Streptosel agar. Edwards modified medium supplemented with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L) allowed growth of all streptococci and streptococci-like organisms, while inhibiting growth of the staphylococci and gram-negative reference strains. Bulk tank milk samples from 114 dairy herds were spiral plated onto Edwards modified medium with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L). A total of 344 isolates (at least three isolates from each sample) were randomly selected and identified to their species. This medium permitted growth of 328 streptococci and streptococci-like organisms belonging to genera Aerococcus, Enterococcus, Gemella, Lactococcus, Streptococcus, and Vagococcus. When Edwards modified medium supplemented with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L) was evaluated using bulk tank milk samples, the sensitivity and specificity of this medium for streptococci and streptococci-like organisms were observed to be 100 and 87.5%, respectively. The positive predictive value for streptococci and streptococci-like organisms was observed to be 99.4%. The results of the study indicate that Edwards modified medium supplemented with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L) can be used as a selective medium for the isolation of streptococci and streptococci-like organisms from bulk tank milk.     

Evaluation of enrichment procedures for recovering Listeria monocytogenes from dairy products

Six different enrichment media and five selective plating media were compared for their suitability for the recovery of Listeria monocytogenes from dairy products. These included media used to test milk products by the U.S. Food and Drug Administration (FDA) and the U.S. Centers for Disease Control (CDC), and media developed by the U.S. Department of Agriculture (USDA) for testing meat and poultry products. Test samples included naturally contaminated goat's milk, cultured milk products and ice cream manufactured with L. monocytogenes, and unpasteurized milk inoculated with heat- and freeze-injured cells of L. monocytogenes. Generally, the media and two-stage enrichment protocol developed by the USDA, with plating of samples after two consecutive 24-h incubation periods, yielded better recoveries than all other enrichment media incubated for 24 h. A modified USDA procedure, incorporating nonselective pre-enrichment of samples by omitting acriflavine and nalidixic acid from the primary USDA enrichment broth, and transfer of a larger volume of the initial culture broth to the secondary enrichment media, significantly increased recoveries of low numbers of sublethally stressed L. monocytogenes. Prolonged incubation of samples in the FDA enrichment broth, for 7 days, did not consistently improve recoveries over the initial 24-h incubation time of the medium. The selective plating medium developed by the USDA, lithium chloride-phenylethanol-moxalactam agar, was the most effective plating agar for isolation of L. monocytogenes following enrichment of samples in any broth culture, and increased recoveries of L. monocytogenes by 19-40% compared with other selective agar media tested.     

Biodiversity of antifungal lactic acid bacteria isolated from raw milk samples from cow, ewe and goat over one-year period

Antifungal lactic acid bacteria (ALAB) biodiversity was evaluated in raw milk from ewe, cow and goat over one year period. Lactic acid bacteria were enumerated using 8 semi-selective media, and systematically screened for their antifungal activity against 4 spoilage fungi commonly encountered in dairy products. Depending on the selective medium, between 0.05% (Elliker agar) and 5.5% (LAMVAB agar) screened colonies showed an antifungal activity. The great majority of these active colonies originated from cow (49%) and goat (43%) milks, whereas only 8% were isolated from ewe milk. Penicillium expansum was the most frequently inhibited fungus with 48.5% of colonies active against P. expansum among the 1235 isolated, followed by Mucor plumbeus with 30.6% of active colonies, Kluyveromyces lactis with only 12.1% of active colonies and Pichia anomala with 8.7% of active colonies. In the tested conditions, 94% of the sequenced active colonies belonged to Lactobacillus. Among them, targeted fungal species differed according to the Lactobacillus group, whose presence largely depended on year period and milk origin. The Lb. casei and Lb. reuteri groups, predominantly recovered in summer/fall, were overrepresented in the population targeting M. plumbeus, whereas isolates from the Lb. plantarum group, predominantly recovered in spring, were overrepresented in the population targeting K. lactis, the ones belonging to the Lb. buchneri group, predominantly recovered in spring, were overrepresented in the population targeting P. anomala. Raw milk, especially cow and goat milks from the summer/fall period appeared to be a productive reservoir for antifungal lactobacilli.     

中式熟食咸鸡主要细菌的分离与初步鉴定

采用嗜冷菌选择性培养基和含盐选择性培养基对咸鸡微生物菌群进行分离筛选。根据细菌的菌落形态、革兰氏染色反应等常见特征,由嗜冷菌选择性培养基分离出6 株菌C1～C6,含盐选择性培养基分离出4 株NaCl 胁迫条件下细菌S1～S4。提取单菌落DNA,对其16S rDNA 序列进行PCR 扩增后测序,结合形态、常规生理生化特性初步鉴定为：松鼠葡萄球菌(Staphylococcus sciuri)C3,土生克雷伯氏菌(Klebsiella terrigena)C6,不动杆菌属(Acinetobacter sp.)C1、C2、C4、C5;日勾维肠杆菌(Enterobacter gergoviae)S1,表皮葡萄球菌(Staphylococcusepidermidis)S2、S3,假蕈状芽孢杆菌(Bacillus pseudomycoides)S4。     

Thin agar layer method for recovery of heat-injured Listeria monocytogenes

A thin agar layer (TAL) method was developed to recover heat-injured Listeria monocytogenes. Modified Oxford medium (MOX), a selective plating medium, inhibits heat-injured L. monocytogenes from growing, whereas tryptic soy agar (TSA), a nonselective medium, does not. In order to facilitate recovery of heat-injured L. monocytogenes cells while providing selectivity of isolation of L. monocytogenes from other bacteria in the sample, a unique TAL procedure was developed by overlaying 5 ml of nonselective medium (TSA) onto prepoured and solidified MOX medium in an 8.5-cm-diameter petri dish. The injured L. monocytogenes repaired and started to grow in the TSA during the first few hours after incubation of the plate. During the resuscitation of injured cells, the selective agents from MOX diffused to the TSA top layer to inhibit other microorganisms. L. monocytogenes showed a typical reaction (black colonies) on TAL after 24 h of incubation at 37 degrees C. The recovery rate for heat-injured L. monocytogenes with the TAL method was compared with those rates associated with TSA, MOX, and the traditional overlay method (OV; pouring selective agar on top of resuscitated cells on TSA agar after 3 h incubation). Milk and 0.1% peptone water that were inoculated with L. monocytogenes (4 to 5 log CFU/ml) were heated for 15 min at 55 degrees C. L. monocytogenes was enumerated on TSA, MOX, OV, and TAL media and procedures. No significant difference occurred among TSA, OV, and TAL (P > 0.05) in terms of enumeration of heat-injured L. monocytogenes, but these media recovered significantly higher numbers than did MOX agar (P < 0.05)-in both samples. The TAL method involves only one step, whereas OV is a more cumbersome two-step procedure.     

Lactococcus Genus: A selective and Differential Agar Medium

An agar medium (Alsan), was selective for Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. lactis biovar. diacetylactis. The former developed white or yellow colonies; the latter gave blue-green or blue colonies. Eleven strains of L. lactis subsp. cremoris did not grow in this medium. The medium contained phenylethanol agar, lactose, glycine anhydride, lithium chloride and trimethoprim. In comparison with M17 medium, Alsan was not significantly different (P > 0.05) in recovering either bacterium. Furthermore, the medium inhibited the common food genera Pseudomonas, Escherichia, Leuconostoc, Enterococcus and Lactobacillus. Bacteriophage plaque sizes of 0.4–0.5 cm were demonstrated on the medium using a lactococcal bacteriophage.