Môi trường nuôi cấy vi sinh Alphachem

Description:
Completed with all its supplements the Agar Listeria Ottaviani & Agosti is a selective and differential medium for the detection of Listeria species and the presumptive identification of Listeria monocytogenes.
The selectivity is achieved by the high concentration of lithium chloride and the mixture of antimicrobics. The differential activity is due to the chromogenic substrate to detect the βglucosidase enzyme that is present in all Listeria species.
The specific identification is obtained by the L-α-phosphatidylinositol, that acts as substratre for a phospholipase C present only in Listeria monocytogenes and some strains of Listeria ivanovii.
The combination of both substrates allows the differentiation L. monocytogenes, which grow in produces colonies blue-green in colour and surrounded by an opaque zone, from the other Listeria species, which blue-green colonies but without any halo. This differentiation is evident after incubating the plates for 24 ± 2 hours at 37 ºC.
Sometimes, especially with highly contaminated samples, it is possible that some colonies, white in colour, are not Listeria growth. In this case an enrichment step is recommended prior to plate inoculation.

Observations:

Most Listeria ivanovii also produce an opaque halo around the colonies after 48 h of incubation. This presumptive evidence must be confirmed by performing the biochemical or serological identification tests (Rhamnose / Xylose sugar fermentation, hemolysis tests, CAMP test, etc.) or any test confirming the species without hesitation.

Technique:
Add 1 botlle supplement Ottaviani & Agosti (L-alpha-phosphatidylinositol) and 1 vial supplemet Ottaviani & Agosti for complete 500 ml medium.
Homogenize by mixing and distribute in Petri dishes. The solidified cool medium appears homogeneously turbid.
There are many standardised methodologies (ISO, FDA-BAM, AOAC, AFNOR, etc.). The technician must follow the protocol validated in his laboratory.

It is used for confirmation after using Listeria Enrichment Broth Base Fraser . This medium is also recommended by ISO 11290-1 for the detection and enumeration for Listeria monocytogenes. Enzymatic digest of animal tissues and enzymatic digest of casein provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is the source of vitamins, particularly of the B-group. Sodium chloride supplies essential electrolytes for transport and osmotic balance. Sodium pyruvate is a source of energy for bacterial metabolism and aids in the resuscitation of stressed organisms. Glucose is the fermentable carbohydrate providing carbon and energy. Magnesium glycerophosphate is a buffering compound. Magnesium sulphate is a magnesium ion required for a large variety of enzymatic reactions, including DNA replication. The differential activity of the medium is due to two factors. Lithium chloride in the base medium and supplementary antimicrobial compounds Ceftazidime, Polymyxin, Nalidixic acid and Cycloheximide provide the medium’s selectivity. Bacteriological agar is the solidifying agent. The presence of the chromogenic component X-glucoside, a substrate for the detection of the enzyme ß-glucosidase, is common to all Listeria species giving the colonies their blue colour. Other organisms that possess this enzyme, for example, Enterococci, are inhibited by the selective agents within the medium and by the selective supplement. The differential activity is also obtained by lipase C substrate, upon which the specific enzyme for L. monocytogenes acts. The lipase is responsible for the opaque white halo which surrounds L. monocytogenes. The combination of both substrates allows us to differentiate the colonies of Listeria monocytogenes from the rest of Listeria spp. since, although all are blue in colour, L. monocytogenes present an opaque white halo surrounding them. It has been observed that some strains of Listeria ivanovii, mostly pathogenic to animals although some have caused infections in humans, also possess lipase activity.

Principles and uses Listeria Half-Fraser Broth Base is a modification of Listeria Fraser Broth Base in which the nalidixic acid and acriflavine concentrations have been reduced to 10 mg/L and 12.5 mg/L respectively. The antibiotics are already included in the formula so it is only necessary to add the Ferric Ammonium Citrate Supplement.

Listeria spp. may be present in small numbers and are often accompanied by considerably larger numbers of other microorganisms, therefore selective enrichment is necessary.

Listeria Half-Fraser broth is used in this selective enrichment and enumeration of Listeria monocytogenes and other Listeria species in all food types, including milk and dairy products, and environmental samples. This formula adheres to ISO 11290. Enzymatic digest of casein, enzymatic digest of animal tissues and meat extract provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is the source of vitamins, particularly of the B-group. Potassium phosphates act as a buffer system. All Listeria species hydrolyze esculin, which reacts with ferric ions producing a blackening of the medium. The addition of ferric ammonium citrate improves the growth of Listeria monocytogenes. Lithium chloride inhibits the growth of enterococci that can hydrolyze the esculin.

Description: Listeria PALCAM selective supplement is added to PALCAM Medium base in order to obtain a complete selective medium used for the detection and the isolation of Listeria monocytogenes from foods. Palcam Agar is based on the formulation described initially by van Netten et al. which has a high selectivity and produces good colonial differentiation. Selectivity is achieved by the inclusion of lithium chloride, acriflavine, polymyxin B and ceftazidime, since they inhibit the growth of almost all the Gram negative and most of the Gram positive companion bacteria. Listeria hydrolyze esculin to esculetin, which reacts with ferric ammonium citrate producing a dark precipitate and green-grey colonies with beige halos. If colonies of enterococci or staphylococci do grow on this medium they can be easily recognized, since they utilise mannitol and produce yellow colonies and haloes, contrasting with the cherry-red colour of medium. However, when there are many Listeria colonies, the entire medium darkens, which can cause interference in differentiation. In these cases it is advisable to perform the inoculation with a more diluted sample.

Technique: Collect, dilute and prepare samples and volumes as required according to specifications, directives, official standard regulations and/or expected results. Reconstitute the vial with the 6 ml sterile distilled water in aseptic conditions and add it to 500 ml of sterilized PALCAM agar base cooled to 50ºC. Do not overheat once suplemented. Pour the complete medium into Petri dishes and, once solidified on a flat surface, spread the plates by streaking methodology or by spiral method. Incubate the plates in aerobic atmosphere at 37 ± 1ºC for 44 ± 4h. (Incubation times longer than those mentioned above or different incubation temperatures may be requied depending on the sample, on the specifications,...) After incubation, enumerate all the colonies that have appeared onto the surface of the agar, observing any blackening of the medium due to esculin hydrolysis, typical for Listeria strains. Presumptive isolation of Listeria sp. must be confirmed by further microbiological and biochemical tests.

Principles and uses Listeria Agar Base Palcam, used with supplements, is a selective and differential medium for Listeria spp. It is recommended by ISO 11290 for the detection and enumeration of Listeria monocytogenes in food products and clinical samples, and can also be used for environmental samples.

It is used after a primary and secondary enrichment stage, using Listeria Enrichment Broth Base (Cat.1120). It allows the easy differential diagnosis of Listeria monocytogenes using a double-system indicator: Esculin/Iron and Mannitol/Phenol red. All Listeria species hydrolyze the esculin to esculetin, which reacts with iron ions producing a blackening of the medium. Lithium chloride included in the medium, along with ceftazidime, polymyxin B sulfate and Acryflavine from the supplement, inhibit the growth of the non-Listeria accompanying bacteria present in foods, which can hydrolyze the esculin. Peptones and maize starch provide a rich nutrient base for growth. Yeast extract is the source of vitamins, particularly of the B-group. Glucose is the fermentable carbohydrate. Ferric ammonium citrate improves the growth of L. monocytogenes. The Mannitol/Phenol red differentiation system is used to differentiate Listeria spp that do not ferment mannitol from other species that occasionally grow in the medium such as enterococci or staphylococci. Differentiation is achieved by the acid increase in the media, causing the phenol red indicator to change the color of the medium from red to yellow. Confirmation of Listeria is done by biochemical and serological identifications tests.

Universal Pre-enrichment Broth is a non-selective medium used for the recovery of Salmonella and Listeria from food products. With traditional methods requiring different pre-enrichments for each microorganism, Universal Pre-enrichment Broth was formulated by Bailey and Cox for the recovery of sublethally injured strains of both Listeria and Salmonella from a single food product specimen. The medium is well buffered, containing Disodium and Monopotassium Phosphates, which allows the recovery of pH-sensitive bacteria. Enzymatic Digest of Casein and Protease Peptones supply necessary growth nutrients such as amino acids, peptides, vitamins, and electrolytes. Dextrose is an energy source to sustain bacterial growth. Sodium Chloride, Magnesium Sulfate and Ferric Ammonium Citrate provide essential electrolytes and maintain the osmotic equilibrium. Sodium Pyruvate stimulates the metabolism of stressed microorganisms.

Developed by Donnelly and Baigent, UVM Modified Listeria Enrichment Broth medium is used for the selective isolation and enrichment of Listeria spp. from clinical specimens and food. Within the formula, the high tolerance to Sodium Chloride of Listeria is used to inhibit the growth of enterococci, Nalidixic Acid (20 mg/L) to inhibit Gram-negative microorganisms, and Acriflavin to inhibit Gram-positive bacteria. The Esculin is utilized by Listeria spp. and results in the blackening of this medium. Casein Peptone, Beef Extract, Yeast Extract and Peptic Digest of Animal Tissue supply necessary growth nutrients such as amino acids, peptides, vitamins, and electrolytes. The medium is well buffered, containing Disodium and Monopotassium Phosphates, which allows the recovery of pH-sensitive bacteria.

Palcam Broth is a liquid medium used for the selective enrichment of Listeria spp. from food and environmental samples. Casein Peptone and Yeast Extract provide essential nutrients like amino acids, peptides, carbohydrates, vitamins and electrolytes. Esculin and Mannitol are the main source of energy for bacterial growth. Mannitol-fermenting species of Listeria give the broth a yellow color due to the presence of the pH indicator Phenol Red. Lithium Chloride provides selectivity to Palcam Broth due to the high salt tolerance of Listeria spp. Most Gram-negative and Gram-positive bacteria are suppressed by the addition of Polymixin B, Acriflavin, and Ceftazimide to the cooled medium. All members of the Listeria group can hydrolyze Esculin to 6,7-dihydroxycoumarine, which reacts with Ferric Ammonium Citrate and results in the blackening of the medium.

TSYEA is a universal medium used in a number of applications or protocols. Given its excellent nutritive value, it is often used for the isolation and purification of microorganisms obtained from selective media (i.e. PALCAM agar, Oxford agar etc.) for detection or enumeration of Listeria, and more specifically, Listeria monocytogenes according to the standards currently in vigor.

The typical composition corresponds to that defined in the standards NF EN ISO 11290-1 and NF EN ISO 11290-2.

PALCAM Agar is a selective medium used for the differentiation and isolation of Listeria monocytogenes and other Listeria, from milk and cheese, as well as in other food products, even highly contaminated.
The media can be used as a second media of choice in the context of the directives concerning the detection of Listeria monocytogenes in food microbiology.

Oxford Agar is a selective medium used for the differentiation, the isolation and the enumeration of Listeria monocytogenes from milk and cheese, as well as in other food samples, even highly contaminated.
The media can be used as the second media of choice in the context of the method of detection of Listeria monocytogenes in food microbiology. 

LEB media (Listeria Enrichment Broth) according to the International Dairy Federation (IDF) is used for the selective enrichment of Listeria in milk and dairy products.

ChromoGel™ Listeria Agar completed with selective and enrichment supplements, is a selective and differential medium for the isolation of Listeria spp. from foodstuffs and other samples and for the identification of L. monocytogenes by ISO 11290

Preparation
# Dissolve 70g in 1000ml of distilled water (purified water)
# Heat and frequent stirring to completely dissolve the medium
# Autoclave at 121°C for 15 minutes
# Cool to 45-50°C, add 2 vials of ChromoGel Listeria Selective Supplement and 2 vials of ChromoGel Listeria Enrichment Supplement
# Mix well and dispense into appropriate sterile containers

Selective supplement
Formula for 500ml of medium Vial
- Nalidixic Acid 10 mg
- Ceftazidime 10 mg
- Amphotericin B 5 mg
- Polymyxin B 38350 UI

Enrichment supplement
Formula for 500ml of medium Vial
- L-α-Phosphatidylinositol :1g