Môi trường nuôi cấy vi sinh Alphachem

Principles and uses Casein Peptone is a pancreatic digest of casein. Pancreatic digestion produces a balanced mixture of amino acids, including essential amino acids, in optimal ration and low molecular peptides. In many cases, this makes for a more nutritious hydrolysate, especially for those organisms that prefer peptides to amino acids. It can be used in the production of toxins, vaccines, enzymes, in fermentation applications and microbiological culture media, especially in blood-containing media.

Principles and uses Bacteriological agar is a gelling agent used in the preparation of culture media and in other bacteriological applications. Its main advantage is the absence of inhibitors which could hinder optimal development of microorganisms. In addition, bacterio-logical agar also possesses other attributes such as transparency, high hysteresis and very reliable reproducibility. Bacteriological agar European Type has higher gel strength and is used in concentrations from 1,0% to 2,0%. Each batch produced by us is thoroughly tested for biological performance against a battery of known bacterial cultures in order to ensure proper growth characteristics and absence of inhibitors. Also, other tests are carried out to be certain that each batch meets established physical and chemical specifications.

This culture medium contains lactose, whose degradation to acid is indicated by the pH indicator phenol red, which changes its colour to yellow. The indicator exhibits a deep red colour in the alkaline range. The growth of the accompanying Gram-positive microbial flora, Salmonella typhi and Shigella is largely inhibited by brilliant green. The growth of Salmonella is, however, improved by the richer nutrient base. Increased growth of accompanying microorganisms is considerably prevented by raising the concentration of brilliant green. Salmonellae are not able to ferment either lactose or sucrose. Thus in contrast to BPL agar, the sucrose contained in this medium allows identification of accompanying, weakly lactose-positive or lactose-negative, but sucrose-positive microorganisms.

Principles and uses Irgasan Ticarcillin and Potassium Chlorate Broth (ITC) is recommended by ISO 10273 as a selective enrichment broth for the detection of the human pathogenic strain of Yersinia enterocolitica in food and water samples. Enzymatic casein digest provides nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is a source of vitamins, particularly of the B-group essential for bacterial growth. Magnesium chloride and malachite green, make the broth highly selective. Irgasan inhibits Gram-positive bacteria, Ticarcillin has bactericide on Gram-negative and Gram-positive bacteria and potassium chlorate has a disinfecting property.

Principles and uses
Sorbitol Peptone Broth And Bile Salts (PSB) is a medium recommended by ISO 10273 for the selective enrichment of Yersinia enterocolitica in food samples. Outbreaks of gastroenteritis associated with Yersinia enterocolitica are increasing. Contaminated food products such as pork, beef, and raw and processed milk have been identified as sources of infection.
Enzymatic digest of casein provides nitrogen, vitamins, minerals and amino acids essential for growth. Sodium chloride supplies essential electrolytes for transport and osmotic balance. Potassium phosphates act as a buffer system. Sorbitol is the fermentable carbohydrate providing carbon and energy.

Preparation
Suspend 31 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Dispense into appropriate containers and sterilize in autoclave at 121°C for 15 minutes.

Yersinia Selective supplement is based on the formulation of Schiemann and is added to Yersinia medium base (CIN) in order to obtain a final selective medium for the isolation and enumeration of Yersinia enterocolitica from clinical specimens and food. Yersinia enterocolitica is becoming increasingly recognised as a cause of diarrhoeal disease of man and also polyarthritis, mesenteric adenitis and septicaemia.

Principles and uses
Yersinia Select Agar Base (ISO 10273) is a selective and differential medium when used with supplements. The formula is based on the CIN Agar described by Schiemann, and is recommended by ISO 10273 for the isolation and detection of presumptive pathogenic Yersinia enterocolitica from a variety of clinical and food samples.
Antibiotics are added as a supplement in order to inhibit the accompanying flora. The growth of Yersinia is promoted by pyruvate as well as by the nutrients content in the base. Yersinia degrades the mannitol of the medium to an acid form; the colonies are turning to red color due to the neutral red indicator. Mannitol fermentation in the presence of neutral red produces a characteristic “bull’s-eye” colony, colorless with a red center.
Mannitol is the fermentable carbohydrate, source of carbon and energy. Enzymatic digest of gelatin and the enzymatic digest of casein and animal tissues provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is a source of vitamins, particularly of the B-group.
Sodium pyruvate is added as a source of energy and as a protective substance in order to overcome oxygen toxicity biologically produced by the organisms. Sodium chloride supplies essential electrolytes for transport and osmotic balance. Magnesium sulphate is an ion required in a large variation of enzymatic reactions, including DNA replication. Neutral red is the pH indicator. Selective inhibition of Gram-negative and Gram-positive organisms is obtained through crystal violet, sodium desoxycholate and Irgasan (triclosan). Cefsulodin and novobiocin improve the inhibition of normal enteric organisms.

Preparation
Suspend 28,5 grams of the medium in 500 ml of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Sterilize in autoclave at 121 ºC for 15 minutes. Cool to 45 ºC and aseptically add one vial of Yersinia Selective Supplement Homogenize gently and dispense into Petri dishes.

Principles and uses A1 Medium, also known as A1 Broth, is used for the detection of fecal coliforms in water samples. The enumeration of coliforms, specifically Escherichia coli, has been used to determine water purity by the most-probable-number method. This medium was created to hasten the recovery time of E. coli and to reduce the number of false positive cultures. A-1 Medium can be used in a single-step procedure, also in foods, not requiring a previous enrichment step. Tryptone provides nitrogen, vitamins, minerals and aminoacids. Lactose is the carbon source and in combinationwith salicin, provides energy for organism growth. Ecosurf is a surfactant and sodium chloride supplies essential electrolytes for transport and osmotic balance. Gas production is a positive reaction indicating the presence of Coliforms. Gas may be produced inside the Durham tubes or may appear as dissolved gas that forms gas bubbles when slightly agitated.

The use of animal tissue for culturing anaerobic organisms was first employed by Theobald Smith in 1890. Von Hibler later used brain tissue for cultivating and classifying anaerobic bacilli. Robertson replaced brain tissue with beef heart and used this medium to differentiate putrefactive and saccharolytic species. The formulation presently used is a modified version of Robertson's formulation. This medium is also referred to as Chopped Meat Medium.  Nutritional requirements needed by most bacteria are provided by beef heart, peptone and dextrose. Dextrose, yeast extract, hemin and vitamin K are added to enhance the growth of anaerobic microorganisms. Amino acids and other nutrients are supplied by the muscle protein in the heart tissue granules. Reducing substances, which permit the growth of strict anaerobes, are supplied by the muscle tissue and the iron filings. It is thought that the meat particles act as a reducing and detoxifying substance, thereby disabling harmful by products that may be produced by the replicating organism. Because reducing substances are more available in denatured protein, the meat particles are cooked before use in the medium. Growth of spore-forming and non-spore-forming obligate anaerobes is supported by this medium. Cooked Meat Medium is also useful as an enrichment broth for cultivating organisms from a very small inoculum. Additionally, researchers have found that Cooked Meat Medium preserves viability of organisms over a long period of time and is useful in maintaining anaerobic stock organisms. The Food and Drug Administration recommends its use in the enumeration and identification of Clostridium perfringens from food.

Principles and uses TGEA Medium (Tryptone Glucose Yeast Extract Agar) is a medium used for the total count of aerobic mesophilic bacteria in water and dairy products. Casein Peptone and Beed Extract provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is source of vitamins, particularly the B-group. Dextrose is the fermentable carbohydrate providing carbon and energy. Bacteriological agar is the solidifying agent

A sterile selective supplement used for isolation and presumptive identification of Clostridium perfringens, according to ISO 7937 and ISO 14189, and other regulations.

Compositon (g/vial) D-Cycloserine....................................................... 0.200 Reconstitute the original freeze-dried vial by adding Sterile Distilled Water...........................................6 ml

Note: each vial is sufficient to supplement 500 ml of medium. TSC Agar Base.

D-cycloserine selective supplement is added to TSC Agar in order to obtain a final selective medium which has the advantage to simplify the counting of plates with high numbers of colonies because smaller colonies of C.perfringrens are formed. Sodium metabisulphite and ferric ammonium citrate are used as an indicator of sulphite reduction made by Clostridium perfringens spp. that produce black colonies in TSC agar.

Principles and uses
Hektoen Enteric Agar is a differential and selective medium used for isolating and differentiating enteric pathogens such as Salmonella and Shigella, both of which cause a variety of serious human gastrointestinal diseases; and other Gram-negative Enterobacteriaceae.
It is used particularly in foods where multi-steps are followed to isolate the pathogens of gastroenteritis. The nutrients for growth are provided by the Meat Peptone and Yeast extract. The increased content of the Peptone and the three fermentable carbohydrates (Lactose, Sucrose, Salicin) as sources of carbon and energy reduce the inhibitory action of the Bile salts on Salmonella and Shigella spp. The lactose concentration in this medium is higher than in many other media used for enterics since this helps the visualization of enteric pathogens and minimizes the problem of delayed lactose fermentation. Bromothymol blue and Acid fuchsin are pH indicators. Sodium thiosulfate provides Sulphur, and Ferric ammonium citrate is the indicator for
H2S production. H2S positive colonies are black-centered. Sodium chloride maintains the osmotic balance.
The norma ISO 21567 recommends the Hektoen Agar as a selective solid media for the enumeration of Shigella spp. Although suppressed, partially inhibited E. coli and other organisms which use lactose, sucrose, and/or salicin with the production of acid, give colonies whose tones vary from yellow to orange to salmon. The Salmonella and Shigella are green or green-blue. Proteus is not inhibited but produces a green-yellow colony when it grows. The colonies of Proteus and Salmonella may present a black center and clear edges if they form iron sulfide as a result of H2S production.

Preparation
Suspend 75,6 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. AVOID OVERHEATING. DO NOT AUTOCLAVE. Cool to 47 ºC and pour into Petri dishes.

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.