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Microbial Culture Media


Know the composition of all microbiological media used for microbiological analysis.

Baird Parker Agar Base

Specification

Solid and selective culture medium for the screening of staphylococci from a variety of samples, acc. to pharma­copeias and ISO standard.

Formula (in g/L)

Tryptone ...................................................10,0
Sodium pyruvate ......................................10,0
Glycine .....................................................12,0
Meat extract ................................................5,0
Lithium chloride ..........................................5,0
Yeast extract ...............................................1,0
Agar .........................................................17,0
Final pH 7,0 ± 0,2

Directions

Suspend 60 g in 950 mL of distilled water. Allow it to soak and then bring to the boil with constant stirring. Sterilize by autoclaving at 121° C for 15 minutes. Cool to 50° C and add 50 mL of Egg’s Yolk Tellurite Sterile Emul­sion (Ref. 06-026). Homogenize and distribute into plates. Once prepared, the medium must not be reheated nor sterilized again.

Description

The Baird Parker Agar Base is specially recommended for the detection and enumeration of staphylococci in food and other material, since it allows a good differen­tiation of coagulase-positive strains. The growth of the accompanying bacteria is usually suppressed by the high concentration in lithium, glycine and pyruvate. Lithium and glycine enhances the growth of staphylococci. Even if its high selectivity does not affect staphylococci it may occasionally permit the growth of some Bacillus species, yeast and very rarely, Proteus. The growth of Proteus species can be suppressed by adding 50 mg/l of sul­phamethazine.
The presence of tellurite and egg’s yolk, which must always be added to the medium after sterilization, allows the differentiation of presumptly pathogenic staphylococ­cal colonies. A high correlation has been found between the coagulase test and the presence of clearing zones of lypolysis in this medium, which is due to the staphylo­coccal lecithinase. On the other hand, studies show that almost 100% of coagulase-positive staphylococci are capable of reducing tellurite, which produces black colo­nies, whereas other staphylococci can not always do so.
When using sterile reagents other than SCHARLAU MICROBIOLOGY, the complete medium may be ob­tained by adding 50 mL sterile egg’s yolk and 10 mL of 1% potassium tellurite solution. Plates should be used on the same day of preparation or within 48 hours, to avoid the loss of definition in the precipitated zones . The basal medium, without the yolk or the tellurite, is perfect­ly stable and therefore can be repeatedly melted.

Technique

The inoculation is done by spreading 0,5 mL of sample over each plate with a Drigalski loop (Ref. 5-010). After 18-24 hours of incubation at 35° C, select the colonies which are black, shiny and convex with regular margins surrounded by a zone of clearing. These can be pre­sumptly identified as coagulase-positive Staphylococcus aureus.
Colonial appearance after 24 h. at 35°C:
Staphylococcus aureus: Black, shiny, convex, regular margins, 1.0-1.5 mm diameter, surrounded by a clearing zone of lipolysis 2-5 mm in width. May produce wide opaque precipitate zones extending into the cleared medium after 48 hours.
Other species of Staphylococcus: Black, usually dully, with regular margins. Sometimes they are brown with zones of clearing but these present wide opaque zones.
Micrococcus spp: Brown, very small and without clear­ing.
Bacillus spp: Various shades of brown, big. May produce clearing after 48 hours.
Yeasts: White, big and smooth.
The egg’s yolk emulsion can be prepared by mixing a fresh egg’s yolk with an equivalent quantity (w/v) of saline solution. Sterilize by filtration and aseptically add to the medium. This reagents´s reference, already steri­lized, is SCHARLAU 6-016.
The potassium tellurite solution is prepared by dissolv­ing 3,5 g potassium tellurite in 100 mL distilled water. Sterilize by filtration. This sterile reagent’s SCHARLAU MICROBIOLOGY reference is 6-011.
Although these solutions can be mixed to be added to the Baird Parker Agar Base forming the commonly known Egg Yolk Tellurite Sterile Emulsion (Ref. 06-026), they are also stable as the separate supplement and can be used in many other culture media.

Blood Agar Base (Columbia)

Specification

Medium especially rich in peptone, appropriate for blood addition or to prepare Chocolate Agar.
Formula (in g/L)
Casein Peptone ........................................12,0
Meat peptone ...........................................11,0
Starch .........................................................1,5
Sodium Chloride .........................................5,0
Agar ..........................................................15,0
Final pH 7,3 ± 0,2

Directions

Add 44,5 g of powder to 950 mL of distilled water and bring it to the boil. Distribute into suitable containers and sterilize at 121°C for 15 minutes. To obtain Blood Agar cool it to 45-50°C and aseptically add sterile defibrinated blood at 5% proportion.

Description

Blood Agar Base contains an equilibrated mixture of meat and casein peptones, being suitable for preparing selective and as diagnostic media with the addition of blood or inhibitors. As it is presented, without additions, it is also an excellent general culture medium.
Generally, Blood Agar base contains a casein peptone, that aids big size colonies formation, or a meat petone, that provides a well defined hemolysis halos or zones. Blood Agar Base is prepared according to the Columbia University formulation, and meets the two conditions mentioned above.

Technique

Some applications for this base are:
Base Agar without either enrichment and inhibitors: This medium supports growth of normal micro­organisms as enterobacteriaand othyers more strengh as Pasteurella, Brucella and Clostridium perfringens.
Clostridium Selective Base Agar: Should a selective clostridium medium be desired, add 240 mg/L Sodium Azide and 180 mg/l Neomycin before the sterilization, or alternatively the contents of SC Inhibitor container (Ref. 06-012CASE).
Blood Agar: Aseptically add to the sterile medium 5% sterile defibrinated sheep blood and cool it to 45°C. This way, medium is enriched and allows the determination of typical hemolytic reactions necessary for the identification of enterococci,staphylococci and other microorganisms.
Selective Gram-positive cocci Blood Agar: Simulta­neously at the time blood addition, also add 10 mg/L of colistine and 15 mg/L of Nalidíxic Acid, or, the contents of a CP Inhibitor container (Ref. 06-013CASE), to obtain an excellent selective medium for gram-positive cocci.
Note: Some authors recommend Blood Agar Base as the maintenance medium for Campylobacter.

Brilliant Green Agar (BGA)

Specification

Medium for Salmonella isolation, according to the Euro­pean Pharmacopoeia

Formula (in g/L)

Meat peptone .......................................5,0000
Casein peptone ....................................5,0000
Sodium chloride ....................................5,0000
Yeast extract .........................................3,0000
Lactose ...............................................10,0000
Sucrose ..............................................10,0000
Phenol Red ...........................................0,0800
Brilliant Green .......................................0,0125
Agar ....................................................15,0000
Final pH 7,0 ± 0,2

Directions

Suspend 53 g of powder in 1 L of distilled water and heat to boiling with constant stirring. Dispense into containers and sterilize at 121°C for 15 minutes.

Description

BGA is a differential selective medium, able to detect the presence of enteropathogenic bacteria in different samples. This medium is a modification to Kauffman’s original formulation, and it complies with the ISO, HMO, Eur. Phar., USP and APHA specifications.
Since it has a high brilliant green concentration, it inhibits notably the growth of most bacteria, except Salmonella. However, Styphi and S. paratyphi are also inhibited. Therefore, when their presence or Shigella is suspected, it is recommended to use other media in parallel, as Deoxycholate Lactose Agar (Ref. 01-057), MacConkey Agar (Ref. 01-118), Salmonella Shigella Agar (Ref. 01-171), Xylose Lysine Deoxycholate Agar (Ref. 01-211 or 1-552) or Endo Agar Base (Ref. 01-589) which are less inhibitory.
Presence of lactose and sucrose allows a good dif­ferentiation between Salmonella, which produce pink or colourless colonies with a red halo or zone, and the companion flora, which produce smaller and green yel­lowish colonies with yellow halo, due to acid created by lactose and/or sucrose fermentation.
Osborn and Stokes suggested the addition of 0,08 g/L of sulfadiacine or 1 g/L of sulfapyridin in order to make this medium more selective for Salmonella and provide suit­able qualities to this medium to perform the examination of food and eggs and their derivatives.

Buffered Peptone Water (Buffered Sodium Chloride-Peptone Solution pH 7.0)

Specification

Diluent for the homogenization of samples according to the European Pharmacopeia and ISO 21149.

Formula (in g/L)

Peptone ....................................................1,00
Sodium chloride ........................................4,30
Disodium phosphate .................................7,23
Potassium phosphate ...............................3,56
Final pH 7,0 ± 0,2

Directions

Dissolve 16 g of powder in 1 L of distilled water, heating up if necessary. Add 1 to 10 mL of Polysorbate 80 (Ref. 06-088) or Polysorbate 20 depending on the type of food to be diluted. Homogenize and distribute into containers. Sterilize by autoclaving at 121ºC for 15 minutes.

Description

This solution is recommended by European Pharmaco­poeia to dilute the sample for microbiological examina­tion. Depending on the amount of fat in the sample to examine it is the technician’s decision regarding the kind and quantity of emulsifying agent to be used.

Casein Lecithin Polysorbate Broth Base

Specification

Liquid medium to dilute and neutralize samples of phar­maceutical, cosmetic, raw material or end-products for the purpose of enumeration

Formula (in g/L)

Casein peptone .....................................20,00
Soya lecithin ............................................5,00
Final pH 7,3 ± 0,2

Directions

Dissolve 25 g of powder in 960 mL of distilled water pre-warmed at 50ºC . Add 40 mL of polysorbate 20, homog­enize and distribute in suitable containers. Sterilize in autoclave at 121ºC for 15 minutes.

Description

This medium is produced according the formulation of the U.S. Pharmacopoeia. In the Section <61> “Microbial Limit Tests” is proposed as alternative system to neutral­ize preservatives and disinfectants before to proceed with the enumeration process, specially by the mem­brane filtration method.

Cetrimide Agar (Pseudomonas Selective Agar)

Specification

Solid culture medium for selective isolation of Pseu­domonas aeruginosa acc. to ISO 22717.

Formula (in g/L)

Gelatin peptone ........................................20,0
Magnesium chloride ...................................1,4
Potassium sulfate .....................................10,0
Cetyltrimethyl-Ammonium Bromide ..........0,3
Agar ..........................................................15,0
Final pH 7,2 ± 0,2

Directions

Suspend 46,7 g of powder in 1 L of distilled water and add 10 mL of Glycerol. Bring to the boil and distribute into suitable containers. Sterilize at 121°C for 15 min­utes.

Description

The Cetrimide Agar is based on the enormous resistance of Ps. aeruginosa strains to the Quaternary Ammonium Compounds (QAC’s). With regard to the Cetyltrimethyl-Ammonium Bromide there has been growth at 1 g/L concentrations, but in such cases it has been very poor and slow.
An inhibitor concentration of 0,3-0,5 g/L does not seem to affect the viability of the pyogenic species. Neverthe­less, it does inhibit the rest of the fastidious accompa­nying bacteria, both gram-positive and gram-negative, as well as other species of Pseudomonas which may develop at lower inhibitory concentrations.
Although Ps. aeruginosa prevails over any other fastidi­ous bacteria after a 48 hour incubation at 35°C, it is recommended to first isolate at 42°C with an incubation of 48 hours. By this method, almost complete inhibition of other microorganisms is obtained.

Dey Engley Neutralizing Agar

Ref. 01-610

Specification

Solid culture medium for the neutralization and testing of antiseptics and disinfectants acc. ISO 22717 and 22718 standards.

Formula (in g/L)

Tryptone ...................................................5,00
Yeast extract .............................................2,50
Dextrose .................................................10,00
Lecithin .....................................................7,00
Sodium thiosulphate .................................6,00
Sodium sulphite ........................................2,50
Sodium thioglycollate ...............................1,00
Polysorbate 80 .........................................5,00
Bromcresol purple ....................................0,02
Agar ........................................................15,00
Final pH 7,6 ± 0,2

Directions

Suspend 54 g of powder in 1 L of distilled water and bring to the boil. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes. The ap­pearance of precipitates is normal and do not interferes the results.

Description

Dey & Engley developed this medium in 1983 to recov­ery chemically damaged staphylococci, At the present its use is generalized for testing by the contact method (RODAC Plates) the efficiency of antiseptics and disin­fectants on impervious surfaces. The present formulation incorporate neutralizing substances for almost all the active products used as antiseptics and disinfectants. Lecithin neutralizes quaternary ammonium compounds (QAC’s); Polysorbate acts on phenolics and formalin; thioglycollate neutralizes the organic-mercurial com­pounds; thiosulfate-sulfite inactive halogen-compounds and lecithin + polysorbate neutralizes ethanol and other alcoholic compounds.

Deoxycholate Citrate Agar

Specification

Differential and moderately selective plating medium for enteric pathogenic bacteria, according the European Pharmacopoeia.

Formula (in g/L)

Peptone .................................................10,00
Meat extract ............................................10,00
Lactose ...................................................10,00
Ferric Citrate .............................................1,00
Sodium Citrate ........................................20,00
Sodium deoxycholate .............................. 5,00
Neutral red ...............................................0,02
Agar ........................................................15,00
Final pH 7,3 ± 0,2

Directions

Suspend 71 g of powder in 1 L of distilled water and bring to the boil. Immediatelly pour into plates. Plates may be used at once or refrigerated for a few days. Do not autoclave or overheat.

Description

The European Pharmacopoeia formulation is one of these modifications to rhr original medium developed by Leifson in 1935. The inhibition of gram-positive microor­ganisms is due primarily to its content of sodium deoxy­cholate, although the two citrate compounds also are active inhibitors. The lactose achieves differentiation of enteric bacilli. Organisms that ferment lactose produce acid that, in presence of neutral red indicator, results in the formation of red colonies. Lactose non-fermenting produces colourless colonies. The black centres due to the ferric sulphide depot detect the production of SH2.

Technique

Inoculate the specimen as soon as possible directly onto surface of medium. Incubate the plates at 35 ± 2ºC for 18-24 hours. Plates can be incubated for an additional 24 hours if no lactose-fermenting are observed.
Typical colonial morphology on Deoxycholate Citrate Agar is as follows:
Escherichia coli: Large, flat, rose-red
Enterobacter / Klesiella: Large, mucoid, pale with pink centre.
Proteus: Large, colourless to tan.
Salmonella: Large, colourless to tan.
Shigella: Colourless to pink
Pseudomonas: Irregular, colourless to brown
Gram-positive bacteria: No growth to slight growth

Dextrose Agar

Specification

General purpose solid culture medium.

Formula (in g/L)

Meat peptone .............................................5,0
Casein peptone ..........................................5,0
Meat extract ................................................3,0
D(+)Glucose .............................................10,0
Sodium chloride ..........................................5,0
Agar ..........................................................15,0
Final pH 6,9 ± 0,2

Directions

Suspend 43 g of powder in 1 L of distilled water and heat to boiling . Sterilize by autoclaving at 121°C for 15 minutes. Should the acid pH is required, add sterile tartaric acid solution when the medium is at 45°C. Do not reheat.

Description

This solid culture medium is suitable for many objectives and it supports growth of most non fastidious microor­ganisms. Adding 5% sterile defibrinated blood to this me­dium, makes it an excellent culture medium which satisfy nearly all kinds of nutritive needs, even for meningococci and pneumococci, however due to its high glucose con­tent it is not suitable for hemolytic reaction studies.

Differential Reinforced Clostridial Medium (DRCM)

Specification

Liquid medium for the enumeration of clostridia in food samples and other products by MPN technique.
Formula (in g/L)
Peptone ................................................10,000
Meat extract ............................................8,000
Yeast extract ...........................................1,000
Starch .....................................................1,000
Glucose ..................................................1,000
L-Cysteine HCl .......................................0,500
Sodium acetate ......................................5,000
Sodium bisulfite ......................................0,500
Ferric-ammonium citrate .........................0,500
Resazurine .............................................0,002
Final pH 7,0 ± 0,2

Directions

Dissolve 27,5 g of powder in 1 L of distilled water. Bring to the boil, distribute in tubes and sterilize in the auto­clave at 121°C for 15 minutes.

Description

This medium is a modification by Freame and Fitzpatrick of the Gibb’s classic medium, to easily detect the pres­ence of sulfite reducing clostridia. The modification is, mainly, an addition of sodium bisulfite and ferric citrate, that make colonies black and thus more conspicu­ously visible. The current version of this medium has no agar in order to facilitate the blackness of the medium. Resazurine,the redox indicator allows the verification of anaerobiosis in the medium in the same assay. L-Cysteine acts as reducing agent in this medium.

Technique

Sample to be examined is distributed in tubes as per the MPN technique, and is covered with paraffin or vaseline oil to help the anaerobiosis. The bank of tubes is kept in a boiling water bath at 75°C for 30 minutes to remove all the dissolved oxygen and vegetative cells. Then, incubate at 30°C up to 7 days before concluding any negative results.
Generally, the spores of sulfate reducing clostridia germinate between the second and fourth day, and the medium turns black, in which case the test is positive.
The medium can be rendered selective by the addition of 70 IU/mL of Polymyxin sulftate.
Prepared tubes without the inoculation may be stored up to 2 weeks provided the resazurine band does not show an excessive oxidation (more than a 1/3 part of the column).
This formulation is also in accordance with the one suggested for the study of frozen food, and also for fruit juices. In this latter case, it is suggested to use it in duplicate, and one of the samples must be acidified in order to facilitate the growth of moulds and yeast in such a selective way.
Acidification can be easily achievevd by adding 7-8 mL of 10% sterile tartaric acid to the sterile, and cooled me­dium at 45°C,producing a pH drop to 3,5 ± 0,2. In these conditions, do not remelt the medium, as then agar tends to get hydrolysed and do not solidify again.

EE Broth

Specification
Liquid culture medium for the enrichment of enterobacte­ria from food samples according to ISO 8523 standard.

Formula (in g/L)

Gelatin peptone ....................................10,000
Dextrose .................................................5,000
Ox bile ..................................................20,000
Di-sodium phosphate .............................6,450
Monopotassium phosphate ....................2,000
Brilliant green .........................................0,015
Final pH 7,2 ± 0,2

Directions

Suspend 43,5 g of powder in 1 L of distilled water and heat at 100°C for 30 min. and cool immediately.

Description

As the name suggests, this medium is for the enrichment of enterobacteria, and is a modification by Mossel(1963) of the classic Brilliant Green Bile 2% Broth (Ref. 02-041). Substitution of lactose by glucose makes it more suitable for enteric bacteria detection, whether gas or non-gas-producer, in food and different samples.
Usual recommended technique is as follows: sample to be studied is added to sterile broth at a proportion of 10%. After strong homogenization, the mixture is incu­bated for a period of18-20 hours at 35-37°C. Afterwards, subcultures are performed on a solid media appropiate for the selective enterobacteria isolation. For this step, Violet Red Bile Agar (Ref. 01-164) is specially recom­mended, though there are also the MacConkey (Ref. 01-118), Deoxycholate or Brilliant green based media.
From the suspected colonies on this media, identification can be performed following the common methodology.

Eosin Methylene Blue Agar (EMB Agar)

Specification

Selective differential medium for the isolation of coliforms from water acc. ISO 21150 Standard.

Formula (in g/L)

Peptone ................................................10,000
Lactose .................................................10,000
Dipotassium Hydrogen phosphate .........2,000
Yellowish Eosin ......................................0,400
Methylene Blue .......................................0,065
Agar ......................................................15,000
Final pH 7,1 ± 0,2

Directions

Add 37,5 g to 1 L of distilled water. Bring to the boil and distribute in suitable containers. Sterilize in the autoclave at 121°C for 15 minutes.

Description

A very versatile medium originally developed for the differentiation of E. coli and Enterobacter aerogenes. It has also proved very effective in the rapid identification of Candida albicans and presents a high correlation with the coagulase test for staphylococci.
It has been repeatedly recommended for the detec­tion, enumeration and differentiation of members of the coliform bacteria.

Technique

The Weld method for the identification of Candida albi­cans uses this medium with Chlortetracycline (100 mg/l) in a 10% CO2 environment. The method’s effectivity has been tested with a variety of samples, such as sputum, oral secretions, faeces, nails and vaginal secretions, all of which provide definite results within 24-48 hours. sta­phylococci are also easily identified, particularly coagu­lase-positive strains. These have a very characteristic appearance: small colourless colonies with a central red nucleus.
Nevertheless, the medium’s prevailing application is in the differentiation of E. coli and E. aerogenes.
The medium should be sterilized once distributed into tubes containing 20 mL of product each, and then be refrigerated. Melt in a boiling water bath before use and stir until it acquires a dark purple colour. Pour a tube into each sterile plate and allow to solidify. It is advisable to dry the medium’s surface before use, leaving the plate open but inverted on a heater.
For each doubtful lactose broth tube,inoculate one plate by streaking , and incubate for 24 to 48 hours at 37°C. Examine afterwards.
Escherichia coli and Citrobacter form flat colonies of 2-3 mm in diameter and are dark violet in colour with a black centre which produces a distinctive green metallic glow when light is reflected on it.
Enterobacter and Klebsiella form convex colonies which are twice as big as the very smooth E. coli , have no metallic glow and are pink in colour with a dark blue centre. Non-lactose fermenting organ­isms produce colourless colonies.
Candida albicans colonies incubated in a CO2 atmos­phere have a very peculiar cotton-like appear­ance which distinguishes them from other Candida species that produce classical yeast like colonies.

Gram Negative Broth (GN Broth)

Specification

Liquid culture medium for enteric bacteria according Hajna’s formulation.

Formula (in g/L)

Peptone ....................................................20,0
Dextrose .....................................................1,0
D-Mannitol ..................................................2,0
Sodium citrate ............................................5,0
Sodium deoxycholate .................................0,5
Di-potassium phosphate .............................4,0
Monopotassium phosphate ........................1,5
Sodium chloride ..........................................5,0
Final pH 7,0 ± 0,2

Directions

Dissolve 39 g of powder in 1 L of distilled water. Dis­pense in tubes or flasks and sterilize in the autoclave at 121°C for 15 minutes.

Description

GN Broth (Gram Negative Broth) is an enrichment and selective medium for enterobacteria, with a strong inhibi­tory action against gram-positive because of its high content of citrate and deoxycholate. On the other hand, mannitol restrains the growth of Proteus and facilitates the proliferation of Salmonella and Shigella.
The medium is strongly recommended for primary enrichment, 14-16 first hours, before going to selective media such as EMB (Ref. 01-068) or MacConkey (Ref. 01-118). Its author, Hajna, declares an extraordinary selectivity of the medium, whatever may the origin of the sample, if everything is kept in a transport medium upto the inoculation.

Lactose Broth

Specification

Medium for the pre-enrichment and the detection of enterobacteria and coliforms in milk and water according ISO 9308-2 and 21150 standards.

Formula (in g/L)

Peptone ......................................................5,0
Meat extract ................................................3,0
Lactose .......................................................5,0
Final pH 6.9 ± 0,2

Directions

Add 13 g of powder to 1 L of distilled water, or in the quantity required for the desired concentration. Dissolve it and distribute into containers fitted with Durham tubes. Sterilize by autoclaving at 121°C for 15 minutes. Avoid any further reheating.

Description

Lactose Broth is a classical medium for use in the presumptive testing for coliforms and for the enrichment of Salmonella. This formulation is as per the standards recommended by APHA, AWWA, USP-NF and Euro­pean Pharmacopoeia.
It is commonly used with Durham fermentation tubes for the examination of gas formation. If the volume of sam­ple to inoculate is important, reconstitute the medium at a concentration such, as to remain normal once the sample has been added to it.
Although it is not the original formulation, this broth pro­vides excellent results in Eijkman assays of gas produc­tion at 45°C, which is a characteristic of Escherichia coli.
While preparing this medium it is important to avoid overheating and to distribute it into tubes before sterili­zation.

MacConkey Agar

Specification

A selective and differential medium for the detection, isolation and enumeration of coliforms from a variety of samples according European Pharmacopoeia and ISO standard.

Formula (in g/L)

Peptone ................................................20,000
Lactose .................................................10,000
Bile Salts #3 ...........................................1,500
Sodium chloride ......................................5,000
Neutral red ..............................................0,030
Crystal violet ...........................................0,001
Agar ......................................................15,000
Final pH 7,2 ± 0,2

Directions

Suspend 51,5 g of powder to 1 L of distilled water. Bring to the boil and sterilize by autoclaving at 121°C for 15 minutes.

MacConkey Broth

Specification

Liquid medium for the detection and enumeration of coliforms by MPN technique.

Formula (in g/L)

Peptone ................................................20,000
Lactose .................................................10,000
Bile Salts ................................................5,000
Sodium chloride ......................................5,000
Neutral Red ............................................0,075
Final pH 7,4 ± 0,2

Directions

Dissolve 40 g of powder in 1 L of distilled water. Bring to the boil and distribute into suitable containers fitted with Durham tubes. Sterilize by autoclaving at 121°C for 15 minutes.

Mannitol Salt Agar (Chapman Agar)

Specification

Selective medium for the isolation of staphylococci
according USP and ISO standard.

Formula (in g/L)

Meat extract ............................................1,000
Casein peptone ......................................5,000
Meat peptone .........................................5,000
Sodium chloride ....................................75,000
D-Mannitol ............................................10,000
Phenol red ..............................................0,025
Agar ......................................................15,000
Final pH 7,4 ± 0,2

Directions

Suspend 111 g of powder in 1 L of distilled water and bring to the boil. Dispense in tubes or flasks and sterilize by autoclaving at 121°C for 15 minutes.

Description

Mannitol Salt Agar is a classical medium for the detec­tion and enumeration of staphylococci. It was described by Chapman and has been adopted by many official or­ganisations. Several modifications have been developed from it with more or less similar effectivity.
This medium uses the advantage of high tolerance of staphylococci to salinity, to use sodium chloride as a se­lective agent, since only the staphylococci and halophilic enterobacteria are able to grow freely at this concentra­tion of salt employed in this medium while other bacteria are inhibited. It also exploits the correlation between the pathogenic and fermentative capacity of mannitol of staphylococci, to establish a presumptive diagnosis. Mannitol fermentation with an accumulation of acid prod­ucts is shown by the phenol red indicator turning yellow, that produces a yellow halo surrounding the presumptive pathogen colonies, meanwhile the rest of the medium remains orange in colour.

Technique

A massive surface inoculation and an incubation at 37°C for 36 hours or at 32°C for 3 days is recommended.
The typical appearance of the colonies after the correct incubation is as follows: Presumptive pathogenic staphy­lococci (coagulase +) are mannitol positive and are big colonies with a yellow halo. Non-pathogenic Staphyloco­cci (coagulase -) are usually mannitol negative and are small colonies without halo or change in colour.
In any case, coagulase presence must be tested by the classical technique, after a pure culture in the liquid me­dium is obtained, in order to establish its true pahogenic potential.

MRS Agar

Specification

Solid culture medium for lactobacilli, according to de Man, Rogosa and Sharpe and ISO standards 9332 and 15214.

Formula (in g/L)

Peptone Proteose ...................................10,00
Meat extract ..............................................8,00
Yeast extract .............................................4,00
D(+)Glucose ...........................................20,00
Sodium acetate ........................................5,00 
Triammonium citrate .................................2,00
Magnesium sulfate ...................................0,20
Manganese sulfate ...................................0,05
Dipotassium phosphate ............................2,00
Polysorbate 80 .........................................1,00
Agar ........................................................14,00
Final pH 6,2 ± 0,2

Directions

Suspend 66 g of powder in 1 L of distilled water. Bring to the boil slowly with gentle stirring until complete dissolu­tion. Dispense into suitable containers and sterilize by autoclaving at 121°C for 15 minutes.

Nutrient Agar

Specification

Solid culture medium for the general purposes according ISO standard.

Formula (in g/L)

Peptone ......................................................5,0
Meat extract ................................................3,0
Agar ..........................................................15,0
Final pH 7,0 ± 0,2

Directions

Suspend 23 g of powder in 1 L of distilled water and heat to boiling. Dispense into suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

Nutrient Broth

Specification

Liquid medium for the cultivation of non fastidious micro­organisms according ISO standard.

Formula (in g/L)

Peptone ......................................................5,0
Meat extract ................................................3,0
Final pH 7,0 ± 0,2
Directions
Dissolve 8 g of powder in 1 L of distilled water heating up only if necessary. Dispense into suitable containers and sterilize by autoclaving at 121°C for 15 minutes.
Description
Nutrient Broth is a modern version of the classical general culture medium based on meat infusion. It is a simple medium that may be used in general purposes (i.e. maintenance of strains) as well as a base for other specialized media. However, in this way, there are other media with more nutrient capacity and better perform­ance.
Nutrient broth is the liquid version of the Nutrient Agar, and it is a classical medium for normal tasks with non fastidious microorganisms. It is the ideal medium for the subculture of general bacteria, especially staphyloco­cci, to carry out later the coagulase and other biochemi­cal tests. It may also be used to determine the Phenol Coefficient by following the technique and microorgan­isms suggested by the AOAC.

Plate Count Agar (PCA)

Specification

Medium for the aerobic plate count by surface inocula­tion method (Standard Plate Count Agar) according ISO 4833 and 17410 standards.

Formula (in g/L)

Casein peptone ..........................................5,0
Yeast extract ...............................................2,5
Dextrose .....................................................1,0
Agar ..........................................................15,0
Final pH 7,0 ± 0,2

Directions

Suspend 23,5 g of powder in 1 L of distilled water. Dissolve by bringing to the boil with frequent stirring. Dis­tribute into final containers and sterilize by autoclaving at 121°C for 15 minutes.

Description

The Plate Count Agar follows the directions given by Buchbinder et al. in their study about media for the plate count of microorganisms.
The original formulation of the standardized agar for dairy microbiology has been modified in order to avoid the addition of milk. This new composition allows the growth of most microorganisms without any further ad­ditions.
This medium’s formulation is equivalent to that pre­scribed by the ‘Standard Methods for the Examination of Dairy products’, the USP’s ‘Tryptone Glucose Yeast Agar’, the ‘Deutsche Landswirtchaft’ and to the APHA and AOAC’s Plate Count Agar. Nowadays this is the medium selected for the plate count of any type of the sample.

Potato Dextrose Agar

Specification

Solid culture medium for the detection and enumeration of yeast and moulds in food, specially recommended in butter and other dairy products.

Formula (in g/L)

Potato peptone .........................................4,00
Glucose ..................................................20,00
Agar ........................................................15,00
Final pH 5,6 ± 0,2

Directions

Suspend 39 g of powder in 1 L of distilled water and bring to the boil. Distribute into suitable containers and sterilize in the autoclave at 121°C for 15 minutes. Do not overheat.

Description

Potato Dextrose Agar is a weakly selective medium for fungi due to its high sugar content and acidic pH. The pigment production and aerial mycelium development is enhanced by the potato peptone, specially in the FusariumAspergillus and Penicillium species.
The selectivity can be increased by adding antibacterial antibiotics like chloramphenicol or tetracyclines, or by simply decreasing the pH to an acidic level. At pH 3,5 the bacterial growth is almost totally inhibited without significant effect on fungi. This acidification can be obtained by the aseptic addition of an adequate amount of organic acid to the medium after sterillization: 10-15 mL/L of a 10% sterile solution of tartaric or lactic acid is usually sufficient.
After its acidification the medium should not be over­heated or reheated since it can hydrolyze the agar and hence there can be a loss in solidification property of the medium.

Technique

Distribute the diluted samples into sterile petri plates. Pour the molten agar melted cooled to 45-50°C and gen­tly mix to homogenize the mixture. After the solidification, plates are incubated for 5-7 days at 20-25°C to permit the complete development of the fungal colonies.
The weak consistency of the agar due to its original acidity makes this medium inadequate for streaking.

R2A Agar

Specification

Solid medium for the enumeration of heterotrophic micro organisms in treated waters

Formula (in g/L)

Yeast Extract ..........................................0,500
Proteose peptone ...................................0,500
Casein hidrolysate ..................................0,500
Glucose ..................................................0,500
Starch .....................................................0,500
Dipotassium hydrogen phosphate .............0,300
Magnesium sulphate, anhydrous ...............0,024
Sodium pyruvate ......................................0,300
Agar ......................................................15,000
Final pH 7,2 ± 0,2

Directions

Suspend 18,1 g of powder in 1 L of distilled water and bring to the boil with constant stirring. Distribute into suit­able containers and sterilize by autoclaving at 121ºC for 15 minutes.

Description

The R2A Agar was proposed in 1979 by Reasoner and Geldenreich and few years later accepted by the APHA as an alternative medium for stressed cells in treated potable water.
The use of nutrient rich media like PCA or TSA allows to the growth of normal microbiota, but do not permits the recuperation of the stressed or chlorine resistant biota. By the use of a medium like R2A of low nutrients in com­bination with a lower temperature and longer incubation time it is possible induce the resuscitation of this dam­aged cells.
In the R2A Agar the source of nitrogen is the peptone and the Yeast Extract supplies the vitamins and growth factors. The source of carbon is the dextrose and mag­nesium sulphate and potassium phosphate maintains the osmotic pressure. The starch is a detoxifier and sodium piruvate increases the recuperations of stressed cells. The agar acts as gelling agent.

Technique

The water sample must be processed as quickly as possible. If it is no possible within the first 6 hours, the sample must be refrigerate, but not for more than 30 hours: then the sample is rejected.
R2A Agar is used with pour plates, streak plates or filtration but must be keep in mind that the pour plates method can affect the recovery capacity of the medium because the thermal shock. The incubation period at 35ºC is of 3-5 days but is more effective a incubation temperature of 20-28ºC an a time of 5-7 days. In any case the plates must be protected against an excessive drying.
The fast-growing or non-stressed microorganisms in these conditions of incubation produce different and minute colonies than in the rich media.

Rappaport Vassiliadis Broth

Specification

Liquid medium for the selective enrichment of Salmo­nella in foodstuffs and other materials.

Formula (in g/L)

Soy peptone ...........................................4,500
Sodium chloride ......................................7,200
Monopotassium phosphate .....................1,260
Dipotassium phosphate ...........................0,180
Magnesium chloride ..............................13,580
Malachite green ......................................0,036
Final pH 5,2 ± 0,2

Directions

Dissolve 26,8 g of powder in 1 L of distilled water, heat­ing if necessary to help dissolve the powder. Dispense into test tubes or flasks and sterilize by autoclaving at 121°C for 15 minutes.

Description

The Rappaport Vassiliadis medium complies with the recommendations of the APHA for the examination of food.
This culture medium is the modificaction of the R10 me­dium (from Rappaport et cols) or RV broth (from Vas­siliadis et cols.)by van Schothort & Renaud. The modi­fications are an adjustement in the magnesium chloride concentration and a buffered reaction of the medium. It shows a higher selectivity towards Salmonella and produces better yields than other similar media, espe­cially after preliminary enrichment and at an incubation temperature of 43°C.
Malachite green and magnesium chloride inhibit the growth of the microorganisms normally found in the in­testine but do not affect the proliferation of most Salmo­nellae. Malachite green inhibits the growth of Shigella. Soy peptone improve the growth of Salmonella. The low pH of the medium increases the selectivity.

Technique

Inoculate the culture medium with the sample or mate­rial from a pre-enriched culture in Buffered Peptone Water (Ref. 02-277) and incubate for up to 18-24 hours at 41±1°C. Streak the sample material from the resulting cultures onto selective culture media.

Reinforced Clostridial Medium

Specification

Fluid medium for the cultivation and enumeration of clostridia by the MPN method.

Formula (in g/L)

Casein peptone ...........................................10,0
Yeast extract ...............................................3,0
Meat extract ...............................................10,0
Dextrose .....................................................5,0
Sodium chloride ..........................................5,0
Sodium acetate ............................................3,0
Soluble starch ..............................................1,0
L-Cysteine HCl ...........................................0,5
Agar ............................................................0,5
Final pH 6,8 ± 0,2

Directions

Suspend 38 g of powder in 1 L of distilled water and heat to boiling with constant stirring. Distribute into suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

Description

Reinforced Clostridial Agar was originally described by Hirsch and Grinstead to initiate the growth of small in­oculums and get a higher Clostridial count. Later, Barnes and Ingram used the medium to develop vegetative cells in assays of Clostridium perfringens. Barnes also used this medium to count clostridia in food, moreover other authors used this medium in enumeration assays of Cl. thermoscharolyticum in sugar, study of intestinal flora, and bacterial count in human or animal faeces, etc.
For the enumeration by the MPN method, the liquid ver­sion is the preferred one.

Technique

Material to be examined is ground in a Turmix or Stom­acher, and a decimal dilution bank is prepared. From each of the dilutions, take an aliquote to Petri plates or tubes, and pour the molten medium at 50°C over them. Let it solidify. Incubate at 30-55°C (depending on the microorganism that is anticipated to be found) for 1-10 days. An anaerobic environment can be achieved if tubes are used and they are covered with Sealing Anaer­obic Agar (Ref. 01-174) immediately after the Reinforced Clostridial Medium is solidified. If the plates are used, they have to be incubated in the anaerobic jars.
Muñoa and Parés added a filter sterilized solution of Nalidixic acid 0,02 g/L, Polymyxin 0,025 g/L, Kanamy­cin sulfate 0,05 g/L, Sodium iodoacetate 0,025 g/L and triphenyl-tetrazolium HCl 0,025 g/L to obtain a selective and differential medium for bifidobacteria in water and wastewater.

Sabouraud Chloramphenicol Agar

Specification

Solid culture medium for the isolation of fungi.

Formula (in g/L)

Casein peptone ...........................................5,0
Meat peptone .............................................5,0
D(+) Glucose ..............................................40,0
Chloramphenicol ..........................................0,5
Agar ...........................................................15,0
Final pH 5,6 ± 0,2

Directions

Suspend 65,5 g of powder in 1 L of distilled water and bring to the boil. Distribute into final containers and sterilize by autoclaving at 121°C for 15 minutes. Do not overheat or reheat the medium since it will affect the solidification.

Description

This culture medium differs from the classical Sabour­aud Agar only in the addition of Chloramphenicol. This thermostable antibiotic has a wide antibacterial spectrum which ensures the selective isolation of fungi from highly contaminated samples, such as eudates, faeces, nails and hair.

Sabouraud Dextrose Agar

Specification

Medium for the enumeration and cultivation of fungi.

Formula (in g/L)

D(+) Glucose ............................................40,0
Casein peptone ..........................................5,0
Meat peptone .............................................5,0
Agar ..........................................................15,0
Final pH 5,6 ± 0,2

Directions

Dissolve 65 g in 1 L of distilled water and bring to the boil with frequent stirring. Distribute into final containers and sterilize by autoclaving at 121°C for 15 minutes. Do not overheat the medium as its acidic pH may partially hydrolize the agar. Alternatively,if the European Pharma­copoeia formulation is desired, add before sterilization 50 mg/L of chloranphenicol (Ref. 06-118CASE)

Description

Sabouraud Dextrose Agar is a modification of the clas­sical Sabouraud medium for the cultivation of fungi. This new formula helps to maintain the morphological aspects of fungi and thus permits a reliable cultivation and dif­ferentiation.
Its selectivity is due to a low pH and a high glucose con­centration, which together with incubation at a relatively lower temperature (25-30°C) favours the growth of fungi while discouraging that of bacteria. Besides, the com­position of this peptone has been studied to provide the fungi with all their nitrogenated nutrient requirements.
Since the Sabouraud medium’s strong acid reaction partially hydrolyzes the agar, only the required amount should be prepared and it should not be remelted. Any overheating will considerably diminish its gelling capac­ity.
Should a higher selectivity be required, a variety of in­hibitors or selective agents may be added after steriliza­tion, while the medium is still in the molten form. It can even be made differential by adding the indicator agents. Some of the inhibitory and differential mixtures most commonly used are listed below:
Penicillin: at 20,000 units/litre, encourages the selectivity of the medium by inhibiting most of the bacteria.
Penicillin and Streptomycin: at 20,000 u/L and 40,000 u/l each, favours the isolation of Histoplasma in dogs.
Penicillin and Neomycin: at 20,000 u/L and 40 mg/L each, is used for the isolation of yeast.
Streptomycin and Chloramphenicol: at 40 mg/L and 500 mg/L each, for the isolation of Trichophyton ver­rucosum
Colistin, Novobiocin and Cycloheximide: at 8 mg/L, 0.1 mg/L and 30 mg/l each, for the isolation of Can­dida albicans .
Potassium Tellurite: at 150 mg/L, is used for the primary isolation of fungi from scales and scabs.
Cupric Sulfate, Crystal Violet and Brilliant Green: at 500 mg, 2 mg and 5 mg each, achieves considerable bacterial inhibition.
Triphenyltetrazolium chloride (TTC): at 100 mg/L, it is the basis of a Pagano-Levin medium for the isola­tion of Candida albicans, unpigmented, among other pathogenic yeast which form pink coloured colonies.

Selenite Broth Base

Specification

Liquid medium for Salmonella and Shigella enrichment.

Formula (in g/L)

Peptone ....................................................5,00
Lactose .....................................................4,00
Potassium phosphate .............................10,00
Final pH 7,0 ± 0,2

Directions

Dissolve 19 g of powder. in 1 L of distilled water and add 4 g of sodium biselenite (Ref. 06-615). Homogenize and bring to the boil. Distribute in suitable containers. Termo­labile medium: Use immediately. Do not autoclave.

Description

Selenite Broth is formulated according to an original formulation by Leifson for selective enrichment of Salmo­nellae from very contaminated samples.
Enrichment is especially effective during the first 12 hours of cultivation, since in this period it seems that only Salmonellae, some Proteus and some strains of Pseudomonas grow easily. For this reason, it is advis­able not to extend the enrichment phase and go quickly for the selective medium, either liquid or solid. According to Bänffer, the efficacy of the medium is improved nota­bly if enrichment is performed at 43°C. Presence of a red precipitate in the medium before inoculation, indicates 138 that there was a overheating in which case the selective properties of the medium are reduced.

Thioglycollate Broth (USP Alternative Thioglycollate Medium)

Specification

A medium for sterility test and the cultivation of microaer­ophilic and anaerobic organisms. It is specially used for viscous or turbid samples.

Formula (in g/L)

Peptone from casein ...................................15,0
Yeast extract ...............................................5,0
Dextrose .....................................................5,5
Sodium chloride ..........................................2,5
Sodium thioglycollate ...................................0,5
L-Cystine ....................................................0,5
Final pH 7,1 ± 0,2

Directions

Dissolve 29 g of powder in 1 L of distilled water, heating if necessary to help dissolution. Distribute into suitable containers and sterilize by autoclaving at 121°C for 15 minutes. This culture medium should always be freshly prepared or heated at 100°C for 10 minutes before use.

Description

The Thioglycolate broth is a standard medium, named also Alternative Thioglycollate Medium, formulated and recommended by USP, NF, NIH and FDA.
It is used for sterility testing of biological products or samples of turbid appearance where Fluid Thioglycol­late Medium (Ref.3-187) is not suitable because of its viscosity.
The formula of Thioglycollate broth is the same as Thioglycollate USP Fluid Medium without resazurin and agar.
Media must be freshly prepared, boiled, sterilised, cooled and used within 4 hours for its inoculation.

Tetrathionate Broth Base

Specification

Medium for the selective enrichment of Salmonellae (AOAC 17th, ICMSF 1968, USP 25th)

Formula (in g/L)

Meat peptone .............................................2,5
Casein peptone ..........................................2,5
Bile salts .....................................................1,0
Calcium Carbonate ...................................10,0
Sodium Thiosulfate ...................................30,0

Directions

Suspend 46 g of powder to 1 L of distilled water, heat to boiling and cool to 40-45°C. Add 20 mL of iodine-iodide solution and 2 vials of the Selective Supplement of Bril­liant Green-Novobiocin ref. 06-017CASE and distribute in sterile tubes.
Do not heat after adding the iodine solution. Medium must be used immediately. Without the iodine solution, medium can be stored in refrigeration for some days. The appearance of white medium precipitate is normal, and it comes from calcium carbonate.

Description

This is the version originally used, which has been modi­fied or improved the Muller-Kauffmann formulation, since the latter one has more efficacy.

Triple Sugar Iron Agar (TSI Agar)

Specification

Differential medium for identification of enterobacteria, according ISO standard.

Formula (in g/L)

Peptone ................................................20,000
Meat extract ............................................3,000
Yeast extract ...........................................3,000
Lactose .................................................10,000
Sucrose ................................................10,000
Dextrose .................................................1,000
Sodium chloride ......................................5,000
Ferric ammonium citrate .........................0,500
Sodium thiosulfate ..................................0,300
Phenol red ..............................................0,025
Agar .....................................................12,000
Final pH 7,4 ± 0,2

Directions

Dissolve 64,8 of powder in 1 L of distilled water and bring to boiling. Dispense into tubes and sterilize at 121°C for 15 minutes. Leave to solidify with short slant and good butts.

Description

TSI Agar is a modification of the classical Kliger’s agar. 1% of sucrose has been added to this medium to dif­ferentiate Proteus and Hafnia (sucrose positive) from Salmonella and Shigella (sucrose negative).
Sugar degradation with acid formation is detected by the turning of an indicator (phenol red) to yellow, whereas if there is alkalinization, it turns to purple. When there is only glucose degradation, the acid production is weak and is evaporated on the surface, so indicator may be reoxidised producing an alkaline surface (red) and an acid butt (yellow). If lactose or sucrose are degradated, acid production is intense and then all of the medium (surface and depth) turns yellow. Gas production is detected by the formation of bubbles and occasionally cracks in the agar.
Hydrogen sulfide production, from thiosulfate or sulfured aminoacids of peptones, is detected by the formation of black FeS precipitate when medium reacts with iron salts.
Use the medium in slanted tubes with good depth and short slant. Inoculate by streaking on surface and stabbing deeply. It is advisable to use tubes with cotton plugs, in order to allow a reoxidation of the indicator. If screw caps are used, they must be loose.
Following will find the table of reading (observations) and interpretation of results in TSI Agar.

Tryptic Soy Agar (TSA) (Casein Soybean Digest Agar)

Specification

General purpose solid medium containing animal and plant peptone according ISO 9308-1 standard.

Formula (in g/L)

Casein peptone ..........................................15,0
Soy peptone ...............................................5,0
Sodium chloride ..........................................5,0
Agar ......................................................... 15,0
Final pH 7,3 ± 0,2

Directions

Mix 40 g of powder in 1 L of distilled water. Let it soak and bring to the boil to dissolve the agar. Sterilize by autoclaving at 121°C for 15 minutes.

Description

TSA is a widely used medium containing two peptones which support the growth of a wide variety of organisms, even that of very fastidious ones such as Neisseria, Listeria, Brucella, etc. It is frequently used for routine diagnostic purposes due to its reliability and its easily reproducible results.
The following list includes some of its most common applications:
1.- Sensitivity testing either by the Kirbky-Bauer system or by following the WHO guidelines. Both the sys­tems recommend the use of the Mueller Hinton Agar (Ref. 01-136) for verification purposes.
2.- The medium provides, with added blood,perfectly de­fined hemolysis zones, while preventing the lysis of the erythrocytes due to its sodium chloride content.
3.- It can be used for the preparation of an exceptionally nutrient ‘chocolate´ agar, thanks to the richness of its peptones.
4.- In a reducing environment or with a CO2 enriched atmosphere, its plates provides an excellent medium for the isolation of Brucella and Neisseria . It may be made selective by using certain additives.
5.- Most streptococci grow in this medium though clear differences can be observed from one species to another.
6.- The Tryptic Soy Agar is the selective medium for the count of urine samples although the differentiation must be done on selective differential media.
7.- Several tests for the differentiation and identification of staphylococci can be obtained in this medium, provided with suitable additives.
8.- Yeast, particularly Candida species, can grow in this medium forming very characteristic colonies.
9.- Chromogenic pseudomonads frequently produce pigmentation on the TSA and are therefore easily recognized.
10.- It is widely used for testing contaminated samples. A vast bibliography documents its applications in the food industry.
11.- It has been frequently used in the Health industry to produce antigens, toxins,etc...
12.- Its simple and inhibitors-free composition makes it suitable for the detection of antimicrobial agents in food and other products.
13.- A balanced and highly nutrient value together with a lack of fermentable carbohydrates make this medium one of the most recommended for the strain mainte­nance.

Tryptic Soy Broth (TSB) (Casein Soybean Digest Broth)

Specification

Highly nutrient liquid medium for the general purposes, formulated according to USP, FDA and Eur. Phar. regula­tions.

Formula (in g/L)

Casein peptone ..........................................17,0
Soya peptone .............................................3,0
Sodium chloride ..........................................5,0
Dipotassium phosphate ................................2,5
Dextrose .....................................................2,5
Final pH 7,3 ± 0,2

Directions

Dissolve 30 g of powder in 1 L of distilled water and sterilize by autoclaving at 121°C for 15 minutes.

Description

The Tryptic Soy Broth was initially developed for the cultivation of very fastidious microorganisms without the addition of serum, blood or any other enrichment agent.
As a general purpose culture medium it supports the growth of most organisms, both aerobes and faculta­tives, even if their requirements are high. Due to its high vitamin content the development of BrucellaPasteurel­la and Streptococcus is perfectly viable, moreover a CO2 enriched atmosphere can further favour it.
In anaerobic conditions this broth will easily bear the growth of Bacteroides and Clostridium species. For this purpose, the best results can be obtained by adding 0.3% agar and 0.05% sodium azide for Clostridium.
The Tryptic Soy Broth’s superior growth-promoting prop­erties makes it particularly suitable for the tube dilution method for antibiotic sensitivity testing. It also achieves good results in the detection of gram-positive cocci. The broth can be used for bile solubility testing in pneumo­cocci, and also used for catalase and coagulase assays and for the preparation of hypersaline broths.
It is a most suitable medium for the preparation of anti­gens and toxins in bacteria, moulds and yeasts.
TSB is used as a primary enrichment medium for food examination. In the dairy industry it is employed for test­ing resazurine reduction.
The medium is not suitable for maintenance purposes since carbohydrate fermentation liberates many acids which may threaten the organisms’ viability. Therefore, though it allows the growth of streptococci and Neisse­ria, these species tend to die if repeatedly subcultured in this medium. Such fastidious organisms are best main­tained on Cystine Tryptone Fluid Medium (CTA) (Ref. 03-045) or even TSA (Ref. 01-200) if it is not suitable or convenient to the use of solid media.

Urea Broth Base

Specification

Liquid diagnostic medium according to Rustigian and Stuart formulation.

Formula (in g/L)

Monopotassium phosphate ........................9,10
Disodium phosphate ..................................9,50
Yeast extract .............................................0,10
Phenol red .................................................0,01
Final pH 6,8 ± 0,2

Directions

Dissolve 19 g of powder into 950 mL of distilled water and sterilize by autoclaving at 121°C for 15 minutes. Let it cool to 50-55°C and then add 50 mL of Urea Sterile Solution 40% (Ref. 06-083). Mix well and dispense in hemolysis tubes (3,0 mL/tube).

Description

According to Rustigian and Stuart, this Urea Broth is excellent for diagnosing enterobacteria, since within this family, only Proteus may alkalinize the medium over pH 8,1. Despite the fact that some authors prefer a buffer of potency 10 or 100 times lower to obtain faster results for saving the time (about 2 hours) does not compensate for the instability of the medium.
Urease production is shown by the indicator turning to dark pink, produced by strong alkalinization by ammo­nium. With plenty of inoculum (2-3 loops in 3-5 mL of medium), Proteus produces the colour change after 6-8 hours, meanwhile other positive enterobacteria need up to 24-48 hours.

Violet Red Bile Dextrose Agar

Specification

Solid medium for the enumeration of enterobacteria ac­cording ISO 8523 standard.

Formula (in g/L)

Yeast extract ...........................................3,000
Gelatin peptone ......................................7,000
Bile salts #3 ............................................1,500
D (+) Glucose .......................................10,000
Sodium chloride ......................................5,000
Neutral red ..............................................0,030
Crystal violet ...........................................0,002
Agar ......................................................13,000
Final pH 7,4 ± 0,2

Directions

Suspend 39,5 g in 1 L of distilled water and let it soak. Bring to the boil and sterilize by autoclaving at 121°C for 15 minutes. If the medium is to be used on the same day of preparation it need not be sterilized. Prolonged heat­ing in thermostatic bath could cause slight precipitates.

Description

This medium is a modification of the Violet Red Bile Agar (Ref.1-164) and the MacConkey Agar (Ref.1-118) as described by Mossel et al. These authors proved that the addition of glucose to the Violet Red Bile Agar favoured both the growth of the most fastidious enterobacteria and the recovery of those having suffered from adverse conditions. Later on, Mossel himself realized that by removing the lactose and keeping the glucose, the medium’s efficiency remained stable. Furthermore, an economic improvement occurred since the same amount of product allows the reconstitution of more litres of the medium.

 Technique

Sample is diluted 1:10 in Lactose Broth (Ref. 02-105) and incubate 2-5 hours at 35-37 ºC. Then a volume of this pre-enrichment is ten fold dilute in EE Broth (Ref. 02-064) and incubate at 35-37ºC for 18-24 hours. From this enrichment the surface of several plates of VRBDL Agar are inoculated. The product passes the test if after 18-24 hours of incubation at 35-37ºC there is no growth of gram negative bacteria in any plate.
In the surface of the VRBDL Agar the Enterobacteriace­ae colonies are deep purple in colour surrounded by a clearing zone. Sometimes are present little colonies from Pseudmonas or Aeromonas that can be easy differenti­ated by the oxidase test.

Vogel Johnson Agar (VJ Agar)

Specification

Solid and very selective medium for isolation and identifi­cation of staphylococci according ISO 22718 standard.

Formula (in g/L)

Casein Peptone .......................................10,000
Yeast Extract ...........................................5,000
Mannitol .................................................10,000
Dipotassium phosphate ............................5,000
Litium chloride ........................................5,000
Glycine ...................................................10,000
Phenol Red .............................................0,025
Agar ......................................................15,000
Final pH 7,2 ± 0,2

Directions

Suspend 60 g of powder in 1 L of distilled water and bring to the boil. Dispense in suitable containers and sterilize at 121°C for 15 minutes. Cool it to 50°C approx. and add aseptically 20 mL of Potassium Tellurite Solu­tion 1% (Ref. 06-089) or 6,0 mL of Potassium Tellurite Solution 3.5% (Ref. 06-011). Do not reheat after tellurite addition.

Description

VJ Agar is a selective medium for detection and enu­meration of pathogenic staphylococci.
The medium’s strong selective action is due to lithium chloride, glycine and potassium tellurite presence. They inhibit almost all the accompanying organisms, mean­while staphylococci are not affected. Although staphy­lococci may reduce tellurite to tellurium, lithium may per­form some action that is compensated by glycine.
Moreover a high correlation between tellurite reduction and mannitol fermentation has been proved, and this is shown in the medium by the indicator turning to yellow due to the amount of acid produced.
The medium’s selectivity avoids, in the first 24 hours, the development of any other bacteria, so massive inocu­lation is permited. Nonetheless, after this period, it is possible that other bacteria may appear like micrococci, which produce tiny colonies, and staphylococci that ferment mannitol and coagulase negative, therefore it is recommended to verify this last test separately.
Due to reduced tellurite, staphylococci generally appear as black colonies over red medium (if they do not fer­ment mannitol) or yellow medium (if they do, and these are presumptive pathogen). Saprophytic staphylococci (S.epidermidis, S.saprophiticus and S.intermedius) have a grey-black colour and are mannitol negative. Complete medium may be stored up to 1 week in the refrigerator. Do not remelt it after tellurite is added.

Xylose Lysine Deoxycholate Agar (XLD Agar)

Specification

Solid medium for the isolation of enteropathogenic species, especially Salmonella according to ISO 6340 standard.

Formula (in g/L)

Xylose .......................................................3,50
L-Lysine ....................................................5,00
Lactose .....................................................7,50
Sucrose .....................................................7,50
Sodium chloride .........................................5,00
Yeast extract .............................................3,00
Phenol red .................................................0,08
Sodium Deoxycholate ................................2,50
Sodium thiosulfate ......................................6,80
Ammonium ferric citrate..............................0,80
Agar ........................................................15,00
Final pH 7,4 ± 0,2

Directions

Suspend 56,68 g of powder in 1 L of distilled water. Heat up constantly with stirring until boiling. Pour it immediate­ly into plates. Do not autoclave and avoid remelting.

Description

Xylose Lysine Deoxycholate Agar is a differential me­dium, slightly selective, very suitable for the detection of pathogenic enterobacteria, especially Shigella. Gram negative flora is inhibited by the low amount of deoxy­cholate, but Shigella grows easier in this medium than in any other selective media.
Xylose, lactose or sucrose fermentation produce the acidification of the medium, and this is seen by an indicator turning to yellow, surrounding the colonies. This colour disappears after 24 hours, so observations must be carried out between 18 and 20 hours.
Hydrogen sulfide production from thiosulfate is easily de­tected because colonies become darker, due to the ferric sulfure precipitate. Lysine decarboxylation to cadaverine may also be observed in the medium, since it produces alkalinization and consequently the indicator turns to red.
All these reactions allow a good differentiation of Shig­ella, which besides Edwardsiella and Proteus inconstans are the single enterobacteria that do not ferment xylose and therefore show negative fermentation reaction. Salmonella type members do ferment xylose, but it is consumed quickly and then alkalinization of the medium, due to lysine decarboxylation, may mask the reaction. The difference between Shigella and Salmonella is that with the latter colonies become darker due to ferrous sulfure precipitates, and this is a common property with Edwardsiella. The other types of enterobacteria do not suffer this phenomenon, since acid acumulation due to lactose and sucrose fermentation is so high that it avoids pH reversion by decarboxylation and even ferrous sul­fure precipitate in the first 24 hours.
In the table below, typical colonial appearances on XLD medium after 24-36 hours of incubation at 37°C are described.

Yeast Extract Agar

Specification

Solid medium for the enumeration of microorganisms from water.

Formula (in g/L)

Tryptone ......................................................5,0
Yeast extract ................................................3,0
Agar ...........................................................15,0
Final pH 7,2 ± 0,2

Directions

Suspend 23 g of powder in 1 L of distilled water and bring to the boil. Distribute into suitable containers and sterilize by autoclaving at 121ºC for 15 minutes.

Description

This medium, formulated according to Windle Taylor, is the most used in the UK for the enumeration of hetero­trophic microorganisms from water. Distinction between bacteria, yeast and filamentous fungi must be carried out by morphology after differential incubations at 35º and 20ºC.

Technique

From the water sample, make a decimal dilution bank with Ringer Solution (Ref. 06-073) and take aliquotes to 2 parallel series of plates. Pour the Yeast Extract Agar, molten and cooled to 45ºC, and homogenize with sam­ple. Once solidified, incubate one of the series at 35ºC for 24 hours and the other one at 20ºC for 3 days.
In order to achieve a good count, select the plates with 30-300 colonies.
Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
Email: .moc.enilediugamrahp@ofni Need Help: Ask Question


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