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, S. typhi 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
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 Fusarium, Aspergillus 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 Brucella, Pasteurella 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.
Wow excellent stuff
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