Agars - the definitive guide

SUMMARY:

 

CYSTINE LACTOSE ELECTROLYTE DEFICIENT (CLED) AGAR

• Recommended for diagnostic urinary bacteriology. Gives clear colonial differentiation and clear diagnostic characteristics.
• The absence of electrolytes inhibits the swarming of Proteus species. Cystine is added for organisms that require it.
• Differentiation of lactose-fermenters and lactose non-fermenters is achieved using bromothymol blue and a pH indicator.
• The medium supports the growth of Streptococcus pyogenes and other fastidious organisms that do not require blood.

FASTIDIOUS ANAEROBE AGAR/ NEOMYCIN FASTIDIOUS ANAEROBE AGAR

• A primary isolation medium capable of growing clinically significant anaerobes.
• Starch and sodium bicarbonate act as de-toxification agents.
• Haemin, cysteine, arginine and soluble pyrophosphate are growth promoting agents. Haemin also encourages pigment production in some anaerobes. Pyruvate helps neutralise hydrogen peroxide and is utilised by some anaerobes as an energy source.
• Vitamin K, sodium succinate and 0.1% glucose are essential growth factors for some anaerobes. A low level of glucose prevents the production of high levels of acids and alcohols which would inhibit colonial development.

GC SELECTIVE AGAR (NEW YORK CITY)

• A selective nutritious medium for the isolation of Neisseria gonorrhoeae and Neisseria meningitidis. The medium is enriched by the addition of lysed blood.
• Yeast autolysate is added as a growth supplement. Starch absorbs toxic metabolites. Phosphates act as buffers to maintain a neutral pH.

SABOURAUD AGAR

• Selective medium for the cultivation of pathogenic and nonpathogenic fungi, particularly dermatophytes, and yeasts.
• The acidic pH (5.6) of the medium inhibits many species of bacteria. The addition of chloramphenicol makes the medium more selective. Diagnostic features such as sporing structures and pigmentation are well developed on this medium

HOYLE’S TELLURITE AGAR

• For isolating, differentiating and identifying Corynebacterium diphtheriae.
• It allows for the rapid growth of all C. diphtheriae types. Other organisms are generally inhibited.
• The medium is enriched by the addition of horse blood and made selective by the addition of potassium tellurite.

PSEUDOMONAS SELECTIVE AGAR

• A selective and differential medium for the isolation of Pseudomonas species.
• Magnesium chloride and potassium sulphate enhance pyocyanin production for the improved detection and differentiation of Pseudomonas species. Pyocyanin is a blue-green pigment that diffuses into the medium around colonies of Pseudomonas aeruginosa.
• Cetrimide is inhibitory to a wide variety of organisms including species of Pseudomonas other than P. aeruginosa.

BURKHOLDERIA CEPACIA SELECTIVE AGAR

• A selective medium for the isolation of Burkholderia cepacia.
• The medium contains bile salts and crystal violet as selective agents. Selective supplements ticarcillin and polymixin B are also added to the medium.

MANNITOL SALT AGAR

• Selective medium for the isolation of staphylococci.
• The high sodium chloride level inhibits most other organisms except some halophilic organisms such as Vibrio species. Most Staphylococcus aureus strains ferment mannitol and produce yellow colonies. The addition of egg yolk emulsion enables the lipase activity of staphylococci to be detected. The high salt concentration in the medium clears the egg yolk emulsion and lipase production is detected as a yellow opaque zone around colonies that produce the enzyme.

Organism	Colour of colony	Comments
S. aureus	Bright yellow
Other staphylococci	White or yellow	Some ferment mannitol
Enterobacteriaceae	No growth
Vibrio species and other halophiles	Usually pink	Yellow if ferment mannitol

CHARCOAL BLOOD AGAR WITH CEPHALEXIN

• With the addition of blood this medium is used to isolate Bordetella pertussis.
• The addition of cephalexin makes the medium more selective and inhibits most other organisms. Cephalexin increases the recovery rate by selecting for B. pertussis and allows for a greater recovery of stressed cells.

HYNE’S DESOXYCHOLATE CITRATE (DCA) AGAR

• Hyne's modification of desoxycholate citrate agar (DCA) has twice the amount of inhibitors as the original formulation by Liefson. It is a selective medium for the isolation of Salmonella species and Shigella species.
• Sodium citrate and sodium desoxycholate are inhibitors.
• Sodium thiosulphate is the substrate for the enzyme thiosulphate reductase being broken down to form sulphite and hydrogen sulphide. The hydrogen sulphide reacts with the ferric ions to produce a black precipitate of ferrous sulphide. This gives a black centre in colonies of hydrogen-producing Salmonella species. Lactose fermentation is used as a differential indicator.
• Lactose-fermenting organisms produce pink colonies and may be surrounded by a zone of precipitated desoxycholic acid which is due to acid production.
• Colonies of lactose nonfermenters are colourless and due to their alkaline reaction they are surronded by a clear orange-yellow zone of medium.

Organism	Colour of colony	Comments
Salmonella species	Colourless	Black centre
Shigella species	Colourless, pale pink
Proteus species	Colourless
Pseudomonas species	Colourless	Green pigment

XYLOSE LYSINE DESOXYCHOLATE (XLD) AGAR

• A selective and differential medium for the recovery of Salmonella and Shigella species.
• It is low in nutrients and contains a small amount of sodium desoxycholate for selectivity. Most enteric organisms except Shigella ferment xylose to produce acid.
• Salmonella also decarboxylate lysine which keeps the pH neutral or slightly alkaline. At this pH Salmonella species can produce hydrogen sulphide from the reduction of thiosulphate. This is indicated by ferric ammonium citrate producing black or black-centred colonies.
• Some organisms, such as Citrobacter, can also decarboxylate lysine. However, they ferment lactose and sucrose which keeps the pH too acid for hydrogen sulphide to be produced.

Organism	Colour of colony	Comments
Salmonella	Red colonies, black centre
Shigella	Red colonies
Escherichia coli	Yellow	Precipitate around colonies; some strains are inhibited
Citrobacter	Yellow	Black centre
Proteus	Red colonies, black centre	Fishy odour
Providencia	Red colonies
Arizona	Red colonies
Edwardsiella	Black centres

 

CEFSULODIN IRGASAN NOVOBIOCIN (CIN) AGAR

• A differential and selective medium for the isolation of Yersinia enterocolitica.
• Fermentation of mannitol in the presence of neutral red produces characteristic "bull's-eye" colonies. These are colourless with a red centre. A zone of precipitated bile may also be present.
• Crystal violet, sodium desoxycholate, cefsulodin, Irgasan (triclosan) and novobiocin are inhibitory agents.
• Typical Y. enterocolitica colonies will have deep-red centres surrounded by a transparent border giving the appearance of a "bull's-eye". Other Yersinia species and Enterobacteriaceae grow on the medium and may look similar to Y. enterocolitica.

THIOSULPHATE CITRATE BILE SALT SUCROSE (TCBS) AGAR

• A selective isolation medium for pathogenic Vibrio species.
• Most Enterobacteriaceae other than Vibrio species are suppressed for at least 24h. Bile salts inhibit Gram-positive organisms. Sodium thiosulphate serves as a source of sulphur which, in combination with ferric citrate, detects hydrogen sulphide production.
• When sucrose is fermented it produces acid which changes the pH. This is indicated by bromothymol blue and thymol blue. The medium is alkaline which enhances the recovery of Vibrio cholerae.

Organism	Colour of colony	Diameter of colony (mm)
V. cholerae	Yellow	2-3
Vibrio parahaemolyticus	Blue-green	3-5
Vibrio alginolyticus	Yellow	3-5
Vibrio metschnikovii	Yellow	3-4
Vibrio fluvialis	Yellow	2-3
Vibrio vulnificus	Blue-green	2-3
Vibrio mimicus	Blue-green	2-3
Enterococcus species	Yellow	1
Proteus species	Yellow-green	1
Pseudomonas species	Blue-green	1

CLOSTRIDIUM DIFFICILE CEFOXITIN CYCLOSERINE EGG-YOLK (CCEY) AGAR

• A selective medium for the isolation of Clostridium difficile from faecal specimens.
• D-cycloserine and cefoxitin are used as selective agents. These inhibit most Enterobacteriaceae, enterococci, staphylococci, Gram-negative non-sporing anaerobic bacilli and Clostridium species, except C. difficile.
• This medium incorporates cholic acid (sodium salt) to enhance spore germination after alcohol treatment.

CAMPYLOBACTER SELECTIVE AGAR

• Two choices of Campylobacter selective agar are recommended for clinical specimens - modified CCDA and Skirrow’s medium.
• Modified CCDA with casein hydrolysate is recommended for the isolation of Campylobacter species from food and water samples.
• Modified CCDA:
  • A blood free medium which will support the growth of enteric Campylobacter species.
  • The selective supplements cefaperazone and amphotericin make the medium selective for Campylobacter jejuni and Campylobacter laridis when incubated at 37^oC. Incubation at 42^oC is no longer necessary and higher recovery rates have been reported at 37^oC.
  • Blood is replaced in the medium with charcoal, ferrous sulphate and sodium pyruvate which enhance the growth and aerotolerance of Campylobacter species.
• Skirrow’s medium:
  • A selective medium which will support the growth of enteric Campylobacter species and suppress faecal flora.
  • Members of the Campylobacter genus requires a wide spectrum of atmospheres for optimum growth.
  • Skirrow antibiotic supplement is designed to be used at 42^oC for optimum selective effect.

BAIRD PARKER AGAR

• A selective medium for the isolation of staphylococci from food samples.
• Sodium pyruvate protects damaged cells and aids their recovery. Egg yolk emulsion assists in the diagnosis of organisms.
• Glycine, lithium and tellurite suppress the growth of other bacteria in foods without inhibiting Staphylococcus aureus.
• S. aureus reduces tellurite to form grey-black shiny colonies and produces clear zones around the colonies due to the proteolytic activity on egg yolk. On further incubation most S. aureus strains produce opaque zones around their colonies due to lipase activity.

Organism	Colour of colony	comments
S. aureus	Grey-black, shiny colony.	Surrounded by a zone of clearing.
Staphylococcus epidermidis	Not shiny black colony	Seldom produce clearing
Staphylococcus saprophyticus	Irregular colony and may produce clearing	Wide opaque zones may be produced in 24h
Micrococcus	Very small colony in shades of brown and black.	No clearing
Bacillus species	Dark matt brown colony with occasional clearing after 48h
Escherichia coli	Large brown-black colony.
Proteus	Brown-black colony	No clearing

CEFIXIME TELLURITE SORBITOL MACCONKEY (SMAC) AGAR

• A selective medium for the isolation of Escherichia coli O157.
• The formulation is based on a modified MacConkey agar and contains 1% D-sorbitol instead of lactose. E. coli O157 is not capable of fermenting sorbitol within 24h.
• The medium is recommended for screening only as other organisms will be found to be non-sorbitol fermenting.
• The medium is made selective by the addition of cefixime and potassium tellurite which suppress other non-sorbitol fermenting organisms with no inhibitory effects on E. coli O157.

ACTINOMYCES SELECTIVE AGAR

• A selective medium for the isolation of Actinomyces species.
• The medium consists of blood agar to which a selective supplement (Metronidazole 10mg/l & Nalidixic acid 30mg/l) is added.

TRYPTOSE SULPHITE CYCLOSERINE (TSC) AGAR

• Selective medium for the isolation and enumeration of vegetative and spore forms of Clostridium perfringens in food and water samples.
• Sodium metabisulphite and ferric ammonium citrate are used as an indicator of sulphite reduction by C. perfringens which produces black colonies. Some strains may produce an opaque zone around the colony due to lecithinase production.

TRYPTONE BILE X-GLUCURONIDE (TBX) AGAR

• A chromogenic medium to aid differentiation between Escherichia coli and other coliforms in food samples.
• The chromogen 5-bromo-4-chloro-3indolyl-β-D-glucuronide (BCIG) is targeted by the enzyme glucuronidase produced by most E. coli strains. The enzyme splits BCIG to release a coloured chromophore which builds up inside the cells causing colonies of E. coli to appear blue/green. Other coliforms which are glucuronidase negative appear colourless.

MANNITOL LYSINE CRYSTAL VIOLET BRILLIANT GREEN (MLCB) AGAR

• Selective medium for the isolation of Salmonella species (not Salmonella typhi or Salmonella paratyphi A) from food samples. MLCB is not suitable for S. typhi or S. paratyphi A due to the inhibitory concentration of brilliant green.
• Salmonella species appear as large purple-black colonies due to hydrogen sulphide production. Mannitol is utilised by the organism causing a drop in the pH which initiates lysine decarboxylation. This is turn causes a further drop in pH and promotes blackening of colonies.

LISTERIA SELECTIVE AGAR

• Inoculate by spreading the sample on the surface of the medium and incubate at 35 °C up to 48 h aerobically.
• Listeria monocytogenes grows as brown-green colored colonies with a black halo (esculin splitting).
• Further biochemical tests should be carried out.

GARDENERELLA AGAR

BRAIN-HEART INFUSION BROTH

ROBERTSON'S COOKED MEAT BROTH

TRICHOMONAS BROTH

GROUP B STREPTOCOCCUS BROTH

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