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Culture Media Supplements

WILKINS-CHALGREN ANAEROBE AGAR

Code: CM0619

A medium for the general growth of anaerobes, recommended for antimicrobial susceptibility testing. See Antimicrobial Susceptibility Testing Section for details of the use of this medium in AST methodology.

Typical Formula*

gm/litre

Tryptone

10.0

Gelatin peptone

10.0

Yeast extract

5.0

Glucose

1.0

Sodium chloride

5.0

L-Arginine

1.0

Sodium pyruvate

1.0

Menadione

0.0005

Haemin

0.005

Agar

10.0

pH 7.1 ± 0.2 @ 25°C

 
* Adjusted as required to meet performance standards

Directions
Suspend 43g in 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes.

Description
Recognising the need for a standard medium for antimicrobial susceptibility testing of anaerobic bacteria, Wilkins and Chalgren1 developed a new medium which would not require the addition of blood. Their formulation included yeast extract to supply vitamins and other growth factors such as purines and pyrimidines, that are necessary for good growth of Peptostreptococcus anaerobius and Prevotella melaninogenica. Arginine was added to ensure sufficient amino acid was available for the growth of Eubacterium lentum. Pyruvate was added as an energy source, for asaccharolytic cocci such as Veillonella2. It also acts similarly to catalase and degrades traces of hydrogen peroxide, which may be produced by the action of molecular oxygen on medium constituents and interfere with the metabolism of anaerobes3. Haemin was found to be essential for the growth of Bacteroides species4 and menadione for Prevotella melaninogenica5.

Peptones derived from the single protein sources casein and gelatin, were used to improve standardisation of the medium. Wilkins and Chalgren1 considered that this medium consistently grew anaerobes as well or better than media such as Brucella Agar or Schaedler Anaerobe Agar. A collaborative study in ten laboratories showed that it could be used in an agar dilution method for susceptibility testing of anaerobic bacteria and recommended a procedure as a reference method 6.

The value of such a procedure was further confirmed by Brown and Waatti7, who found that the incidence of resistance of anaerobic bacteria to frequently used antibiotics had increased. They considered it essential that diagnostic laboratories should have the capability of carrying out susceptibility tests on anaerobic bacteria.

Wilkins-Chalgren Agar (CM0619) has been recommended for the susceptibility testing of anaerobic bacteria using the Receiver Operating Characteristic (ROC) procedure.

Wilkins-Chalgren Anaerobe Agar is also recommended for the isolation of anaerobic organisms from clinical specimens. It has been shown to function well both in Petri dishes and roll tubes. (B.S. Drasar, personal communication).

References
1. Wilkins T. D. and Chalgren S. (1976) Antimicrob. Agents Chemother. 10. 926-928.
2. Rogosa M. (1964) J. Bacteriol. 87. 162-170.
3. Hoffman P. S., George H. A., Kreig N. R. and Smibert R. A. (1979) Can. J. Microbiol. 25. 8-16.
4. Quinto G. and Sebald M. (1964) Am. J. Med. Technol. 30. 381-384.
5. Gibbons R. J. and MacDonald J. B. (1960) J. Bacterio. 80. 164-170.
6. Sutter V. L., Barry A. L., Wilkins T. D. and Zabransky R. J. (1979) Anti-Microb. Agents Chemother. 16. 495-502.
7. Brown W. J. and Waatti P. E. (1980) Antimicrob. Agents Chemother. 17. 629-635.
8. Castel O., Grollier G., Agius G. et al (1990) Eur. J. Clin. Microbiol. Inf. Dis. 9. 667-671.
9. Drasar B.S. Personal communication.


N-S ANAEROBE SELECTIVE SUPPLEMENT

Code: SR0107

For the selective isolation of non-sporing anaerobes.

Vial contents (each vial is sufficient for 500 ml of medium)

per vial
per litre

Haemin

2.5mg

5.0mg

Menadione

0.25mg

0.5mg

Sodium pyruvate

500mg

1000mg

Nalidixic acid

5.0mg

10.0mg

G-N ANAEROBE SELECTIVE SUPPLEMENT

Code: SR0108

For the selective isolation of Gram-negative anaerobes.

Vial contents (each vial is sufficient for 500ml of medium)

per vial
per litre

Haemin

2.5mg

5.0mg

Menadione

0.25mg

0.5mg

Sodium succinate

1.25g

2.5g

Nalidixic acid

5.0mg

10.0mg

Vancomycin

1.25mg

2.5mg


Directions
To prepare Non-Selective Medium for all anaerobic organisms
Suspend 21.5g of Wilkins-Chalgren Anaerobe Agar (CM0619) in 475ml of distilled water. Bring to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 50°C and aseptically add 25ml defibrinated blood (SR0050/SR0051). Mix gently, and pour into sterile Petri dishes (see plate 1).

To prepare Selective Medium for Non-Sporing Anaerobes
Suspend 21.5g of Wilkins-Chalgren Anaerobe Agar (CM0619) in 475ml of distilled water containing 0.5ml `Tween 80’. Bring to the boil to dissolve completely and sterilise by autoclaving at 121°C for 15 minutes. Cool to 50-55°C and aseptically add the contents of 1 vial of N-S Anaerobe Supplement (SR0107) rehydrated as directed, together with 25ml of defibrinated blood (SR0050/SR0051). Mix gently and pour into sterile Petri dishes (see plate 2).

To prepare Selective Medium for Gram-Negative Anaerobes
Suspend 21.5g of Wilkins-Chalgren Anaerobe Agar (CM0619) in 475ml of distilled water. Bring to the boil to dissolve completely and sterilise by autoclaving at 121°C for 15 minutes. Cool to 50-55°C and aseptically add the contents of 1 vial of G-N Anaerobe Supplement (SR0108) rehydrated as directed, together with 25ml defibrinated blood (SR0050/SR0051). Mix gently and pour into sterile Petri dishes (see plate 3).

Wilkins-Chalgren Anaerobe Agar (CM0619) is recommended but other media may be used satisfactorily, e.g. Columbia Agar Base (CM0331) and Blood Agar Base No.2 (CM0271). Sufficient haemin and menadione are contained in N-S and G-N Supplements to provide adequate levels in these media when used as directed.

Note
Use N-S Supplement with Wilkins-Chalgren Medium for NAT medium. Use G-N Supplement with Wilkins-Chalgren Medium for NAV Medium1.

Discussion
Selective Medium for Non-Sporing Anaerobes
This medium is referred to in the published literature1 as NAT Medium and is recommended for the isolation of non- sporing anaerobes from clinical specimens.

The recovery of non-sporing anaerobes from clinical material may sometimes prove difficult in specimens containing mixtures of aerobic and anaerobic bacteria. A medium which contains nalidixic acid as the selective agent was described by Wren1 for isolating these organisms. It was shown to be virtually non-inhibitory to most non-sporing anaerobes whilst retaining good selectivity for these organisms when present in mixed cultures. The medium is particularly useful for the recovery of non-sporing Gram-positive anaerobes since the presence of `Tween 80’ stimulates their growth2.

Another advantage of this medium is the earlier colonial pigmentation of the Prevotella melaninogenica group due to the slow lysis of the blood by `Tween 80’ during incubation. It is also a less inhibitory medium than aminoglycoside- containing media for non-sporing anaerobes in general.
The NS Anaerobe Supplement (SR0107) for non-sporing anaerobes contains nalidixic acid as the selective agent, together with haemin, menadione and sodium pyruvate as an additional energy source1,4.

Haemin was found to be essential for the growth of Bacteroides species5 and menadione for Bacteroides melaninogenicus.6 Pyruvate, in addition to being an energy source, acts similarly to catalase and degrades traces of hydrogen peroxide which may be produced by the action of molecular oxygen on media constituents. Hydrogen peroxide is known to affect the metabolism of anaerobes7.

Downes et al.8 showed that NAT Medium was superior to kanamycin agar (KA) and neomycin agar (NA) in the recovery of all non-clostridial anaerobes. The major superiority was in the recovery of anaerobic, Gram-positive cocci.

Selective Medium for Gram-Negative Anaerobes
This medium is described 9 as NAV Medium and is recommended for the isolation of Gram-negative anaerobes from clinical specimens.

NAV Medium is a modification of NAT Medium1 in which `Tween 80’ and sodium pyruvate have been replaced by sodium succinate. Vancomycin has been added, thus making the medium totally selective for Gram-negative anaerobes.

G-N Anaerobe Supplement (SR0108) contains nalidixic acid and vancomycin as selective agents; haemin, menadione and sodium succinate as growth factors. Haemin was found to be essential for the growth of Bacteroides species5 and menadione for Prevotella melaninogenica6. Some Gram-negative anaerobes require succinate as a source of energy10.

The recovery of Gram-negative anaerobes on NAV Medium has been shown8 to be superior to that on media containing neomycin and kanamycin as selective agents.

In order to isolate the maximum non-sporing anaerobic bacteria from clinical specimens the following scheme must be followed.

Specimen
Inoculate on to each of the following media and incubate anaerobically for 48 hours.

Plate 1

Plate 2

Plate 3

Wilkins-Chalgren Anaerobe Agar CM0619 + 5% (v/v) defibrinated blood

CM0619 + `Tween 80’ +5%(v/v) defibrinated blood + SR0107

CM0619 + 5% (v/v) defibrinated blood + SR0108

All bacteria capable of growing under anaerobic conditions

(NAT Medium) Non-Sporing Gram +ve and Gram -ve anaerobic bacteria

(NAV Medium) Gram-negative
anaerobic bacteria

A non-selective plate is included for attempted isolation of any strain, in particular Bacteroides corrodens which is sensitive to the selective agents.

Technique
1. Prepare supplies of Plate 1 (CM0619 + blood) Plate 2 (CM0619 + blood + Tween 80 + SR0107) and Plate 3 (CM0619 + blood + SR0108) as described in the section marked Directions.
2. Inoculate the specimens on to plates of each medium. Best results are obtained if freshly prepared plates are used but plates may be stored at 4°C for up to 3 days.
3. Incubate the plates anaerobically at 35°C for 48 hours. The Oxoid Anaerobic System with a Gas Generating Kit (BR0038) is recommended. Alternatively use Anaerogen (AN0025 or AN0035). Anaerogen does not require the addition of water or a catalyst.
4. Examine the plates. If no growth has occurred then incubation should be continued up to 5 days before plates are discarded, as up to 20% of non-sporing anaerobes require prolonged incubation under unbroken anaerobic conditions.
5. Carry out confirmatory tests on the isolates and record the results as follows:
(i) all facultative anaerobes and obligate anaerobes isolated on the Wilkins-Chalgren Anaerobe Agar plate.
(ii) all non-sporing anaerobes isolated on the medium for non-sporing anaerobes.
(iii) all Gram-negative anaerobes isolated on the medium for Gram-negative anaerobes.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared plates of medium at 2-8°C away from light.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Straw coloured gel

Quality control

Positive controls:

Expected results

Clostridium perfringens ATCC® 13124

Good growth; straw coloured colonies

Bacteroides fragilis ATCC® 25285 *

Good growth; straw coloured colonies

Negative control:

 

Uninoculated medium

No change

NAT Medium modification 
Positive controls:  
Prevotella loescheii ATCC® 15930 †Good growth; grey/white colonies
Peptostreptcoccus anaerobius ATCC® 27337 *Good growth; grey/white colonies
Negative control:  
Escherichia coli ATCC® 25922 * Inhibited
NAV Medium modification 
Positive controls:  
Bacteroides fragilis ATCC® 25285 *Good growth; grey/white colonies
Fusobacterium necrophorum ATCC® 25286 *Good growth; grey/white colonies
Negative control:  
Escherichia coli ATCC® 25922 *Inhibited
* This organism is available as a Culti-Loop®
† Formally known as Bacteroides melanogenicus

References
1. Wren M. W. D. (1977) J. Med. Microbiol. 10. 195-201.
2. Holdeman L. V and Moore W. E. C. (1977) Anaerobe Lab. Manual (4th edition).
3. Wren M. W. D. (1980) J. Clin. Pathol. 33. 61-65.
4. Rogosa M. (1964) J. Bacteriol. 87. 162-170.
5. Quinto G. and Sebald M. (1964) Am. J. Med. Technol. 30. 381-384.
6. Gibbons R. J. and MacDonald J. B. (1960) J. Bacteriol. 80. 164-170.
7. Hoffman P. S., George H. A., Krieg N. R. and Smibert R. M. (1979) Can. J. Microbiol. 25. 8-16.
8. Downes J., Stern L. and Andrew J. H. (1986) Pathology 18. 141-144.
9. Wren M. W. D. (1981) Personal Communication.
10. Lev M., Keudell K. C. and Milford A. F. (1971) J. Bact. 108. 175-178.

 
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