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Dehydrated Culture Media

Further Information related to  CM0367

GC SELECTIVE MEDIA

Isolation techniques for Pathogenic Neisseria.

NEISSERIA ISOLATION MEDIA AND TECHNIQUES

Introduction
The probability of success in the isolation and identification of pathogenic Neisserii from clinical specimens is related to five factors:
1 The amount of care taken in obtaining good specimens, their transport to the laboratory and correct inoculation on culture medium.
2 The provision of a culture medium capable of growing small inocula of demanding strains.
3 The provision of optimal incubation temperature and gaseous environment.
4 The inclusion of selective agents in the medium which are capable of preventing overgrowth of commensal organisms but which will not inhibit the growth of the species required.
5 Confirmatory tests to identify the species.

Importance of Accurate Diagnosis
Diagnosis of gonorrhoea is achieved by microscopic examination and cultivation of material from infected sites. Presumptive gonococci have then to be distinguished from other organisms of the Neisseria group which have a similar morphology and staining characteristics.

Sampling Sites
The diagnosis is achieved by examining smears and cultures from a number of sampling sites. These include the urethra and rectum of males, the urethra, cervix and rectum of females and occasionally also the ducts of Bartholin's glands. The oropharynx of both sexes may also require sampling1,2. Repeated examination may be necessary before a diagnosis is achieved. The calcium alginate or cotton wool swabs that are used must be of low toxicity for bacteriological purposes.
As with meningococcal infection, gonorrhoea may present as septicaemia with signs of rash, fever and/or arthritis and the responsible organism may be isolated from the blood, joints or (rarely) the cerebrospinal fluid. Ocular infections can occur in neonates and occasionally in adults.

Inoculation
The plates are inoculated by rolling the culture swab across a segment of a moist plate or preferably in a large 'Z' pattern so that an adequate area of the plate is inoculated. Streaking the plate with a sterile inoculating loop is carried out to ensure adequate dispersion of the organisms.

Media
Media used for the cultivation of gonococci are agars of high peptone and starch content enriched with fresh horse blood (lysed) or soluble haemoglobin and GC growth supplements (Yeast Autolysate Supplement SR0105 and Vitox SR0090) which have been shown to stimulate growth from small inocula.
Several combinations of selective antibiotics (VCNT, VCN, LCAT and VCAT) have been described that can be added to culture media in order to suppress Gram-positive and Gram-negative contaminants.
The choice of the selective supplement is dependent upon the preference of the laboratory as well as regional and strain differences of the organism. See table below.

Medium Constituents
Soluble Haemoglobin or Blood Growth Supplement Selective Supplement
Thayer Martin Medium
(non-selective)
Oxoid GC Agar Base CM0367 Soluble Haemoglobin LP0053 Vitox SR0090 -
Thayer Martin Medium Oxoid GC Agar Base CM0367 Soluble Haemoglobin LP0053 Vitox SR0090 VCN Antibiotic Supplement SR0101
Thayer Martin Medium(modified) with Vitox Oxoid GC Agar Base CM0367 Soluble Haemoglobin LP0053 Vitox SR0090 VCNT Antibiotic Supplement
SR0091
Thayer Martin Medium(modified) with Yeast Fractions Oxoid GC Agar Base CM0367 Soluble Haemoglobin LP0053 GC Supplement
SR56*
GC Supplement
SR56*
'Transgrow' Medium Oxoid GC Agar Base CM0367 +
1% Agar
Soluble Haemoglobin LP0053
Supplement SR56*
Vitox SR0090 or
GC Supplement
SR56*
VCN SR0101 or
VCNT SR0091 or
GC Supp. SR56*
GC Medium
derived from the New York City formulation
Oxoid GC Agar Base CM0367 Lysed Defibrinated horse blood or Laked horse
blood SR0048
Yeast Autolysate
Supplement
SR0105
LCAT Antibiotic
Supp. SR0095 or VCAT Antibiotic Supp. SR0104

Directions for preparation of the Variant Medium

Thayer Martin Medium with GC Supplement
1 Suspend 18g of Oxoid GC Agar Base in 235ml of distilled water and bring to the boil to dissolve the agar. Sterilise by autoclaving at 121° C for 15 minutes.
2 Prepare a 2% solution of Soluble Haemoglobin Powder LP0053 by adding 250ml of warm distilled water to 5g of haemoglobin powder. Continually stir the solution during the addition of water. Sterilise by autoclaving at 121° C for 15 minutes.
3 Dissolve the contents of a vial of sterile GC Supplement SR56* in 15ml of sterile distilled water.
4 Aseptically add the GC Supplement Solution SR56* to the 235ml of sterile GC Agar Base cooled to 50° C and mix gently. Add the 250ml of sterile haemoglobin solution, cooled to 50° C, to the GC Agar Base-Supplement solution. Mix gently to avoid trapping air bubbles in the agar and pour into sterile petri dishes.

Thayer Martin Medium with Vitox and either VCN or VCNT Antibiotic Supplement.
1 Suspend 18g of GC Agar Base in 240ml of distilled water and bring gently to the boil to dissolve the agar. Sterilise by autoclaving at 121° C for 15 minutes.
2 Prepare a 2% solution of Soluble Haemoglobin Powder LP0053 by adding 250ml of warm distilled water to 5g of haemoglobin powder. Continually stir the solution during the addition of water. Sterilise by autoclaving at 121° C for 15 minutes.
3 Dissolve the contents of one vial of Vitox SR0090 as directed on the vial label.
4 Dissolve the contents of a vial of either VCN Antibiotic Supplement SR0101 or VCNT Antibiotic Supplement SR0091 as directed on the vial label.
5 Aseptically add the Vitox solution to 240ml of sterile GC Agar Base cooled to 50° C.
6 Aseptically add the reconstituted antibiotic supplement VCN or VCNT to the GC Agar Base-Vitox solution.
7 Aseptically add the 250ml of sterile haemoglobin solution, cooled to 50° C to the GC Agar Base-Vitox-Antibiotic Supplement solution. Mix gently to avoid trapping air bubbles in the agar and pour into sterile petri dishes.

'Transgrow' Medium
'Transgrow' Medium is prepared in the same manner as described for Thayer Martin Medium variants, except that 5g of Agar is added to the 18g of GC Agar Base. The medium is dispensed as agar slants in glass bottles. The extra glucose required is contained in the growth supplements.

GC Medium derived from the New York City Formulation with LCAT or VCAT antibiotic supplement
1 Suspend 18g of Oxoid GC Agar Base in 425ml of distilled water and bring gently to the boil to dissolve the agar. Sterilise by autoclaving at 121° C for 15 minutes.
2 Dissolve the contents of a vial of sterile Yeast Autolysate Supplement SR0105 in 15ml of sterile distilled water.
3 Dissolve the contents of either LCAT SR0095 or VCAT SR0104 in 10ml of sterile distilled water.
4 Aseptically add 50ml laked horse blood (SR0048), Yeast Autolysate Supplement and the Antibiotic Supplement (LCAT or VCAT) to the sterile GC Agar Base cooled to 50° C. Mix gently to avoid trapping air bubbles in the agar and pour into sterile petri dishes.
This is a derivative of NYC Medium11,12,13 based on Young's publication14 where the higher level of glucose recommended by the originators was reduced to allow sugar fermentation test to be carried out15.

Technique
1 Prepare the medium as directed and pour into petri dishes.
2 Inoculate by rolling the culture swabs across a segment of the plate or preferably in a large 'Z' pattern so that an adequate area of the plate is inoculated. Streaking of the plate is carried out with a sterile loop to ensure adequate dispersion of the organisms.
3 Plates are incubated at 37° C in a sealed jar with at least 70% humidity and 5-10% carbon dioxide provided by the introduction of the gas into the incubator or by the use of a candle jar. The above incubation conditions can be conveniently achieved by using the Oxoid Carbon Dioxide Gas Generating Kit BR0039 in the Oxoid Gas Jar HP0011.
4 Examine the plates after 24 hours incubation and if negative reincubate for a further 24 hours.
5 Presumptive gonococcus colonies are identified by the Gram stain, oxidase and sugar fermentation reactions.

Typical reactions of N. gonorrhoeae
Gram negative cocci, usually arranged in pairs with long axes parallel. Oxidase positive.

Fermentation Reactions of the Neisseria Group

  Glucose Maltose Lactose Sucrose
N. meningitidis + + - -
N. gonorrhoeae + - - -
Bran. catarrhalis - - - -
N. flava and
related types
+ ± - ±

+ = Acid production
±= Variation in reaction among different types

List of products available from Oxoid for the Growth and Identification of Neisseria Species
GC Agar Base CM0367
VCN Selective Supplement SR0101
Agar No.1 LP0011
VCNT Selective Supplement SR0091
Soluble Haemoglobin Powder LP0053
LCAT Selective Supplement SR0095
Sterile Yeast Autolysate Supplement SR0105
VCAT Selective Supplement SR0104
Vitox SR0090
GC Supplement SR56*
(Yeast Fractions plus VCNT Antibiotics)
Defibrinated Horse Blood SR0050
Laked Horse Blood SR0048

* PLEASE NOTE: GC Supplement SR56 is no longer available and can be substituted by a combination of VCNT Selective Supplement SR0091 plus Yeast Autolysate Supplement SR00105.

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

Appearance
Dehydrated medium: Light straw coloured, free-flowing powder.
Prepared medium(before additions): Straw coloured gel.

Quality Control

Positive control: Expected result
with antibiotics  
Neisseria gonorrhoeae ATCC® 19424 * Good growth; grey-brown colonies.
Neisseria meningitidis ATCC® 13090 * Good growth; grey-brown colonies.
Negative control:  
with antibiotics  
Proteus hauseri ATCC® 13315 * Inhibited
Staphylococcus aureus ATCC® 25923 * Inhibited
without antibiotics  
Uninoculated medium. No change

* This organism is available as a Culti-Loop®

Precautions
Use Dacron® or calcium alginate swabs for specimen collection, avoid cotton wool.
Any suspected Neisseria-containing specimen should be inoculated onto primary isolation media immediately on collection. If this is not possible then N. gonorrhoeae swabs are better held at 4° C for not more than 3 hours. N. meningitidis swabs should not be chilled and they should be inoculated on to pre-warmed media.
It is a wise precaution to inoculate GC media with and without antibiotics in parallel because some strains of neisseriae may fail to grow in the presence of antibiotics, especially vancomycin16.
Humidity is essential for the successful isolation of gonococci. If the prepared plates look dry moisten the surface with a few drops of sterile broth and allow it to soak into the agar before inoculation. Do not flood the plate with broth. Place damp gauze or paper towels in the CO2 container before incubation.
Hosty et al.17 showed that the usual transport media are not totally reliable for N. gonorrhoea. Inoculation of the sample on to the surface of medium slants in bottles is preferable. The incubator temperature should be set at 35° C because many strains of N. gonorrhoea will not grow well at 37° C on culture media18.
It has been shown that agars vary widely in their toxicity for N. gonorrhoea and may be a major factor preventing the growth of gonococci on solid media19.

The Gram morphology, colony characteristics and oxidase tests form the presumptive identification for the genus Neisseria. All presumptive neisseriae should be confirmed by carbohydrate fermentation tests and fluorescent antibody or other serological tests20.
An ONPG test will detect lactose-utilising strains of neisseriae e.g. N. lactamica21.

This product is not suitable for use as the base agar when preparing the medium described in CLSI standard M0222 as GC agar + 1% defined growth supplement for the disc susceptibility testing of N. gonorrhoeae i.e. the medium cannot be used with Vitox as the sole supplement.

References

  1. Willcox R. R. (1979) Euro Reports and Studies, 12, World Health Organization, Regional Office for Europe, Copenhagen.
  2. 'Neisseria gonorrhoeae and gonococcus infections' (1978) Report of a WHO Group, Technical Report Series 616, World Health Organization, Geneva.
  3. Thayer J. D. and Martin J. E. (1966) Public Health Rep., 81. 6. 559-562.
  4. Seth A. (1970) Brit. J. Vener. Dis. 46. 201-202.
  5. Riddel R. H. and Buck A. C. (1970) J. Clin. Path. 23. 481-483.
  6. Odegaard K. (1971) Acta Path. Microbiol. Scand., Sect. B. 79. 545-548.
  7. Martin J. E. and Lester A. (1971) HSMHA Health Reports. 86. No.1 30-33.
  8. Young H. (1978) Brit. J. Vener. Dis. 54. 36-40.
  9. Reyn A. and Bentzon M. W. (1972) Brit. J. Vener. Dis. 48. 363-368.
  10. Mirrett S., Reller L. B. and Knapp J. S. (1981) J. Clin. Microbiol. 14. 94-99.
  11. Faur Y. C., Wiesburd M. H., Wilson M. E. and May P. S. (1973) Health Lab. Sci. 10. 44-54.
  12. Faur Y. C., Weisburd M. H. and Wilson M. E. (1973) Health Lab. Sci. 10. 55-60.
  13. Faur Y. C., Weisburd M. H. and Wilson M. E. (1977) Health Lab. Sci. 15. 22-27.
  14. Young H. (1978) Brit. J. Ven. Diseases 54. 36-40.
  15. Young H. (1978) J. Clin. Microbiol. 7. 247-250.
  16. Faur Y. C., Weisburd M. H. and Wilson M. E. (1978) Health Lab. Sci. 15. 22-26.
  17. Hosty T., Freear M., Baker C. and Holston J. (1974) Amer. J. Clin. Path. 62. 435-438.
  18. Finegold S. M. and Martin W. J. (1982) Bailey and Scott's Diagnostic Microbiology 6th Edn. C. V. Mosby & Co. St. Louis. p. 107.
  19. Jones R. T. and Talley R. S. (1977) J. Clin. Microbiol. 5. 9-13.
  20. CDC (1975) 'General information to aid in handling Neisseria gonorrhoea specimens in the laboratory' US DHEW Center for Disease Control. Atlanta. Ga.
  21. Hollis D. G., Wiggins G. T. and Weaver R. E. (1969) Appl. Microbiol. 17. 71-77.
  22. M02-A12: Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard – Twelfth Edition CLSI document A02-A12. Wayne, PA: Clinical and Laboratory Standards Institute 2015
 
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