Labratory Methods
Identification of Cryptococcus gattii
Environmental Sampling
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Tree swabbing |
A sterile medical transport swab is used to swab over the surface of the tree, particularly in sheltered areas such as in hollows, cracks or scars, under loose areas of bark. In the lab, the swab is transferred to Staib media |
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Soil and debris sampling |
Soil and small woody debris can be collected in a clean zip-lock bag. Collect soil from the top 6 inches (15 cm) of the ground, since this is where most fungi can be found. Use a trowel or turn the bag inside out and use 'doggy-bag' technique to pick up the soil. In the lab, weigh out approx 2 grams of soil into a 50-mL Falcon tube. Add 10 mL of sterile water. Vortex for 30 seconds. Allow the sediment to settle, and then spread 100 µL of the |
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Air sampling |
Air sampling is conducted using either an RCS-plus system or the Andersen 6-stage air sampler. The RCS-plus sytem is lightweight, portable and fast easy to set up and operate. In both systems Staib media is used to detect airborne Cryptococcal cells or spores. |
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Water sampling |
Approximately 500 mL of water is collected either in clean screw-top jars, or in strong zip-lock bags (double bagged). In the lab, 100 mL aliquots of water are filtered through a sterile 0.45 µm nitrocellulose filter. The filter membrane is then placed on a Staib plate. |
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Identification of Cryptococcus gattii
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Staib media For differential identification of Cryptococcus sp. Recipe |
The swab is transferred to a Staib (niger seed) media plate, and incubated at 30 ºC for up to 10 days. Cryptococcus colonies will appear dark brown, round and smooth on this media. The plates should be checked daily for these colonies, as they can quickly be overgrown by contaminating filamentous fungi. |
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India Ink stain For visualising capsule |
This stain can be used either on fresh culture or on CSF if cryptococcal meningitis is suspected. A drop of india ink is mixed with a loopful of liquid culture or CSF on a glass microscope slide. A coverslip is placed over the mixture, and then viewed under a microscope. |
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CGB media For distinguishing Cryptococcus gattii from Cryptococcus neoformans |
Canavanine-Glycine-Bromothymol Blue media is commonly used to distinguish between Cryptococcus gattii and the closely related species Cryptococcus neoformans. Cryptococcus gattii is resistant to canavanine and is able to utilise glycine as a sole carbon source, and will therefore grow on CGB media and trigger a bromothymol blue colour reaction. Cryptococcus neoformans is sensitive to canavanine and cannot utilise glycine as a sole carbon and nitrogen source, and will therefore not grow, and the media remains yellow in colour. Note that false positive or false negative results may occur occasionally. Cryptoccus laurentii may occasionally trigger a green-blue colour change. |
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Serotyping Crypto-chek slide agglutination kit Mitsubishi Kagaku Iatron, Tokyo Japan) |
The Crypto-chek serotyping kit groups Cryptococcus gattii or Cryptococcus neoformans strains according to carbohydrate antigens expressed on the surface of the capsule surrounding the cell. Cryptococcus gattii serotype B agglutinates with sera 1 and 5, and serotype C agglutinates with sera 1 and 6. Cryptococcus neoformans agglutinates with sera 1 and 7 (serotype A), 1 and 8 (serotype D), or sera 1, 7, and 8 (serotype AD). Note: The Crypto-chek slide agglutination serotyping kit manufactured by Mitsubishi Kagaku Iatron, Tokyo Japan, is no longer distributed. There are currently no other methods available that specifically determine serotype, although genotyping methods are arguably more informative. |
Genotyping
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PCR-fingerprinting |
PCR-fingerprinting utilises a single primer specific to a hypervariable repetitive DNA motif, in a PCR reaction. The primer anneals to these sites in the genome and amplifies the DNA regions lying between them. These amplified fragments are viewed on an agarose gel. Because of the high mutation frequency in hypervariable DNA, variation in the amplified fragments is common, and may be exploited to distinguish between strains, or species. |
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URA5-RFLP |
The URA5 gene of Cryptococcus gattii is amplified in a PCR reaction. The PCR product is then digested using specific restriction enzymes. Differences in the DNA sequence in this gene means that the restriction enzymes will cut the DNA into differently sized fragments, which can be identified on an agarose gel. |
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Multilocus Sequence Typing (MLST) |
A selection of unrelated, phylogenetically informative genes are amplified and sequenced. Differences in the DNA sequence of these genes represent different sequence types (alleles). Therefore different strains can be assigned a 'barcode' of sequence types, which can be used for identifying or distinguishing between strains. |
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Commonly Used Media
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Adapted from
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To make 1 Litre 70 g Guizotia abyssinica (niger seed) Grind seeds of Guizotia abyssinica as finely as possible using a coffee grinder. Place ground seeds in a beaker and add 1 L of distilled water. Stir to remove clumps. Autoclave for 20 min at 110 ºC (230 ºF) and allow to cool. Remove as much oil as possible from the top of the seed extract. Pass the seed extract through several layers of cheese cloth, and adjust volume to 1000 mL. Add the seed extract to a beaker containing the creatinine, glucose, and KH2PO4. Dispense into two flasks containing 7.5 g each of agar. Autoclave at 110 ºC (230 ºF) for 20 min. Cool the media to 50 ºC, and swirl to mix all components. Add 0.5 mL chloramphenicol solution to each flask. Pour the media into 90 mm petri dishes. Allow the plates to solidify and dry before use. |
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From a
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To make 1 Litre: Make up solution A and B in advance. Solution A: 10 g Glycine Dissolve ingredients in small beaker and adjust pH to 5.6. Filter sterilise solution using a 0.22 µm filter membrane. Store at 4 ºC until ready to use. Solution B: 0.4 g Bromothymol blue Dissolve the Bromthymol Blue in the NaOH and add to the water. To make 1 Litre of the media mix together the following: 880 mL Distilled water Autoclave to 121 ºC (250 ºF) for 15 minutes, cool to 50 ºC. Add 100 mL of solution A and mix. Dispense into petri-dishes or slopes. |
