Category Archives: MYCOLOGY

MYCOBIOTIC OR MYCOSEL AGAR

Mycobiotic or Mycosel selective medium principally formulated for the isolation of dermatophytes but also used for the isolation of other pathogenic fungi from specimens contaminated with saprophytic fungi and bacteria.

The medium consists primarily of peptones from a pancreatic digest of soybean meal and dextrose. The selective agents are cycloheximide and chloramphenicol.

Cycloheximide inhibits the faster-growing saprophytic fungi. Chloramphenicol inhibits Gram-negative and Gram-positive organisms. Susceptibility to cycloheximide may be used for identification of fungi.

WATER AND CORNMEAL AGAR

WATER AGAR


Water agar is a nutritionally-deficient media known to enhance production of spores and conidia of sporulation. Media Preparation


• Mix 20 grams of agar in 1 litre of distilled water.


•Bring reagents to a boil.


• Autoclave at 15 lb/in2 for 15 minutes.


• Cool in slanted position or store as butts to be melted down and used as slants or plates as needed.

CORNMEAL TWEEN-80 AGAR


CMT agar is a well-established medium used for cultivation of fungi as well as to study chlamydospores production by Candida species and other yeasts.

CHROMOGENIC BISMUTH GLYCINE GLUCOSE YEAST (BIGGY)

Agar BiGGY agar is a partially selective and differential medium for the cultivation and identification of Candida species from pure cultures or clinical specimens.

Candida species, through a process of substrate reduction, reduces the bismuth salt to bismuth and sulfite to sulfide. Bismuth and sulfide combine to form a brownish to black precipitate that stains colonies and may diffuse into the medium.

Also bismuth and sulfur compounds are inhibitory to many bacteria. Yeast extract and glucose provide essential nutrients for growth. Glycine is an additional nutrient, but also inhibits many bacterial species at the high concentration used in this medium.

POTATO DEXTROSE AGAR (PDA)

Is a general purpose medium for a wide range of yeasts and molds.

The nutritionally rich base (potato infusion) encourages mold sporulation and pigment production in some dermatophytes like Trichophytan rubrum.

Most fungi thrive on Potato Dextrose Agar (PDA), but this can be too rich for many fungi, so that excessive mycelial growth is obtained at the expense of sporulation.

Is composed of dehydrated potato infusion, dextrose and agar. Many standard procedures use a specified amount of sterile tartaric acid (10%) to lower the pH of this medium to 3.5 +/- 0.1, inhibiting bacterial growth.

It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.

BRAIN HEART INFUSION (BHI) BROTH AND AGAR

It is a non-selective fungal culture medium that permits the growth of virtually all clinically relevant fungi. It is used for the primary recovery of saprophytic and dimorphic fungi.

The medium contains proteose peptone and infusions from calf brain and beef heart which serve as sources of carbon, nitrogen, essential growth factors, amino acids and vitamins. Dextrose is used as a source of energy. Disodium phosphate helps in maintaining the buffering action of the medium whereas sodium chloride maintains the osmotic equilibrium of the medium.

BHI with 10% defibrinated sheep blood is useful for isolation and cultivation of Histoplasma capsulatum and other fungi.

One can culture food, blood and water samples at 35–37°C for 24–48 hours for the isolation of Candida albicans. Some fungi might require more incubation time.

After the incubation period, examine tubes for turbidity which is an indication of growth. If desired, a loopful of the culture broth can then be sub-cultured onto an appropriate solid medium to observe colony morphology.

It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.

MALT EXTRACT AGAR (MEA)

Malt Extract Agar is used for the cultivation of fungi and is not intended for use in the diagnosis of disease or other conditions in humans. It is frequently used for culturing fungi from soil, wood, basidiomycetes etc. However, clinical specimens such as skin scrapings can be processed on the medium.

It is recommended for the detection, isolation, maintenance and enumeration of yeasts and moulds such as Aspergillus brasiliensis, Candida albicans and Saccharomyces cerevisiae.

An acidic medium which will support the growth of most yeasts and molds whilst inhibiting most bacteria.

Selectivity can be increased by further lowering the pH with the addition, after sterilization, of Lactic Acid (X037).

It should be noted that excess heating of this medium together with its low pH can easily result in hydrolysis of the agar gel producing soft plates.

The medium consists of malt extract, peptone and agar.

Cultural characteristics observed after an incubation at 25-30°C for 48-72 hours at a pH of 5.4±0.2.

Further biochemical tests must be carried out for further identification

SABOURAUDS DEXTROSE AGAR (SDA)

Sabouraud’s agar is sufficient for the recovery of dermatophytes from cutaneous samples and yeasts from genital cultures.

It is nutritionally poor with acidic pH (5.6).

Peptone (Enzymatic Digest of Casein and Enzymatic Digest of Animal Tissue) provide the nitrogen and vitamin source required for organism growth in SDA. Dextrose is added as the energy and carbon source. Agar is the solidifying agent.

Not recommended as a primary isolation medium because it is insufficiently rich to recover certain fastidious pathogenic species, particularly most of the dimorphic fungi.

It does not promote conidiation of filamentous fungi.

Sabouraud’s dextrose agar (2%) is most useful as a medium for the subculture of fungi recovered on enriched medium to enhance typical sporulation and provide the more characteristic colony morphology.

Identification of fungi is performed by observing various aspects of colony morphology, characteristic microscopic structures, rate of growth, media which supports the organism’s growth, and source of specimen.

Yeasts will grow as creamy to white colonies. Molds will grow as filamentous colonies of various colors.

PREREQUISITES FOR MYCOLOGY SPECIMEN COLLECTION

  • Always consult the SOP manual of the diagnostic laboratory for the particular specimen and test.
  • Aim to collect sample in a sterile kit under aseptic conditions.
  • Ensure that you have the appropriate swab kit (alcohol swabs, gloves, transportation tube and media).
  • If it is a special or non-routine test, coordinate with the laboratory about specific requirements for collection and handling.
  • Document the following:
  1. Patient identifiers (e.g. name, age, medical record number);
  2. Sample identifiers (e.g. swab, smear, scrape, urine, blood);
  3. Location and type (e.g. lower limb wound, nail scraping, and hair sample);
  4. Date and time of collection;
  5. Deviations from standard protocol during collection (e.g. not performed under aseptic conditions if large traumatic wound);
  6. Relevant clinical information including recent and current antimicrobial therapy.
  • Ensure that adequate material (at least 2 ml of bodily fluids) is sent to the laboratory for proper yield. An inadequate specimen may lead to a false negative result.
  • Transport the sample within 2 hours and process promptly for optimum recovery of fungi.
  • If a delay is anticipated, refrigerate specimens at 4°C (exceptions: blood, bone marrow, CSF and sterile tissues should be stored at 35-37°C).
  • Ideally, collect specimens as soon as symptoms appear and whenever possible before antifungal therapy is initiated.
  • Staff must take all precautions to avoid inadvertent contamination of sample as well as for their own personal safety.

DIFFERENT TYPES OF CULTURE MEDIA


For a culture medium to be successful in growing the pathogen sought it must provide all essential nutrients, ions, and moisture, maintain the correct pH and osmotic pressure, and neutralize any toxic materials produced. It is also essential to incubate the inoculated medium in the correct atmosphere, at the optimum temperature and for an adequate
period.

The main types of culture media are:
● Basic
● Enriched
● Selective
● Indicator
● Transport
● Identification

BASIC MEDIA

These are simple media such as nutrient agar and nutrient broth that will support the growth of microorganisms that do not have special nutritional requirements.
They are often used in the preparation of enriched media, to maintain stock cultures of control strains of bacteria, and for subculturing pathogens from differential or selective media prior to performing biochemical and serological identification tests.

ENRICHED MEDIA

Enriched media are required for the growth of organisms with exacting growth requirements such as
H. influenzae, Neisseria species, and some Streptococcus species. Basic media may be enriched with whole or lyzed blood, serum, peptones, yeast extract, vitamins and other
growth factors. An enriched medium increases the numbers of a pathogen by containing all the necessary ingredients to promote its growth. Such a medium is often used for specimens collected from sites which are normally sterile to ensure
the rapid multiplication of a pathogen which may be present only in small numbers.

ENRICHMENT MEDIA

Enrichment media: This term is usually applied to fluid selective media which contain substances that inhibit the growth of unwanted organisms,e.g. Rappaport-Vassiliadis broth which is often used as an enrichment medium for Salmonella serovars in faeces.

SELECTIVE MEDIA

These are solid media which contain substances (e.g. bile salts or other chemicals, dyes, antibiotics)
which inhibit the growth of one organism to allow the growth of another to be more clearly demonstrated. A selective
medium is used when culturing a specimen from a site having a normal microbial flora to prevent unwanted contaminants
overgrowing a pathogen. Media made selective by incorporating antibiotics are usually expensive.

Other ways to select organisms:
Incubation conditions may be used to select organisms, e.g. P. aeruginosa is inhibited by anaerobic conditions. Also the
pH of a medium may make it selective for a particular
organism, e.g. V. cholerae can be isolated on an alkaline medium such as TCBS agar. Temperature may also help to
select an organism e.g. Listeria monocytogenes can grow at 4° C whereas other organisms are inhibited. Growth, however, is slow.

INDICATOR MEDIA

These are media to which dyes or other substances are added to differentiate microorganisms. Many differential media distinguish between bacteria by incorporating an indicator which changes colour when acid is produced following fermentation of a specific
carbohydrate e.g. MacConkey agar.

Many media used to isolate pathogens are both selective and enrichment or both selective and differential.

TRANSPORT MEDIA

These are mostly semisolid media that contain ingredients to prevent the overgrowth of commensals and ensure the survival of aerobic and anaerobic pathogens when specimens cannot be cultured immediately after collection. Their use is particularly important when transporting microbiological specimens from health centres to the district microbiology laboratory or specimens to the Regional Public Health Laboratory.

Examples of transport media include
Cary-Blair medium for preserving enteric pathogens and Amies transport medium for ensuring the viability of gonococci.

IDENTIFICATION MEDIA

These include media to which substrates or chemicals are added to help identify bacteria isolated on primary cultures. Examples include peptone water sugars, urea broth, and Kligler iron agar. Organisms are mainly identified by a change in the colour of the medium and or the production of gas. Organisms used to inoculate identification media must be first isolated in pure culture.

USING INDIAN INK FOR FUNGAL DETECTION

This procedure is employed to observe capsule around the yeasts especially Cryptococcus neoformans in CSF. Some bacteria with capsule eg klebsiella stain with Indian ink but are one fourth the size of yeast and are not budding.

Indian ink is used as definitive evidence of Cryptococcus neoformans but other studies such as dark brown pigment production on caffeic acid agar, direct antigen testing are also used.

1. Place a small drop of India ink on a grease free slide containing the test sample

Process different specimens as follows:

2. Fluid samples like CSF should be centrifuged and the sediment used on the slide.

3. In thick specimens like pus and sputum add drop of 10% KOH along with India ink to improve visualisation.

4. Leukocytes and tissue cells can be dissolved by adding a drop of 10% KOH.

5. Observe under ×10 and confirm by ×40

6. Record the findings