Getting the most out of your in-house fungal cultures (Proceedings)

Bio-Hazard Considerations

• In practices this consists of common sense and wearing laboratory coats and gloves and following basic good laboratory practices

• Do not eat, drink, chew gum, use tobacco, apply cosmetics, or handle contact lenses in the work area

• Do not store food for human consumption in the mini lab area

• Do not store personal items (clothes, boots, etc) in the min lab area

• Wash hands frequently after specimens

• Keep hands away from mouth, nose, eyes, face, and hair

• Wear protective clothing: laboratory coat and gloves

• Fungal cultures should be kept in a specific area in a sealed container

• Cover the counter top with a disposable paper to absorb any spills (paper towels)

• Routinely wipe off work area with a disinfectant (Routine disinfectant spray followed by 1:100 bleach)

• Dispose of fungal cultures and inoculation materials in a biohazard container, not regular trash

• Dispose of glass slides in a container for just glass slides

In House Supplies

• Individually wrapped toothbrushes(for culture)

• Sterile forceps

• Alcohol wipes

• Sterile cotton tipped swabs (for pressing specimens onto medium surface or collecting specimens from jars)

• Fungal culture medium

• Acetate tape (clear or frosted-clear is preferred and easier to use)

• Glass cover-slips

• Lactophenol cotton blue or new methylene blue stain

• Markers

• Plastic self closing sandwich size bags

• Clear plastic storage box

• Inexpensive digital fish tank thermometer

• Clear finger nail polish (for sealing edges if review by other or outside laboratory, makes a semi-permanent mount)

• Disinfectant for counter/work area

Fungal Culture Medium

• Criteria for selection: most volume of medium for the money, how easily the plate can be inoculated, how easy it is to sample the plate, does the plate have a red indicator, shelf life, and cost

• Recommendations

• Avoid glass jars because they can be difficult to inoculate and sample, if sealed too tightly can have increased growth of contaminant bacteria and fungi

• Avoid mini culture plates as there is too little surface and volume of medium for veterinary patients. These were designed for use in human medicine.

• Small medium plates will often turn red very rapidly making it difficult to monitor colonies.

• Use flat culture plates with phenol red colour indicator

• Plates that require refrigeration are at risk for getting ruined if they are accidently frozen, keep the plates in the door or vegetable crisper

Fungal Culture Sampling Options

• Samples can be obtained using the toothbrush fungal culture technique or by plucking individual hairs.

• Toothbrush cultures are recommended in cats and kittens. Toothbrush an area for 20 swipes; hairs should be visible.

• Individual hair sampling is most commonly used when glowing hairs can be identified and plucked; wiping area with alcohol is not recommended. ALWAYS pluck in the direction of growth as it is the proximal (hair bulb) area that is desired for culture.

• Wiping the area with alcohol is recommended if the visual contamination with organic debris is indicated or if a previous fungal culture was swarmed by contaminant growth.

• When in doubt, use the toothbrush fungal culture technique.
• If sampling the WHOLE body, culture suspect lesions LAST to avoid mechanical spread of infectious material.

Inoculation Tips

• Toothbrush Fungal Cultures: Gently stab bristles in the plate STARTING at the center of the plate and moving out to the edges. Do not press too hard or the medium will be pulled off the plate

• Plucked hairs: Press firmly onto the surface of the medium but do not embed; use sterile cotton swabs to press to surface if necessary. Be sure to disinfect and sterilize the forceps.

Optimizing Incubation

• Label plate and outside sandwich bag.

• Seal Cultures in Self Sealing Sandwich Bags: This will increase humidity and minimize premature dehydrate of the plates. It will prevent the plates from cross contamination and from contamination by media mites. It increases biohazard safety in small in house laboratories.

• Incubate fungal culture plate medium side UP. If moisture accumulates it will deposit on the lid and not on the plate minimizing overgrowth of bacteria and fungi.

• Incubate Plates at 75 to 80°F. This will increase growth and sporulation. This is warmer than room temperature, especially in the summer if air conditioning is used.

• Place a small inexpensive digital fish tank thermometer near the plates to monitor temperature. Place thermometer in a plastic bag so it does not become contaminated.

Practice Pearls!

• Fungal cultures can be incubated in small clear plastic shoe box. This will contain specimens and often be adequate for incubation at optimum temperatures. It is also easily decontaminated.

• If room temperature is still too cold a “mini incubator” can be made using a “PlayMate” cooler and a fish tank water heater. Remove drain from side of cooler and place fish tank incubator into the hole.

Optimizing Monitoring of In-House Fungal Cultures

• Examine plates daily. Print off a copy of the calendar month and tape it to the top of the container with the fungal culture plates. Place your initials in the box when this task is done.  Use this sheet to alert other technicians and staff doctors to fungal culture plates that are suspect.

• Dermatophyte Test Medium

• Originally developed for use in Vietnam to help rapidly identify dermatophytosis in soldiers. The goal was to develop a fungal culture medium that could be used as a visual “positive” or “negative” tool. It was very rapidly shown that DTM was helpful in decreasing the growth of contaminants but false positives were common in people. Shortly afterward this was shown in animals.
• DTM Medium is a basic fungal culture medium (Sabouraud's medium) which contains antimicrobials to inhibit/slow the growth of contaminant bacteria and fungi. It also contains a colour indicator that is ph sensitive. The ph of the media is 5.5 (straw yellow). Pathogens turn the medium alkaline (red) as they grow. So do many contaminants.

• A red colour change in dtm medium is not diagnostic for dermatophytosis. 

• Rules of thumb for dtm:
• Red does not mean i am positive. Red means look at me.

• Skip me if i am heavily pigmented, i am not a pathogen. I am a contaminant.

• Skip me if i am heavily pigmented even if i develop a red ring of colour around me as i grow. I am a contaminant.

• Skip me if i am pale or buff and do not have a red ring of colour developing around me as i grow. I am a contaminant.

• Do not skip me if i am pale/buff and a red ring of colour is developing around me as i grow.  I may be a pathogen. Examine me microscopically.

Practice Pearls

• Red colour change alone does not indicate a positive fungal culture

• The benefit of DTM is lost once the plate fully changes colour.

Microscopic Examination of Fungal Cultures

Microscopic examination is necessary to positively identify dermatophytes; gross examination of the colony is not sufficient.

The number of colonies and the amount of time spent performing microscopic examinations is markedly decreased by focusing on only highly suspect colonies.

Highly suspect colonies are grossly pale or buff and have a red colour change developing around them as they grow.

Scotch tape examination of colonies is used for microscopic examination.

Reporting Fungal Culture Data

• Simple reporting of fungal culture results as “positive” or “negative” is insufficient for proper record keeping, legal issues, and most importantly proper care of the pet. Reports of fungal culture results in the medical record can be reported as “negative” or “positive with identification of the pathogen” provided detailed data is present in the Excel Spreadsheet (see below). The point is that there is a wealth of information that can be obtained from the fungal cultures and it needs to be captured.

• It is optimum to report results as no growth, contaminant growth (add description see below), heavy contamination, or specific identification of a pathogen and an exact count or estimation of the number of colony forming units per plate.
• No growth: Specifically identifying a plate as no growth may be critical when animals are being treated. Also, if no growth is found yet there is high suspicion that it should have been, this allows for identification of a possible problem. For example, no growth can occur on what should be positive fungal cultures if the sampling culture is poor, the materials are over heated, or there are problems with incubation.

• Contaminants: It is not necessary to report the genus and species of a contaminant. It is helpful to report the color of the gross colonies and/or  if the colony is suspect (pale colony with red color change as it grows) and microscopically examined report what is seen: pigmented colonies, no growth consistent with Microsporum or Trichophyton, or unsporulated hyphae.

• Heavy Growth: Often culture plates are swarmed by contaminant growth in the first 7 days depending upon where the animal was prior to the sampling. Heavy growth of contaminants can suppress or hide pathogen growth and it is important to the clinician to know this piece of information.

• Pathogens: The most important pathogen to recognize is Microsporum canis. Recognition of the Microsporum genus morphology is important as these are the most important pathogens in small animal practice. Trichophyton spp are most commonly isolated from horses, cows, hedge hogs, rats, guinea pigs, and some dogs with very inflammatory lesions.  Report Microsporum colony growth to genus and species and Trichophyton spp to genus (identification to species level often requires sending the specimen out to diagnostic laboratory).
• Colony Forming Units: If pathogens are identified, the number of colony forming units is important to report as this may determine the course of treatment or be critical in monitoring of treatment. An abbreviation for ‘pathogen score is used commonly by the author'.

• P-score: This is the pathogen score and corresponds to the number of colony forming units on the plate.

• <4 colonies on the plate

• 5-9 colonies on the plate

• >10 colonies, too many to count colonies or a “swarmed plate”

Use of an Excel spreadsheet is recommended.

• This allows for easy recording of data on a weekly basis, tracking of fungal culture results, sorting and searching, and cost capture.  (CTRL-f will bring up search function).

• This form of recording of data becomes critical when animals are under treatment.

• This can be arranged on a continuous sheet or via month.

• Fungal culture results are entered into a simple column spread sheet and data recorded using standard comments and abbreviations.

• A centralized location of data allows for rapid reporting to staff veterinarians and clients.

• A centralized location of data allows for easy and rapid back tracking if needed.

• The number of fungal culture plates purchased versus the number of plates used and charged to clients can easily be tracked.

Key to Abbreviations and Explanation of Use

• DC: date cultured-date the pet was sampled

• DI: date the fungal culture was set up, in most cases DC and DI will be same

• Wk 1, Wk2, Wk 3: Record results of fungal culture examination during the appropriate week or after 7, 14, or 21 days. If a culture is heavily contaminated or culture positive, report results in the appropriate week. In other words, if cultures are examined daily and are culture positive 10 days after culture, report that within week 2. Otherwise report readings at 7, 14, and 21 days.

• Abbreviations

• C-contaminant growth

• HC-heavy contaminant growth (if this occurs in the first week notify staff doctor that a re-culture may be indicated)

• Ng-no growth

• M. canis, M. gypseum, Trichophyton (Microsporum canis, Microsporum gypseum, Trichopthyon are the most common pathogens). Recommend highlighting text in RED to make for easy recognition on the spread sheet.

• Results: Use the above abbreviations (NG, C, HC, or ID the pathogen)

• Results Final: Results are finalized after 21 days so this column will have an “N” in the column from the first read of the culture until finalized at day 21.

• P-score: This is the pathogen score and corresponds to the number of colony forming units on the plate.

•  <4 colonies on the plate

•  5-9 colonies on the plate

•  >10 colonies, too many to count colonies or a “swarmed plate”

• Culture comments: place to list comments such as lesions on pet, findings on the plate (e.g. exact number of cfu)

Example Name Results Final DC DI Wk 1 Wk 2 Wk 3 P score Comments