Dalmau Plate Technique: Introduction, Principle, Test Requirement

Introduction

Table of Contents

The Dalmau plate technique is a classic mycological method used to identify and differentiate yeast species, specifically Candida species, by observing their unique morphological features.

Fig. Cornmeal agar containing 1% Tween 80 in a 90-mm plate after yeast colony streaking

The technique is a specialized culture method that uses a “starvation medium” to induce the formation of characteristic structures like pseudohyphae, blastoconidia, and chlamydospores. It remains a valuable, cost-effective tool for preliminary identification in clinical laboratories.

Principle

The principle relies on using a nutrient-deficient medium (starvation medium) and creating a microaerophilic environment (reduced oxygen) by placing a coverslip over the inoculated area. This stress induces yeasts to switch from their vegetative budding state to filamentous growth or spore formation.

Corn Meal Agar (Dalmau plate) after 4 days incubation showing yeast growth — Fig. Corn Meal Agar (Dalmau plate) after 4 days of incubation, showing yeast growth

Test Requirements

Media: Cornmeal Agar (CMA) is most common; Rice extract agar or Pea agar can also be used.
Additive: Tween 80 (a surfactant) is often added to reduce surface tension and enhance the formation of pseudohyphae and chlamydospores.
Equipment: Sterile inoculation wire or loop, sterile glass coverslips, and a light microscope.

Fig. Dalmau Plate Microscopy at low power field (10X objective)

Procedure

Prepare Cornmeal agar containing 1% Tween 80 in a 90-mm plate.
Divide the plate into 4 quadrants and label each quadrant.
Using a sterile needle or straight wire, lightly touch the yeast colony and then make 2-3 streaks of approximately 3.5 – 4 cm long and 1.2 cm apart.
Place a flame-sterilized and cooled 22 mm square cover glass over the control part of the streak.
This will provide a partially anaerobic environment at the margins of the cover slip. Incubate the plates at 25°C for 3-5 days.
Remove the lid of the petri plate and place the plate on the microscope stage, and observe the edge of the cover glass using the low-power objective (10X) first and then the high-power objective (40X).
Note morphological features like hyphae, pseudohyphae, blastospores, ascospores, chlamydospores, basidiospores, or sporangia, and find out the organism as shown in the application table.

Fig.Dalmau Plate Microscopy at high power field (40X objective)

Applications

Speciation of Candida: Differentiating C. albicans (which produces large, thick-walled terminal chlamydospores) from non-albicans species.
Morphological Studies: Observing the arrangement of blastoconidia along pseudohyphae, which is unique to different yeast genera.
Starvation Response: Studying how yeast-like fungi adapt to nutrient-poor environments.

Feature	Possibility
Terminal chlamydoconidia	Candida albicans, Candida dubliniensis
Yeasts only	Candida glabrata, Candida famata, Pichia anomala, Pichia augusta (Hansenula polymorpha), Cryptococcus neoformans
Giant hyphae, blastospores at nodes	Candida parapsilosis
Abundant pseudohyphae, pine forest arrangement, blastoconidia formed at or in between septa	Candida tropicalis
Elongated yeasts, abundant pseudohyphae (matchstick-like appearance)	Candida krusei (Pichia kudriavzevii)
Scant pseudohyphae with chains of blastoconidia	Candida guilliermondii
Short, distinctly curved pseudohyphae with occasional blastoconidia at septa	Candida lusitaniae
Arthroconidia without blastoconidia	Geotrichum species
Arthroconidia with blastoconidia	Trichosporon species

Candida albicans in Dalmau culture plate microscopic footage at a magnification of 100x with 4x optical enhancement — Fig. Candida albicans in Dalmau culture plate, microscopic footage at a magnification of 100x with 4x optical enhancement

Candida albicans in Dalmau culture plate microscopic footage at a magnification of 400x with 4x optical enhancement — Fig. Candida albicans in Dalmau culture plate, microscopic footage at a magnification of 400x with 4x optical enhancement

Candida glabrata in Dalmau culture plate microscopic footage at a magnification of 100x with 4x optical enhancement — Fig. Candida glabrata in Dalmau culture plate, microscopic footage at a magnification of 100x with 4x optical enhancement

Candida glabrata in Dalmau culture plate microscopic footage at a magnification of 400x with 4x optical enhancement — Fig. Candida glabrata in Dalmau culture plate, microscopic footage at a magnification of 400x with 4x optical enhancement

Fig.Candida glabrata in Dalmau plate technique microscopic footage at a magnification of 400x with 4x optical enhancement

Candida tropicalis in Dalmau culture plate microscopic footage at a magnification of 100x — Fig. Candida tropicalis in Dalmau culture plate, microscopic footage at a magnification of 100x

Candida tropicalis in Dalmau culture plate microscopic footage at a magnification of 400x with 4x optical enhancement — Fig. Candida tropicalis in Dalmau culture plate, microscopic footage at a magnification of 400x with 4x optical enhancement

Fig. Pichia kudriavzevii (Candida krusei) in Dalmau culture plate microscopic footage at a magnification of 100x with 4x optical enhancement

Fig. Pichia kudriavzevii (Candida krusei) in Dalmau culture plate microscopic footage at a magnification of 400x with 4x optical enhancement

Fig. Pichia kudriavzevii in Dalmau culture plate microscopic footage at a magnification of 400x with 4x optical enhancement

Fig. Pichia kudriavzevii( formerly Candida krusei) in Dalmau culture plate microscopic footage at a magnification of 100x with 4x optical enhancement

Candida krusei in Dalmau culture plate microscopic footage at a magnification of 400x with 4x optical enhancement — Fig. Candida krusei in Dalmau culture plate, microscopic footage at a magnification of 400x with 4x optical enhancement

Fig. Typical Pichia kudriavzevii( formerly Candida krusei) in Dalmau culture plate microscopic footage at a magnification of 400x with 4x optical enhancement

Fig. Pichia kudriavzevii(formerly Candida krusei) in Dalmau culture plate microscopic footage at a magnification of 400x with 4x optical enhancement

Fig. Candida krusei (Pichia kudriavzevii) in Dalmau culture plate microscopic footage at a magnification of 400x with 4x optical enhancement

Fig. Candida krusei (Pichia kudriavzevii) in Dalmau culture plate microscopic footage at a magnification of 100x with 4x optical enhancement

Candida parapsilosis in Dalmau culture plate microscopic footage at a magnification of 100x with 4x optical enhancement — Fig. Candida parapsilosis in Dalmau culture plate, microscopic footage at a magnification of 100x with 4x optical enhancement

Candida parapsilosis in Dalmau culture plate microscopic footage at a magnification of 400x with 4x optical enhancement — Fig. Candida parapsilosis in Dalmau culture plate, microscopic footage at a magnification of 400x with 4x optical enhancement

Fig. Candida parapsilosis in Dalmau plate technique microscopic footage at a magnification of 400x with 4x optical enhancement

Keynotes

Observation Point: The most diagnostic morphology is typically found at the edge of the coverslip, where the oxygen gradient is ideal.
Chlamydospores: Candida dubliniensis often produces multiple terminal chlamydospores in clusters, while Candida albicans typically produces them singly.
Alternative Methods: While reliable, it is slower than modern methods like the Germ Tube Test or MALDI-TOF MS.

Dalmau Plate Technique: Introduction, Principle, Test Requirements, Procedure, Application, and Keynotes

Introduction

Principle

Test Requirements

Procedure

Applications

Keynotes

Further Readings

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