Biochemical Tests for Bacteria and Fungi

Dr. Parag Chauhan

Introduction

  • Biochemical tests are laboratory methods used to identify microorganisms based on their metabolic activities.
  • They help differentiate bacteria and fungi that may have similar microscopic or colony characteristics.
  • These tests detect specific enzymes, metabolic reactions, and nutrient utilization patterns.
  • Common bacterial tests include catalase, oxidase, coagulase, indole, and urease tests.
  • Important fungal tests include germ tube, carbohydrate assimilation, and urease tests.
  • Biochemical tests are commonly performed on pure microbial cultures.
  • Results are interpreted along with microscopy and culture characteristics.
  • They are important for accurate microbial identification and diagnosis of infections.

Biochemical Tests for Bacteria

  • Biochemical tests help to identify bacteria by evaluating their metabolic properties, enzymatic activity, and reaction to specific chemicals.
  • These tests are essential for distinguishing species, particularly those with similar morphological characteristics.
  • Here’s a more detailed explanation of the key biochemical tests used in bacteriology:

Catalase Test

  • The catalase test is a biochemical test used to detect the enzyme catalase in microorganisms.
  • It is mainly used for the identification and differentiation of Gram-positive cocci.
  • The test is especially useful for differentiating Staphylococcus from Streptococcus species.

Principle

  • Catalase enzyme breaks down hydrogen peroxide (H₂O₂) into water and oxygen.

2H₂O₂ → 2H₂O + O₂↑

  • The release of oxygen produces visible bubbles.
  • Bubble formation = Catalase positive
  • No bubble formation = Catalase negative

Requirements

  • Fresh bacterial culture
  • Clean glass slide
  • Wooden applicator stick or sterile loop
  • 3% hydrogen peroxide
  • Dropper
  • Positive and negative controls

Specimen

  • A fresh, pure bacterial culture is generally used.
  • The test should preferably be performed from a suitable non-blood-containing medium because blood can contain catalase and may interfere with interpretation.

Procedure

  1. Place a drop of 3% hydrogen peroxide on a clean glass slide.
  2. Using a sterile wooden applicator stick, transfer a small amount of the bacterial colony.
  3. Mix the bacterial growth with the hydrogen peroxide.
  4. Observe immediately for bubble formation.
  5. Record the result as positive or negative.

Interpretation

Positive Catalase Test

  • Immediate bubbling is observed.
  • Indicates the presence of catalase enzyme.
  • Example: Staphylococcus spp.

Negative Catalase Test

  • No bubble formation is observed.
  • Indicates absence of detectable catalase activity.
  • Example: Streptococcus spp.

Result

Observation Result
Immediate, obvious bubbles Catalase positive
No bubbles Catalase negative

Clinical Significance

  • The catalase test is an important primary identification test in bacteriology.
  • It is commonly used to differentiate:
    • Staphylococcus → Catalase positive
    • Streptococcus → Catalase negative
  • It can also help in the identification of other catalase-producing bacteria.

Coagulase Test

  • The coagulase test is a biochemical test used to detect the enzyme coagulase produced by certain bacteria.
  • It is mainly used to differentiate Staphylococcus aureus from other staphylococci.
  • S. aureus is typically coagulase positive, while most coagulase-negative staphylococci are negative.

Principle

  • Coagulase promotes the conversion of fibrinogen to fibrin, resulting in clotting of plasma.
  • The test is performed as:
    • Slide coagulase test – detects bound coagulase (clumping factor).
    • Tube coagulase test – detects free coagulase.

Requirements

  • Fresh bacterial culture
  • Clean glass slide
  • Sterile loop or wooden applicator stick
  • Rabbit plasma or appropriate plasma reagent
  • Test tubes
  • Normal saline
  • Incubator

Specimen

  • A fresh, pure bacterial culture suspected to be Staphylococcus is used.
  • Clinical specimens such as pus, wound swabs, or blood are first cultured to obtain isolated colonies.

Slide Coagulase Test

  1. Place two drops of normal saline on a clean slide.
  2. Emulsify the bacterial colony in each drop to prepare two suspensions.
  3. Add a drop of plasma to one suspension.
  4. Mix gently and observe for visible clumping.
  5. The saline suspension serves as a control for auto-agglutination.

Interpretation

  • Visible clumping within a short period → Positive
  • No clumping → Negative

Tube Coagulase Test

  1. Add an appropriate amount of plasma to a sterile test tube.
  2. Prepare a suspension of the test organism in the plasma.
  3. Incubate according to the laboratory’s validated protocol.
  4. Examine the tube periodically for clot formation.
  5. A known positive and negative control should be included when appropriate.

Interpretation

  • Clot formation → Coagulase positive
  • No clot formation → Coagulase negative

Result

Observation Result
Plasma shows clotting Coagulase positive
Plasma remains liquid Coagulase negative

Clinical Significance

  • The coagulase test is an important test for identifying S. aureus.
  • S. aureus is an important cause of:
    • Skin and soft-tissue infections
    • Wound infections
    • Abscesses
    • Bacteremia
    • Pneumonia
    • Food poisoning
  • Correct identification is important for appropriate clinical management and antimicrobial testing.

Oxidase Test

  • The oxidase test is a biochemical test used to detect the enzyme cytochrome c oxidase in bacteria.
  • It is particularly useful for identifying and differentiating Gram-negative bacteria.

Principle

  • Cytochrome c oxidase is involved in the bacterial electron transport chain.
  • The enzyme oxidizes the oxidase reagent, producing a characteristic dark purple or blue color.
  • Color development within the specified reaction time = Oxidase positive.
  • No color development = Oxidase negative.

Requirements

  • Fresh bacterial culture
  • Oxidase reagent or oxidase test strip
  • Sterile wooden applicator stick or appropriate non-reactive tool
  • Filter paper or commercial oxidase strip
  • Positive and negative controls

Procedure

  1. Place a piece of filter paper or an oxidase test strip on a clean surface.
  2. Moisten it with the appropriate oxidase reagent, if required.
  3. Using a sterile wooden stick, transfer a small amount of the bacterial colony.
  4. Rub the colony onto the reagent area.
  5. Observe for color development within the manufacturer’s specified time.
  6. Record the result as positive or negative.

Interpretation

Oxidase Positive

  • A purple/blue color develops rapidly within the specified time.
  • Indicates the presence of detectable cytochrome c oxidase.
  • Examples:
    • Pseudomonas
    • Neisseria
    • Vibrio
    • Aeromonas

Oxidase Negative

  • No characteristic color develops within the specified time.
  • Examples:
    • Escherichia coli
    • Klebsiella
    • Salmonella

Clinical Significance

  • The oxidase test is particularly useful for differentiating oxidase-positive non-fermenting Gram-negative bacteria from Enterobacterales.
  • It is usually performed along with Gram staining, colony morphology, and other biochemical tests for accurate identification.

Indole Test

  • The indole test is a biochemical test used to determine whether a bacterium can produce indole from tryptophan.
  • It is commonly used for the identification and differentiation of Gram-negative bacteria.

Principle

  • Some bacteria produce the enzyme tryptophanase.
  • Tryptophanase breaks down tryptophan to form indole.
  • After incubation, Kovac’s reagent is added.
  • If indole is present, a cherry-red/pink layer develops at the surface.

Tryptophan → Indole + other products

Requirements

  • Fresh bacterial culture
  • Tryptophan-containing medium, commonly tryptone broth
  • Kovac’s reagent
  • Sterile inoculating loop
  • Incubator
  • Appropriate positive and negative controls

Procedure

  1. Inoculate the test organism into tryptone broth.
  2. Incubate according to the laboratory’s validated conditions.
  3. Add the required amount of Kovac’s reagent.
  4. Allow the reagent to form a layer on the surface.
  5. Observe the surface layer for red or pink color development.
  6. Record the result.

Interpretation

Indole Positive

  • A cherry-red/pink ring develops at the top of the medium.
  • Indicates production of indole.
  • Examples:
    • Escherichia coli
    • Proteus vulgaris
    • Morganella morganii

Indole Negative

  • No red/pink ring develops.
  • The reagent layer remains yellow or unchanged.
  • Examples:
    • Klebsiella pneumoniae
    • Enterobacter spp.

Clinical Significance

  • The indole test is useful for differentiating closely related Gram-negative enteric bacteria.
  • It is commonly used along with other biochemical tests for bacterial identification.

Methyl Red and Voges-Proskauer Tests

1. Methyl Red (MR) Test

  • The MR test detects the production of stable acidic end products during glucose fermentation.
  • It helps differentiate enteric Gram-negative bacteria based on their fermentation patterns.

Principle

  • Some bacteria ferment glucose through the mixed-acid fermentation pathway.
  • This produces stable acids that significantly lower the pH of the medium.
  • Addition of methyl red indicator produces a red color when the pH is sufficiently acidic.

Procedure

  1. Inoculate the organism into MR-VP broth.
  2. Incubate according to the laboratory’s validated protocol.
  3. Add methyl red reagent to the culture.
  4. Observe the color change.
  5. Record the result.

Interpretation

  • Red color → MR positive
  • Yellow color → MR negative
  • An intermediate orange color should be interpreted according to the laboratory’s validated procedure.

Examples

  • Escherichia coli → MR positive
  • Klebsiella pneumoniae → MR negative

2. Voges–Proskauer (VP) Test

  • The VP test detects the production of acetoin, an intermediate product of the 2,3-butanediol fermentation pathway.

Principle

  • Some bacteria ferment glucose and produce acetoin.
  • In the presence of oxygen and appropriate VP reagents, acetoin is converted to a compound that produces a red color.
  • A red color therefore indicates a VP-positive reaction.

Procedure

  1. Inoculate the organism into MR-VP broth.
  2. Incubate according to the laboratory’s validated protocol.
  3. Transfer or use the appropriate portion of the culture as specified by the test procedure.
  4. Add the required VP reagents.
  5. Mix and expose the reaction to oxygen as specified.
  6. Observe for red color development within the recommended time.

Interpretation

  • Red color → VP positive
  • No red color → VP negative

Examples

  • Klebsiella pneumoniae → VP positive
  • Escherichia coli → VP negative

Urease Test

  • The urease test is a biochemical test used to detect the enzyme urease produced by microorganisms.
  • It is useful for identifying and differentiating several bacterial species.

Principle

  • Urease hydrolyzes urea into ammonia and carbon dioxide.

Urea + H₂O → 2NH₃ + CO₂

  • Ammonia increases the pH of the medium.
  • A pH indicator, commonly phenol red, changes color in an alkaline environment.
  • Pink/red color → Urease positive
  • No color change → Urease negative

Requirements

  • Fresh bacterial culture
  • Urea-containing medium, such as Christensen’s urea agar or urea broth
  • Sterile inoculating loop
  • Incubator
  • Appropriate controls

Procedure

  1. Inoculate the test organism into the urea-containing medium.
  2. Incubate according to the laboratory’s validated protocol.
  3. Examine the medium for color change.
  4. Compare the result with appropriate positive and negative controls.
  5. Record the result.

Interpretation

Urease Positive

  • The medium develops a pink to reddish-purple color.
  • Indicates production of urease.
  • Common examples:
    • Proteus spp.
    • Morganella spp.
    • Providencia spp.
    • Corynebacterium urealyticum

Urease Negative

  • The medium shows no characteristic alkaline color change.
  • Examples include:
    • Escherichia coli
    • Salmonella spp.

Clinical Significance

  • The urease test helps differentiate bacteria with different urea-hydrolyzing abilities.
  • It is particularly useful in the identification of Proteus, Morganella, and Providencia species.
  • Urease-producing organisms can contribute to alkaline urine and certain urinary tract infections, and some are associated with urinary stone formation.

Carbohydrate Fermentation Tests

  • Carbohydrate fermentation tests are biochemical tests used to determine whether a microorganism can ferment specific carbohydrates.
  • They help identify and differentiate bacteria based on their carbohydrate metabolism.

Principle

  • Bacteria ferment carbohydrates such as glucose, lactose, sucrose, or mannitol to produce acid, with or without gas.
  • The acid lowers the pH of the medium and causes a color change in the pH indicator.
  • Gas production may be detected using a Durham tube.

Carbohydrate → Organic acids ± Gas

Requirements

  • Carbohydrate fermentation broth
  • Specific carbohydrate, such as glucose or lactose
  • Suitable pH indicator
  • Durham tube for gas detection, when required
  • Fresh bacterial culture
  • Sterile inoculating loop
  • Incubator
  • Appropriate positive and negative controls

Procedure

  1. Prepare or use the appropriate carbohydrate fermentation medium containing the selected sugar and indicator.
  2. Place a Durham tube in the broth if gas detection is required.
  3. Inoculate the medium with the test organism.
  4. Incubate according to the laboratory’s validated conditions.
  5. Observe the medium for color change.
  6. Check the Durham tube for gas production.
  7. Record the result.

Interpretation

Positive Fermentation

  • A change in indicator color due to acid production indicates carbohydrate fermentation.
  • A bubble in the Durham tube indicates gas production.
  • Results may therefore be reported as:
    • Acid positive
    • Acid and gas positive

Negative Fermentation

  • No characteristic color change indicates that the organism did not ferment the tested carbohydrate under the test conditions.
  • No gas is observed in the Durham tube.

Common Carbohydrates Tested

  • Glucose
  • Lactose
  • Sucrose
  • Mannitol
  • Maltose
  • Inositol
  • Other carbohydrates depending on the organism and identification system

Examples

  • Escherichia coli: commonly ferments glucose and lactose with acid and gas production.
  • Salmonella: commonly ferments glucose but generally does not ferment lactose.
  • Shigella: generally ferments glucose without gas production.
  • Staphylococcus aureus: commonly ferments mannitol, producing acid.

Clinical Significance

  • Carbohydrate fermentation patterns are useful for identification and differentiation of microorganisms.
  • They are particularly helpful in differentiating enteric Gram-negative bacteria.
  • They are often used together with other biochemical tests such as indole, citrate, urease, MR, and VP tests.

Triple Sugar Iron Test

  • The Triple Sugar Iron (TSI) test is a biochemical test used mainly to differentiate Gram-negative enteric bacteria based on their ability to ferment sugars and produce gas and hydrogen sulfide (H₂S).
  • It is commonly used for the identification of members of the Enterobacterales.

Principle

  • TSI agar contains three carbohydrates:
    • Glucose – 0.1%
    • Lactose – 1%
    • Sucrose – 1%
  • It also contains phenol red as a pH indicator.
  • Fermentation of sugars produces acid, causing the medium to turn yellow.
  • If only glucose is fermented, the limited glucose is rapidly depleted on the slant and the slant may become alkaline again, producing a red slant/yellow butt (K/A).
  • Gas production causes cracks, bubbles, or lifting of the agar.
  • H₂S production produces blackening, mainly in the butt.

Requirements

  • TSI agar slant
  • Fresh bacterial culture
  • Sterile inoculating needle
  • Incubator
  • Appropriate positive and negative controls

Procedure

  1. Take a fresh, pure bacterial colony.
  2. Use a sterile inoculating needle to pick the organism.
  3. Stab the butt of the TSI agar to near the bottom of the tube.
  4. Streak the slant surface while withdrawing the needle.
  5. Leave the cap loosely closed to allow appropriate gas exchange.
  6. Incubate according to the laboratory’s validated protocol.
  7. Examine the slant and butt for color changes, gas production, and H₂S production.

Interpretation

TSI results are generally reported using slant/butt reactions.

Reaction Meaning
A/A Glucose + lactose and/or sucrose fermentation
K/A Glucose fermentation only
K/K or K/NC No carbohydrate fermentation
Gas + Cracks, bubbles, or lifting of agar
H₂S + Blackening of the butt

A = Acidic (yellow)
K = Alkaline (red)
NC = No change

1. A/A Reaction

  • Slant: Yellow
  • Butt: Yellow
  • Indicates fermentation of glucose plus lactose and/or sucrose.
  • Example: Escherichia coli

2. K/A Reaction

  • Slant: Red
  • Butt: Yellow
  • Indicates glucose fermentation only.
  • Example: Salmonella spp.

3. K/K or K/NC Reaction

  • Slant: Red
  • Butt: Red or unchanged
  • Indicates no carbohydrate fermentation.

4. H₂S Production

  • Blackening of the butt indicates hydrogen sulfide production.
  • When blackening occurs, the butt should generally be considered acidic.

5. Gas Production

  • Gas is indicated by:
    • Cracks in the agar
    • Bubbles
    • Separation or lifting of the agar

Clinical Significance

  • TSI testing is useful for the presumptive identification of enteric Gram-negative bacteria.
  • It helps differentiate organisms based on:
    • Sugar fermentation
    • Gas production
    • H₂S production
  • It is commonly interpreted together with indole, citrate, urease, MR-VP, oxidase, and other biochemical tests.

Biochemical Tests for Fungi

  • For fungi, the identification is generally based on morphology, growth patterns, and specific metabolic reactions.
  • Many biochemical tests for fungi focus on their ability to assimilate nutrients or produce specific enzymes.
  • Here’s a detailed look at some common biochemical tests for fungi:

Germ Tube Test

  • The Germ Tube Test is a rapid microscopic test used mainly for the presumptive identification of Candida albicans and Candida dubliniensis.
  • It detects the ability of yeast cells to produce germ tubes under suitable conditions.

Principle

  • When certain Candida species are incubated in a suitable protein-rich medium, they form germ tubes.
  • Germ tubes are tube-like extensions that arise from yeast cells without a constriction at their point of origin.
  • A positive germ tube test supports the identification of C. albicans/C. dubliniensis.

Requirements

  • Fresh yeast culture
  • Serum or validated germ-tube test medium
  • Sterile test tube
  • Incubator
  • Microscope
  • Clean glass slide and coverslip

Procedure

  1. Prepare a suspension of the yeast colony in sterile serum or an appropriate test medium.
  2. Incubate under the laboratory’s validated conditions, commonly for a short period.
  3. Place a drop of the suspension on a clean glass slide.
  4. Cover with a coverslip.
  5. Examine microscopically using appropriate magnification.
  6. Look for germ tube formation.

Interpretation

Positive Germ Tube Test

  • Tube-like extensions are seen emerging from yeast cells.
  • The extension has no constriction at its base.
  • A positive result supports Candida albicans or Candida dubliniensis.

Negative Germ Tube Test

  • No germ tubes are observed.
  • Yeast cells remain predominantly oval or budding without true germ tube formation.
  • Most other Candida species are germ-tube negative.

Clinical Significance

  • The test provides a rapid presumptive identification of C. albicans/C. dubliniensis.
  • It is useful in clinical microbiology laboratories for the preliminary identification of Candida isolates.
  • It can help guide further identification and testing.

Chlamydospore Formation

  • Chlamydospore formation is a characteristic feature used in the identification of certain fungi, particularly Candida species.
  • Chlamydospores are thick-walled, rounded survival structures formed under unfavorable or nutrient-limited conditions.
  • The test is mainly associated with the identification of Candida albicans.

Principle

  • Under suitable nutrient-limited conditions, Candida albicans can produce large, thick-walled chlamydospores.
  • These structures are usually formed at the ends or sides of pseudohyphae.
  • Their presence provides a useful presumptive identification feature.

Requirements

  • Fresh yeast culture
  • Suitable chlamydospore-inducing medium, such as cornmeal agar
  • Sterile inoculating needle
  • Petri plate
  • Incubator
  • Microscope

Procedure

  1. Inoculate the suspected yeast onto an appropriate chlamydospore-inducing medium.
  2. Incubate under suitable laboratory conditions.
  3. Examine the culture microscopically after the recommended incubation period.
  4. Look for pseudohyphae with large, round, thick-walled chlamydospores.
  5. Record the findings.

Microscopic Appearance

  • Chlamydospores appear as:
    • Large
    • Round or oval
    • Thick-walled
    • Usually associated with pseudohyphae
  • They may occur terminally or laterally along the pseudohyphae.

Interpretation

Positive

  • Characteristic chlamydospores are observed.
  • Supports presumptive identification of Candida albicans.

Negative

  • No characteristic chlamydospores are observed.
  • Suggests that the isolate may be another yeast species, although the result must be interpreted with other identification tests.

Clinical Significance

  • Chlamydospore formation is a useful traditional mycological identification test.
  • It can help differentiate Candida albicans from many other yeast species.
  • It is particularly useful when combined with the germ tube test, colony morphology, and other identification methods.

Sugar Assimilation Tests

  • Sugar assimilation tests are biochemical tests used mainly for the identification and differentiation of yeasts and other fungi.
  • They determine whether a microorganism can utilize specific sugars as a source of carbon for growth.

Principle

  • Different fungi have different abilities to utilize carbohydrates.
  • The organism is tested against individual sugars as the sole carbon source.
  • If the organism can assimilate the sugar, growth occurs around or in the corresponding test system.
  • The pattern of sugar utilization helps identify the microorganism.

Common Sugars Tested

  • Glucose
  • Maltose
  • Sucrose
  • Lactose
  • Galactose
  • Raffinose
  • Trehalose
  • Other carbohydrates depending on the identification system

Requirements

  • Pure yeast culture
  • Suitable basal medium without another carbon source
  • Individual carbohydrate sources
  • Sterile test materials
  • Incubator
  • Appropriate control strains

Procedure

  1. Prepare a standardized suspension of the test yeast.
  2. Inoculate the organism into or onto the appropriate assimilation test system.
  3. Provide different sugars separately as potential carbon sources.
  4. Incubate under suitable conditions.
  5. Observe for growth associated with each sugar.
  6. Record the assimilation pattern.

Interpretation

Positive Assimilation

  • Visible growth occurs in the presence of a particular sugar.
  • Indicates that the organism can utilize that sugar as a carbon source.

Negative Assimilation

  • No significant growth occurs with the tested sugar.
  • Indicates that the organism does not assimilate that carbohydrate under the test conditions.

Clinical Significance

  • Sugar assimilation patterns help differentiate clinically important yeasts.
  • They are useful in the identification of organisms such as:
    • Candida species
    • Cryptococcus species
    • Other medically important yeasts
  • The results can be compared with standard identification databases or biochemical identification systems.

Advantages

  • Useful for differentiating yeast species with similar morphology.
  • Can provide a characteristic biochemical profile.
  • Helpful when combined with microscopy and colony characteristics.

Urease Test for Yeasts

  • The urease test detects the ability of yeast to produce the enzyme urease.
  • It is useful for differentiating certain medically important yeasts.
  • It is particularly helpful in the identification of Cryptococcus species.

Principle

  • Urease breaks down urea into ammonia and carbon dioxide.

Urea + H₂O → NH₃ + CO₂

  • Ammonia increases the pH of the medium.
  • A pH indicator changes color in an alkaline environment.

Procedure

  1. Inoculate the yeast culture into an appropriate urea-containing medium.
  2. Incubate under suitable laboratory conditions.
  3. Observe the medium for a color change.
  4. Compare the result with appropriate controls.

Interpretation

  • Positive: Pink/red color develops due to alkalinization of the medium.
  • Negative: No characteristic color change occurs.

Examples

  • Cryptococcus neoformans → Urease positive
  • Candida albicans → Usually urease negative

Clinical Significance

  • The test is useful as a supportive identification test for yeasts.
  • Urease positivity can help differentiate Cryptococcus from many other medically important yeasts.
  • Results should be interpreted together with colony morphology, microscopy, and other identification tests.

Potassium Hydroxide (KOH) Preparation

  • KOH preparation is a simple and rapid microscopic test used to detect fungal elements in clinical specimens.
  • It is commonly used for the preliminary diagnosis of superficial fungal infections.

Principle

  • Potassium hydroxide, commonly 10–20% KOH, dissolves keratin, cellular debris, and other tissue components.
  • Fungal structures such as hyphae, pseudohyphae, and yeast cells are more resistant and remain visible under the microscope.
  • This makes fungal elements easier to detect.

Specimens

Common specimens include:

  • Skin scrapings
  • Hair
  • Nail clippings or nail scrapings
  • Oral or vaginal specimens
  • Other appropriate clinical materials suspected of fungal infection

Requirements

  • Clinical specimen
  • 10–20% KOH solution
  • Clean glass slide
  • Coverslip
  • Microscope
  • Appropriate collection materials

Procedure

  1. Place a small amount of the clinical specimen on a clean glass slide.
  2. Add a drop of KOH solution.
  3. Place a coverslip over the specimen.
  4. Allow the preparation to clear for an appropriate period.
  5. Examine the preparation microscopically using low and high-power objectives.
  6. Look for characteristic fungal elements.

Microscopic Findings

Positive KOH Preparation

Fungal structures may appear as:

  • Septate or non-septate hyphae
  • Pseudohyphae
  • Budding yeast cells
  • Other characteristic fungal structures

Negative KOH Preparation

  • No fungal elements are observed in the examined preparation.

Clinical Significance

  • KOH preparation provides a rapid preliminary diagnosis of fungal infection.
  • It is particularly useful for detecting dermatophytes in skin, hair, and nail specimens.
  • It can also demonstrate yeast and pseudohyphal elements in appropriate specimens.
  • A negative result does not completely exclude fungal infection because fungal elements may be sparse or absent in the examined sample.