How to Identify Yeast by using different Techniques

Introduction

Yeast is a type of fungus that belongs to the kingdom of Fungi. It is a unicellular microorganism that is commonly found in soil, water, and on plant surfaces. Yeast is an important organism in various industries, including baking, brewing, and pharmaceuticals, due to its ability to convert simple sugars into carbon dioxide and ethanol.

Characteristics of yeast

Yeast is characterized by its unicellular nature, small size (typically less than 5 micrometers in diameter), and the presence of a cell wall. Yeast cells can reproduce by budding, which involves the growth of a small outgrowth from the mother cell that eventually separates to form a new daughter cell. Yeast cells also contain a nucleus, which contains the genetic material of the cell.

Application

One of the most well-known applications of yeast is in the baking industry. Yeast is used as a leavening agent in bread, where it ferments sugars present in the dough to produce carbon dioxide gas, which causes the dough to rise. Yeast is also used in the brewing industry to ferment sugars in grains to produce beer, wine, and other alcoholic beverages.

In the pharmaceutical industry, yeast is used as a host for the production of therapeutic proteins, such as insulin and human growth hormone. This is accomplished by genetically engineering yeast cells to produce the desired protein, which can then be harvested and purified.

Other applications of yeast include the production of enzymes, such as amylase and invertase, which are used in the food industry to break down complex sugars into simpler forms. Yeast is also used as a probiotic, due to its ability to improve gut health by modulating the microbiome.

Sample collection 

Sample collection and preparation is a critical step in the identification of yeast. This process involves selecting an appropriate sample type, sterilizing equipment and media, and using appropriate culture techniques to isolate yeast from the sample.

Choosing appropriate sample types

Choosing appropriate sample types is important for the successful identification of yeast. Common sample types include soil, water, and food. For example, soil samples can be collected from areas with high microbial activity, such as compost piles or decaying organic matter. Water samples can be collected from lakes, rivers, or other bodies of water, while food samples can be collected from a variety of sources, including dairy products, fruits, and vegetables.

Sterilization of equipment and media 

Once the sample has been collected, it is important to sterilize the equipment and media to prevent contamination from other microorganisms. This involves using techniques such as autoclaving, which involves subjecting the equipment and media to high temperatures and pressure to kill any microorganisms present. Sterilization ensures that the yeast isolated from the sample is pure and not contaminated by other microorganisms.

Culture techniques

Culture techniques are used to isolate and identify yeast from the sample. Selective media, such as Sabouraud agar, can be used to isolate yeast from other microorganisms present in the sample. These media contain ingredients that inhibit the growth of bacteria and promote the growth of yeast. Dilution plating is another common technique used to isolate yeast. This involves diluting the sample in a liquid media and then spreading the diluted sample onto agar plates. The yeast cells grow into colonies on the agar plates, which can then be isolated and identified.

Other culture techniques used to identify yeast include biochemical tests, such as the utilization of specific substrates, and molecular techniques, such as DNA sequencing. These techniques are used to identify specific characteristics of the yeast, such as its ability to utilize certain sugars or its genetic makeup.

Macroscopic characteristics 

The macroscopic examination is a key method used in the identification of yeast. It involves observing the appearance of yeast colonies on agar plates and the general morphology of the yeast cells. These observations can provide important clues about the identity of the yeast species.

The appearance of yeast colonies

The appearance of yeast colonies on agar plates can vary depending on the species and environmental conditions. Common characteristics used to describe yeast colonies include size, shape, texture, and color. Yeast colonies are typically small and round, with smooth or wrinkled edges. The texture of the colonies can vary from smooth to rough, and the color can range from white to cream to yellow, depending on the species and the medium used.

Observation of yeast morphology under a microscope

In addition to the macroscopic examination of the yeast colonies, microscopic examination is also important. This involves observing the morphology of the yeast cells under a microscope. Common characteristics used to describe yeast cell morphology include shape, size, and budding pattern.

Microscopic examination 

The shape of yeast cells can vary depending on the species. Some yeast cells are spherical, while others are oval or cylindrical. The size of yeast cells can also vary, with some species having smaller cells than others.

The budding pattern under the microscope is another important characteristic used to identify yeast species. Yeast cells reproduce asexually through budding, where a small outgrowth or bud forms on the parent cell and eventually detaches to form a new daughter cell. The pattern of budding can vary depending on the species. For example, some yeast species may produce multiple buds, while others may produce a single bud.

Observation of yeast cell structure is important in the identification of yeast. Yeast cells have several distinct structures, including the cell wall, vacuole, and nucleus. The cell wall is a key feature of yeast cells and provides structural support and protection. The cell wall can be observed using staining techniques such as the Calcofluor White stain. The vacuole is a membrane-bound organelle that is involved in the storage and transport of various molecules. The nucleus contains the genetic material of the yeast cell and can be observed using staining techniques such as DAPI (4′,6-diamidino-2-phenylindole).

Identification of specific structures

Identification of specific structures is also important in the identification of yeast species. Pseudohyphae are structures that can be observed in some yeast species and are formed when cells remain attached after budding. They can be observed using staining techniques such as lactophenol cotton blue. Ascospores are another structure that can be used to identify yeast species. These are reproductive structures that are formed in some yeast species and can be observed using staining techniques such as the ascospore stain.

Physiological and biochemical tests

Physiological and biochemical tests are important methods used in the identification of yeast species. These tests examine the growth requirements, substrate utilization, and enzyme production of the yeast, providing important information about the metabolic characteristics of the yeast species.

Growth requirements

Growth requirements refer to the environmental conditions necessary for the yeast to grow. These conditions include temperature, pH, and oxygen availability. Yeast can grow over a wide range of temperatures, but the optimal temperature for growth can vary between species. Similarly, the optimal pH for growth can vary between species, with some yeast species requiring acidic conditions and others requiring neutral or alkaline conditions. The availability of oxygen can also affect the growth of yeast, with some species being obligate aerobes, requiring oxygen for growth, while others are obligate anaerobes, growing only in the absence of oxygen.

Utilization of specific substrates

The utilization of specific substrates is another important characteristic used in the identification of yeast species. Yeast can utilize a wide range of substrates for energy production, including sugars, amino acids, and fatty acids. The ability to utilize specific substrates can vary between species, with some species being able to utilize a broader range of substrates than others. For example, Saccharomyces cerevisiae, commonly used in the production of beer and wine, is able to utilize a variety of sugars, while Candida albicans, a pathogenic yeast species, are able to utilize a range of nitrogen sources.

Production of specific enzymes

The production of specific enzymes is another characteristic used in the identification of yeast species. Yeast produces a range of enzymes, including amylase, lipase, and protease, which can be used for various metabolic processes. The production of specific enzymes can vary between species, providing important clues about the metabolic characteristics of the yeast. For example, the production of lipase is important in the spoilage of dairy products, making it an important characteristic to consider in the identification of yeast species involved in food spoilage.

Molecular techniques 

Molecular techniques are powerful methods used in the identification of yeast species. These methods involve the use of DNA extraction and amplification, sequence analysis, and comparison to existing databases for the accurate identification of yeast species.

DNA extraction and amplification

DNA extraction is the first step in the molecular identification of yeast species. This involves breaking open the yeast cells and isolating the DNA. There are several methods of DNA extraction, including the use of commercial kits and traditional methods such as phenol-chloroform extraction. Once the DNA is extracted, it can be amplified using polymerase chain reaction (PCR) or other amplification techniques.

Sequence analysis

Sequence analysis is a crucial step in the molecular identification of yeast species. The most commonly used sequences for yeast identification are the 16S rRNA gene and the internal transcribed spacer (ITS) region. The 16S rRNA gene is a conserved region of DNA that is present in all prokaryotes and eukaryotes, while the ITS region is a variable region of DNA found between the small and large ribosomal RNA genes in fungi. The amplified DNA can be sequenced using Sanger sequencing or Next-Generation Sequencing (NGS) technologies.

Comparison to existing databases and phylogenetic analysis

Comparison of the obtained sequence data to existing databases is an important step in the identification of yeast species. Several databases, such as GenBank, NCBI, and UNITE, contain sequences of identified yeast species, and these databases can be used to match the obtained sequence data to a known yeast species. Phylogenetic analysis can also be performed to identify the evolutionary relationships between the yeast species.

MALDI-TOF

The identification of yeast species using MALDI-TOF involves the extraction of proteins from the yeast cells, followed by the application of a matrix that facilitates the ionization of the proteins. The ionized proteins are then separated based on their mass-to-charge ratio and detected by a mass spectrometer. The resulting mass spectrum provides a unique protein profile for each yeast species, which can be used for identification.

MALDI-TOF mass spectrometry has several advantages over traditional identification methods, including its speed and accuracy. The method can provide identification results within minutes, compared to several days required for traditional methods such as physiological and biochemical tests. The method is also highly accurate, with reported identification rates of up to 99% for yeast species.

The use of MALDI-TOF mass spectrometry for yeast identification has been shown to be effective for a wide range of yeast species, including commonly encountered species such as Candida albicans and Cryptococcus neoformans, as well as less common species such as Geotrichum candidum and Trichosporon asahii.

Common species for identification

Identification of common yeast species is crucial for various industries and medical fields. Accurate identification of these species is important for the diagnosis and treatment of yeast infections and for selecting appropriate strains for use in fermentation processes. In this section, we will discuss four common yeast species and their identification methods.

Saccharomyces cerevisiae (baker’s yeast)

Saccharomyces cerevisiae is a commonly used yeast species in the food and beverage industry, particularly in baking and brewing. It is a unicellular, round yeast that reproduces through budding. Identification of Saccharomyces cerevisiae can be achieved through microscopic examination, physiological and biochemical tests, and molecular techniques such as the sequencing of the 18S rRNA gene and ITS region.

Candida albicans (human pathogen)

Candida albicans is an opportunistic pathogen that can cause a range of infections in humans, including thrush and systemic candidiasis. It is a unicellular, oval yeast that reproduces through budding. Identification of Candida albicans can be achieved through microscopic examination, physiological and biochemical tests, and molecular techniques such as sequencing of the 18S rRNA gene and ITS region.

Cryptococcus neoformans (human and animal pathogen)

Cryptococcus neoformans is a pathogenic yeast that can cause serious infections in humans and animals, particularly those with weakened immune systems. It is a unicellular, round yeast that reproduces through budding. Identification of Cryptococcus neoformans can be achieved through microscopic examination, physiological and biochemical tests, and molecular techniques such as the sequencing of the 18S rRNA gene and ITS region.

Rhodotorula spp. (food spoilage organism)

Rhodotorula spp. is a yeast species that can cause spoilage of various food products, including dairy and meat products. It is a unicellular, round yeast that reproduces through budding. Identification of Rhodotorula spp. can be achieved through microscopic examination, physiological and biochemical tests, and molecular techniques such as sequencing of the 18S rRNA gene and ITS region.

Future perspective

In recent years, there have been several emerging trends in yeast identification techniques and research. These include the development of rapid and high-throughput sequencing technologies, such as Next-Generation Sequencing (NGS) and MinION sequencing, which can provide rapid and accurate identification of yeast species. The use of MALDI-TOF mass spectrometry is also becoming more common for the identification of yeast species. This technology allows for the rapid and accurate identification of yeast species based on their protein profiles.

In addition to these emerging technologies, there is also ongoing research in the identification of new yeast species and their metabolic capabilities. This research aims to identify yeast species with unique metabolic capabilities that can be used in various biotechnological applications, such as the production of biofuels and the development of novel drugs. This research is also important for the identification of yeast species with potential medical and industrial applications.

References 

  • D. H. Pincus, S. Orenga, S. Chatellier, Yeast identification — past, present, and future methods, Medical Mycology, Volume 45, Issue 2, March 2007, Pages 97–121, https://doi.org/10.1080/13693780601059936
  • Hierro, N., González, Á., Mas, A. and Guillamón, J.M. (2004), New PCR-based methods for yeast identification. Journal of Applied Microbiology, 97: 792-801. https://doi.org/10.1111/j.1365-2672.2004.02369.x
  • Nerurkar V, Khan S, Kattungal S, Bhatia S. Identifying Candida and other yeast-like fungi: utility of an identification algorithm in resource limited setting. J Clin Diagn Res. 2014 Dec;8(12):DC01-4. doi: 10.7860/JCDR/2014/9753.5259. Epub 2014 Dec 5. PMID: 25653943; PMCID: PMC4316249.
  • Qvirist, L. A., De Filippo, C., Strati, F., Stefanini, I., Sordo, M., Andlid, T., Felis, G. E., Mattarelli, P., & Cavalieri, D. (2016). Isolation, Identification and Characterization of Yeasts from Fermented Goat Milk of the Yaghnob Valley in Tajikistan. Frontiers in Microbiology, 7. https://doi.org/10.3389/fmicb.2016.01690
  • https://www.mdpi.com/2311-5637/8/10/538
Mubashir Iqbal
Mubashir Iqbal

Mubashir Iqbal is a highly dedicated and motivated Microbiologist with an MPhil in Microbiology from the University of Veterinary and Animal Sciences. Currently, he is researching the efficacy of commercially available SARS Cov-2 vaccines to neutralize the omicron variant in Pakistan. He holds a Bachelor's degree in Microbiology and has experience in chemical and microbiological analysis of water samples, managing SOPs and documents according to standard ISO 17025. Additionally, he has worked as an internee in BSL 3, Institute of Microbiology, UVAS, where he gained experience in RNA extraction, sample processing, and microscopy.

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