7.2: Microbial Identification

Biotech Focus

Artificial Intelligence (AI) is increasingly being used in healthcare to identify bacteria. AI can process a large amount of data faster than traditional lab methods, allowing for faster diagnosis and treatment. AI-drive automated systems utilize databases, algorithms, and artificial intelligence to interpret test results rapidly, providing reliable identification within hours. Using these systems, AI-driven image analysis is also being developed for recognizing colony morphology and analyzing microscopic images, offering new avenues for rapid and accurate identification. It can be used to analyze medical images, such as microscope slides, to detect bacterial presence and classify the species involved. AI can process vast amounts of data more quickly than traditional lab methods. One of the applications of AI in microbiology is to test whether bacteria isolated from a patient are resistant or not to an antibiotic, which helps in determining which antibiotic treatment could be used.

Watch the video below to learn more about AI is being used detect antibiotic resistance.

Introduction

Microbes are everywhere—from the human body to the depths of the oceans, from sterile medical devices to harsh environments like volcanic springs. Identifying microbes is fundamental to microbiology, biotechnology, medicine, environmental science, and many other fields. Understanding which microorganisms are present in a sample helps researchers make important decisions—whether diagnosing infections, understanding ecosystem dynamics, or developing new biotechnologies. Microbe identification involves isolating and characterizing them in order to determine their identity, using a variety of traditional and modern methods.

Microbe identification is critical for various purposes:

• Healthcare: Identifying pathogens causing diseases is crucial for appropriate treatment. Knowing whether an infection is bacterial, fungal, or viral helps determine the best therapeutic approach.
• Biotechnology: Identification helps in selecting the right microbe for industrial applications, such as enzyme production or waste treatment
• Ecology: Identifying microbes in soil or aquatic environments helps scientists understand nutrient cycling, plant-microbe interactions, and environmental changes.
• Food Safety: Identifying spoilage organisms and pathogens is essential for food safety and quality control.

Microbial identification involves understanding not only the taxonomy of organisms but also their physiology and capabilities. Advances in technology have transformed how we approach this task, making identification faster, more precise, and accessible.

Learning Objectives

Being able to correctly identify a microbe is essential to fields like microbiology, medicine, and biotechnology. By the end of this section, you will be able to:

• Describe some of the stains used in identifying microbial morphology, including bacteria and yeast
• Describe the major types of media used culturing microbes
• Explain the difference between a broth, a plate, a slant, and a deep
• Describe some of the metabolic tests used in identifying microbes like bacteria and yeast
• Describe some of the molecular tests used in studying microbes
• Describe some of the chemotaxic and spectrometry tests used in studying microbes

Traditional Methods of Identification

Traditional methods of identification have formed the foundation of microbiology. These methods include morphological, cultural, biochemical, and immunological approaches.

Morphological Identification

Morphology is the study of the form, structure, and shape of organisms, cells, or biological components. By observing the morphology of bacteria (e.g., rod, cocci, spirilla), fungi (e.g., molds, yeasts), and other microbes, scientists can classify them into broad categories. For identification using morphology, microscopy is often the first step. Microbes like bacteria and yeast can be visualized using bright-field, dark-field, phase-contrast, or electron microscopy. For more information on microscopy, go to Chapter 1.4 Advanced Biotechnology Equipment.

Morphology can be studied and identified under the microscope using specific staining techniques. Depending on the objective, different staining protocols can be used. Some common stains used are:

1. Simple stains
2. Gram stain (differential stain)
3. Acid-fast stain (differential stain)
4. Spore stains (differential stain)

Simple stains can be used to determine the cellular shape and arrangement. They are used in a simple staining protocol. Simple staining involves staining a heat-fixed sample with only one dye in order to increase contrast and allow visualization of cell shape, size, and arrangement under a microscope. Basic dyes, such as crystal violet (purple), methylene blue (blue), malachite green (green), or safranin (red/pink), work well for simple staining of bacteria and yeast. These positively charged dyes bind to the negatively charged components in the cell walls and cytoplasm. Go to Chapter 13.15: Bacterial Identification Methods to learn how to perform a simple stain.

Concept in Action

Video: Simple Staining with Methylene Blue

A differential stain, like a Gram stain, uses a minimum of two stains. Each stain is specific to a structure or an organism in the sample. The first stain used is called the primary stain. Following staining with the primary stain, a mordant may then be used. A mordant is not a stain but is used to fix the primary stain and enhance its staining ability. A decolorizer is then used to selectively remove the primary stain from certain structures or organisms before a second stain, called a counterstain, is applied. Common decolorizing solutions are ethanol, acetone, or a combination of both.

The Gram stain stains the peptidoglycan component of bacterial cell walls (Figure \(\PageIndex{1}\)). For more information about the bacterial cell wall, go to Chapter 7.1 Microbial Classification. Based on how well they retain this Gram stain, bacteria are classified into Gram-positive and Gram-negative groups. The identification of bacteria as either Gram-positive or Gram-negative is critical to determining what antibiotics are chosen to treat a bacterial infection. The Gram stain uses a primary stain called crystal violet, combined with an iodine treatment as the mordant. The iodine forms a complex with the crystal violet stain which then allows it to bind the peptidoglycan layer of the cell wall more efficiently. An acetone or alcohol wash step "decolorizes" the bacteria, washing away the Gram stain. The bacteria are then counterstained with the counterstain, safranin O, which will stain the bacteria pink. Gram-positive bacteria, with their thick peptidoglycan layer, will efficiently retain the Gram stain and cannot be decolorized. As a result, they take on a purple color that masks the pink counter-stain. In contrast, Gram-negative bacteria are easily decolorized because the outer membrane prevents Gram staining of the peptidoglycan layer. The counter-stain is easily visualized in Gram-negative bacteria and these bacteria appear pink under the microscope. Go to Chapter 13.15: Bacterial Identification Methods to learn how to perform a Gram stain.

Figure \(\PageIndex{1}\): Gram stained bacteria. Gram staining of a bacterial smear containing Pseudomona aeroginosa and Staphylococcus epidermidis is shown in this figure. The pink rods are Pseudomona aeroginosa, a Gram-negative bacteria. The purple cocci are Staphylococcus epidermidis, a Gram-positive bacteria. (Yaritza Castellanos, CC BY-NC 4.0)

Acid-fast staining is differential staining technique used to identify bacteria with cell walls rich in mycolic acid, a "waxy" lipid that makes the cell wall impermeable to the Gram stain. Bacteria stained using this technique are Mycobacterium, a genus that contain pathogens such as Mycobacterium tuberculosis and Mycobacterium leprae that cause tuberculosis and leprae, respectively. In acid-fast staining bacteria are stained with a primary stain, called carbol fuschin, that binds to the mycolic acid and stains the bacteria reddish red or dark pink. Heat is used to help this stain penetrate the cell wall. The bacteria are decolorized to wash away the stain and then counter-stained with methylene blue. Acid-fast bacteria will retain their red color, while non acid-fast bacteria will lose the stain and appear blue as a result of the counter-stain. Go to Chapter 13.15: Bacterial Identification Methods to learn how to perform an acid-fast stain.

Spore staining is a differential staining technique used to view endospores. Endospores, also referred to as spores, are dormant structures that help bacteria survive unfavorable conditions. Endospores can germinate back into active bacterial cells when conditions improve. Endospore staining can be used to identify bacterial species and understand their ability to survive extreme conditions like high heat or desiccation, or to assess their pathogenicity. Due to their resistance to extreme conditions, it is important to take the presence of endospores into consideration when choosing conditions used to sterilize equipment as inadequate conditions will allow the endospores to survive. Common endospore forming bacteria include Bacillus anthracis, the bacteria that causes anthrax, and many bacteria in the Clostridium genus. The presence of high levels of keratin in the outer coat of the endospore makes conventional stains, like Gram stain, inadequate. Spore staining uses a primary stain, called malachite green, to stain spores and mature bacterial cells green. A decolorizing step is followed by counter-staining with safranin (Figure \(\PageIndex{2}\)). Spores are resistant to decolorizing and will remain green, while mature bacteria will display the pink color of the counter-stain. Go to Chapter 13.15: Bacterial Identification Methods to learn how to perform an endospore stain.

Figure \(\PageIndex{2}\): Endospore stained bacteria. Endospore staining of a bacterial smear containing Bacillus megaterium is shown in this figure. The red rods are vegetative cells of Bacillus megaterium. The green structures are its endospores. (Kareen Martin, CC BY-NC 4.0)

Yeast can be stained using several stains. India Ink/Nigrosin detects the polysaccharide capsule surrounding specific yeast species. Yeast cells appear to have a clear halo around them. The presence of polysaccharides in the yeast cell wall can be detected using a Periodic Acid-Schiff/PAS (bright pink or red). Chitin within the fungal cell wall can be stained using a Lactophenol Cotton Blue (LPCB) stain (blue). Several fluorescent stains are also available, with acridine orange staining live yeast cells green and dead cells orange. The fluorescent stain Calcofluor white is highly sensitive and specific to yeast, binding to the chitin in their cell walls and fluorescing blue/green under UV light. In addition, yeast cells can also be stained using many bacterial stains. Simple stains like methylene blue can be used to distinguish between live yeast cells (colorless) and dead cells (blue). Gram staining is also used to identify yeast cells because their thick cell wall structure efficiently retains the Gram stain. As a result, yeast cells will stain dark purple. Distinguishing Gram-positive bacteria from stained yeast cells in a mixed sample is relatively straightforward owing to the larger size of the yeast cell.

Identification Using Culture Techniques

Culturing microbes, like bacteria and yeast, is essential for isolating and identifying individual species from a sample and also for the maintenance of stock cultures. A culture medium is a specially formulated mixture of nutrients and salts used for this purpose. Some culture media are chemically defined, meaning their exact chemical composition is known. Others are more complex, with their chemical composition is unknown. These undefined media usually consist of extracts and digests of yeasts, meat or plants. Different types of media are used depending on the need of the microbiologist. Table \(\PageIndex{1}\) below gives a few examples of common bacterial media.

Table \(\PageIndex{1}\): Culture Media Types

Media Type	Purpose	Examples
Non-selective media	Provides essential nutrients to allow for a wide variety of non-fastidious bacteria to grow (i.e., those bacteria that do not require special nutrients)	LB (Luria-Bertani) medium, Nutrient agar and Tryptic Soy Agar (TSA)
Selective media	Used for the growth of specific bacteria (i.e., fastidious bacteria). Includes compounds that inhibit the growth of unwanted species	MacConkey Agar - selects for Gram-negative bacteria Mannitol Salt Agar - selects for Staphylococcus species Eosin Methylene Blue (EMB) Agar - selects for Gram-negative gut bacteria
Differential media	Used to produce specific reactions (i.e. color changes) that distinguish between species based on metabolic characteristics.	Blood Agar
Enriched media	Contain extra nutrients for difficult to grow organisms	Chocolate Agar (contains lysed red blood cells)

Culture media can be prepared as a liquid (broth), as a solid (plate or slant) or as a semi-solid (deep tube) (Figure \(\PageIndex{3}\)). A broth is used to establish a liquid culture. Liquid cultures can be used to produce a desired volume of cells that can then be used in the laboratory in downstream applications, such as DNA isolation and metabolic assays. In a liquid culture, an individual cell, like a bacterium, is added to the broth and then incubated for a specific amount of time in order to allow them to "grow" (i.e. increase in number). The cloudier the broth, the more cellular growth. Hence, the amount of growth can be assessed by measuring the turbidity of the broth. To learn how to start a liquid culture of bacteria using a broth, go to Chapter 13.6: Bacterial Transformation.

Culture plates are prepared using a liquid medium that contains agar, a polysaccharide that polymerizes to a gel-like solid. When added to a broth, agar will cause the the liquid medium to solidify when a specific temperature is reached. To make a plate, the agar-containing liquid medium is poured into a petri dish and then the plate is allowed to cool. Culture plates are used to create individual colonies, using an inoculation loop and a technique called "streaking". A bacterial colony is a visible mass of bacteria that has grown from a single bacterial cell. Culture plates are used to observe the characteristics of colonies, such as size, color, shape, and texture. When the culture plates are stored properly, they can be used for short-term culture maintenance. The colonies that form on the plate can also be chosen to start liquid cultures in a broth. To learn how to streak a culture plate, go to Chapter 13.16: Basics of Bacterial Culture.

A slant is prepared in a test tube using an agar-containing medium. The test tube is tilted at an angle while the medium solidifies, leading to a slanted surface. The slant provides a larger surface area for cell growth. Slants are used to generate stocks for long-term maintenance. The cells (e.g. bacteria or yeast) can be streaked onto the slanted surface for observation of morphology or the inoculation loop can be "stabbed" into the agar to test for anaerobic growth. Cells grown in or on slants can survive for weeks to months and can be easily and safely transported.

A deep is prepared in a test tube that remains upright during solidification. The inoculation loop is stabbed into the column of agar. Depending on the media used, the deep can be used for biochemical analysis, along with an assessment of cell motility through the deep and their oxygen requirements. Those bacteria and yeast cells that can only grow on the surface of the deep require oxygen and are considered to be obligate aerobes. Cells that can survive throughout the deep are known as facultative aerobes (can growth with or without oxygen). Growth at the bottom of the deep classifies the cells as obligate anaerobes, cells that can grow without oxygen.

To learn how to make a culture plate, slant, or deep, in addition to learning about how to streak a plate and inoculate a bacterial culture, go to Chapter 13.16: Basics of Bacterial Culture.

Figure \(\PageIndex{3}\): Bacteria Culture Techniques. Techniques for culturing bacteria include, from left to right, liquid broth, plates, slants, and deep stabs. Each culture is created using tools such as an inoculation loop. (Bacteria Culture Techniques by Patricia Zuk, CC BY 4.0; figure created in BioRender. Zuk, P. (2025); photo credits (left to right): K. rhizophila liquid culture by Alexandre.Cz, CC BY-SA 3.0; Bacterial growth on blood agar by Ajay Kumar Chaurasiya, CC BY-SA 4.0 ; Actinomycetes by CDC/Dr. David Berd, public domain; Stab culture by Todd Parker, public domain)

Identification Using Biochemical Tests

Biochemical tests help determine the metabolic and enzymatic properties of bacteria and other microbes. Common tests include:

1. Catalase test
2. Oxidative test
3. Carbohydrate fermentation
4. Analytical Profile Index (API) strips

The catalase test is used to determine whether a cell produces catalase, an enzyme that breaks down hydrogen peroxide into water and oxygen (O₂). Aerobic cellular respiration (biological reactions that require O₂) releases hydrogen peroxide (H₂O₂) as a toxic byproduct that can damage or kill cells. Some cells produce catalase to protect themselves against the damages caused by hydrogen peroxide. The catalase test is a simple test that involves transferring cells (e.g. bacteria) into a drop of hydrogen peroxide. The presence of bubbles indicates a positive test - the production of oxygen, meaning that hydrogen peroxide has been split by catalase. The absence of bubbles means the test is negative and the cell doesn't produce catalase.

The oxidase test, also known as the oxidative test, is used to determine whether a cell produces the enzyme cytochrome c oxidase, the last enzyme within the electron transport chain, that catalyzes the transfer of electrons to oxygen. Microbes, like bacteria or yeast, are smeared onto filter paper and a few drops of an oxidase reagent are added. The presence of cytochrome c oxidase will cause the reagent to turn purple or blue. The oxidative test is particularly useful in the identification of oxidase-positive bacteria, including Neisseria sp. (which includes Neisseria gonorrhoeae and Neisseria meningitis), Vibrio sp (which includes Vibrio cholera), and Pseudomonas sp.

Carbohydrate fermentation tests are used to determine whether a microbe, like a bacteria or yeast cell, can ferment specific sugars. Because fermentation release acids and gases, the carbohydrate fermentation test detects the production of these components using a pH indicator for acid production and an inverted glass tube to capture gas production. In the test, a liquid medium containing nutrients, a pH indicator, and a specific sugar is dispensed into a sterile test tube. A small, thin Durham tube is inverted and submerged completely in the test tube. The tube is then inoculated with the organism of interest (Figure \(\PageIndex{4}\)).

Figure \(\PageIndex{4}\): The carbohydrate fermentation test. To test for sugar fermentation, bacteria are inoculated into test tubes containing a Carbohydrate broth composed of nutrients, a pH indicator, and a specific sugar (e.g. glucose, maltose, sucrose). A Durham tube is inverted and submerged in the broth so that it fills completely with broth. The tube is incubated at 37C for 24 to 48 hours to allow for growth and potential fermentation. The production of carbon dioxide will result in the presence of an air bubble in the Durham tube. The production of acids will be indicated by the pH indicator. The three tubes in this figure show positive fermentation (acids and gas) in the tube containing glucose and negative fermentation (no acids, no gas) in the tubes containing maltose and sucrose. (Carbohydrate Fermentation by Patricia Zuk, CC BY 4.0; adapted from Carbohydrate Fermentation by NewDancinShoes, CC BY-SA 4.0)

As the organism grows in the liquid medium, it will begin to ferment the added sugar and produce carbon dioxide gas and specific acids. The presence of a gas bubble in the Durham tube indicates the production of gas. The pH indicator will confirm the production of acids. Go to Chapter 13.15: Bacterial Identification Methods to learn how to perform a carbohydrate fermentation test.

Concept in Action

Video: Carbohydrate Fermentation Test

Analytical Profile Index (API) strips are composed of several biochemical tests on a single strip (Figure \(\PageIndex{5}\)). Each API strip is made of multiple "cups" or cupules. Each cupule serves as mini-testing chamber that contains a specific substrate. These substrates test for a specific metabolic function or for the production of specific enzyme. API testing strips can be used to analyze microbes, like bacteria or yeast. To use, a cellular suspension is added to each of the cupules and the strips are incubated. During incubation, reactions with each cupule produce color changes that are either spontaneous or revealed by the addition of reagents. The results are recorded as positive or negative depending on the color reaction on the strip. API strips allow for quick screening of multiple metabolic capabilities. For example, the API 20E test strip tests Gram-negative bacteria, like the Enterobacteriaceae family, for a variety of enzymatic reactions associated with fermentation. The use of API strips provide numerous advantages including fast reliable results for many clinical and environmental bacterial strains, standardized results, and no need for complex equipment. Furthermore, some API strips are compatible with automated readers that can compile the results and store them in a database. For the API strip protocol, go to Chapter 13.15: Bacterial Identification Methods.

Figure \(\PageIndex{5}\): An API strip. This figure shows results after incubating a suspension of E.coli cells in an API 20E test strip. The results in each well are classified as either positive or negative based on the colored product produced. (API 20E Escherichia coli by A doubt, CC BY-SA 4.0)

Molecular Methods for Identification

Advances in molecular biology have revolutionized microbial identification, providing fast, highly specific, and accurate methods for characterizing microorganisms, like bacteria, without needing extensive culture-based work.

Polymerase Chain Reaction (PCR) has become a cornerstone in microbial identification, allowing for the amplification of specific DNA sequences from small samples. By using species-specific primers, it is possible to amplify unique regions of bacterial DNA (and other microbes), confirming the presence of a particular organism. Variants such as qPCR (quantitative PCR) can be used to not only identify but also quantify the presence of microbial DNA in a sample. For more about PCR, go to Chapter 3.2 Replication of DNA. To learn about the PCR protocol, go to Chapter 13.2: Polymerase Chain Reaction (PCR).

Gene sequencing is one of the most common molecular methods for microbe identification. Bacterial identification can be achieved through the sequencing of the 16S rRNA gene. The 16S rRNA gene codes for the 16S ribosomal protein found within the 30S small subunit. The 16S rRNA gene contains several variable regions that are unique to different bacterial taxa, making it a reliable marker for identification of bacteria at the genus or species level. In addition to 16S sequencing, whole-genome sequencing (WGS) has gained popularity. WGS provides comprehensive information about an organism’s entire genetic makeup, offering insights not only into its taxonomy but also into its resistance genes, virulence factors, and potential metabolic capabilities. Identification of multiple microbial species in a sample through the sequencing of their genomes is being made possible through Next-Generation Sequencing (NGS). This approach has been used to study complex microbiomes, such as those in soil, the human gut, or in environmental samples. By directly sequencing DNA from an entire community, researchers can characterize microbial diversity and understand the functional roles of different species. For more about DNA sequencing, including WGS and NGS, go to Chapter 5.3 Genome Sequencing. To learn about the DNA sequencing protocol, go to Chapter 13.9: DNA Sequencing.

Chemotaxonomic and Spectroscopy Methods

Another set of methods used in identification involves chemotaxonomy (i.e., chemical taxonomy), which consists on classifying organisms based on their biochemical composition, and spectroscopic techniques.

Chemotaxonomy can classify bacteria and other microbes based on the presence of proteins, lipids, carbohydrates and other metabolic compounds. Gram staining and its identification of the presence of peptidoglycans in Gram-positive bacteria could be considered a chemotaxonomic technique. Fatty Acid Methyl Ester (FAME) analysis and its identification of the unique fatty acid "signatures" of a cell is another. Profiling by FAME can help differentiate microbial species, including bacteria, by examining the types and proportions of fatty acids unique to each group. FAME is a cheap, reliable and easy technique.

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry is a powerful tool for rapid identification of bacteria and other microbes. It works by analyzing the unique protein fingerprint of an organism, primarily focusing on ribosomal proteins, which are highly conserved across species. Results are then compared against a reference database to identify the microbe. MALDI-TOF has become popular in clinical and research laboratories for its speed, accuracy, and cost-effectiveness.

Immunological tests like serological tests use antibodies to detect specific antigens associated with an organism. In addition, the ELISA (Enzyme-Linked Immunosorbent Assay) is an example of an immunological technique used for microbial identification. These methods are particularly useful for detecting pathogenic microorganisms in clinical settings and are often used to identify bacteria, viruses, and fungi based on their specific surface markers.

Challenges and Future Directions

Despite the impressive advances in identification, challenges remain in identifying bacteria and other microbes. Identifying "unculturable" microorganisms, those that cannot be grown in a lab, is still a major obstacle. Another challenge is the presence of mixed microbial communities where multiple species are present in a single sample. Differentiating between closely related species requires precise methods, often involving multi-gene or whole-genome analysis. Next generation sequencing, together with metagenomics, the study of genetic material recovered directly from an environmental sample rather than from a cultured organism, and single-cell genomics, the sequencing of an entire genome from a single cell, are addressing these challenges, allowing researchers to sequence genomes directly from mixed populations, environmental samples or individual cells, respectively.

The future of microbial identification lies in continued advancements in sequencing technologies, machine learning, and bioinformatics. Developing more rapid, cost-effective, and high-throughput identification techniques will open doors to deeper insights into the microbial world and lead to new discoveries that impact health, biotechnology, and environmental management.

Lab Protocols

• Lab Technique: Bacterial Transformation
• Lab Technique: Polymerase Chain Reaction (PCR)
• Lab Technique: DNA Sequencing
• Lab Technique: Bacterial Identification Methods
• Lab Technique: Basics of Bacterial Culture

Key Concepts

Microbial identification is a cornerstone of many scientific and applied disciplines, providing critical insights into health, biotechnology, and ecology. From traditional microscopy and biochemical testing to cutting-edge molecular techniques and artificial intelligence, the journey of identifying microbes, such as bacteria allows us to explore microbial diversity in greater detail, understand complex communities, and harness the potential of microbes for various applications.

Some important concepts to remember are:

• Microbes like bacteria can be identified through morphology, the study of the form, structure, and shape of an organism
• Staining techniques enhance the morphological identification of microbes
• Simple stains use only one stain; differential stains uses multiple stains
• Media used to grow bacteria and other microbes can range from liquid broths to solid media like plates, slants, and deeps
• Non-selective media can be used to grow a wide variety of bacteria and other microbes
• Selective media compositions can be used to grow a specific type of microbe
• Molecular methods of microbe identification include PCR and DNA sequencing
• Chemical methods for identification include biochemical tests like catalase and oxidase tests
• More advanced techniques like FAME analysis and MALDI-TOF Mass Spectrometry identify microbes by examining their unique biochemical compositions

Glossary

 

Acid-fast staining - a staining technique used for staining bacteria with large amounts of mycolic acid in their cell walls

Aerobe – a microorganism that requires oxygen for growth

Agar - a polysaccharide isolated from red algae; when added to media it causes the solidification at a specific temperature

Anaerobe – a microorganism that does not require oxygen for growth and may even be harmed by it

Analytical Profile Index (API) test strip - a strip composed of several biochemical tests contained within small wells called "cupules"

Bacillus (plural = bacilli) – a rod-shaped bacterium

Bacterium (plural = bacteria) – a single-celled prokaryotic microbe that can be found in diverse environments and classified based on shape, Gram stain, and metabolism

Coccus (plural = cocci) – a spherical-shaped bacterium

Colony - a visible cluster of microorganisms, like bacteria, growing on a solid medium and originating from a single cell or a group of identical cells

Counter-stain - a secondary stain used in a staining technique; provides contrast to the primary stain

Classification – the organization of microorganisms into hierarchical groups based on characteristics such as genetics, morphology, and metabolism

Defined medium - a culture medium in which all chemical components are known and present in specific amounts

Differential medium - a type of culture medium that allows different types of microbes to be distinguished based on their biological characteristics

Differential staining - a staining technique that uses two or more contrasting stains to distinguish between different types of microbes or different structures within a single organism

Endospore – a highly resistant, dormant structure formed by some bacteria to survive harsh conditions

Endospore staining - a technique used to specifically stain endospores

Enriched medium - a microbial medium that provides additional nutrients to support the growth of fastidious organisms that require specific growth

Facultative anaerobe – a microorganism that can grow with or without oxygen

Fastidious - organisms that require special nutrients to grow

Genus – a taxonomic rank above species that groups related organisms based on common traits

Gram staining – a differential staining technique used to classify bacteria as Gram-positive (purple) or Gram-negative (pink) based on cell wall composition

Inoculation - the process of introducing microbes like bacteria, fungi, viruses, or other microbes into a culture medium

Medium - a mixture of nutrients used to support the growth of a cell; also called culture medium or growth medium

Microscopy - the technique of using a microscope to observe objects and structures that are too small to be seen with the naked eye

Mordant - a compound that enhances the staining effect of a dye by forming a complex with it

Morphology – the study of microbial shape, structure, and form

Non-fastidious - organisms that do not require special nutrients to grow

Obligate aerobe – a microorganism that requires oxygen for survival and metabolism

Obligate anaerobe – a microorganism that cannot survive in the presence of oxygen

Pathogen – a microorganism that causes disease in a host organism

Peptidoglycan – a polymer found in bacterial cell walls; providing structural strength and protection to the bacterial cell

Primary stain - the first dye applied in a staining procedure

Simple staining - a technique that uses a single stain to color microbes like bacteria

Stain - a chemical dye used to color cells, tissues, or cellular components in order to make them more visible under a microscope

Turbidity - the cloudiness of a liquid culture, indicating microbial growth