UNDER MICROBIOLOGICAL INVESTIGATION Microbiologists begin by sampling the object of their interest. It could be nearly any thing or place on Earth (or even Mars). Very common sources are body fluids, foods, water, solid, plants, and animals, but even places like icebergs, volcanoes, and rocks can be sampled.

SPECIMEN COLLECTION Note: The process begins with obtaining a sample from a patient, environment, or object.

UNDER MICROBIOLOGICAL INVESTIGATION The sample is placed into a container of medium that will support its growth. The medium may be in solid or liquid form, and held in tubes, plates, flasks, and even eggs. The delivery tool is usually a loop, needle, swap or syringe.

INOCULATION Note: The collected specimen is introduced into a sterile culture medium to encourage microbial growth.

UNDER MICROBIOLOGICAL INVESTIGATION Inoculated media are placed in a controlled environment (incubator) to promote growth. During the hours or days of this process, a culture develops as the visible growth of the microbes in the container of medium.

INCUBATION Note: The inoculated sample is placed in an incubator at an optimal temperature, oxygen level, and humidity. This allows microbes to grow and form visible colonies over time (usually 24–48 hours).

UNDER MICROBIOLOGICAL INVESTIGATION Some inoculation techniques can separate microbes and spread them apart to create isolated colonies that each contain a single type of microbe. This is invaluable for identifying the exact species of microbes in the sample, and it paves the way for making pure cultures.

ISOLATION Note: If multiple microbes are present, they must be separated into individual colonies for further study. The streak plate method or other isolation techniques help separate different species.

UNDER MICROBIOLOGICAL INVESTIGATION Cultures are observed for the macroscopic appearance of growth characteristics. Cultures are examined under the microscope for basic details such as cell type and shape. This may be enhanced through staining and use of special microscopes.

INSPECTION Note: The isolated colonies are examined for color, shape, texture, and microscopic structure. Gram staining, biochemical tests, and genetic analysis may be performed to classify the microbes.

UNDER MICROBIOLOGICAL INVESTIGATION Additional tests for microbial function and characteristics are usually required. This may include inoculations into specialized media that determine biochemical traits, immunological testing, and genetic typing. Such tests will provide specific information unique to a certain microbe.

INFORMATION GATHERING Note: Additional tests are performed to gather biochemical, genetic, and immunological data. Examples include: Biochemical Tests – Sugar fermentation, catalase test, oxidase test, etc. Genetic Analysis – PCR (Polymerase Chain Reaction), sequencing, etc. Immunological Tests – ELISA, antibody-antigen reactions, etc.

UNDER MICROBIOLOGICAL INVESTIGATION One goal of these procedures is to attach a name or identify to the microbe, usually to the level of species. Any information gathered from inspection and investigation can be useful. It is accomplished through the use of keys, charts, and computer programs that analyze the data and arrive at a final conclusion.

IDENTIFICATION Note: The final step where the exact species or strain is determined. The organism is compared against known microbial databases. The identified microbe can then be classified and, if necessary, an appropriate treatment plan can be developed (in clinical settings).

WHAT IS THIS? This is the extent of enlargement of an object when viewed through a microscope.

MAGNIFICATION Note: This occurs due to the interaction between visible light waves and the curvature of a lens.

In the Final Image formation ___________ of the final image is a product of the separate magnifying powers of the two lenses.

Total Magnification Note: This is determined by the combination of objective lens magnification and ocular lens magnification.

The Methods for Studying Microorganisms Flashcards by Athea Ordis

What are the 7 steps of microbiological investigation in order?

Specimen Collection
Inoculation
Incubation
Isolation
Inspection
Information Gathering
Identification

REMEMBER THE ACRONYM SIIIIII(6)

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UNDER MICROBIOLOGICAL INVESTIGATION

Microbiologists begin by sampling the object of their interest. It could be nearly any thing or place on Earth (or even Mars).
Very common sources are body fluids, foods, water, solid, plants, and animals, but even places like icebergs, volcanoes, and rocks can be sampled.

SPECIMEN COLLECTION

Note:

The process begins with obtaining a sample from a patient, environment, or object.

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UNDER MICROBIOLOGICAL INVESTIGATION

The sample is placed into a container of medium that will support its growth. The medium may be in solid or liquid form, and held in tubes, plates, flasks, and even eggs. The delivery tool is usually a loop, needle, swap or syringe.

INOCULATION

Note:

The collected specimen is introduced into a sterile culture medium to encourage microbial growth.

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UNDER MICROBIOLOGICAL INVESTIGATION

Inoculated media are placed in a controlled environment (incubator) to promote growth. During the hours or days of this process, a culture develops as the visible growth of the microbes in the container of medium.

INCUBATION

Note:

The inoculated sample is placed in an incubator at an optimal temperature, oxygen level, and humidity.
This allows microbes to grow and form visible colonies over time (usually 24–48 hours).

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UNDER MICROBIOLOGICAL INVESTIGATION

Some inoculation techniques can separate microbes and spread them apart to create isolated colonies that each contain a single type of microbe. This is invaluable for identifying the exact species of microbes in the sample, and it paves the way for making pure cultures.

ISOLATION

Note:

If multiple microbes are present, they must be separated into individual colonies for further study.
The streak plate method or other isolation techniques help separate different species.

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UNDER MICROBIOLOGICAL INVESTIGATION

Cultures are observed for the macroscopic appearance of growth characteristics. Cultures are examined under the microscope for basic details such as cell type and shape. This may be enhanced through staining and use of special microscopes.

INSPECTION

Note:

The isolated colonies are examined for color, shape, texture, and microscopic structure.
Gram staining, biochemical tests, and genetic analysis may be performed to classify the microbes.

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UNDER MICROBIOLOGICAL INVESTIGATION

Additional tests for microbial function and characteristics are usually required. This may include inoculations into specialized media that determine biochemical traits, immunological testing, and genetic typing. Such tests will provide specific information unique to a certain microbe.

INFORMATION GATHERING

Note:

Additional tests are performed to gather biochemical, genetic, and immunological data.

Examples include:

Biochemical Tests – Sugar fermentation, catalase test, oxidase test, etc.
Genetic Analysis – PCR (Polymerase Chain Reaction), sequencing, etc.
Immunological Tests – ELISA, antibody-antigen reactions, etc.

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UNDER MICROBIOLOGICAL INVESTIGATION

One goal of these procedures is to attach a name or identify to the microbe, usually to the level of species. Any information gathered from inspection and investigation can be useful. It is accomplished through the use of keys, charts, and computer programs that analyze the data and arrive at a final conclusion.

IDENTIFICATION

Note:

The final step where the exact species or strain is determined.
The organism is compared against known microbial databases.
The identified microbe can then be classified and, if necessary, an appropriate treatment plan can be developed (in clinical settings).

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A reliable microscope has these two characteristics that work together to help scientists and researchers study microorganisms effectively.

What are these?

Magnification
Resolving Power

REMEMBER THE ACRONYM MR

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WHAT IS THIS?

The ability of the microscope to enlarge objects so they appear bigger than their actual size. This is achieved through the ocular lens (eyepiece) and objective lens (closer to the specimen).

Magnification

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WHAT IS THIS?

The ability of the microscope to show detail. It determines the clarity and sharpness of the image. A higher of this means finer details can be observed.

Resolving power

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WHAT IS THIS?

This is the extent of enlargement of an object when viewed through a microscope.

MAGNIFICATION

Note:

This occurs due to the interaction between visible light waves and the curvature of a lens.

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STUDY THE PARTS OF THE MICROSCOPE (Make sure to have the picture of it)

Interpupillary adjustment
Ocular (eyepiece)
Body
Nosepiece
Arm
Objective lens (4)
Mechanical stage
Substage condenser
Aperture diaphragm control
Base with light source
Field diaphragm lever
Light intensity control
Course adjustment knob
Fine focus adjustment knob
Stage adjustment knobs

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Magnification occurs in two phases and this is through these lenses. What are these?

Objective lens and Ocular lens

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The objective lens (located near the specimen) captures light and magnifies the image.

What image does the objective lens form?

Real image

Note:

This is larger than the actual specimen but still inside the microscope.

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The ocular lens (eyepiece) takes the real image and magnifies it again.

This produces what kind of image?

Virtual image

Note:

This is the image we see when looking through the microscope.

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In the Final Image formation

___________ of the final image is a product of the separate magnifying powers of the two lenses.

Total Magnification

Note:

This is determined by the combination of objective lens magnification and ocular lens magnification.

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What is the formula for Total Magnification?

Objective power x Ocular power = Total Magnification

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The capacity to distinguish or separate two adjacent objects and depends on what?

RESOLUTION

Note:

Resolution is the ability of a microscope to distinguish two closely spaced objects as separate rather than a single blurred image.

ADD:

Resolution vs. Magnification

Magnification makes objects larger but does not improve detail.
Resolution determines how clear and sharp the image appears.
High magnification without good resolution results in a blurry image.

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Under QUANTIFYING RESOLUTION

What is the formula for Resolving Power (RP)?

Wavelength of light in nm / 2 x Numerical Aperture of objective lens

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Quantifying Resolution in Microscope

Visible light wavelength is 400 nm–750 nm

Light microscopes use visible light to illuminate specimens, and the wavelength of this light affects the clarity of the image. Shorter wavelengths, closer to 400 nm, provide better resolution because they can capture finer details, while longer wavelengths, closer to 750 nm, may produce a less sharp image.

Numerical aperture of lens ranges from 0.1 to 1.25

The numerical aperture (NA) measures a lens’s ability to collect light and resolve fine details. A higher NA, closer to 1.25, allows more light to enter and improves image sharpness. A lower NA, around 0.1, gathers less light and results in a less detailed image.

Shorter wavelength and larger numerical aperture will provide better resolution

Resolution is the ability to distinguish two close objects as separate. A shorter wavelength improves resolution because it minimizes diffraction, while a larger numerical aperture gathers more light, enhancing image clarity. Combining both factors results in a sharper and more detailed image.

Oil immersion objectives resolution is 0.2 μm

Oil immersion lenses improve resolution by reducing light refraction, allowing more light to pass through the specimen. This technique enhances image clarity, making it possible to see structures as small as 0.2 micrometers, which is essential for viewing tiny microorganisms.

Magnification between 40x and 2000x

The total magnification of a microscope depends on the combination of objective and ocular lenses, typically ranging from 40x to 2000x. While higher magnification enlarges the image, resolution must also be high to maintain clarity; otherwise, the image may appear blurry or distorted.

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Purpose of Oil in Microscopy

Oil immersion is used in high-power microscopy to improve resolution by reducing light refraction. When light passes from glass to air, it bends and scatters, leading to image distortion. Immersion oil has the same refractive index as glass, preventing light from bending and allowing more light to enter the objective lens. This enhances clarity and detail, making it possible to see very small structures more clearly, especially in specimens like bacteria.

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What are the 3 Variations on the Optical Microscope?

Bright-field
Dark-field
Phase-contrast

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WHAT IS THIS?

Most widely used; specimen is darker than surrounding field; used for live and preserved stained specimens.

Bright-field microscope

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WHAT IS THIS? * Brightly illuminated specimens surrounded by dark field; used for live and unstained specimens.

**Dark-field** microscope

WHAT IS THIS? * Transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures.

**Phase-contrast** microscope

WHAT IS THIS? * Modified microscope with an **ultraviolet radiation** source and filter. * Uses **dyes** that emit visible light when bombarded with shorter UV rays — fluorescence. * Useful in diagnosing infections.

**Fluorescence Microscope**

WHAT IS THIS? It is an advanced optical microscope that improves image clarity and depth perception. * Uses a **laser beam** of light to scan the specimen. * Integrates images to allow focus on multiple depths or planes.

Scanning Confocal Microscope

WHAT IS THIS? * Forms an image with a **beam of electrons** that can be made to travel in wavelike patterns when accelerated to high speeds. * Electron waves are 100,000 times shorter than the waves of visible light. * Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength. * Magnification between 5000X and 1,000,000X

Electron Microscopy

What are the **2 Types of Electron Microscopes**?

* Transmission electron microscopes (TEM) * Scanning electron microscopes (SEM)

WHAT IS THIS? * Transmit electrons through the specimen. Darker areas represent thicker, denser parts and light areas indicate more transparent, less dense parts.

**Transmission Electron Microscopes** (TEM)

WHAT IS THIS? * Provide detailed three-dimensional view. It bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it.

**Scanning Electron Microscopes** (SEM)

In SPECIMEN PREPARATION FOR OPTICAL MICROSCOPES * This allow Examination of characteristics of **live cells**: size, motility, shape, and arrangement.

**Wet mounts** and **hanging drop mounts**

In SPECIMEN PREPARATION FOR OPTICAL MICROSCOPES * ________ are made by drying and heating a film of specimen.

Fixed mounts

In SPECIMEN PREPARATION FOR OPTICAL MICROSCOPES * This ______ is stained using dyes to permit visualization of cells or cell parts.

**smear** Note: **Fixed Mounts (Smear Preparation & Staining)** Process: 1️⃣ *Smear* – A thin film of specimen is spread on a slide. 2️⃣ *Drying & Heat-fixing* – Slide is air-dried and heat-fixed to kill the cells and secure them. 3️⃣ *Staining* – Dyes are applied to enhance visualization of cell parts.

WHAT IS THIS? * ______ are used to **increase contrast** and make microbes more visible.

**Dyes** Note: * Staining highlights **cell structures, shapes, and arrangements.**

UNDER STAINING * Cationic, positively charged chromophore

Basic dyes

UNDER STAINING * Surfaces of microbes are negatively charged and attract basic dyes.

Positive staining

UNDER STAINING * Anionic, negatively charged chromophore.

Acidic dyes

UNDER STAINING * Microbe repels dye, the dye stains the background.

Negative staining

UNDER STAINING * One dye is used; reveals shape, size, and arrangement.

Simple stains

UNDER STAINING Use a primary stain and a counterstain to distinguish cell types or parts. Examples: * Gram stain * Acid-fast stain * Endospore stain

Differential stains

UNDER STAINING * Reveal certain cell parts not revealed by conventional methods: capsule and flagellar stains.

Structural stains

What are the **6 I's of Culturing Microbes**?

* Inoculation * Isolation * Incubation * Inspection * Information gathering * Identification

WHAT IS THIS? * Introduction of a sample into a container of media to produce a **culture** of observable growth.

Inoculation

WHAT IS THIS? * Separating one species from another.

Isolation

WHAT IS THIS? * Under conditions that **allow growth**.

Incubation

WHAT IS THIS? * If an individual bacterial cell is separated from other cells and has space on a nutrient surface, it will grow into a mound of cells called?

**Colony** Note: * A colony consists of one species.

What are the **3 Isolation Techniques**?

* **S**treak plate technique * **P**our plate technique * **S**pread plate technique REMEMBER THE ACRONYM ***SPS***

ISOLATION TECHNIQUES * Bacteria are spread in a **zigzag or quadrant pattern** on an agar plate to isolate individual colonies.

Streak Plate Technique

ISOLATION TECHNIQUE * A diluted bacterial sample is **mixed with melted agar** and poured into a dish, allowing bacteria to grow inside and on the surface.

Pour plate technique

ISOLATION TECHNIQUE * A liquid bacterial sample is evenly spread across an agar surface using a **sterile spreader**, leading to surface colony growth.

Spread plate technique

Under Inspection in Microbiology, a species could grow into a single or more species. What are the **3 microbiological cultures**?

* Pure culture * Mixed culture * Contaminated culture

WHAT IS THIS? * Contains only **one species** of microorganism.

Pure culture

WHAT IS THIS? * Contains **two or more species** in the same container.

Mixed Culture

WHAT IS THIS? * Contains **unwanted or unknown microbes** that accidentally entered the culture.

Contaminated culture

FAMILIARIZE ONLY! **Ways to Identify a Microbe**

🔬 1. **Morphology & Staining** – Observing **cell shape, size, and arrangement** under a microscope using staining techniques. 🧬 2. **DNA Sequencing** – Analyzing the **genetic material** of the microbe for precise identification. 🧪 3. **Biochemical Tests** – Detecting **chemical reactions and metabolic activities** to classify the organism. 🩸 4. **Immunological Tests** – Using **antibodies** to detect specific microbes based on their surface markers.

Under "MEDIA: Providing Nutrients in the Laboratory" Media can be classified according to **three properties**. What are these?

* Physical State * Chemical composition * Functional type

What properties of media is this? * **Liquid, semisolid, and solid.**

Physical state

What properties of media is this? * **Synthetic** (chemically defined) and **complex**.

Chemical composition

What properties of media is this? * **General purpose, enriched, selective, differential, anaerobic, transport, assay, enumeration.**

Functional Type

UNDER **PHYSICAL STATES OF MEDIA**, what is this? * **Broth; does not solidify.** A. Liquid B. Solid C. Semisolid

A. Liquid

UNDER **PHYSICAL STATES OF MEDIA**, what is this? * **Contains solidifying agent.** A. Liquid B. Solid C. Semisolid

C. Semisolid

UNDER **PHYSICAL STATES OF MEDIA**, what is this? * **Firm surface for colony formation**. — Contains solidifying agent — Liquefiable and nonliquefiable A. Liquid B. Solid C. Semisolid

B. Solid

WHAT IS THIS? * **The most commonly used solidifying agent.**

**Agar** Note: * Solid at room temperature, liquefies at boiling (100°C), does not re-solidify until it cools to 42°C * Provides framework to hold moisture and nutrients. * Not digestible for most microbes.

What are the **2 Most Commonly Used Media**?

**Nutrient broth** and **Nutrient agar**

WHAT IS THIS? * A **liquid medium** containing **beef extract** and **peptone**, used for growing a wide variety of bacteria.

Nutrient broth

WHAT IS THIS? * A **solid medium** containing **beef extract, peptone, and agar**, commonly used for bacterial culture and isolation.

Nutrient agar

What are the **4 Types of Media Based on Chemical Content**?

* Synthetic * Complex or nonsynthetic * General purpose media * Enriched media

WHAT IS THIS? * Contains **pure organic and inorganic compounds** in an exact chemical formula.

Synthetic

WHAT IS THIS? * Contains at least **one ingredient** that is **not chemically definable**.

Complex or nonsynthetic

WHAT IS THIS? * Grows a **broad range of microbes**, usually nonsynthetic.

General purpose media

WHAT IS THIS? * Contains **complex organic substances** such as blood, serum, hemoglobin, or special growth factors required by fastidious microbes.

Enriched media

WHAT IS THIS? * Contains one or more agents that inhibit growth of some microbes and **encourage growth of the desired microbes**.

**SELECTIVE MEDIA**

WHAT IS THIS? * Allows growth of several types of microbes and displays **visible differences** among those microbes.

Differential media

**Some media can be both Selective & Differential** TRUE OR FALSE

TRUE

**Miscellaneous media** refers to specialized types of culture media that **do not fit into the common categories** (such as general-purpose, selective, or differential media). Instead, these media serve **specific functions** based on unique bacterial growth needs. What are the **2 Examples of Miscellaneous Media**?

* Reducing medium * Carbohydrate fermentation medium

WHAT IS THIS? * Contains a substance that **absorbs** oxygen or **slows** pentration of oxygen into medium; used for growing anaerobic bacteria.

Reducing Medium

WHAT IS THIS? * Contains **sugars** that can be fermented, converted to acids, and a **pH indicator** to show this reaction; used to detect acid production from fermentation.

Carbohydrate fermentation medium