Microbiology W1-2

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Questions and Answers

What is the primary purpose of using staining techniques in light microscopy?

To reduce the limit of resolution

To visualize three-dimensional cell structures

To improve the contrast between specimens and their surroundings (correct)

To increase the magnification of specimens

Which scientist is credited with the first description of microbes in 'Micrographia'?

Joseph Lister

Antoni van Leeuwenhoek

Louis Pasteur

Robert Hooke (correct)

What is the limit of resolution for a light microscope?

0.2 μm (correct)

0.1 μm

0.5 μm

1 μm

Which type of microscope uses two sets of lenses to form an image?

Compound microscope Signup and view all the answers

What is the primary charge of basic dyes and their role in staining?

Positively charged, bind strongly to negatively-charged cell components Signup and view all the answers

How do gram-positive bacteria appear under a gram stain?

Purple-violet Signup and view all the answers

What does the term 'total magnification' refer to in microscopy?

The product of the objective magnification and ocular magnification Signup and view all the answers

Which microscopy method allows for observing living cells with minimal alteration?

Phase-contrast microscopy Signup and view all the answers

What is the primary advantage of phase-contrast microscopy?

Improves image contrast of unstained, live cells Signup and view all the answers

What characteristic of a light microscope limits its resolution?

The wavelength of visible light Signup and view all the answers

What is a key characteristic of dark-field microscopy?

Only scattered light from the specimen reaches the lens Signup and view all the answers

Which type of microscopy is best for observing motility?

Dark-field Signup and view all the answers

What function does the objective lens serve in a microscope?

It magnifies the specimen at various levels Signup and view all the answers

Which microscopy technique is best suited to visualize the internal structures of cells?

Electron microscopy Signup and view all the answers

What effect does the phase ring have in phase-contrast microscopy?

It amplifies differences in the refractive index of cell and surroundings Signup and view all the answers

What is the main purpose of fluorescence microscopy?

To visualize specimens that emit light after specific illumination Signup and view all the answers

What type of light microscope enhances contrast by using differences in the phase of light waves?

Phase-contrast microscope Signup and view all the answers

Which dye is used to show dead cells in fluorescence microscopy?

Propidium iodide (PI) Signup and view all the answers

How do gram-negative bacteria appear in a gram stain?

Pink Signup and view all the answers

Which microscopy technique enhances contrast using light that has been scattered from the sides?

Dark-field microscopy Signup and view all the answers

What characteristic of differential interference contrast (DIC) microscopy contributes to its three-dimensional imaging capability?

It utilizes a polarizer to create distinct beams of polarized light. Signup and view all the answers

Which microscopy technique involves the use of a laser coupled with a computerized microscope?

Confocal scanning laser microscopy (CSLM) Signup and view all the answers

Why must specimens be very thin when using transmission electron microscopy (TEM)?

To facilitate the passage of electrons through the specimen. Signup and view all the answers

What is a unique feature of cryo-electron microscopy (cryo-EM) in terms of specimen preparation?

Specimens are rapidly frozen in liquid ethane. Signup and view all the answers

What advantage does scanning electron microscopy (SEM) have compared to transmission electron microscopy (TEM)?

It can visualize thicker specimens. Signup and view all the answers

What characteristic is common to both differential interference contrast microscopy and confocal scanning laser microscopy?

They create three-dimensional images. Signup and view all the answers

What type of image is created by transmission electron microscopy (TEM)?

Electron micrographs that show molecular structures. Signup and view all the answers

What is the primary limitation of electron microscopes compared to light microscopes?

They operate under vacuum conditions. Signup and view all the answers

What is the main purpose of using high atomic weight substances in transmission electron microscopy (TEM)?

To scatter electrons and improve the contrast of images. Signup and view all the answers

Which microscopy technique is demonstrated to visualize protein structures in detail by analyzing numerous particle images?

Cryo-electron microscopy (cryo-EM) Signup and view all the answers

What is the role of bile salts in MacConkey agar?

To selectively inhibit most Gram-positive bacteria Signup and view all the answers

Which component in MacConkey agar is responsible for the color change indicating lactose fermentation?

Neutral red Signup and view all the answers

What is the significance of the color pink to mauve in CHROMagar VRE?

Reflects the growth of Vancomycin-Resistant Enterococci Signup and view all the answers

What makes CHROMagar a unique culture medium compared to traditional agar plates?

It employs chromogenic differentiation for specific bacterial detection Signup and view all the answers

What is the main function of the proteose peptone in MacConkey agar?

To provide a source of carbon and nitrogen for bacterial growth Signup and view all the answers

Which component in CHROMagar allows for detecting E.coli specifically?

β-galactosidase Signup and view all the answers

What should be the pH adjustment for MacConkey agar before use?

7.1 Signup and view all the answers

What is the purpose of following aseptic techniques in microbial culture?

To avoid contamination of the culture medium Signup and view all the answers

Which aspect of the CHROMagar patented system distinguishes it from other media?

Being the first chromogenic medium available commercially Signup and view all the answers

What type of bacteria are primarily inhibited by crystal violet in MacConkey agar?

Some Gram-positive bacteria Signup and view all the answers

What is the primary feature of enriched media?

It contains complex media plus highly nutritious materials. Signup and view all the answers

What do selective media achieve in the context of microbial growth?

They contain compounds that inhibit the growth of certain microbes. Signup and view all the answers

Which of the following best describes differential media?

Media that contains indicators to detect metabolic reactions. Signup and view all the answers

How does the composition of solid media differ from liquid media?

Solid media contain gelling agents like agar. Signup and view all the answers

What metabolic byproducts are generated by E. coli during anaerobic fermentation of lactose?

A mixture including lactate, acetate, and ethanol. Signup and view all the answers

What is a critical factor for successful cultivation of microbes in culture media?

Understanding and supplying the correct nutritional requirements. Signup and view all the answers

What role does β-galactosidase play in the metabolism of lactose by E. coli?

It converts lactose into glucose and galactose. Signup and view all the answers

Which characteristic is essential to consider when using differential media?

The presence of indicators that signal specific metabolic processes. Signup and view all the answers

Which of the following statements is true about the agar used in solid media?

Agar is derived from plant fibers and serves as a solidifying agent. Signup and view all the answers

What role do the acidic byproducts of fermentation play in the growth of E. coli?

They inhibit the growth of competing bacteria by lowering pH. Signup and view all the answers

What is the primary reason for diluting bacterial samples before measuring Optical Density (OD) when they are too dense?

To maintain linear correlation as per Beer-Lambert Law Signup and view all the answers

Which material is recommended for cuvettes when measuring absorbance in the ultraviolet region?

Quartz Signup and view all the answers

Optical Density (OD) is defined mathematically using which formula?

$-log10(T)$ Signup and view all the answers

Which of the following factors does NOT influence the Beer-Lambert Law relationship?

Rate of vibration of light waves Signup and view all the answers

What is a significant disadvantage of using a spectrophotometer for measuring microbial turbidity?

Results can be affected by cell clumping Signup and view all the answers

Which value indicates a region where the Beer-Lambert law is typically valid?

OD < 1.0 Signup and view all the answers

What does Optical Density correlate with in a spectrophotometric analysis of bacterial cells?

Concentration of bacterial cells Signup and view all the answers

The equation for calculating absorbance directly includes which component?

Transmittance (T) Signup and view all the answers

Which of the following is NOT an advantage of using a spectrophotometer?

High specificity for all microbes Signup and view all the answers

What is the correct relationship between intensity and absorbance as described in the equations?

Absorbance increases as transmittance decreases Signup and view all the answers

What is the main reason for requiring a standard curve when using turbidity measures to estimate microbial cell numbers?

To convert optical density readings to actual cell counts Signup and view all the answers

Which condition is least likely to affect the accuracy of viable plate counts?

Measurement of turbidity Signup and view all the answers

What does the term 'the great plate count anomaly' refer to regarding microbial counts?

The discrepancy between direct and indirect measurement methods Signup and view all the answers

Why should plates with fewer than 30 colonies be excluded from viable plate count analysis?

They may not represent a statistically valid sample Signup and view all the answers

In which phase of bacterial growth does E.coli typically double every 30 minutes under optimal conditions?

Exponential phase Signup and view all the answers

What limitation does the plate counting method have when analyzing mixed microbial cultures?

It cannot quantify organisms that do not grow under the conditions used Signup and view all the answers

What main factor contributes to the turbidity in a culture of unicellular organisms?

Light scattering by cells Signup and view all the answers

What is primarily accounted for when reporting plate counts in colony-forming units (CFU)?

Clumping of cells Signup and view all the answers

Why is the measurement of absorbance at 600 nm commonly used for E.coli density estimations?

E.coli absorbs light maximally at that wavelength Signup and view all the answers

What influence does a mixed culture have on the accuracy of viable plate counting?

Affects the growth rate of individual organisms Signup and view all the answers

Study Notes

Light Microscopy and the Discovery of Microorganisms

Robert Hooke, in 1665, was the first to describe microbes using a microscope.
Hooke's book, Micrographia, illustrated the fruiting structures of molds.
Antoni van Leeuwenhoek was the first to describe bacteria using a microscope.

Improving Contrast in Light Microscopy

Magnification is the ability to make an object appear larger.
Resolution is the ability to distinguish two adjacent objects as distinct and separate.
The limit of resolution for a light microscope is approximately 0.2 micrometers, which is half the wavelength of visible light.
Several types of light microscopy exist:
- Bright-field
- Phase-contrast
- Differential interference contrast
- Dark-field
- Fluorescence
Compound microscopes use two sets of lenses to form an image: an objective lens (magnifies 10-100x) and an ocular lens (magnifies 10-30x).
The total magnification of a compound microscope is the product of the objective lens magnification and the ocular lens magnification.
A magnification of 1,000x is needed to reach the 0.2-micrometer resolution limit of most light microscopes.
Bright-field microscopy uses normal light and visualization depends on differences in contrast or density between the specimen and its surroundings.

Microscopy & Staining

Staining can improve contrast by using dyes that bind to specific cellular materials.
Basic dyes, like methylene blue, crystal violet, and safranin, are positively charged and bind to negatively charged cell components, such as nucleic acids, acidic polysaccharides, and cell surfaces.
Simple staining uses dried cells to highlight the overall shape, size, and arrangement of cells.
Differential stains differentiate between cell types by staining them different colors.
Gram staining is a differential stain widely used for identifying bacteria, which differentiates them into two main groups: gram-positive and gram-negative.
- Gram-positive bacteria stain purple-violet due to a thick peptidoglycan layer in their cell walls.
- Gram-negative bacteria stain pink because they have a thinner peptidoglycan layer and an outer membrane.

Phase-Contrast Microscopy

Phase-contrast microscopy enhances image contrast of unstained, live cells.
A phase ring amplifies differences in refractive index between the cell and its surroundings.
An image with dark cells on a light background is produced.

Dark-Field Microscopy

Light reaches the specimen from the sides.
Only scattered light reaches the lens, resulting in an image that appears light on a dark background.
Better resolution than bright-field microscopy.
Excellent for observing motility, such as flagella.

Fluorescence Microscopy

Used to visualize specimens that fluoresce, meaning they emit light after being illuminated at a different wavelength.
Cells appear to glow on a black background due to filters.
Some cells fluoresce naturally (autofluorescence), while others can be stained with fluorescent dyes like DAPI.
Widely used in microbial ecology to enumerate bacteria in natural samples.

Differential Interference Contrast (DIC) Microscopy

Uses a polarizer to generate two polarized light beams
Provides a three-dimensional appearance to structures like nuclei, endospores, vacuoles, and inclusions

Confocal Scanning Laser Microscopy (CSLM)

Uses a computerized microscope with a laser source to create a 3D image
Computer focuses the laser on individual layers of the specimen
Different layers can be compiled for a complete 3D image

Electron Microscopy

Uses electrons instead of visible light (photons)
Electromagnets act as lenses
Operates in a vacuum
Images are captured using a camera, resulting in an electron micrograph
Two primary types: Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM)

Transmission Electron Microscopy (TEM)

Offers significantly higher resolving power (0.2 nm) than light microscopes (200 nm)
Enables visualization of structures at the molecular level
Requires thin specimens (20–60 nm) stained with high atomic weight substances for contrast

Scanning Electron Microscopy (SEM)

Provides detailed, three-dimensional images of surfaces

Cryo-Electron Microscopy (Cryo-EM)

Used to dissect protein structure
Involves rapidly freezing a specimen in liquid ethane
Cryo-samples are then visualized at -170 °C
3D structures are determined by analyzing thousands of individual protein particle images
Applicable for very large protein complexes

Enriched Media

Contain complex media plus highly nutritious materials, for example serum or blood
Used to culture fastidious microbes, which are nutritionally demanding

Selective Media

Contain compounds selectively inhibiting the growth of some microbes but not others

Differential Media

Contain an indicator, typically a dye, that detects particular metabolic reactions during growth

Considerations for Growth Media

For successful microbial cultivation, it is crucial to know the nutritional requirements and supply them in the proper form and proportions in a culture medium.
Cells can be cultivated in liquid or solid culture media.
Solid media are prepared by adding the gelling agent agar to liquid media.
When grown on solid media, cells form isolated masses called colonies.

Properties of Bacteria Utilized for Differential Media

Example: Lactose utilization and anaerobic fermentation in E. coli
Involves the conversion of lactose to glucose by β-galactosidase
Under anaerobic fermentative conditions, a mixture of succinate, formate, acetate, lactate, and ethanol is produced
The products, including e.g., acetate and lactate, are acidic

MacConkey Agar

A selective and differential medium
Selective: Bile salts inhibit most Gram-positive bacteria, and crystal violet inhibits certain Gram-positive bacteria.
Differential: Detects lactose fermentation
Lactose-fermenting bacteria: Consumes lactose or other six-carbon sugars and metabolizes them.
Acid production: Reduces the pH, turning the neutral red dye red.
Composition: Peptone, proteose peptone, lactose, bile salts, sodium chloride, neutral red, crystal violet, agar.

CHROMagar™

Enzyme-based chromogenic detection system
Invented by Dr. Alain Rambach in 1979
Pioneered chromogenic differentiation culture media for detecting E.coli
Patented a chromogenic system for detecting Salmonella in 1989, known as Rambach Agar
Established CHROMagar Microbiology in 1993

Typical Appearance of Microorganisms on CHROMagar™

VRE (Vancomycin-Resistant Enterococci) faecalis/faecium: Pink to mauve
E. gallinarum/E. casseliflavus: Blue or inhibited
Other Bacteria: Inhibited

CHROMagar™ E. coli and CHROMagar™ VRE

CHROMagar™ E. coli: β-galactosidase-based
Most chromogenic mixes are proprietary

Growth Media and Laboratory Culture

Microbes are ubiquitous.
Sterilization of media is crucial.
Aseptic technique should be followed to prevent contamination.

Viable Plate Counts

Measures living, reproducing microbial populations
Two main methods:
- Spread-plate method
- Pour-plate method
Serial dilution is key for accurate counts, which are typically between 30 and 300 colonies

Sources of Error

Inoculum size, viability, culture medium, and incubation conditions affect accuracy
Mixed cultures grow at different rates, leading to inaccuracies
Colony-forming units (CFUs) are reported instead of viable cell numbers to address clumping

Applications of Plate Counts

Quick and easy
Used in food, dairy, medical, and aquatic microbiology, as well as water analyses
High sensitivity
Can target specific species in mixed samples

The Great Plate Count Anomaly

Direct microscopic counts often reveal far more organisms than plate counts
Dead cells are counted microscopically, but not by viable methods
Different organisms have vastly different growth requirements, biasing culture methods

Turbidimetric Measures of Microbial Cell Numbers

Cell suspensions are turbid (cloudy) due to cells scattering light.
Turbidity is measured with a spectrophotometer, and the measurement is called optical density (OD)
OD is proportional to cell number for unicellular organisms within a certain range.
A standard curve must be established to relate OD to cell count.

Bacterial Growth Phases

E. coli can grow quickly, doubling every 30 minutes under optimal conditions.
Growth typically goes through three phases:
- Lag phase
- Exponential phase
- Stationary phase

E. Coli Culture and Turbidity

E. coli is typically cultured at 37°C with shaking for aeration.
Absorbance at 600 nm is commonly used to measure bacterial density, particularly for E. coli.
Absorbance is correlated to bacterial cell concentration.

Mechanism of a Spectrophotometer

A beam of light is passed through the sample, and the intensity of the transmitted light (I) is measured, compared to the initial light intensity (Io).
Transmittance (T) = I/Io
Optical density (OD) or Absorbance (A) = -log10T

How to Calculate Concentration

The Beer-Lambert Law relates absorbance (A) to concentration, path length (ℓ) and absorptivity (ɛ):
A = ɛℓC*
Absorbance is linearly correlated with concentration within a certain range, typically absorbance < 1.0

Advantages of Turbidimetric Methods

Quick and easy to perform
Does not require sample destruction or significant disturbance
Allows repeated measurements on the same sample

Disadvantages of Turbidimetric Methods

Can be problematic for microbes that form clumps or biofilms in liquid medium.

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Description

Explore the fundamentals of light microscopy and its role in the discovery of microorganisms, illustrated famously by Robert Hooke and Antoni van Leeuwenhoek. This quiz covers different types of microscopy, magnification, and resolution. Test your knowledge on the essential concepts that paved the way for modern microbiology.