Fundamental Techniques 2018 (1)

Microscopy

The technology of making very small things visible to the human eye.

Total Magnification

The product of the eyepiece magnification times the objective lens magnification.

Resolution

The ability of a lens to distinguish two points as separate and discrete units.

Numerical Aperture

A measure of a microscope objective's ability to gather light and resolve fine specimen detail.

Working Distance

The distance between the front edge of the objective lens and the specimen surface.

Bright-Field Microscope

A light microscope that produces a dark image against a brighter background.

Gram Stain

A differential stain that distinguishes Gram-positive cells which turn purple from Gram-negative cells which turn pink.

Aseptic Technique

A set of specific practices and procedures performed under controlled conditions to minimize contamination.

Sterilization

The process of removing or killing all living or viable organisms from a medium.

Culture Media

Mixtures of nutrients that support microbial growth.

Selective Media

Growth media that favor one organism while suppressing others through added components.

Differential Media

Growth media that allow identification of microorganisms based on phenotypic properties.

Turbidity Measurement

An indirect method of measuring microbial growth, related to the cloudiness of a culture.

Exponentially Growth

Growth pattern where bacteria double in number through binary fission.

Plate Count

Method of measuring viable cells by counting colonies formed on a nutrient agar surface.

Fluorescence Microscopy

A microscopy technique that uses UV light to excite molecules in a specimen to produce visible fluorescence.

Electron Microscope

A type of microscope that uses beams of electrons for greater resolving power than light microscopes.

Heat Sterilization

A sterilization method using heat to destroy microorganisms, including endospores.

Phase Contrast Microscope

A type of light microscope that enhances contrast in transparent and colorless objects by altering the phase of light passing through the specimen.

Confocal Microscopy

A technique that provides increased optical resolution by using a spatial pinhole to eliminate out-of-focus light.

Sample Preparation

The process of treating samples to prepare them for observation under a microscope, which may include fixation, sectioning, and staining.

Staining Techniques

Methods used to enhance contrast in microscopic specimens by adding dyes or stains that attach to specific parts of the cells.

Biosafety Level (BSL)

A set of biocontainment precautions required to isolate dangerous biological agents in an enclosed laboratory facility.

Autoclaving

A method of sterilization using high-pressure steam to kill all forms of microbial life, including spores.

Petri Dish

A shallow, flat dish made of glass or plastic that is used to culture microorganisms on a solid medium.

Colony Forming Unit (CFU)

A unit used to estimate the number of viable bacteria or fungal cells in a sample, based on the number of colonies that develop.

Inoculation

The introduction of microorganisms into a culture medium for growth.

Broth Culture

A liquid growth medium used for cultivating organisms in a nutrient-rich environment.

Direct Microscopic Count

A method of measuring bacterial growth by directly counting cells under a microscope.

Optical Density Measurement

A method that assesses the turbidity of a culture by measuring light absorbance.

Fermentation Test

A technique to measure bacterial growth and metabolism based on gas production or pH changes during fermentation.

Most Probable Number (MPN) Method

A statistical method used to estimate the concentration of viable microorganisms in a sample based on dilution.

Flow Cytometry

A technology that analyzes individual cells in a fluid as they pass through a laser, allowing for growth measurement by counting.

Pros of Direct Microscopic Count

Immediate results; allows for the examination of cell morphology.

Cons of Direct Microscopic Count

May include dead cells in count; requires a well-prepared sample and skilled technician.

Pros of Plate Count Method

Measures only viable cells; provides a visual representation of colony growth.

Cons of Plate Count Method

Time-consuming; requires incubation periods and may not represent actual population sizes.

Pros of Most Probable Number (MPN) Method

Useful for estimating low microbial counts; quick and easy setup.

Cons of Most Probable Number (MPN) Method

Less precise than direct counts; relies on statistical estimations and dilutions.

Pros of Optical Density Measurement

Provides rapid results; allows for continuous monitoring of growth.

Cons of Optical Density Measurement

Does not distinguish between live and dead cells; may be affected by particulate matter in the culture.

Cell Counting

The process of determining the number of cells in a given volume of culture.

Hemocytometer

A specialized glass slide device used for counting cells in a specific volume under a microscope.

Cell Viability,

The ability of cells to survive and maintain their functions, often assessed alongside cell counting.

Cell Density

The number of viable cells per unit volume, usually expressed in cells/mL.

Flow Cytometry Analysis

The method of counting cells as they pass through a laser, providing quick and precise measurements.

Colony Count

A method of quantifying the number of microorganisms based on the number of colonies formed after incubation.

Dilution Series

A procedure used to reduce the concentration of cells in a sample, facilitating accurate counts.

Total Cell Count

An estimation of all cells present, including both viable and non-viable cells.

Live/Dead Staining

A technique that uses dyes to differentiate between live and dead cells for counting purposes.

Automated Cell Counters

Devices that use various technologies to count

Doubling Time

The time required for a population of cells to double in number during the exponential growth phase.

Lag Phase

The initial phase of bacterial growth where cells adapt to a new environment before division begins.

Exponential Phase

The period of rapid cell division and population growth, characterized by a constant doubling time.

Stationary Phase

The stage of bacterial growth where the rate of cell division equals the rate of cell death, leading to a stable population.

Death Phase

The final phase of bacterial growth where nutrients are depleted and waste products accumulate, leading to a decline in viable cells.

Cell Growth Curve

A graphical representation of the growth of a microbial culture over time, showing lag, exponential, stationary, and death phases.

Batch Culture

A type of culture where microorganisms are grown in a closed system with limited nutrients.

Continuous Culture

A culture system where nutrients are continuously supplied, and waste products are removed, allowing for constant growth.

Biofilm Formation

The process where microbial communities adhere to surfaces and produce a protective matrix, affecting growth and nutrient access.

Generation Time

The time it takes for a bacterial population to double in number, crucial for assessing growth rates.

Enrichment Media

Growth media that boost the growth of specific microorganisms while inhibiting others.

Transport Media

Media used to maintain and preserve viable microorganisms during transport.

Nutrient Agar

A general-purpose medium that supports the growth of a wide range of non-fibrous organisms.

MacConkey Agar

A selective and differential agar that isolates Gram-negative bacteria and differentiates lactose fermenters.

Blood Agar

A richly nutritious medium that supports the growth of fastidious organisms and allows for the observation of hemolysis.

Mannitol Salt Agar

A selective media used to isolate Staphylococci, differentiating mannitol fermenters from non-fermenters.

Cetrimide Agar

A selective medium for Pseudomonas aeruginosa that contains cetrimide to inhibit the growth of other bacteria.

XLD Agar

A differential medium used for isolating enteric Gram-negative bacteria, especially Salmonella and Shigella.

Potato Dextrose Agar (PDA)

A rich medium for the cultivation of fungi and yeasts.

Luria-Bertani (LB) Broth

A nutrient-rich media used for the growth of bacteria, especially Escherichia coli.

Microbial Ecology

The study of the interactions between microorganisms and their environment.

Bioremediation

The use of microorganisms to remove or neutralize contaminants from the environment.

Pathogenic Microorganisms

Microbes that can cause disease in hosts.

Probiotics

Live microorganisms that provide health benefits to the host when consumed in adequate amounts.

Antibiotics

Medicinal compounds that inhibit the growth of or kill bacteria.

Infection Control

Strategies used to prevent the spread of infectious diseases in healthcare settings.

Plasmid

A small, circular piece of DNA found in some bacteria that can replicate independently of chromosomal DNA.

Biotechnology

The use of living systems or organisms to develop or make products, often involving genetic manipulation.

Horizontal Gene Transfer

The transfer of genetic material between organisms without sexual reproduction, significant in microbial evolution.

Microbial Fermentation

The metabolic process that converts sugars to acids, gases, or alcohol in the absence

Types of Sterilization Methods

Includes physical methods (like heat and radiation) and chemical methods (like ethylene oxide and hydrogen peroxide).

Biological Indicator

A test system containing viable microorganisms used to monitor the efficacy of sterilization processes.

Chemical Sterilization

The use of chemical agents to achieve sterilization, effective for heat-sensitive items.

Radiation Sterilization

Utilizes ionizing radiation to kill microorganisms by damaging their DNA.

Filtration Sterilization

A method that removes microorganisms from solutions by passing them through a filter.

Sterility Assurance Level (SAL)

The probability of a non-sterile product being released from a sterilization process, ideally 10^-6.

Detergent Sterilization

Using detergents for cleaning and sterilizing surfaces; typically part of a two-step process.

Steam Sterilization

Also known as autoclaving; effective method using high-pressure steam to destroy microorganisms.

Aseptic Processing

Techniques used to maintain sterility of products during manufacturing and packaging.

Sterilization Validation

The process of proving that a sterilization method consistently yields sterility.

What is a differential stain?

A staining technique that allows for the differentiation of microorganisms based on their physical and chemical properties.

Examples of differential stains

Common examples include Gram staining and acid-fast staining, which help identify various types of bacteria.

Purpose of differential staining

To classify organisms and provide information on their morphology and cellular structure.

Mechanism of Gram stain

Differential staining that uses crystal violet and safranin to identify Gram-positive (purple) and Gram-negative (pink) bacteria.

Significance of acid-fast stain

A differential stain used to identify bacteria like Mycobacterium, which resist decolorization after staining.

Staining protocol

The process often involves fixing the specimen, applying the stain, rinsing, decolorizing, and counterstaining.

Dark-Field Microscopy

A microscopy technique that enhances the contrast of unstained samples by using a special condenser to scatter light.

Polarized Light Microscopy

A technique that uses polarized light to observe samples, useful for studying crystalline materials and biological tissues.

Phase Contrast Microscopy Application

Allows observation of living cells and tissues without staining, enhancing visibility of cellular structures.