Bacterial Genetics: DNA, Gene Expression, and Reproduction

• Bacterial genetics focuses on genetic processes/mechanisms in bacteria and studies the structure, function, and regulation of genetic material.

Basic Concepts

• DNA structure is a circular, double-stranded molecule within the cytoplasm in a compacted protein-bound state called a nucleoid.
• Gene expression synthesizes proteins which are regulated by transcriptional, translational, and post-translational controls.
• Mutations alter bacteria's genetic material, resulting in altered traits from spontaneous events or environmental factors (radiation/chemicals).
• Horizontal gene transfer transmits genetic material between bacteria other than parent to offspring, done through transformation, transduction, and conjugation.

Variation and Heredity

• Bacteria are haploid so reproduction happens mainly via asexual mode.

Asexual Reproduction

• Asexual reproduction is the most common and important bacterial growth cycle, like fission.
• There is no genetic recombination and changes result from mutations.

Sexual Reproduction

• Bacteria aren't differentiated into somatic or gametic cells.
• There are two functional mating types of a donor (male) and recipient (female).
• There’s a unidirectional DNA transfer from donor to recipient, leading to variations.

Methods of Genetic Transfer

Conjugation

• Conjugation transfers genetic information via cell-to-cell contact involving plasmids which are extra-chromosomal and independently replicating DNA often found in yeasts and bacteria.

Transduction

• Transduction transfers bacterial genes by viruses and is the most common transfer mechanism.
• Two types of transduction are generalized and specialized.

Generalized Transduction

• Generalized transduction happens during the lytic cycle of virulent and temperate viruses.
• Host DNA from any part of the host genome replaces the virus' genome to become part of the virion's DNA.

Specialized Transduction

• Specialized transduction occurs in some temperate viruses.
• Transfers specific host chromosome regions into the virus, replacing some viral genes.

Transformation

• Transformation exists when bacterial cells take up free DNA and incorporate it in their genome.
• Transformation is facilitated by specific proteins and the recipient cell's competence state.
• Transformation occurs naturally in some bacteria, and artificial methods facilitate transformation in others.

Protoplast Fusion

• Protoplast fusion is a lab technique that merges genetic material from two+ bacterial cells without a known conjugation system.
• Enzymes digest the cell wall to form protoplasts.
• Steps include protoplast isolation, fusion mixture preparation, protoplast fusion using polyethylene glycol (PEG), and screening/selection of hybrid cells.

Microbial Growth and Reproduction

• Microbial growth increases in the number of microorganisms (bacteria, fungi, viruses) in culture/environment.
• Microbial growth is important in fermentation, bioremediation, and antibiotic/enzyme production.

Factors Affecting Microbial Growth

• A favorable condition for growth of foodborne pathogens: [FAT TOM]

Food

• Nutrient availability/concentration in food affects microbial growth.
• Protein-rich (poultry, fish, dairy, eggs) and carbohydrate-rich foods (rice, pasta) are susceptible to microbial growth and contamination.

Acid

• Acidic conditions inhibit bacterial growth, which is why low-acid foods require proper storage.
• Environmental pH affects microbial growth where bacteria thrives with neutral pH levels of 6.5 to 7.5. Some fungi/bacteria prefer more acidic or alkaline pH.
• FDA regulations require acidified foods be brought to pH 4.5 (or less).

Time

• Food shouldn't stay in temperature danger zone (5-60°C or 41-140°F) for two+ hours, requiring cooling/heating.
• Food shouldn't be left out for one+ hour if temperature is beyond 32°C or 90°F.

Temperature

• Microorganisms need specific temperature for best growth and the temperatures range is known a growth temperature range.

Oxygen

• Microorganisms require different oxygen for growth like anaerobes.

Moisture

• Microbial growth affected by their environment's water activity.
• The water activity is the measurement of unbounded water for microbial growth.

Phases of Growth

• Microorganisms have specific growth stages

Lag Phase

• The initial growth stage requires new environment adjustments and prep for growth.
• During this phase, the population and metabolic activity are low.

Exponential (log) Phase

• Microorganisms grow at max rate, population size doubles at regular intervals with increased growth and population.

Stationary Phase

• It is characterized by a plateau reached once the growth rate slows. Balance between new cells vs dying or inactive cells, or death = growth.

Death Phase

• The phase starts when population decreases due to the accumulation of wastes, depletion of nutrients and stress factors, causing growth < death.

Prolonged Decline Phase

• Also called stationary phase II and some cultures enter a prolonged decline during which the cells are viable but not actively growing.

Mathematical Expressions of Microbial Growth

• Bacterial population increases by geometric progression during exponential growth by binary fission.
• The generation/doubling time is the interval for cells to divide.

Measurement of Microbial Growth

Plate Count

• Also known as a standard plate or viable cell count (most common).
• Indirectly done, using serial dilution and plating.
• Plate counts can determine the number of live cells.
• Disadvantage: Underestimation of cell population
• Countable ranges: yeasts and molds is 10-150 colonies/plate and bacteria is 25-250 colonies/plate

Direct Cell Count

• A direct cell count involves counting cells in a sample using a microscope and counting chamber.
• Both viable and non-viable cells are counted, leading to overestimation.

Turbidimetric Method

• the turbidimetric method easily and rapidly measures cell mass indirectly.
• Uses a spectrophotometer or colorimeter that determines turbidity/cloudiness measuring the amount of light passing through a cell suspension.
• With increased cells and a turbid/cloudy suspension, less light will pass through the suspension.
• Culture must be dense enough to register turbidity on the instrument.

Determination of Cell Dry Weight

• This involves measuring a culture's cell weight after extracting all water.

Determination of Cell Metabolic Activity

• This involves measuring the enzymatic activity or from cellular components that are involved in metabolism.

Isolation and Cultivation of Microorganisms

Pure Cultures

• A pure culture is a population of cells from a single cell, free from contamination.

Cultivation

• The cultivation process involves growing microorganisms with nutrients and environmental factors.

Culture Media

• Culture media encompasses nutrient solutions tailored for growing microorganisms in the lab.

Colony

• A colony is a visible mass of cells from a single cell or cell group (divided and grown) on solid agar.

Culture Media

• Serve specific purposes and support the growth of different microorganisms in lab settings.

Types of Culture Media

• Culture media differ based on their physical state and composition.

• Liquid (Broth): aqueous form without solidifying agents.

• Semi-Solid: has 0.1-0.5% solidifying agent, this media allows for motility.

• Solid: 1.5-2.0% solidifying agent like an agar or gelatin.

• Synthetic: chemically defined media from pure chemicals like minimal agar medium.

• Complex: media from natural sources like animal or plant with undefined composition like a brain heart infusion agar medium.

• In food microbiology, combining selective, non-selective, and differential media helps analyze microorganisms in samples.

Selective Media

• Selective media possesses certain components which suppresses the growth of certain microorganisms while letting others grow.
• Selective media isolates and enriches target microorganisms like pathogens from complex food matrices.

Non-Selective Media

• Non-selective media grows a wide range of microorganisms for overall enumeration and cultivation of bacteria in food samples without any components to inhibit the growth of particular organisms.

Differential Media

• Differential media have components that separate microorganisms using metabolic characteristics or colony appearance to distinguish them.
• Differential media identifies microorganisms or differentiate strains or species.

Examples of Selective Media.

MacConkey Agar

• Selective for Gram-negative bacteria, especially Enterobacteriaceae, and lactose metabolism.
• MacConkey also classified as differential media to differentiate organisms with lactose metabolism.

Brilliant Green Agar

• Brilliant Green inhibits Gram-positive bacteria and used to select Salmonella species.
• Brilliant Green can isolate Salmonella from meat.

VRBD (Violet Red Bile Dextrose) Agar

• Violet Red Bile Dextrose is selective for isolation and enumeration of Enterobacteriaceae.

Examples of non-selective media.

Nutrient Agar

• It is a general-purpose medium which supports the growth of a wide range of bacteria and fungi.

Tryptic Soy Agar (TSA)

• It provides a rich nutrient base commonly used for total bacterial counts in food samples.

Plate Count Agar (PCA)

• PCA media supports the growth of most bacteria, and it's often used to determine total viable count in food.

Comparison of Spread and Pour Plate Techniques

• There are a number of different advantages and disadvantages for these two techniques.

Serial Dilution

• Serial dilution simplifies manageable concentrations of a desired organism paired with petri dish streaking for systematic reduction of an entity through resuspension into fixed liquid volumes.

Membrane Filter Technique

• Especially useful for detecting low levels of microorganisms in liquids such as drinking liquids by using a sterile membrane filter.

Single Cell Isolation Technique

• Single-Cell Isolation characterizes individual microbial cells with unique properties not in the bulk commonly using microcapillary manipulation, or laser capture microdissection.

Culture Preservation Methods

• Culture preservation ensures long-term availability, maintaining integrity of microbial strains valuable in research and supporting industrial quality and safety.

Different Culture Preservation Methods

• Periodic Subculturing (transfer to fresh media): Requires time interval transfers between the culture medium and microorganisms that are very stable.
• Overlaying with Mineral Oil: Limits the availability of O2 to reduce metabolic rate. Simple, limited.
• Freeze-Drying (Lyophilization): Freezing then sublimation to remove water achieves storage in containers, less opportunity for changes in the culture.
• Freezing with Liquid Nitrogen: Specimens are frozen along with agent glycerol in nitrogen refrigerators, long term,

Microbial Repositories

• Also called culture collections, is specialized facilities that store and maintain various collections of them for research, education, and more.
• Includes bacteria, fungi, viruses, and protozoa, and used to help study microbial diversity, evolution, and biotechnology.

Microscopy

• Microscopy scientifically uses microscopes to watch/research small objects or specimens not visible otherwise.

Microscopy features:

• Magnification: Magnification range enables the user to change specimen levels of viewing.
• Resolution: Clarity and sharpness in viewing image's details and structures.
• Contrast: Enhances the specimens for clearer views.
• Durability: Should function through the years.
• Ease of use: Instructions and easy control.

Features of a microscope:

• Head

• Nosepiece

• Scanning

• Low-high power

• Stage clip

• condenser

• coarse adjustment

• fine adjustment

Different Types of Microscopes

• Light microscope: Uses light to magnify that's simple or compound developed by Anton van Leeuwenhoek.
• Bright Field Microscopy: Microscopic field is brightly lit objects are dark, uses gross morphology.
• Dark Field Microscopy: Black background makes object bright or luminous.
• Phase Contrast Microscopy: Internal structure in detail.
• UV Microscopy: Makes use of ~180-400 nm, and images are on displays detection.
• Fluorescence Microscopy: antigen-antibody reaction.
• Electron microscope: Beams of electrons creates an image with a higher magnification for ultrastructure and molecule viewing. Ernst Ruska's enhancement helps with enhanced resolution:
• Transmission Electron Microscope (TEM): It examines viruses and internal ultrastructure.
• Scanning Electron Microscope (SEM): Focused beam to highly detail specimen's surface, and see surface features.

Examination of Specimen

• Specimen: the object/material observed. Biological sample (cell, tissue, organism) or non-biological (metal, mineral, polymer) in living or natural state vs stained preparation. Wet mount and hanging drop techniques examine natural states.
  • State of the specimen
    • Living or In Natural State: Advantages being studying cellular processes and unaltered characteristics with a disadvantage of cells being similar to water.
    • Stained Preparation: Advantages of provides contrast, slides can be preserved. Disadvantages of complicated preparations and expensive.

Staining

• It enhances visibility and microscope contrast with a dye. Dye being an organic compound with color groups. Acidic, neutral, and basic stain types are from adsorption, absorption, osmosis, capillary action, and ion exchange. Follow these basic steps to stain.

1. Obtain specimen.
2. Mount on slide, an example being smear preparation.
3. Fix by preventing decay, heat fixation is an example.
4. Use the dyes
5. Observe.

Staining Procedures

Simple staining

• Simple can directly (cells in same color with dye) or indirectly (dye with colors background.

Differential straining

• Differential staining Uses 2+ reagents for differentiating between cell types.

Structural Straing

• Improve visualization of cellular structures.

Control and Destruction of Microorganisms

• Microbial control inhibits microbial with growth and destruction to: prevent destruction of valuable substances and/or the person, environment, and more.

Terms

• Sterilization - Barren, removal of microbes.
• Disinfection - Killing/removal organisms that cause disease by inanimate object usage.
• Sanitization- Reduces microbial to safe levels in standards of public health.
• Antisepsis - Prevents infection in tissues via living by antiseptic chemical usages (not toxic).

Physical Agents Used to Control Microbial Growth

• Moist Heat: With boiling or flowing steam for 10 minutes (vegetative/eukaryotic spores). Denaturation of coagulation on proteins.
• Dry Heat: For incineration (flame use) and hot air with mechanical convection for one hour. Oxidation of components for heating to boiling points.
• Low Temperature: refrigeration for 4°C and -95°C freezing. Limits and changes to cell activity.
• Filtration: Removes 99.97% particles. By physical exclusion on enzymes, vitamins, and etc.
• Desiccation: With the sun or freeze drying/lyophilization, microorganism growth is inhabitable.
• Osmotic pressure: From the microbes' concentration increases and inhibit growth from high pressure (inhibition).
• Radiation: High energy causes DNA deconstruction (sterilizing) and thymine (lethal) dimers.
• Ionizing: causes formation with gamma sterilizing use.
• Non-Ionizing: causes formations with DNA, at 260 nm

Chemical Agents Used to Control Microbial Growth

• Phenol: Used for standard effectiveness, disruption of membrane, denaturing, and inactivation which can control growth of microbes.
• Alcohol: Bacterial and fungi, but protein denatures and resolves membrane which causes oxidation of amino acids.
• High Concentration: more effective. For example, over 75+ for alcohol solutions.
• Temperature - The activity is enhanced.
• Local environment - Maybe protected by a sterilization.