Introduction what are microbes

Introductory Microbiology (MIC 121) Study Summary

Course Instructors

• DR. Uzeh, R.E. (Coordinator)
  • Dept. of Microbiology, Faculty of Science, UNILAG
  • Email: ruzeh@unilag.edu.ng
• DR. Adelowotan, A.O.
  • Dept. of Microbiology, Faculty of Science, UNILAG.
  • Email: aadelowotan@unilag.edu.ng

Course Outline

• Apparatus & equipment in Microbiology
• Techniques & experimental Methods (in Bacteriology, Virology & Mycology)
• Laboratory Reports & Procedures
• Laboratory safety, ethics & disposal of organisms.
• Aseptic Technique: sterilization, disinfection & handling of Microorganisms.

Course Objectives

• Identify and describe tools used in Microbiology.
• Describe general protocols for the inoculation, incubation and description of microbes in or on media.
• Identify the safety precautions required to take in a general laboratory setting.
• Understand general concepts of aseptic technique.

Course Goal

At the end of this course, the student will be able to:

• Identify and describe tools used in Microbiology.
• Describe the general concept of aseptic techniques used in laboratory preparation and analysis.
• Explain and differentiate between sterilization, disinfection, and sanitization.
• Discuss inoculation, incubation, and isolation.
• Describe the various fixation and staining techniques to identify microbes with the light microscope.
• Describe the fixation and staining methods used in electron microscopy.

INTRODUCTION: WHAT ARE MICROBES?

• A microbe, or microorganism, is a microscopic organism that is extremely small.
• The study of microorganisms is called microbiology, a subject that began with Anton van Leeuwenhoek's discovery in 1675.
• Microorganisms are diverse, including bacteria, fungi, algae, and protozoa. Some microbiologists also include viruses (though some consider them nonliving).
• They are ubiquitous in nature and vital to humans and the environment, fulfilling roles like recycling dead remains and waste products through decomposition.

TOOLS USED IN MICROBIOLOGY

Microbiology equipment includes:

• Microscopes, incubators, autoclaves, Bunsen Burner, inoculation loops, slides, Refrigerator, Deep-fridge, Incinerator, oven, digital shakers, Water Bath, Colony Counter, Magnetic Stirrer, Vortex Mixer, Homogenizer, test tubes, petri dishes, pH Meter, Distilled Water Plant.
• Growth media (solid and liquid), pipettes and tips, and laminar flow hoods.
• Some equipment (microscopes, hoods) are permanent, while others (pipette tips) are disposable.

MICROSCOPE

• Used to examine objects too small to be seen by the naked eye.
• Different types are used in Microbiology.
• The basic principle is magnification.
• Can be classified by what interacts with the sample:
  • Light or photons (optical microscopes)
  • Electrons (electron microscopes)
  • A probe (scanning probe microscopes)

Types of Light Microscopes:

1. The light microscope:
   1. A compound microscope uses two types of lenses to magnify an object.
   2. Dark-field microscope: Used to observe live spirochetes. Uses a condenser that scatters light, making the object appear light against a dark background.
   3. Phase-contrast microscope: Uses special condensers to throw light "out of phase", causing it to pass through the object at different speeds. Live, unstained organisms and internal cell parts are seen clearly.

Uses of Microscope:

• Observation of minute particles not visible to naked eyes.
• Study of microscopic algae, fungi, and biological specimens.
• Used by watchmakers, jewelers to see small or fine parts.
• To see enlarged images of text, fabric textures, soil particles.
• Used by palmists, skin specialists, and to see details of stamps and engravings.

INCUBATOR

• Definition: A device used in laboratories for the growth and maintenance of microorganisms and cultures.
• Provides optimal temperature, pressure, moisture, etc., for microbial growth.
• Principle: Maintains a proper atmosphere for organism growth.
• Has a heating system for temperature adjustment.
• Adjustments for CO2 concentration, pH, and humidity.
• Shaking incubators allow continuous movement for cell aeration and solubility studies.

Use of Incubator:

• Growing cell cultures.
• Reproduction of germ colonies for germ count in food industry and biochemical oxygen demand determination (wastewater monitoring).
• Reproduction of microorganisms (bacteria, fungi, yeast, viruses).
• Breeding of insects and hatching of eggs in zoology.
• Controlled sample storage.
• Growing of crystals/protein crystals.

AUTOCLAVE

• Definition: A device that provides a physical method of sterilization by killing bacteria, viruses, and spores using steam under pressure.
• Sterilizes by subjecting items to high-pressure steam at 121° C or more for 15-20 minutes.
• Considered more effective than dry heat due to moist heat sterilization.
• Invented by Charles Chamberland in 1879, building on Denis Papin's steam digester concept (1679).

Types of Autoclave:

• Stove top autoclaves: Simplest, resemble pressure cookers, require external heat source, dangerous for untrained users.
• Front loading autoclaves: More widely used, box-shaped, self-contained with heating unit, adjustable temperature and operation time, and pressure gauge.

Principle of Autoclave:

• Same working principle as a domestic pressure cooker.
• Uses moist heat sterilization: steam under pressure sterilizes material.
• High pressure increases water's boiling point, allowing higher sterilization temperatures.
• Boiling water in an open container reaches 100°C; in a closed, pressurized container, steam temperature can exceed 100°C.
• High temperatures kill all bacteria, including heat-resistant spore-formers.
• Steam temperature increases with increased steam pressure.

Why Steam? (Principle of action)

• Heat kills cells by breaking down and coagulating proteins in the cell wall.
• Steam is an efficient medium for heat transference.
• Air is inefficient due to its low heat of vaporization.
• Steam at 100°C contains significantly more energy (540 kcal/liter) than water at 100°C (80 kcal/liter).
• When steam contacts a cooler object, it condenses, transferring its latent heat directly into the water, heating cells more efficiently than hot air.
• All trapped air must be removed for effective sterilization, as hot air is poor at achieving sterility. Steam at 134°C achieves the same sterility in 3 minutes that hot air at 160°C takes two hours to achieve.

Uses of Autoclave:

• Sterilization of materials containing water that cannot be sterilized by dry heat.
• Decontamination of specific biological waste and sterilization of media, instruments, and lab ware.
• Inactivation of regulated medical waste containing bacteria, viruses, and other biological materials before disposal.
• Sterilization of medical equipment, glassware, surgical equipment, and medical wastes in labs.
• Sterilization of culture media, autoclavable containers, plastic tubes, and pipette tips.

HOT AIR OVEN

• Definition: Electrical devices used for sterilization using dry heat.
• Uses extremely high temperatures over several hours to destroy microorganisms and bacterial spores.
• Temperature maintained and energy conserved by double-walled insulators (inner layer poor conductor, outer metallic).
• Sterilizes by conduction: heat from outside surfaces moves towards the center of the item.

Commonly-used temperatures and times:

• 180°C for 20 minutes
• 170°C for 30 minutes
• 160°C for 60 minutes
• 150°C for 150 minutes

Factors for Sterilization (Dry Heat):

• Thermal Death Point (TDP): Lowest temperature to kill all microorganisms in a specific time.
• Thermal Death Time (TDT): Minimum time to kill all microorganisms at a specific temperature.
• TDP and TDT vary by species (e.g., Clostridium botulinum is more heat resistant).

Principle of Hot Air Oven:

• Utilizes dry and hot air convection (circulation of heated air), conduction, and radiation.
• Hot air convection types:
  1. Gravity convection process: Heated air rises, displacing cooler air. Produces inconsistent temperature and slow turnover.
  2. Mechanical convection: Uses a blower or fan to force heated air throughout the chamber, ensuring uniformity.
• Dry heat damages by oxidizing molecules, destroying essential cell constituents.
• High temperature for almost an hour destroys resistant spores and bacterial endotoxins (pyrogens).
• Kills by oxidation, protein denaturation, and toxic effects of elevated electrolyte levels; it is more efficient.

Applications of Hot Air Oven:

• Baking, curing, drying, and annealing applications by removing moisture.
• Sterilizing heat-stable glass objects (flasks, pipettes, petri dishes, test tubes).
• Sterilizing metal items (scissors, forceps, spatulas, scalpels).
• Sterilizing pharmaceutical products (liquid paraffin, fats, grease, dusting powder).
• Sterilizing non-volatile compounds (sulphonamide, zinc, starch powder).
• Widely used in research-based operations.

Advantages of Hot Air Oven:

• Easy to operate.
• Small size, minimal space, easy to install.
• Non-corrosive for metal equipment (no water needed), safer.
• Efficiently sterilizes heat-stable and non-aqueous materials.
• Reaches higher temperatures.
• Kills bacterial endotoxin (unlike all other treatments).
• Effective for heat-stable articles, only method for sterilizing oils and powders.
• Protective of sharps/instruments with cutting edges (less dulling).
• Does not leave chemical residue.
• Non-toxic and does not harm the environment.

BUNSEN BURNER

• Definition: A standard laboratory tool, gas-fueled single open flame, named after Robert Bunsen.
• Gas can be natural gas (methane) or liquefied petroleum gas (propane, butane, or mixture).

Principle of the Bunsen Burner:

• Made of a metal tube on a flat base with a gas inlet and an adjustable valve.
• Sides of the tube have openings with a collar to adjust air intake.
• Gas is forced by pressure from a source to the top, where it's ignited.

Uses of Bunsen Burner:

• Sterilization (e.g., micro-loop sterilization, flaming test tube lips during inoculations).
• Combustion.
• Heating.

CENTRIFUGE

• Definition: A device that rotates an object about a single axis, applying an outward force perpendicularly.
• Many types varying by intended use and rotor design.

Types of Centrifuges by Intended Use:

1. Laboratory centrifuges: General-purpose instruments (clinical, superspeed, preparative ultracentrifuges).
2. Analytical ultracentrifuges: Designed for sedimentation analysis of macromolecules.
3. Haematocrit centrifuges: Measure volume percentage of red blood cells.
4. Gas centrifuges: For isotopic separations (e.g., Zippe-type).

• Motor-based laboratory centrifuges rotate liquid samples to separate components.

Further Applications:

• Very high-speed centrifuges and ultracentrifuges can separate fine particles down to nano-scale and molecules of different masses.
• Large centrifuges simulate high gravity for testing (e.g., pilot training).
• Medium-sized centrifuges in washing machines to draw water out of fabrics.
• Gas centrifuges for isotope separation (e.g., enriching nuclear fuel).

Principle of Centrifuge:

• Works on the principle of sedimentation.
• High-speed rotation causes denser particles to move away from the center, while lighter, less dense particles are forced towards the center.
• Denser particles settle at the bottom; lighter particles are collected at the top.
• Laboratory tabletop centrifuges align sample tubes at an angle for a shorter travel distance to the bottom.

Uses of Centrifuge:

• Primary application: Separation of particles suspended in a suspension.
• Separation of cell organelles, nucleic acid, blood components, and isotopes.

REFRIGERATOR / DEEP FREEZER / ULTRA-LOW TEMPERATURE FREEZER

• Refrigerator ("fridge"): Cooling appliance maintaining temperatures a few degrees above freezing point of water (0°C to 7°C).
• Deep freezer: Maintains temperatures below the freezing point of water.
• Ultra-low temperature freezer: Stores cultures for long periods at temperatures of -70°C or lower, or even -196°C in liquid nitrogen tanks.
• Designed with cooling compressors and CFC-free refrigerants.

Types of Laboratory Refrigerators:

1. Explosion-proof refrigerators: Store flammable/hazardous chemicals. Lack electrical equipment to prevent sparks.
2. Laboratory fridges: Maintain consistent temperatures, digital displays. General lab use, lockable, easy-to-clean, cool samples, preservation.
3. Blood bank refrigerators: Critical reliability, temperature monitoring, separate compartments for different blood sample types.
4. Chromatography refrigerators: For research experiments requiring precise temperature settings and stability (e.g., setting up chromatography apparatus).

Working Principle:

• Refrigeration/Cooling: Simple principle of removing heat from one region and depositing it elsewhere. Low-temperature liquid (coolant/refrigerant) evaporates as it absorbs heat from objects, taking the heat away.
• Deep freezers: Based on the principle that extremely low temperatures minimize microbial growth, protecting and preserving substances.
• Cultures preserved over long periods without changes in microorganism concentration.

Refrigeration and Freezing for Microbial Control:

• Low temperatures control microbial growth, except for psychrophiles (cold-loving organisms).
• Refrigerator temperatures (0-7°C) inhibit microbial metabolism, slowing growth and preserving products.
• Freezing below -2°C may stop growth and kill susceptible organisms.
• Bacterial cultures and medical specimens for long-term storage are often frozen at ultra-low temperatures (-70°C or lower).
• Ultra-low temperatures achieved using dry ice in freezers or liquid nitrogen tanks.

Uses:

• Refrigerator: Repository for thermo-labile chemicals, solutions, antibiotics, serums, biochemical reagents.
• Deep-freezer: Stores chemicals and preserves samples at very low sub-zero temperatures.
• Ultra-low temperature freezer: Stores cultures for a long period of time.

ANALYTICAL BALANCE

• Definition: Used in laboratories for accurate measurement, particularly for small samples.
• Detects the slightest increase in weight.
• Enclosed to protect from external factors like dust and air.
• Measures samples in the sub-milligram range.
• Often includes a counting feature for small samples.
• Automatic calibration when ambient temperature changes ensures accuracy.

Principle of Analytical Balance:

• An electric analytical balance uses the force necessary to counteract the mass rather than measuring the mass itself.
• An electromagnet generates the force needed to balance the mass of the substance, and this force is displayed as the weight.

Uses of Analytical Balance:

• Accurately weighing reagents, laboratory media, and drugs.
• Weighing test materials, sampling amounts, formulation, density determination, purity analysis, quality control testing, and material conformance testing.

COLONY COUNTER

• Definition: Used to estimate the density of a liquid culture by counting the number of CFU (colony forming units) on an agar plate, gel, or Petri dish.
• A CFU is a precise unit to estimate viable bacteria or fungal cells capable of multiplying.
• Distinguishes viable cells from dead cells (unlike microscopic examination).

Importance of Colony Counting:

• Microorganisms are difficult to count directly due to their small size, hence the usefulness of CFUs.
• Provides a better idea of existing microorganism concentration and potential future presence.

Method and Types:

• Requires culturing microbes to ensure only viable cells are counted.
• Counting can be manual (with touch pressure and digital counter), semi-automatic, or fully automatic.
• Types: Manual Colony Counter, Digital Colony Counter.

Working Principle & Uses:

• Accommodates different plate sizes, scanned with UV, white light, and/or fluorescent illumination.
• Counting can be manual (touch pressure) or with a digital counter.
• Primarily used for counting colonies on a culture plate to estimate microorganism concentration in liquid culture.

Applications for Colony Counters:

• Used in various research laboratories: Life sciences, cell and molecular biology, food and beverage, pharmaceutical research.
• Environmental testing.
• Medical-based environments.
• Helps inform professionals about patient health by determining blood microorganism concentration.
• Tracks infectious disease progression or immune system effectiveness based on bacteria concentration in blood.

DISTILLED WATER PLANT

• Produces distilled water.
• Types: Glass Water Distiller, Unit Wall Mount Distiller, Tabletop Water Distiller.

Working Principle (Distillation):

• Tap water is fed into a boiling chamber.
• A heating element boils the water, producing steam.
• Volatile contaminants (gases) are discharged via a vent. Minerals and salts remain in the boiling chamber as scale.
• Steam enters a coiled tube (condenser) and is cooled by water.
• Condensation forms water droplets, collected in a storage tank.
• Summary: Boils water into vapor, condenses it, and collects it in liquid state.

Uses of Distilled Water:

• Widely used industrially, medically, and scientifically for experiments and cleanliness.
• Medical procedures: washing wounds, sterilizing surgical instruments, cleaning hospital equipment.
• Used in autoclaves, batteries, and other miscellaneous equipment.
• Hydroponic systems: provides a "blank slate" for precise nutrient/mineral mixture.

Advantages of Distilled Water:

• Guaranteed water purity: Free of bacteria, viruses, inorganic compounds, protecting against harmful contaminants.
• Wide range of applications.
• Quicker and more convenient access to purified water.
• Remineralizing cartridges can raise alkalinity and mineral content if desired.

Disadvantages of Distilled Water:

• Generates a significant amount of wastewater.
• More complex installation and maintenance.
• High energy costs: Requires considerable energy to heat water, making it less energy-efficient.

WATER BATH

• Definition: A device for regulating the temperature of substances by immersing a vessel containing the substance into another vessel containing water kept at a desired temperature.
• Used for incubating samples and heating substances indirectly (not directly on a Bunsen burner or hot plate).
• Only materials whose boiling point is less than that of water can be heated.
• Consists of a heating unit, a stainless-steel chamber, and a control interface.
• Used to allow chemical reactions at specific temperatures.
• Can be used up to 99.9°C; above 100°C, oil, silicone, or sand baths are used.

Types of Water Bath:

1. Circulating water baths (stirrers):
   • Constantly circulate water for rapid heating and cooling.
   • Enable efficient temperature maintenance over a wide range.
   • Ideal for applications requiring temperature uniformity and consistency (enzymatic, serologic experiments).
   • Thorough circulation results in uniform temperature.
2. Non-circulating water baths:
   • Rely primarily on convection for heating.
   • Less accurate in temperature control.
   • Can have add-ons for stirring to improve heat transfer.
3. Shaking water baths:
   • Feature for shaking to move liquids around (can be turned on/off).
   • In microbiology, constant shaking allows liquid-grown cell cultures to mix with air.
   • Ideal for thawing, heating, mixing, shaking samples.
   • Applications include hybridization, bacterial culturing, cell aeration, molecular biology assays.

Working Principle:

• A sensor in the device transfers water temperature to a reference value.
• This signal is amplified, and a control system generates a signal for the heating system, which then heats the water to the desired temperature.

Uses of Water Bath:

1. Provides indirect heat for samples under controlled temperatures.
2. Maintains cell lines.
3. Heats flammable chemicals that might combust if exposed to open flame.
4. Warms blood bags.
5. Used for incubation tests, such as activated partial thromboplastin time (aPTT) to evaluate blood clotting ability.