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General Microbiology

In this unit:


History of Microbiology

Microscopy and Staining

Morphology and Physiology of Bacteria

Sterilization and Disinfection

Bacterial Cultures

Microbial Genetics

Microbial Biotechnology

Microbial Pathogenicity

Antimicrobial Compounds

This course is under construction and is being updated in real time


Microbiology is the study of microorganisms including bacteria, viruses, fungi, and some parasites. Most of these organisms are studied within the concept of causing diseases in humans, animals and plants. Broadly, microbiology also includes the study of microbial pathogenesis, disease pathology, immunology, and epidemiology of the resultant diseases.

History of Microbiology

Microbes are the most significant biological forms in the planet interacting with humans because of their pervasive presence. Microbes may have either beneficial roles in maintaining life or undesirable consequences causing disease in humans, animals and plants. Diseases caused by microbes vary in severity from mild infections to severe diseases such as tuberculosis.

Microbial diseases have shaped global history for many ways. In 1347, Bubonic plague or Black Death started to spread in Europe and about 4 years later, (1351) the plague had killed one-third of the population (an estimate of 25 million people). Over the next 80 years, the disease reemerged frequently and had killed about 75% of the European population.

Another example started towards the end of 2019. A viral disease (later named Covid-19) caused by a coronavirus strain started to ravage the global population causing millions of deaths across the globe. At the time of this article, there has been 3.3 million deaths globally.

These are just examples from many such epidemics and pandemics, which resulted in extensive deaths of people irrespective of economic and social stature. Other epidemics that have had significant consequences include smallpox, malaria, measles and polio.

The first person to observe and describe microorganisms was Antony van Leeuwenhoek who spent most of his spare time tinkering with convex glasses to make simple microscopes. The microscopes could magnify organisms 50-300 times and he was able to observe both bacteria and protozoa.

Although microorganisms had been observed under the microscope, there was significant diagreement as to their origin. Spontaneous generation was one of the theories proposed. In this theory, microorganisms were believed to originate from non-living things.

Louis Pasteur is known by most as the father of Microbiology. He even coined the name 'micro'-'biology' as the study of microscopic organisms. He proposed the 'Germ theory of disease' after several experiments and proved that all life arose only from their like, thereby disproving the theory of spontaneous generation.

(Photograph originally posted by Albert Edelfelt)

Louis Pasteur also developed the pasteurization process, a process used to kill microbes in liquids by heating followed by rapid cooling. This technique is now commonly used in preservation of fruit juices and milk.

Louis also observed that some organisms required oxygen for their survival and growth, while others did not require oxygen. He called these aerobic and anearobic microorganisms respectively.

Louis Pasteur was the first to demonstrate the concept of vaccination. He attenuated the bacillus anthracis organisms by incubation at 42oC and then innoculated the attenuated bacteria into animals. He later observed that innoculated animals developed a certain level of protection against Anthrax. In 1885, Louis developed the Rabies vaccine.

Joseph Lister is known for his contributions in the development of antiseptics. He designed antiseptic surgery to prevent wound infection using heat sterilization.

The Koch's Postulates

(Photograph from Wilhelm Fechner - Public Domain.)

Robert Koch demonstrated that Anthrax was caused by a bacteria (Bacillus anthracis). His criteria for demontrating a microorganism as the cause of a disease are published as the Koch's postulates, which include:

Solid Media for bacterial growth

Koch pioneered the use of agar as a base for culture media. This enabled the isolation of pure cultures that contained only one type of bacteria. Koch also developed meat based or protein based culture media for isolating bacteria from the body. These media resembled the composition of body fluids and are used to develop nutrient broths or nutrient agar that are still in widespread use today.

Microscopy and Staining

A microscope is an instrument that uses one or multiple lenses to produce a magnified image of an object that would have been invisible to the naked eye.

There are three main types of microscopy techniques that will be covered in details in this course.

Light Microscopy

As the name suggests, light microscopy uses natural or artificial light to illuminate the object for viewing. Light increases the resolving power of the microscope (Resolution is the ability of the microscope lens to distinguish two closely adjacent objects as distinct separate entities. )The resolving power of a light microscope can also be anhanced by changing the medium through which light passes. For example, the use of emersion oil helps to improve the resolution of the microscope. There are 4 types of light microscopy as described below:

1. Bright-Field Microscopy: Also called 'Ordinary light microscopy' being the most common type of light microscopy. It contains compound lens, containing multiple objective lens and a fixed ocular lens. The total magnification is the product of the maginifications of objective lens and ocular lens (such as X10 and X40 to make a total magnification of X400).

Dark-field microscopy: (also called Dark-ground microscopy. This makes use of a dark-ground microscope, which uses reflected light rather than transmitted light. This prevents light from directly falling on the objective lens. The object appears brightly stained against a dark background.
An image as it appears under a dark field microscope. - Source Wikipedia. Under Creative Commons Licence - Public Domain. No alterations. Dark field microscopy is good for demonstrating flagellated bacteria and protozoa, or observing very thin bacteria such as spirochetes, not visible under ordinary light microscopes.

Phase-contrast microscopy: Uses a specific optical system that converts differences in density in an organism into differences in light intensity resulting in light and dark contrast within the image. It is useful for examining the internal structures of a living cell. Left: Image under an ordinary miscroscopy. Right: Same image as it appeared under a phase-contrast microscopy. Source Wikipedia. Under Creative Commons Licence - Public Domain. No alterations.

Interference microscopy: This is used for demonstrating cell organelles or quantitative measurement of the chemical constituents of the cells, such as proteins, lipids, and nucleic acids.

Fluorescence microscopy

Fluorescence microscopy uses flourescent dyes to stain the object / specimen, and once viewed under UV light, the object appears as brightly glowing against a dark background.

Some examples of flourescent dyes used to visualize bacteria include: Auramine O, acridine orange, and rhodamine.

Electron microscopy (EM)

Electron microscopy utilizes a beam of electrons (instead of light). The object is placed on the path of the electron beam causing the electrons to scatter and produce an image, which is displayed on a screen. Because electrons have a very low wavelength (0.005nm) as compared to the wavelength of visible light (5000 nm), electron microscopes can have a resolution about 100,000 times higher than light microscopes. (

There are several techniques that have been developed for electron microscopy including:

Bacterial Microscopy

Bacteria can be visualized under a microscope in either stained or unstained (wet mount) preparations. Unstained preparations can be used to demonstrate motility, while stained preparations are best for demonstrating structural details due to the color contrast.

Staining Methods

1. Simple Stains: Such as Methylene Blue provide color contrast but cannot be used to differentiate different bacterial species on the same specimen.

2. Negative Staining: In this staining technique, the bacteria remain unstained but their background is. Using stains such as Indian ink, the background becomes dark. It can be used to demonstrate bacterial capsules that usually cannot take stains when using simple staining techniques.

3. Impregnation Stains: This technique is used to observe cells or structures that are too thin. Silver impregnation is used to make such structures visible.

4. Gram Staining: Is a differentiation staining method used to identify Gram positive and Gram negative bacteria groups. This technique consists of four steps:
a. Primary Staining using a basic dye such as methyl violet
b. Addition of dilute iodine solution as a mordant
c. Decolorization using Ethanol, acetone or aniline
d. Counterstaining with acidic dye such as carbol fuchsin, safranine or neutral red.

Gram-positive bacteria are those that resist decolorization and retain the primary dye-iodine complex, appearing violet. Gram-negative bacteria are decolorized by organic solvents and take the counterstain, appearing red.

There are some bacterial species such as Mycobacterium tuberculosis and Mycobacterium leprae that have a waxy envelope containing complex glycolipids that make them impervious to the Gram stain. Therefore it is impossible to classify them as either Gram +ve or -ve.

Morphology and Physiology of Bacteria

Living things can be classified into three kingdoms; Plant, Animals and Protista. Microorganisms belong to the kingdom Protista. This kingdom consists of single celled organisms such as bacteria, fungi, algae and protozoa. The kingdom is further divided into three groups: Prokaryotes, Eukaryotes and Archaebacteria.

Bacteria are very small, their size is measured in microns (micrometer). 1 micron (µ) is equal to 1/1000 millimeters. Most bacteria of medical importance measure 2-5 microns in length.

Bacteria Shape

1. Cocci: Cocci are oval or spherical shaped cells. These may be arranged in pairs (such as, pneumococci), tetrads (such as, micrococci), chains (such as, streptococci), and clusters (such as, staphylococci).

2. Bacilli: Bacilli (singular bacillus) are rod shaped. Bacilli occur in one of the following arrangement: (a) Coccobacilli: Length of the bacteria is approximately the same as its width, for example, Brucella. (b) Streptobacilli: These are arranged in chains, such as, Streptobacillus. (c) Comma shaped: These occur in a curved shape, such as, Vibrio. (d) Spirilla: These are rigid spiral shapes, such as, Spirillum.

3. Spirochetes: Spirochetes are slender, flexuous spiral shapes, such as, Treponema.

4. Actinomycetes: Actinomycetes are branching filamentous bacteria resembling the shape of fungi.

Image created by Tensai.org. All rights reserved.

Bacterial Cell Wall

Bacteria are bound by a fairly rigid cell membrane and well wall. The cell wall is composed of peptidoglycan and is responsible for the shape of the cells. Peptidoglycan is a disaccharide with two sugar derivatives joined together by a peptide chain. The cell wall of gram +ve bacteria is thick and is made up of multiple layers of peptidoglycan. The gram negative cell wall is more complex but contains less peptidoglycan at about 1-2 layers. The gram negative cell wall also contains lipoprotein, outer membrane and lipoplysaccharides.

Bacterial Cell Membrane

The plasma membrane is thin and acts as an osmotic barrier. It lies underneath the cell wall. It contains phospholipids and proteins such as enzymes. However, unlike eukaryotic plasma membranes, the bacterial cell membrane does not contain cholesterol.

Ribosomes

Located within the cytoplasm, bacterial ribosomes are smaller (70S) than eukaryotic ribosomes (80S). They have two subuntis (30S and 50S). They function in protein synthesis (translation).

Mesosomes

Mesosomes are convoluted vesicles formed as invagination of the plasma membrane into the cytoplasm. They are analogs of mitochondria in eukaryotes and they perform similar functions related to energy metabolism.

Inclusion bodies

The main function of these vesicles is believed to be storage. Some vesicles store organic molecules such as glycogen while others store inorganic substances such as sulfur. Different types of granules can be detected by special staining techniques. Starch granules can be detected using iodine stains, while lipid granules can be demonstrated using Sudan black dye.

Bacterial Nucleus

Unlike eukaryotic cells, the bacterial nuclues is not bound by a nuclear membrane. There is also no nucleolus. The nucleus is basically the region where the DNA is located. Bacterial DNA is a circular double stranded structure measuring about 1000(µ) when stretched straight. The DNA is located in a region called the nucleoid, or sometimes referred to as the chromosome. Bacteria also contain extrachromosomal DNA called plasmids. Plasmids are circular double stranded DNA molecules that replicate independently. They are not required for growth but sometimes they carry genes that result in antibacterial resistance, or the ability for bacteria to produce toxins.

Capsule

The bacterial capsule is a gel-like layer formed on top of the cell wall. When its narrower, its called a microcapsule. The colloidal material secreted by some bacteria is called slime or glycocalyx. The bacterial capusle is made up of polysaccharides referred to as exopolysaccharides. In some species (such as Bacillus anthracis, the capsule is made up of polyamino acids.

The capsule has several functions including:

The capsule can be observed under the microscope using special staining methods such as the Welch method, which uses Copper as a mordant. The M'Fadyean capsule stain, which uses polychrome methylene blue stain, is used to observe the Bacillus anthracis capsule. Other methods used to observe the bacterial capsule include Negative stianing with India ink and Other Serological staining methods.

Bacterial Flagella

These are thread-like appendages embedded in the cell envelope. These structures are responsible for motility and may a role in bacterial pathogenicity because they are highly antigenic. There are 4 main types of flagella:

Pili

They are also called Fimbriae and play an important role in the adherence of bacteria on the host, a step that is needed for a bacteria to cause infection. Sex pili are involved in Conjugation, i.e., the transfer of DNA between bacterial cells.

Bacterial Spores

Some bacteria species form spores when they are exposed to environmental conditions that are not ideal for growth, such as nutritionally deficient conditions. Spores are highly resistant dormant of bacteria such as Bacillus and Clostridium species.

An illustration of the sporulation process. Source: Wikipedia - Under the Creative Commons Licence. No alterations made.

Bacterial spores are resistant to extreme environmental conditions including boiling, heating, disinfectants, freezing and drying. However, they can be destroyed by autoclaving at 121oC.

The conversion of a spore to a vegetative form is called germination.

Spores can be observed under a microscope as refractile bodies as shown here observed under phase-contrast microscopy.

Source: Wikipedia - Under the Creative Commons licence - No alterations made.

Spores can also be observed through gram staining or by using malachite green stain.

Bacterial Growth and Multiplication

Bacterial growth is simply the increase in all the chemical components of the cell. Multiplication occurs when the cell components reach a maximum level. Bacterial growth is expressed as the exponential increase in the number of cells. And the growth rate is the change in the number of bacterial cells per unti time. The Generation time can be defined as the amount of time it takes for a bacteria to give rise to 2 daughter cells. Most pathogenic bacteria have a generation time of about 20 minutes. Mycobacterium tuberculosis has a generation time of 20 hours, while Mycobacterium leprae has one of the longest generation times of 20 days. Because of the exponential nature of the division, an E. coli cell can undergo 30 generations in 10 hours resulting in 1 billion cells

Bacterial Growth Curve

The bacterial growth curve (increase in cell count) shows 4 distinct phases:

Effects of Oxygen on bacterial growth

The effects of oxygen on bacterial growth depends on whether the bacterial utilize oxygen in their metabolism or not. Bacteria can be classified into:

Other factors that influence bacterial growth include:

Bacterial Nutrition

Bacteria require an energy source such as glucose. They also require sources of elements such as Nitrogen and sulfur (they use to synthesize proteins, DNA, RNA and ATP. Several other elements/ions are needed for normal functioning of the bacterial cells including K, Na, Ca, Fe, Cl, H, C etc. C and H is usually obtained from inorganic compounds supplied in the media. Other bacteria can only grow in media that contain blood, or meat digests or certain supplementary amino acids. Finally, some bacteria, such as Mycobacterium leprae can only grow if inoculated into living animals, they cannot grow in any in vitro cultures.

Sterilization and Disinfection

Microorganisms can cause food spoilage and diseases so it is necessary to develop ways to inhibit their growth.

Sterilization is defined as the elimination of all living microorganisms including spores. Materials that have undergone sterilization are described as Sterile.

Disinfection is the process of using a chemical (disinfectant) to destroy all pathogenic microbes excluding spores. Disinfectants are used only in inanimate objects. Antiseptics are substances that can be applied directly on the body surface such as skin, wounds, mucous membranes, to inhibit the growth of pathogenic microbes.

Sepsis is defined as the presence of microbes or microbial toxins in the body or in the blood and other tissues. Procedures that prevent the access of microbes into tissues is called Asepsis.

Sanitization defines techniques that mechanically remove microbes along with other organic contaminants. This process is achieved using sanitizers, which include soaps and detergents.

Sterilization

Sterilization can be achieved by two major approaches, using physical (such as sunlight, dry heat, moist heat, filtration, radiation and sound waves) or chemical processes.

1. Sunlight: Direct sunlight works due to the presence of ultraviolet and heat rays. Its the natural method that reduces the microbial growth in lakes, rivers and tanks.

2. Moist Heat: Heat is the most common and reliable method of sterilization. High temperatures are microbicidal (they kill the microbes) while lower temperatures (such as freezing) are microbistatic (inhibit microbial growth but do not kill the microbes). Heat sterilization can be through the following techniques:

3. Dry Heat: There are several processes that use dry heat for sterilization. The advantage is that one is not limited to water's boiling point, dry heat can achieve upto several thousand oC.

3. Filtration: Solutions that cannot be heated can be sterilized through filtration. They can be used to sterilize pharmaceutical substances, ophthalmic solutions, liquid culture media, oils, antibiotics, and other heat-sensitive solutions. Filtration can also be used to obtain bacteria-free filtrates of samples/specimens for virus isolation. Air can also be sterilized by filtration such as using HEPA filters to remove nearly 99% of particles from the air.

Radiations: Both ionizing and non-ionizing radiations can be used to sterilize objects. X-rays, Gamma rays, Cosmic rays all have microbicidal effects and can also destroy spores. Non-ionizing radiations such as UV radiation is used primarily for disinfection of closed areas in microbiology laboratory, inoculation hoods, laminar flow, and operating theaters. It kills most vegetative bacteria but not spores, which are highly resistant to these radiations. However, it does not penetrate glass, dirt films, water, and other substances very effectively.

Sound waves: High frequency sound waves can generate heat that has antimicrobail action. Gram-negative rods are most sensitive to ultrasonic vibrations, while Gram-positive cocci, fungal spores, and bacterial spores are resistant.

Disinfection

We have already defined a disinfectant, but what are the properties of an ideal disinfectant:

Factors influencing the efficacy / activity of disinfectants:

The various types of disinfectants include:

Bacterial Cultures

Cultures are used as a way to confirm the presence of specific bacterial species in a sample. It is most commonly used to confirm the causative agent of a disease. Bacterial cultures can either be solid or liquid (broth). KIch pioneered the use of agar as a base for culture media. Agar is an ideal base because it doesnt melt until it is exposed to tempratures above 100oC, and also because it is not metabolized by most bacteria. Agar is obtained from a variety of sea weeds and is processed into powder.

Other ingredients in bacterial cultures include:

Liquid Media

Bacteria grown in liquid media form colloidal suspensions and need to be subcultured onto solid media to be able to process the colonies for identification. Growth in liquid media cannot be quantified since it doesnt occur as separate colonies.

Solid Media

Uses agar as the solidifying agent. Some bacterial species can be recognized on the basis of colony morphology.

Nutrient agar is the simplest and most common type of growth agar. It is prepared by adding 2% agar to a nutrient broth. Other examples of solid media include Blood agar, Chocolate agar, MacConkey agar and others.

Semi-Solid agar Is made using a lower concentration of agar. Semi-solid media allow bacteria to spread more easily in the media.

Enrichment Media is a liquid media that stimulates the growth of certain bacteria or suppress the growth of others. Enrichment media is used when culturing specific bacterial species from samples that are expected to have too many bacterial species. For example, if you intend to isolate E coli from a sample of feces, which has many other baccterial contamination.

Selective Media: contain substances that inhiobit the growth of certain bacterial species. For example;

Differential or indicator media: distinguish one microorganism from another growing on the same media by their growth characteristics.