The Subject of Microbiology

The Subject of Microbiology 

Definition of “microbiology” and “microorganism”

Microbiology is the study of all living organisms (biological objects) that are too small to be visible to the naked eye.

The discipline is used to learn about all aspects of the organisms to not only determine how they live in their environment,

but also, how they impact their respective surroundings and thus other organisms around them (human beings, animals, etc.).

 

Classification of microbiological sciences

In general, the field of microbiology can be divided into the more fundamental branch

-         pure microbiology,

-         such as General Microbiology,

-         Immunology,

-         Bacteriology,

-         Mycology,

-         Protozoology and

-         Virology

-         and the applied microbiology (for E.g.- Medical, Sanitary, Veterinary and Industrial Microbiology).

 

The tasks of Medical Microbiology

This is the branch of microbiology that is concerned with the

-         diagnosis,

-         prevention and

-         treatment of human diseases caused by different types of organisms (infection agents).

There are four kinds of microorganisms that cause diseases in human beings:

-         bacteria,

-         fungi,

-         parasites and

-         viruses,

-         and one type of infectious protein called prion.

Methods of microbiological diagnostics

Several diagnostic methods can be used ranging from direct methods,

01. by directly detecting the microorganism causing the infection,

-         such as microscopy,

-         cultures,

-         specific gene detection and antigen detection,

02. to indirect methods,

-         such as serology, in which the levels of specific antibodies against certain microorganism is detected.

03. Animal testing,

-         also known as animal experimentation, animal research and in vivo testing

-         the use of non-human animals in experiments

-         can be used at microbiological diagnostics as well.

 

History of Microbiology: descriptive period, physiological (Pasteur’s) period, immunological period and modern period

In the 1670s and the decades thereafter,

a Dutch merchant named Anton van Leeuwenhoek made careful observations of microscopic organisms,

which he called animalcules.

Until his death in 1723, van Leeuwenhoek revealed the microscopic world to scientists of the day and is regarded as one of the first to provide accurate descriptions of protozoa, fungi, and bacteria.

From these works the descriptive period was started.

Louis Pasteur worked in the middle and late 1800s and put the beginning of the physiological (Pasteur’s) period.

Many of the etiologic agents of microbial disease were discovered during that period.

During the next – immunological – period (from the end of XIX century) the immune response was discovered.

The modern period was stared from the middle of XX century with the transition of microbiological investigations on the molecular level.

Scientific contribution of Pasteur

He found that each type of fermentation is carried out by a living microorganism.

However, before his discovery, people had a misconception about fermentation that it was generated by a series of chemical reactions in which enzymes are produced.

Using his work with fermentation, Pasteur was able to devise a process, now known as pasteurization, to kill microbes and preserve certain products.

Pasteurization prevents fermenting and spoilage in beer, milk, and other goods.

Pasteur successfully identified the organisms that cause diseases in humans:

-         Staphylococcus,

-         Pneumococcus (Streptococcus pneumoniae) and

-         Clostridium.

He was the first scientist to create live vaccines for

-         fowl cholera;

-         anthrax, a major livestock disease    and rabies.

 

Scientific contribution of Koch

A German physician and microbiologist.

As one of the main founders of modern bacteriology,

he identified the specific causative agents of

-         tuberculosis,

-         cholera,

-         and anthrax

-         and also gave experimental support for the concept of infectious disease, which included experiments on humans and other animals.

Koch created and improved laboratory technologies and techniques in the field of microbiology

-         (solid agar media for cultivation of microorganism,

-         aniline dyes for staining microorganisms and

-         equipped the light microscope with immersion objective).

His research led to the creation of Koch's postulates,

a series of four generalized principles linking specific microorganisms to specific diseases that remain today the "gold standard" in medical microbiology.

For his research on tuberculosis, Koch received the Nobel Prize in Physiology or Medicine in 1905.

 

Classification of microorganisms

All living organisms are classified into groups based on very basic, shared characteristics.

Classification of microorganisms is based on their,

-         morphological,

-         biochemical,

-         physiological (cultural),

-         serological and

-         molecular biological features.

 In science, the practice of classifying organisms is called taxonomy

-         (Taxis means arrangement and nomos means method).

The classification of living things includes 7 levels:

1.     kingdom,

2.     phylum (or division),

3.     classes,

4.     order,

5.     families,

6.     genus,

7.     and species.

Microorganisms belong to kingdoms

1.     “Monera” (bacteria),

2.     “Plantae” (fungi) and

3.     “Animalia” (protozoans).

The basic taxon used for classification of microorganisms is species the species includes intraspecies subdivisions:

-         variant,

-         strain and

-         clone.

 

Methods of microscopy

There are 3 main microscopic techniques that are used;

1.     optical microscopy,

2.     electron microscopy and

3.     scanning probe microscopy.

Optical microscopy, otherwise known as light microscopy, involves the useage of visible light and one or more lens to produce an enlarged image of an object that is placed in the focal plane of the lens.

There are many applications to optical microscopy.

-         In light microscopy,

-         oil immersion is a technique used to increase the resolving power of a microscope.

This is achieved by immersing both the objective lens and the specimen in a transparent oil of high refractive index, thereby increasing the numerical aperture of the objective lens.

01. Oil immersion microscopy is the main microscopic technique used in bacteriology.

02. Dark field microscopy is a technique for improving the contrast of unstained, transparent specimens.

Dark field illumination uses a carefully aligned light source to minimize the quantity of directly transmitted (unscattered) light entering the image plane, collecting only the light scattered by the sample.

As a result, the field around the specimen (i.e., where there is no specimen to scatter the beam) is generally dark.

Dark field can dramatically improve image contrast – especially of transparent objects – while requiring little equipment setup or sample preparation.

03. Phase-contrast microscopy is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image.

Phase shifts themselves are invisible, but become visible when shown as brightness variations.

04. A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances.

"Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a more simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, (which uses optical sectioning to get better resolution of the fluorescence image).

 

Electron microscopy uses electron beams to create an image of the object being used.

Electron microscopes have a much higher magnification than light microscopes and so a much higher resolution as a result, this allows us to see smaller specimens in greater detail.

The resolution is able to be increased because as the electrons travel faster their wavelength becomes shorter so there is a direct correlation between reducing wavelength and increasing resolution.

There are 2 types of electron microscopes used,

1.     Transmission and

2.     Scanning electron microscopes.

TEM involves shooting a high voltage beam through a thin layer of specimen and gathering information about the structure.

SEM in contrast produces images by detecting secondary electrons that have been emitted off the surface due to excitation by the primary electron beam.

 

Scanning probe microscope (SPM) is a branch of microscopy that forms images of surfaces using a physical probe that scans the specimen.

Basically, it works by being moved around in a rectangular pattern known as raster scanning.

A type of scanning probe microscopy is called STM (scanning tunneling microscopy, an instrument for imaging surfaces at the atomic level) this is when a very sharp conducting tip is brought to the surface and a voltage is applied between them and we are able to find out the tunnel current and if this is maintained we can trace the elevation of the surface and thus produce it on an x-ray.

 

Methods of staining

Single stain with use of only one dye is applied for simple staining techniques:

-         methylene blue,

-         aqueous fuchsine or other.

Several stains with use of several dyes applied in certain order are used for differential staining techniques: i.e.

-         Gram stain,

-         Ziehl-Neelsen stain,

-         Neisser stain and

-         Burry-Hines (using India ink) stain.