CHAPTER 1: THE SCIENCE OF MICROBIOLOGY

MICROBIOLOGY  2. Some microorganisms are essential in

biotechnology and a wide range of industries.
 Derived from the Greek words “mikros” (small),
“bios” (life”), and “logia” (study of).  This includes food and beverage,
pharmaceuticals, mining, genetics, and
 It is therefore the study of organisms that are so
many more. Much of the knowledge
small they cannot be seen with the naked eye.
available in the study of genetics and
MICROBIOLOGY CLASSIFICATION biochemistry utilize microorganisms as
model organisms.
 These organisms are called microorganisms or
microbes and are categorized into two:  3. Some microorganisms, especially bacteria
and fungi, are important sources of microbial
1. Cellular agents.
 Prokaryotes (Bacteria, Cyanobacteria,  For example, penicillin was derived
and Archeans) from the fungus Penicillium.
 Eukaryotes (Fungi, Protozoa, and  4. Some microorganisms act as saprophytes
Algae) or decomposers of waste products and dead
2. Acellular organisms, making them essential in
maintaining a balanced ecosystem.
 Viruses.
 5. Better understanding of how
MICROBIOLOGY FURTHER CLASSIFICATION microorganisms produce disease, paving the
way to better disease management and
 Microbiology is further classified into different
control.
fields of study:
 6. Certain diseases which were thought to
1. Bacteriology (Study of bacteria)
have been eradicated are now re-emerging.
2. Virology (Study of virus)
 Some have the potential as biological
3. Mycology (Study of fungi) warfare agents.

4. Parasitology (Study of protozoa and parasitic worms)  At the same time, there are now a
number of pathogens that are developing
5. Phycology (Study of algae) resistance to antibiotics.
6. Immunology (Study of immune system and immune  In this context, the study of
response) microbiology is relevant for better
WHY STUDY MICROBIOLOGY? understanding of the negative instances
in which science can be used.
 1. Microbiology has an impact in the daily
live of humans.
 Microorganisms are everywhere- in the
air one breathes, in the environment, and EVOLUTION OF MICROBIOLOGY
even in one’s body. About a thousand or
more organisms inhabit the human body.  Different types of fossils of primitive
These are collectively called normal microorganisms have been found in ancient rock
flora or indigenous flora which only formations, dating back to as early as 3.5 billion
produces disease in persons with years ago, long before the existence of animals
compromised immune systems. and humans.
 Infectious diseases have existed for thousands of
years.
 CHAPTER 1: THE SCIENCE OF MICROBIOLOGY

 In 3180BC, an epidemic known as the  Louis Pasteur in the middle and late 1800s,
“plague” broke out in Egypt. performed countless experiments that led to his
germ theory of disease.
 In 1122BC, an outbreak of a smallpox-
like disease that originated in China  He postulated that microorganisms were
spread worldwide. The exhumed in the environment and could cause
mummified remains of Rameses V infectious diseases.
showed skin lesions resembling
 Developed the process of pasteurization.
smallpox.
(A technique that kills microorganisms
 In the mid-1600s, the microscope was in different types of liquids which
discovered with the use of this instrument. became the basis for aseptic techniques.)
 Robert Hooke was able to discover the cell- the  He also introduced the terms aerobes
basic unit of living organisms. and anaerobes and developed the
fermentation process.
 CELL THEORY

 Anton von Leeuwenhoek, a Dutch merchant,

created a single-lens microscope that he used to  Robert Koch to prove that microorganisms
make observations of microorganisms which he caused certain diseases through a series of
then called animalcules. scientific steps which led to his formulation of
the Koch’s postulates.
 He became known as the “Father of
Microbiology” and was the one  Edward Jenner discovered the vaccine for
provided accurate descriptions of smallpox.
bacteria, protozoa, and fungi.  Joseph Lister applied the theory to medical
 Anton von Leeuwenhoek, a Dutch merchant, procedures paving the way to the development
created a single-lens microscope that he used to of aseptic surgery.
make observations of microorganisms which he  After WW2, antibiotics were introduced to the
then called animalcules. medical world.
 He became known as the “Father of  Paul Ehrlich discovered Salvarsan for
Microbiology” and was the one the treatment of syphilis.
provided accurate descriptions of
bacteria, protozoa, and fungi.  This drug was heralded the
“magic bullet” of chemotherapy,
 CHAPTER 1: THE SCIENCE OF MICROBIOLOGY

which is treatment of disease by  Visible light is its main source of illumination.

using chemical substances. As such, it is also known as the compound light
microscope.
 Alexander Fleming discovered the antibiotic
penicillin from the mold Penicillium notatum.  The compound microscope utilized today
EVOLUTION OF MICROBIOLOGY
 With the discovery of antibiotics, the incidence
of infectious diseases like tuberculosis,
pneumonia, meningitis, and others was
significantly reduced.
 In the 1930s, electron microscope was
developed that experimentations in
microbiology became more complex.
 It was also this time have led the
discovery of viruses.
 In the 1940s and 1950s; microbiologists together consists of two magnifying lens systems.
with the discovery of other vaccines have led to
better prevention and control of numerous  The eyepiece (or ocular) contains what is called
potentially fatal infectious diseases. the ocular lens that has a magnifying power of
10x.
MICROSCOPY
 The second lens system is located in the
 Microorganisms are miniscule organisms that objective that is positioned directly above the
cannot be seen with the naked eye. The organism to be viewed.
discovery of the microscope has led to their
close observation, allowing microbiologists and BRIGHTFIELD MICROSCOPE
other scientists to study them further.  Made up of a series of lenses and utilizing
 A microscope is an optical instrument that can visible light as its source of illumination, the
magnify organisms a hundredfold or even a brightield microscope can magnify an object
thousand fold. 1,000 to 1,500 times. This is used to visualize
bacteria and fungi.
 From the time of its initial discovery in the
1600s, the microscope has undergone great  Objects less than or thinner than 0.2 um cannot
revolutionary changes. Making it more advanced be visualized by this type of microscope.
and complex throughout time.  The term "brightfield" is derived from the fact
 The following are the different types of that the specimen appears dark against the
microscopes that have evolved from von surrounding bright viewer field of this
Leeuwenhoek’s simple prototype. microscope. However, it has very low contrast
and most of the cells need to be stained to be
COMPOUND MICROSCOPE properly viewed.
 The compound microscope is a type of DARKFIELD MICROSCOPE
microscope that contains more than one
magnifying lens.  This microscope utilizes reflected light instead
of transmitted light, with a special condenser
 It can magnify objects approximately a thousand that has an opaque disc that blocks the light,
times their original size. such that only the specimen is illuminated.
 CHAPTER 1: THE SCIENCE OF MICROBIOLOGY

 The specimen to be studied appears bright  The specimen under study fluoresces or appears
against a dark background. This type of to shine against a dark background.
microscope is ideal for studying specimens that
 Fluorescence microscopy is based on the
are unstained or transparent and absorb little or
principle that certain materials emit energy that
no light.
is detectable as visible light when they are
 It is also useful in examining the external details irradiated with the light of a given wavelength.
of the specimen such as its outline or surface.
 It uses a higher intensity of light source and this
This type of microscope is used to view
in turn excites a fluorescent species. The
spirochetes.
fluorescent species then emits a lower energy
PHASE-CONTRAST MICROSCOPE light of a longer wavelength which produces the
magnified image instead of the original light
 Phase-contrast microscopy is based on the
source.
principle that differences in refractive indices
and light waves passing through transparent  Fluorescence microscopy can be used to
objects assume different phases. visualize structural components of small
specimens such as cells and to detect the
 This type of microscopy was first introduced by
viability of cell populations.
Frits Zernike, a Dutch physicist, in 1934.
 It may also be used to visualize the genetic
 The phase-contrast microscope has a contrast-
material of the cell (DNA and RNA).
enhancing optical technique in order to produce
high-contrast images of specimens that are CONFOCAL MICROSCOPE
transparent which include thin tissue slices,
 Also known as the confocal laser scanning
living cells in culture, and subcellular particles
microscope (CLSM) or laser confocal scanning
(such as nuclei and organelles).
microscope (LCSM), the confocal microscope
DIFFERENTIAL INTERFERENCE uses an optical imaging technique that increases
CONTRAST MICROSCOPE optical resolution and contrast of the micrograph
by using a spatial pin-hole to block out-of-focus
 The differential interference contrast microscope
sight in image formation.
is similar to the phase-contrast microscope
except that it utilizes two beams of light instead  The specimen is stained with a fluorescent dye
of one and therefore has higher resolution. to make it emit or return light. The object is
scanned with a laser into planes and regions.
 The resulting contrasting colors of the specimen
This is used, together with computers, to
being studied are due to the prisms that split the
produce a three-dimensional image.
light beam.
 It is also useful in the study of cell physiology.
 It was developed by Georges Nomarski in 1952
as an improvement to the phase-contrast ELECTRON MICROSCOPE
microscope.
 The electron microscope utilizes a beam of
 It is useful in examining living specimens when electrons to create an image of the specimen.
normal biological processes might be inhibited The electron beams serve as the source of
by standard staining procedures. illumination and magnets are used to focus the
beam.
FLUORESCENCE MICROSCOPE
 It is used to visualize viruses and subcellular
 The fluorescence microscope makes use of
structures of the cell.
ultraviolet light and fluorescent dyes called
fluorochromes.  There are two types of electron microscopes-
transmission electron microscope and scanning
electron microscope.
 CHAPTER 1: THE SCIENCE OF MICROBIOLOGY

 The transmission electron microscope  This method of staining is a quick and easy way
(TEM) is the original form of the to visualize cell shape, size, and arrangement of
electron microscope. It produces two- bacteria.
dimensional, black and white images,
 It uses basic dyes such as safranin, methylene
and magnifies objects up to 200,000
blue, or crystal violet. These stains give up or
times.
accept hydrogen ion, leaving the stain positively
 The scanning electron microscope charged. Most bacterial cells and cytoplasm are
(SEM) relies on interactions at the negatively charged and since the dye is
surface rather than transmission. It can positively charged, it adheres readily to the cell
magnify bulk samples with greater depth surface enabling the visualization of bacterial
of view so that the image produced cell morphology.
represents the 3-D structure of the
DIFFERENTIAL STAINING
sample, but the image is still only black
and white. Generally, it can magnify the  Differential Stains
object 10,000 times.
 Differential stains are used to differentiate one
SCANNING PROBE MICROSCOPE group of bacteria from another.
 The scanning probe microscope was developed  There are two types of differential staining
in the 1980s by the Swiss scientists Dr. Gerd procedures commonly used, namely:
Binnig and Dr. Heinrich Rohrer.
1. Gram stain - distinguishes gram-positive bacteria
 It is used to study the molecular and atomic from gram-negative bacteria.
shapes of organisms on a nanoscale. A physical
probe is used to scan back and forth over the  gram-positive bacteria stain blue or
surface of a sample. purple, while gram-negative bacteria
stain red or pink.
 A computer then gathers data that are used to
generate an image of the surface. lt can also be  As a general rule, all cocci are gram-positive
used to determine the variations in temperature except Neisseria, Veilonella, and Branbamella.
inside the cell as well as its chemical properties On the other hand, all bacilli are gram-negative
except Corynebacterium, Clostridium, Bacillus,
STAINING and Mycobacterium.
 Most microorganisms besides being very tiny 2. Acid-fast stain -stain used for bacteria with high
are also devoid of any color and are thus lipid content in their cell wal1, hence cannot be
difficult to see, even with the use of the stained using Gram stain.
microscope.
 Two methods are used, namely Ziebl-Neelsen
 To facilitate visualization, staining procedures stain-also known as the "hot method" because
have been developed by various scientists. These it requires steam- a. bathing the prepared smear
staining procedures are meant to give color to after addition of the primary dye.
the organisms, making them easier to see
under the microscope.  This is because the primary stain used is
aqueous and will not bind to the cell wall of the
SIMPLE STAINING organism. Acid-fast organisms will appear red
on a blue background.
 Simple Stains
 b. Kinyoun stain also known as the "cold
 Simple stains make use of a single dye which
method" as it does not utilize heat after addition
can either be aqueous (water-based) or alcohol-
of the primary stain, which is oil-based. The
based.
acid-fast organisms will appear red on a green
background.
 CHAPTER 1: THE SCIENCE OF MICROBIOLOGY

SPECIAL STAINS  Exhibit a clot-like consistency at

ordinary room temperature and contain
 Special Stains.
agar at concentrations of 0.5% or less
 These are used to demonstrate specific structures that allows thickening of the media
in a bacterial cell. without producing a firm substance.

 For instance, metachromatic granules can be  They have a soft consistency similar to
visualized using the LAMB (Loeffler Alkaline custard and are best suited for culture of
Methylene Blue) stain. microaerophilic bacteria or for the study
of bacterial motility.
 Other special stains include Hiss stain (capsule
or slime layer);  3. Solid media - contain a solidifying agent such
as 1.5%-2% agar, giving them a firm surface on
 Dyer stain (cell wall), Fischer-Conn stain which cells can form discrete colonies.
(flagella), Dorner and Schaeffer-Fulton stain
(spores), and India ink or nigrosine (capsule of  They are used for isolation of bacteria
the fungus Cryptococcus neoformans). and fungi or for determining the colony
characteristics of the organism under
SUMMARY OF STAINING TECHNIQUES study.
CULTURE MEDIA Solid media come in two forms:
 Staining procedures only give clues as to the  (a) liquefiable (or reversible) solid media and
probable organism being studied.
 (b) non-liquefiable (or non-reversible) solid
 To identify a specific organism, culture using media.
specific culture media is the most ideal.
CLASSIFICATION OF CULTURE MEDIA
 A Media (sing.medium) are used to grow INTO THREE PRIMARY LEVELS
microorganisms.
ACCORDING TO CHEMICAL COMPOSITION
 A culture medium is basically an
aqueous solution to which all the 1. Synthetic media
necessary nutrients essential for the
 - contain chemically-defined substances
growth of organisms are added.
which are pure organic and/or inorganic
CLASSIFICATION OF CULTURE MEDIA INTO compounds.
THREE PRIMARY LEVELS
 The precise chemical composition of a
ACCORDING TO PHYSICAL STATE: synthetic medium is known. They may
be simple or complex, depending on
 1. Liquid media - commonly called broths, what supplement is added to it.
milk, or infusions, these are water-based
solutions that do not solidify at temperatures 2. Non-synthetic media
above the freezing point.
 – complex media that contain at least
 These contain specific amounts of one ingredient that is not chemically
nutrients but do not contain gelling defined, which means that it is neither a
agents such as gelatine or agar. simple or pure compound.

 Liquid media are suited for the  It is not representable by an exact

propagation of a large number of chemical formula. Most are extracts of
organisms, fermentation studies, and animals, plants, or yeasts.
other tests.
 2. Semi-solid media
 CHAPTER 1: THE SCIENCE OF MICROBIOLOGY

 Non-synthetic media can  It is designed to prevent the growth of

support the growth of more unwanted contaminating bacteria or
fastidious organisms. commensals so only the target bacteria
will grow.
ACCORDING TO FUNCTIONAL TYPE:
 Examples of approaches that will make
1. General Purpose media
the medium selective include changing
 - are designed for primary isolation of a the pH of the culture medium or adding
broad spectrum or microbes and contain substances such as antibiotics, dyes, or
a mixture of nutrients that support the other chemicals.
growth of both pathogenic and non-
 These are usually agar-based solid
pathogenic organisms.
media that allow isolation of individual
 Examples are peptone water, nutrient bacterial colonies.
broth, and nutrient agar.
Examples of this type of culture medium include the
2. Enrichment media following:

 - contain complex organic substances  a. Thayer-Martin agar -contains the antibiotics

such as blood, serum, or special growth trimethroprim, nystatin, vancomycin, and
factors, and are designed to increase the colistin. It is used for the isolation of Neisseria.
number of desired microorganisms
 b. Mannitol Salt agar - contains 10% NaCl and
without stimulating the rest of the
used for the isolation of Staphylococs aureus.
bacterial population.
 c. MacConkey's agar - promotes the growth of
 These are used to grow fastidious or
gram-negative bacteria, primarily those
nutritionally exacting bacteria.
belonging to the family Enterobacteriaceae, and
 There are two commonly used enrichment inhibits the growth of gram- positive bacteria
media, namely: through the addition of bile salts. It is both
selective and differential.
 a. Blood agar - contains general nutrients with
56-10% (by volume) blood added to a blood  d. Löwenstein -Jensen medium - a selective
agar base. Certain gram-positive bacteria medium used to recover Mycobacterium
produce exotoxins that cause hemolysis of red tuberculosis. It is made selective by the
blood cells contained in the blood agar. incorporation of malachite green.

 Their hemolytic reaction is categorized  e. Saboraud's dextrose agar - used for the
into three, which is useful in the isolation of fungi.
classification of these bacteria.
4. Differential media
 B. Chocolate agar – a type of nutrient medium
 Allow the growth of several types of
that is used for the culture of fastidious
microorganisms. These are designed to
organisms such as Haemophilus sp. Heat is
show visible differences among certain
applied to lyse the red blood cells, causing the
groups of microorganisms.
medium to turn brown.
 The differences may be in the form of
3. Selective media
variations in colony size or color,
 Contain one or more substances that changes in color of culture media, or
encourage the growth of only a specific formation of precipitates or gas bubbles.
target microorganism and inhibit the
 Differential media allow the growth of
growth of others.
more than one target microorganism that
 CHAPTER 1: THE SCIENCE OF MICROBIOLOGY

demonstrate morphologic variations in

colony morphology.
 Examples include MacConkeys agar and
Triple Sugar Iron agar.
5. Transport media
 Used for clinical specimens that need to
be transported to the laboratory
immediately after collection.
 These media prevent the drying of
specimen and inhibit the overgrowth of
commensals and contaminating
organisms.
 Charcoal is added to neutralize
inhibitory factors.
 Examples are the Cary Blair transport
medium for transport of feces of
suspected cholera patients and Pike's
medium which is used to transport throat
specimens of patients with streptococcal
infection.
6. Anaerobic media-
 Media used specifically for organisms
that cannot survive in the presence of
oxygen and require reduced oxidation-
reduction potential and other nutrients.
 These are supplemented with nutrients
such as vitamin K and hemin.
 They underggo boiling to remove
dissolved oxygen. To reduce the
oxidation-reduction potential,
substances such as 1% glucose, 0.1%
ascorbic acid, 0.1% thioglycolate, or
0.05% cysteine are added.
 Methylene blue or resazurin is added as
an indicator of the oxidation- reduction
potential.
 Examples are chopped cooked meat and
thioglycolate broth.