INTRODUCTION

TO MICROBIOLOGY

By: Ma’am Jill

At the end of this chapter, the student should be able to:

1. define Microbiology and give the importance of the study of Microbiology;

2. name important persons with significant contributions to the field of
Microbiology;
3. differentiate the various types of microscopes and their uses;
4. compare the various staining methods used to visualize microorganisms;
and
5. classify the different types of culture media based on their physical state,
chemical composition,and functional type.
Myth busters vs. Microbes" Quiz:
Answer the following questions whether it is a myth or fact.

1. All germs are bad and will make you sick.

2. You can only see microbes under a microscope.
3. Antibiotics are like magical germ-zappers that kill all bacteria, good and bad.
4. Eating raw cookie dough is safe because the heat from baking is the only thing that kills
harmful bacteria in the dough.
5. The five-second rule is a legitimate guideline for determining whether food is safe to eat
after it falls on the floor.
6. If a surface looks clean, it means there are no microbes present.
7. Hand sanitizers are just as effective as washing hands with soap and water in eliminating all
types of germs.
8. Bacterial infections can always be treated with antibiotics.
9. The majority of the Earth's oxygen comes from trees and plants, not from microbes like algae
in oceans.
10. Probiotics are only beneficial for gut health and have no impact on other aspects of well-
being.
11. If a food item doesn't have an expiration date, it means it will last indefinitely without spoiling
or posing any health risks.
12. Drinking alcohol kills all the bacteria in your mouth, making it a good substitute for
mouthwash.
MICROBIOLOGY
Microbiology
is derived from the Greek words mikros (“small”),
bios (“life”), and logia or logos (“study of”)

Microbiology is the study of all living organisms

that are too small to be visible with the naked eye.

(1) Cellular- prokaryotes (bacteria, cyanobacteria, and

archaeas) or eukaryotes (fungi, protozoa, and
algae);

(2) Acellular- viruses

Bacteria are small single-celled organisms.
Come in various shapes. They can be spheres, they can be rods, or
they can be spirals
Pathogenic and Non-pathogenic
 Cyanobacteria

Cyanobacteria are aquatic and photosynthetic, that is, they live in the
water, and can manufacture their own food.

They are one of the largest and most important groups of bacteria on
earth.
Eukaryotic microorganisms

Protists are a diverse group of eukaryotic

microorganisms
•Algae: Protists such as diatoms, dinoflagellates,
and green algae are classified as algae. They can be
both unicellular and multicellular and are often
found in aquatic environments.
•Protozoa: These are unicellular, heterotrophic
protists. Examples include amoebas, paramecia,
and flagellates. They are often found in soil and
aquatic habitats.
•Slime Molds: Some protists, like slime molds,
exhibit characteristics of both fungi and amoebas.
They are often found in damp environments.
Fungi form their own kingdom, separate from plants, animals, and bacteria. Fungi
play crucial roles in various ecosystems, including decomposition, nutrient cycling,
and symbiotic relationships with other organisms.

•Examples:
•Yeasts: Single-celled fungi that often
reproduce by budding.
•Molds: Multicellular fungi with a fuzzy
appearance, commonly found on decaying
organic matter.
•Mushrooms: Reproductive structures of
certain fungi that release spores for dispersal.
Viruses are smaller than bacteria
and are not technically alive on
their own — they must infect a
host cell to survive.
Viruses are made up of some
genetic material surrounded by a
viral coat, but they lack all the
machinery necessary to make
proteins and catalyze reactions.
Why study microbiology?
1. Microbiology has an impact in the daily lives of humans. Microorganisms are
everywhere—in the air one breathes, in the environment, and even
in one’s body. About a thousand or more organisms inhabit the
human body. These are collectively called normal flora or indigenous
flora which only produce disease in persons with compromised
immune systems
2. Some microorganisms are essential in biotechnology and a wide range of
industries which include food and beverage, pharmaceuticals, mining, genetics,
and many more. Much of the knowledge available in the study of genetics and
biochemistry utilize microorganisms as model organisms.

3. Some microorganisms, especially bacteria and fungi, are important sources of

antimicrobial agents. For example, penicillin was derived from the fungus
Penicillium.

4. Some microorganisms act as saprophytes or decomposers of waste products and

dead organisms, making them essential in maintaining a balanced ecosystem.
Uses of Microbes in Biotechnology
Published August 21, 2020
Posted in: Cell Culture

1. Beer, Bread, and Wine

 Biotechnology used for food and drink production is called yellow

biotechnology.

 Yeast forms were likely the first “domesticated” organisms used to

make edible products through the process of fermentation.
 bread, beer, and wine
 Other fermented food
Uses of Microbes in Biotechnology
Published August 21, 2020
Posted in: Cell Culture

2. Milk Products

Milk is fermented by Lactobacillus

some cheeses, like blue cheese are made with a fungi (Penicillium).
Similarly, certain dessert wines are made from the “noble rot” of the
fungus Botrytis cinerea
Uses of Microbes in Biotechnology
Published August 21, 2020
Posted in: Cell Culture

3. Antibiotic Production
Red biotechnology or Medical Biotechnology- biotechnology applied to manufacture
pharmaceuticals like enzymes, antibiotics and vaccines, and its use for molecular
diagnostics.

Fungi are most commonly associated with cheese and wine production. However,
they have a long history in traditional medicine as moldy bread, overgrown with the
fungi Penicillum and Aspegillum, would regularly be applied to wounds for their
antimicrobial effect. The jump from antimicrobial compounds to the antibiotics we
know now was ushered in by Scottish scientist Alexander Fleming, who accidentally
discovered Staphylococcus growth suppression by penicillin mold in 1928. However,
it took another 12 years and a world war to start mass-producing penicillin using
deep fermentation techniques
Uses of Microbes in Biotechnology
Published August 21, 2020
Posted in: Cell Culture

4. Protein Production

Protein production in E.coli was the new stage in biotechnology

because it used recombinant DNA technology instead of traditional
selection techniques. The first human protein produced in E. coli in
1978 was insulin, followed by human growth hormones.
Uses of Microbes in Biotechnology
Published August 21, 2020
Posted in: Cell Culture

6. Microalgae

Blue biotechnology uses sea resources to create products and industrial

applications, including medicine, cosmetics and personal care products,
wound treatment, waste clean-up, aquaculture (as in fish farming).

The leading application of blue biotechnology is producing renewable bio-oils

with photosynthetic microalgae to replace oil extraction.
Uses of Microbes in Biotechnology
Published August 21, 2020
Posted in: Cell Culture

7. Agrobacterium Tumefaciens

Green biotechnology, also known as plant biotechnology or

agricultural biotechnology, is a rapidly evolving field that applies scientific
tools and techniques to improve plants, agriculture, and the environment. Its
main focus is on developing sustainable solutions for food production,
environmental protection, and resource management.
Uses of Microbes in Biotechnology
Published August 21, 2020
Posted in: Cell Culture

8. CRISPR
CRISPR - Clustered Regularly Interspaced Short Palindromic Repeats.
 It’s a naturally occurring system found in bacteria and archaea that helps
them defend themselves against viruses.

Scientists have adapted CRISPR into a powerful tool for editing DNA in living
organisms, including humans.
CRISPR has the potential to revolutionize medicine, agriculture, and
even bioremediation. Here are some of its potential applications:

•Gene therapy: Correcting genetic mutations that cause diseases like cystic
fibrosis and sickle cell anemia.

•Cancer treatment: Developing new personalized therapies by editing genes in

cancer cells.

•Agriculture: Creating crops that are more resistant to pests and diseases, or
have higher yields.

•Bioremediation: Cleaning up polluted environments by using CRISPR-edited

microbes to break down harmful chemicals.
Evolution of Microbiology
Zacharias Janssen (1570-1638)
Robert Hooke (1635-1703)
Antony van Leeuwenhoek (1632-1723)
Van Helmont (1580-1644)
Francesco Redi (1626-1697)
Louis Joblot (1645-1723)
Lazzaro Spallanzani (1729-1799)
Edward Jenner (1749-1823)
Joseph Lister (1827-1912)
 Louis Pasteur (1822-1895)

• He performed countless experiments that led to his germ

theory of disease.
• He postulated that microorganisms were in the environment
and could cause infectious diseases.
• He also developed the process of pasteurization, which kills
microorganisms in different types of liquids, and which
became the basis for aseptic techniques.
• He also introduced the terms aerobes and anaerobes and
developed the fermentation process.
Robert Koch (1843-1910)
This led to an increased effort by other scientists to prove and illustrate further the germ
theory that was initially formulated by Louis Pasteur. Thus, the late 1800sand the first
decade of the 1900s came to be known as the Golden Age of Microbiology.
Since then, numerous scientists have made significant contributions to the field of
Microbiology.
Paul Ehrlich (1854-1915)
Hans Christian Gram (1853-1938)
Alexander Fleming (1881-1955)
 Most of the experiments conducted in the field of microbiology during the early20th
century involved the study of bacteria. During this time scientists were not yet equipped
with advanced technology in their study of microorganisms.
It was only in the 1930s when the electron microscope was developed that
experimentations in microbiology became more complex. It was also during that time
when viral culture was introduced paving the way for rapid discoveries on viruses. The
vast knowledge gained from the experiments performed by microbiologists together with
the discovery of other vaccines in the 1940s and 1950s have led to better prevention and
control of numerous potentially fatal infectious diseases.
THE FIELDS OF
MICROBIOLOGY
• Aquatic, soil, and agricultural microbiology study the microorganisms associated
with aquatic (including wastewater treatment systems), soil, and agricultural
environments, respectively.
• Bacteriology is the identification and characterization of bacterial species.
• Immunology is the study of the body’s response to infection by microorganisms.
Included within this field is the area of vaccine research, which aims to develop more
and better ways of immunizing people from microorganisms that cause life
threatening infections.
• Industrial microbiology applies the large-scale use of microorganisms to make
things like antibiotics or alcohol.
• Medical microbiology is the study of pathogenic microorganisms that cause
infectious disease in humans and animals and ways to prevent and treat infections.
• Microbial biochemistry aims to understand the enzymes and chemical reactions
inside microbial cells.
• Microbial biotechnology is genetically engineering microorganisms to produce a
foreign gene or pathway so that it may either make a product for human use (for
example, human insulin) or perform a function that we need (for example, degradation
of environmental contaminants).
• Microbial ecology is the study of microbial diversity in nature, as well as microbial
populations and microbial communities and their effects on their environments. This
includes nutrient cycling and biogeochemistry (biological, chemical, and physical
processes that control the composition of the natural environment).
• Microbial genetics is the study of the genomes of microorganisms, including how the
genetic code varies between microbes and how genes are passed on.
• Microbial systematics is the study of how microorganisms diversified through time. It
includes the naming and organizing of microbial groups with respect to one another.
 • Mycology is the study of fungi, both in terms of their natural habitats and
genetics, and in terms of their ability to cause disease in humans, other
animals, and plants.
• Parasitology is the study of parasites of animals and humans. These are all
eukaryotic (not bacterial or archaeal) and include protists and worms.
• Virology is the study of viruses and simple nonviral entities, such as viroids
(RNA molecules that behave like infectious agents) and prions (proteins that
behave like infectious agents).
MICROSCOPY
• Microscopes are instruments designed to produce
magnified visual or photographic images of small
objects.

The microscope must accomplish three tasks

1. produce a magnified image of the specimen
2. separate the details in the image,
3. render the details visible to the human eye or camera.
Simple Microscope
 Light passes through only 1 lens.

Example: magnifying glass

Compound Microscope
The compound microscope is a type of microscope that contains more
than one magnifying lens. It can magnify objects approximately a
thousand times their original size.

Visible light is its main source of illumination.

As such, it is also known as the compound light microscope.

Brightfield Microscope
 Can magnify an object 1,000 to 1,500
times.
 This is used to visualize bacteria and
fungi.
 Objects less than or thinner than 0.2 μm
cannot be visualized by this type of
microscope.
 The term “brightfield” is derived from
the fact that the specimen appears dark
against the surrounding bright viewer
field of this microscope.
Darkfield Microscope
 Utilizes reflected light instead of transmitted
light, with a special condenser that has an
opaque disc that blocks the light, such that only
the specimen is illuminated.

 The specimen to be studied appears bright

against a dark background.
 This type of microscope is ideal for studying
specimens that are unstained or transparent and
absorb little or no light.
 It is also useful in examining the external details
of the specimen such as its outline or surface.

 This type of microscope is used to view spirochetes

 Phase-contrast Microscope

 was first introduced by Frits Zernike, a Dutch physicist, in 1934.

 The phase contrast microscope has a contrast enhancing optical technique in
order to produce high contrast images of specimens that are transparent which
include thin tissue slices, living cells in culture, and subcellular particles (such as
nuclei and organelles)
Phase-contrast Microscope

 utilizes two beams of light instead of one

 has higher resolution
 It was developed by Georges Nomarski in 1952 as
an improvement to the phase contrast
microscope.

 It is useful in examining living specimens when

normal biological processes might be inhibited by
standard staining procedures.
Fluorescence Microscope
 The fluorescence microscope makes use of ultraviolet
light and fluorescent dyes called fluorochromes.

The specimen under fluoresces or

appears to shine against a dark
background.

 Fluorescence microscopy can be used to visualize structural components of

small specimens such as cells and to detect the viability of cell
populations.
 It may also be used to visualize the genetic material of the cell (DNA and
RNA)
Confocal Microscope

 Also known as the confocal laser scanning microscope

(CLSM) or laser confocal scanning microscope (LCSM
 Uses an optical imaging technique that increases optical
resolution and contrast of the micrograph by using a spatial pin
hole to block out of focus light in image formation

 This is used, together with computers, to produce a three dimensional

image.

 It is also useful in the study of cell physiology.

Electron Microscope

 Utilizes a beam of electrons to create

an image of the specimen

 German Engineer Ernst Ruska in 1933

 Modern electron microscopes are

capable of magnifying objects up to 2
million times.

 Transmission electron microscope

 Scanning electron microscope
 Transmission Electron Microscope (TEM)

 is the original form of the electron microscope. It

produces two dimensional, black and white images,
and magnifies objects up to 200,000 times

Scanning Electron Microscope (TEM)

 relies on interactions at the surface rather than
transmission. It can magnify bulk samples with greater
depth of view so that the image produced represents
the 3 D structure of the sample, but the image is still
only black and white.
Scanning Probe Microscope

 Developed in the 1980s by the Swiss scientists Dr.

Gerd Binnig and Dr. Heinrich Rohrer.

 It is used to study the molecular and atomic shapes of

organisms on a nanoscale.

 It can also be used to determine the variations in

temperature inside the cell as well as its chemical
properties.
Staining procedures are meant to give color to the organisms,
making them easier to see under the microscope
Simple Stains

 make use of a single dye (aqueous or water based or alcohol based).

 Basic dyes (methylene blue, or crystal violet). These stains give up or

accept hydrogen ion, leaving the stain positively charged. Most bacterial
cells and cytoplasm are negatively charged and since the dye is positively
charged, it adheres readily to the cell surface enabling the visualization of
bacterial cell morphology.

 quick and easy way to visualize cell shape, size, and arrangement of
bacteria.
Differential Stains
1. Gram-stain- distinguishes gram positive bacteria from gram negative
bacteria.

 gram positive bacteria

- all cocci except Neisseria, Veilonella, and Branhamella
 gram negative bacteria- all bacilli except Corynebacterium,
Clostridium, Bacillus, and Mycobacterium.
2. Acid-fast stain– stain used for bacteria with high lipid content in their cell
wall, hence cannot be stained using Gram stain.

a. Ziehl-Neelsen stain– also known as the “hot method” because it requires steam bathing the
prepared smear after addition of the primary dye. Acid fast organisms will appear red on a
blue background.

b. Kinyoun stain– also known as the “cold method” as it does not utilize heat after addition of
the primary stain, which is oil based. The acid fast organisms will appear red on a green
background
Special Stains
 used to demonstrate specific structures in a bacterial cell

 LAMB (Loeffler Alkaline Methylene Blue) stain

 Hiss stain (capsule or slime layer)
 Dyer stain (cell wall)
 Fischer-Conn stain (flagella)
 Dorner and Schaeffer Fulton stain (spores)
 India ink or nigrosine (capsule of the fungus Cryptococcus neoformans)
 Culture media
This is the most ideal method in identifying a
specific organism.

 Media are used to grow microorganisms. A culture medium is basically an aqueous solution to
which all the necessary nutrients essential for the growth of organisms are added.

 These are classified into three primary levels: physical state, chemical composition, and
functional type
 According to Physical State

1. Liquid media– commonly called broths, milk, or infusions, these are water based solutions
that do not solidify at temperatures above the freezing point.
 These contain specific amounts of nutrients but do not contain gelling agents such as
gelatin or agar.
 Liquid media are suited for the propagation of a large number of organisms, fermentation
studies, and other tests.
Escherichia coli (E. coli): This common Staphylococcus aureus (Staph aureus): This
inhabitant of the human gut readily grows in opportunistic pathogen can be cultured in liquid media
various liquid media like Luria-Bertani (LB) like trypticase soy broth (TSB), revealing its
broth, providing a workhorse for research and characteristic golden pigment and aiding in antibiotic
biotechnology. susceptibility testing.
Streptococcus pneumoniae (Pneumococcus): This
causative agent of pneumonia thrives in broth media
like brain-heart infusion (BHI) broth, allowing for its
isolation and identification from clinical samples.

Saccharomyces cerevisiae (Baker's yeast): This workhorse of the

fermentation world readily grows in broth media like Yeast Extract-
Peptone-Dextrose (YPD) broth, producing bubbles of carbon dioxide
as it feasts on the sugars available.
2. Semi-solid media– exhibit a clot like consistency at ordinary room temperature and contain
agar at concentrations of 0.5% or less that allows thickening of the media without producing a
firm substance.
 They have a soft consistency and are best suited for culture of microaerophilic bacteria or for
the study of bacterial motility.

Proteus mirabilis Vibrio cholerae Campylobacter jejuni

3. Solid media – contain a solidifying agent such as 1.5%–2% agar, giving them a firm surface on which
cells can form discrete colonies.
 Used for isolation of bacteria and fungi or for determining the colony characteristics of the organism under
study.
Solid media come in two forms:
(a) Liquefiable (or reversible) solid media
 Contain an agar-based solidifying agent that melts at a specific temperature (typically around 100°C) and
resolidifies when cooled down (around 45°C).
Examples include nutrient agar and blood agar.
(a) non liquefiable (or non reversible) solid media
 Contain solidifying agents that do not melt readily, even at high temperatures. Common examples
include solidified egg, serum, potato slices, and rice grains.
Escherichia coli (E. coli) Staphylococcus aureus (Staph Bacillus subtilis
aureus)
According to Chemical Composition
1. Synthetic media– contain chemically defined substances which are pure organic and/or inorganic
compounds. The precise chemical composition of a synthetic medium is known. They maybe simple or
complex, depending on what supplement is added to it.

2. Non-synthetic media– complex media that contain at least one ingredient that is not chemically
defined, which means that it is neither a simple or pure compound. It is not representable by an exact
chemical formula.
Most are extracts of animals, plants, or yeasts. Non-synthetic media can support the growth of more
fastidious organisms
According to Functional Type
1. General Purpose media – are designed for primary isolation of a broad spectrum of microbes and
contain a mixture of nutrients that support the growth of both pathogenic and non-pathogenic
organisms. Ex: peptone water, nutrient broth, and nutrient agar.
2. Enrichment media– contain complex organic substances such as blood, serum, or special growth
factors, Designed to increase the number of desired microorganisms without stimulating the rest of the
bacterial population. These are used to grow fastidious or nutritionally exacting bacteria.
Two commonly used enrichment media
a. Blood agar– contains general nutrients with 5%–10% (by volume) blood added to a blood agar base.
Certain gram positive bacteria produce exotoxins that cause hemolysis of red blood cells contained in
the blood agar. The hemolytic patterns are:
i. Beta hemolysis– shows complete lysis of red blood cells resulting in complete clearing around
the colonies.
ii. Alpha hemolysis– shows incomplete lysis of red blood cells, producing a greenish
discoloration of the blood agar around the colonies.
iii. Gamma hemolysis– shows no hemolysis, resulting in no change in the medium

b. Chocolate agar– a type of nutrient medium that is used for the culture of fastidious organisms.
Heat is applied to lyse the red blood cells, causing the medium to turn brown.
3. Selective media– contain one or more substances that encourage the growth of only a specific
target microorganism and inhibit the growth of others. It is designed to prevent the growth of
unwanted contaminating bacteria or commensals so only the target bacteria will grow.
Examples of approaches that will make the medium selective include changing the pH of the
culture medium or adding substances such as antibiotics, dyes, or other chemicals. These
are usually agar based solid media that allow isolation of individual bacterial colonies.
Ex:
a. Thayer-Martin agar
b. Mannitol Salt agar
c. MacConkey’s agar
d. Löwenstein-Jensen medium
e. Saboraud’s dextrose agar
a. Thayer-Martin agar– contains the antibiotics trimethroprim, nystatin, vancomycin, and
colistin. It is used for the isolation of Neisseria.
b. Mannitol Salt agar– contains 10% NaCl and used for the isolation of Staphylococcus aureus.
c. MacConkey’s agar– promotes the growth of gram negative bacteria, primarily those
belonging to the family Enterobacteriaceae, and inhibits the growth of gram positive bacteria
through the addition of bile salts. It is both selective and differential.
d. Löwenstein-Jensen medium– a selective medium used to recover Mycobacterium
tuberculosis. It is made selective by the incorporation of malachite green.
e. Saboraud’s dextrose agar– used for the isolation of fungi
4. Differential media– allow the growth of several types of microorganisms. These are
designed to show visible differences among certain groups of microorganisms. The
differences may be in the form of variations in colony size or color, changes in color of culture
media, or formation of precipitates or gas bubbles.
 Differential media allow the growth of more than one target microorganism that
demonstrate morphologic variations in colony morphology.
 Ex: MacConkey’s 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.
 Ex: Cary Blair transport medium for transport of feces of suspected cholera patients
 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
undergo 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.

Ex: chopped cooked meat and thioglycolate broth.

Thank you!