MICRO PARA

INTRODUCTION

JONALD P. REGIO, RN, RMT

 UNIT 1

• Unit 1: Intro to Microbiology

• Lesson 1: The Science of Microbiology
• 1.            Fields of Study
• 2.            Why Study Microbiology?
• 3.            Evolution of Microbiology
• a.            Microscopy
• b.            Staining of Microorganism
• c.             Culture Media
 LEARNING OBJECTIVES

•After 3 hours of discussion leaners / students may be able to:

•Define the meaning of common terms related to the topic
•Differentiate / enumerate the different studies of
Microbiology
•Explain the significance of Microbiology in the environment
•Know the evolution of Microbiology
•Familiarized with the microscope, different culture media and
staining methods
 MICROBIOLOGY: THE SCIENCE

•Microbiology is the study of living organisms of

microscopic size.
•“Study of microbes”
• Medical microbiology is the subdivision concerned
with the causative agents of infectious disease of man,
the response of the host to infection and various
methods of diagnosis, treatment and prevention.
MICROBIOLOGY THE SCIENCE
 MICROBIOLOGY: THE SCIENCE

• Disease-causing microorganisms are technically known as

pathogens (also referred to as infectious agents)

• WHY STUDY MICROBIOLOGY?

• Indigenous microflora – these are microbes that live on

and in the body

• Opportunistic pathogens - Microbes that colonize

(inhabit) our bodies
 MICROBIOLOGY: THE SCIENCE

WHY STUDY MICROBIOLOGY?

Opportunistic Pathogen eg Escherichia coli
Photosynthesis
As decomposers
Bioremediation (genetic engineering)
Microbial ecology
Food chain
Biotechnology
Antibiotics
Microbes as cell models
 MICROBIOLOGY: THE SCIENCE

• Pathogens cause 2 major types of diseases”

• INFECTIOUS DISEASES
• Results when a pathogen colonizes the body and causes
disease.

• MICROBIAL INTOXICATIONS
• A person ingests a toxin (poisonous substance)
 MICROBIOLOGY: THE SCIENCE

• The earliest known account of a “pestilence” oc-

curred in Egypt about 3180 BC. This may
represent the first recorded epidemic, although
words like pestilence and plague were used
without definition in early writings. Around 1900
BC, near the end of the Trojan War, the Greek
army was decimated by an epidemic of what is
thought to have been bubonic plague. The Ebers
papyrus, describing epidemic fevers, was
discovered in a tomb in Thebes, Egypt; it was
written around 1500 BC. A disease thought to be
smallpox occurred in China around 1122 BC.
Epidemics of plague occurred in Rome in 790, 710,
and 640 BC and in Greece around 430 BC.
 MICROBIOLOGY: THE SCIENCE

• In addition to the diseases already mentioned,

there are early accounts of rabies, anthrax,
dysentery, smallpox, ergotism, botulism, measles,
typhoid fever, typhus fever, diphtheria, and
syphilis. The syphilis story is quite interesting. It
made its first appearance in Europe in 1493.
Many people believe that syphilis was carried to
Europe by Native Americans who were brought
to Portugal by Christopher Columbus. The
French called syphilis the Neapolitan disease; the
Italians called it the French or Spanish disease;
and the English called it the French pox. Other
names for syphilis were Spanish, German, Polish,
and Turkish pocks. The name “syphilis” was not
given to the disease until 1530.
 PIONEERS IN THE SCIENCE OF
MICROBIOLOGY

Anton Van Leeuwenhoek (1632-1723)

1st person to see bacteria and protozoa
Father of Microbiology, Father of Bacteriology and Protozoology
Not a trained Scientist
Animalicules / animalcules – tiny living creatures
 PIONEERS IN THE SCIENCE OF
MICROBIOLOGY

Louis Pasteur (1822-1895)

French Chemist
Discovery or “aerobic and anaerobic” microbes
Introduced and discovered ”pasteurization” at 55C
Discovery of Silk worm disease and how to prevent it
Contributions to “Germ Theory of Disease”
Championed hospital practices to minimize spread of diseases
Development of vaccines against ( chicken cholera, anthrax, swine
erysipelas)
Development of rabies vaccines in dogs and used the vaccs in
humans
 PIONEERS IN THE SCIENCE OF
MICROBIOLOGY

Robert Koch (1843-1910)

German Physician
Germ theory of Disease (proved B. anthracis as CA of Anthrax)
Koch’s postulates
Koch developed methods of fixing, staining, photographing
bacteria
Discovered M. tuberculosis and V. cholerae
Tuberculin ( a protein derived from M. tuberculosis) led to the
development of a skin test valuable in diagnosing tb.
 PIONEERS IN THE SCIENCE OF
MICROBIOLOGY

• Koch’s Postulates
• During the mid- to late-1800s, Robert Koch and his col- leagues established an experimental procedure to prove that a
specific microbe is the cause of a specific infectious disease. This scientific procedure, published in 1884, be- came known as
Koch’s Postulates (Fig. 1-10).
• Koch’s Postulates (paraphrased):
• 1. A particular microbe must be found in all cases of the disease and must not be present in healthy animals or humans.
• 2. The microbe must be isolated from the diseased animal or human and grown in pure culture in the laboratory.
• 3. he same disease must be produced when microbes from the pure culture are inoculated into healthy sus- ceptible
laboratory animals.
• 4. The same microbe must be recovered from the exper- imentally infected animals and grown again in pure culture.
• After completing these steps, the microbe is said to
• have fulfilled Koch’s Postulates and has been proven to be the cause of that particular infectious disease. Koch’s Postulates
not only helped to prove the germ theory of disease, but also gave a tremendous boost to the develop- ment of
microbiology by stressing laboratory culture and identification of microbes.
 PIONEERS IN THE SCIENCE OF
MICROBIOLOGY
• Koch’s Postulates – exception
• Circumstances do exist in which Koch’s Postulates can- not be fulfilled. Examples of such circumstances are as
follows:
• • To fulfill Koch’s Postulates, it is necessary to grow (cul- ture) the pathogen in the laboratory (in vitroc) in or
on artificial culture media. However, certain pathogens will not grow on artificial media. Such pathogens include
viruses, rickettsias (a category of bacteria), chlamydias (another category of bacteria), and the bac- teria that
cause leprosy and syphilis.Viruses, rickettsias, and chlamydias are called obligate intracellular pathogens (or
obligate intracellular parasites) because they can only survive and multiply within living host cells. Such
organisms can be grown in cell cultures (cultures of living human or animal cells of various types), embry-
onated chicken eggs, or certain animals (referred to as laboratory animals). In the laboratory, the leprosy
bacterium (Mycobacterium leprae) is propagated in ar- madillos, and the spirochetes of syphilis (Treponema
pallidum) grow well in the testes of rabbits and chim- panzees. Microbes having complex and demanding
nutritional requirements are said to be fastidious (meaning fussy). Although certain fastidious organisms can
be grown in the laboratory by adding special mix- tures of vitamins, amino acids, and other nutrients to the
culture media, others cannot be grown in the labo- ratory because no one has discovered what ingredient(s) to
add to the medium to enable them to grow.
 PIONEERS IN THE SCIENCE OF
MICROBIOLOGY

•Koch’s Postulates – exception

• To fulfill Koch’s Postulates, it is necessary to infect laboratory animals with the pathogen being studied. However, many
pathogens are species-specific, mean- ing that they infect only one species of animal. For ex- ample, some pathogens that
infect humans will only infect humans. Thus, it is not always possible to find a laboratory animal that can be infected with a
pathogen that causes human disease. Because human volunteers are difficult to obtain and ethical reasons limit their use, the
researcher may only be able to observe the changes caused by the pathogen in human cells that can be grown in the
laboratory (called cell cultures).
• • Some diseases, called synergistic infections, are caused not by one particular microbe, but by the com- bined effects of
two or more different microbes. Examples of such infections include acute necrotizing ulcerative gingivitis (ANUG; also
known as “trench mouth”) and bacterial vaginosis. It is very difficult to reproduce such synergistic infections in the
laboratory.
 PIONEERS IN THE SCIENCE OF
MICROBIOLOGY

•Koch’s Postulates – exception

• • Another difficulty that is sometimes encountered while attempting to fulfill Koch’s Postulates is that certain pathogens
become altered when grown in vitro. Some become less pathogenic, whereas others become non- pathogenic. Thus, they will
no longer infect animals after being cultured on artificial media.
• It is also important to keep
in mind that not all diseases
are caused by microbes. Many
diseases, such as rickets and
scurvy, result from dietary de-
ficiencies. Some diseases are inherited because of an ab- normality in the chromosomes, as in sickle cell anemia. Others, such
as diabetes, result from malfunction of a body organ or system. Still others, such as cancer of the lungs and skin, are
influenced by environmental factors. However, all infectious diseases are caused by microbes, as are all microbial
intoxications.
 PIONEERS IN THE SCIENCE OF
MICROBIOLOGY
• Pasteur—Fermentation
Pasteur—Disproved spontaneous generation
• Pasteur—Pasteurization
Lister—Aseptic surgery
Koch*—Germ theory of disease
• Neisser—Neisseria gonorrhoeae
Koch*—Pure cultures
Finlay—Yellow fever
Koch*—Mycobacterium tuberculosis
• Hess—Agar (solid) media
Koch*—Vibrio cholerae
 PIONEERS IN THE SCIENCE OF
MICROBIOLOGY

• Metchnikoff*—Phagocytosis
• Gram—Gram-staining procedure
• Escherich—Escherichia coli
Petri—Petri dish
Kitasato—Clostridium tetani
von Bering*—Diphtheria antitoxin
• Ehrlich*—Theory of immunity
• Winogradsky—Sulfur cycle
Shiga—Shigella dysenteriae
Ehrlich*—Syphilis treatment
• Chagas—Trypanosoma cruzi
• Rous*—Tumor-causing virus (1966 Nobel Prize)
 CAREERS IN MICROBIOLOGY

MICROBIOLOGIST
BACTERIOLOGIST
PHYCOLOGIST (ALGOLOGIST)
PROTOZOOLOGIST
MYCOLOGIST
VIROLOGIST
 MICROSCOPY

• Microscope is an optical instrument used to

magnify (enlarge) minute objects or micro-
organisms which cannot be seen by naked
eye.
 MICROSCOPY

Microscopy: Instruments
Microscopic Methods
1.Light Microscopy
1. Brightfield (light) microscopy
2. Darkfield microscopy
3. Phase-contrast microscopy
4. Fluorescent microscopy
5. Confocal
2.Electron Microscopy – Transmission– Scanning
3.Scanning Probe Microscopes
 MICROSCOPY

A. Light Microscopy

Light microscopy refers to the use of any kind of microscope

that uses visible light to observe specimens. A modern
compound light microscope (LM) has a series of lenses and
uses visible light as its source of illumination.
 MICROSCOPY

Principles of Light Microscopy: the Brightfield

Microscopy
In light microscopy, light typically passes through a specimen
and then through a series of magnifying lenses. The most
common type of light microscope, and the easiest to use, is the
brightfield microscopy, which evenly illuminates the field of
view.
 MICROSCOPY
1. compound Light Microscopy
A series of finely ground lenses forms a clearly focused image that is many times larger than the specimen
itself. This magnification is achieved when light rays from an illuminator, the light source used to illuminate
the specimen positioned on a stage, pass through a condenser, which has lenses that direct the light rays
through the specimen. From here, light rays pass into the objective lenses, the lenses closest to the speci-
men. The image of the specimen is magnified again by the ocular lens, or eyepiece. Three different objective
lenses are commonly used: low power (x10); high dry (x40); and oil immersion (x100).
 MICROSCOPY

Resolution
The limitation of brightfield microscopy is the resolution (also
called resolving power) of the image (i.e. the ability to
distinguish that two objects are separate and not one). The
resolving power of a microscope is determined by the
wavelength of light used to illuminate the subject and the angle
of light entering the objective lens (referred to as the
numerical aperture).
 MICROSCOPY

Immersion oil: Immersion oil is placed between the glass

slide and the oil immersion objective lens. The immersion oil
has the same refractive index as glass, so the oil becomes part
of the optics of the glass of the microscope. The oil enhances
the resolution by preventing light rays from dispersing and
changing wavelength after passing through the specimens. A
specific objective lens, the oil immersion lens, is designed for
use with oil; this lens provides 100x magnification on most
light microscopes.
 MICROSCOPY

• 2. darkground (darkfield) Microscopy

• Darkfield microscopy is frequently performed on the same microscope on which
brightfield microscopy is performed. Instead of the normal condenser, a darkfield
microscopy uses a darkfield condenser that contains an opaque disc. The disc blocks
light that would enter the objective lens directly. Only light that is reflected off (turned
away from) the specimen enters the objective lens. Because there is no direct
background light, the specimen appears light against a dark background. This creates a
‘dark- field’ that contrasts against the highlighted edge of the specimens and results
when the oblique rays are reflected from the edge of the specimen upward into the
objective of the microscope.
• Use
• This technique is particularly valuable for observing organisms such as Treponema
pallidum, a spirochete which cannot be observed with direct light.
 MICROSCOPY
3. Phase-contrast Microscopy
• Principle: In a phase-contrast microscope, one set of light rays comes directly from the light source. The other set comes
from light that is reflected or diffracted from a particular structure in the specimen. When the two sets of light rays—direct
rays and reflected or diffracted rays—are brought together, they form an image of the specimen on the ocular lens,
containing areas that are relatively light (in phase), through shades of gray to black (out of phase). Through the use of
annular rings in the condenser and the objective lens, the differences in phase are amplified so that in-phase light appears
brighter than out-of-phase light. The special phase condenser consists of annular diaphragms on a rotating disk fitted to the
bottom of the condenser.
• Uses
• To study unstained living cells.
• Detailed examination of internal structures in
• living microorganisms.
• To study flagellar movements and motility of
• bacteria and protozoans.
• To study intestinal and other live protozoa such
• as amoebae and Trichomonas.
• To examine fungi grown in culture.
 MICROSCOPY

• Fluorescence Microscopy
• Fluorescence microscopy takes advantage of fluorescence, the ability of
substances to absorb short wavelengths of light (ultraviolet) and give off light at a
longer wavelength (visible). If tissues, cells or bacteria are stained with a
fluorescent dye and are examined under the microscope with ultra- violet light
instead of ordinary visible light, they become luminous and are seen as bright
objects against a dark background.
• Principal Use
• The principal use of fluorescence microscopy is a diagnostic technique called the
fluorescent antibody (FA) technique, or immunofluorescence employed
for detection of antigen (direct fluorescent antibody technique) and
antibodies (indirect fluorescent antibody methods).
 MICROSCOPY

• B. ELEctron MIcroScoPY
• ∙  Electron microscopy is in some ways comparable to light microscopy. Rather than using
glass lenses, visible light, and the eye to observe the specimen, the electron microscope
uses electromagnetic lenses, electrons, and a fluorescent screen to produce the magnified
image. That image can be captured on photographic film to create an electron
photomicrograph.
• ∙  The electron beam is focused by circular electro- magnets, which are analogous to the
lenses in the light microscope. The object which is held in the path of the beam scatters
the electrons and produces an image which is focused on a fluorescent viewing screen.
• ∙  The wavelength of electrons used in an EM is 0.005 nm as compared to 500 nm with
visible light, i.e. about 100,000 times shorter than that of ordinary light. Theoretically, the
resolving power of the EM should be 100,000 times (reso- lution down to 0.0025 nm). In
practice, the best resolution that can be obtained is 0.3–0.5 nm, a hundred times better
than that of the light microscope.
 MICROSCOPY

•types of Electron Microscopes

•There are two types of electron microscopes in general use:
• (i) Transmission Electron Microscope (TEM)
•In transmission electron microscope (TEM), electrons like light pass
directly through the specimen that has been prepared by thin
sectioning, freeze fracturing, or freeze etching. It is used to observe
fine details of cell structure.
• (ii) Scanning Electron Microscope (SEM)
•A scanning electron microscope scans a beam of electrons back and
forth over the surface of a specimen producing three-dimensional
views of the surfaces of whole microorganisms.
 MICROSCOPY

•c. Scanning – Probe Microscope

•Scanning probe microscopes map the bumps and valleys of a surface
on an atomic scale. Their resolving power is much greater than the
electron microscope, and the samples do not need special
preparation as they do for electron microscopy. Among the new
scanned-probe microscopes are: 1. Scanning tunneling
microscopy (STM): There are used to provide incredibly detailed
views of molecules, such as DNA.
2. Atomic force microscopy (AFM): These produce
three-dimensional images of the
surface of a molecule.
 MICROSCOPY

• Microscopy: A simple microscope consists of one lens; a compound microscope has multiple lenses.
• compound Light Microscopy
• ™ The most common microscope used in microbiology is the compound microscope/bright field microscope/light microscope.
• ™ Darkfield microscopy: It is most useful for detecting the presence of extremely small organisms.
• Phase-contrast microscopy: It allows the detailed observation of the living organisms.
• ™ Fluorescence microscopy: Fluorescence microscopy is used primarily in a diagnostic procedure called fluorescent-antibody
(FA) technique, or immunofluorescence.
• ™ Electron microscopy: Electron microscopes use electromagnetic lenses instead of glass lenses, electrons and fluorescent
screens to produce a magnified image. There are two types (i) Transmission electron microscopes (TEMs); and (ii) Scanning
electron microscopes
• ™ Scanned-probe microscopy: Their resolving power is much greater than the electron microscope.
 CULTURE MEDIA

• Culture medium: A nutrient material prepared for the growth of microorganisms in a laboratory is called a culture
medium.
• COMMON INGREDIENTS OF CULTURE MEDIA
• Water:
• Agar:
• Peptone:
Meat extract is also available commercially and is known as Lab-Lemco.
• Yeast extract:.
• Malt extract:
• Blood and serum:
 CULTURE MEDIA

CLASSIFICATION OF MEDIA
• Media have been classified into many ways
• A. Phases of Growth Media
• Growth media are used in either of the two phases: liquid (broth) or solid (agar).
• 1. Liquid (Broth) Media
• In broth media, nutrients are dissolved in water, and bacterial growth is indicated by a change
• in broth’s appearance from clear to turbid (i.e. cloudy).
• 2. Solid (Agar) Media
• Solid media are made by adding a solidifying agent to the nutrients and water. Agarose is the
most common solidifying agent. The Petri dish containing the agar is referred to as agar.
• 3. Semisolid Media
• For special purposes where agar is added to media in concentrations that are too low to
solidify them.
 CULTURE MEDIA

•B. Based on Nutritional Factors

•1. Simple media (Basal media): Simple media are those which
contain only basic nutrients required for the growth of ordinary
organisms, and used as a general purpose media, e.g. peptone water,
nutrient broth and nutrient agar
•2. Complex media: Media that contain some ingredients of
unknown chemical composition are called complex media. One
common ingredient is peptone. Extracts, which are the
water-soluble components of a substance, are also used.
•Nutrient broth: A commonly used complex medium, nutrient
broth (in liquid form). It is a simple basal liquid medium, supports
growth of many organisms.
 CULTURE MEDIA

• Types of Nutrient Broth

• There are three types of nutrient broth:
1. Meat infusion broth; 2. Meat extract broth; 3. Digest broth.

• Nutrient Agar
• Nutrient agar is prepared by adding agar at a concentration of 2% to the nutrient broth.
It is simplest and most common medium used routinely in microbiology laboratories, to
grow nonfastidious bacteria nutrient agar is commonly referred to as “agar medium”.
• Semisolid agar: If the concentration of agar is reduced to 0.2–0.5%, semisolid or
sloppy agar is obtained which enables motile organisms to spread but not non-motile
bacteria.
• Firm agar: If the concentration of agar is increased to 6%, it is called firm agar.
• 3. Synthetic or Chemically Defined Media
• They are prepared exclusively from pure chemical substances and their exact
composition is known.
CULTURE MEDIA
 CULTURE MEDIA

• C. Special Media
• These are prepared to meet the nutritional requirements of more exacting bacteria by the addition of substances such as
blood, serum or egg to a basal medium.
• Fig. 6.1: Nutrient agar Examples of Enriched Media
• 1. Blood agar (Fi: Many medically important bacteria are fastidious, requiring a medium that is even richer than nutrient agar
commonly used in clinical laboratories is blood agar. Blood agar is used for isolation of streptococci, pneumococci,
Haemophilus
• 2. Chocolate agar (Fig. 6.3): A medium used to culture even more fastidious bacteria is chocolate agar, (heated blood
agar). It is used for isolation of Neisseria (meningococci and gonococci) and Haemophilus.
CULTURE MEDIA
 STAINING METHODS

B. Staining Reactions
A number of staining techniques for the identification of bacteria, are available. Of
these, Gram stain and Ziehl-Neelson stain are most important.
Gram stain: The Gram stain divides bacteria into gram positive and gram
negative.

Ziehl-Neelsen staining: With Ziehl-Neelsen staining divides bacteria into acid

fast and non-acid fast. Numerous other stains are used for special purposes, such
as demonstration of flagella, capsule, spores, and metachromatic granules. The
fluorescent antibody technique enables one to
identify them according to their surface antigens.
 STAINING METHODS

Staining simply means coloring the microorganisms with a dye that

emphasizes certain structures. Before the microorganisms can be
stained, however, they must be fixed (attached) to the microscope
slide. Fixing simultaneously kills the microorganisms and fixes them to
the slide. It also pre- serves various parts of microbes in their natural
state with only minimal distortion.