Chapter 1 All of the following may be

considered Microorganisms
Scope of Microbiology  Bacteria (eubacteria, archaebacteria)
 Fungi
 Protozoa
Microbiology  Microscopic Algae
 Study of Micro-organism,  Viruses
 must be viewed individually with the aid  Various Parasitic Worms
of a Microscope.
 Is the study of Microbes. Microbiology
 Germs are the microbes that cause
 Study of microorganism
Disease.
 Found in Modern Biotechnology
 Micro means very small anything so
 Among the many Specialized fields of
small that it must be viewed with a
microbiology
Microscope (an optical instrument used
to observed very small objects)
Virology, Mycology, Bacteriology, Immunology,
 Microbes are mostly beneficial or have a
Microbial Ecology, Biotechnological
neutral effect on our lives.
Microbiology,
 Microbiology is the scientific study of Environmental Microbiology, Food Microbiology,
these microorganisms. Microorganisms Forensic Microbiology, Molecular Biology.
are those organisms that are too small to
see with the naked eye and include
things like Bacteria, fungi and Viruses. Microorganism And
Microbiology Cont’d
1. EXIST ( Webster) to continue to be, have life,
live HALLMARKS OF LIFE
Two Main Themes involved in Microbiology
Metabolism ( Nutrient uptake, biomass, waste
output)
Differentiation (Bacillus spp. Caulobacter) 1. Basic – Cellular Process
Reproduction (Binary fission) 2. Applied – concerning agriculture,
Communication ( Pseudomonas Aeruginosa) industry and health
Evolution ( Antibiotic resistance, Pathogen)
The Two major categories of microbes
Small Microorganism include: are called:
 Bacteria
 Acellular Microbes also called
 Algae
Infectious Particles (Virus and Prions)
 Protozoa
 Fungi  Cellular Microbes also called
 Viruses Microorganism ( Bacteria, all Archae,
all Protozoa and some Fungi)
Often called: Microbes that cause disease are known as
 Microbes Pathogens ( disease causing microorganism )
 Single-celled organism 3% those that do not cause disease are called
 Germs Non-Pathogens ( microbes that’ do not cause
disease) majority of known microbes, some
Microorganism and Non-pathogens are beneficial to us. Microbes
that help us microbial allies and those that harm
Microbiology us is Microbial Enemies.

Microorganism

 Living things which individuals are too

small to be seen with naked eye.

Themes in Microbiology and

its Field
 Microbiology disease and improvement of yields.
Basic
By organism – Bacteriology, Phycology,
Aquatic Microbiology
Myocology, Virulogy, Parasitology, Protozoalogy.  Aquatic microbiology is the study of
By Process – Microbial Metabolism, Microbial microorganism and their activity in the
Genetics fresh and marine water including lakes,
Disease related – Immunology, Epidemiology, rivers, bays, estuaries and seas.
Etiology.  It also includes water purification,
microbiological examination and
Applied biological degradation waste.
Disease related – Infection control,
Chemotherapy
Environmentally related – Environmental
Air Microbiology
Microbiology  It deals with the role of aerosphere in
Industrial – Food and Beverage, Tech contamination and spoilage of food
Pharmaceutical microbiology, Genetic  It also deals with the spread of plant and
Engineering animal diseases through air.

Epidemiology
Scope of Microbiology  It is concerned with the monitoring,
( Branches ) control and spread of disease in
communities.
1. Pure Science
Bacteria – Bacteriology
Algae – Phycology Microbes in our Lives
Fungi – Mycology
Protozoa – Protozoology  Some are pathogenic ( disease- causing)
Parasites – Parasitology  Decompose organic waste
Genetics – Study of heredity and variations  Produces through photosynthesis (e.g
involved in the development ofresistance by Purple sulphur bacteria must fix CO2 to
body to infectious disease. Live)
Virus – Virology
 Play role in industry (Fermentation to
produce ethanol and acetone)
Medical Microbiology  Produce fermented food (vinegar,cheese
and bread)
 It deals with the study if causative  Produce production used in
agents of infectious diseases in human manufacturing ( cellulase) and treatment
beings. (Insulin)
 Medical Microbiology has close links
with other disciplines such as pathology,
clinical medicine, pharmacology and
Microorganism and Food
Therapeutics
Microorganism and Food
Phamaceutical Microbiology 1. Prevent Spoilage ( tempeh, Salted fish)
2. Assist in manufacturing of food
 It deals with the study of
microorganism which are responsible Microorganism and Energy
for the production of antibiotics, 1. Natural Gas (Methane)
enzymes, vaccines, vitamins, and other 2. Ethanol (Biofuel)
pharmaceutical substances 3. Bioremediation
 Also it includes the method of
sterilization and disinfection, Microbes and the Future
microbiological testing of 1. Genetic Engineering
pharmaceuticals, sterile product Microbes and Human Disease
preparation and diagnosis of disease  Bacteria were once classified as plants
and treatment. which gave rise to use of the term Flora
for Microbes
Agricultural Microbiology  This term has been replaced by
 It is the study of relationship between Microbiota
microorganism and crops with on  Microbes normally present in and on the
emphasis on the control of the plant human body are called Normal
 Microbiota
1. Eukaryotes (pre-nucleus) Simple cells
Introduction to Microbiology Organisms with a true nucleusand more
 Organisms that make up the Microbial complex e.g Algae, Fungi, and Protozoa
World 2. Prokaryotes (true nucleus)
 Because of their characteristics, Complex cells
microorganisms join all other living Organisms without true nucleus and less
organisms in 2 major groups complex cells e.g Archae and Bacteria

 Prokaryotic Note: Viruses are microbes but theyare not

 Eukaryotic considered to be cells because they are acellular,
hence they are not included on the family tree.

Prokaryotes Characteristics Classification of Bacteria

Domain
Size – 0.2-2 in diameter
Nucleus – none  Bacteria
Organelles with Phospholipid membrane -  Archaea
None  Eukarya
Glycocalyx – capsule (organized)
Slime layer (unorganized) Traditional Whittaker Classification
Motility – rotating Flagella (some)
Flagella - some Five Kingdom
Cillia - none  Prokaryotae ( Monera)
Fimbriae & Pill - some  Protista – Eukaryotic, unicellular,
Cell Wall – most, bacteria (peptidoglycan) multicellular
Plasma membrane – lacking carbs and sterols  Fungae – multicellular,eukaryotic
Cytosol - all  Plantae - multicellular,eukaryotic
Inclusions - all  Animalia - multicellular,eukaryotic
Endopores - some Based on:
Ribosomes – cytoplasm (70s)
 Morphology
Chromosomes – single,circular,lack histones
 Metabolism ( Biochemical Activity)
Eularyotes Characteristics  Molecular Techniques
Fatty Acid Profiles
Size – 10-100 in diameter Protein Differentiation
Nucleus – all DNA Finger Printing
Organelles with Phospholipid membrane – Er,
Golgi bodies, Lysosome, Mitochondrials, Taxonomy: Naming,Classifying and
Chloroplast Identifying Microorganism
Glycocalyx – surround some animal cells
Motility – undulated Flagella & Cillia (‘9+’2)  Microbial Nomenclature – naming
arrangement microtubes others by amboid action microorgani
Flagella - some  Taxonomy – classifying living things
Cillia - some  Identification – discovering and
Fimbriae & Pill - none recording the to be named and
Cell Wall – most classified.
protein,cellulose,algin,agar,carrageenan,silicate, Scientific Names
glucomanna.chitin
Plasma membrane – has glycoproteins,
 Italicized or Underlined
glycolipids sterols
The genus is capitalized, and the specific
Cytosol - all
epithet is with lower case
Inclusions - all
 Could be as an honor for a scientist
Endopores – none
Ribosomes - Cytoplasm (80s)  A latin origin
mitochondria &chloroplast (70s) e.g Escherichia coli ( E.coli) - In
Chromosomes - more than one, linear, conatain Intestine
histones discoverer: Theodor Escherich
disribes the habitat ( colon/intestine)
Family Tree of Microorganism: e.g Staphylococcus aureus (S.aureus) –
Primitive cells are divided into On Skin
 - Clustered (staphylo), spherical (cocci) reproduction
- Gold colored colonies (aureus) 3. Obligate Anaerobe – anaerobe that
grows only in an anaerobic environment
Microbial Structure (no oxygen)

Two cell lines 4. Aerotolerant Anaerobe – does

Prokaryote – microscopic,unicellular not require oxygen and grows better on
organism, lack nuclei and membrane bound the absence of oxygen but can survive in
organelles atmosphere containing molecular
Eukaryote – unicellular (microscopic) and oxygen (room air)
multicellular, nucleus and membrane bound 5. Facultative Anaerobe – capable
organelles. of surviving in either the presence or
Viruses – Acellular, parasitic particles composed absence of oxygen, anywhere from
of nucleic acid and protein. 0%noxygen to 20-21% oxygen.
6. Caprophytes – bacteria that grow
better in the presence of increase
Microbial Classification concentrations of carbon dioxide.
Bacteria (s.bacterium) are a type of biological cell.
Morphology – size,shape, and cell arrangement

Shapes
1. Spherical or coccoid bacteria – COCCI
(s.coccus)
2. Rod – shaped bacteria BACILLI
(s.bacillus)
3. Curved or spiral shaped – SPIRILLI

Arrangement: COCCI
1. Diplococci – Pairs
2. Streptococci – Chains
3. Staphylococci – Clusters
4. Tetrads – Packets of four
5. Sacrinae – Packets of eight

Arrangement: BACILLI

1. Pairs - Diplobacilli
2. Chains - Streptobacilli
3. Long Filaments or Branched
4. Short – Coccobacilli or resembled
5. Curved shaped or Comma shaped
e.g Vibros – Vibro cholera
6. Spiral shaped
Classification of bacteria on BRIEF HISTORY OF
the basis of their MICROBIOLOGY
Relationship to Oxygen and
Carbon Dioxide DEVELOPMENT OF
MICROBIOLOGY

1. Obligate Aerobe – requires an • Aristotle and other believed that living

atmosphere containing oxygen in organism could develop from non-loving
concentration comparable top that
materials. (384-322)
foundn in room air (20-21% oxygen)
e.g mycobacterium and certain fungi
Microaerophilic aerobe – requires • Hans and Zacharias Janssen
oxygenlower than that found room air mounted two lenses in a tube to produce the first
(5% oxygen) compound microscope. (1590)
2. Anaerobes – organisms that do not
requires oxygen for life and
• Robert Hooke published
Martinus Beijernick (1884-1885)
Micrographia drawings and detailed
• Discovered “viruses” (toxins and poisons)
observations of biological materials made with the
• Infectious agents in tobacco plant fluids
best compound microscope and illumination
system of the time. (1660)
Paul Ehrlich (1910)
• Introduced concept of chemotherapy
• Anton van Leeuwenhoek the very • Use of salvarsan for treatment of syphilis
first person to observe microorganism (1676) Alexander Fleming (1928)
• Discovered the first antibiotic (penicillin)
• Carl Zeiss and Ernst Abbe
pioneered developments in microscopy (such as
immersion lenses and apochromatic lenses which PROOF THAT MICROBES
reduce chromatic aberration) exist until the present
day. (1883)
CAUSE DISEASE

1546: Hieronymus Fracastorious

• Ernst Ruska constructed the 1sr electron (Girolamo Fracastoro) wrote “On Contagion” the
microscope (1931)
Ist known discussion of the phenomenon of
contagious infection.
SPONTANEOUS GENERATION
CONTROVERSY 1835: Agostino Bassi de Lodi showed that
a disease affecting silkworms was caused by
fungus – the first microorganism to be recognized
• (1688) Francesco Redi was an as a contagious agent of animal disease.
Italian physician who refuted the idea of
spontaneous generation by showing that rotting 1847: Ignaz Semmelweiss (1818-1865) a
meat carefully kept from flies will not Hugarian physician who decided that doctors in
spontaneously produces maggots. Vienna hospitals were spreading childbed fever
• (1836) Theodore Schwann while delivering babies. He started forcing doctors
helped developed the cell theory of living under his supervision to wash their hands before
organisms, namely that all living organisms, touching patient.
namely that all living organisms are composed of
one or more cells and that the cell is the basic
functional unit of living organisms.
• (1861) Louis Pasteur’s famous 1857 : Louis Pasteur proposed the Germ
experiments with swan necked flasks finally Theory of Disease, ancients believed that disease
proved that microorganisms do not arise by was the result of a divine punishment. Pasteur
spontaneous generation. fought to convince surgeons that germs existed
THE GOLDEN AGE OF and carried diseases, and dirty instruments and
MICROBIOLOGY (1857-1914) 50 hands spread germs and therefore disease.
Pasteur’s pasteurization killed germs and
YEARS OLD prevented the spread of disease.

Beginning with Pasteur’s work, discoveries 1876 : Robert Koch (1843-1910) a German
included relationship between microbes and bacteriologist and was the first to cultivate anthrax
disease, immunity and antimicrobial drugs. bacteria outside the body using blood serum at
body temperature.
Robert Koch
• Identified a bacterium as cause of anthrax
• Introduced agar, inoculating loop to transfer
KOCH’S POSTULATES
bacteria and prepare cure cultures
• Introduced “Koch’s Postulates” and the concept Robert Kotch demonstrated the first direct role of
that a disease is caused by a single organism a bacterium in disease.

Joseph Lister (1865) Koch’s postulate (1884) the critical test for
• Introduced the “antiseptic technique” the involvement of a microorganism in a disease.
• Use phenol (carbolic acid) as disinfectant
1. The agent must be present in every case of the that give the bacterium some advantage over
disease. other bacteria. For example, it may contain a
2. The agent must be isolated and cultured in vitro gene that make the bacterium resistant to a
3. The disease must be reproduced when a pure certain antibiotic.
culture of the agent is inoculated into a susceptible
host. BACTERIA are classified into five
4. The agent must be recoverable from the groups according to their basic shapes:
experimentally infected host.
1. Spherical (COCCI)
This will eventually lead to: 2. Rod (BACILLI)
• Development of pure culture techniques 3. Spiral (SPIRILLA)
• Stains, agar, culture media, petri dishes. 4. Comma (VIBRIOS)
5. Corkscrew (SPIROCHAETES)
MICROORGANISM AND
DIVISION OF MICROBIOLOGY They can exist as single cells, in pairs, chains
or clusters.

What is virus? • Bacteria are found in every habitat on Earth,

• A virus is another microorganism that caused soil, rock, oceans, and even arctic snow.
diseases Some live in or one other organism including
• A virus is even smaller than bacteria, it must plants and animals including humans. (the
use a living cell to grow and reproduce rest is nasa ppt, basahin niyo na lang doorn)
• A virus can cause colds
RUDIMENTARY FORMS
VIRUS OF BACTERIA
• A virus is not a cell. A virus are replicated • Rickettsia, Chlamydia, and Mycoplasmas
only when they are in a living host cell. are bacteria but they do not possess all of the
• Consist of DNA or RNA core attributes of typical bacterial cells, they are
• Core is surrounded by a protein coat also small and difficult to isolate.
• Coat may be enclosed in a lipid envelop. CHARACTERISTICS

1. Coccoid, rod shaped or pleomorphic

VIRAL INFECTIONS are usually limited
(irregular)
by the body defense: phagocytes and
2. Gram-negative
INTERFERON antiviral proteins produced
3. Obligate intracellular parasite – a
by virus infected cells.
parasite that must live within a host cell.
BACTERIOPHAGES viruses that infect
bacteria or phages.
VIROID and PRIONS smaller than viruses FUNGI
and less complex infectious agents. Viroid
infect plants and Prions infect livestock or • Fungi exist in either yeast or mold forms.
humans. • The smallest of yeasts are similar in size to
bacteria, but most are larger (2 to 12 um) and
What are bacteria? multiply by budding.
• Bacteria are a large group of unicellular • Molds form tubular extensions called hyphae,
microorganism. Typically a few micrometers which, when linked together in a branched
in length. network, form the fuzzy structure seen on
• Bacteria have a wide range of shapes, neglected, bread.
ranging from spheres to rods and spirals. • Fungi are eukaryotic, and both yeasts and molds
• Bacteria are microbes with a cell structure have a rigid external cell wall composed of their
simpler than that of many other organisms. own unique polymers, called glucan, mannan, and
They control center, containing the genetic chitin. Their genome may exist in a diploid or
information, is contained in a single loop of haploid state and replicate by meiosis or simple
DNA. Some bacteria have an extra circle of mitosis.
genetic material called a plasmid rather than • Most fungi are free-living and widely distributed
a nucleus. The plasmid often contains genes in nature.
• Generally, fungi grow more slowly than bacteria, chain as they release large amounts of
although their growth rates sometimes overlap. oxygen on the planet.
• The fungi probably represent an evolutionary o Algae are Photosynthetic
offshoot of the protozoa; they are unrelated to the o Algae could be Multicellular or
actinomycetes, mycelial bacteria that they unicellular
superficially resemble. o Algae arrange in colonies which are
• The major subdivisions (phyla) of fungi are: found in water and soil Algae is an
Chytridiomycota, Zygomycota (the zygomycetes), important source of food
Ascomycota (the ascomycetes). Basidiomycota o Algae damage water system by clogging
(the basidiomycetes), and the "deuteromycetes" filter and piper In microbiology the roles
(or imperfect fungi). of mutation and selection in evolution
are coming to be better understood
Singular : Fungus through the use of bacterial cultures of
mutant strains (Edward Lawrie Tatum)
1.
Eukaryotes
2.
Chitin cell walls In microbiology the roles of mutation and
3.
Use organic chemicals for energy selection in evolution are coming to be better
4.
Molds and mushrooms are multicellular, understood through the use of bacterial cultures
consist of mycelia (composed of of mutant strains (Edward Lawrie Tatum)
filaments called hyphae)
5. Yeasts are unicellular
6. Found anywhere on earth
7. Maybe harmful or beneficial BASIC MICROBIOLOGY AND
8. Live on unlikely materials like leather EQUIPMENT AND
and plastics PROCEDURES IN THE STUDY
9. Beneficial fungi are important in the
OF BACTERIA
production of cheese, yoghurt, beer,
wine and drugs and antibiotics.
What is PPE (Personal Protective
CHARACTERISTICS
Equipment?)

1. Eukaryotic and includes mushroom, molds and  PPE is a precautionary step to protect
yeast. yourself and the people around you.
2. Saphropytes which is the main source of food in  PPE is specialized clothing or equipment
dead and decaying organic matter.’ worn for protection against dangerous or
3. Yeast helps in fermentation process of infectious materials
wine/beer and used to leaven light bread.  PPE prevents contact with an infectious agent
by creating a barrier between the potentially
Eg. Pathogenic yeast such as candida infectious material and the public health
albicanscandidiasis. practitioners.

Algae are any of the various groups of PPE EQUIPMENTS

chlorophyll-containing. mostly eukaryotic MASK
organisms that range from infinitesimal single-
celled forms to multicellular kinds 100 feet (30
meters) or longer. Algae are mainly distinguished  Place over nose, mouth, and chin
from plants because they lack genuine roots,  Standard Mask used when there is risk of
stems, and leaves and have an absence of non- exposure to droplets containing infectious
reproductive cells in the reproductive structures. agents
 N95 Masks used when there is risk of
CHARACTERISTICS exposure to airborne infectious agents such
as M. Tuberculosis (Prevent 95% of particles
less)
o Algae are characteristically autotrophic
since they derive their energy and food
LAB ATTIRE
from their environment in the form of
 PPE: Hand Protection
sunlight.
o Gloves are required while you work with
o Algae play a significant role in the food
chemical or biological agents in the lab.
o Use the gloves provided to you in the lab, immune system lowers down and is always at
they are compatible with agents you work risk of being affected by any microbe, can
with. remain safe and healthy when he is in the
o Don’t touch face with gloves hands of a nursing professional.
o You will be asked ti leave if you do not abide  Microbiology helps a nursing professional to
by safety protocol. understand the basic concepts of
reproduction, morphology, biochemical
characteristics and genetics. Microbiology
TYPES OF PPE
makes aware about new diseases and modern
o Direct-Ventilated: Resist direct passage
molecular identification methods. The role of
of large particles into the goggle. Prevents
microorganism in development of certain
fogging by allowing air circulation.
medications and vaccinations cannot be
o Indirect-Ventilated: Prevents fogging
ignored either.
by allowing air circulation. Protects against
 To prevent spread of infection; Nurses should
liquid or chemical splash entry,
have knowledge about the mode of spread of
o Non-Ventilated: Does not allow the
infection some of the infections are spread by
passage of air into the goggle. Prevents
contact (touch), air (air-borne), droplets
splash entry. May fog and require frequent
(sneezing, coughing). Some by eating
lens cleaning.
contaminated food or drink (food borne),
sexual contact (STDs), by arthropod bite
(vector born) and others by contaminated
PERSONAL SAFETY (CLOTHING)
blood transfusion, etc.
o Wear: Gloves, Long Lab Coat, Goggles,
 This knowledge would help a nurse to look
Closed-Toed Shoes, Tie back long hair. for specific control of spread of infection. If a
o Do Not Wear: Sandals, Jewelry, Loose or nurse knows how the disease producing
Baggy Clothing, Contact Lenses organism to enter into the body, how they are
discharged from the body and how they
 LABORATORY SAFETY spread from person to person, the knowledge
o Correct dispose of biohazardous waste would help a nurse to use particular measures
(double bag) to save community and hospital spread of
o Causes of infectious airborne transmission: infection e.g. in tuberculosis case a negative
 Removing rubber stoppers pressure room works in a positive way for
 Splashing during transfer of blood or other patient health.
body fluids
 Centrifuging without covering with HOW DO WE VIEW
biological hood MICROORGANISM?
 Not wearing a proper face shield when
working with specimens Units of measurement
 Exposure to sharps, such as needles and When talking about cells and microscopic
lancets organisms, you would be measuring using
MICROMETRE (abbreviated: p --micron) or
THE IMPORTANCE OF BEING
stated as: um (micrometer).
FAMILIAR WITH LABORATORY
1 μm = 1 x 10 meters/ 1 x 10 mm
EQUIPMENT AND THEIR USES
1 mm 1 x 10' nanometers/ 1 x 10' pm
To give you the idea of how small a micro
 Microorganisms as the name suggests are metre is.
microscopic organisms which have an ability 1- a human hair is about 100 um, wide,
to adapt according to environment for 2- a red blood cell would be around 8 um wide
instance, commensals change pathogenic in 3- typical size of an animal cell would be from 10-
certain changed environment. 100 μm
 The adaptation of microorganisms to certain
treatments or medicines remains a challenge MICROSCOPE
for a healthcare professional like a nurse. Light microscope
Microbiology touches life of a nurse wild Uses light
he/she works in a medical health care setting.
Microbiology helps a healthcare professional Few types
to have better understanding of these
 Compound Light Microscopy
microorganisms so that a patient whose
 Darkfield Microscopy
 Phascontrast Microscopy MICROSCOPE
 Differential Interference Contrast
Microscopy  RESOLVING POWER
 Fluorescence Microscopy o The limit of resolution of a microscope is the
 Confocal Microscopy smallest distance between 2 points that can
be seen using a microscope
PURPOSE OF MICROSCOPE o This is a measure of the clarity of the image
o A microscope with a high resolving power
 Bacteria is one of the small microorganisms will allow 2 small objects whichare close
that can't be seen with the naked eyes, most together to be seen as 2 distinct objects.
bacteria range in size between 0.5-2.0 MICROSCOPY: THE
micrometers (um) so, there is a need to INSTRUMENTS
magnify the bacteria several times by using a
microscope in order to see it.
 Refractive Index: is the light-bending
 There are different types of microscopes ability of a medium.
which are used in microbial life.
 The light may bend in air so much that it
 In this lab, you will become familiar with the misses the small high-magnification lens
use of the light compound microscope
 The refractive indexes of oil and glass are
(particularly oil immersion microscopy) and
similar
will compare the relative size and shape of
 Immersion Oil is used to keep light from
various microorganisms.
bending.
COMPOUND LIGHT MICROSCOPE
Depth of Field: the range of distance at the
The image is magnified again by ocular lens
specimen parallel to the illuminating beam in
which the object appears to be in focus.
TOTAL MAGNIFICATION =
OBJECTIVE LENS X OCULAR Depth of Focus: the range of distance at the
LENS image plane (i.e. the eyepiece, camera, or
photographic plate) in which a well-focused object
 Resolution- ability of lenses to distinguish appears to be in focus.
two points c.g. RP of 0.4 nm can distinguish
between 2 points ≥ 0.4 nm Consider the following in the
 Shorter light wavelength provides greater
resolution
use of the Microscope
 Refractive index- Light bending ability of a
medium ED SH  The light microscope utilizes a
 Light may bend in air that it misses the small convex lenses to magnify the image
high-magnification lens  Lenses are used to focus the image in a
 Immersion oil is used to keep the air from compound light microscope.
bending.  The light microscope has a greater
resolving power.
TERM MAGNIFICATION  The size of a virus is less than 1000 nm and
is best viewed using a microscope with a
 Ratio of image size to actual size large resolution.
 Ocular: 10X .  The total magnification of an object viewed
("X" means times; for example, the ocular under the light microscope is derived by
multiplying the magnification of the ocular
magnifies something 10 times)
and the objective.
4 objectives  The CONTRAST in a phase-contrast
1. Scan: 4X microscope is due to the scattering light by
2. Low Power: 10X the opaque ring in condenser.
3. High Power: 40X  A DARK FIELD MICROSCOPE produces
4. Oil immersion: 100x an image where the object is bright against a
 Multiply ocular x objective to get overall dark background and is useful in viewing
magnification unstained specimens.
 FLUORESCENCE MICROSCOPES work
NUMERICAL APERTUR OF A by observing the emission of light from the
 specimen.
 The FINE ADJUSTMENT KNOBS should
not be used when focusing a specimen under
high power and oil immersion objectives.
 DIAPHRAGM
o It is located on the lower surface of the
stage. It is used to control the amount
TYPES OF MICROSCOPE
of light that reaches the specimen
through the hole in the stage.
 Light Microscope - found in most
 ILLUMINATOR
schools, use compound lenses and light to
o Simple compound microscopes have a
magnify objects. The lenses bend or refract
mirror that can be moved to adjust the
the light, which makes the object beneath
amount of light that can be focused on
them appear closer.
the specimen. However, some
 Stereoscope this microscope allows for
advanced types of compound
binocular (two eyes) viewing of larger
microscopes have their own light
specimens. (The spinning microscope at the
source.
top of this page is a stereoscope)
 THE ADJUSTMENT
 Scanning Electron Microscope allow
o 2 adjustment knobs: fine adjustment &
scientists to view a universe too small to be
course adjustment knob: refine the
seen with a light microscope. SEMs do not
focus of the lenses. The course
use light waves; they use electrons
adjustment knob helps in improving
(negatively charged electrical particles) to
the focus of the low powers whereas
magnify objects up to two million times.
the fine adjustment help in adjusting
 Transmission Electron Microscope
the focus of the lenses with higher
also uses electrons, but instead of scanning
magnification
the surface (as with SEM's) electrons are
passed through vers thin specimens.
Specimens may be stained with heavy metal MAGNIFICATION
salts
Your microscope has 3 magnifications:
PARTS OF MICROSCOPE
Scanning. Low and High. Each objective will have
written the magnification. In addition to this, the
 EYEPIECE
ocular lens (eyepiece) has a magnification.
o To observe specimen. Contain two or
more lenses. The most common
The total magnification is the ocular x
magnification for the eyepiece is 10X.
objective.
There are also 2x and 5x. An eye piece
is a removable, can be interchanged for
TOTAL MAGNIFICATION =
different magnification
MAGNIFICATION OF EYEPIECE X
 OBJECTIVE LENSES MAGNIFICATION OF OBJECT LENSE
o More than one objective lenses. They
are the primary lenses of a compound STAINING
microscope and can have magnification Device to examine bacteria smeared on a slide and
of 4x. 5x, 10, 20s, 40s. 30% und 100s allowed to air dry. Specific stains and techniques
 STAGE are used to observe bacterial morphology.
o The platform below the objective lens
on which the object in he viewed is PREPARING SMEARS FOR STAINING
placed. A hole in the stage allows light  Staining- coloring microbe with a dye to
ham to pass and illuminate the emphasize certain structure
specimen.
 Smear- A thin film of a microbe solution
 STAGE CLIPS on a slide, a smear is usually fixed to attach
o These are two stage clips one on each microbes to the slide and kill microbes.
side of the stage. Once the slide  STAINS are classified broadly as basic,
containing the specimen is placed on acidic, or neutral stains. The chemical nature
the stage, the stage clips are used to of the cells under examination determines
hold the slide in place which stain is selected for use.
 CELL STAINING is important in the STAIN)
diagnosis of microorganisms because bacteria 4 STEPS IN GRAM STAINING
can be identified by the color differentiation PROCESS
of stains (dyes).
 Flood the fixed and air dried smear with
Crystal Violet (a purple dye) for 30 minutes
SIMPLE STAIN  Rinse gently with water and cover the smear
 Staining with one dye. Mordant may be used with Grams Iodine Solution
to hold the stain or to coat the specimen to  After 30 seconds, wash off iodine with water
enlarge it. and decolorize with Ethanol
GRAM STAIN  Counterstain with Safranin (a bright red dye)
 Distinguished for 1 minute, rinse, dry and examine under
 Gram +ve and gram -ve oil.
 Gram +ve bacteria are prone to penicillin and
detergents GRAM STAIN TECHNIQUES
 Gram-ve are more resistant to antibiotics  Prepare bacterial smear on the clean slide.
 Pass the slide through over the flame 2-3
DIFFERENTIAL STAIN times. (Heat fixing)
 Distinguish  Apply Crystal Violet (Primary stain) on
 Gram stain smear for 1 minutes & rinse with water.
 Acid-fast stain  Apply Gram's iodine (Mordant) for 1 minute
& wash with water.
ACID-FAST STAIN  Then wash with 95% alcohol (Decolorizer)
Stained waxy cell wall is not decolorized by acid- for 10-20 seconds & rinse with water.
alcohol  Apply Safranin (Secondary stain) for 1
• Mycobacterium minute & wash with water.
• Nocardia  Air dry, Blot dry & Observe under
Microscope.
SPECIAL STAIN
 Distinguish special parts of cells RESULT:
 Capsule  Gram Positive bacteria retain the purple color
 Endospore (Malachite green and safranin) of the crystal violet
 Flagella (carbolfuchsin . simple stain)  Gram Negative purple color is removed by
the alcohol and the cells are stained red by
THE GRAM STAIN is the most important safranin.
staining procedure in microbiology. It is used to  Neither purple nor red-gram variable bacteria
differentiate between gram positive organisms and  eg. Mycobacterium Tuberculosis and
gram negative organisms. Hence, it is a Tuberculosis Leprae
differential stain. Gram negative and gram
positive organisms are distinguished from each ACID FAST STAIN
other by differences in their cell walls.  The acid-fast stain is a differential stain used
to identify acid-fast organisms such as
members of the genus Mycobacterium. Acid-
TYPES OF STAINING
fast organisms are characterized by wax-
TECHNIQUES like, nearly impermeable cell walls; they
contain mycolic acid and large amounts of
 Simple Staining use of single stain fatty acids, waxes, and complex lipids.
o for visualization of morphological  Carbol Fuchsin (a bright red dye) is driven
shape and arrangement to the bacterial cell wall with heat so that
 Differential Staining use of two decolorizing agent (a mixture of acid and
contrasting stains separated by a decolorizing alcohol) does not remove the red color from
agents mycobacteria, they are said to be ACID-
FAST.
o Identification ( GRAM STAIN
AND ACID FAST STAIN)
o Visualization of Structure
(SPORE STAIN AND CAPSULE BACTERIAL GROWTH
 Early civilizations practiced salting, smoking,
Motility prickling, drying, and exposure of food and
clothing to sunlight to control microbial growth.
 Ability of an organism to move by itself. Use of spices in cooking was to mask the taste of
 Bacterial motility is associated with flagella spoiled food. Some spices prevented spoilage. In
(whip-like organelle of motility) or axial mid 1800s Semmelweis and Lister helped
filaments. developed aseptic techniques to prevent
 Most spiral-shape bacteria and ½ of the contamination of surgical wounds.
bacilli are motile but cocci are generally non Before then: Nosocomial infections caused death
motile. in 10% of surgeries. Up to 25% mothers
delivering in hospitals died due to infection.
Growth
 Variable depending on the medium. BACTERIAL GROWTH RATE

CULTURE MEDIA USED IN What is Growth Rate of Bacteria?

MICROBIOLOGY The Growth Rate of Bacteria formula is defined as
the rate of exponential growth of a bacterial
GENERAL PURPOSE MEDIA culture which is expressed as generation time, also
Supports the growth of many microorganisms the doubling time of the bacterial population and
(Nutrient Agar) is represented as G = T/n or generation_time =
Time/No. of generation.
ENRICHED MEDIA
Has a special nutrients to encourage the growth of  Lag Phase - phase when bacteria absorb
fastidious heterotrophs. (Blood Agar) nutrients, synthesize enzyme and prepare for
reproduction.
SELECTIVE MEDIA
 Logarithmic Growth Phase or
Favors the growth of one type of microorganisms
Exponential Growth Phase-phase
and inhibits the growth of others (Lunia + when bacteria multiply so rapidly that the
Penicillin Agar) population number doubles with each
generation time.
DIFFERENTIAL MEDIA
 Generation Time - time between the
Distinguishes between different groups of bacteria formation of a new bacterium and its division
on the basis of biochemical characteristics (Eosine into two daughter cells. Growth rate is
Methylene Blue Agar) greatest during the logarithmic phase.
 Stationary Phase - phase when the rate
of division slows and the number of bacteria
dividing equals the number of dying. It is
during this phase that its culture maintain its
greatest population density.
 Death Phase or Decline Phase -
microorganism die at rapid rate because of
overriding, the toxic waste products increase
and the nutrients and oxygen supply
decreases.

CONTROL OF MICROBIAL
GROWTH: Definitions

 Sterilization – killing or removing all

MICROBIAL GROWTH AND forms of microbial life (including
endospores) in a material or an object.
CONTROL
 Heating is the most commonly used
 method of sterilization Complete destruction patient during surgery. Aseptic techniques are
of all living organisms including cells, viable also used to prevent bacterial contamination
spore and viruses. in food industry.
 BACTERIOSTATIC AGENT: An agent
Sterilization is accomplished by: that inhibits the growth of bacteria, but does
not necessarily kill them.
1. Heat  SUFFIX STASIS: To stop or steady.
2. Heat and steam under pressure  GERMICIDE : An agent that kills certain
3. Gas (ethylene acide) microorganisms.
4. Chemicals (formaldehyde)  BACTERICIDE: An agent that kills
5. Radiation of the ultraviolet rays bacteria. Most do not kill endospores.
or gamma rays  VIRICIDE: An agent that inactivates
viruses.
 COMMERCIAL STERILIZATION –  FUNGICIDE: An agent that kills fungi.
Heat treatment that kills endospores of  SPOROCIDE: An agent that kills bacterial
Clostridium botulinum the causative agent of endospores of fungal spores
botulism, in canned food. Does not kill
endospores of thermophiles, which are not Rate of Microbial Death When bacterial
pathogens and may grow at temperatures populations are heated or treated
above 450C. antimicrobial chemicals, they usually
 DISINFECTION reducing the number of die at a constant rate.
pathogenic microorganisms to the point
where they no longer cause diseases. Usually Several factors influence the effectiveness of
involves the removal of vegetative or non- antimicrobial treatment.
endospore forming pathogens.
 it is the destruction or removal of infections NUMBER OF MICROBES:
or harmful microorganism from non-living o The more microbes present, the more time it
objects by physical or chemical means. takes to eliminate population.
 PASTEURIZATION use of disinfectants
and use of antiseptic TYPE OF MICROBES:
o Endospores are very difficult to destroy.
NOTE: Disinfection is not sterilization Vegetative pathogens vary widely in
because not all microbes are destroyed in susceptibility to different methods of
disinfection microbial control.
 DISINFECTANTS chemicals used to  ENVIRONMENTAL INFLUENCES:
disinfect inanimate objects like bedside or o Presence of organic material (blood, feces,
OR equipment saliva) tends to inhibit antimicrobials, pH etc.
Ex: Cydex, Chlorox, Phenol, Carbolic  TIME OF EXPOSURE:
Acid, Eresol, Xylenols, and o Chemical antimicrobials and radiation
Orthophenylphenol treatments are more effective at longer times.
 DISINFECTANT applied to inanimate In heat treatments, longer exposure
objects compensates for temperatures
 ANTISEPTIC applied to living tissue
(antiseptics)
 DEGERMING mechanical removal of PHYSICAL METHOD OF
most microbes in a limited area. MICROBIAL CONTROL
 SANITIZATION use of chemical agent on
food-handling equipment to meet public
health standards and minimize chances of  HEAT: Kills microorganisms by denaturing
disease transmission. *hot soap and water” their enzymes and other proteins. Heat
 SEPSIS comes from the Greek work decay resistance varies widely among microbes.
(bacterial contamination)  THERMAL DEATH POINT (TDP):
 ASEPSIS: Absence of significant Lowest temperature at which all of the
contamination. microbes in a liquid suspension will be killed
 ASEPTIC TECHNIQUES are used to in ten minutes.
prevent contamination of surgical  THERMAL DEATH TIME (TDT):
instruments, medical personnel, and the Minimal length of time in which all bacteria
 will be killed at a given temperature.  BY HEAT: Kills by oxidation effects.
 DECIMAL REDUCTION TIME  DIRECT FLAMING: Used to sterilize
(DRT): Time in minutes at which 98% of inoculating loops and needles. Heat metal
bacteria at a given temperature will be killed. until it has a red
Used in canning industry Moist Heat: Kills  INCINERATION: Effective way to
microorganisms by coagulating their
sterilize disposable Items (paper cups,
proteins. In general, moist heat is much more
dressings) and biological waste.
effective than dry heat.
 HOT AIR STERILIZATION: Place
 BOILING: Heat to 188 of or more at sea
level. Kills vegetative forms of bacterial objects in an oven. Require 2 hours at 1700
pathogens, almost all viruses, and fungi and for sterilization. Dry heat is transfers neat less
their spores within 18 minutes or less. effectively to a cool body, than moist heat.
Endospores and some viruses are not  FILTRATION: Removal of microbes by
destroyed this quickly. However brief boiling passage of a liquid or gas through a screen
will kill most pathogens. like material with small pores. Used to
 HEPATITIS VIRUS: Can survive up to sterilize heat sensitive materials like
30 minutes of boiling. vaccines, enzymes, antibiotics, and some
 ENDOSPORES: Can survive up to 20 culture media
hours or more of boiling.  HIGH EFFICIENCY PARTICULATS
 HOIST HEAT (CONTINUED): AIR FILTERS (HEPA)
Reliable sterilization with moist heat requires Used in operating rooms and burn units to
temperatures above that of remove bacteria from air.
 MEMBRANE FILTER: Uniform pore
AUTOCLAVE: Chamber which is filled with
size. Used in industry and research. Different
hot steam under pressure. Preferred method of
sizes: Used to filter most bacteria. Don't
sterilization, unless material is damaged by heat,
retain spirochetes, mycoplasmas and 0.01 am
moisture, or high pressure. Autoclave: Closed
Chamber with High Temperature and Pressure Press Retain all viruses and some large
proteins.
 LOW TEMPERATURES EFFECT
 Temperature of steam reaches 1210C at tutce
depends on microbe and treatment applied.
atmospheric pressure.
 Most effective when organisms contact steam  REFRIGERATION: Temperatures from 0
directly or are contained in a small volume of to 7 oc. Bacteriostatic effect. Reduces
liquid. metabolic rate of most microbes so they
 All organisms and endospores are killed cannot reproduce or produce toxins.
within 15 minutes. u Require more time to  FREEZING: Temperatures below 0oC.
reach center of solid or large volumes of  FLASH FREEZING: Does not kill most
liquid microbes. u Slaw Freezing: More harmful
PASTEURIZATION: Developed by Louis because ice crystals disrupt cell structure.
Pasteur to prevent the spoilage of beverages. Used  Over a third of vegetative bacteria may
to reduce microbes responsible for spoilage of
survive 1 year. Most parasites are killed by a
beer, milk, wine, juices, etc.
few days of freezing
 DESIRATION: In the absence of water,
CLASSIC METHOD OF PASTEURIZATION: microbes cannot grow or reproduce, but some
MIIk was exposed to 650C for 38 minutes, may remain viable for years. After water
becomes available, they start growing again.
 HIGH TEMPERATURE SHORT Susceptibility to desiccation varies widely:
TIME PASTEURIZATION (HTST): o Neisseria gonorrhea: Only survives about one
Used today. Milk is exposed to 72°C for 15 hour.
seconds. o Mycobacterium tuberculosis: May survive
 ULTRA-HIGH TEMPERATURE several months,
PASTEURIZATION (UHT): o Clostridium spp. and Bacillus spp.: May
 Milk is treated at 1480c for 3 seconds and survive decades
then cooled very quickly in a vacuum o Viruses are fairly resistant to desiccation
chamber. Advantage: Milk can be stored at
room temperature for several months.  OSMOTIC PRESSURE: The use of high
 concentrations of salts and sugars in foods is
used to increase the osmotic pressure and
create a hypertonic environment.
 PLASMOLYSIS: As water leaves the cell,
plasma membrane shrinks away from cell
wall. Cell may not die, but usually stops
growing.
 YEASTS AND MOLDS: More resistant
to high osmotic pressures. Staphylococci spp.
that live on skin are fairly resistant to high
osmotic pressure.

 RADIATION: Three types of radiation kill

microbes:
1. SONIZING RADIATION: Gamma
rays, x rays, electron beams, or
higher energy rays.
a. Have short wavelengths (less than 1
nanometer). Dislodge electrons from atoms
and form ions. Cause mutations in DNA
and produce peroxides. Used to sterilize
pharmaceuticals and disposable medical
supplies. Food Industry Is Interested in
using ionizing radiation.

Disadvantages: Penetrates human tissues. May

cause genetic mutations in humans.