BACTERIOLOGY
THE USE AND CARE OF MICROSCOPE
MICROSCOPE - instrument widely use to
magnify and resolve the image of an object
that is otherwise invisible to the naked eye.
HISTORY OF MICROSCOPE
● 1590 - Zacharias Jansen and his father
Hans developed the first compound
microscope.
● ANTHONY VAN LEEUWENHOEK – Father
of Microscopy.
→ First person to observe and describe single
celled organisms.
● Dental plaque
● Animalcules
→ First to record and observe muscle fibers,
bacteria, spermatozoa, and blood flow in
capillaries.
PARTS OF THE MICROSCOPE
PARTS OF MICROSCOPE: BODY
BASE – support
BODY TUBE – holds the objective lenses and
the ocular lens at the proper distance.
NOSE PIECE – holds the objective
ARM – supports the microscope when carried.
→ Connects the base to the binocular head -
revolving nosepiece assembly.
MECHANICAL BASE – where the specimen to
be examined is placed.
LECTURE/LABORATORY
PARTS OF A MICROSCOPE: LENS
OBJECTIVES – perform the initial
magnification of the object.
● SCANNER - 4X
● LOW POWER (LPO) - 10X
● HIGH POWER (HPO) - 40×
● OIL IMMERSION (010) - 100X
INSCRIPTIONS:
● TYPE OF OBJECTIVE (S, LP, HPO, OIO)
● MAGNIFICATION (10x,40x,100x)
● NUMERICAL APERTURE
● MICROSCOPE TUBE LENGTH
● COVERSLIP THICKNESS TO BE USED
COARSE KNOB – for initial focusing, moves
the mechanical stage.
FINE KNOB – to sharpen or clarify the image.
EYEPIECE – where the ocular lens system is
located.
→ OCULAR – designed to further magnify the
object that has been enhanced by the
objectives. (10x)
→ DIOPTER ADJUSTMENT KNOB – used to
compensate for the variations in vision
between the operator's eyes.
PARTS OF A MICROSCOPE:
ILLUMINATION SYSTEM
LIGHT SOURCE → RHEOSTAT – regulates
the intensity of the light
FIELD DIAPHRAGM - controls the light beam
reaching the slide and is adjusted for optimal
illumination.
CONDENSER - focuses the light on the
specimen and controls the light for a uniform
illumination.
APERTURE DIAGHRAGM - controls the
amount of light and the angle of light that pass
the specimen and lens.
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TERMINOLOGIES

RESOLVING POWER - ability to distinguish

two small objects that are a specific distance
apart.

RESOLUTION - ability to visualize fine details.

PARFOCAL - lens that stays in focus when

magnification/focal length is changed.

FIELD OF VIEW - the diameter of the circle of

view when looking through the oculars.
→ the higher the magnification the smaller the
field of view.

FINAL MAGNIFICATION – Ocular

magnification (10) X Objective Magnification

HOW TO USE THE MICROSCOPE

● Place the slide on the stage and examine it

with the scanning objective (4x).
● Rotate the revolving nosepiece to place the
lower-power objective (10x) in the optical
axis.
● When turning the nosepiece, grasp the
nosepiece itself or the part of the objective
adjacent to the nosepiece to avoid excess
stress on the objective.
● Proceed to high dry (40x).

CARE FOR THE MICROSCOPE

● Carry microscope with two hands, supporting

the base with one hand.
● Always hold the microscope in a vertical
position.
● Only clean optical surfaces with a good
quality lens tissue and commercial lens
cleaner.
● Do not use the 10X and 40X objectives with
oil.
● Clean the oil immersion lens after use.
● Always remove slides with the low-power
objective raised.
● Store the microscope with the low-power
objective in position and the stage centered.

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MICROBIOLOGY – is about cells and how
they work, especially the bacteria, a large
group of cells of enormous basic and practical
importance.
→ is about diversity and evolution, about how
different kinds of microorganisms arose and
why.
→ It is about what microorganisms do in the
world at large, in soils and waters, in the
human body, and in animals and plants.
→ microbiology may as the foundation of the
biological sciences.
● Microorganisms differ from the cells of
macroorganisms.
→ The cells of macroorganisms such as plants
and animals are unable to live alone in nature
and exist only as parts of multicellular
structures, such as the organ systems of
animals or the leaves of leafy
plants.
→ By contrast, most microorganisms can carry
out their life processes of growth, energy
generation, and reproduction independently of
other cells.
PATHWAYS OF DISCOVERY
● ROBERT HOOKE (1635-1703) – English
mathematician and natural historian.
● Micrographia (1665) - the first book devoted
to microscopic observations, fruiting structures
of molds the first known description of
microorganisms.
● ANTON VAN LEEUWENHOEK (1632-1723)
– Dutch draper and amateur microscope
builder.
→ First person to see bacteria in 1676.
● FERDINAND COHN (1828-1898) – botanist
and became an excellent microscopist.
→ Study of unicellular plants the algae and
later to photosynthetic bacteria.
→ Discover the important group of bacteria
that form endospores.
LECTURE/LABORATORY
LOUIS PASTEUR (1822-1895) – trained as a
chemist.
→ detailed study of the mechanism of the
alcoholic fermentation, classic experiments on
spontaneous generation
Other works:
→ development of vaccines for the disease’s
anthrax, and rabies vaccine to a human.
ROBERT KOCH (1843-1910) – “The Germ
Theory of Disease.”
● The disease-causing organism must always
be present in animals suffering from the
disease and should not be present in healthy
animals.
● The organism must be cultivated in a pure
culture away from the animal body
● The isolated organism must cause the
disease when inoculated into a healthy
susceptible animal
● The organism must be reisolated from these
experimental animals and cultured again
in the laboratory, after which it should still be
the same as the original organism.
TOOLS:
STEP 1: Microscopy, staining
STEP 2: Laboratory Culture
STEP 3: Experimental animal
STEP 4: Laboratory reisolation
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GENERAL CHARACTERISTICS OF
MICROORGANISMS
● PROKARYOTES – no nucleus and
organelles
● EUKARYOTES – Membrane bound nucleus
and organelles.
● VIRUSES – genomes contain either DNA or
RNA
→ newer agent is proteinaceous
SIZE OF MICROORGANISMS
VIRUSES – less than 1um
BACTERIA – less than 2.5 um
HELMINTHS – 10 mm
NOMENCLATURE
→ both italicized / underlined
GENUS – always underlined
specie – always lowercase
Staphylococcus aureus
Staphylococcus aureus
IDENTIFICATION:
LECTURE/LABORATORY
The process of microbial identification and
placing them in a taxonomic scheme includes:
● Microscopic morphology and colony
appearance
● Physiological/biochemical characteristics
● Chemical analysis
● Serological analysis
● Genetic and molecular analysis
→ G + C base composition
→ DNA analysis using genetic probes
→ Nucleic acid sequencing and rRNA analysis
MAJOR GROUPS OF MICROORGANISMS:
● Bacteria → Bacteriology
● Fungi → Mycology
● Algae → Phycology
● Virus → Virology
● Protozoa → Protozoology
SHAPES OF BACTERIA
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COCCI → LOPHOTRICHOUS: tuft at one end

→ AMPHITRICHOUS: both ends
→ PERITRICHOUS: all around bacteria

BACTERIAL GROWTH
BACILLI
● Culture: Increase in the population of cells
● Generation time: The time cell takes to divide
(double) is called reproduction
● Reproduction: Binary Fission
→ Division exactly in half
COCCOBASILLI → Most common means of bacterial
reproduction
→ Forming two equal size progenies
→ Genetically identical offspring
→ Cells divide in a geometric progression
STAPHYLOCOCCI doubling cell number
(CLUSTER)
BINARY FISSION

STREPTOCOCCI
(CHAIN)

SPIROCHETE

FLAGELLA

● Motility - movement
● Arrangement basis for classification
→ MONOTRICHOUS: 1 flagellum
BACTERIAL CULTURE GROWTH

LAG PHASE:
● Organisms are adapting to the environment

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● Synthesizing DNA, ribosomes, and enzymes → Photoautotrophs

to breakdown nutrients, and to be used for → Photoheterotrophs
growth. ● Chemosynthetic Bacteria:
→ Chemoautotrophs
LOG PHASE: (growth) → Chemoheterotrophs
● Division is at a constant rate
Basic bacterial requirements:
STATIONARY PHASE: ● Water: Used to dissolve materials to be
● Dying and dividing organisms are at an transported across the
equilibrium cytoplasmic membrane
● Death is due to reduced nutrients, pH ● Carbon: required for the construction of all
changes, toxic waste, and reduced oxygen organic molecules
● Cells are smaller and have fewer ribosomes → Autotrophs use inorganic carbon (CO2) as
their carbon source
DEATH PHASE: → Heterotrophs: use organic carbon
●The population is dying in a geometric fashion ● Nitrogen: Many organisms use nitrogen gas
so there are more deaths than new cells by nitrogen fixation to
● Deaths are due to sever reduced nutrients, produce ammonia
pH changes, toxic waste, and reduced oxygen. → Inorganic source: e.g., Nitrogen gas (N2),
Nitrate (NO3), Nitrite (NO2), and Ammonia
(NH3)
→ Organic source: e.g., Proteins, broken down
to amino acids

TEMPERATURE:
● PSYCHROPHILES (cryophiles/cryophilic):
Some exist below 0 °C if liquid water is
available e.g., Oceans, refrigerators, and
freezers
● MESOPHILES: Most human flora and
pathogens.
● THERMOPHILES: Hot springs, effluents from
Laundromat, and deep ocean thermal vents.

OXYGEN:
● Required for aerobic respiration and energy
production
● Organisms are classified according to their
FACTORS INFLUENCING BACTERIAL gaseous requirements
GROWTH: → Obligate aerobes – O2
→ Facultative anaerobes
NUTRITION: → Obligate anaerobes
● Bacteria are found in almost every
environment because they can use widely
different energy sources.
Based on their energy source bacteria can be SALINITY:
grouped into 4 MAJOR TYPES: ● Halophiles: Bacteria that specifically require
NaC1 for growth
●Photosynthetic Bacteria:

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● Moderate Halophiles: Grow best at 3%
NaC1 solution, many oceans dwelling bacteria
● Extreme Halophiles: Grow well at NaC1
concentrations of greater than 15% e.g., salt
lakes, pickle barrels
PH:
● Acidophiles: Some bacteria can grow in
acid substrates
● Neutrophiles: most microbes prefer a pH
near neutrality
● Alkalinophiles: Microbes which can grow in
very alkaline substrates
CONTROL OF BACTERIAL GROWTH
PHYSICAL METHODS:
MOIST HEAT:
● Coagulation (denaturing) of proteins
● Hydrogen bonds are broken
BOILING:
● Not always effective
● Kills most vegetative pathogens, viruses,
fungi, and spores within 10minutes
● Some microbes resistant to boiling e.g.,
endospores (20 Hours)
AUTOCLAVE:
● Preferred method
● Moist heat (steam) and pressure
→ Limitations: Material must be able withstand
heat and moisture 15 psi (121°C) for 15mins
will kill all organisms
PASTEURIZATION:
● Mild heating (Initially 63°C for 30 minutes)
● Kills most pathogens and bacteria that cause
spoilage
● Lowers bacterial numbers
● Preserves taste of product
● High Temperature Short - time pasteurization
(HTST)
→ Kills pathogens
→ Lowers bacterial numbers, milk keeps while
refrigerated
DRY HEAT STERILIZATION:
● Flaming
● Incineration
LECTURE/LABORATORY
● Hot air sterilization: Placed in oven (170 ° C
for 2 hours)
FILTRATION:
● Liquids and heat sensitive materials
● Filters composed of cellulose or plastic
polymers. Vacuum assists gravity
● Small pores prevent passage of bacteria (0.1
um -1mm)
REFRIGERATION:
● Bacteriostatic (stop microbial growth)
● Psychrotrophs still present and grow
● Slow freezing more harmful to bacteria than
rapid.
● Ice disrupts the cell structure
● Thawing damages bacteria as well
HIGH PRESSURE:
● Applied to liquid suspensions
● Alters protein shape
● Endospores are resistant
DESICCATION:
● Removal of water
● Microorganisms cannot grow but still survive
● Reintroduce water microorganisms resume
growth and division
● Effectiveness varies between organisms
● Removal of water
● Microorganisms cannot grow but still survive
● Reintroduce water microorganisms resume
growth and division
● Effectiveness varies between organisms
→ Neisseria withstand dryness for one hour
→ Mycoplasmas withstand dryness for months
→ Endospores remain for centuries
OSMOTIC PRESSURE:
● High concentrations of salt and sugar
● Creates hypertonic environment
● Water leaves microbes’ cell
● Molds and yeasts can grow better than
bacteria in high osmotic pressure or low
moisture
RADIATION:
● Ionizing radiation (gamma rays)
● High energy short wavelength
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● Radioactive elements
● X-rays
● Penetrate deeply
● Require longer times
● Ionizes water to form hydroxyl radicals
● Food preservation in other countries

CHEMICAL METHODS:

ALCOHOLS:
● Protein denaturation and lipid dissolution.

HEAVY METALS:
● Denaturation of enzymes and other essential
proteins.

SURFACE-ACTIVE AGENTS:
● Soaps and acid-anionic detergents
→ Mechanical removal of microbes through
scrubbing.
● Acid-anionic detergents
→ Not certain; may involve enzyme
inactivation or disruption.

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