Tortora, Funke, & Chase (2016)

Colorless Most microorganisms appear almost __________ when viewed

through with brightfield microscopy, so we must prepare them for
observation.
Staining It simply means coloring the microorganisms with a dye that
emphasizes certain structures.
Fixed (Attached) Before the microorganisms can be stained, however, they must be
__________ to the microscope slide.
Smear A thin film of material containing the microorganisms which is spread
over the surface of the slide.
Chromophore Stains are salts composed of a positive and a negative ion, one of which
is colored and is known as the __________.
Cation; Anion The color of so-called basic dyes is in the __________; in acidic dyes, it
is in the __________.
Crystal Violet, Methylene Blue, Examples of basic dyes.
Malachite Green, Safranin
Acidic dyes Dyes which are not attracted to most types of bacteria because the
dye’s negative ions are repelled by the negatively charged bacterial
surface, so the stain colors the background instead?
Negative Staining Preparing colorless bacteria against a colored background is called
__________.
Cell Shapes, Sizes, and Capsules The answer from the previous question is valuable for observing overall
__________, __________, and __________ because the cells are made
highly visible against a contrasting dark background.
Eosin, Acid Fuchsin, Nigrosin Examples of acidic dyes.
Simple, Differential, and Three kinds of staining techniques.
Special
Simple Stain It is an aqueous or alcohol solution of a single basic dye.
Mordant A chemical added to the solution to intensify the stain.
Increase the affinity of a stain; Functions of the answer from the previous question.
To coat a structure
Differential Stains Stains which react differently with different kinds of bacteria and thus
can be used to distinguish them.
Crystal Violet The primary stain in Gram method.
Alcohol or Alcohol-Acetone The decolorizing agent in Gram method.
Solution
Gram-positive; Gram-negative The purple dye and the iodine combine in the cytoplasm of each
bacterium and color it dark violet or purple. Bacteria that retain this
color after the alcohol has attempted to decolorize them are classified
as __________; bacteria that lose the dark violet or purple color after
decolorization are classified as __________.
Counterstains Stains such as safranin that have a contrasting color to the primary stain
are called __________.
Crystal violet–Iodine (CV–I) Different kinds of bacteria react differently to the Gram stain because
Complex structural differences in their cell walls affect the retention or escape
of a combination of crystal violet and iodine, called the __________.
Because the size of CV-I Why do gram-positive cells retain the color of the crystal violet dye?
complex is larger than the
Tortora, Funke, & Chase (2016)

crystal violet molecule that

enters the cells, it cannot be
washed out of the intact
peptidoglycan layer of gram-
positive cells by alcohol.
The alcohol wash disrupts the Why are gram-negative cells colorless until counterstained with
outer lipopolysaccharide layer, safranin?
and the CV–I complex is
washed out through the thin
layer of peptidoglycan.
Young The Gram reaction is most consistent when it is used on __________,
growing bacteria.
Penicillins and Cephalosporins Gram-positive bacteria tend to be killed easily by __________ and
__________.
Lipopolysaccharide Layer Gram-negative bacteria are generally more resistant because the
antibiotics cannot penetrate the __________.
Acid-Fast Stain Type of differential stain which binds strongly only to bacteria that
have a waxy material in their cell walls.
Mycobacterium and Nocardia Bacteria in the genera __________ and __________ are acid-fast.
Carbolfuchsin In the acid-fast staining procedure, the red dye __________ is applied
to a fixed smear, and the slide is gently heated for several minutes.
(Heating enhances penetration and retention of the dye.)
Blue Non–acid-fast cells appear __________ after the counterstain is
applied.
Special Stains Stains which are used to color parts of microorganisms, such as
endospores, flagella, or capsules.
Virulence In medical microbiology, demonstrating the presence of a capsule is a
means of determining the organism’s __________, the degree to
which a pathogen can cause disease.
Because capsular materials are Why is capsule staining more difficult than other types of staining
water soluble and may be procedures?
dislodged or removed during
rigorous washing.
India Ink or Nigrosin To demonstrate the presence of capsules, a microbiologist can mix the
bacteria in a solution containing a fine colloidal suspension of colored
particles, usually __________ or __________ to provide a contrasting
background and then stain the bacteria with a simple stain, such as
safranin.
Halos Because of their chemical composition, capsules do not accept most
biological dyes, such as safranin, and thus appear as
__________surrounding each stained bacterial cell.
Because the dyes don’t Why endospores are cannot be stained by ordinary methods, such as
penetrate the endospore’s simple staining and Gram staining?
wall.
Schaeffer-Fulton endospore The most commonly used endospore stain.
stain
Malachite green The primary stain in the endospore staining.
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Safranin The counterstain in the endospore staining.

Mordant and Carbolfuchsin Flagella staining procedure uses a __________ and the stain
__________ to build up the diameters of the flagella until they
become visible under the light microscope.
Tortora, Funke, & Chase (2016)
Micrometers and Nanometers
van Leeuwenhoek
Zaccharias Janssen
Joseph Jackson Lister
Light Microscopy
 Compound Light Microscopy
 Darkfield Microscopy
 Phase-Contrast Microscopy
 Differential Interference
Contrast (DIC) Microscopy
 Fluorescence Microscopy
 Confocal Microscopy
Compound Light Microscope
Illuminator
Condenser
Diaphragm
Coarse Adjustment
Fine Adjustment
Objective lenses
2000 X
Resolution or Resolving Power
* If the resolution of the lens system is 0.5 μm, anything larger than
Numerical Aperture
By shortening the wavelength of How can the resolution of a microscope be increased?
light used and by increasing the
size of the cone of light (the
numerical aperture)
Refractive index
A drop of immersion oil is placed
in the space between the glass
slide and the glass lens. Immersion
Microorganisms are measured in even smaller units, such as
__________ and __________.
The first person to see bacteria.
He is credited with making the first compound microscope
around 1600.
It was not until about 1830 that a significantly better microscope
was developed by __________.
It refers to the use of any kind of microscope that uses visible
light to observe specimens.
Several types of the answer from the previous question.
It has a series of lenses and uses visible light as its source of
illumination.
Light source
Has lenses that direct the light rays through the specimen.
It opens and closes to permit more or less light into the viewing
area.
It is used with the scanning or low power lenses.
It is used with the high- power and oil immersion lenses.
The lenses closest to the specimen.
Some compound light microscopes can achieve a total
magnification of __________ with the oil immersion lens.
It is the ability of the lenses to distinguish fine detail and
structure.
Specifically, it refers to the ability of the lenses to distinguish two
points that are a specified distance apart.
0.5 μm can be seen clearly, but anything smaller cannot be
observed with clarity.
This denotes the size of the cone of light entering the aperture of
the lens.
It refers to the light- bending ability of a medium such as air or
glass.
How can the problem posed by the refractive index be solved by
the light microscope?
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oil has the same refractive index

as the glass.
Small To achieve high magnification (1000 X) with good resolution, the
objective lens must be __________.
* The oil has the same effect as increasing the objective lens
diameter; therefore, it improves the resolving power of the
lenses. If oil is not used with an oil immersion objective lens, the
image has poor resolution and becomes fuzzy.
 Scanning Electron Microscope Two types of electron microscopy.
 Transmission Electron
Microscope
 Scanning Tunneling New scanned-probe microscopes.
Microscope
 Atomic Force Microscope
Tortora, Funke, & Chase (2016)

The Growth of Bacterial Cultures

Bacterial Growth It refers to an increase in bacterial numbers, not an increase in

the size of the individual cells
Binary fission Bacteria normally reproduce by __________.
Budding It refers to the formation of a small initial outgrowth (a bud)
that enlarges until its size approaches that of the parent cell,
and then it separates.
Conidiospores Some filamentous bacteria (certain actinomycetes) reproduce
by producing chains of __________ (an asexual spore) carried
externally at the tips of the filaments.
Generation Time The time required for a cell to divide (and its population to
double.
Logarithmic scales It is difficult to graph population changes of such enormous
magnitude by using arithmetic numbers. This is why
__________ are generally used to graph bacterial growth.
Bacterial Growth Curve It shows the growth of cells over time.
Lag Phase, Log Phase, Stationary Four basic phases of growth.
Phase, Death Phase
Lag Phase Period of little or no cell division.

The microbial population is undergoing a period of intense

metabolic activity involving, in particular, synthesis of enzymes
and various molecules.
Log Phase The cells begin to divide and enter a period of growth, or
logarithmic increase.

Cellular reproduction is most active during this period, and

generation time (intervals during which the population
doubles) reaches a constant minimum.

This is the time when cells are most active metabolically and is
preferred for industrial purposes where, for example, a product
needs to be produced efficiently.
Stationary Phase This is the time when the growth rate slows, the number of
microbial deaths balances the number of new cells, and the
population stabilizes.

Period of equilibrium.
Death Phase This is the time when the number of deaths eventually exceeds
the number of new cells formed. This phase continues until the
population is diminished to a tiny fraction of the number of
cells in the previous phase or until the population dies out
entirely.
 Plate Counts Direct Measurements of Bacterial Growth
 Filtration
Tortora, Funke, & Chase (2016)
 The Most Probable Number
(MPN) Method
 Direct Microscopic Count
 Turbidity
 Metabolic Activity
 Dry Weight
Plate Count
It measures the number of viable
cells.
It takes some time.
Colony-Forming Units
25-250; 30-300
Serial Dilution
 Heat-sensitive microorganisms
may be damaged by the
melted agar.
 Colonies that form beneath
the surface of a pour plate are
not satisfactory for diagnostic
purposes.
Spread Plate Method
Filtration
Most Probable Number (MPN)
Method
95%
Direct Microscopic Count
Petroff-Hausser cell counter
No incubation time is required.
Indirect Methods of Estimating Bacterial Numbers
The most frequently used method of measuring bacterial
populations.
An important advantage of the answer in the previous
question.
Disadvantage.
The most frequently used method of measuring bacterial
populations is often reported as __________.
The U.S. Food and Drug Administration convention is to count
only plates with __________ colonies, but many microbiologists
prefer plates with __________ colonies.
The original inoculum is diluted several times in a process called
__________.
Drawbacks of the pour plate method.
This method positions all the colonies on the surface and avoids
contact between the cells and melted agar.
When the quantity of bacteria is very small, as in lakes or
relatively pure streams, bacteria can be counted by
__________ methods. In this technique, at least 100 ml of
water are passed through a thin membrane filter whose pores
are too small to allow bacteria to pass.
This statistical estimating technique is based on the fact that
the greater the number of bacteria in a sample, the more
dilution is needed to reduce the density to the point at which
no bacteria are left to grow in the tubes in a dilution series.
The answer in the previous question is only a statement that
there is a (percentage) chance that the bacterial population
falls within a certain range and that the MPN is statistically the
most probable number.
In the method, a measured volume of a bacterial suspension is
placed within a defined area on a microscope slide. Because of
time considerations, this method is often used to count the
number of bacteria in milk.
A specially designed slide which is used in the answer to the
previous question.
Advantages.
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 Motile bacteria are hard to Disadvantages.

count.
 Dead cells are about as likely
to be counted as live ones.
 A rather high concentration of
cells is required to be
countable—about 10 million
bacteria per milliliter.
Turbidity It is a practical way of monitoring bacterial growth.
Spectrophotometer (or The instrument used to measure the answer in the previous
Colorimeter) question.
Percentage of Transmission (%T) As bacterial numbers increase, less light will reach the detector.
This change of light will register on the instrument’s scale as
the __________.

Metabolic Activity This method assumes that the amount of a certain metabolic
product, such as acid or CO2, is in direct proportion to the
number of bacteria present.
Dry Weight One of the better ways to measure the growth of filamentous
organisms is by __________. In this procedure, the fungus is
removed from the growth medium, filtered to remove
extraneous material, and dried in a desiccator. It is then
weighed. For bacteria, the same basic procedure is followed.
Tortora, Funke, & Chase (2016)

Culture Media, Obtaining Pure Cultures, Preserving Bacterial Cultures

Culture Medium A nutrient material prepared for the growth of microorganisms

Inoculum
Culture
 Right nutrients
 Sufficient moisture
 Properly adjusted pH
 Suitable level of oxygen or
none at all
 Sterile
 Should be incubated at the
proper temperature
100°C; 40°C
50°C
Chemically Defined Medium
Fastidious
Complex Media
Peptones
Nutrient Broth; Nutrient Agar If a complex medium is in liquid form, it is called __________.
Reducing media
Sodium thioglycolate,
Rickettsias and Chlamydias
Capnophiles
Selective media
Bismuth Sulfite Agar
Sabouraud’s Dextrose Agar
in a laboratory.
Microbes that are introduced into a culture medium to initiate
growth.
The microbes that grow and multiply in or on a culture medium
Criteria must the culture medium meet.
Agar liquefies at about __________ and at sea level remains
liquid until the temperature drops to about __________.
For laboratory use, agar is held in water baths at about
__________. At this temperature, it does not injure most
bacteria when it is poured over them.
A medium whose exact chemical composition is known.
Organisms that require many growth factors are described as
__________.
Most heterotrophic bacteria and fungi, such as you would work
with in an introductory lab course, are routinely grown on
__________ made up of nutrients including extracts from
yeasts, meat, or plants, or digests of proteins from these and
other sources. The exact chemical composition varies slightly
from batch to batch.
Partial digestion by acids or enzymes reduces proteins
to shorter chains of amino acids called __________. These
small, soluble fragments can be digested by most bacteria.
When agar is added, it is called __________.
Because anaerobes might be killed by exposure to oxygen,
special media called __________ must be used.
The answer in the previous question contain ingredients, such
as __________ that chemically combine with dissolved oxygen
and deplete the oxygen in the culture medium.
Obligate Intracellular Bacteria; Do not grow on artificial media.
Microbes that grow better at high CO2 concentrations.
Media which are designed to suppress the growth of unwanted
bacteria and encourage the growth of the desired microbes.
One medium used to isolate the typhoid bacterium, the gram-
negative Salmonella typhi, from feces.
Agar which has a pH of 5.6, is used to isolate fungi that outgrow
most bacteria at this pH.
Tortora, Funke, & Chase (2016)
Differential Media
Blood Agar
Mannitol Salt Agar
Enrichment culture
Streak Plate Method
Refrigeration
Deep-freezing and Lyophilization Two common methods of preserving microbial cultures for long
(freeze-drying)
Deep-freezing
Lyophilization (freeze-drying)
This media make it easier to distinguish colonies of the desired
organism from other colonies growing on the same plate.
A medium that microbiologists often use to identify bacterial
species that destroy red blood cells.
Example of agar which is both selective and differential.
This is also a selective medium, but it is designed to increase
very small numbers of the desired type of organism to
detectable levels.
The isolation method most commonly used to get pure
cultures.
It can be used for the short-term storage of bacterial cultures.
periods.
It is a process in which a pure culture of microbes is placed in a
suspending liquid and quick-frozen at temperatures ranging
from -50°C to -95°C.
During this process, a suspension of microbes is
quickly frozen at temperatures ranging from -54°C to -72°C, and
the water is removed by a high vacuum (sublimation).
Tortora, Funke, & Chase (2016)

The Requirements for Growth

Carbon It is the structural backbone of living matter; it is needed for all

the organic compounds that make up a living cell.

Half the dry weight of a typical bacterial

cell is __________.
Nitrogen; Sulfur Protein synthesis requires considerable amounts of
__________ as well as some
__________.
Nitrogen; Phosphorus The syntheses of DNA and RNA also require __________ and
some __________, as does the synthesis of ATP.
Nitrogen It makes up about 14% of the dry weight of a bacterial cell.
Sulfur and Phosphorus Constitute about another 4%.
Nitrogen Organisms use __________ primarily to form the amino group
of the amino acids of proteins.
 Decomposition of proteins Sources of the answer in the previous question.
 Ammonium Ions
 Nitrates
 Gaseous Nitrogen
Sulfur It is used to synthesize sulfur-containing amino acids and
vitamins such as thiamine and biotin.
 Sulfate Ion Important natural sources of sulfur.
 Hydrogen Sulfide
 Sulfur-containing amino acids
Phosphorus It is essential for the synthesis of nucleic acids and
the phospholipids of cell membrane
Phosphate Ion A source of phosphorus.
K, Mg, Ca Require often as cofactors for enzymes
Fe, Cu, Mo, Zn Trace Elements
Obligate aerobes Organisms that require oxygen to live.
Facultative anaerobes Organisms that can use oxygen when it is present but are able
to continue growth by using fermentation or anaerobic
respiration when oxygen is not available.
E. coli, yeasts Examples of the answer in the previous question.
Nitrate ions Substitute for oxygen as electron acceptors.
Obligate anaerobes Bacteria that are unable to use molecular oxygen for energy-
yielding reactions.
 Singlet oxygen Toxic forms of oxygen
 Superoxide radicals or
superoxide anions
 Peroxide anion
 Hydroxyl radical
Singlet oxygen It is a normal molecular oxygen (O2) that has been boosted into
a higher-energy state and is extremely reactive.
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Superoxide radicals or superoxide These are formed in small amounts during the normal
anions respiration of organisms that use oxygen as a final electron
acceptor, forming water.
Superoxide dismutase Neutralizes the answer in the previous question.
 Aerobic bacteria Types of bacteria that produce SOD
 Facultative anaerobes growing
aerobically
 Aerotolerant anaerobes
O2 and H2O2 SOD converts the superoxide radical into __________ and
__________.
Peroxide anion It is the active principle in the antimicrobial agents hydrogen
peroxide and benzoyl peroxide.
Water and Oxygen Catalase converts H2O2 into __________ and __________.
Oxygen When a drop of hydrogen peroxide is added
to a colony of bacterial cells producing catalase, __________
bubbles are released.
Water Peroxidase converts H2O2 into ______.
Hydroxyl radical It is another intermediate form of oxygen and probably the
most reactive. It is formed in the cellular cytoplasm by ionizing
radiation.
Aerobic respiration Type of respiration that produces traces of the answer in the
previous question.
Obligate anaerobes Type of bacteria that usually produce neither superoxide
dismutase nor catalase.
Aerotolerant anaerobes Microorganisms that cannot use oxygen for growth, but they
tolerate it fairly well.
* Many of the aerotolerant bacteria characteristically ferment
carbohydrates to lactic acid.
Lactobacilli Common examples of lactic acid–producing aerotolerant
anaerobes
Microaerophiles Microorganisms that grow only in oxygen concentrations lower
than those in air.
Superoxide radicals; Peroxides The limited tolerance of the answer in the previous question is
probably due to their sensitivity to __________ and
__________, which they produce in lethal concentrations under
oxygen-rich conditions.
Organic Growth Factors Essential organic compounds an organism is unable to
synthesize are known as __________; they must be directly
obtained from the environment.
Amino acids, purines, pyrimidines The answer in the previous question required by some bacteria.