Microscopy and Staining Procedure Flashcards
Historically, the science of microbiology has taken its greatest leaps forward as _________ are developed and _____.
new tools for the study of microorganisms are developed and old tools are improved
The microscope is? (Overview)
the microbiologist’s oldest and most basic tool for studying microbial structure.
It is the microbiologist’s oldest and most basic tool for studying microbial structure.
Microscope
Many types of microscopes are used and some are ______ ________.
Extremely powerful
(Overview) Microorganisms are tiny organism - organism _______________.
Cannot be seen by an unaided eye.
Generally some type of microscope is required to see them; thus, microorganisms are said to be _____.
Microscopic
Why are microorganisms said to be microscopic
Because generally some type of microscope is REQUIRED to see them
It is the technology of making very small things visible to human eye.
Microscopy
Microscopy is?
The technology of making very small things visible to the human eye.
In microscopy, the ______ as well as the ______ is expressed in metric system.
Size of microorganism, resolving power of optical instrument
The _______ (primarily the __ and __) are used to express the sizes of microorganism.
Metric units; micrometer; nanometer
The metric units are used to?
Express the sizes of microorganisms, primarily micrometer and nanometer
The basic unit of length in the metric system
Meter (m)
Meter is equivalent to approximately ___ and is therefore about ____ longer than a ___.
39.4 inches; 3.4 inches; yard
A meter may be divided into 10 equally spaced units which is called?
decimeter (dm)
A meter divided into 100 equally spaced units?
centimeter (cm)
Meter divided into 1000 equally spaced units
millimeter (mm)
meter divided into 1 million equally spaced units
micrometer (um) 10^6
a meter divided into 1 billion equally spaced units
nanometer (nm) 10^9
the sizes of bacteria are expressed in?
micrometers
whereas the size of viruses are expressed in?
nanometers
for example, a typical spherical bacterium is approximately?
1 um in diameter
a typical spherical bacterium is called a?
coccus or cocci
a coccus or cocci is decribed as?
a typical spherical bacterium
The smallest virus has the size of?
20 nm
20 nm is the size of?
smallest virus
Give the metric and English equivalent of the following:
1 meter
standard unit of length; 39.37 inches, 3.38 feet; 1.09 yards
Give the metric and English equivalent of the following:
1 dm
0.1 meter or 10^-1 m; 3.9 inches
Give the metric and english equivalent of the following:
1 cm
0.01 = 10^-2 m; 0.39 inch
Give the metric and English equivalent of the following:
1 mm
0.0001 = 10^-3 m; .039 inch = 3.9 x 10^-2
Give the metric and English equivalent of the following:
1 micrometer
10 ^-6 meter; 3.9 x 10^-5 in
Give the metric and English equivalent of the following:
1 nanometer
10^-9 m ; 3.9 x 10^-8 in
Give the metric and English equivalent of the following:
1 angstrom (A)
10^-10 m; 3.9 x 10^-9 in
The existence of microorganisms was ___ until the invention of the microscope
unknown
Is an optical instrument that is used to observe tiny objects, often objects cannot be seen at all times with the unaided human eye.
Microscope
Microscope definition in The Microscope section
optical instrument used to observe tiny objects cannot be seen by unaided eye.
In seeing microorganisms, a microscope of some sort is needed and it can be either ____ or ____.
a light microscope or an electron microscope
In general, these microscopes are used to examine cells at relatively low magnifications
light microscopes
In general, these microscopes are used to examine cells and cell structure at very high magnifications
electron microscopes
what is the difference between a light microscope and an electron microscope
light - relatively low magnifications ; electron - very high magnifications
In the microbiology laboratory, the sizes of cellular microbes are measured using an ____.
ocular micrometer
an ocular micrometer is defined as
a tiny ruler within the eyepiece (ocular) of the compound light microscope.
It is used to measure the dimensions of objects being viewed with a compound light microscope.
ocular micrometer
an ocular micrometer is used as …
used to measure the dimensions of objects being viewed with a compound light microscope.
number of time that microscope enlarge the original size of the object is
magnification power of the microscope
what is magnification power?
the number of time that microscope enlarge the original size of the object
the limit in each optical instrument of what can be seen in using the instrument
resolution power or resolving power
Resolution power is defined as…
the ability of the lenses to distinguish fine details and structure
What is the specific definition of resolution power
Specifically, it is the ability of the lenses to distinguish between two points, a specified distance apart.
It is the ability of the lenses to distinguish between two points, a specified distance apart.
Resolution power
the ability of the lenses to distinguish fine details and structure
resolving power
A microscope with a resolving power of 0.4 nm can distinguish?
between two points if they are at least 0.4 nanometer apart.
Who developed the simple microscope and at what time?
Anton van Leeuwenhoek; 16th century
The simple microscope has only one lens and is similar to?
a magnifying glass
Anton van Leeuwenhoek was considered to the best what?
best lens grinder in the world in his day
Leeuwenhoeks lenses were grounded with such precision that a single lens could magnify a microbe at?
300 x its original size
a microscope that contains more than one magnifying lense
compound microscope
a compound microscope is defined to be
a microscope that contains more than one magnifying lense
The first compound microscope was developed by … and when?
Hans Jansen and his son Zaccharias Jansen in the late 16th century
What was the issue with the first compound microscope?
The microscope was of poor quality and could not be used to observe live bacteria.
At what time did a significantly better microscope was developed and who developed it?
1830; Joseph Jackson Lister (father of Joseph Lister)
A compound microscope has usually a magnification of … and has a resolving power of …
magnify objects about 1000 times; 0.2 um
0.2 um resolving power is about …
1000 times better than the resolving power of the unaided eye
what is the source of illumination in compound microscope?
visible light
visible light is the … of compound microscope?
source of illumination
Because it uses visible light, compound microscope is also referred as?
compound light microscope
what limits the size of the object that can be seen in compound light microscope?
wavelength of the visible light
THe compound light microscope contains two magnifying lens systems. These are?
Ocular lens found in eyepiece and the other found in the objective
The ocular lens is found within? and usually has a magnifying power of?
eyepiece or ocular; x10
the second magnifying lens is found in —— which is positioned immediately ____ the ____ to be _______
objective; immediately above the object to be viewed
Each objective lens system has its own what?
magnification power
If the LPO and HPO of the microscope is ____, the image remains in approximate focus when objective is shift from LPO to HPO.
parfocal
When the LPO and HPO of a microscope is parfocal, the image remains in?
approximate focus when objective is shift from LPO to HPO
The product of the magnification power of its objective and ocular lenses
total magnification
what is total magnification?
The product of the magnification power of its objective and ocular lenses
How do you calculate the total magnification of the microscope?
multiplying the magnifying power of the ocular lens (x10) and the magnifying power of the objective being used
So a specimen is usually first observed using what part of a microscope?
a low power objective with a magnification of x10
Once the specimen is in focus after it was subjected in low power objective, the _____ is then swung into position.
high power or high dry objective
The high powr objective can be used to study what microorganisms?
algae, protozoa and other large microorganisms.
in bacteriological examination, what kind of objective should be used?
oil-immersion objective
oil-immersion objective is used in?
bacteriological examination
How do you prepare an oil-immersion objective?
a drop of immersion oil is placed between the specimen and the objective
it reduces the scattering of light and ensures that the light will enter the oil-immersion lens
immersion oil
This is made up of a series of lenses and utilizes TRANSMITTED visible light as its source of illumination.
brightfield microscope
In brightfield microscope objects are observed against ______
a bright background
A bright background or transmitted light is used to visualized the _____ of ____ and ____ .
morphology of bacteria and fungi
The morphology of bacteria and fungi is visualized in what states?
in living (unstained) or nonliving (stained) state
The brightfield microscope cannot be used to observe organism measures as ?
less than 0.2 micrometer in diameter of thickness
Examples of such organisms that a brightfield microscope cannot observe are:
spirochetes and virus
this microscope utilizes reflected light instead of transmitted light.
darkfield microscope
The reflected light used in darkfield microscope goes through a ____ with an _____ that blocks the light such that only the object is illuminated.
special condenser; opaque disc
So the dark field microscope observes an object that appears to be bright under a _____
dark background
The darkfield microscope are used to observe miroorganisms that are _____ under ordinary light microscope.
invisible
dark field microscope is routinely used to view spirochetes such as ____ which cauase what disease?
treponema pallidum; causative agent of syphilis
microscope that has a special condenser and objective lenses that accentuate small difference in the refractive index of various structures within theorganism.
phase-contrast microscope
In a phase-contrast microscope, what is the use of the special condenser and objective lenses?
to accentuate small difference in the refractive index of various structures within the organism.
What happens to the light passing through the object of different refractive indexis?
the light is slowed down and diffracted
the change in the speed of light in a phase-contrast microscope is seen as what?
different degrees of brightness
what causes the different degrees of brightness in a phase-contrast microscope?
the changes in the speed of light
phase-contrast microscopes can be used to observe what type of living microorganisms?
unstained living microorganisms
Why is it possible for unstained living microorganisms to be studied under a phase contrast microscope?
Because the light refracted by living cells are different from the light of the refracted medium thus the contrast is increased and the organisms are more easily seen.
what internal structure example is examined by the phae-contrast microscope?
endospores
why is phase contrast especially useful in examining LIVING microorganisms?
because it permits the examination of internal structures
It is also not necessary to fix (attach the specimen to the slide) or stain the specimen because it remains living disallowing the possibility of?
distortion or kill the microorganism
what procedures are not necessary in using a phase-contrast microscope with a living microorganism?
fixing (attaching the specimen in a slide) and staining the specimen
what is phase contrast microscope’s resolving power and useful magnification?
0.2000 um and 1000x
this microscope uses ultraviolet light and flourescent dye
fluorescence microscope
what do you call fluorescent dyes?
fluorochromes
What happens to the object under study in a fluorescence microscope?
Fluoresces against the dark background
Fluorescence microscopy is often used in what laboratories?
Immunology laboratories
What does fluorescence microscopy’s role in immunology laboratories?
Detection of specific antigen and antibodies
What microscope uses an electron beam as a source of illumination and electromagnet to focus the beam?
Electron Microscope
In electron microscope what is used as source of illumination?
Electron beam
In electron microscope what is used to focus the electron beam?
Electromagnet
Electron microscope can magnify an object at ______
10,000x or more
Objects smaller than ____ must be examined with an electron microscope
0.2 um
Objects smaller than 0.2 um that should be examined by an electron microscope includes examples like?
Viruses and internal structure of cells
What are the two types of electron microscope?
Transmission electron microscope
Scanning electron microscope
This type of microscope uses electromagnetic lenses instead of glass lenses that is used in a light microscope.
Transmission electron microscope
What lenses is used in a transmission electron microscope, what’s the difference in light microscope?
Transmission - electromagnetic
Light - glass
What are the three things an electromagnetic lens controls?
Illumination, focus, magnification
This microscope is used to study the molecular and atomic shape of organism.
Transmission electron microscope
Transmission electron microscope is used to study the _____ and _____ of organism
Molecular and atomic shape of organism
A transmission electron microscope can also determine the _______ inside the cell as well as its ________.
Temperature; chemical properties
This microscope is used to examine internal structure of viruses in thin section of cells
Transmission electron microscope
Transmission electron microscope is used to examine internal structure of viruses in what?
Thin section of cells.
How much is the resolving power and magnification power of a transmission electron microscope
2.5 nm; 10,000-100,000x its original size
This microscope provides three dimensional views of the specimen.
Scanning electron microscope
Scanning electron microscope is provides ______ view of the specimen.
Three-dimensional
Microscope used to examine SURFACE features of cells and viruses.
Scanning electron microscope
What does a scanning electron microscope examines in cells and viruses
Surface features
What is the resolution and magnification power of a scanning electron microscope
20 nm and 1,000-10,000x its original size
What is the difference in terms of use and components of the 5 types of compound microscope?
Answer in module
What is the difference between the two types of electron microscope
Answer in module
What is the difference between the simple and compound microscope?
What is the difference between light and electron microscope
Answer in module
What is the commonly used microscope in microbiology laboratory?
Compound light microscope
What do you call a compound microscope with single eyepiece (ocular)
Monocular
A compound microscope with two eyepieces
Binocular
What is the difference between monocular and binocular?
Monocular - single eyepiece or ocular
Binocular - two
Where does light enters in a compound microscope?
From the source in the base
As light enters the microscope it often passes through a what?
Blue filter
What does the blue filter do to the light passing through it?
It filters out the long wavelength of the light, leaving the shorter wavelengths
As the blue filters the long wavelength of light what in turn happens?
Shorter wavelengths are left thus improving resolution
How many does a compound light microscope have up to in terms of lenses?
Up to 5 lenses with different magnification powers.
What are the four objectives used in most compound light microscope?
x4, x10, x40, x100 objectives
As mentioned above to get the total magnification, it is calculated by multiplying the magnifying power of the _____(_) by the magnifying power of the ______
Ocular (x10) ; objective you are using
What objective is rarely used in microbiology?
x4
Usually specimens are first observed using the ____ objective
x10
Once the specimen is in focus the _____ objective is then swung into position
High power or high dry
This swung high power or high dry objective is used to observe ___, ____, and _____.
Algae, protozoa, and other large microorganisms
Oil-immersion objective has a total magnification of?
x1000
Why should an oil immersion objective be used to study bacteria?
Because they are so tiny
What does the immersion oil do to the microscope?
It reduces the scattering of light and ensure that the light will enter the oil immersion lens
The oil immersion objective cannot be used without ?
Immersion oil
This oil is not required in _____
Other objectives
controls the amount of light entering through the condenser
collector lens with field diaphragm
used to move the stage and microscope slide
stage adjustment knobs
contains a lens system that focuses light onto the specimen
condenser
used to adjust the amount of light passing through the condenser
iris diaphragm control arm
used to adjust the height of the condenser
condenser control knob
turns the light source on and off
on/off switch
controls the amount of light emitted from the light source
rheostat control knob
used to adjust the amount of light passing through the collector lens
field diaphragm lever
supports the binocular body and the revolving nosepiece
arm
used to focus the objective lenses
fine and coarse adjustment knobs
contains the light source
base
held with one hand when carrying the microscope, with the other hand beneath the base to support the weight of the microscope
arm
used to magnify objects placed on the stage
objective lenses
flat surface on which the specimen is placed
stage
holds the ocular lenses in their proper place
binocular body
holds the objective lenses
revolving nosepiece
is a x10 magnifying lens
ocular lens
LOCATION: front side of the base
rheostat control knob
LOCATION: on the side of the base
on/off switch
LOCATION: at the top of the microscope
ocular lens
LOCATION: on the condenser
iris diaphragm control arm
LOCATION: on the arm of the microscope near the base
fine and coarse adjustment knobs
LOCATION: beneath the stage
condenser; stage adjustment knobs
LOCATION: attached to the field diaphragm
field diaphragm lever
LOCATION: beneath and behind the condenser
condenser control knob
LOCATION: above the stage
revolving nose piece
LOCATION: beneath the condenser
collector lens with field diaphragm
LOCATION: held in place above the stage by the revolving nosepiece
objective lenses
LOCATION: directly beneath the objective lenses and the revolving nosepiece
stage
What can be used to stain cells and increase the contrast so that they can be more easily seen in the brightfield microscope?
dyes
Dyes are used to stain cells and increase the contrast so that they can be more easily seen in the what microscope?
brightfield microscope
Dyes are ____ compounds and has an affinity for specific ______.
organic; cellular materials
Since in microbiology many dyes used are positively charged thus they are called?
basic dyes
examples of basic dyes (3)
methylene blue, crystal violet, safranin
So basic dyes bind strongly with negatively charged cell components such as?
nucleic acids, and acidic polysaccharides
cells also have surfaces that tends to be negatively charged therefore basic dyes have a high affinity to the surface of the cell making them very useful ____.
general-purpose stains
Describe a bacteria in its living state
colorless, transparent, and difficult to see under the microscope.
To make bacteria visible under the microscope a method has been devised that enables scientists to examine bacteria.
staining method
the process by which the organic dye is applied to the smeared organism for them to become visible under the microscope
staining
staining is the process by which the______ is applied to the ______ for them to become visible under the microscope
organic dye, smeared organism
In preparation for staining, the bacteria are smeared unto a _____, _____ and then ____.
glass slide, air-dried, fixed
a thin film of material containing microorganisms spread on the surface of the glass slide
smear
THe process that proceeds after the smear has been prepared where the specimen has to be attached or secured in a slide.
fixation
what is the process of fixation?
the process of attaching or securing the smear in a slide
two common method of fixation:
heating fixation and methanol fixation
is usually accomplished by passing the smear through a bunsen burner flame
heat fixation
If heat fixation is not properly done, ____ can distort the _____.
excess heat; morphology of the cell
how is heat fixation accomplished?
by passing the smear through the bunsen burner flame
this method is accomplished by flooding the smear with absolute methanol for 30 seconds.
methanol fixation
this method is accomplished by flooding the smear with absolute methanol for 30 seconds.
methanol fixation
how is methanol fixation accomplished? BY?
flooding the smear with absolute methanol for 30 sec
what are the three purposes of the fixation method
- it kills the organism
- It preserves the morphology of the organism
- it anchors the smear to the slide
is the application of dye or stain to microbial smears for them to become visible under the microscope.
staining
the purpose of staining is to increase the contrast between microorganisms and the background so that they are more readily seen in the ______.
light microscope
This type of staining is used to observe or determine the bacterial shape and morphologic shape.
Simple staining
the simple staining utilizes how many dyes?
single dye
the single dye used in simple staining can either be?
water or alcohol based
what are the commonly used stains for simple staining are?
crystal violet and methylene blue
simple staining procedure is commonly used to observe?
bacterial capsule, spores, flagella
simple staining procedure that is commonly used to observe bacterial capsule, spores, and flagella are collectively known as?
structural staining procedure
The six steps of simple staining procedure
Smear loopful of microbes onto slide
Air dry
Drip methanol onto specimen to fix
Flood with methylene blue
Rinse with water and blot dry
Examine under oil immersion objective
this staining procedure is most frequently used to differentiate Gram-staining bacteria and Acid-fast bacteria.
Differential Stain
Differential staining is used commonly to differentiate between a ______- bacteria and _______.
Gram-staining bacteria and acid fast bacteria
The Gram-stain and Gram-staining procedure was developed in ___ and by the ______.
1884, Danish bacteriologist Dr. Hans Christian Gram
Gram staining is one of the most useful staining procedures because it classifies into two large groups: ____ and _____.
Gram-positive and Gram-negative bacteria
State the gram staining procedure’s four steps
The color of the bacteria at the end of the gram staining procedure depends upon the _____?
chemical composition of their cell wall
What is the color of a gram-positive bacteria?
blue-to-purple in color
the thick layer of _____ in the cell wall of Gram-positive bacteria makes it difficult to remove the crystal violet-iodine complex during decolorization.
peptidoglycan
the thick layer of peptidoglycan in the cell wall of Gram-positive bacteria makes it difficult to remove the ____.
crystal violet-iodine complex during decolorization.
Gram-negative bacteria is in what color at the end of the procedure?
pink-to-red in color
during the gram-staining procedure in a gram-negative bacteria, the _____ was removed from the cells during the _____, and the cell was subsequently stained by the final stain ______.
crystal violet; decolorization; safranin
What’s the difference between the layer of peptidoglycan in a gram-positive or -negative bacteria?
thick layer -positive
thin layer -negative
The ____ layer of ____ makes it easier to remove the ____ during decolorization
thin; peptidoglycan; crystal violet-iodine complex
a stain that binds strongly only to bacteria that have a waxy material in their cell.
Acid-fast stain
The technique of acid-fast staining is used to identify all bacteria in the genus ____, including the two most important pathogens which are? and what disease they cause.
mycobacterium; mycobacterium tuberculosis, the causative agent of tuberculosis and Mycobacterium leprae, the causative agent of leprosy
acid-fast stain was developed in ___ by ____.
1882; Paul Ehrlich
acid-fast stain is especially useful in tuberculosis laboratory where the acid fast mycobacterium are readily seen as ____ against a ____ background in _____ from a tuberculosis patient.
red bacilli; blue or green background; sputum specimen
The two methods of acid-fast staining:
Ziehl-Neelsen stain
Kinyoun stain
Ziehl_Neelsen stain is also known as the “___” because it requires steam-bathing the organism with the aqueous dye
hot method
Ziehl_Neelsen stain is also known as the “hot method” because it requires ____ the organism with the ____.
steam bathing; aqueous dye
Kinyoun stain is also known as “____” because it does not utilize heat for staining
cold method
what decolorizing agent is used in acid-fast stain?
mixture of acid and alcohol
what red dye is used in the acid fast staining method?
carbol fuchsin
the red dye ____ is first driven from the bacterial cell wall with ____ so that the decolorizing agent does not remove the red color from the mycobacterium cell wall.
carbol fuchsin; heat
the heat is necessary because the cell wall of the mycobacterium contains ___, which prevents the stain from ____.
waxes: penetrating the cell
the heat is necessary because the cell wall of the mycobacterium contains ___, which prevents the stain from ____.
waxes: penetrating the cell
what does the heat do to the waxes so that the stain can penetrate?
the heat softens the waxes
bacterias that retain the red color particularly mycobacterium’s after it is decolorized by a mixture of acid and alcohol then it is said to be?
acid-fast
most bacteria that are decolorized by the acid-alcohol treatment are sai to be?
non-acid fast
These type of staining method are used to demonstrate specific structure in a bacterial cell.
Special stain
Metachromatic granules can be visualized using the?
Loeffler alkaline methylene blue stain (LAMBS)
Stain for demonstration of bacterial capsule or slime layer
Hiss stain
Stain for demonstrating bacterial endospores
Schaeffer-Fulton endospore stain
Stain that add layers of dye or metal to the surface of the flagella to make those surfaces visible.
Flagellar stains
What is visualized using the Loeffler alkaline methylene blue stain?
Metachromatic granules
What is demonstrated using Hiss stain?
Bacterial capsule/slime layer
What is demonstrated using Schaeffer-Fulton stain?
Bacterial endospores
Flagellar stains add layers of dye or metal to the surface of the _______ to make those surfaces visible.
Flagella
Flagellar stains add layers of ________ to the surface of the flagella to make those surfaces visible.
Dye or metal
Flagellar stains add layers of dye or metal to the ______ of the flagella to make those ______.
Surfaces; surfaces visible
This solution is an aqueous solution used for staining bacteria, fungi, and blood parasite in blood smear
Loeffler alkaline methylene blue solution
Using the solution in an oil immersion objective, the bacterial cell stain is ______ color and the background stain _______.
Medium blue, lighter blue
A type of stain that stains the capsule or slime layer and the bacterial cell with a lighter background
Hiss stain
With hiss stain the capsule appears as a ______ color between a _____ bacterial cell and _____ background
Light violet, dark violet colored, colorless
The Schaeffer-Fulton endospore stain is designed to isolate endospores by staining endospore _____ and any other bacterial bodies ____.
Green, red
The primary stain in Schaeffer-Fulton endospore stain is _______.
Malachite green
The counterstain in Schaeffer-Fulton endospore stain is?
Safranin
The counterstain dyes any other bacterial bodies?
Red
The ____ allows observation of bacterial flagella under the light microscope.
Flagellar stain
_____ are normally too thin to be seen in conditions with the flagellar stain.
Bacterial flagella
The flagellar stain employs a _____ to coat the flagella with stain until they are thick enough to be seen under the microscope.
Mordant
