Lucky Lynn R.

Abrera Microbiology and Parasitology

BSN1 Tools of the Labatory

1. When buying a microscope, what features are most important to check for?
Explain the importance of each feature.
 According to research, there are generally two types of microscopes to
look out for: stereo or dissecting microscopes (low power), and biological
or compound microscopes (high power). Each type has their own
features and is used for very different tasks. So this features are the most
important to check for:

Magnification
o There are two sources of magnification in a microscope. First you
have the objective lens and the other is the eyepiece. To get an
understanding of an overall magnification, you will need to multiply
the magnification power of the objective lens to the eyepiece.

For example: a regular eyepiece magnification strength is 10x and the

objective lens is 4x. Multiply those together and the total
magnification is 40x.

Biological Microscope: Most microscopes have at least 3-4 objective

lenses usually at 4x, 10x, 40x and 100x. Some microscopes can have
up to 5 objective lenses which can be found in clinics and labs.

Stereo Microscopes: the low magnification range of stereo

microscopes are very manageable and they can come with either
fixed or zoom magnification. Some fixed magnification microscopes
are dual power, i.e. they allow users to switch between two fixed
magnification settings, e.g. 10x and 30x, or 20x and 40x, while zoom
magnification allows users to use any magnification within the range
of magnification.
Microscope Head
o For biological Microscopes, there are 3 configurations to consider.
Monocular, binocular and trinocular. In general, young children find
monocular configurations more comfortable, while adults prefer
binocular microscopes. Trinocular allows user to connect a camera or
webcam onto the 3rd eyepiece to take pictures or videos of the
specimen. For stereo Microscopes, most are fitted with a binocular
 configuration but trinocular is also an option. This allows the
attachment of a camera or webcam to the microscope for teaching,
digital imaging or demonstration purposes.

Coarse vs Fine Focus

 The focus knob is another important feature on a microscope

since everyone’s has a different point of focus. The little knob
attached to the microscope allows you to find the right focus for
your eyes. Some focus knob are on it’s own (coarse focus) while
some has another tiny knob on top of it (fine focus). Using coarse
focus alone will usually give you a clear view while having the fine
focus feature allows you to further focus on smaller particles on
the slide. To get a better understanding of coarse and fine focus.

Light Source
 There are many factors to consider when it comes to the type of
light bulbs built in a microscope. Color accuracy, heat, lamp life
and ease of replacement, just to name a few. Here is a breakdown
of light bulbs found in microscopes

Tungsten/Incandescent Light

 Warm white light

 Heats up quickly
 Usually does not come with a dimmer
 Short lamp life
 Inexpensive to buy but hard to find replacement bulbs
 Found less in newer model microscopes

Halogen

 Bright warm light

 Easy to find replacement bulbs
 Heats up quickly
 Usually comes with a dimmer
 Medium Lamp Life
 Fluorescent

 White Light with little heat

 Low lamp life
 Found in specialist, epi-fluorescent microscopes

LED

 Bright cool light with no heat

 Comes with a dimmer
 High Lamp Life
 Inexpensive and easy to find replacement bulbs
 Off white with slight blue tint, not favorable for researchers

Mirror
 Some microscopes do not have any light bulbs as it uses natural
light as the light source. This works by having a small mirror
(instead of a light bulb) reflecting natural sunlight into the
objective lens and eyepiece. However this method is favorable
only at a well-lit environment. You will also need to manually align
the mirror to get the best reflection into the objective lens.

Diaphragm

 The diaphragm is usually found above the light source and just
below the stage. It is responsible for controlling the amount of
light passing through the slide before entering the objective lens
and eyepiece. This is particularly useful when there is insufficient
contrast to your specimen. It may look washed out to begin with
and by controlling how much light you want passing through will
help detecting the specimen better.

The most common diaphragm is a rotating disk diaphragm that

sits under the microscope stage. The disk has different sized
holes, which controls the amount of light projecting upwards. To
adjust the disk, simply rotate to a larger hole for more light, or a
smaller hole for less light.

The iris diaphragm is another popular diaphragm that looks like a

pupil of an eye. It manipulates light by adjusting the size of the
 opening with a simple lever at the side. The iris diaphragm is
easier to adjust and can be found on high quality microscopes.

Condenser

 A condenser collects light from the light source and focuses it like
a cone of light onto the specimen. By having a condenser on your
microscope, the image of your observation is sharper compared
to those without a condenser. This is useful when you are using
400x magnification and above.

Microscope stage
 Microscope stage holds your specimen in place and there are two
types to consider. The first is a stage with stage clips and you will
need to manually move the slide to observe different parts of the
specimen. The other is a mechanical stage where you can move
the slide at a X and Y axis with just the twist of two knobs. The
mechanical stage also has a measurement print, known as
graduated locator markings on the side if you need to mark a
location of the specimen. Most mechanical stages can be found
on higher-end microscopes but they can also come as an
accessory and be mounted manually on a microscope that does
not come with a mechanical stage.

Portability
 Newer models of microscopes are capable of being powered by
batteries, giving them versatility in outdoor applications and while
on the move. Most microscopes are now LED lighted as they can
be powered by batteries. Size and weight microscope may also be
a consideration for those seeking to use a portable microscope.

2. What is probably true of a $20 microscope that claims to magnify 1,000X.?

 It is possible to have "empty" magnification, with the object being
enlarged but not resolved. Beware of a cheap microscope that
claims to have 1000X magnification, because it probably cannot
resolve what it magnifies. Also it does indeed magnify 1000x -
with massive distortion. Anybody can get high magnification by
 putting in lenses with a lot of curvature. That doesn't mean the
final image quality is usable.

3. Differentiate between microscopic and macroscopic methods of observing

microorganisms, citing a specific example of each method.
 The microscopic observation of specimen involves preparation of
smear. A drop of sample is added on a glass slide and is heated
gently to fix the specimen and kills the microorganisms present in
it. The fixation preserves cellular components in natural state.
Further the smear is stained with an appropriate dye and viewed
under microscope. In positive staining, the cells appear in the
color of the dye on a white background.

In the negative staining, the background is stained gray or black

and the cells appear colorless on the dark background. The
macroscopic growth of a culture can be observed by inoculation
of sample into a suitable medium. The medium is either solid or
liquid. A solid medium is plated and the sample is inoculated
either by streaking or spreading. A liquid culture is poured into a
test tube or flask and the sample is introduced with a pipette.

The inoculated sample on solid or liquid media is incubated for a

specific period and later observed for visible growth. On solid
media, the culture forms macroscopic growth with visible colonies
and isolated colonies appear in fine streak or at the edges of the
plate. In liquid media, the broth becomes turbid or cloudy or
appears as a precipitate indicating the presence of microbial
growth.

4. Describe the steps of the Gram stain, and explain how it can be an important
diagnostic tool for infections.
 According to research:

A Gram stain is a test that checks for bacteria at the site of a

suspected infection such as the throat, lungs, genitals, or in skin
wounds. Gram stains may also be used to check for bacteria in
certain body fluids, such as blood or urine.

There are two main categories of bacterial infections: Gram-

positive and Gram-negative. The categories are diagnosed based
on the how the bacteria react to the Gram stain. A Gram stain is
colored purple. When the stain combines with bacteria in a
sample, the bacteria will either stay purple or turn pink or red. If
the bacteria stay purple, they are Gram-positive. If the bacteria
turns pink or red, they are Gram-negative. The two categories
cause different types of infections:

Gram-positive
 infections include methicillin-resistant Staphylococcus
aureus (MRSA), strep infections, and toxic shock.
Gram-negative
 infections include salmonella, pneumonia, urinary tract
infections, and gonorrhea.
Knowing whether bacteria is Gram-positive or Gram-negative can
help your provider identify the type of infection you have and
which antibiotics will be most effective in treating it.

Other names: Gram's stain

The Gram stain, the most widely used staining procedure in

bacteriology, is a complex and differential staining procedure.
Through a series of staining and decolorization steps, organisms in
the Domain Bacteria are differentiated according to cell wall
composition. Gram-positive bacteria have cell walls that contain
thick layers of peptidoglycan (90% of cell wall). These stain purple.
Gram-negative bacteria have walls with thin layers of
peptidoglycan (10% of wall), and high lipid content. These stain
pink. This staining procedure is not used for Archeae or
Eukaryotes as both lack peptidoglycan. The performance of the
Gram Stain on any sample requires 4 basic steps that include
applying a primary stain (crystal violet) to a heat-fixed smear,
followed by the addition of a mordant (Gram's Iodine), rapid
decolorization with alcohol, acetone, or a mixture of alcohol and
acetone and lastly, counterstaining with safranin.

The Gram stain is the most important staining procedure in

microbiology. It is used to differentiate between gram positive
organisms and gram negative organisms. Hence, it is a differential
stain. Gram negative and gram positive organisms are
 distinguished from each other by differences in their cell walls.
Also, gram stain is the primary test to perform directly on some
special samples such as cerebrospinal fluid and positive cultures
which serves as the most rapid and simplest test to characterize
microorganisms.

5. Evaluate the following preparations in terms of providing information on microbial size,

shape, motility, and differentiation: (a) spore stain, (b) negative stain, (c) simple stain, (d)
hanging drop slide, and ( e )Gram stain.
 Spore stain provides vital information in differentiating spores
and vegetative cells that make them. It allows us to specify which
bacteria have endospores. Endospores can be larger or smaller in
diameter than the vegetative cells which makes it easier to
determine which is which. Because of their tough protein coats
made of keratin, spores are highly resistant to normal staining
procedures. The primary stain in the endospore stain procedure,
malachite green, is driven into the cells with heat. Since malachite
green is water-soluble and does not adhere well to the cell, and
since the vegetative cells have been disrupted by heat, the
malachite green rinses easily from the vegetative cells, allowing
them to readily take up the counterstain.
 Negative stain employs the use of an acidic stain and, due to
repulsion between the negative charges of the stain and the
bacterial surface, the dye will not penetrate the cell. Negative
staining allows us to view the cells without risk of them being
damaged or distorted as they might be with a positive stain. The
dark stained background provides contrast making it easier to
view the bacteria. Negative stains will not penetrate and stain the
bacterial cell wall because they have a negative charge and
therefore are repelled by the negative charge of the bacterial cell.
The stain will produce a deposit around the bacteria or produce a
dark background making the bacteria appear to be clear or
unstained.
 Simple stain creates a contrast between the bacteria and the
background. Basic dyes have a positively charged chromogen that
forms an ionic bond with negatively charged bacterial cells and
thus colorize the bacterium; the advantage of using basic dyes is
that basic dyes allow you to directly see the cell. In simple staining
time is important because improper time can produce false
reactions. You can over stain the bacteria making the bacteria too
 dark. You can under stain the bacteria making the bacteria too
light.
 Hanging drop slides is a well-established method for examining
living, unstained, very small organisms. The traditional procedure
employs a glass slide with a circular concavity in the center into
which a drop of fluid, containing the 'microorganisms', hangs from
a coverslip. The hanging drop is a more complex technique, but it
allows for longer-term observation and more reliable observation
of motility. This technique is usually performed without the
addition of any stains; therefore, the organisms can be difficult to
see. Reduce the illumination on your microscope as much as you
can while still allowing yourself enough light to observe the
organism. If you use this technique to observe motility, be sure
you can tell the difference between motility and Brownian
motion. Vibration of the cellist caused by the cell colliding with
water molecules. True motility allows the cell to move in different
directions and across larger areas.
 Gram stain is the most common differential stain used in
microbiology. Differential stains use more than one dye. The
unique cellular components of the bacteria will determine how
they will react to the different dyes. The Gram stain procedure
has been basically unchanged since it was first developed in 1884.
Almost all bacteria can be divided into two groups, Gram negative
or Gram positive. A few bacteria are gram variables. Trichomonas,
Strongyloides, some fungi, and some protozoa cysts also have a
Gram reaction. Very small bacteria or bacteria without a cell wall,
such as Treponema, Mycoplasma, Chlamydia, or Rickettsia do not
have a gram reaction. The characterization of any new bacteria
must include their gram reaction. Typically, a differential stain has
four components; the primary stain, a mordant that sets the stain,
a decolorizing agent to remove the primary stain, and a
counterstain. In the Gram stain, the primary stain is crystal violet.
This gives the cell an intense purple color. The mordant, iodine,
forms a complex with the crystal violet inside the cell wall. The
cell is then washed with either Gram's decolorizer or 95% ethanol.
Gram positive cells will retain the dye complex and remain purple.
The dye rinses out in gram negative cells. The counterstain,
safranin, is used to color the cells that lost the primary stain,
otherwise they would remain colorless and you wouldn't be able
to see them. The large iodine-crystal violet complex is retained
within the cell walls of gram positive cells because of the
molecular structure of the many layers of peptidoglycan in the cell
wall. There are lots of cross-linked teichoic acids and the iodine-
dye complex cannot physically get out. There is also less lipid in
the membrane and the decolorizing agent cannot get to it as well.
Gram negative cells have an outer membrane and only one layer
of peptidoglycan, with more lipid. The crystal violet dye is easily
washed out.