Condenser Lens

Module 2: Review of Microscopy - Gathers the beam of light and

focuses it so that it converges on
Microscope the specimen with high intensity in
- is one of the principal tools of the a small area
biologist. It was invented through
the efforts of Dutch scientist Anton Specimen
Van Leeuwenhoek. - Usuallly is on a glass slide that
- Serves as a very useful tool to help rests on a platform called stage
you discover the fascinating
secrets of the living world which Objective Lens
the unaided eye cannot see. - Performs the bulk of the
- has been of crucial importance for magnification of the specimen
the development of microbiology
as a science. It is the most Eyepiece
important instrument used in the - Receives the light beam from the
laboratory as it functions to objective lens and refocuses it to
magnify the image of the objects recreate the image in the viewer’s
that cannot be seen by the naked eye
eye.
(2) Phase-contrast Microscope
Light Microscopes - Some unpigmented living cells are
- Useful for studying specimens not visible in the light microscope
ranging in size from 1mm to about because it cannot create
20 nm differences in contrast between
cells and water. Only
Light microscopes are classified in these phase-contrast microscope can
following groups: create contrast difference between
cell and water that is why these
(1) Bright Field Microscopy cells only visible in phase-contrast
- The most common form of light microscope. This is suitable for
microscopy studying the shape and motility of
- The light beam passes through the microorganisms.
sample and into the objective lens
- In a bright-field microscope, the (3) Fluorescent Microscope
specimen appears as dark against - In this type, the specimen is
the bright background. They are stained with fluorescent dyes and
used in the laboratory for studying then exposed to ultraviolet rays
the outer structure of (UV). Fluorescent dyes will absorb
microorganisms. low wavelength light and become
excited as a result they will release
Parts: a high wavelength light. This is
usually used in medical
Lamp laboratories for the identification of
- Is where the light beam is initiated pathogens. This is also used for
- This beam if divergent as it localization of specific proteins.
approaches the condenser lens
In the laboratory, the microscope serves - You should only need to do minor
as a very useful tool to help you unravel adjustments at each magnification
the fascinating secrets of the living world - When focusing, move the slide just
which the unaided eye cannot see. In this a little bit back and forth with the
module, you will review the basic parts slide holder knobs
and functions of the bright-field - Your eyes can see motion better
microscope. Its properties of bringing than stationary objects
magnification, resolution and actual - If you are observing unstained
measurements of the objects being specimens, use the stage
viewed under this microscope will be diaphragm to reduce the amount of
presented. light passing through
- If you are observing unstained
specimens, try to find a bubble or
How to use the Microscope other easily observed object under
the cover slip and focus on that
Using the Microscope
1. Open the clip on the stage with “The specimen becomes blurry when I
one hand and slide a microscope move to a higher power lens”
slide to the back of the stage - You may need to clean the lens
2. Be sure the slides doesn’t go - Take a piece of lens paper with
under the clips that hold it in place lens cleaner and wipe the lens,
3. Use the knobs on the lower right then wipe dry with clean lens
side of the stage to center the paper.
specimen over the condenser
lense Magnification
4. Start with the low power objective - Ability of the microscope to enlarge
lens (4x) and the stage in its an image.
highest position. The specimen - It depends on the degree of
should be nearly in focus curvature of the glass lens.
5. Once you found the specimen on - Highly curved lens will increase the
lower power, be sure it is centred magnification.
in the field of view then move to
the highest objective lense (10x)
Ocular Lens Objective Lense
6. Your specimen should be nearly in
focus, and you should only need to 10x (Scanner) 4x
use the smaller, fine-focus. Use
only the smaller fine-focus knob. 10x (LPO) 10x
7. Again, centre the specimen and 10x (HPO) 40x
you are ready to move to the
highest power lens (40x) 10x (OIO) 100x

Troubleshooting The eyepiece, or ocular magnifies the

“I’m having difficulty focusing on the image formed by the objective lens.
specimen
- Make sure specimen is centred As a result, the total magnification seen by
- Start over at the lower power and the observer is obtained by multiplying the
move the stage back to its highest
position
magnification of the objective lens by the A typical microscope is constructed of
magnification of the ocular, or eyepiece. several important pieces you will need to
be aware of during general use
*Magnification – the ratio of the size of a
magnified object created by an optical Objective Lens
system.

Example: Object seen under the LPO was

100X larger than its original size.

Resolution
- Ability of the lens to distinguish two
points as clear and as distinct.
- One of the most important parts of
Depends on:
the microscope are the lenses
Wavelength of light
used to actually view the specimen
- ↓λ= ↑R
- The microscope uses the eyepiece
lens or the lens you look through,
Numerical Aperture (NA)
to present the image of the
- characteristic of the lens system.
specimen
- engraved on the condenser and
- Eyepiece lenses are typically 10X,
objective.
but may also come in 5X, 15X, or
- refers to the light-gathering ability
20x
of the lens
- The power is generally found
- expressing mathematically the
labeled on the eyepiece itself
solid cone of light delivered to the
specimen
Immersion Oil Lense

- Most lenses are used “dry” or

without oil. Some lenses use a
special type of oil to increase their
Refractive Index (RI)
resolution
- the amount light bends
- Oil is typically used with higher
- if the RI of two materials are the
powered lens such as 100x.
same, then there would be no
- These lenses typically have special
bending of light.
markings on the side of the
housing to indicate it is designed to
n oil = n glass
be used with oil
Microscope Care - Include the eyepiece and objective
- Remove the microscope from the lenses, but not the focusing block
cabinet by gripping the arm firmly
with one hand while placing the C-mount
other hand underneath the base of - Is an adapter for use with a video
the device for support camera
- Do not remove the dust cover until
the microscope is placed and Coarse Focus
positioned on the table where it will - The larger of two adjustment knobs
be used that moves to the objective lenses
closer to, or farther away from, the
Terminology Abbe Condenser specimen in large steps
- Specially-designed lens, with an
iris-type aperture, and mounted Coaxial Focus
under the stage, moves vertically - This focusing system features the
to adjust the beam of light entering coarse and fine focus knobs on a
the lens system single rotation axis
- Changing the size of the iris and - With this setup, the coarse focus
adjusting its position in relation to knob is generally larger and on the
the stage control the diameter and outside while the fine focus knob is
focal point of cone of light as it smaller and on the inside
passes through the specimen
Condenser Lens
Achromatic lenses - This lens focuses or “condenses:
- Are used to correct the bending or the light into a specimen
refraction of light as it passes - The use of a condenser helps
through the prism or lenses in the increase illumination and resolution
microscope
Arm Contrast Plate
- Is the area of the microscope - Used on low-power microscopes,
between the tube and the base this circular opaque plate is placed
on the stage, and can be flipped
Articulated Arm between white or black side
- This stand holds the microscope depending on the coloration of the
body, clamps to a table, and specimen
provides movement in three
dimensions Cover Slip
Base - Is a very thin square or plastic or
- The base is the bottom support glass used to cover the specimen
structure of the microscope on a slide

Binocular Head Diaphragm

- Features a head with two eyepiece - Typically a five-hole disc housed
lenses, one eyepiece for each eye under the stage adjusts the
Body amount of light passing through the
- Refers to the main section of the stage opening
microscope, minus the stand
(base) or any illuminators
Diopter Adjustment Illuminator
- Used on microscopes with a - This light source mounted
binocular head underneath the stage commonly
- This adjustment allows each uses one of three types of lights:
eyepiece to be focused tungsten, fluorescent, and halogen
independently to compensate for
the difference in vision between Immersion oil
your two eyes - Special oil used exclusively with a
100x higher objective lens, at
Dual Head typically 1000x total power
- Found on high-power microscopes,
this unit features a single eyepiece Inclination Joint
on one side and an additional - Some microscopes feature a
eyepiece on the top or opposite rotating arm pivot point can be
side adjusted to allow for more
comfortable viewing
Eyepiece lens
- Also known as ocular lenses Interpupillary Adjustment
- Are typically 10X - Allows the distance between the
- Is what you actually look through to eyepiece lenses, to be set either
see your specimen closer or farther apart for more
comfortable viewing
Fine Focus
- The smaller of two adjustment Mechanical Stage
knobs moves the objective lens - A slide holder featuring two knobs
closer to, or farther away from, the that allow the slide position to be
specimen in very small steps adjusted forwards and backwards
from side to side
Field of View
- Is the diameter of light visible when Micrometer
looking into the eyepiece lens - Or micron, is the metric linear
measurement used for microscopy
Fixed Arm Mirror
- A type of stand used for - This reflective surface directs light
lower-power microscopes where through the opening in the stage to
the arm and the body are integral illuminate a specimen
parts and fixed firmly to the base
Monocular Head
Focus - A microscope head featuring a
- The method by which the single eyepiece lens
specimen’s distance to the
objective lens is adjusted to Nosepiece
provide a sharp image - Also called a revolving nosepiece
or turret, this structure holds the
Head objective lenses
- Upper portion of the microscope,
contains the eyepiece tube or
tubes and prisms
Numerical Aperture (N.A.) Resolution
- This number is an expression of - A lens system’s ability to resolve
the lens’ ability to resolve fine fine details of the observed object
detail in the observed object
Reticle
Objective Lens - This special grid patter is inserted
- The lens closest to the object into the eyepice lens for taking
being viewed is known as the measurements
objective lens
Revolving Nosepiece
Oil Immersion Lens - Also called a nosepiece or turret,
- An objective lens, generally 100x this structure holds the objective
or more, designed to work with a lenses
drop of immersion oil placed on the
slide to increase the resolution of Ring Light
the lens - A separate light connected to the
microscope body that produces a
On/Off Switch ring of light
- Controls the power to the
microscope Semi-Plan Lenses
- This type of lens improves image
Percentered quality by presenting a sharper
- A microscope that is calibrated this image with less distortion in the
way will maintain the specimen in perimeter of POV
the center of the view when the - Provides better image quality;
objective lens is rotated more expensive

Parfocal Slide
- A microscope that is calibrated this - This rectangular, plate, made of
way will maintain the specimen in either plastic or glass, is used to
focus when the objective lens is hold the specimen
rotated
Slip Clutch
Pointer - This mechanical device prevents
- Some microscopes feature a gear damage to the microscope
pointer that is visible when looking when the focus adjustment
through the eyepiece of lens reaches its maximum travel

Post Stand Stage

- Typically used with low-power - This area is the main, flat surface
microscopes, this type of stand that holds the slides for
features a single post attached to observation
the base allowing the body of the
microscope to be rotated or Stage Adjustment Knob
adjusted vertically - This mechanical stage allows for
the adjustment of the slide position
forward, backward, and left or right
Stage Clips Universal Stand
- The stage has several of these - A long, boom-type arm used to
clips that are used to hold a slide in support a low power microscope
place body

Stage Plate Widefield eyepiece lenses

- Found on low-power microscopes, - These wide-diameter eyepiece
this frosted circular glass place fits lenses offer a broader FOV when
in over the lower illuminator viewing a specimenC

Stand X
- For lower-power microscopes, - This represents the magnification
there are typically three types of factor of a lens or view
connections between the
microscope body and the base XR
including Post Stand, Fixed Arm, - In this notation, the X represents
and Universal Stand the magnification of the lenses
while the R stand for retractable
Stereo - XR lenses contain a spring-loaded
- When using two objectives slightly mechanism that allows them to
offset, the image is presented in telescope inwards to prevent
3D damage to the lens

Sub-Stage
- This area is located directly below Other important features of the
the stage itself microscope objectives
- Focal length (mm) is an optical
T-mount constant of the lens system, is the
- This type of adapter is useful for distance from the center of the lens
attaching still cameras to the to the point where parallel rays
microscope entering the lens are brought to a
focus.
Tension Adjustment - Numerical aperture (N.A.) is a
- A factory adjustment of the measure of the resolving power of
focusing mechanism that makes an objective. An objective with 0.25
the microscope easy to focus N.A. allows the viewer to
distinguish as separate 25000 lines
Trinocular Head per inch.
- This type of microscope features - Working distance (mm) is the
two eyepieces for viewing and a free space between the specimen
third for attaching a camera surface and the objective.

Turret Microscopic objects can be measured by

- Also called a revolving nosepiece, means of an ocular micrometer or filar
or simply, nosepiece, holds the micrometer. It is a glass disc with mounted
objective lenses scale but no unit of measurement. It is
inserted into the eyepiece and must be
calibrated for each objective, eyepiece
and tube length of the particular See Figure 6 for the proper way of
microscope unit before measurements are alignment.
made. - Count the number of divisions on
the ocular micrometer subtended
Calibration of Ocular Micrometer (covered end to end)by the
number of divisions on the
stage micrometer. The lines must
coincide properly.
- Use the formula below to
determine the value of one ocular
micrometer division.

Stage micrometer
- is used for calibrating ocular or filar
micrometer. It is a glass slide with
graduations of known intervals.
The length of the smallest division
is 0.01 mm or 10 µm and is a
formula constant. There are 100
divisions in a stage, which gives it
a total length of 1000 µm. The unit
of linear measurement in
microbiology is the micrometer
(µm), which is equivalent to 1/1000
mm or 1/25400 inch.

Procedure for Calibration

- A compound microscope with
ocular micrometer is usually
provided in the lab.
- Place the stage micrometer on the
stage and put to focus initially
under scanner.
- The eyepiece can be rotated to
align the lines to the stage.
Likewise, the stage micrometer
can be moved so that lines
coincide perfectly with the lines on
the ocular scale. In general, we are
measuring the total length of the
ocular micrometer using our ruler,
the stage micrometer and then
diving the total measurement to the
number of lines in the ocular to get
the measure per ocular division.
 staining, acid-fast staining,
Module 3: Staining Technique endospore staining, flagella
staining, and capsule staining
Observation of microorganisms under the - Gram staining is an effective
light microscope is very difficult because method to differentiate types of cell
they are translucent. The development of walls of bacteria based on their
synthetic dyes for staining cells was then a color retention. Gram-positive
major force that led to modern cytology bacteria retain the primary stain
because they made cellular structures crystal violet; gram-negative – the
distinct. Natural dyes such as indigo and counterstain safranin.
saffron were found to react with certain
cell parts, making them far more visible These techniques often require fixing
under the microscope. specimens on the slide (called a smear) to
immobilize and kill the microbes with just
Simple stains enough heat to preserve the integrity of
- can be used as a quick and easy their cellular components for observation.
way to determine cell shape, size
and arrangements of cells like Materials, Reagents, And Cultures
bacteria. The colors absorbed by
the cells are dependent on the
charge of the colored ion, the
chromophore of the dye.

Basic dyes
- such as methylene blue, safranin,
or crystal violet, the chromophore
is positively charged and stick to
cell walls, which typically have
negative charges making the
former a positive stain.

Acidic dyes
- The reverse Preparation of Smears
- with the chromophores negatively 1. Properly label slides corresponding
charged resulting in the staining of to your 24-hr bacterial culture.
the specimen surroundings and 2. Using a sterile wire loop, place a
producing silhouettes of the loopful of water on a clean glass
organisms as in negative slide
staining. 3. From the slant culture, get a
loopful of your specimen by lightly
Differential Staining scraping the surface of the agar,
- is another type of staining then mix it with the water on your
technique that uses two or more slide until turbid as in 1.b,d. Be
dyes to distinguish organisms sure to follow aseptic technique to
based on their interactions with prevent contamination such as
multiple stains. Examples of this flaming loop before and after use,
technique commonly used in and flaming the mouth of the
clinical settings include Gram
 culture tube before and after taking - Air dry only.
your sample. - Focus under the OIO.
4. Air Dry, then fix by heat by passing - The microorganisms should
the slide quickly over the flame for appear colorless with a dark
2 to 3 times. Set aside for staining. background.

Positive Staining Differential Staining: Gram Staining

- Stain a smear with methylene blue - Stain the bacterial smears with
for 30s. ammonium oxalate crystal violet
- Wash with water and blot dry. for 1 minute. Rinse thoroughly and
Focus under the OIO. gently with tap water.
- The microorganisms should be - Cover the smears with iodine
colored blue with a bright solution (mordant) for 1 min. Rinse
background. with tap water.
- Add with 95 % ethanol drop by
Negative Staining drop for about 15 sec. or until the
alcohol becomes clear. Rinse with
tap water.
- Counterstain with safranin solution
for 1 min. Rinse with tap water and
blot dry. Air dry or blot with tissue
to dry. Focus under the OIO.
- The microorganisms should be
either blue to violet for gram
positive and pink to red for gram
negative.
- Put a drop of nigrosine dye on a
clean slide.
- Using a wire loop to take a loopful
of your bacterial culture, mix with
the dye and spread thinly. Perform
this aseptically.
 Developing a thorough understanding and
Module 4: Culture Media Preparation knowledge of aseptic techniques and
culture transfer procedures is a
Medium (media) prerequisite to working with
- in microbiology, the liquid or solid microbiological cultures. You will save
nutrient mixture(s) used to grow yourself a lot of time and energy and avoid
microorganisms erroneous results if a few simple and
common sense rules are observed when
Pure Culture working with cultures.
- a laboratory culture containing a
single species of organism. 1. Disinfect the work area before and
after use.
Mixed Culture 2. Do not touch or breathe into sterile
- one that contains more than one culture media or the stock cultures.
type of organism growing in a 3. When working with tubes, the tube
sterile medium. caps should not be placed on the
table top, they should be held in
Aseptic technique your hand while inoculating.
- a series of steps taken to prevent 4. Always sterilize your inoculating
contamination of laboratory loop by flaming before using it to
cultures and media. enter any culture material.

Contamination
- the presence of unwanted
organisms in the culture.

Aseptic Techniques

(also called sterile techniques) are defined

as the processes required for transferring Diagrammatic representation of a
a culture from one vessel to another typical flame
without introducing any additional
organisms to the culture or contaminating
the environment with the culture.

The following conditions must exist for

aseptic technique to be successful:

1. The work area must be wiped with

an antiseptic to reduce the number
of potential contaminants.
2. The transfer instruments must be
sterile.
3. The work must be accomplished
quickly and efficiently to minimize - Always flame the lip of the culture
the time of exposure during which tube before inserting your sterile
contamination of the culture or loop into the culture. This destroys
laboratory worker can occur.
 any contaminating cells that may - Closed-toe shoes
have been inadvertently deposited
near the lip of the tube during First Aid
previous transfers or by other - In case of contact with eyes,
means. This heats the air inside immediately wash the eyes with
the tube so the air moves out of large amounts of water for 15
the tube, preventing contaminants minutes, while holding eyelids
from entering the tube. open. Get medical attention
- Keep all culture materials covered immediately. If contact lenses are
with their respective caps and lids worn remove them immediately.
when not making transfers. Do not - In the event of skin contact, wash
lay tube caps or petri dish lids on the area thoroughly.
the tabletop, thereby exposing - In the event of skin contact with a
cultures to possible contamination. hot beaker, seek medical attention.
When transferring colonies from - Report any incident to the
petri plates, use the lid as a shield laboratory manager. Disposal
by slightly raising it enough so that Requirements
your loop can be inserted but the - Spills can be cleaned up with a
agar surface is still protected from paper towel and disposed of in the
contaminants falling upon it. trash. Use caution with hot media.
- Do not allow tube closures and - Unused plates (if not reusable) can
petri dish lids to touch anything be disposed of in the waste to be
except their respective culture autoclaved trash cans.
containers. This will prevent
contamination of closures and Sterilization and the autoclave
therefore of cultures. - When microbiological media has
- Minimize the time that cultures and been made, it still has to be
growth media are open to the sterilized because of microbial
environment. contamination from air, glassware,
hands, etc. Within a few hours
Bacteria are found in all parts of the there will be thousands of bacteria
laboratory environment--on the reproducing in the media so it has
workbench, in the air, on your hands, etc. to be sterilized quickly before the
The precise methods for handling sterile microbes start using the nutrients
materials, for taking samples, for making up. The sterilization process is a
cultures, and for disposing of 100% kill, and guarantees that the
contaminated materials after use are all medium will stay sterile UNLESS
designed to prevent the spread of bacteria exposed to contaminants by less
from one area to another. Close attention than adequate aseptic technique to
to details in the written procedure and with exposure to air.
instructor’s demonstrations in the lab will - Media sterilization is carried out
prevent contamination and infection. with the autoclave, basically a
huge steam cooker. Steam enters
Safety into a jacket surrounding the
- Safety Goggles or Glasses chamber. When the pressure from
- Lab Coat or Long Sleeves the steam is at a certain point in
- Gloves the jacket, a valve allows the
- Long Pants steam to enter the chamber. The
 pressure will go up over 15 pounds 1. Pellicle – mass of
per square inch (psi): at this point organisms floating on top of
the timer begins to count down--- the broth
usually for 15 minutes, depending 2. Turbidity – organisms
on the type of media. The high appear as a general
pressure in a closed container cloudiness throughout the
allows the temperature to go above broth
the highest temperature one could 3. Sediment – mass of
get by just boiling, around 121⁰C. organisms appears as a
Therefore, the parameters for deposit at the bottom of the
sterilization with an autoclave are tube
121⁰C at >15 psi for 15 minutes.
Fifteen minutes is the thermal Agar Slant
death time for most organisms - nutrient medium plus a solidifying
(except some really hardy agent, agar-agar. The medium has
sporeformers). been allowed to solidify at an angle
in order to get a flat inoculating
Culture Media surface.

Growth Media Agar Stab/Agar Deep

- To study bacteria and other - tubes of hardened agar medium
microorganisms, it is necessary to which are inoculated by stabbing
grow them in the laboratory under the inoculum into the agar
controlled conditions. Growth
media contain a variety of nutrients Agar Plates
that is necessary to sustain the - sterile petri plates that are
growth of microorganisms. aseptically filled with a melted
sterile agar medium and allowed to
Basic requirement of a culture media solidify. Plates are much less
- Nutrients confining than slants and stabs,
- Energy source and are commonly used in
- Carbon source culturing, separating and counting
- Nitrogen source microorganisms.
- Mineral salts
- Suitable pH
- Growth factors

Types of Culture Media

Broth Tube Classification

- tubes containing liquid medium. A Defined Media (Chemically-defined)
typical nutrient containing broth - are prepared by adding precise
medium such as Trypticase Soy amounts of pure inorganic or
broth, nutrient broth. After organic chemicals to distilled
incubation, growth may be water. Therefore, the exact
observed as one or a combination composition of a defined medium
of the three forms:
 (in both a qualitative and For example, on the container of the
quantitative sense) is known. nutrient agar, the following directions are
listed:
Complex Media
- are made from digests of microbial, Suspend 28g of powder in 1000mL
animal, or plant products, such as distilled water, then dissolve by heating
milk protein (casein), beef (beef until boiling. Autoclave for 20 minutes then
extract), soybeans (tryptic soy cool and pour in petri dishes.
broth), yeast cells (yeast extract),
or any of a number of other highly - So if we need to prepare 1000 mL
nutritious substances. (1L) of nutrient agar medium, we
should dissolve 28g of media
Summary of other culture media powder in 1000mL distilled water.
categories typically encountered in - Ideally, the amount of media
other references: poured in each Petri-dish is about
25-30mL (about 1/3 to ½ of the
petri dish height); and in test tubes,
broths around 5-7mL, agar slants
around 7-10mL
- If we do not need 1000mL of
media, multiply the number of
dishes needed by 20, then
Considerations for media preparations: consider it as the total volume of
tools and calculations media needed in the following
equation:
Tools, glasswares, and instruments

- All volumes should be converted to

mL and weights to g before
beginning calculations.
- From L to mL: multiply by 1000;
while from mL to L: divide by 1000
- From Kg to g: multiply by 1000;
while from g to Kg: divide by 1000

Practical example: suppose you only need

14 petri dishes with nutrient agar:
 In the laboratory, microorganisms are
Module 5: Microorganisms in the widely distributed--- on the surface of
environment objects, tables, sinks, and floors and in the
circulating air as suspended bioaerosols.
The Earth is known as a “closed system” Microbes are also present in the soil, dust,
where materials cycle between lithosphere and on the surface tissues and orifices of
(rocks), atmosphere (air), hydrosphere the human body. The human body, as well
(water), and biosphere (organism). as the Microbiology Laboratory, represent
Together, they make up all the potential sources of microorganisms that
components of our planet, both living and may contaminate cultures handled in
nonliving. Earth produces everything it common working areas. These include but
needs to ensure the survival and growth of are not limited to laboratory tables,
its residents. biosafety cabinets, etc. Contamination
might progress to human infection,
Environment especially if proper care is not observed
- is defined as the circumstances or during laboratory activities (aseptic
conditions that surround an technique)
organism or group of organisms.
The environment is the complex of Microorganisms are microscopic,
social or cultural conditions that ubiquitous organisms. They are widely
affect an individual or community. distributed anywhere. In your own home
Since humans inhabit the natural environment, they can be present on the
world as well as the built or surface of objects, clothes, tables, and
technological, social, and cultural floors. They are also present in soil, dust,
world, they all constitute an and on the surface tissues of the body. In
important part of our environment. the laboratory where microbial cultures are
prepared, the environment and the
Microorganisms individuals represent potential sources of
- One important component of the microorganisms that may contaminate
environment cultures in the working areas and may
- There is a wide range of microbes also unlikely find their way to humans that
present in our biosphere eventually lead to disease. Microbial
depending on their physical and contamination occurs when these
other characteristics. Microbes fall microorganisms enter a culture medium in
into two groups, prokaryotes and which a pure culture of microorganism is
eukaryotes, depending upon growing.
whether they have a nucleus or
not. Prokaryotes lack this Colony Morphology
membrane around their genetic - is simply the appearance or the
material, and this group includes visual culture of the colony once it
viruses, bacteria, and related grows on an agar plate. It is an
archaea. The other category of important way to identify and
microbes includes algae, fungi, isolate them via colony picking for
protists, and other microscopic other applications.
animals, having cell nuclei. - Some virulent microbial species
manifest characteristic
morphological features that are
distinguishable from non-virulent
 counterparts. It is important to note Elevation
colony characteristics such as - Describes the “side view” of a
form, margin, elevation, surface, colony
consistency, and transparency to
identify whether a microbial colony
has the potential to cause disease.
In bacteria, some quick
morphological indicators of
potential virulence include a form
(typical virulent bacteria may
Consistency
appear rhizoidal and irregular) and
- Describes the texture of the colony
consistency (viscid colonies are
- Rough colonies have a granular
potentially pathogenic due to the
- Flattened surface and are often
presence of bacterial capsule) of
associated with loss of virulence
colonies growing on a solid culture
- Smooth colonies have a glistening,
medium.
rounded surface
- Other terms used to describe
Colony Morphology Guide
colony texture include mucoid
Form
(gummy, viscous), butyrous
- Refers to the shape of the colony
(buttery texture), or dry (brittle or
or its overall appearance
powdery colonies)

Pigmentation
- Some bacterial species produce
Surface pigments that can be either
- “How does the surface of the water-soluble or soluble in fat.
colony appear” - The production of pigments may
vary depending on the
environmental conditions or age of
the colony

How to Observe And Describe Bacterial

Colony Morphology
Margin
- Refers to the edge of a colony Observing bacterial colony morphology
needs to be a systematic process. The
steps below act as a good guideline.

- Before beginning to observe the

grown colonies, note down the
medium that was used in the agar
plate to grow the bacterial
 colonies. It will act as a good Chromogenesis
reference when comparing the - Color of colonies, pigmentation:
bacteria morphology in a different white, buff, red, purple, etc.
type of medium. - Some pigments are water-soluble,
- Look at the agar plate and observe others are not.
the different colonies on it. Pick a - If you take a large inoculum and
colony that is well-isolated and place it in a tube of water or saline,
well-sized so it’s easy to look at. do you see color?
- Take a look at the colony’s form - Do you see any pigment if the
and color, followed by opacity and organism is growing in a broth
surface appearance. You can use medium?
a magnifying glass to do this if you - Does incubation temperature affect
wish. Alternatively, to get the best the color?
view of the colony, use a - Does the entire colony have the
dissecting/stereoscopic color, or is it more like a bull’s eye?
microscope and put the agar plate
under the lens with the cover Opacity of Colony
closed. Wipe down the inside of - Is the colony transparent (clear),
the cover if it is condensed before opaque (not transparent or clear),
observation. translucent (almost clear, but
- Tilt the plate to view the colony distorted vision–like looking
from the side to observe elevation through frosted glass), iridescent
and margin. Often, the odor is an (changing colors in reflected light)?
optional morphology to note down,
but you can take a quick whiff from Elevation of Colony
a bit of a distance if you’d like to - How much does the colony rise
note this down (and if it’s safe). above the agar (turn the plate on
- Lastly, to determine texture, use an end to determine height)?
inoculating loop or a needle to pick
at the colony to observe its Surface of Colony
consistency as it leaves the - Smooth, glistening, rough, dull
medium. Once you’ve noted down (opposite of glistening), rugose
all of the above, you can (wrinkled)
successfully describe the
morphology of the colony you’ve Consistency or Texture
chosen. - Butyrous (buttery), viscid (sticks to
loop, hard to get off), brittle/friable
Whole Shape of Colony (dry, breaks apart), mucoid (sticky,
- Varies from round to irregular to mucus-like)
filamentous and rhizoid (root-like)

Size of Colony
- Can vary from large colonies to
tiny colonies less than 1mm =
punctiform (pin-point). Measure
with a millimeter rule.