Microbiology Laboratory

MEDIA PREPARATION, STERILIZATION & – solid and semi-solid media contain a solidifying
ASEPTIC TECHNIQUE agent such as agar or gelatin.
– agar, which is a polysaccharide derived from red
Preparation and Sterilization of Bacterial Culture seaweed (Rhodophyceae) is preferred because it is an
Media inert, non-nutritive substance.
– agar provides a solid growth surface for the bacteria.
Bacteria – Antoni Van Leeuwenhoek – colonies: the formation of the distinctive lumps of
– Robert Koch, Louis Pasteur, and friends cells that bacteria reproduce.

The methods of these scientists are still widely used

today. Most of these methods involved isolating single
bacteria derived from a natural source (such as a
diseased animal or human) and cultivating them in an
artificial environment as a pure culture to facilitate
additional studies.

Growing bacteria in pure culture

– most widely used methods in microbiology.
– bacteria are heterotrophic, which means that they
rely on organic compounds as food to provide energy Types of Culture Media:
and carbon. • Synthetic – pure compounds in exact chemical
– some even require added nutritional components like formula
vitamins in their diet. • Complex/Non-synthetic – contains at least one
– thus, an appropriate physical environment must be ingredient that is not chemically definable
created
• General Purpose – grows a broad range of
– where important factors such as temperature, pH,
microbes
and the concentration of atmospheric gases
(particularly oxygen) are controlled and maintained.

Bacteriological culture media can be prepared as:

• A liquid (broth)
• A solid (plate media or slant media)
• A semisolid (deeps)

• Differential – differentiates colonies of desired

microbes from others

• Selective – inhibits the growth of certain

microbes
 • Enriched – contains complex organic
substances such as blood

Culture Media Preparation Heat Sterilization

Reminders and Tips: UV & Filtration Sterilization

1. When preparing broths, heat the flask with
constant stirring prior to sterilization. This is to
make sure the media dissolves properly and
evenly.
2. When preparing agar for plates, most
manufacturers require the media to be heated
up to boiling with constant stirring prior to
sterilization. This is to make sure the agar will
solidify and media dissolves properly and
evenly.
3. When preparing agar slants, heat to boiling the
media then distribute ahead to the tubes
before agar solidifies. Sterilize the tubes in the
autoclave and slant it after sterilization.
4. Never overfill a container for sterilization, this
might lead to spillage in the autoclave. The Autoclave
Example: Preparing 800 ml nutrient agar in a
1,000 ml flask.
5. Always read the manufacturer’s directions in
the bottle, instructions for media preparation
can vary depending on the brand or use.
6. Always label the date when media was
prepared.
7. Never use mineral water, always used distilled
or deionized water.

Sterilization
– the process of making any material entirely free from
living microorganisms.
– can be done by physical means such as heat,
filtration, and UV light, or by chemical means such as
chemicals and antibiotics (disinfection).
Procedure: – always remember that bacteria are present on all
surfaces in the laboratory, as well as on your own
hands and clothing.
– these techniques are designed to prevent the
transfer of bacteria from the surrounding environment
into a culture medium.

1. Dissolved the desired ingredients or the

complete dehydrated medium into an
appropriate volume of distilled water.
2. Then determine the pH of the medium by
using a pH meter and adjust it if necessary.
3. Add agar to this medium and boiled it to
dissolve the agar in case of solid medium is
required.
4. Then dispensed the medium into tubes or
flask.
5. After that, sterilized them by using an
autoclave. Some heat-labile media or specific Describing Colonial Morphology of Bacteria
ingredients are sterilized by using filtration. – looking closely at the colonial growth on the surface
of a solid medium (agar plate), characteristics such as
surface texture, transparency, and the color or hue of
the microbial growth can be described.
Aseptic Techniques and Growth Media • Texture - describes how the surface of the
colony appears (smooth, glistening, mucoid,
Growth Media slimy, dry, powdery, flaky etc.)
– can be categorized based on their chemical • Transparency - colonies may be transparent
constituents, or the purpose for which they are used. (you can see through them), translucent (light
• Complex growth media - contain ingredients passes through them), or opaque (solid-
whose exact chemical composition is unknown appearing).
(e.g. blood, yeast extract, etc.). • Color/Pigmentation - many bacteria produce
• Synthetic (also called chemically defined) - intracellular pigments which cause their
growth media are formulated to an exactly colonies to appear a distinct color, such as
defined chemical composition. yellow, pink, purple or red. Also, many bacteria
• General purpose growth medium (e.g. do not produce any pigment and appear white
tryptic soy agar (TSA) or Luria broth (LB) - is or gray.
used to grow a wide variety of non-fastidious – as the bacterial population increases in number, the
bacteria. This type of medium is often a colonies get larger and begin to take on a shape or
complex growth medium. form.
• Selective growth medium - contains – these can be quite distinctive and provide a good
chemicals that allow some types of bacteria to way to tell colonies apart when they are similar in color
grow, while inhibiting the growth of other types. or texture.
• Differential growth medium - is formulated
such that different types of bacteria will grow
with different characteristics (e.g. colony
color).

Blood Agar
– an example of differential growth medium which
differentiates among bacteria based on their ability to
break down red blood cells and hemoglobin.
– also a complex growth medium because it contains
blood.

Inoculation
– the purposeful introduction of bacteria into a sterile
growth medium.
– a material is sterile when it has no living organisms
present; contamination is the presence of unwanted
microorganisms.

Aseptic Techniques
– are practices that prevent the contamination of
growth media.
– these techniques require care and concentration.
Cultural Characteristics of Microbes Subculturing Procedure:

Plate Methods:
• Pour plate
- still liquid agar is poured over a liquid sample,
mixed, solidified and incubated.
- colonies grow within the nutrient agar.
• Spread plate
- agar is first poured and solidified before the
liquid sample is spread over the surface.
- colonies grow on the surface of the nutrient
agar.

Bacterial Growth through time

Sample Dilution for Plating

Bacterial Isolation
Using the SFIC (Subculturing For Isolated Colonies)
Method
 BACTERIAL SMEARING AND STAINING – used to determine a
bacterial species’ morphology
Preparation of Bacterial Smears and Introduction and arrangement, but they do
to Staining not give any additional
information.
Bacterial Smearing
1. Preparing the slides
2. Labelling
3. Smearing – different techniques, depending on
where they are grown
4. Heat fixation

• Differential Staining – use of two contrasting

stains separated by a
decolorizing agent.
Procedure: – cells will have a different
appearance based on their
chemical or structural
properties.
– For identification: Gram
stain, acid-fast stain
– For Visualization of
structure: Capsule stain, spore
stain

Gram Stain
– very commonly used staining procedure was first
developed by the Danish bacteriologist Hans Christian
Gram in 1882 (published in 1884) while working with
tissue samples from the lungs of patients who had died
from pneumonia.
– results reflect differences in cell wall composition.
• Gram Positive cells
- have thick layers of a peptidoglycan (a
carbohydrate) in their cell walls.
- also have teichoic acids.
• Gram negative cells
- have very little peptidoglycan layers.
- no teichoic cells.
- have an outer membrane that resembles the
phospholipid bilayer of the cell membrane.
– Gram stains are best performed on fresh cultures—
older cells may have damaged cell walls and not give
Staining the proper Gram reaction.
Most types of cells do not have much natural pigment – Some species are known as Gram-variable, and so
and are therefore difficult to see under the light both Gram positive and Gram-negative reactions may
microscope unless they are stained. be visible on your slide.
Specific staining techniques can be used to determine
the cells’ biochemical or structural properties, such as
cell wall type and presence or absence of endospores.

Types of Staining Techniques:

• Simple Staining – use of a single stain.
– all types of bacteria appear
as the color of that stain.
– include crystal violet,
safranin, and methylene blue.
 Understanding the Stain:
• Bacterial cell walls are made up of
peptidoglycan - repeating disaccharide
attached by polypeptides in a lattice
• Gram-positive cell walls contain more
peptidoglycan layers compared to Gram-
negative cell walls.
• Crystal violet (CV) stains all cells because it
enters the cytoplasm.
• Iodine forms large crystals with CV so it cannot
escape through the cell walls.

In Gram positive bacteria

– Alcohol dehydrates peptidoglycan making it more
impermeable to the primary dye.

In Gram negative bacteria

– Alcohol dissolves the outer membrane, and can
leave holes in the peptidoglycan, and allows CV to
escape.

Procedure:
Gram Stain: Streak Plate:

result:

result: