Laboratory 1: Media, Culture & Staining

Laboratory 1
Media, Culture &

Staining
Presented by: Mirna Rajeh

This week …
The purpose of this lab is to
expose you to:
- Culture media
- Aseptic techniques
- Differential staining
You will begin to understand the ubiquitous nature

of bacteria.
Culture Media
• Microorganisms must have a constant
nutrient supply to survive.
• In order to successfully grow bacteria in the lab,

we must provide an environment suitable for growth, and this can be
established using culture media (singular = medium).
• Media = mixtures of nutrients that bacteria require in order to live.
• Media may be liquid (broth), semi-solid, or solid (agar).
• Agar is the solidifying material used in solid media.

How is media made?
• Media is made by measuring out a certain quantity

of dry powdered nutrient media and adding a
certain volume of distilled water according to the
manufacturer.
• Autoclave
• The autoclave exposes the media to high

temperature (121°C) and pressure (15 psi) for 20
minutes.
• Once the media is autoclaved, it is considered

sterile (all life forms killed).
Aseptically pouring
agar plates
Agar plates are stored upside down to
prevent condensation.
Types of
Culture Media
General Purpose Media:
Nutrient Broth and Nutrient Agar
Enriched Media
Chocolate Agar
Differential
and
Selective Media
1. What Does Selective Mean?
2. What Does Differential Mean?

MacConkey (MAC)
MacConkey is both selective & differential.
1. MacConkey is selective because crystal violet & bile salts

inhibit the growth of Gram positive bacteria & allows the
isolation of Gram negative bacteria.
2. MacConkey is differential because the incorporation of the carbohydrate lactose and

the pH indicator neutral red permits the differentiation of bacteria on the basis of
their ability to ferment lactose.
- Bacteria that ferment lactose (a type of sugar), produce acidic metabolites which cause the
pH indicator neutral red to turn red.
- These “lactose fermenters” will grow in pink colonies while non-lactose fermenters will be
colorless and clear.
Blood agar (BAP)
Most specimens received in a clinical microbiology lab

are plated onto Blood Agar, since it supports the
growth of most organisms (contains 5% sheep
blood).
This media is not selective, only differential:
Certain bacteria produce enzymes (hemolysins) that

act on the red cells to produce either:
* Beta hemolysis: Enzymes lyse the blood cells
completely, producing a clear area around the
colony.
* Alpha hemolysis: Incomplete hemolysis produces

a greenish discoloration around the colony.
* Gamma hemolysis: No effect on the red cells.

Mannitol Salt (MSA)
Is a selective media with a high concentration of
NaCI (7.5%).
Most bacteria cannot survive in this

environment.
The genus Staphylococcus, however, grows well

in this media.
Mannitol Salt is also differential because it

contains a dye that identifies organisms that
ferment mannitol. The organic acids
produced change the dye from red to
yellow.
This medium works well for identifying

pathogenic staphylococci, such as
Staphylococcus aureus, which will ferment
mannitol.
Most non-pathogenic staphylococci will not

ferment mannitol.
Gel media (in-between liquid broth and solid
agar) is used to test for motility
Spreading out
indicates motility
ASEPTIC TECHNIQUE
• Microorganisms are very
ubiquitous
• Aseptic technique prevents
environmental organisms from
entering a culture
– Wearing gloves
– Cleaning benches
– Working near the flame
– Use sterile transfer loops
CULTIVATION
• When working with
microorganisms it is
desirable to work with a
pure culture.
• A pure culture is composed
of only one kind of
microorganism.
• In a mixed culture there
are two or more organisms
that have distinct
characteristics.
• To get the final desired
separation, we use the
streaking plate technique.
STREAKING PLATE TECHNIQUE
1
Nice isolation!
1
2
4
3
Streak pattern 4
3
A dilution technique
that involves
spreading a loopful of
culture over the
Bacterial growth pattern
surface of an agar
plate.
Starting with a mixed
culture, isolation of
different species may be
obtained by streaking
STAINING
• Bacteria have almost the same refractive
index as water.
• Staining is done for making microbial cells
easier to visualize.
• Simple stains use only one dye that stains
the cell wall of bacteria.
• Differential stains use two stains and
categorize cells into groups.
• Both staining techniques allow the
detection of cell morphologies or shape:
cocci or spherical, bacilli or rods,
coccobacilli, spiral…
• The most common differential stain used in
microbiology is the Gram stain.
GRAM STAIN
Distinguishes between two large groups of microorganisms:
- purple staining, Gram-positive cells
- pink staining, Gram-negative cells
These two types of cells differ significantly in the chemical and physical structure
of their cell wall.
• Bacterial film or smear is made on a glass slide & fixed with gentle heating
• Procedure
1. Flood the smears with crystal violet & let stand for one minute
2. Wash with tap water
3. Flood smears with the Gram’s iodine mordant & let stand for 2 minutes
5. Decolorize with 95% ethyl alcohol. Caution: Do not over decolorize. Add reagent drop by drop
until crystal violet fails to wash from smear.
7. Counter stain with safranin for 1 min
8. Blot dry with tissue paper & examine under oil immersion
Acid-Fast Stain
This stains the cells of the genera Mycobacterium and Nocardia, which
cause many diseases in humans, including tuberculosis, leprosy, and
other lung and skin infections.
Cells of these bacteria have large amounts of waxy lipid in their cells
walls, so the Gram stain and other water-based stains don’t work well on
them.
Cells are determined to be acid-fast or not, based on whether they retain

their primary stain after decolorization.
ACID FAST STAIN
1. Ziehl Neelsen acid fast stain:
It is used for the identification Of Mycobacteria.
Because of the presence of the waxy cell wall,
heat is applied to allow the stain to penetrate
the cell wall.
Reagents
Primary stain: Carbolfuchsin

Decolorizer: Acid alcohol (HCL, and concentrated ethanol)
Counter stain: Methylene blue
PROCEDURE
• Prepare a heat fixed smear
• Flood the smear with carbolfuchsin
& heat slowly for 3-5 min, do not
allow the slide to boil or dry out
• Rinse with tap water
• Flood the smear with acid alcohol &
let stand for 2 min
• Flood the smear with methylene
blue & let stand for 1 min
• Air dry & examine under oil
immersion
Acid fast organism stain pink-red, other

organisms stain blue.
ACID FAST STAIN
2. Kinyoun acid fast stain (cold
method):
It is used for the identification of
Mycobacteria. No heat is applied
here, but higher concentration of
carbolfuchsin & phenol is used to
allow the stain to penetrate the cell
wall
Reagents
Primary stain: modified carbolfuchsin
Decolorizer: acid alcohol (HCL,
concentrated ethanol)
Counter stain: methylene blue
PROCEDURE
1. Prepare a heat fixed smear
2. Flood the smear with carbolfuchsin & let it
stand for 5 min at room temp
3. Rinse with tap water
4. Flood the smear with acid alcohol and let
stand for 2 min
6. Flood the smear with methylene blue & let
stand for 1-3 min
8. Air dry & examine under oil immersion
Acid fast organism stain pink-red, other organisms

stain blue.

Laboratory 1: Media, Culture & Staining

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Laboratory 1

Media, Culture &

Presented by: Mirna Rajeh

You will begin to understand the ubiquitous nature

• In order to successfully grow bacteria in the lab,

• Media = mixtures of nutrients that bacteria require in order to live.

• Media may be liquid (broth), semi-solid, or solid (agar).

• Agar is the solidifying material used in solid media.

• Media is made by measuring out a certain quantity

• The autoclave exposes the media to high

• Once the media is autoclaved, it is considered

1. What Does Selective Mean?

2. What Does Differential Mean?

MacConkey is both selective & differential.

1. MacConkey is selective because crystal violet & bile salts

2. MacConkey is differential because the incorporation of the carbohydrate lactose and

Most specimens received in a clinical microbiology lab

This media is not selective, only differential:

Certain bacteria produce enzymes (hemolysins) that

* Alpha hemolysis: Incomplete hemolysis produces

* Gamma hemolysis: No effect on the red cells.

Most bacteria cannot survive in this

The genus Staphylococcus, however, grows well

Mannitol Salt is also differential because it

This medium works well for identifying

Most non-pathogenic staphylococci will not

Cells are determined to be acid-fast or not, based on whether they retain

Primary stain: Carbolfuchsin

Acid fast organism stain pink-red, other

Acid fast organism stain pink-red, other organisms

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