Enhanced Biology 8

• Escherichia coli and Salmonella cause food

poisoning.
• Helicobacter pylori cause gastritis and ulcers.
• Neisseria gonorrhoeae causes the sexually
transmitted disease gonorrhea.
• Neisseria meningitis causes meningitis.
• Staphylococcus aureus causes a variety of
infections in the body, including boils, cellulitis,
abscesses, wound infections, toxic shock syndrome,
pneumonia, and food poisoning.
• Streptococcal bacteria cause a variety of infections
in the body, including pneumonia, meningitis, ear
infections, and strep throat.
 Good bacteria

• Bifidobacterium bifidum can help to repair

stomach ulcers caused by Helicobacter pylori.
• Streptococcus thermophilus is a starter culture in
the manufacturing process of yogurt, mozzarella
cheese and other fermented dairy products.
• Bacillus coagulans may be useful in the treatment
of gastrointestinal disorders such as diarrhea
associated with an antibiotic regimen, traveler's and
children's diarrhea, inflammatory bowel disease and
irritable bowel syndrome.
• As they exist in nature, most bacteria are colorless,
transparent, and difficult to see.

• Therefore, different staining techniques are devised

to enable scientists to examine bacteria.

• In preparation for staining, the bacteria are smeared

onto a glass slide, air-dried and then “fixed”.
Scientist uses different procedures or techniques in
identifying organisms.

Fixation
Staining
• What is Fixation?
 Fixation?????

• It is the process to
preserve or fix into
position.
 Types of Fixation
• Heat Fixation
- Is usually accomplished by passing the smear through
a Bunsen burner flame.
 Methanol Fixation

- Is accomplished by flooding the smear with absolute

methanol for 30 seconds, is a more satisfactory
fixation technique.
 Three purposes of fixation

• It kills the organisms.

• It preserves their morphology (shape).
• It anchors the smear to the slide.
 Simple Staining

• Determines bacterial shape and morphological

arrangement.
• For this method, a dye (such as methylene blue) is
applied to the fixed smear, rinsed, dried, and
examined under the microscope.
 Gram staining

• In 1883, Dr. Hans Christian Joachim Gram

developed a staining procedure in the bacteriology
laboratory.
• The procedure he developed-now called the Gram
stain- demonstrated two general categories of
bacteria cause pneumonia: some of them stained
blue and some of them stained red.
Hans Gram
• The Gram stain is a very important preliminary step
in the initial characterization and classification of
bacteria.
• It is also a key procedure in the identification of
bacteria based on staining characteristics, enabling
the bacteria to be examined using a light microscope.
• If a bacterium is blue to purple at the end of the
Gram staining procedure, it is said to be Gram-
positive.
• If, on the other hand, it ends up being pink to red, it
is said to be Gram-negative.
Gram positive
bacteria
Staphylococcus aureus
Gram negative
bacteria
Escherichia coli
The Gram stain has four steps:
• 1. crystal violet, the primary stain: followed by

• 2. iodine, which acts as a mordant by forming a crystal violet-

iodine complex, then

• 3. alcohol, which decolorizes, followed by

•
4. safranin, the counterstain.
 Steps in Gram staining

• Heat fix specimen to the slide. Flood slide with

crystal violet solution; allow to act for 1 minute.
• Rinse the slide, then flood with iodine solution; allow
iodine to stand for 1minute.
• Before acetone decolorization (next step), all
organisms appear purple, that is gram positive.
 Steps in gram staining

• Rinse off excess iodine. Decolorize with acetone,

approximately 5 seconds.
• Wash slide immediately in water. After acetone
decolorization, those organisms that are gram-
negative are no longer visible.
• Apply safranin counterstain for 30 seconds.