BACTERIOLOGY

Two Types of Cell

1. Eukaryotic
2. Gram’s Iodine (Mordant)
- eu; true, karyo; nucleus
- Mordant makes dye less soluble so it adheres to
- cells with true nucleus
cell walls
- animal cell and plant cell
- Iodine is added to enhance the attachment of
2. Prokaryotic
the dye to the peptidoglycan layer of the
- pro; before, karyo; nucleus
bacteria
- cells without true nucleus, but only have
- Gram (+) = absorbed; purple
chromosomes
- Gram (-) = absorbed; purple
- bacterial cell
3. 95% Alcohol (Decolorizer)
- Decolorizer washes away stain from gram (-)
Bacteria cell walls
- single-celled organisms (prokaryotic cell) - Gram (+) = not decolorized because of thick
- cause disease to man peptidoglycan layer; purple
- Gram (-) = decolorized because of thin
Three ways how bacteria cause diseases:
peptidoglycan layer; colorless
1. producing toxic substances
4. Safranin (Counter Stain)
2. compete with the body’s resources
- Counterstain allows dye adherence to gram (-)
3. displacing normal tissues of the body during
cell walls
their replication
- Gram (+) = not absorbed because it still has the
initial stain; purple
Classification of Bacteria - Gram (-) = absorbed; red because of red
According to shape safranin
a. Coccus – “berry,” round/spherical shaped
b. Bacillus – “stick or rod”
According to Acid Fast Stain Reaction
c. Spirals – corkscrew
- Acid fast staining is particularly done on
d. Pleomorphic – no definite shape because of
bacteria (bacilli) with mycolic acid in its cell
lack of cell wall
wall, which is only seen in genus
Mycobacterium.
According to arrangement
- Opposite of Gram’s Reaction
Arrangement Prefix Example
a. Acid Fast Bacilli or Acid Fast Positive (red
single
or pink) – initial stain has an affinity to the
pairs diplo Diplococcus pneumoniae mycolic acid giving it a red or pink color
chains strepto Streptococcus pyogenes b. Non-acid Fast Bacilli or Acid Fast Negative
cluster staphylo Staphylococcus aureus (blue or violet) - bacteria are stained with
4’s tetrad Gaffkya tetragena the counter stain giving it a blue or violet
8’s sacrina Sacrina lutaea color
palisade Corynebacterium
Acid Fast Staining Technique
(side-by-side) diphtheriae
1. Carbol Fuchsin (Initial Stain)
- Acid Fast Bacilli = absorbed; red
According to Gram’s Reaction - Non-acid Fast Bacilli = absorbed; red
- Average size of 1 micrometer, that is 1/1000 of 2. Tergitol (Mordant)
a millimeter - Acid Fast Bacilli = absorbed; red
- Hans Christian Gram introduced the use of this - Non-acid Fast Bacilli = absorbed; red
method of staining the peptidoglycan of the 3. Acid Alcohol (Decolorizer)
cell wall to distinguish between 2 kinds of - Acid Fast Bacilli = not decolorized because of
bacteria: the strong attachment to mycolic acid; red
a. Gram positive (blue or violet) – thick - Non-acid Fast Bacilli = decolorized; colorless
peptidoglycan layer which retain the initial 4. Methylene Blue (Counter Stain)
stain - Acid Fast Bacilli = not absorbed; red
b. Gram negative (pink or red) – thin - Non-acid Fast Bacilli = absorbed; blue
peptidoglycan layer thus washed off when
exposed to the decolorizer, making it
stained with the counter stain Other Classifications
1. Oxygen Requirement
Gram Staining Technique - Aerobic
1. Crystal Violet (Initial Stain) - Anaerobic
- Primary stain added to specimen smear 2. Osmotic Pressure
- Gram (+) = absorbed; purple - Halophiles
- Gram (-) = absorbed; purple - Facultative Halophiles
- Non Halophiles
 BACTERIOLOGY

3. pH
- Acidophiles
- Alkaliphiles
- Neutrophiles
4. Temperature Requirement
- Psychrophiles
- Thermophiles
- Mesophiles
5. Nutritional Requirement
- Photoautotrophs
- Chemoautotrophs
- Chemoheterotrophs

Processing of Bacterial Samples

Routine bacteriologic processes are divided into 3
stages. The objective of these tests is to have as much
evidences of the characteristics of the bacteria being
identified.

1. Macroscopic Evaluation
- Purpose: to observe for cultural characteristics
that are visible to the eyes
- Tests performed: appearance on culture media,
color, odor, pigments produced, elevation,
hemolytic pattern
2. Microscopic Evaluation
- Purpose: uses the microscope and staining
techniques to observe bacterial morphology or
microscopic characteristics (like shape,
arrangement, and grams pre-action)
- Tests performed: Gram’s Reaction, Acid Fast
Reaction, and Morphology (shape and
arrangement)
3. Biochemical Tests
- Purpose: tests are done to determine chemicals
that are utilized or produced by bacteria (like
carbohydrates, proteins, lipids)
- Tests performed: tests for CHO, CHON, Lipids,
and Anti-microbial Susceptibility

Steps on Processing of Bacterial Samples

1. Specimen collection
2. Initial Gram’s stain – to see if bacteria is really
present in the specimen
3. Macroscopic evaluation
– Involves inoculating of bacteria in a culture
medium (petri-dish)
– Cultural characteristics (isolation and
subculture)
– Subculturing: getting a part of the grown
bacteria to make it a pure culture
4.