PHARMACEUTICAL MICROBIOLOGY & - surgical technique in which all
PARASITOLOGY microorganisms are excluded
from the surgical field
2. Contaminant
MICROSCOPY - an unwanted organism in an
otherwise pure culture
3. Culture
- the technique of isolating and
growing one species of
microorganisms on a growth
medium
4. Medium
- a nutrient substance used to
grow microorganisms
STAINING PROCEDURES:
o Gram Staining:
- method of staining bacteria
- Hans Christian Joachim Gram
o Steps in Gram Staining:
1. Smear is heat fixed and stained with
(Microscope) Gentian violet (primary stain)
2. Treated with Gram’s solution (1part
CHARACTERISTICS OF THE VARIOUS TYPES OF iodine, 2 parts potassium
MICROSCOPES: iodides, 300 parts water)
3. Washed with ethyl alcohol
Brightfield (decolorizing agent)
- used to observe morphology of 4. Bacteria retain the strong blue
microorganisms such as bacteria, color of gentian violet (gram
protozoa, fungi and algae in living positive)
(unstained state) and non-living 5. If completely decolorized, counter
(stained state) stained with safranin turns
Darkfield reddish color (gram negative)
- useful for examining spirochetes
- example: Treponema pallidum o Staining Procedures:
- syphilis and yaws Gram + Gram -
Phase Contrast Color at end blue to pink to red
purple
- can observe dense structures in living
Peptidoglycan thick layer thin layer
prokaryotic and eukaryotic
in cell walls
microorganisms Lipopolysaccharide absent present in
Fluorescence cell walls
- fluorescent dye attached to specimen
- primarily a diagnostic technique to o Acid Fast Stain (carbol fuchsin)
detect microorganisms in cells, tissue - Mycobacteria
and clinical specimens
Transmission Electron Microscope DIFFERENTIAL STAINING PROCEDURES:
- viruses 1. Gram Staining
Scanning Electron Microscope - to differentiate between gram-
- viruses positive and gram-negative
Resolving Power or Resolution bacteria
- a limit as to what can be seen using 2. Acid Fast Stain
the instrument (microscope) - to differentiate between acid
Total magnification fast and non-acid fast bacteria
- ocular lens x magnifying lens
- example: 10 x 100 equals x1000 Types of Stains
Low Power Objective 1. Simple Stain
- used to locate microorganisms to be - employing usually methylene blue,
studied crystal violet or carbol fuchsin
High Power or High Dry - only single color so that it reveal/differ
- used to study algae, protozoa, and in size, shape and arrangement of cells
other large microorganisms 2. Differential Stains
Oil Immersion Objective
, - if alcohol is added, proteins are
STAINING REACTIONS: coagulated which results to a
corresponding increase in pore resize
1. GRAM STAIN which will increase in permeability
- effectively divide bacteria into two (2) c. Magnesium ribonucleate
large groups - protein complex of which gram staining is
- devised by Hans Christian Gram associated
A. Gram positive - responsible for gram positive staining
- those that will retain the
primary stain and are colored CONDITIONS when Gram-positive
violet becomes Gram-negative:
RULE: all cocci are gram positive 1. whether cell wall is physically damage or
except the Neisseria group and physically disrupted
Branhamella (Moraxella) 2. when cells that you are staining is “old”
catarrhalis culture, therefore, there is deterioration of
B. Gram negative cell wall
- cells which are decolorized and 3. when cells are exposed to antibiotics
are stained by the counterstain especially those whose target sight are cell
pink or red wall
RULE: all bacilli are gram 4. the cells themselves have autolytic
negative except the Acid group enzymes which acts on cell wall
(Mycobacterium, Nocardia), Loss of integrity of the cell wall
Spore formers (Bacillus and would lead to organisms being
Clostridium) and unable to retain the primary stain of
Corynebacterium species CVI complex
Gram negative cells are always gram
NOTE: Spirals are difficult to stain, and if negative
stained, they will be gram negative.
2. ACID FAST CHAIN
STEPS IN GRAM STAINING: - walls of certain bacteria contain long
1. make a smear chain of mycolic acid which gives
2. add crystal violet organisms the ability to resist
- primary or initial stain decolorization by acid alcohol
- both gram-positive and gram-negative - presence of mycolic acid leads to a
cells are violet difficulty in staining
3. add Gram’s iodine - to make dye to enter, apply heat of
- serve as mordant steaming
- enhance color of the primary stain
- once added, insoluble complex is STEPS IN ACID FAST STAINING:
formed within the cell known as Crystal 1. make a smear
violet iodine complex 2. add carbolfuchsin
4. add 95% alcohol - allowed to stay for some time in the
- act as decolorizer smear
- or use 50/50 mixture of alcohol and 3. apply acid alcohol
acetone - as decolorizer
- gram-positive are still violet while 4. apply methylene blue
gram negative is colorless - as counterstain
5. add safranin (pink) - if color of primary stain is taken up by
- color of the gram negative and gram acid fast organism, non-acid fast
positive are still violet organism which is decolorized by acid
alcohol takes the color of counterstain
FOUR BASIC STAINS:
1. methylene blue
2. crystal violet
3. carbol fuchsin
4. safranin
REASONS why there is different staining:
a. Gram positive
- has more protein in the cell envelope
- if alcohol is added, proteins are
coagulated which leads to a decrease in
pore size which results to a decrease in
PARASITOLOGY microorganisms are excluded
from the surgical field
2. Contaminant
MICROSCOPY - an unwanted organism in an
otherwise pure culture
3. Culture
- the technique of isolating and
growing one species of
microorganisms on a growth
medium
4. Medium
- a nutrient substance used to
grow microorganisms
STAINING PROCEDURES:
o Gram Staining:
- method of staining bacteria
- Hans Christian Joachim Gram
o Steps in Gram Staining:
1. Smear is heat fixed and stained with
(Microscope) Gentian violet (primary stain)
2. Treated with Gram’s solution (1part
CHARACTERISTICS OF THE VARIOUS TYPES OF iodine, 2 parts potassium
MICROSCOPES: iodides, 300 parts water)
3. Washed with ethyl alcohol
Brightfield (decolorizing agent)
- used to observe morphology of 4. Bacteria retain the strong blue
microorganisms such as bacteria, color of gentian violet (gram
protozoa, fungi and algae in living positive)
(unstained state) and non-living 5. If completely decolorized, counter
(stained state) stained with safranin turns
Darkfield reddish color (gram negative)
- useful for examining spirochetes
- example: Treponema pallidum o Staining Procedures:
- syphilis and yaws Gram + Gram -
Phase Contrast Color at end blue to pink to red
purple
- can observe dense structures in living
Peptidoglycan thick layer thin layer
prokaryotic and eukaryotic
in cell walls
microorganisms Lipopolysaccharide absent present in
Fluorescence cell walls
- fluorescent dye attached to specimen
- primarily a diagnostic technique to o Acid Fast Stain (carbol fuchsin)
detect microorganisms in cells, tissue - Mycobacteria
and clinical specimens
Transmission Electron Microscope DIFFERENTIAL STAINING PROCEDURES:
- viruses 1. Gram Staining
Scanning Electron Microscope - to differentiate between gram-
- viruses positive and gram-negative
Resolving Power or Resolution bacteria
- a limit as to what can be seen using 2. Acid Fast Stain
the instrument (microscope) - to differentiate between acid
Total magnification fast and non-acid fast bacteria
- ocular lens x magnifying lens
- example: 10 x 100 equals x1000 Types of Stains
Low Power Objective 1. Simple Stain
- used to locate microorganisms to be - employing usually methylene blue,
studied crystal violet or carbol fuchsin
High Power or High Dry - only single color so that it reveal/differ
- used to study algae, protozoa, and in size, shape and arrangement of cells
other large microorganisms 2. Differential Stains
Oil Immersion Objective
, - if alcohol is added, proteins are
STAINING REACTIONS: coagulated which results to a
corresponding increase in pore resize
1. GRAM STAIN which will increase in permeability
- effectively divide bacteria into two (2) c. Magnesium ribonucleate
large groups - protein complex of which gram staining is
- devised by Hans Christian Gram associated
A. Gram positive - responsible for gram positive staining
- those that will retain the
primary stain and are colored CONDITIONS when Gram-positive
violet becomes Gram-negative:
RULE: all cocci are gram positive 1. whether cell wall is physically damage or
except the Neisseria group and physically disrupted
Branhamella (Moraxella) 2. when cells that you are staining is “old”
catarrhalis culture, therefore, there is deterioration of
B. Gram negative cell wall
- cells which are decolorized and 3. when cells are exposed to antibiotics
are stained by the counterstain especially those whose target sight are cell
pink or red wall
RULE: all bacilli are gram 4. the cells themselves have autolytic
negative except the Acid group enzymes which acts on cell wall
(Mycobacterium, Nocardia), Loss of integrity of the cell wall
Spore formers (Bacillus and would lead to organisms being
Clostridium) and unable to retain the primary stain of
Corynebacterium species CVI complex
Gram negative cells are always gram
NOTE: Spirals are difficult to stain, and if negative
stained, they will be gram negative.
2. ACID FAST CHAIN
STEPS IN GRAM STAINING: - walls of certain bacteria contain long
1. make a smear chain of mycolic acid which gives
2. add crystal violet organisms the ability to resist
- primary or initial stain decolorization by acid alcohol
- both gram-positive and gram-negative - presence of mycolic acid leads to a
cells are violet difficulty in staining
3. add Gram’s iodine - to make dye to enter, apply heat of
- serve as mordant steaming
- enhance color of the primary stain
- once added, insoluble complex is STEPS IN ACID FAST STAINING:
formed within the cell known as Crystal 1. make a smear
violet iodine complex 2. add carbolfuchsin
4. add 95% alcohol - allowed to stay for some time in the
- act as decolorizer smear
- or use 50/50 mixture of alcohol and 3. apply acid alcohol
acetone - as decolorizer
- gram-positive are still violet while 4. apply methylene blue
gram negative is colorless - as counterstain
5. add safranin (pink) - if color of primary stain is taken up by
- color of the gram negative and gram acid fast organism, non-acid fast
positive are still violet organism which is decolorized by acid
alcohol takes the color of counterstain
FOUR BASIC STAINS:
1. methylene blue
2. crystal violet
3. carbol fuchsin
4. safranin
REASONS why there is different staining:
a. Gram positive
- has more protein in the cell envelope
- if alcohol is added, proteins are
coagulated which leads to a decrease in
pore size which results to a decrease in
