WK 3 Flashcards
PURPOSE OF STAINING SPECIMEN
- Increase visibility of specimen
- Differentiate one organism from another
o Some microorganism will take color under a given
condition. In that case, we will be able to differentiate
one bacteria to another
o Just like in the use of differential stains like AFB stains
or Gram stains - Accentuate specific morphological features
o There are the so-called differential stains, which react
only with particular structures of bacteria.
o Example: specific stain will only color the spore; stain
to color the metachromatic granules - Preserve specimen
o By putting on cover slip with an adhesive and it will
preserve the morphology
react
only with particular structures of bacteria
DIFFERENTIAL STAINS
HOW TO PRESERVE SPEIMEN
By putting on cover slip with an adhesive and it will
preserve the morphologY
coloring agent used for general purposes
DYE
dye used for biological purposes (specific)
STAIN
derived from coal tar
rendered synthetically to become a stain
ANILINE DYE
Colorless organic compound that can bind to the nitro
group of chromophores.
HIGHLY TOXIC SILA
BENZENE
Chemical groups with conjugated double bonds,
imparts color to the colorless benzene
CHROMOPHORE
CHROMOPHORE PLUS BENZENE MAKES
CHROMOGEN
EXAMPLE OF CHROMOPHORE
NITRO GROUP
Groups that intensify the color of chromophore by
conveying the property of ionization to the chromogen
and enabling it to form salts and binding to the
biological substance
AUXOCHROME
EXAMPLE OF AUXOCHROME
HYDROXYL GROUP
organic compound
containing both chromophores and auxochrome link to the
benzene.”
STAIN
responsible for transferring the color of the
dye to a substance or material to which the dye will act upon
AUXOCHROME
STAIN BASED ON RIGIN
NATURAL AND SYNTHETIC
NATURAL = HEMATOXYLIN AND CARMINE
SYNTHETIC = SAFRANIN, METHYLENE BLUE, CRYSTAL VIOLET
STAIN BASED ON PURPOSE
DIRECT, INDIRECT, SELECTIVE AND DIFFERENTIAL
DIRECT = ANILINEDYES
INDIRECT = INDIA AND NIGROSIN
Selective – particular parts of the organism like the flagella or spore of bacteria
Differential – differentiate two groups of bacteria in mixture
STAIN BASED ON STAINING ACTIVITY
NUCLEAR, CYTOPLASMIC AND HISTOLOGIC
NUCLEAR = HEMATOXYKLINE AND CARMINE
CYTOPLASMIC= ANILINE BLUE AND EOSIN
HISTOLOGIC = SAFRANIN
STAIN BASED ON CHARGE
ACIDIC , BASIC AND NEUTRAL
negatively-charged chromophore, anionic;
more affinity to positively-charged cellular components
such as protein
ACIDIC STAIN
positively-charged chromophore, cationic,
more affinity for negatively-charged cellular
constituents such as the DNA and RNA
BASIC STAIN
WHAT ISTHE CHARGE OF BACTERIA
NEGATIVE
EXAMPLE OF BASIC DYE
CYRSTAL VIOLET, METHYLENE BLUE, HEMATOXYLIN, GENTIAN VIOLET
both having positive and negative charge
NEUTRAL STAIN
NEUTRAL STAIN EXAMPLE
GIEMSA, LISHMAN, WRIGHT AND ROMANOWSKY WHICH IS USED FOR HEMATOLOGY
DISTRIBUTION OF BACTERIAL CELL IN A SLIDE IN ORER TO VIEW THEM IN A MICROSCOPE
SMEAR
WHAT HAPPENS IF BACTERIAL INOCULUM IS SPREAD TOO THICK OR TOO THIN
If too thick, you will not appreciate the morphology because
they are too crowded. And if it is too thin, you will not be
able to see it
Process by which internal and external structures are
preserved and fixed in position
FIXATION
Process by which organism is killed and firmly attached to
microscope slide
FIXATION
Protein will be coagulated and the bacterial cell will
adhere to the slide
HEAT FIXZATION
Preserves overall morphology but not internal
structures
HEAT FIXATION
Protects cellular structure and morphology of
larger and more delicate organisms
CHEMICAL FIXATION
CHRMICAL FIXATION IS DONE VIA
Done through application of methanol or any
alcohol (dipping it for 5 to 6 times); it has to be
dried first before staining
- Direct stains the bacteria
positive stain
Where the actual cells are being imparted with color. They
will appear in a clear background having color so they are
being contrasted from the environment. Colored bacterial
cell stands out.
positive stain
kind of dye used in simpole staining
basic dye
mpart different colors to different bacteria, utilizes two or
more stains
dfferential staining
- Allows bacterial cells to be categorized into groups or types
- Divides microorganisms into groups based on their staining
properties (e.g. gram stain, acid-fast stain
differential stianing
o Most widely used differential staining procedure
gram stainign
gram staining was innovated by
hans christian gram
added safranin
karl weigert
Divides bacteria into two groups based on differences
in cell wall structur
gram staining
staining process
▪ Crystal violet (primary stain) for 30 sections,
wash rinse for two seconds
▪ Gram’s iodine (mordant) for 1 minute, water rinse
▪ Wash with 95% ethanol and acetone
(decolorizer) for 10-30 seconds, water rinse
▪ Safranin (counterstain) for 30-60 seconds, water
rinse and blo
, a substance that
will form an insoluble compound with a stain and helps to fix
the color to the cell. Acts like glue
Mordant
: is the most critical step in staining
Decolorization
staining concept of gram positive explain
For gram positive bacteria, if the peptidoglycan layer is
treated with the mixture of acetone-alcohol, the initial reaction
is that the layer will only shrink because it is thick, trapping the
crystal violet stain at the beginning not allowing other stains to
penetrate the cell.
staining concept of gram nega6i8veq
for the gram negative bacteria, it contains an outer
membrane composed of lipopolysaccharide (high lipid content)
and the alcohol is a lipid solvent, it will increase the porosity of
the cell wall, hence the crystal violet iodine complex that was
formed earlier can still pass through the wall because of the
increase porosity, leaving the cell wall colorless. Now, taking
the counter stain safranin – red.
- Aid in particular structures
special staining
Indirect staining, cells remain clear, only the background is
colored for easy visualization of image
negative staining
reporting of gram stain
Gram reaction (positive or negative; red or violet),
morphology (cocci, bacilli, etc.) arrangement (chain, pairs,
etc.)
Cell wall damage of bacteria is due to what
antibiotic therapy or
excessive heat fixation of the smear
gram negative bacteria may not
be fully decolorized during decolorization steps and appear as gram positive
When a smear is too thick,
stain weakly with gram stain (they are gram
resistant
mycobacteria
do not take up the dyes used in Gram stain or are too small
to be seen with light microscopy
Mycoplasma, Rickettsiae, Chlamydiae
To be visible on a slide, organisms that stain by the Gram
method must be present in concentrations of about
104 to
105 organisms per ml of uncentrifuged fluid
In an appropriately stained
specimen, the nuclei of neutrophils are _____.
Nuclei are
____, decolorization is insufficient.
RED
BLUE
All cocci are gram positive except
Neisseria, Moraxella,
Veillonella
All bacilli are gram negative except
Bacillus,
Bifidobacterium, Actinomyces, Nocardia, Streptomyces,
Clostridium, Corynebacterium, Erysipelothrix, Listeria,
Lactobacillus
HOT METHOD AKA
ZIEHL NEELSEN
HOT METHOD EXPLAIN PROCEDURE
o Make a smear. Air dry. Heat fix.
o Flood smear with Carbol Fuchsin stain (primary stain).
o Cover flooded smear with filter paper.
o Steam (mordant) for 10 minutes. Add more Carbol
Fuchsin stain as needed.
o Cool slide.
o Rinse with DI water.
o Flood slide with acid alcohol (leave 15 seconds). The
acid alcohol (decolorizer) contains 3% HCl and 95%
ethanol, or you can decolorize with 20% H2SO4.
o Tilt dry 45 degrees over the sink and add alcohol drop
wise (drop by drop) until the red color stops streaming
from the smear.
o Once there’s no more color coming out, it can already
be rinsed with water.
o Counterstain: methylene blue, malachite green
o Add Loeffler’s Methylene Blue (counterstain) stain.
This adds blue color to non-acid fast cells. Leave it on
smear for 5 minutes.
o Rinse slide. Blot dry.
o Use oil immersion objective to view.
IN HOT METHOD
: Acid fast bacilli – ???
: Non-acid fast bacilli - ???
: Acid fast bacilli – red
: Non-acid fast bacilli - blue
COLD METHOD AKA
KINYOUN
COLD METHOD REQUIREMENTS
Reagents required:
▪ Carbol Fuchsin stain (filtered)
▪ Acid alcohol 3% v/v (or 20% sulfuric acid)
▪ Malachite green 5 g/l (0.5 w/v) or Methylene blue
5g/l
o There is no steaming process involved.
heated to enable dye to
penetrate the waxy
mycobacterial cell wall IN HOT METHOD
Phenol-carbol fuchsin stain
Stain is not heated but the
penetration is achieved by
increasing concentration of
basic fuchsin and phenol
and incorporating a ‘wetting
agent’ chemical
COLD METHOD - KINYOUN METHOED
FLOURESCENT METHOD VIA F MICRO
o More sensitive and rapid method
o Very specific
o Mycobacteria will glow; no need to look for them
o Quite expensive
When any red bacilli are seen, report the smear as
“AFB
positive
Does not distinguish between viable and dead organisms.
Follow-up specimens from patients on treatment may be
smear positive yet culture negative
AFB microscopy
AFB (sputum specimen) are being collected
three times;
preferably every morning
Many TB patients have
negative AFB smears with a
subsequent positive culture. Negative smears do not
exclude TB disease
Acid-fast organisms other than Mycobacterium
- Nocardia spp. – partial acid fast
- Rhodococcus spp. – partial acid fast
- Legionella micdadei – partial acid fast in tissue
- Cyst of Cryptospordium – acid fast
- Cyst of Isospora – acid fast
EXPLAIN NEGATIVE STAINING
used to reveal negatively-charged bacterial
capsules, the encapsulated cells will have a halo
appearance under the microscope
- Capsules are colorless against a stained background.
Particularly useful for determining cell size and
arrangement. It can also be used to stain cells that
are too delicate to be heat-fixed
o India ink, Nigrosin
Organisms are not stained, only the background is
stained
INDIA INK, NIGROSIN
Used to demonstrate the capsule of Cryptococcus
neoformans, Streptococcus pneumoniae
INDIAN INK, NIGROSIN
- Double staining technique
- Bacterial endospore is one color and vegetative cell is a
different color
SPORE STAINING
- For C. diphtheria’s metachromatic or volutin granules
ALBERT STAINING
C. DIPTHERIA
e thin gram-positive bacilli, straight or
slightly curved and often enlarged (clubbing) at one or
both ends and are arranged at acute angles, giving
shapes of Chinese letters or V-shape which is
characteristic of these organisms. Present in the body
of the bacillus are numerous metachromatic granules
which give the bacillus beaded or barred appearance.
These granules are best demonstrated by Albert’s stai
- How the C. diptheria appear
To demonstrate metachromatic granules in C.
diphtheria, the granules appear bluish black whereas
the body of bacilli appear green or bluish green
FLAGELLAR STAINING
- Mordant applied to increase thickness of flagella (e.g. tannic
acid, potassium alum) - Flagella are usually invisible under light microscopy, but
their identification and anatomy are important in
determining some pathogens. Certain chemicals that bind
to the flagella are used in the staining process. The flagella
color may change or an increase in contrast should make
them visible
genera Bacillus andClostridium, cause diseases such as
anthrax, tetanus, and
gangrene
- Types of spore locations
o Terminal (Clodtrisium tetani)
o Subterminal (Bacillus subtilis)
o Central (Bacillus cereus)
Schaeffer-Fulton staining
This technique designed to isolate endospores by staining
any present endospores green, and any other bacterial
bodies red. The green stain is malachite green (primary),
and the counterstain is safranin, which dyes any other
bacterial bodies red.
– used to highlight microorganisms to determine
cellular shapes and arrangements. Aqueous or alcohol
solution of a single basic dye stains cells. Sometimes, a
mordant is added to intensify the stain
SINPLE STAIN
ACID FAST STAIN
– used to distinguish Mycobacterium species
and some species of Nocardia. Acid-fast bacteria, once
stained with carbol fuchsin and treated with acidalcohol, remain red because they retain the carbol
fuchsin stain. Non-acid fast bacteria, when stained and
treated the same way and then stained with methylene
blue, appear blue because they lose the carbol fuchsin
stain and are then able to accept the methylene blur
stain.
used to color and isolate various structures such
as capsules, endospores and flagella, sometimes used as a
diagnostic aid
SPECIAL
