PURPOSES OF STAINING OF - Chemical groups with conjugated

double bonds that give dye its color.

SPECIMENS - Greek word “chromo” color “phorus” to
bear
 To increase visibility of specimen
- Most example are those with nitro
 To differentiate one organism from another -
groups
some microorganism will take color under a
c) Auxochrome
given conditions so in that case we will be able
- Groups that intensify the color of
to differentiate one bacterium from the other.
chromophore
Just like in the in the case in the humus of
- Greek “auxein” to increase and
differential stains like AFB stains or Gram stain
“chroma” color
 To accentuate specific morphological features -
- Most example are those with hydroxyl
there are the so-called stains that which can
groups
react only with particular structures of bacteria.
- Responsible for transferring the color of
Like when to see just like the spore so this
the chromogen to a substance to which
specific stain will just color the spore or maybe
the dye will act upon
you would want to appreciate some
metachromatic granules and then so these Stain- can be chemically defined as an organic
granules will be stain. compound containing both chromophores and
 To preserve- once the specimen is stained, we auxochrome link to the Benzene ring.
can preserve this by putting coverslip with an
adhesive so the morphology that you can see
under the microscope will also be preserve CLASSIFICATION OF STAINS BASED ON ORIGIN

 Natural
DYE vs STAIN - Obtained from the natural resources or
product such as from the heart of the
DYE
wood tree.
 Used to refer a coloring agent used for general - Example is hematoxylin and carmine
purposes  Synthetic
 Examples of dyes are actually natural products - When it is artificially produced usually
made of coal, this are rendered synthetic and coal-tar are used for fractionation and
transformed to become stains. recombination so that you could come
up with safranin, methylene blue,
STAIN
crystal violet etc.
 Used to refer to that dye which is used for
Based on purpose
biological purposes
 A specific dye for staining bacteria,  Direct
microorganism - Also known as general stain
 Most stains are analyne dyes they derived from - They stain bacteria directly and mostly
coal-tar distillation. this are analyne leus
 Familiar stains are methalene blue, stain. - The bacteria itself will be stained
 Indirect
COMPONENTS OF STAINS- Have complex molecular
- It will only stain the background
structures
- Example Indian ink, negrosin dye.
a) Benzene  Selective- it will only stain particular organism it
- Colorless organic compound that can can maybe only flagella or the spore
bind the nitro group of chromophore  Differential- you want to differentiate maybe
- Highly two groups’ bacteria in a mixture so that is the
b) Chromophore classification of the differential stain.
Based on staining Activity
 Nuclear- if you only want to stain the nucleus
like hemotoxyline, carmine; acid in nature
 Cytoplasmic- if you only want to stain just the
cytoplasm usually eosin blue, eosin can be used;
basic in nature
 Histologic- if you want to stain particular tissues
like maybe you want to employ safranin for
staining tissues.
Based on Charge
 Acidic stains
- Negatively charge stains
- Anionic the chromophore is negatively
charge if the stain is negatively charge
of course opposite attract if the acidic
stains negatively they have the affinity
to positively charge constituents like
proteins.
 Basic stains
- Positively charge stains
- Cationic the chromophore is positively
therefore they have the affinity for the
negatively charge constituents like DNA,
RNA. Bacteria themselves are negatively
charge class so that is why we often
used basic stains in the lab.
- Example crystal violet, hematoxylin,
methylene blue
 Neutral stains
- Both cationic and anionic
- It will impart different colors to
different components
- Example giemsa stain
FIXATION
-Process by which external and internal structures are
preserved and fixed in position
-Process by which organism is killed and firmly attached
to microscope slide
a. Heat fixing- preserves over all morphology but
not internal structures
b. Chemical fixing- protects fine cellular
substructure and morphology of larger, more
delicate organisms.
METHODS OF STAINING
POSITIVE STAINING
-here the actual cells are being imparted with color they
will appear in a clear background having colors so they
are being contrasted from the environment
-what will standout is the colored bacterial cells
 Simple staining- only used a single dye that
produce a single color, uniform color
 Differential staining- it will impart different
color to different bacteria because it
neutralizes two or more stain like in gram
stains.
 Special staining (flagella, spores, granules)-
to aid visualization of the particular
structures of the bacteria
NEGATIVE STAINING
-indirect were the cell remained clear. You will only
color the background what will stand out here is the
colorless bacteria.
SIMPLE STAINING
 Single staining is used
 Basic dyes are frequently used
 Dyes with negative charges
 e.g. crystal violet
DIFFERENTIAL STAINING
 uses two or more stains that allow 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
GRAM STAINING
-most widely used differential staining procedure
-divides bacteria into two groups based on differences
in cell wall structures.
STEPS IN GRAM STAINING
1. Flood the slide with crystal violet
2. Use a mordant a substance that forms a
insoluble compound with a stain that helps to
fix the color of the crystal violet to the bacterial
cell (e.g Gram iodine) after 1 minute washed the
 slide in the running tap water to washed out the
iodine
3. Decolorize the slide with 95% ethanol or
acetone. Decolorizer component 50% acetone
and 50% alcohol. You will only stop decolorizing
if there is already no color. But it is still depend
on the structure of the microorganism. After
that wash it again in a tap running water.
4. After decolorized submerge it in the secondary
color which is the safranin again it depends on
the structure of the microorganism. After
washed it again then air dry.
GRAM STAIN REACTION
REPORTING OF GRAM’S STAIN
 Take note of the gram reaction
 Add the morphology (shape, cocci, bacilli)
 Add the arrangement (chain, diplo, pair)
VARIATIONS/ DISCREPANCY IN GRAM REACTION
 Cell wall damage of bacteria due to antibiotic
therapy or excessive heat fixation of the smear
 Over decolorization of the smear
 Used of an iodine solution which is too old;
yellow instead of brown in color (always store in
brown glass or another tight opaque container)
 Smear has been prepared from old culture
 When smear is too thick, gram-negative
bacteria may not be fully decolorized during
decolorization steps and appear as gram
positive bacteria.
 Old culture can lose its integrity.
LIMITATIONS OF GRAMS STAIN
 Mycobacteria bacteria because of its cell wall
resistant it is weakly stain with gram stain or
called ass gram resistant
 Bacteria such as mycoplasma, rickettsae,
chlamydiae do not take up the dyes used in
gram stain or too small to be seen with light
microscopy.
SENSITIVITY OF GRAMS STAIN- to be visible on a
slide organism that stain by the gram method must
be present in concentrations of about 10^4 to 10^5
organisms per mL of uncentrifuged fluid.
ADEQUACY OF GRAMS STAIN
 MICROBIOLOGIST/TECHNICIAN – should be
the first to determine whether the gram
stain is adequate. In an appropriately
stained specimen, the nuclei of the
neutrophils are red. If the nuclei are blue,
the decolorization is insufficient.
ACID FAST STAINING
-particularly useful for staining members of the genus
Mycobacterium and Nocardia which are acid-fast
bacteria
- high lipid content in cell walls is responsible for
staining characteristics.
a. Mycobacterium tuberculosis- causes tuberculosis
specimen sputum
b. Mycobacterium leprosy- causes leprosy
AFS METHODS
 Hot Method (Ziehl Neelsen)
 Cold Method (Kinyoun’s)
 
Fluorescent Method- the one that used  Carbolic acid- act as mordant
fluorescent microscope
 Others
A. HOT METHOD B. COLD METHOD
1. Carbol fuchsin stain (filtered)
-in AFB there are only 3 reagents, in gram stain 4 here
2. Acid alcohol 3% v/v (20% sulfuric acid)
we have the (Primary, Secondary, and Decolorizer)
3. Malachite green 5g/L ( 0.5%w/v) or methylene
 Primary stain: it is the Carbol Fuchsin blue 5g/L
combination of the analyne dye with phenol HOT vs. COLD METHOD
 Decolorizer: acid alcohol combination of 1. In the Hot ZN technique, the phenol- carbol
hydrochloric acid and ethanol fuchsin stain is heated to enable the dye to
 Counter Stain/ Secondary: methalene blue or penetrate the waxy mycobacterial cell wall
malachite green 2. In the cold method known as the Kinyoun
technique, stain is not heated but the
Steps: penetration is achieved by increasing
1. Make a smear, air dry, heat fix concentration of basic fuchsin and phenol
2. Flood smear with carboxyl fuchsin stain and incorporating a wetting agent chemical.
3. Cover flooded smear with filter paper Strongly Acid-Fast: mycobacterium tuberculosis,
4. Steam for 10 minutes. Add more carboxyl mycobacterium ulcerans.
fuchsin stain as needed
5. Cool slide Weakly Acid-fast: mycobacterium leprae – used only .
6. Rinse with DI water DI- Deionize/ Distilled 5% decolorizer acid solution
7. Flood slides with acid alcohol (leave 15 minutes)
the acid contains 3% HCL and 95% ethanol or
you can decolorize with 20% H2SO4 C. FLUORESCENT METHOD
8. Tilt slide 45 degrees over the sink and add acid  Uses fluorochrome dye more sensitive, more
alcohol drop by drop until the red color stops rapid method of doing AFS (acid-fast staining)
streaming from the smear  Very specific because you are using fluorescent
9. Rinse with DI water microscope. It just glows bacteria will just only
10. Add Loefflers Methylene blue stain (counter fluores no need to look for them
stain). This stain adds blue color to non- acid-  Quite expensive because it requires fluorescent
fast cells. Leave for 5 minutes. microscope equipment
11. Rinse slide blot dry
PRIMARY AFB FLUORESCES
12. Use OIO to view
FLUOROCHROME
Non-Acid-Fast Bacilli- blue Auramine O Green
Auramine O- Yellow/ orange
Acid- Fast Bacilli- red rhodamine B
Steaming- is a drastic measure so that application of will Acridine orange Yellow/ orange
allow this analyne dye to be absorb in the wall of the
mycobacterium. Remember its waxing so heat softens REPORTING OF AFB (ACID-FAST BACILLI)
the wax then allow basic fuchsin to enter and since the
fuchsin dye is more soluble in phenol carbolic acid than 1. When no AFB after examining 300 fields, report
in water soluble therefore the stain will be retained in the smear as No AFB seen/ 300 visual fields
the cell wall. 2. When very few AFB are seen (when only 1 or 2
AFB are seen after examining 100 fields, request
Note: a further specimen to examine, those AFB might
have come from tap water saprhonytic
 Once stained it resist decolorization that is why
mycobacteria or it maybe scratch glass slide or
it is called Acid-fast
 by the use of same piece of paper of blotting
paper while drying.
3. When many red bacilli are seen report the
smear as AFB positive and give an indication of
the number of bacteria present as follow (the
greater the number the more infectious the
patient)
INTERPRETATION OF AFB SMEARS
# of AFB seen (A+ Reported as follows
HPF-1000x
magnification)
0 AFB per 300 field AFB not seen
1-2 AFB per 300 doubtful; repeat with
fields another specimen
(+1 to +9)
1-9 AFB per 100 field 1+
1-9 AFB per 10 field 2+
1-9 AFB per field 3+
>9 AFB per field 4+
LIMITATION OF AFB MICROSCOPY
-does not distinguish between viable and dead
organisms follow up specimens from patients on
treatment may be smear positive yet culture negative
SENSITIVITY OF AFB SMEARS
 High bacterial load 5,000-10,000 AFB/mL is
required for detection (in contrast to 10 to 100
bacilli for a positive culture)
 Many TB patients have negative AFB smears
with a subsequent positive culture. Negative
smears do not exclude TB disease
 AFB should be collected 3x a day best time is
early in the morning
 It is also important to put the specimen into a
very tightly sealed container
SPECIFICITY OF AFB MICROSCOPY
 All mycobacteria are acid-fast
 Does not provide species information
 Local prevalence of MTB and NTM determine
the predictive values of a positive smear for
MTB
MTB- (mycobacterium tuberculosis)
NTM- (non-tuberculosis mycobacterium)
LIST OF ACID-FAST ORGANISMS OTHER THAN
MYCOBACTERIUM
1. Nocardia spp: partial Acid-fast
2. Rhodococcus spp: partial Acid-fast
3. Legionella nicoladei: partially acid-fast in tissue
4. Cyst of cryptosporidium: Acid-fast
5. Cyst of isopora: Acid-fast
SPECIAL STAINING: STAINING SPECIAL STRUCTURES
 Capsule stain (negative staining)- capsules are
colorless against a stained background
 Spore stain
 Granule stain
SPORE STAINNING
 Double staining technique
 Bacterial endospore is one color and vegetative
cell is different color that is why double staining
FLAGELLA STAINING- because of the thin nature of the
flagella mordant is applied to increase thickness of
flagella. (e.g tanic acid, potassium allum)
Methods of flagellar staining:
 Grey method
 Basic fuchsin- is the stain
 Lietson’s method- analyne
 West method – silver nitrate reagent
 Deep cuss method – crystal violet
ENDOSPORE STAINING
-the cell wall of endospore is impermeable to most
chemicals and being in the genera Bacillus and
Clostridium, cause diseases such as anthrax, tetanus
and gangrene.
-the staining process involves both a primary stain and a
counter stain
LOCATION OF SPORES:
a. Central spore- centrally located spore (bacillus
cerus)
b. Terminal spore- spores at the end
c. Sub terminal spore- spores in between central
and end (bacillus subphylis)
SCHAEFFER-FULTON STAIN 0.20gm 3.0gm
Glacial acetate acid Distilled water
-spore stain technique designed to isolate endospore by 1.0mL 300mL
staining any present endospores green and any other Alcohol (95%) 2.0mL
bacterial bodies red. The green is malachite green and Distilled water
the counterstain is safaranin which dyes any other 100mL
bacterial bodies red.

CAPSULE STAINING (indirect)- a stain used to reveal ABERT STAINING PROCEDURE

negatively charged bacterial capsules the encapsulated
cells will have a halo appearance in the microscope.  Cover the heat-fixed smear with Albert Stain I.
let it stand for 2 minutes
NEGATIVE STAINING:  Wash with water
 Indian ink, negrosin (black in color) expect a red  Cover the smear with Albert stain II. Let it stand
background for 2 minutes
 Organism are not stained only the background  Wash with water blot dry and examine
is stained HOW THE C. diphtheria APPEARANCE
 Unstained organisms stand out in contrast
 Use to demonstrate the capsule of cryptococcus -to demonstrate metachromatic granules in C.
neoformans, streptococcus pneumoniae diphtheria these granules appear bluish black whereas
the body of bacilli appear bluish green or green.
ALBERT’S STAINING FOR: C. diphtheria’s
metachromatic or Volutin granules Metachromatic granules- this are reserved granules

 Diphtheria is a serious disease when you FLAGELLAR STAINING

suspect diphtheria
 In all cases of suspected cases of diphtheria,
stains one of the smears in gram stain
 If gram stain smear shows morphology
suggestive of C. diphtheria proceed to the
Albert staining which demonstrates the
presence or absence of melachromatic granules
-Flagella are usually invisible under the light microscopy,
but their identification and anatomy are important in
determining some pathogens. Certain chemicals that
bind the flagella are used in the staining process. The
flagella color may change or an increase in contrast
make them visible.

Diphtheria- notice the pseudo membrane in the

posterior pharynx. It can become very large and may
obstruct the airway.

APPEARANCE OF C. diphtheria

-C. diphtheria are thin gram positive bacilli, straight or

slightly curved and often enlarged(clubbing) at one or
both ends arranged at acute angles giving shapes of
Chinese letters or V shape which is characteristics of
these organisms. Present in the body of the bacillus are
numerous metachromatic granules which gives the
bacillus beaded or barred appearance these granules
are best demonstrated by Albert’s stain.

ALBERT STAIN I ALBERT STAIN II

(SOLUTION) (SOLUTION)
Toluidine blue Iodine 2.0 gm
0.15gm
Malachite green Potassium iodide