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Post Lab

Discussion
Examination of Microorganisms
and Staining Techniques

Preparation of Specimens for Compound


Light Microscope Examination:

1. Wet Mount
drop of medium with microorganisms is
placed on a slide and covered with
coverslip
shape, movement of microorganisms
Advantage: live organisms are observed
Disadvantage: dries out quickly

2.Hanging drop
drop of liquid containing microorganism is
placed on a cover slip and inverted and
suspended on a slide with a
concavity/depression
Shape, movement of microorganisms
(organisms with flagella)
more complex technique
Advantage: allows for longer-term observation
and more reliable observation of motility

3. Preparation of Bacterial Smear


thin film material containing microorganisms
spread on a slide
heat fixed to kill microbes
study the morphology

Preparation and Staining of


Specimens
specimens must be fixed and stained to
enhance their visibility and to distinguish
morphological properties

Purpose of Fixation:
Preservation of internal and external
features of cells
Cellular enzymes are inactivated
Cell structures are hardened
Organism dies AND adheres strongly to
the glass slide

2 Types of Fixation:
1.Heat fixation
flame heating bacterial smear
preservation of morphology but NOT internal
structures
2.Chemical fixation
chemical fixatives penetrate cells and preserves
intracellular components
Acetone
Ethanol
Acetic acid
Mercuric chloride
Formaldehyde
Glutaraldehyde

Dyes and Simple Staining


Stains and bind to cells by ionic, covalent and
hydrophobic forces
Basic dyes (positively charged) bind to
negatively charged groups
Work best at high pH

Acidic dyes (negatively charged) bind to


positively charged groups
Work best at low pH

Common Dyes
Basic Dyes
Safranin
Carbol fuchsin
Crystal violet
Methylene blue
Malachite green
- commonly used

Acidic Dyes
Eosin
Acid fuchsin
Congo red

Staining Techniques
1. Simple Stain
basic dye
Purpose:
cell shape and basic structures become
visible
Highlight microorganisms shapes and
arrangements

Morphology
Shape
cocci
bacilli
Curved and spiral
Arrangement
cocci in pairs, in clusters,
in chains, tetrads and
sarcinae
bacilli in singles, in pairs
and in chains
spirillum in singles

Morphologic arrangement of Cocci:


Cocci in cluster
Staphylococci

Cocci that remain in


pairs after dividing
are called diplococci
Neisseria gonorrhoea

Cocci in chainlike
patterns are called
streptococci

Cocci in groups of
four are called tetrads

Cocci attached in
groups of 8 are called
sarcinae

Bacilli in singles, in
pairs or in chains
Diplobacilli
streptobacilli

2. Differential Stain
differentiates between types of organisms
uses 2 or more dyes to stain organism
a. Grams Stain
- Hans Christian Gram
- Gram positive and Gram negative
b. Acid fast stain
- Acid fast organisms
- Non-acid fast organisms

Gram Positive

Gram Negative

Color after G. staining


procedure

purple

red

Peptidoglycan in c. wall

Thick layer

Thin layer

Lipopolysaccharide in cell
walls

Absent

Present

Gram positive
CV-I complex is difficult to
remove during
decolorization
Purple in color

Gram negative
decolorizer dissolves lipids
in cell wall of Gram
negative bacteria
CV-I Complex is removed
Colorless
retain the color of
counterstain
red

Gram Positive

Gram negative

Erroneous Result
1.
2.
3.
4.

Film is too thick


Decolorization not completed.
Film is over decolorized.
Use of old culture which gives variable
results.

Common Gram positive bacteria


Streptococcus pyogenes
Streptococcus pneumoniae
Staphylococcus aureus
Enterococcus fecalis
Bacillus cereus
Bacillus subtilis
Clostridium sp.

Common Gram negative bacteria:

Salmonella sp.
Shigella sp.
Neisseria meningitidis
Haemophilus influenzae
Escherichia coli
Klebsiella pneumoniae
Proteus sp.
Pseudomonas aeruginosa

Acid-Fast Staining

Some organisms do not stain well with conventional dyes


(e.g. Mycobacterium and Nocardia)
Mycobacterium tuberculosis
Mycobacterium leprae
Harsher treatment required (slide is heated for several
mins.)
Steps:
1. Slide is heated while using carbol fuchsin (ZiehlNeelsen technique)
2. Decolorize: Acid alcohol
3. Counterstain with methylene blue
Acid-fast cells remain red due to high mycolic acid
content (lipid)
Non-acid-fast cells = blue (counterstain)

Ziehl-Neelsens Method
Acid fast organism
retain red color because
carbolfuchsin is soluble
in cell wall lipids (red)

Non-acid fast organism


cell wall lacks lipid
components and carbol
fuchsin is removed
retain the color of the
counterstain (blue)

3. Special Stains
Used to color and isolate specific parts of
microorganisms
Endospores, flagella, capsule

a. Negative Staining for Capsules


CAPSULE
gelatinous covering of microorganisms
Mix bacteria with colloidal suspension of
colored particles (India ink or nigrosin) to
provide dark background
Bacteria stained with simple stain
Capsules appear as halos surrounding stained
bacterial cell

B. Endospore (spore) staining


Spore staining
detects endospores produced by
Bacillus and Clostridium
various sizes, shapes and locations
dormant structures within the cell
endospores protect the organism from
adverse environmental conditions
endospores cannot be stained by
ordinary methods

Steps (Schaffer-Fulton Method)

Preparation of a Bacterial Smear


Air dry
Heat fix
Application of Primary Dye (Malachite Green)
5 minutes w/ concurrent application of heat
Rinse with water
Application of Counterstain (Safranin) 1minute

Endospore
Most commonly used endospore stain
(Schaeffer-Fulton endospore stain)
Malachite green (primary stain)
safranin (counterstain)
Spores ( green) , bacterial cells (red)

Spore Staining: Bacillus subtilis stained using


Schaeffer Fulton Method. Note the central elliptical
green spores within the red cells (X 1,000)

MOTILITY TEST

Positive result - test tube exhibits growth radiating from


the stab; E. coli
Negative result - test tube exhibits growth along the stab
- Staphylococcus aureus

CATALASE TEST
Test to detect whether the bacteria
produce enzyme catalase
Differentiation between Staphylococcus
and Streptococcus genera
Catalase is an enzyme used by bacteria to
induce the reaction of reduction of
hydrogen peroxide into water and oxygen

CATALASE TEST

Positive result - evolution of gas bubbles


- Staphylococcus aures

COAGULASE TEST
Pathogenicity test
Tests to detect if bacteria produce
coagulase enzyme
Coagulase is an enzyme produced by
microorganism that enables the
conversion of fibrinogen to fibrin
Two methods:
1. slide method
2. tube method

+ Result-

coagulation or forming a clot


Staphylococcus aureus
- Result the plasma remains liquid
Staphylococcus epidermidis