Post on 05-Apr-2018
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Gram staining by Hans Christian Gram
is a method of differentiating bacterialspecies into two large groups (Gram-positiveand Gram-negative).
It is based on the chemical and physical
properties of their cell walls. Primarily, itdetects peptidoglycan, which is present in athick layer in Gram positive bacteria.
A Gram positive results in a purple/bluecolor while a Gram negative results in apink/red color.
The Gram staining method
1. A small sample
of a bacterial
culture isremoved from a
culture.
The bacterial
suspension is
smeared ontoa clean glass
slide.
The bacterial
smear is thendried slowly at
first and then,
when dry,
heated for a
few seconds
Once cool, the slide
is flooded with a
stain called Crystal
Violet . The stain is
left on the slide forabout 1 minute. This
stains all the
bacteria on the slide
The Crystal
Violet is gently
washed off theslide with
running water
The bacterial
smear is then
treated with
Gram's solutionwhich consists of 1
part iodine, 2 parts
potassium iodide,
and 300 parts
water.
After about 30 seconds
the slide is gently
rinsed with ethyl
alcohol which causesthe dye-iodine complex
to be washed out of
some bacteria but not
others. This is called
decolourisation.
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a colour which
contrasts with the
blue-black colour of
the Gram-positive
cells. The staincommon used is
safranin which is
red. This is called
counterstain.
The counter
stain is left on
the smear for
about 30-60seconds and
then gently
rinsed away
with running
water.
After the
counterstain has
been rinsed off, the
slide is placed
between some
absorbent paper
and the excess
water gently
blotted off.
a drop of immersion
oil is placed on the
stained bacterial
smear.
The slide is then
placed on a
microscope stage
focus under oil
immersion objective
Typical Gram-positive bacteria
staphylococci such as Staphylococcus
epidermidis and Staphylococcus aureus which
is a common cause of boils streptococci such as the many species of oral
streptococci, Streptococcus pyogenes which
causes many a sore throat and scarlet fever
and Streptococcus pneumoniae which causes
lobar pneumonia
clostridia such as Clostridium tetani
which cause tetanus (lockjaw)
actinomyces such asActinomycesodontolyticus which is found in mouths
species of the genus Bacillus such as
Bacillus subtilis which are common
microbes living in soil
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Typical Gram-negative bacteria
the bacilli that cause
whooping cough, Bordetella pertussis
typhoid, Salmonella typhi
cholera, Vibrio cholerae gut-dwelling Escherichia coli
Acid Fast Staining by Ziehl Neelsen
Acid-fastness is a physical property of certain
bacteria, specifically their resistance to
decolorization by acids during staining
procedures. The high mycolic acid content of certain
bacterial cell walls, like those of
Mycobacteria is responsible for the staining
pattern of poor absorption followed by high
retention.
Mycobacterium tuberculosis
d. Lipases attacks fats and break them downinto glycerol and fatty acids
e. Zymases changes sugars to alcoholf. Oxidases catalyse oxidation, ex. Alcohol to
acetic acid
g. Dehydrogenases catalyze anaerobicoxidations, which removes hydrogen
h. Coagulases produce coagulation in liquidprotein
i. Reductases changes hydrogen peroxide towater and molecular oxygen
2. BASED ON RESULTS OF MICROBIAL GROWTH
a. Acid production in
media containing milk or
carbohydrates certain
bacteria form Lactic acid,
Acetic acid, Butyric acid,
Formic acid, or Proprionic
acid acids
Yellow Acid fermentation
of sugar
Red Alkaline due to
deamination of protein
IMViC TEST
- used to distinguish between different enteric
bacteria (Family Enterobacteriaceae)
- E. coli and Klebsiella are lactose fermenters
- Salmonella and Shigella are lactose
nonfermenters
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. Indol production
Bacteria that contain the
enzyme tryptophanase
can hydrolyze
tryptophan to indole,
pyruvic acid andammonia, can be
detected by adding
Kovacs reagent
- production of bright red
compound on the
surface of the medium
Methyl Red Test
Based upon the pH
concentration upon the
addition of Methyl red
Orange red color is (+) Yellow is (-)
Distinguishes:
Aerobacter aerogenes (-)
Escherichia coli (+)
Voges-Proskauer test
Based upon the
production of
acetylmethylcarbinol from
dextrose .
Useful to distinguish
Aerobacter aerogenes (+)
from Escherichia coli(-)
Citrate test
Determines ability of
bacteria to use citrate as a
sole carbon source for energyneeds
Bromothymol blue is used as
indicator
Escherichia coli (+)
b. Gas production
Observed in media
containing
carbohydrate such as
lactose or dextrose,
the gases formed are
carbon dioxide,
hydrogen, nitrogen,
hydrogen sulfide,
ammonia and
methane
c. Urease test
Bacteria containing
enzyme urease uses
nitrogen and carbon in
amide compounds such as
Urea.
Distinguish:
Proteus (+ ) from other
Nonlactose-fermenting
enteric bacteria like
Salmonella and Shigella (-)
d. Proteolysis
Liquefies gelatin,
coagulate serumEx. Proteus
Pseudomonas
d. Alcohol production
Produced by yeast,
molds and a few
bacteria
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e. Pigment Production
Carotenoids
produces yellow, red,
orange pigments
Ex. Sarcina,
Micrococcus Anthocyanins
produces red, blue and
intermediate shades
Ex. Actinomyces
Melanins
produces black,
brown
Ex. Azobacter,
Actinomyces
SMEAR PREPARATION AND STAINING
A BACTERIAL SMEAR IS
A DRIED PREPARATION
OF BACTERIAL CELLS ON
A GLASS SLIDE.
A SMALL AMOUNT OF
BACTERIAL GROWTH IS
TRANSFERRED TO A
DROP OF WATER ON A
GLASS SLIDE AND
MIXED, THE MIXTURE IS
SPREAD OUT EVENLY
OVER THE LARGE AREA
ON THE SLIDE
1. HANGING DROP
SLIDE AND BACTERIAL
MOTILITY
- Bacteria that possessflagella exhibit
flagellar motion
- Helically shaped
spirochete moves in a
corkscrew and
bending-type motion
- Gliding motion- slides
over moist surface
2. NEGATIVE STAINING
- uses India ink or
Nigrosin that will
stain around the
bacteria to produce adark background
3. GRAM STAINING
Is a method of
differentiating bacterial
species into two large
groups (Gram-positive
and Gram-negative).
Uses crystal violet as
Primary stain and
Safranin as counterstain
4. ACID-FAST STAINING
Ziehl-Neelsen techniqueemploys heat to drivecarbol fuchsin into the
cell, once stained theyare not easlydecolorized. The acidfastness is due tomycolic acid
Kinyountechniqueemploys a wettingagent (Tergitol 7)
5. ENDOSPORE
STAINING
SCHAEFFER-FULTON
uses malachite green
as primary stain and
safranin as counter
stain
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6. CAPSULE STAINING
- Anthonys method
employs crystal
violet as primary
stain and Coppersulfate as
decolorizer
PREPARATION OF CULTURE MEDIA
CULTURE MEDIUM refers to any material in
which microorganisms find nourishment.
- When the microorganisms in a culture are all of
the same species, it is called pure culture.- When two or more organisms are present it is
called mixed culture .
FORMS OF CULTURE MEDIA
- SYNTHETIC MEDIA
- NONSYNTHETIC MEDIA- DEHYDRATED MEDIA
May contain meat extract and agar
For tubes = 6 - 7 ml are necessary
For Plates = 10 ml are necessary
TYPES OF CULTURE MEDIA
Classification/Types Of Culture Media.
1. According to physical state.
a) Liquid media: fluid in nature, usuallyplaced in test tubes, for
example, nutrient broth.
b) Solid Media: Prepared by addingsolidifying agents like gelatin and agar tothe liquid medium, forexample, nutrient agar.
2. According To Composition
a)Simple Media: it contains only basicsubstance such as nitrogen , carbon andminerals that are essential for bacterialgrowth, for example, nutrient broth,
nutrient agar, peptone water .
b) Enriched Media: Some nutritionallyenriched material like blood, serum orasctic fluid is added to the medium,repuired for proper growth of somebacteria, for example, blood agar,chocolate agar.
c) Differential Media) it differntiate betweentwo groups of bacteria, for example, blood
agar, MacConkey's Medium
d) Selective Media: In this media an inhibitory
substance is added to the media whichprevents growth of all organisms except theone for which it is designed. for example,Lowenstein Jensen's medium.
e) Media used for biochemical reaction: Thismedia is used to detect different biochemicalreactions produced by different organisms. forexample, simmon citrate medium .
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Important Culture Media:
a) Nutrient agar
b) Blood agar
c)Chocolate agar
d)McConkey's Medium
e) Lowenstein jensen (LJ) mediumf) Loeffler's Coagulated Medium
g )Nutrient Broth
h) Mueller Hinton
i) Brain Heart Infusion
METHODS OF OBTAINING PURE CULTURES
1. POUR PLATE
- A series of dilution of bacterial culture in a
medium is made and then pouring in Petri
dish2. STREAK PLATE
- Melted agar is poured into Petri dishes and
allowed to harden