Macarambon, Abdul Malic A.

    CULTURAL
  MICROSCOPY STAINING BIOCHEMICAL
(example)
DESCRIPTI It is the most  Staining is a  Biochemical
ON common method technique used to tests are the tests
to detect enhance contrast used for the
microorganisms in samples, identification of
directly in clinical generally at the bacterial species
specimens and microscopic level. based on the
for the Stains and dyes differences in the
characterization are frequently biochemical
of organisms used in histology activities of
grown in culture. and in the different bacteria.
medical fields of Bacterial
  histopathology, physiology differs
hematology, and from one type of
It allows us to cytopathology organism to
observe that focus on the another.
microorganisms study and
through its diagnoses The ability of
magnification disease at a bacteria to form
lenses. microscopic level. organic
compounds by
metabolizing
certain
carbohydrates
and related
compounds is a
widely used
method for the
identification of
microorganisms.

TEST/S There are  There are  There are

different different Staining different
microscopy techniques: Biochemical
techniques: techniques:
Macarambon, Abdul Malic A.

1. Bright field 1.Gram Staining Test used to

Identify Gram
2. Dark field 2. Acid-fast Positive Bacteria:
Staining
3. Phase- 1. Catalase Test
Contrast 3. Capsule
Staining 2. Mannitol Salt
4. Differential Agar
Interference 4. Endospore
Staining 3. Blood Agar
5. Fluorescence Plates (Streak-
5. Flagella stab technique)
6. Transmission Staining.
Electron 4. Optochin
sensitivity testing
7. Scanning
Electron 5. Bacitracin
sensitivity testing

6. CAMP Test

7. Nitrate Broth

8. Spirit Blue
Agar

9. Starch
Hydrolysis Test

10. Motility Agar

11. Coagulase
Test

Test used to
identify Gram
Negative
Bacteria:

1. Oxidase Test
Macarambon, Abdul Malic A.

2. Sugar broth
with Durham
tubes

3. Methyl
Red/Voges-
Proskauer

4. Kliger’s Iron
Agar

5. Nitrate Broth

6. Motility Agar

7. MacConkey
Agar

8. Simmon’s
Citrate Agar

9. Urease test

10. Sulfur Indole

Motility Media
POSSIBLE Magnify objects  Gram staining:  Catalase
RESULT/S 100x, 400x, and test:
1000x from its Gram-positive
original size, Catalase positive
which render -purple (purplish)
microorganisms -bubble formation
to be visible by Gram-negative
the naked eye. Catalase
-pink negative
 
Acid fast -no bubble
Morphology of formation
-red
bacteria:
Mannitol Salt
Non-acid fast Agar:
1. Cocci(chains,
clusters,
Macarambon, Abdul Malic A.

diplococci) -blue -yellow colonies

surrounded by
2. Bacilli Capsule stains yellow zone
appear clearer if
3. Spirilla presented. Staphylococcu
aureus
4. Comma Endospores
shaped - pink/red colony
-bluish-green
5. Gram Staining Staphyloccocus
result Others epidermidis
confirmation:
- pink/red - Red
1. Blue -
positive Flagella are Micrococci
2. Red/pink - visible if present
negative - No Growth

  Eschericihia coli

6. Acid Fast  Blood Agar

Staining Result: Plates

1. Pink bacilli Beta-hemolysis

in blue
background -transparent zone
2. Blue bacilli that surrounds
in blue the colony.
background
Staphylococcus
7. Semi- aureus,
Quantitave Streptococcus
Result pyogenes, and
streptococcus
1. GS: Few, agalactiaeare b-
moderate, hemolytic
abundant/O
IF Alpha-hemolysis
2. AF: 0, 1+,
2+, 3+, 4+, -colonies are
5+, 6+ surrounded by
Macarambon, Abdul Malic A.

  green, opaque
zone.

Streptococcus
pneumonia and
Streptococcus
mitis

Gamma-
hemolysis

-no zones

Staphylococcus
epidermidisis

 Optochin
sensitivity
testing

Positive

-zone of
inhibition has
6mm disk with
14mm or higher
in diameter.

Negative

-No zone of
inhibition

Equivocal

-any kinds of
zone for
inhibition but less
than 14 mm is
questionable as
pneumococci,
Macarambon, Abdul Malic A.

the strain in
pneumococcus
could be
identified if the
bile is soluble.

 Bacitracin
sensitivity
testing

Susceptible

-any zone of
growth inhibition

Resistant

-no zone of
growth inhibition

 CAMP test

Positve

-enhanced
hemolysis
indicated by an
arrow shape
zone of beta
hemolysis at the
junction of 2
organism

Negative

-no hemolysis
enhancement

 Nitrate
Broth
Macarambon, Abdul Malic A.

Positive

-developed red
cherry colour with
a reagent of A
and B added on
it.

-Absence of red
color if Zn powder
is added

Negative

-Develop red
color with Zn
powder added on
it.

 Spirit Blue
Agar

Positive

Negative

 Starch
Hydrolysis
Test

Positive

-clear zone
around the
growth line with
iodine solution
added on it that
indicates that the
organism was
successfully
Macarambon, Abdul Malic A.

hydrolysed.

Negative

-appears black,
purple, or blue
colored in its
medium
depending on the
concentration of
iodine added

 Motility
Agar

Positive

-has hazy
growths that
spread through
the medium
making it slightly
opaque

Negative

-Growth is
confined within
the stab-line

 Coagulase
Test

Positive

-any size of Fibrin

Clot
Macarambon, Abdul Malic A.

Negative

- No Clot

 Oxidase
Test

Positive

-Developed a
deep purple-blue
colour which
indicates oxidase
production within
5-10 seconds.

Negative

-No purple-blue
color or any
change

 Sugar
broth with
Durham
tubes

Acid production:

Positive/Negative

Gas production:

Positive/Negative

 Methyl
Red/Voges
-Proskauer

Positive MR test:
Macarambon, Abdul Malic A.

-When methyl red

is added to MR-
VP broth that has
been inoculated
with E. coli, it will
stay red

Negative MR test:

-When methyl red

is added to MR-
VP broth that has
been inoculated
with Enterobacter
cloacae

Positive VP test:

-When VP
reagents are
added to the test
that has been
inoculated with
Enterobacter
cloacae

Negative VP test:

Reagents VP are
added to the test
that has been
inoculated with E.
coli, the media
turns copper
colored.

 Kliger’s
Iron Agar

Carbohydrate
Macarambon, Abdul Malic A.

fermentation

For slant and butt

Positive

-yellow (acid)

Negative:

-red (alkaline)

For butt:

Positive

-yellow

Negative

-red slant/yellow
butt (dextrose
positive, lactose
negative).

H2S Production

Positive

-black colour
throughout the
medium or
junction between
slant and butt

Negative

-no black colour

Gas production
Macarambon, Abdul Malic A.

Positive

-presence of
bubbles/cracks in
the medium

Negative

-absence of
bubbles/cracks in
the medium

 MacConke
y Agar

-acidic
environment
appear pink

-non fermenters
will produce
normal colour
colonies

 Simmons
Citrate
Agar

Positive

-medium turns
blue

Negative

-no colour is
changed

 Urease
Test
Macarambon, Abdul Malic A.

Positive

-
Pink,Orange,Mag
neta

Negative

-No Color change

,Yellow/orange
coloration

 Sulfur
Indole
Motility
Media

Positive

Pink-red colour
band is formed at
the top of
medium after
adding the
provided reagent
(Kovacs
Reagent)

Negative

-Growth confined
within the stab
line
Macarambon, Abdul Malic A.

ADVANTA v  Convenient; no  -gives quick  -shortens the

GE/S more tedious results when time required to
procedures; fast examining identify microbes
result infections.
-reduce costs
v  Less errors
-simple and cost
effective -ensure or
v  Provides enhance the
confirmation for -helps with accuracy of
bacterial determining identification of
identification appropriate an unknown
treatments for sample
infection
- identifying
-it allows for bacteria relied
various methods heavily in
of testing biochemical
testing
-it is basically a
key procedure in
identifying
bacteria
DISADVAN Some  -has significant  -labour intensive
TAGE/S microscopy limitations when
techniques are used for -must be
inconvenient to environmental performed on
use, such as the microbiology solid cultures only
TEM and SEM, and that the
and are also -has a few side same phenotypic
expensive which effects pattern may
may not be sometimes be
present in all -it comes with exhibited by
clinical certain types of different species.
laboratories. risk
-expensive
-might lead to
misinterpretation -need trained
personnel and
facilities
Macarambon, Abdul Malic A.

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