Name: 1 minutes and rinse with water. 5.

Name:                                                Kavie Tyrell


Course:                                               Clinical

Code:                                   MTCH331

Lecturer:                                            Mr.
Delbert Robinson


Lab Date                                                           22
Jan. 2018

Title:                                           Gram Staining


Principles of Gram staining:

Gram staining is a method
used in microbiology to differentiate between microorganisms and place them
into two distinct groups; gram positive and gram negative bacteria. This
procedure involves the staining of the microorganism which will retain
characteristic color of the stain based on their cell was constituency. It is
performed on body fluids and biopsies when an infection is suspected. The main
components of gram stain is a; primary stain (crystal violet), Mordant
(Iodine), Decolorizer (ethyl alcohol, acetone), and counterstain (safranin or
neutral red). The mordant iodine binds to crystal violet and traps it in the
cell. When a decolorizer such as
alcohol or acetone is added, it interacts with the lipids of the cell membrane
causing the gram negative bacteria cells to lose its out lipopolysaccharide
membrane leaving the inner peptidoglycan layer exposed, while gram positive
cells become dehydrated and traps the stain withing the cells due to the
multilayered nature of the petodoglycan present. This step is critical and must
be done with caution to avoid over decolorizing.   The
gram stain will stain gram positive bacteria crystal violet (primary stain) due
to the presence of peptidoglycan in their cell walls, while it will stain gram
negative bacteria red (counterstain) due to thinner peptidoglycan in the cell
walls, therefore they do not retain the crystal violet color. The gram staining
technique was named after a Danish bacteriologist Hans Christain Gram
(1853-1938) who originally devised it in 1882 to discriminate between pneumococcal
and Klebsiella pneumonia bacteria in the lungs.

Procedures of Gram Staining:

Take a clean, grease free slide.

Prepare the smear of suspension on the clean slide with a loopful
of sample.

Air dry and heat fix

Crystal Violet was poured and kept for about 30 seconds to 1
minutes and rinse with water.

Flood the gram’s iodine for 1 minute and wash with water.

Then ,wash with 95% alcohol or acetone for about 10-20 seconds and
rinse with water.

Add safranin  for about 1 minute and wash with water.

Air dry, Blot dry and Observe under Microscope.

of Novobiocin test:

test is used for the treatment of infections due to staphylococci and other
susceptible organisms. Novobiocin is an
aminocoumarin antibiotic, produced by the actinomycete Streptomyces nivens,
with antibacterial property. It interferes with the unpackaging and
repackaging of DNA during DNA replication and the bacterial cell cycle. This
test determines whether the bacterium is either susceptible to novobiocin or resistant to the
drug. Susceptible microorganisms are determined by placing a novobiocin-impregnated paper disk on
a agar
plate that is inoculated with the
microbe that is under investigation. The microbe is streaked unto the agar
media covering as much of it as possible, then a novobiocin disk is placed in
the center of the streaked media. As the microbe multiplies to for colonies
during incubation cells are exposed to the antibiotic diffusing into the agar
from the paper disk. If the microbes are susceptible to novobiocin, there will be a
visible zone of inhibition forming
around the disk, representing an area where the antibiotic concentration has
prevented bacterial growth, and if the microbe be resistant, there will be a
visible growth up to the margin around the disk.


Procedures of Novobiocin test:

Allow containers to come
to room temperature before use.

Using a pure 18-24 hour
culture, prepare a suspension of the organism; equivalent to a McFarland 0.5
opacity standard; to be identified in Tryptic Soy Broth , Sterile Water,
 or Brain Heart Infusion (BHI) broth

Inoculate Mueller Hinton Agar, 5% Blood Agar, or Tryptic Soy agar
plate with a sterile swab to obtain confluent growth.

Aseptically apply one 5ug
novobiocin disk onto the inoculated agar surface and lightly press down to
ensure full contact with the medium.

Incubate plate aerobically for
18 to 24 hours at 35 to 37°C.

Measure (in millimeters)
the diameter of the zone of inhibition around the novobiocin disk, and record
as susceptible or resistant.

Principles of Catalase Test:

Catalase is an enzyme produced
by bacteria that respire using oxygen, it catalyses the reduction of hydrogen
peroxide. The test is performed to differentiate between catalase-positive
staphylococci and catalase-negative streptococci bacteria. The enzyme initiates
the breakdown of hydrogen peroxide into oxygen and water as shown in the equation

In routine culture 3% H2o2
is used, while in 15% H2o2 is used for the detection of
catalase in anaerobes. Catalase-positive bacteria are determined when small inoculums
are introduced into hydrogen peroxide and rapid effervescence of oxygen bubbles
occurs. Catalase-negative bacteria will show a lack/weak oxygen bubbles
production when introduce to hydrogen peroxide. Catalase test is also used to differentiate aerotolerant strains
of Clostridium, which
are catalase negative, from Bacillus species, which are
positive, it can be used as an aid to the identification of Enterobacteriaceae,
valuable in differentiating aerobic and obligate anaerobic bacteria, also used
for the identification of Mycobacterium tuberculosis.


The test can be done by
two methods.
a)    Slant method
b)    Slide method 

Slant Method:

1.      Using a sterile
technique, inoculate each experimental organism into its appropriately labeled
tube by means of a streak inoculation.

2.      Incubate all cultures
for 24-48 hours at 37?C.

3.      Allow three or four
drops of the 3% hydrogen peroxide to flow over the entire surface of each slant

4.      Examine each culture for
the presence or absence of bubbling or foaming.


Slide Method:

1.      Divide a clean glass
slide into two sections with grease pencil. One should be labeled as “test” and
the other as “control”.

2.      Place a small drop of
normal saline on each area.

3.      With a sterilized and
cooled inoculating loop, pick up a small amount of the culture from the
nutrient agar slant or Petri plate.

4.      Emulsify one or two
colonies on each drop to make a smooth suspension. The smear should be about
the size of a pea.

5.      With a Pasteur pipette,
place one drop of hydrogen peroxide over the test smear. Be careful not to run
the drops together.

6.      Do not put anything in
the other drop that serves as control.

7.      Observe the fluid over
the smears for the appearance of gas bubbles.

8.      Discard the slide in a
jar of disinfectant.


Principles of Coagulase Test:

Coagulase is an enzyme produced by S. aureus that converts
(soluble) fibrinogen in plasma to (insoluble) fibrin. The free coagulase secreted by S.aureus
reacts with coagulase reacting
factor (CRF) in plasma to form a complex, which is thrombin. This converts
fibrinogen to fibrin resulting in clotting of plasma. It can be produced
in two forms; bound and free coagulase. A coagulase test is performed in the
efforts to differentiate coagulase positive Staphylococcus aureus bacteria from
coagulase negative bacteria Staphylococcus. Free coagulase is an extracellular
enzyme that can be detected in tube coagulase test while bound coagulase is a
cell wall associated protein that can be detected in slide coagulase test.
According to an article from Microbiology and infectious disease; Bound
coagulase is also known as clumping factor. It cross-links ? and ? chain of
fibrinogen in plasma to form fibrin clot that gets deposited on the cell wall
of the cocci. As a result, individual coccus sticks to each other and clumping
can be observed. The
free coagulase secreted by S. aureusreacts with coagulase reacting
factor (CRF) present in plasma to form a complex, thrombin. This converts
fibrinogen to fibrin resulting in clotting of plasma.


The enzyme coagulase is
demonstrated invitro by two methods
a)    The Slide coagulase test
b)    The Tube coagulase test

The Slide coagulase test

1.      Divide the slide into
two sections with grease pencil. One should be labeled as “test” and the other
as “control

2.      2Place a small drop of
distilled water on each area

3.      Emulsify one or two
colonies of Staphylococcus on blood agar plate on each drop to make a smooth

4.      The test suspension is treated
with a drop of citrated plasma and mixed well with a needle

5.      Do not put anything in
the other drop that serves as control. The control suspension serves to rule
out false positivity due to auto agglutination

6.      Clumping of cocci within
5-10 seconds is taken as positive. 

Some strains of
S.aureus may not produce bound coagulase, and such strains must be identified
by tube coagulase test.

The Tube Coagulase Test

1.      Three test tubes are
taken and labeled “test”, “negative control” and “positive control”.

2.      Each tube is filled with
1 ml of 1 in 10 diluted rabbit plasma.

3.      To the tube labeled
test, 0.2 ml of overnight broth culture of test

4.      bacteria is added.

5.      To the tube labeled
positive control, 0.2 ml of overnight broth culture of known S.aureus is added

6.      To the tube labeled
negative control, 0.2ml of sterile broth is added.

7.      All the tubes are
incubated at 37oC and observe the suspensions at half hourly intervals for a
period of four hours.

8.      Positive result is
indicated by gelling of the plasma, which remains in place even after inverting
the tube.

9.       If the test remains
negative until four hours at 37oC, the tube is kept at room
temperature for overnight-incubation.


Heamolysis in microbiology
is the lysing of red blood cells due to the bacterial protein known as
hemolysin which result in the release of hemoglobin from the RBCs. It is
observed in blood agar to determine the different bacteria that produces these
enzymes. The degree at which hemolysis occur during bacteria culture helps to
distinguish between the different types of bacteria. The three types of heamolysis
that can be observed are Beta, Gama, and Alpha hemolysis. According to; “Alpha hemolysis is a greenish
discoloration that surrounds a bacterial colony growing on the agar. This type of hemolysis represents
a partial decomposition of the hemoglobin of the red blood cells. Alpha
hemolysis is characteristic of Streptococcus
pneumonia and so can be used as a diagnostic feature in the
identification of the bacterial strain. Beta hemolysis represents a complete
breakdown of the hemoglobin of the red blood cells in the vicinity of a
bacterial colony. There is a clearing of the agar around a colony. Beta
hemolysis is characteristic of Streptococcus
pyogenes and some strains of Staphylococcus
aureus. The third hemolysis Gamma hemolysis is a lack of hemolysis in
the area around a bacterial colony. A blood agar plate displaying gamma
hemolysis actually appears brownish. Gamma hemolysis is a characteristic
of Enterococcus faecalis”.



Morphologies (Table 1)


































Gram Stain,
Coagulase and Catalase Test (Table 2)





S. aureus



Gram Positive C.

S. epidermidis



Gram Positive C.

S. saprophyticus



Gram Positive C.


Novobiocin Test
(Table 3)

S. aureus


S. epidermidis


S. saprophyticus





streaking was done on blood agar plate to culture S.aureus, S.epidermidis, and
S.saprophyticus bacteria which was then incubated and stained using the gram
staining method. After incubation the colonial morphologies of each bacteria
was recorded as shown in Table 1 of the result which also shows that
s.epidermidis and s.saprophyticus where negative for hemolysis, while s.aureus
was positive for beta hemolysis. Beta hemolysis is describes as the straw halo
that appears around bacteria such as s.aureus. It is
a complete lysis of red cells in the media around and under the colonies.
Streptolysin, an exotoxin, is the enzyme produced by the bacteria which causes
the complete lysis of red blood cells. The Gram stain of the
microorganisms method was carried out by first selecting a slide, label and
date it with necessary information such as name of microbe and initials, after
which a sterile loop was used to place a drop of water onto the slide, the was
then sterilize and used to touch a colony of the cultured bacteria that was incubated
and suspended in the drop of water on the slide, the slide was left to air dry
after which it was heat fixed and stained. After staining the slide is view
under oil immersion and it was determined that s.aureus, s.saprophyticus and
s.epidermidis was GPCs due to their crystal violet appearance observed under
the microscope. Catalase and Coagulase test was also performed on the microbes
and the results that were obtained showed that S.aureus was positive for
catalase and coagulase, S. epidemidis and s. saprophyticus was negative for
coagulase and positive for catalase test. The coagulase test was carried out by
inoculating there plasma tubes with each of the bacteria and incubating the
tubes at 370c. After incubation it was observed that S.aureus was coagulase
positive, while S. epidermidis and S. saprophyticus were coagulase negative as
shown in Table 2 of the results. The three bacteria were test against
novobiocin to determine if they are susceptible or resistant against the drug
and it was confirmed that S.areus and S.epidermidis were susceptible and
S.saporphyticus was resistant. The test was performed by inoculated a biplate
agar via streaking (which covers the entire agar section in contrast to
quadrant streaking) with the different bacteria and placing a novobiocin disk
in the center of each sections of the biplate. The inoculated biplate were then
incubated and observed to determine the growth of each bacterium around the
novobiocin disk. Susceptible microbe’s growth shows a clear bacteria free zone
around the novobiocin disk which give a suggestive conclusion. The zone is
measured to confirm susceptibility and the zone should be greater than or equal
to 14mm to confirm susceptibility while a less that 14mm would indicate that
the microbe is resistant to the drug. Resistant microbes grow all the way up to
the novobicin disk which shows that the drum does not have any effect against
the bacteria as was observed with S.saprophyticus.



S.aureus, and S.saprophyticus was identified.



“Gram Staining.” Microscopy, 3 Nov.

“Gram stain.” Medical microbiology,

Catalase Test. (n.d.). Retrieved January
22, 2018, from

Blood Agar, Hemolysis, and Hemolytic Reactions.
(n.d.). Retrieved January 23, 2018, from
Agar, Hemolysis, and Hemolytic Reactions. (n.d.). Retrieved January 23, 2018,

Aryal, S. (2017, May 08). Catalase Test-
Principle, Uses, Procedure, Result Interpretation with Precautions. Retrieved
January 23, 2018, from