Title: Identification of Gram Positive Organisms using various techniques and tests.

Objectives:
 To familiarize ourselves with blood agar plates and the various equipments used in the
microbiology lab.
 To familiarize ourselves with how to streak a plate properly and why it is done a
particular way.
 To be able to know what a good plate is from one that is contaminated.
 To familiarize ourselves with the chemicals used when doing a Gram Stain and how to
perform a Gram Stain on a smear.
 To be able to produce isolated colonies on a plate using the organisms provided.
 To familiarize ourselves with the Staphylococci and Streptococci species.
 To perform the various tests needed to determine the type of organism it is.
 To ensure that we can perform the Bacitracin Test and be able to have the knowledge of
its principle.
 To ensure that we can perform the Optochin Test and be able to the principle of the test.
 To be able to perform the Camp Test and also state why it is done.
 To be able to perform the Coagulase Tube Test and state why it is done.
 To be able to perform the Novobiocin Test and state why it is being done.
 To be able to read the morphology of all the smears stained and be able to differentiate
between the two species of organisms.
 To be able to say the type of organism it is after doing the differentiating tests.

Introduction:
Microbiology is the study of microorganisms and the effects whether positive or negative

they have on humans, animals, plants and the environment. These organisms are too small to be

seen with the unaided eye so therefore a microscope is needed for them to be seen.

Microorganisms may be Prokaryotes or Eukaryotes and by using techniques of microbiology we

can isolate these organisms, study their characteristics and by using various tests, distinguish

them accordingly.
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 One technique used in microbiology is the streaking plate technique. This technique of

streaking a plate with an inoculating wire loop is used to spread millions of cells over the surface

of a solid medium so that some individual cells are deposited at a distance from all others. These

cells grow and reproduce, forming an isolated colony. One or more colonies will be separated

from all others and represent a source of pure culture. The streak plate method is a rapid and

simple technique of mechanically diluting a relatively large concentration of microorganism to a

small scattered population of cells. The goal is to obtain isolated colonies on a large part of the

agar surface so that desired species can be processed appropriately. In order for isolated colonies

to be obtained the agar must be of solid medium.

An agar is of different types based on the nature of the organism to be grown. Agar plates

can either be selective, differential and non-selective or it can be both. There are a wide variety

of plates used in the microbiology lab but the most common ones that are of use are as follows:

The blood agar plate is a rich non selective, differential medium which supports the

growth of a wide variety of microbes. Blood plates are made with sheep blood as well as other

selected nutrients. On the blood agar plate there are three types of hemolysis that can be seen.

Bacteria that destroy blood cells (produced by enzymes) around them are said to be beta

hemolytic because there is complete breakdown of the red blood cells. Alpha hemolysis can also

be seen on blood agar plates with the partial breakdown of the red blood cells because some

bacteria cannot destroy all the red blood cells. This type of hemolysis can be seen with a greenish

coloring of the colonies. The last is gamma hemolytic or no breakdown of the red blood cells.

Other plates used in microbiology are the Eosin Methylene Blue Agar which is both selective

and differential. Another common plate used is the Mackoney Agar which is a differential plate,

selective for the Gram Negative Bacteria used primarily for the detection and isolation of the

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Enterobacteriace family. The Mannitol Salt Agar is a selective medium that favors the growth of

staphylococcus and differentiates between pathogenic and non- pathogenic staphylococcus

organisms. The Sabourad Dextrose Agar is a medium that promotes the growth of fungi. Another

vital and important technique used in identifying the organisms is the gram stain. The Gram

Stain involves four processes:

 Staining with a water soluble dye called crystal violet.

 Iodine

 Decolonization

 Counterstaining

Due to the differences in the thickness of the peptidoglycan layer in the cell membrane

between Gram Positive and Gram Negative Bacteria, Gram Positive Bacteria retains the crystal

violet stain during the decolorization process while Gram Negative Bacteria lose the crystal

violet stain and are instead stained by the safranin in the final staining process.

Gram Positive Organisms describes a diverse class of bacteria. It is one of the basic

classifications of bacteria by separating it from Gram Negative. Gram Positive organisms have

several characteristics that make them unique from other bacteria and microorganisms. They

have a cytoplasmic membrane which serves to transport materials, nutrients and waste in and out

of the cell. They have a capsule which serves as a protective layer (not all but many). Some also

have a flagellum. Gram Positive Organisms can take on a number of shapes. Many of them form

coccus or circular shapes. Under the microscope they can be further identified by their staining.

Some have slightly flattened edges and come in pairs. These are known as diplococci. Other

circular organisms will from clusters or lines. These organisms such as Streptococcus and

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Staphylococcus are often pathogenic in humans and will cause disease in the right circumstances.

While some are harmless, it is important to know the potential for disease. The organism can also

form a bacillus or rod shaped. The rod shaped Gram Positive Organisms are usually divided into

spore forming and non-spore forming. Gram Positive Organisms describes a group of organisms

that is diverse in shape and function.

The Staphylococcus species of Gram Positive Organisms can cause many forms of

infections. Staphylococcus aureus causes superficial skin lesions and localized abscesses in other

sites. It is a major cause of hospital acquired (nosocomial) infection of surgical wounds. It also

causes food poisoning and can cause toxic shock syndrome. Staphylococcus epidermidis causes

infection associated with indwelling medical devises. Staphylococcus saprophyticus causes

urinary tract infections especially in young women and girls.

The Streptococcus species of Gram Positive Organisms are composed of about 20 different

species that are gram positive spherical cells occurring in chains of varying length. They are non-

motile and non-spore forming as well as capsulated. They can be classified in five ways: pattern

of hemolysis on the blood agar plate, antigenic differences in the cell wall known as the

Lancefield Grouping, growth characteristics, biochemical reactions and genetic analysis.

Streptococci organisms have anti-phagocytic components and release various toxins streptolysin

O and S and enzyme streptokinase, hyaluronidase and proteinase.

Distinguishing between Staphylococcus organisms and Streptococcus organisms are done by

the catalase test. It is one of the major differences between members of the Staphylococcus

species which are catalase positive and the Streptococcus species which are catalase negative.

The principle behind these tests is the hydrogen peroxide which is a highly toxic substance to

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bacteria is produced by many strains of Staphylococcal organisms in the presence of oxygen.

Catalase under the same aerobic conditions breaks down the hydrogen peroxide to water and

oxygen. The enzyme is necessary for the life of bacteria growing anaerobically and it is found in

large quantities in strict aerobics, however strict anaerobes since they cannot grow in air do not

form hydrogen peroxide and therefore do not produce catalase. Catalase production by an

organism is detected by the evolution of gas indicated by bubbles when hydrogen peroxide is

added to the culture. Based on whether it is a Staphylococcus species or a Streptococcus species

further identification tests are done. The morphology of organisms on the plate, the cellular

morphology under the microscope and the catalase tests differentiates between the organism

being a Staphylococcus or a Streptococcus.

All Staphylococcus organisms are Catalase Positive. The three most common

Staphylococcus known are the Staphylococcus aureus, the Staphylococcus epidermidis and the

Staphylococcus saprophyticus. There are many more Staphylococcus organisms but these three

are the ones we dealt with and the ones we were introduced to. The Tube Coagulase Test is one

of the tests used to differentiate between Staphylococcus aureus from other commonly isolated

Staphylococcus species. Another test used for the identification of Staphylococcus species is the

Novobiocin Test. This test is used to differentiate Staphylococcus saprophyticus from other

coagulase negative Staphylococci.

If the organism is Catalase Negative then it is a Streptococcus. The most common

Streptococci organisms that we were introduced to were the Streptococcus pyogenes,

Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus viridians and the

Enterococcus faecalis. In order to differentiate and identify these organisms the hemolysis on the

blood agar plate must be observed. The hemolysis hints at the type of organisms it can be but

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further identification must be done for confirmation. If beta hemolysis is seen on the blood agar

plate then the Bacitracin Test is done. This test is useful for differentiating beta hemolytic Group

A Streptococcus from beta hemolytic non- Group A Streptococcus. This is important because

most Streptococcal diseases are caused by Group A Streptococcus. Bacitracin is a true antibiotic

in that it is an antimicrobial compound which is naturally produced by a microorganism. It is

produced by the Bacillus lichenformis and acts to interrupt the formation of the bacterial cell

wall. The next identifying step is the CAMP test. This test is used to differentiate between Group

B beta hemolytic Streptococci. Streptococcus agalactiae produces an extracellular diffusible

protein referred to as CAMP factor which interacts with the beta toxins of some strains of

Staphylococcus aureus. Group B beta hemolytic Streptococci form part of the normal

oropharagenael and vaginal flora but can cause infections in the meninges and septicemia in

infants. Another differentiating test is the Optochin Test which is used to differentiate between

two types of alpha hemolytic Streptococci that is Streptococcus pneumoniae and Streptococcus

viridians. Most strains of Streptococcus pneumoniae are sensitive to the drug ethylhydrocupine

hydrochloride whereas Streptococcus virdan is not. Optochin causes the bacterial cells of

Streptococcus pneumoniae to lyse. This test is also an alternative presumptive test for the bile

solubility test. The Bile Solubility Test on the other hand is used to differentiate between the

Streptococcus virdans and the Streptococcus pneumoniae both of which is alpha hemolytic. The

majority of the strains of the Streptococcus virdans are insoluble in bile however the majority of

the Streptococcus pneumoniae are soluble. The source of the bile used for this test is the sodium

desoxycholate. The bile solubility test is used to determine the ability of bacterial cells to lyse in

the presence of bile salts within a specified time. The Streptococcus pneumoniae possesses an

autolytic enzyme which lyses the cell’s own wall during division. The addition of the bile salts

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activates the autolytic enzyme and the organisms rapidly autolyze. To differentiate between the

Enterococcus and Group D the bile esculin tests done. This test basically is the ability of some

organisms to grow in the presence of bile and also be able to hydrolyze esculin.

Materials:
 2 wire loops
 1 Bunsen burner
 12 slides
 Candle jar
 Incubator
 Slide holder
 Sharpie
 11 blood agar plates
 1 Muller Hinton plate
 Microscope
 Disposable pipettes
 Immersion oil
 Cotton swab
 Applicator stick
 Ruler
 Test tubes

Chemicals:
 Hydrogen peroxide
 10% bleach
 Saline
 Crystal violet
 Iodine
 Acetone
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  Saffarin
 Human plasma (should use rabbit plasma but none was available)
 Bacitracin Disc
 Optochin Disc
 Novobiocin Disc
 Sodium Deoxecolyate
 Bile Esculin

Organisms:
 Staphylococcus aureus
 Staphylococcus epidermidis
 Staphylococcus saprophyticus
 Streptococcus pyogenes
 Streptococcus pneumoniae
 Streptococcus viridians
 Streptococcus agalactiae
 Enterococcus faeclis

Procedure/ Result:
Day 1:

1) Label one of the blood agar plates Staphylococcus aureus together with the date and the
group number.
2) Streak the plate by following the steps outlined below:

How to streak a plate:

 Sterilize a wire loop by heating it until red hot in a flame and allow the loop to cool.
 Pick u a loop full of bacterial growth from the surface of the agar plate and starting
about one inch from the edge of the plate, streak lightly back and forth with the loop
being flat. This must be done close to each other as well as parallel.
 Sterilize the loop again and let cool. Turn the plate about 20 degrees and lightly make
another set of parallel streaks in only one direction to the uninoculated agar.

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  Flame and cool the loop again and make another set of streaks in one direction
allowing it to be perpendicular to and crossing the second set of streaks made but
ensuring that the first set made is not touched.
 Flame and cool loop again and make a third set of streaks away from the last set.
 Flame and let the loop cool again. From the last set of streaks made using the loop
make squiggle lines out the centre of the plate without touching any of the streaks.
3) Incubate the plate at 35-37 degrees for 18-24 hours.
4) A slide was taken and labeled with the group number as well as the name of the organism
and the date.
5) Using a china marker a circle was drawn in the centre of the slide.
6) One loop full of saline was added to the centre of the slide.
7) The loop was flamed and allowed t dry and one colony of the organism Staphylococcus
aureus was touched and then it was smeared into the water on the slide.
8) The slide was allowed to air dry.
9) After the slide was dried it was heat fixed b simply passing it through the flame of the
Bunsen burner two to three times for about 2 seconds.
10) The above steps were repeated using the remaining organisms for both the making of the
smears and the streaking of the plates.
11) All plates were placed in the incubator for 18-24 hours at a temperature of 35-37 degrees.
12) The slides were placed on a slide holder to dry. It should be noted that a control was also
made by taking a loop full of broth and smearing it on slide.

Day 2:

1) The Gram Stain was done on all the slides that were made.
The Gram Stain was done by following the outlines given below:
 One slide was taken and placed on a rack inside the sink.
 The slide was flooded with Crystal violet and left for one minute.
 The slide was then rinsed gently with distilled water and flooded again with
iodine and left for another one minute.
 After the one minute, the slide was rinsed with distilled water three drops of
acetone was added to the slide but was quickly washed off.

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  The slide was then flooded with saffron and allowed to sit for thirty seconds. This
was then washed off with distilled water and left to dry.
2) All the plates were taken out from the incubator and the morphology of the organisms
were recorded.

The following table shows the results of the organisms’ morphology.

Organism Observations
Staphylococcus aureus Golden brown colonies
Moist on the plate
Edges of colonies appear to be smooth
Size of colonies are medium
Plate showed beta hemolysis
Staphylococcus epidermidis Colonies appeared white in color
It was dry on the plate
Colonies were of medium size
It was non-hemolytic
Staphylococcus saprophyticus Colonies appeared to be medium
Colonies were white in color
Colonies were moist and contained smooth
edges.
Streptococcus pyogenes Beta hemolytic
With colonies with smooth edges
Slightly moist
Pin point colonies
Streptococcus agalactiae White colonies
Small with smooth edges
Beta hemolytic
Enterococcus faeclis Non hemolytic
White colonies
Medium colonies

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 3) The Gram Stain was done on all the slides that were stained and the results recorded.
The following table shows the results of the Gram Stain done on all the organisms.

Organism Gram Stain/ Observations

Control Purple clusters accompanied by pink short chains
with some attached to each other. This is both
Gram Positive and Gram Negative.
Staphylococcus aureus Purple clusters/ bunches
Round in shape
Gram Positive
Staphylococcus epidermidis Purple bunches
Cocci
Gram Positive
Staphylococcus saprophyticus Purple clusters
Round cocci
Gram Positive
Streptococcus pyogenes Purple chains
Large oval shaped
Gram Positive
Streptococcus pneumoniae Purple chains
Fine, diplococcic
Gram Positive
Streptococcus virdans Purple chains
Gram Positive
Streptococcus agalactiae Purple
Small cocci in clusters
Gram Positive
Enterococcus faeclis Purple
Fine cocci in chains
Gram Positive

4) The Catalase Test was done on all the organisms that were inoculated on the blood agar
plates. The Catalase Test is done by following the outlines given below:
 On a slide, one drop of hydrogen peroxide was added.

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  Using the wooden applicator stick, one colony of the organism was touched and
placed on the hydrogen peroxide.
 It was observed for the formation of bubbles.
 The above steps were repeated for all the organisms.

The table below shows the results for the Catalase Test that was done:

Organism Interpretation of test

Staphylococcus aureus Positive
Staphylococcus epidermidis Positive
Staphylococcus saprophyticus Positive
Streptococcus pyogenes Negative
Streptococcus pneumoniae Negative
Streptococcus agalactiae Negative
Streptococcus virdans Negative

5) The Coagulase Tube Test was set up on all the Staphylococcus organisms that were
catalase positive. This test is done by following the steps outlined below:

 A wire loop was flamed and allowed to cool.

 Three tubes containing human plasma (should have been rabbit plasma) were
labeled Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus
saprophyticus.
 A loop full of Staphylococcus aureus was taken and inoculated into the respective
tube.
 The above step was done for the remaining two organisms.
 All three tubes were incubated at 35-37 degrees for 18-24 hours.
6) The Novobiocin Test was then set up in order to differentiate which from the set of
Coagulase negative test is the Staphylococcus saprophyticus. This test is done by
following the steps outlined below:

 In a tube containing 5 ml of saline (0.85% sodium chloride) add two the

three colonies of the Staphylococcus saprophyticus organism and invert
gently to mix.

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  Compare the suspension made with a 0.5 MacFarlane Standard.
 If the suspension is stable and matches the McFarlane Standard, a cotton
swab was taken and placed inside the suspension. The swab was swirled
and then removed to be lawned on a Muller Hinton Plate.
 After the organism was lawned on the plate a 30micrograms disc was
placed in the middle of the plate.
 The plate was then incubated at 35-37 degrees for 18-24 hours.

7) The Streptococcus species was done next with further testing being done to identify the
organism.
8) The Bacitracin Test was done to determine between Group A beta hemolytic
streptococcus from other beta hemolytic streptococcus organisms. The following is the
steps outlined on how to perform the Bacitracin Test:
 A blood agar plate was divided into half.
 It was labeled Streptococcus Group A and the other half Streptococcus Group B.
 Streptococcus pyogenes was inoculated on the plate labeled Group A while the
Streptococcus agalactiae was inoculated onto the plate labeled Group B.
 A Bacitracin disc was placed on both halves of the plate.
 The plate was incubated for 18-24 hours at a temperature of 35-37 degrees.
9) The Camp Test was then set up in order to differentiate between Group B beta hemolytic
Streptococci from other beta hemolytic Streptococci. The following steps outlined how
the test is done:
 A blood agar plate was divided into two.
 On one half two lines were drawn parallel to each other.
 The middle line was labeled Staphylococcus aureus while the two parallel lines
were labeled as Group A streptococcus and Group B Streptococcus.
 Two colonies of the Staphylococcus aureus were touched with the wire loop and
were streaked down the middle of the blood agar plate.
 One colony of the Streptococcus agalactiae was touched and a single streak was
made on the line labeled Group B ensuring that the streak does not touch the
Staphylococcus streak.

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  One colony of the Streptococcus pyogenes was touched and streaked on the line
labeled Group A ensuring that the streak does not touch the Staphylococcus
streak.
 The plate was incubated at a temperature of 35-37 degrees for 18-24 hours.
10) The Optochin Test was set up next to differentiate between the two types of alpha
streptococci organisms that were grown. The following is the steps outlined as to how
this test is performed:
 Label two blood agar plates as Streptococcus pneumoniae and the other as
Streptococcus viridians.
 Inoculate both plates with each respective organism.
 Place an Optochin Disc on the middle of each plate.
 Incubate both plates at a temperature of 35-37 degrees for 18-24 hours.

Day 3:

1) All the plates that were set up on the previous day were removed from the incubator
and the results were interpreted.
2) The blood agar plate containing the Optochin Test was observed.
Results: It was observed that there was a clearing around the disc on the plate of the
Streptococcus pneumoniae and that clearing had a measurement of 15 mm known as
the zone of inhibition. Considering that the zone size distinguishing it from being
sensitive or resistant is less than 14 this is considered to be sensitive. There was no
clearing around the disc for the Streptococcus viridians so therefore it is considered to
be resistant to the Optochin disc. Some Streptococcus pneumoniae is resistant so
therefore the Optochin test is followed by the Bile Solubility Test.

Based on the above results from the Optochin Test, the Bile Solubility Test was done.
This test is a follow up to the Optochin Test in that it is used to differentiate between
the Streptococcus viridians and the Streptococcus pneumoniae which happens to be
both alpha hemolytic. The following outlines the steps used when performing the Bile
Solubility Test:

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  Label two tubes containing saline Streptococcus pneumoniae and
Streptococcus virdans.
 Add about 5 colonies to the respective tubes and make a suspension.
 Compare with the MacFarlane Standard to compare the turbidity.
 With each tube containing the sodium deoxecolyate add 1 ml of the
suspension.
 With the remaining suspension add 1 ml of saline so that the control can be
made.
 Place in incubator for 2-3 hours at 37 degrees Celsius.

Result of Bile Solubility Test: Tube containing the Streptococcus pneumoniae was clear.

Tube containing the Streptococcus virdans was cloudy.

Controls: Positive control: Streptococcus pneumoniae

Negative control: Streptococcus virdans.

Result of Bacitracin Test: No zone of inhibition was seen on any of the organism. Both were
resistant but because it was known organisms the Streptococcus pyogenes was supposed to be
sensitive.

Positive Control: Streptococcus pyogenes

Negative control: Streptococcus agalactiae

Result of the CAMP Test: No arrowhead as seen at the junction of any of the two organisms
that was plated. This was due to the composition of the blood plate was not made with sheep
blood but rather cow’s blood. Since the organisms were known the arrowhead should have
appeared at the junction of the Group B which was the Streptococcus agalactiae.

Positive Control: Streptococcus agalactiae

Negative Control: Streptococcus pyogenes.

Result of the Novobiocin Test: A clearing or zone of inhibition was seen around the
Staphylococcus saprophyticus indicating that it is sensitive. However the concentration of the

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disc was 30mg when in fact it was suppose to be 5mg. The zone of inhibition was supposed to be
seen around the Staphylococcus epidermidis.

Positive Control: Staphylococcus epidermidis

Negative Control: Staphylococcus saprophyticus

Result of the Coagulase test: Staphylococcus aureus – positive formation of clot

Staphylococcus saprophyticus – negative no clot formation

Staphylococcus epidermidis – negative no clot formation

Result of the Bile Esculin Test: Tube containing the esculin slant turned black confirming the
Group D or the Enterococcus faecalis.

Positive Control: Enterococcus faecalis

Discussion:
These labs done were only on the common Gram Positive Organisms known and are
commonly mentioned in the clinical laboratory. Identification of Gram Positive Organisms
follows a flow chart that best describes how to go about identifying these organisms. Before
identification can begin one must know about the various organisms, their characteristics
whether it is the morphology on the plate or the cellular morphology found when the Gram Stain
is done. In this lab that was processed, it was noted that the Gram Stain was very vital and
important when it comes to starting with the identification process. Gram Positive Organisms
stain purple in color. Gram Positive Organism can either carry the shape of cocci in pairs, rods in
chains or can be both known as diplococcic. These characteristics along with the various
differentiating test helps in the identification of the organisms. With each test that is done a
control must be done with the test. A control tells whether the test was accurately performed or
not. The Flow chart for the Identification of Gram Positive Organisms is shown below:

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 As long as the Gram stain is done accurately then proceeding to the next step is simple. In
this lab after the gram stain was done and the morphology of the plate was observed the Catalase
test was done in order to distinguish which organisms belong to the Staphylococci species and
which organisms belong to the Streptococci species as all Staphylococci are Catalase positive
whereas all Streptococci are Catalase negative. The Gram satin indicates if it can be
Staphylococci or a Streptococci but the confirmation is done by performing the Catalase test. The
control for the Catalase test is the use of a Streptococci organism as they are Catalase negative
and the positive control will be an organism from the Staphylococci species. After distinguishing
between The Staphylococci and the Streptococci we proceed further. We did the tests required
for the differentiation of the different Staphylococcus organisms as it was confusing to do both
sets if identification tests. There were only three Staphylococcus organisms that we worked with
and they were the Staphylococcus aureus, the Staphylococcus saprophyticus and the
Staphylococcus epidermidis. To confirm it is a Staphylococcus aureus, the tube Coagulase test
was one. Once the organism is Staphylococcus aureus a clot would form in the tube containing
the rabbit plasma which is the ideal substance used. However in this test human plasma was used
and the formation of the clot were faint but still present. It I not recommended to use human
plasma. The negative control that was done was the Staphylococcus epidermidis which had no
fibrin strands in the plasma. This is because Staphylococcus epidermidis is a Coagulase negative
organism just like Staphylococcus saprophyticus. After the identification of the staphylococcus

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aureus the Novobiocin test was done to identify between the Staphylococcus epidermidis and the
Staphylococcus saprophyticus. This uses a disc that tells which organism is sensitive and which
is resistant. The disc is five micrograms and it is impregnated with the Novobiocin.

The Streptococci was done next and the identifying of these organisms starts with not
only the characteristics of the Gram stain but it is of the utmost importance that the hemolysis on
the blood agar plate is read. There are three types of hemolysis shown on a blood agar plate. The
first being beta hemolysis: if this type of hemolysis is seen then the Bacitracin Test is done
which identifies between Group A beta hemolytic Streptococci from non -hemolytic
Streptococci. From the four organisms that we worked with for the lab, the positive control for
this test was the Streptococcus pyogenes while the negative control was the Streptococcus
agalactiae. While the Bacitracin Test is for Group A Streptococcus, Group B beta hemolytic is
identified using the CAMP Test. The Group B which is the Streptococcus agalactiae produces a
toxin that interacts with strains of the Staphylococcus aureus in order to produce a reaction. This
reaction is in the form of an arrowhead at the junction of where both organisms meet. The bile
solubility test was next done on the alpha hemolytic organism which was the Streptococcus
pneumoniae and the streptococcus virdans. The positive control was the streptococcus
pneumoniae while the negative control was the streptococcus virdans. The streptococcus
pneumoniae was clear confirming that the colonies dissolved clearly the initial turbidity while
the negative control remained turbid. To confirm that it was streptococcus pneumoniae the
Optochin test was done. This test resulted in the Streptococcus pneumoniae giving a zone of
inhibition of 15mm making it sensitive while the Streptococcus virdans had no zone of
inhibition. The Bile Esculin test on the other hand is used to identify bacteria that can hydrolyze
esculin in the presence of bile mainly the Enterococcus or Group D. Performing this lab one
needs to take certain precautions in order to ensure that accurate results are produced and also
precautions to protect ourselves. Some of these precautions are as follows:

 Ensure that the blood agar plates are pre- dried.

 Ensure that the wire loop is sterilized before each use.
 When flaming the wire loops be careful of the organisms turning to aerosols and infecting
someone.

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  Always ensure that when grams staining you do not over decolorize as you will cause a
false positive while under decolorizing can produce a false negative.
 Always ensure that the incubators are at 37 degrees Celsius for optimum growth of the
organisms.
 Ensure that the agar is not included in your colonies when doing the catalase tests or else
false positives will occur.
 The ideal substance used for the coagulase test is rabbit plasma. Any other reagent used
will be difficult to interpret.
 Ensure that the Novobiocin disc is of the concentration of 5 micrograms.
 Bacitracin disc should have a concentration of 0.04 units.
 Sheep blood is best used to prepare blood agar as the cow’s blood was used and the
CAMP was unsuccessfully.
 Optochin disk should be of the accurate concentration.
 Bile Solubility tests should use sodium deoxecolyate.
 The suspensions made should match the corresponding MacFarlane standard for the
particular test.
 All tests should be accurately followed and done.

Conclusion:

Within the limits of experimental error it can be concluded that the aim of this lab which
was the identification of Gram Positive Organisms was a success. All the objectives that were
put forward were achieved. Techniques such as streaking a plate and gram staining was
performed adequately and accurately while all the identifications tests required for the
Staphylococci species and the Streptococci species were done .After this lab was completed the
most vital and important things that I learnt was:

 The method of streaking a plate.

 Gram Positive Organisms stain purple and can either be in pairs and cocci as well
as in chains in rods.
 All Staphylococci organisms are catalase positive while all Streptococci
organisms are catalase negative.

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  For Staphylococci organisms the identifying tests are the coagulase test and the
Novobiocin test. Staphylococcus aureus is Coagulase positive while
Staphylococcus epidermidis is sensitive with a zone size of 15 mm.
 Streptococci organisms can either be differentiated into beta hemolytic, alpha
hemolytic or non-hemolytic from their blood agar plate.
 Identification test for beta hemolytic Streptococci are the Bacitracin test and the
CAMP test which identifies Group A and Group B. Group A is Streptococcus
pyogenes while Group B is Streptococcus agalactiae.
 Alpha hemolytic tests are the Optochin test and the Bile solubility test that
identifies Streptococcus pneumoniae as bile soluble and Optochin sensitive.
 The Bile Esculin was positive for the Enterococcus faecalis.

It should be noted that all the tests performed where done using controls to ensure that our media
was working and that all our tests were accurately performed. All the organisms were identified
using the various tests and techniques outlined throughout the lab.

References: Microbiology Standard Operating Procedures and Identification Tests.

Microbiology Theory Notes.

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