Introduction:

The purpose of these experiments was: to perform biochemical tests on known

Staphylococcus Enterococcus and Micrococcus species, as well as Streptococcus

and Enterococcus species. These experiments are important to people today because they help

distinguish the different types of bacteria. For example, Staphylococcus aureus is the most

pathogenic in the family of Micrococcaceae. It causes boils, impetigo, food poisoning,

osteomyelitis, toxic shock syndrome, and other life threatening infections. Meanwhile,

Staphylococcus saprophyticus caused urinary tract infection, particularly in young

women, and is second to only E. coli in causing UTI. Knowing what bacteria is in a host can

help doctors figure out the proper treatments to cure a patient.

In order to distinguish bacteria from each other, one must go through several tests to

make an accurate conclusion. Micrococcaceae, such as Staphylococcus and Micrococcus

may be distinguished from Streptococcus and Enterococcus by the catalase test.

Staphylococcus and Micrococcus are catalase positive while Streptococcus and

Enterococcus are catalase negative. A catalase positive tests yields vigorous bubbling when

hydrogen peroxide is converted to water and oxygen gas. It’s usually the first test done to a gram

positive coccus specimen.

Micrococcus can be distinguished from Staphylococcus, Streptococcus, and

Enterococcus by its positive oxidase reaction. In an oxidase positive test, the enzyme

cyctochrome oxidase reduces oxygen. An oxidase disc that contains p-aminodimethylaniline is

reduced by this enzyme and makes the color change from colorless to pink to purple in about

five minutes.
 On a TSA plate, the colonial morphology of Staphylococcus are small and white to gold

colored. Streptococcus colonies are pinpoint sized and translucent to grayish-white in color.

Micrococcus colonies are small, and yellow or pink colored.

Beta hemolytic Staphylococcus aureus is distinguished from other staphylococcus

by it coagulase positive, DNase positive, and positive mannitol fermentation, unlike most other

bacteria. One exception is Staphylococcus saprophyticus, which also ferments mannitol.

The coagulase test shows how Staphylococcus aureus can clot fresh blood plasma. The

DNase test is similar to the coagulase test; DNA bound to methyl green is the medium, and when

an organism such as Staphylococcus aureus excretes DNase, it will hydrolyze the DNA in

the medium and create a zone of clearing in the growth. The mannitol salt test shows that

Staphylococcus aureus can grow in the presence of a high concentration of salt and ferment

mannitol. Staphylococcus aureus will be able to grow in the NaCl medium and show it can

ferment mannitol by changing the pH indicator phenol red and turning it into a yellow color.

Other staphylococci and micrococci species can grow in NaCl medium but can’t ferment the

mannitol.

When characterizing Streptococci, there are 5 groups: Group A, B, C, and D.

Streptococcus pyogenes is the only Group A strep. These groupings are based on the

chemical differences of cell surface components. The carbohydrate surface components are

antigenically distinct based on the ability of specific antibodies to recognize a specific antigen.

Group A beta hemolytic Streptococcus pyogenes produces two types of beta hemolysins,

streptolysin S and streptolysin O. Streptococcus pyogenes is sensitive to bacitracin, while

other streptococci are resistant. Streptococcus pneumoniae is sensitive to optochin.

 Group B, beta, or gamma hemolytic Streptococcus agalactiae are distinguished through

the CAMP test, which stands for Christie, Atkins, Munch, Peterson. Group B streptococci

excrete an extracellular factor that enhances the beta hemolysis of Staphylococcus aureus when it

is streaked at right angles to the Staphylococcus aureus. A positive result of the CAMP test will

show and arrowhead-shaped zone of hemolysis at the intersection of the two strains pointing

toward the Staphylococcus aureus. An exception to this is that Streptococcus pyogenes also

excrete the CAMP factor. Streptococcus agalactiae can be distinguished from Streptococcus

pyogenes by having long chains of paired cocci.

Group D Enterococcus faecalis and Enterococcus faecium may be alpha, beta or gamma

hemolytic. Group D streptococci are difficult to treat because they are resistant to most

antibiotics. They can grow in high salt concentrations and can ferment mannitol and give a weak

positive fermentation reaction on a mannitol salt plate. When gram stained, these enterococci

come in clusters, chains, or singles. The DNA sequence of group D streptococci (or

Enterococcus) are different from other streptococci, so they can also be distinguished through

DNA testing.

Fermentation is another useful test to distinguish species of Streptococcus, especially

alpha hemolytic Streptococcus. Streptococci are strict fermenters, meaning they produce mostly

one end-product, lactic acid. CT agar, which is Cystine Tryptic Agar, supplies the necessary

nutrient to aid in the growth of fastidious organisms without the need for additional carbon

dioxide. It is a semisolid so so motile organisms such as the fastidious Streptococci and

Neisseria will move away from the line of inoculation. Staphylococcus and the enterics are not

good to use because they are vigorous growers, so phenol red broths are best used for these
organisms. A yellow color in the upper one-third or throughout is a positive reaction in a CT agar

test.

Results:

Staphylo Catalase Oxidase Colonial Hemoly Novo DNa Fer Gro

cocci Test (Taxo N) Morphol sis bioci se me wth
(+ pink) ogy n nta
(- no color Sensi tio
change) tivity n

Enteroco - -
ccus
faecalis

Staphylo + - round beta R + + A

coccus
aureus

Staphylo + - round gamma R - + A

coccus
saprophy
ticus

Staphylo + - round beta S _ - K

coccus
epidermi
s
Staphylo Catalase Oxidase Colonial Hemoly Novo DNa Fer Gro
cocci Test (Taxo N) Morphol sis bioci se me wth
(+ pink) ogy n nta
(- no color Sensi tio
change) tivity n

Micrococ + +
cus
luteus

Streptococci Gram Lancefield Group Hemolysis Bacitracin Optochin

Stain

Pyogenes + alpha beta S R

agalactiae + beta beta R R

pneumoniae + - alpha R S

salivarius + k alpha R R

faecalis + D sigma R R

Salt Mannitol Glucose Lactose Mannitol Inulin

Tolerance Fermenta (Strep) (Strep) (Strep) (Strep)
tion

Staph. + A
aureus

Strep. - K A A (K) K K
pyogenes
 Salt Mannitol Glucose Lactose Mannitol Inulin
Tolerance Fermenta (Strep) (Strep) (Strep) (Strep)
tion

Strep. - K A A (K) K K
agalactiae

Strep. - A A A K A
salivarius

E. faecalis + K A A A K

Strep. - K A A K A
pneumoni
ae

Procedures:

27

For the catalse test, use a sterile loop or wooden applicator stick, and place a clump of

the organism on a dry slide. Add a drop of 3% hydrogen peroxide to the culture and

observe. For the oxidase test place Taxo N disc on a dry slide. Moisten with a drop of

water and obtain a visible amount of culture with a sterile applicator stick. Observe any

color change. Also, prepare 3 blood agar plates and streak the given cultures for

isolation with the quadrant method. Place antibiotic disks on each plate and incubate.

Also, obtain one DNase plate and divide it into thirds. Using the given cultures, spot
each organism with a single line streak. Do the same procedure with a mannitol salt

plate and incubate both.

29

As a group, gram stain all the given bacteria and find the colonial morphology. Also,

label five blood plates with the given cultures and streak each plate for isolation using

the quadrant method. Before flaming, stab the adjacent to the primary sector to the

bottom of the plate in the center of each blood plate. Next, place bacitracin on the 1st

sector of each blood plate. Incubate the blood plates in a candle jar for a couple days.

For the CAMP test, obtain a loopful of Staphylococcus aureus and streak a single line

vertically on one edge or center of one blood agar plate. Inoculate the other given

cultures about 1 cm from and perpendicular to the S. aureus. Incubate the CAMP test

plate for 24 hours and then refrigerate. For the mannitol salt plate test, divide 2 mannitol

salt plates into 3 sectors and spot the given cultures. Incubate for a couple days and

examine growth and fermentation, if any. For the CT agar test, label CT tubes of

glucose, mannitol, and inulin with the given cultures. Inoculate them by stabbing with a

loop one half the depth of CT agar and incubate for 2 days and examine growth.
Discussion:

The three pathogens in yellow can be distinguished quite easily from each other. For one,

Staphylococcus saprophyticus is the only one of the three that is resistant to novobiocin,

while the others are not. E. faecilis is also a gram negative bacteria, making it different from the

gram positive Staphylococcus saprophyticus. Being able to figure out what kind of

bacteria one is dealing with can help a great deal with the strategy to deal with it. Knowing that

novobiocin has no effect to S. saprophyticus will tell doctors that they must use another

antibiotic that is actually effective against it. Without proper identification methods, many people

would be infected with these bacteria and would become seriously ill or even die due to the lack
of treatment options because the bacteria is unknown. Overall the experiments were a success;

all results were accepted and met.

References:

Barnett, Margaret. 2008. Selected Materials from Microbiology Laboratory

Exercises, 2nd Edition, McGraw-Hill, pp. 157-159, 183-186