MT 111 LABORATORY NOTES 9: STAPHYLOCOCCI

OBJECTIVES:

 Review on the morphology of the organism

 Compare and contrast the species (Staphylococcus aureus vs Staphylococcus epidermidis) of the genus in terms of:
o Pigment production on potato agar
o Catalase production on trypticase soy agar (TSA)
o Hemolysis and colonial morphology on blood agar (BA)
o Fermentation on mannitol salt agar (MSA)
o Coagulase production
 In the investigation of any unknown organism in microbiology laboratory, a particular step/procedure is followed. The
procedure entails: screening test, presumptive test and the confirmatory tests
o Screening test- will only screen for the presence or absence of that organism in that particular specimen/sample
 Identification of the unknown organism cannot be limited at the screening test only because the screening test will
only help identify if the organism of interest is present/absent in the specimen received in the microbiology
laboratory
 If proven that the screening test says that there is the presence of the microorganism in the sample that is
processed, proceed to the next step which is performing the presumptive test
o Presumptive test- will only presume the identification of the unknown organism in that particular sample
 These tests are termed presumptive because whatever the result of the presumptive test might be correct to
several organisms. The result of the presumptive test might be indication that there is really the presence of the
organism of interest in the sample. A positive presumptive test would indicate the performance of confirmatory
tests
o Confirmatory tests- will confirm the presence of the organism of interest in the sample. It will rule in that there is
really the organism of interest in that particular sample and the patient is really infected of that particular
microorganism.
 Staphylococci are ubiquitous of all organisms. There two species associated with man are Staphylococcus aureus and
Staphylococcus epidermidis. S. aureus produces yellow pigment known as lipochrome and S. epidermidis produces
white pigment.
o They are normally found on the skin, mouth, intestines and genitor-urinary tract of men and animals. Staphylococcus
aureus is pathogenic than S. epidermidis.
 For microbiology laboratory, in the investigation of any unknown organism
o Screening tests- microscopic evaluation of the morphology of the organism
 Microscopic evaluation of the morphology of the organism- will help deduce/conclude that the organism of
interest might be present or absent in the sample. If the screening test says otherwise, there is absence of
microorganism then the confirmatory test may not be performed.
o Presumptive tests- helps support the result of the screening test; a specific test makes use of a specific culture
medium
 Pigment production on potato agar- uses potato agar medium, contained in a test tube; turbid and yellow
 Catalase test catalase on trypticase soy agar (TSA)- uses trypticase soy agar; clear and yellow
 Hemolysis pattern and colonial morphology on blood agar (BA)- uses blood agar, prepared in a petri dish in a
plate, red
o Confirmatory tests- two confirmatory tests for the Staphylococcus
identification
 Fermentation on mannitol salt agar (MSA)- pinkish in color (light pink)
 Coagulase test- does not use any specific culture medium
MORPHOLOGY EVALUATION OF THE ORGANISM

 Staphylococci are gram-positive organisms arranged in clusters (clustered circular organisms colored purple, cocci in
clusters)
 It is akin looking to grapefruit
 Staphylococcus aureus and Staphylococcus epidermidis cannot be differentiated by just looking at the morphology of
the organism because both Staphylococcus aureus and Staphylococcus epidermidis appears to be cocci in clusters in
their morphology evaluation.
o Looking at the morphology, the term representative organism is used because from the staphylococcus family, all
the species under that genus will appear cocci in clusters under the microscope.
 The morphology evaluation is only a screening test that will help detect if Staphylococcus aureus or Staphylococcus
epidermidis is present or not in the processed sample.
 Further tests are performed to positively identify the identity of the unknown organism in the sample.

PRESUMPTIVE TESTS

 Catalase Test
o To detect the ability of the organism to produce the enzyme catalase by using hydrogen peroxide (H2O2)
o Used to differentiate Staphylococci from Streptococci
o Culture medium used: trypticase soy agar (TSA)
o Color: clear and yellow
o In the trypticase soy agar (TSA), the sample received known to have an unknown organism is inoculated to the
trypticase soy agar. Incubate the TSA at 37°C in an incubator for 24 hours. After 24 hours, there will be grown
colonies in the surface of the TSA.
o The grown colonies on the TSA are used to perform the screening test (morphology evaluation). If the morphology
evaluation revealed Staphylococci because it is Gram-positive cocci in clusters then a presumptive test is needed to
know if the colonies are really Staphylococci.
o The grown colonies in the TSA is treated with drops of hydrogen peroxide and if the organism produces the enzyme
catalase, the appearance of gas bubbles in the TSA after the reagent hydrogen peroxide is added to the grown
colonies of the culture medium, indicates that the catalase test is positive and the organism treated with H 2O2
produces catalase
o (+)- appearance of gas bubbles
o Principle:
 Hydrogen peroxide is a disinfectant (causes death/destruction to the organism). In
organisms that produces the enzyme catalase, instead of the organism being harmed by
hydrogen peroxide, the organism will convert the hydrogen peroxide to other harmless end
products so that the endpoint is the organism is not harmed at all by the hydrogen peroxide
because the organism has a defense mechanism against the hydrogen peroxide which is the
enzyme catalase.
 If the H2O2 is added to the grown colonies of a known organism that produces the enzyme
catalase, it will convert the hydrogen peroxide into water and oxygen. Oxygen is responsible for the gas bubble
appearance.
H 2 O 2 Catalase H 2 O+O 2
→
o Clinical Significance: a (+) catalyst test would mean that the unknown organism in the TSA is of Staphylococci family
o Presumptive tests which are aligned to identifying species of Staphylococci
are to be performed so that the identity of the Staphylococcus causing
infection to the sample can be identified.
 Colonial Morphology
 o Culture medium used: blood agar
o Characteristics of blood agar: enrichment medium and differential medium
o Staphylococcus aureus always produces small circular colonies which are yellow/golden yellow in color. It produces
yellow/golden yellow colonies because it produces the golden yellow pigment lipochrome. If it appears
yellow/golden yellow, it presumptively identifies Staphylococcus aureus.
 Presumptive because there might be other organisms in the microbiology world that will present also golden
yellow colonies in the blood agar medium. Its identification cannot be confirmed yet using only the appearance of
its colonies in the blood agar medium.
o Staphylococcus epidermidis produces white colonies in the blood agar medium.
 Hemolysis Pattern
o Culture medium used: blood agar
o Characteristics of blood agar: enrichment medium and differential medium
o The main content/base content/ingredient of blood agar is trypticase soy
agar
o Before the blood is added to the blood agar medium, it appeared as clear
and yellow as well because its base content/main ingredient is TSA. It only
appeared red because after the TSA is plated in a petri dish, 5-10% Sheep’s
blood/human blood to the TSA already plated.
o The blood agar contains sheep’s blood or RBCs and the trypticase soy agar.
o Enrichment medium- blood agar can support the growth of all organisms, whether gram-positive organisms, gram-
negative organisms, aerobic organisms anaerobic organisms, generally all organisms can grow in blood agar
 The Trypticase Soy Agar content is responsible for its enrichment medium characteristic.
 Whatever the number of organisms about to be inoculated in the blood agar, after some time of incubation, it will
double the number because it will enrich the number of the organism in that sample.
o Differential medium
 When an organism is inoculated in the blood agar medium and that particular organism will utilize a particular
substance found, inside the red blood cell→ cause destruction/lysis of the red blood cells in the blood agar
medium so that there can be different hemolytic patterns that can be observed in the blood agar medium
depending on the ability of the organism to cause lysis of the red blood cells because it is also depending how
much of the substance inside the red blood cell the organism needs.
 The organism would utilize the hemoglobin molecule (hemoglobin is always found inside the red blood cell being a
small, protein molecule, conjugated protein). The unknown Staphylococci will destroy the red cells and will cause
different hemolytic patterns.
 Since different hemolytic patterns is associated to different Staphylococcus organisms, that means one
Staphylococcus organism can be differentiated from the other Staphylococcus species based on the hemolytic
pattern.
 The hemolytic pattern observed in the blood agar medium is responsible for its differential characteristic.
o Brown’s Classification- three different hemolytic patterns that can be observed in the blood agar medium
 Beta-hemolysis- complete hemolysis/complete destruction/complete lysis of the RBCs in the blood agar medium
 Yellow discoloration can be imparted to the blood agar medium. When BA appears red at first and after
sometime of incubation, the organism has caused beta hemolysis in the blood agar medium, the areas of the
colonies of the unknown organism causing beta-hemolysis are growing has yellow discoloration. The yellow
discoloration reveals the base content/main ingredient of the BA medium (trypticase soy agar) because all the red
cells in those areas with colonies of beta hemolytic organism are completely destroyed.
 Alpha-hemolysis- incomplete destruction of RBCs
 Brown discoloration can be imparted to the blood agar medium. It is the formation of methemoglobin. When
few of the red cells are destroyed incompletely in the blood agar medium, the heme gets liberated in the surface
 of the blood agar medium and gets converted to oxidized form so that methemoglobin is also seen in the surface
of the blood agar medium.
 Gamma-hemolysis- no hemolysis of RBCs (absence of destruction of RBCs); no discoloration can be imparted in
the blood agar medium (appears red)
o Staphylococcus aureus produces beta-hemolysis whereas Staphylococcus epidermidis produces gamma-hemolysis.

CONFIRMATORY TESTS

 Mannitol Fermentation
o
Culture medium used: mannitol salt agar (MSA)
o
Color: pink/peach/light peach/light pink
o
Characteristics of blood agar: selective medium and differential
medium
o Selective medium- contains mannitol and salt. The salt content is 7.5%
sodium chloride.
 Its salt content is high that only salt-loving organisms can survive in this culture medium. The mannitol salt agar
will only select those organisms that can tolerate its high salt content. The culture medium is only selecting the
organisms that can tolerate its high salt content.
 The 7.5% sodium chloride content of the culture medium is responsible for its selective property.
o Differential medium- the other content of the mannitol salt agar is the carbohydrate/sugar content mannitol
 If the culture medium contains any carbohydrate/sugar and if the unknown organism inoculated to that culture
medium containing that particular sugar, if that organism ferments (utilizing) the sugar, there is always acid
products that will happen.
 When the organism ferments the sugar mannitol in the mannitol salt agar, the organism starts to produce acid in
that culture medium. When the culture medium is uninoculated yet, its pH is in the neutral range. But when the
organism is inoculated in the mannitol salt agar and the sugar mannitol is fermented/utilized by the organism, it
produces acid and there will be changes of pH.
 Any culture medium that has carbohydrate content in it also has a pH indicator content in it. Whatever changes of
pH inside that culture medium will be indicated by the pH indicator content by changing the color of the medium
from its original color to another color to determine for acid products in the culture medium.
 pH indicator: Phenol red
 When mannitol is fermented, the pH indicator will change the color of mannitol salt agar from light pink to yellow.
 If the organism does not ferment mannitol, the organism will grow in the mannitol salt agar and will also change
the pH of the culture medium from neutral to basic pH, the pH indicator will change the color of the medium from
peach/light pink to bright pink/hot pink.
 If one Staphylococcus will produce a positive mannitol fermentation test and the other Staphylococcus will produce
a negative mannitol fermentation test, the two staphylococcus organisms are differentiated from each other.
 The mannitol content of the mannitol salt agar is responsible for its differential property.
 (+)- Staphylococcus aureus; (-)- Staphylococcus epidermidis
 Coagulase Test
o Coagulase is an enzyme. Coagulase test detects the ability of the organism to produce the enzyme coagulase
o It differentiates Staphylococcus aureus from the other species of Staphylococcus because only Staphylococcus
aureus is coagulase positive since only Staphylococcus aureus produces the enzyme coagulase
o COAG= blood clot
o Coagulase test class has two types- slide coagulase and the tube coagulase
 Slide coagulase- detects for the clumping factor/bound coagulase enzyme
 Tube coagulase- detects for the extracellular coagulase enzyme or the free coagulase enzyme the
free coagulase enzyme.
 oReagent used in the coagulase test is plasma.
 Plasma contains a lot of fibrinogen whereas serum does not contain any fibrinogen because it is already clotted.
 Fibrinogen is a coagulation protein. If the organism produces the enzyme coagulase and the reagent used in the
coagulase test is plasma, if the grown colonies of the unknown organism producing the enzyme coagulase is mixed
with the reagent plasma, the coagulation enzyme will convert the fibrinogen coagulation protein to fibrin with
clot formation at the bottom of the tube.
o (+)- clot formation or gel formation
o Slide coagulase test- positive (+) is agglutination or clumping of the organism
o (+)- Staphylococcus aureus; (-)- Staphylococcus epidermidis
 Novobiocin Sensitivity
o Differentiates Staphylococcus epidermidis (susceptible) from Staphylococcus saprophyticus (resistant)

Staphylococcus Staphylococcus epidermidis

aureus

Catalase Test

Morphology

Hemolytic Pattern

Mannitol
Fermentation Test

Coagulase Test