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Microbiology of the Respiratory Tract: STAPHYLOCOCCI

Staphylococci are ubiquitous in our environment and in the normal flora of our bodies. They are

particularly numerous on skin and in the upper respiratory tract. Some are also associated with human

infectious diseases.

Staphylococci are gram-positive cocci, characteristically arranged in irregular clusters. They are hardy,

facultatively anaerobic organisms that grow well on most nutrient media. There are three principal clinically

important species: Staphylococcus epidermidis, Staphylococcus saprophyticus, and Staphylococcus aureus.

S. epidermidis,as its name implies, is the most frequent inhabitant of human surface tissues, includingskin and mucous membranes. It is not usually pathogenic, but it may cause serious infections if it has an unusual

opportunity to enter past surface barriers. S. saprophyticushas been implicated in acute urinary tract infections

in young women. It has not been found among the normal flora and is not yet known to cause other types of

infection.

Like S. epidermidis, S. aureusis often found among the normal flora of healthy persons, but in contrast,

most staphylococcal disease is caused by strains of this species. S. aureus strains produce a number of toxins and

enzymes that can exert harmful effects on the cells of the infected host: hemolysins, coagulase, leukocidin,

hyaluronidase, staphylokinase, enterotoxin, TSST-1. Strains of S. epidermidis and S. saprophyticus do not

produce these toxic substances.

Common skin infections caused by S. aureus include pimples, furuncles (boils), carbuncles, and impetigo.

Serious systemic (deep tissue) infections that result from S. aureus invasion include pneumonia, pyelonephritis,osteomyelitis, meningitis, and endocarditis. In addition to pneumonia, S. aureus may also produce infections of

the sinuses (sinusitis) and middle ear (otitis media).

Laboratory Exercise 1

Isolation and Identification of Staphylococci

The laboratory diagnosis of staphylococcal disease is made by identifying the organism (usually S.

aureus) in a clinical specimen representing the site of infection (pus from a skin lesion, sputum when pneumonia

is suspected, urine, spinal fluid, or blood).

The principal features by which staphylococci are recognized and distinguished in the laboratory includetheir microscopic morphology, colonial appearance on blood agar (especially hemolytic activity), coagulase

activity, reaction to the carbohydrate mannitol, and susceptibility to the antimicrobial agent novobiocin. Also, a

rapid latex agglutination test is available for identifying S. aureus from characteristic colonies growing on agar

media. The antibody-coated latex beads react with two surface proteins typically found onS. aureus strains.

One is a type of coagulase bound to the staphylococcal surface, and the other is a surface protein known as

protein A. The species are indistinguishable microscopically.

Purpose: To characterize the different staphylococci according to their culture and biochemical properties

To isolate and identify staphylococci from clinical specimens

Materials: Blood agar plates (BAP), Mannitol salt agar plates (MSA), Novobiocin disks (5 g), calliper/ruler,

Dropping bottle containing 3% hydrogen peroxide, glass slides,

Tubed plasma (0.5-ml aliquots), sterile 1.0-ml pipettes, aspirator,

Latex agglutination kit for S. aureus, alcohol lamps, inoculating loops, forceps

24-hour broth cultures of S. epidermidis, S. aureus, S. saprophyticus, and E. coli

Procedures:

A.

BAP, MSA culture/ Test for Susceptibility to Novobiocin

1.

With your marking pen, divide the bottom of a BAP and an MSA plate into two (or four) segments each.

2.

Using the grown broth cultures, inoculate/ streak one section of each plate with S. aureus, (one with S.

epidermidis, one with S. saprophyticus),and one with E. coli.

3.

With heated and cooled forceps, pick up a novobiocin disk, place it in the center of one of the streaked

areas of the BAP, and press it gently onto the agar with the forcep tips. Do the same for the remaining

three organisms on the streaked BAP.

4.

Incubate the plates for 24 hours at 35C.5.

After 24 hours of incubation, examine and record colonial morphology of BAP and MSA plates.

Interpret MSA plates for growth and fermentation using the following:

a. Growth w/o fermentation: MSA plate retains color w/ no yellow halo around the growth

b. Growth and fermentation: yellow halos around the colonies

c. No Growth: non-staphylococcal species or other gram negative organisms

6.

Make Gram stains of each culture on the BAP and record microscopic morphology. Draw typical

colonies.

7.

Measure and record the diameter of the zone of inhibition around the novobiocin disks. A zone size

greater than 16 mm in diameter is considered susceptible.

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B.

Catalase Test: Perform a catalase test on each of the three Staphylococcuscolonies from MSA plates.

1.

Place a drop of hydrogen peroxide on a clean microscope side.

2.

Pick up a portion of a colony w/ a loop (from a non-blood containing medium) and mix with hydrogen

peroxide on the slide.

3.

Observe for bubble formation

C.

Coagulase Test: Perform a coagulase test on each of the three Staphylococcus broth cultures as follows:

1.

Using a sterile pipette, measure 0.1 ml of the S. aureus broth culture with the aspiration device. Transferthis inoculum to a tube of 0.3-0.5 ml citrated human plasma. Discard the pipette in disinfectant. Label

the tube.

2.

Inoculate a second and third tube of plasma with 0.1 ml of the S. epidermidis and S. saprophyticus broth

cultures, respectively, as in step B1.

3.

Place all inoculated plasma tubes in the 35C incubator. After 30 minutes, remove and examine them.

Hold the tubes in a semihorizontal position to see whether the plasma in the tube is beginning to clot

into a solid mass (sign of coagulation). If so, make a record of the tube showing coagulase activity.

Return unclotted tubes to the incubator.

4.

Repeat procedure B3 every 30 minutes for 4 hours, if necessary.

D.

Rapid latex agglutination test

1.

Place one drop of the latex agglutination reagent onto each of two circles on the card provided.

2.

With the special stick contained in the kit or a sterile inoculating loop, pick up several colonies of S.

aureus from the blood agar plate you inoculated at the previous laboratory session. Emulsify the

colonies in the latex reagent, being careful not to scratch the card.

3.

Repeat this procedure with colonies of S. epidermidis. *Do not use colonies from the mannitol agar plate

as these are difficult to emulsify.

4.

Rotate the card gently for 20 seconds, observing the circles for a clearly visible clumping of the latex

particles and a clearing of the milky background.This reaction signifies a positive test. Record the

results.

5.

Dispose the reaction card in the disinfectant provided.

Study Questions (Answer on a separate sheet of bond paper):1.

Differentiate the microscopic morphology of staphylococci and streptococci as seen by Gram stain.

2.

What is in S. aureusthat is responsible for a beta-hemolytic reaction in BAP?

3.

What is coagulase? How is the presence of coagulase related to the pathogenicity of the organism?

4.

Why do you have to use colonies from a non-blood containing medium for catalase test?

5.

Why is mannitol salt agar used as a differential media in the isolation of Staphylococcal species?

6.

What is protein A? What properties of S. aureus distinguish it from S. epidermidis and S. saprophyticus?

7.

What diseases are commonly associated with each staphylococcal species?

8.

From what specimen type would S. saprophyticus most likely be isolated?

9.

What is a nosocomial infection? Who acquires it? Why?

10.

Why are staphylococcal infections frequent among hospital patients?

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Schematic Diagram for the Isolation of Staphylococcal species

Differentiating Characteristics of the Three Major Staphylococcal Species:

Test S. aureus S. epidermidis S. saprophyticus

Blood Agar Plate

Hemolysis Generally Beta-hemolytic (-hemolytic) (-hemolytic)

Colonial Pigmentation Generally golden yellow white whiteMannitol Salt Agar Plate

Growth + + +

Fermentation +

Catalase +

Coagulase +

Novobiocin susceptibility Resistant Resistant Sensitive

Final Species

Identification

S. epidermidis

Final Species

Identification

S. saprophyticus

Susceptible?

Final Species

Identification

S. aureus

Novobiocin

SusceptibilityPositive?

Micrococcaceae Coa ulase TestNo

StreptococcaceaePositive?

Presumptive Species:

S. aureusPresumptive Species:

S. saprophyticus, S. epidermidis

Yellow halo around

colonies:

positive fermentation

Pink/red plate w/o yellow

halo around colonies:

negative fermentation

Subculture on Nutrient

Agar or other non blood

containing medium

Observe Culture CharacteristicsObserve Culture Characteristics

Gram Positive?

Plate and Inoculate on BAP and MSA

Gram Positive?

Broth culture of Staphyloccus species or clinical specimen with unknown organism

MSABAP

Yes

No

NoYes

Yes No

No

Yes

No

Yes

Catalase Test