LAB 15: ISOLATION AND IDENTIFICATION OF STAPHYLOCOCCI

DISCUSSION

Staphylococci (see Fig. 6) are often found in the human nasal cavity (and on other mucous membranes) as well as on the skin. They are Gram-positive cocci 0.5-1.0 µm in diameter and occur singly, in pairs, in short chains, and most commonly, in irregular grape-like clusters. The staphylococci are strongly catalase positive and generally tolerate relatively high concentrations of sodium chloride (7.5-10%). This ability is often employed in preparing media selective for staphylococci.

Staphylococcal capsules play a major role in the ability of the bacteria to adhere to and colonize biomaterials.

There are five species of staphylococci commonly associated with clinical infections: Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. hominis and S. saprophyticus.

A. Staphylococcus aureus (coagulase-positive staphylococci)

Staphylococcus aureus is the most pathogenic species and is implicated in a variety of infections. S. aureus is with some frequency found as normal human flora in the anterior nares (nostrils). It can also be found in the throat, axillae, and the inguinal and perineal areas. Approximately 30% of adults and most children are healthy periodic nasopharyngeal carriers of S. aureus. Around 15% of healthy adults are persistent nasopharyngeal carriers. The colonization rates among health care workers, patients on dialysis, and people with diabetes are higher than in the general population.

In the majority of S. aureus infections the source of the organism is either:

The portal of entry is usually the skin. S. aureus causes pus-filled inflammatory lesions known as abscesses. Depending on the location and extent of tissue involvement, the abscess may be called:

1. a pustule
A pustule is an infected hair follicle where the base of the hair follicle appears red and raised with an accumulation of pus just under the epidermis. Infected hair follicles are also referred to as folliculitis.

2. a furuncle or boil
Furuncles appear as large, raised, pus-filled, painful nodules having an accumulation of dead, necrotic tissue at the base. The bacteria spread from the hair follicle to adjacent subcutaneous tissue.

3. a carbuncle
Carbuncles occur when furuncles coalesce and spread into surrounding subcutaneous and deeper connective tissue. Superficial skin perforates, sloughs off, and discharges pus.

S. aureus also causes impetigo, a superficial blister-like infection of the skin usually occuring on the face and limbs and seen mostly in young children. S. aureus may also cause cellulitis, a diffuse inflammation of connective tissue with severe inflammation of dermal and subcutaneous layers of the skin. S. aureus is also a frequent cause of accidental wound and postoperative wound infections.

Less commonly, S. aureus may escape from the local lesion and spread through the blood to other body areas, causing a variety of systemic infections that may involve every system and organ. Such systemic infections include septicemia, septic arthritis, endocarditis, meningitis, and osteomyelitis, as well as abscesses in the lungs, spleen, liver, and kidneys. S. aureus pneumonia may also be a secondary respiratory complication of viral infections such as measles, and influenza and is a frequent cause of nosocomial pneumonia in patients who are debilitated. Finally, S. aureus is frequently introduced into food by way of abscesses or the nasal cavity of food handlers. If it is allowed to grow and produces an enterotoxin, it can cause staphylococcal food poisoning.

In a 1990-1992 National Nosocomial Infections survey, CDC found S. aureus to be the most common cause of nosocomial pneumonia and operative wound infections, as well as the second most common cause of nosocomial bloodstream infections. Antibiotic resistant S. aureus is a common problem. For example, a survey conducted by CDC reported the proportion of methicillin-resistant isolates S. aureus (MRSA) with sensitivity only to vancomycin increased from 22.8% in 1987 to 56.2% in 1997.

Virulence factors for S. aureus include exotoxins such as leukocidin (kills leukocytes), alpha and delta toxins (damage tissue membranes), microcapsules (resist phagocytic engulfment and destruction), coagulase and protein A (both help resist phagocytic engulfment). Some strains also produce TSST-1 (toxic shock syndrome toxin-1) and cause toxic shock syndrome, usually associated with wounds. Approximately 25% of all S. aureus strains are toxigenic and approximately 6000 gases of toxic shock syndrome occur each year in the U.S. Some strains also produce exfoliatin, an exotoxin that causes scalded skin syndrome, an infection usually seen in infants and young children.

 

For further information on virulence factors associated with S. aureus, see the following Learning Objects in your Lecture Guide:

 

Since most S. aureus strains produce the enzyme coagulase (see the coagulase test described below), they are often referred to as coagulase-positive staphylococci.

 

 

B. Coagulase-Negative Staphylococci

Clinically common species of staphylococci other than S. aureus are often referred to as coagulase-negative staphylococci. These staphylococci are normal flora of the skin and, as such, frequently act as opportunistic pathogens, especially in the compromised host. S. saprophyticus is a relatively common cause of urinary tract infections, especially in healthy young women, but is seldom isolated from other sources. The great majority of infections caused by other coagulase-negative staphylococci, including S. epidermidis, S. haemolyticus, and S. hominis, are associated with intravascular devices (prosthetic heart valves and intra-arterial or intravenous lines) and shunts. Also quite common are infections of prosthetic joints, wound infections, osteomyelitis associated with foreign bodies, and endocarditis.

Although certain reactions may vary from strain to strain, a series of biochemical tests will usually differentiate the most common clinically encountered species of staphylococci. Today we will use a number of tests to determine if an unknown is S. aureus, S. epidermidis, or S. saprophyticus.

Medscape articles on infections associated with organisms mentioned in this Lab Exercise. Registration to access this website is free.

ISOLATION AND IDENTIFICATION OF STAPHYLOCOCCI

1. Blood agar with a novobiocin (NB) disc

To isolate staphylococci, clinical specimens are usually grown on Blood agar (described in Lab 14). Staphylococci produce round, raised, opaque colonies 1-2mm in diameter. The novobiocin disc is used to detect sensitivity or resistance to the antibiotic novobiocin.

Test

Staphylococcus aureus,
pigmented strain

S. aureus,
non-pigmented strain

Staphylococcus epidermidis Staphylococcus saprophyticus
Hemolysis (*) Usually beta(1) Usually gamma(2) Usually gamma(2)
Pigment Often creamy gold(1) Usually white(2) Usually white(2)
Novobiocin test Sensitive Sensitive Resistant

(*) see Lab 14 for descriptions of hemolysis
(1) some strains do not show hemolysis and/or pigment
(2) some strains do show hemolysis and/or pigment
sensitive = zone of inhibition around disc
resistant = no zone of inhibition around disc

2. Gram stain

All staphylococci appear as Gram-positive cocci, usually in irregular, often grape-like clusters (see Fig. 6).

3. Mannitol fermentation on Mannitol Salt agar (MSA)

Staphylococci are able to tolerate the high salt concentration found in Mannitol Salt agar and thus grow readily. If mannitol is fermented, the acid produced turns the phenol red pH indicator from red (alkaline) to yellow (acid).

Test Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus
Mannitol fermentation Positive Negative Usually positive

positive = acid end products turn the phenol red pH indicator from red to yellow
negative = prenol red remains red

4. Production of deoxyribonuclease (DNase) on DNase agar

DNase agar contains 0.2% DNA. To detect DNase production, the plate is inoculated and incubated. After growth, the plate is flooded with 1N hydrochloric acid (HCl). DNase positive cultures show a distinct clear zone around the streak (see Fig. 1) where the DNA in the agar was broken down by the bacterial DNase. DNase negative cultures appear cloudy around the growth where the acid caused the DNA in the agar to precipitate out of solution.

Test Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus
DNase production Positive Negative Negative

positive = clear zone around growth after adding 1N HCl (no DNA remaining in the agar)
negative = cloudy around growth after adding 1N HCl (DNA remains in the agar forming a precipitate)

5. Production of coagulase

The staphylococcal enzyme coagulase will cause inoculated citrated rabbit plasma to gel or coagulate. The coagulase converts soluble fibrinogen in the plasma into insoluble fibrin.

Test Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus
Coagulase production Positive Negative Negative

positive = plasma will gel or coagulate
negative = plasma will not gel

6. The Staphyloslide® Latex Test for cell-bound coagulase (clumping factor) and/or Protein A

The Staphyloslide® Latex Test is an agglutination test that detects cell-bound coagulase (clumping factor) and/or Protein A. Approximately 97% of human strains of S. aureus possess both bound coagulase and extracellular coagulase. Approximately 95% of human strains of S. aureus possess Protein A on their cell surface (see Fig. 7). This test uses blue latex particles coated with human fibrinogen and the human antibody IgG. Mixing of the latex reagent with colonies of the suspected S. aureus having coagulase and/or Protein A bound to their surface causes agglutination of the latex particles.

Test Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus
Cell-bound coagulase (clumping factor) and/or Protein A Positive Negative Negative

positive = clumping of latex particles
negative = no clumping of latex particles

 

 

For further information on coagulase and Protein A associated with S. aureus, see the following Learning Objects in your Lecture Guide:

 

Staphylococci are also being identified using chemiluminescent labelled DNA probes complementary to species-specific bacterial ribosomal RNA (rRNA) sequences as well as by other direct DNA techniques.

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SCENERIO FOR TODAY'S LAB

Choose either unknown #1 or unknown #2 as your unknown for this Case Study.

A 57 year old female who is diabetic, a smoker, and who 30 days ago had hip replacement surgery presents with pain and signs of inflammation at the surgical site. Examination shows she has a fever of 101 °F and an increased total white blood cell count with a left shift. Radiologic examination shows a deep pelvic abcess. A culture of the implant site was taken.

Assume that unknown you are given is the culture from this patient.

 

MATERIALS

1 plate of blood agar, 1 novobiocin (NB) disc, 1 plate of mannitol salt agar, 1 DNase agar plate, 1 tube of citrated rabbit plasma (coagulase test), materials to perform a Gram stain, inoculating loop

 

PROCEDURE (to be done in groups of 3)
[Keep in mind that in a real clinical situation other lab tests and cultures for bacteria other than those upon which this lab is based would also be done.]

 

CAUTION: TREAT EACH UNKNOWN AS A PATHOGEN!. Inform your instructor of any spills or accidents. WASH AND SANITIZE YOUR HANDS WELL before leaving the lab.

1. Do a Gram stain on the unknown. Since the bacterium today is growing in a broth culture rather than on a petri plate, do the following:

a. Using a sharpie, draw a circle about the size if a nickel on the bottom of your microscope slide.

b. Turn the slide over. Using your sterile inoculating loop, aseptically place 2 or 3 loops of the culture within this circle on the top of the slide. Do not use water.

c. Using your inoculating loop, spread the suspension over the area delineated by the circle to form a thin film.

d. Allow this thin suspension to completely air dry.

e. Heat-fix the bacteria to the slide and perform the Gram stain. Be careful not to over decolorize! Remember bacteria from a broth culture are much less concentrated than those taken off of an agar plate. Watch the slide carefully while decolorizing. Stop decolorizing and immediately wash the slide with water as soon as the decolorizer coming off the bacterial smear looks a very light blue.

 

2. Using your loop, streak your unknown for isolation on a plate of Blood agar as described below.

a. Using a sterile inoculating loop, streak your unknown for isolation on a blood agar plate so as to get single, isolated colonies (Fig. 2, step 1, Fig. 2, step 2, and Fig. 2, step 3).

b. Using your inoculating loop,  stab the agar several times in each of the growth areas in order to detect oxygen-sensitive hemolysins (Fig. 2, step 4).

c. Place a novobiocin antibiotic disc in the center of the area of the plate that you streaked first and where you expect to see heaviest growth (Fig. 2, step 5).

d. Incubate upside down and stacked in the petri plate holder on the shelf of the 37°C incubator corresponding to your lab section until the next lab period.

3. Streak your unknown for isolation on a plate of Mannitol Salt agar (MSA) as shown in Fig. 3. Incubate upside down and stacked in the petri plate holder on the shelf of the 37°C incubator corresponding to your lab section until the next lab period.

4. Streak a single line of your unknown down the center of a plate of DNase agar as shown in Fig. 4. Incubate upside down and stacked in the petri plate holder on the shelf of the 37°C incubator corresponding to your lab section until the next lab period.

5. Inoculate a tube of citrated rabbit plasma with your unknown and incubate your test tube rack at 37°C.

 

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RESULTS

 

Case Study Lab Report for Lab 15:
Staphylococci

 The concept behind the case studies presented in Lab 15 used to illustrate the genus Staphylococcus is for you and your lab partners as a group to:

1. Come up with a valid diagnosis of the surgical wound infection and identify the bacterium causing that infection.

2. Support your group’s diagnose based on:

a. Any relevant facts in the patient’s history
b. The patient’s symptoms (reliable on-line sources can be used to support this)
c. Each of the individual lab tests given in the case study
d. All microbiological lab tests you performed as part of the lab

You will hand in the Lab Report section of this lab at the completion of today’s lab. Remember, you are working as a group to solve a problem.  Your grade for this lab is based on the completeness of your report and written evidence of the critical thinking process that went into making and supporting your diagnosis, therefore, it is critical that all members of the group participate, question any conclusions being made by the group, and contribute to the report. Remember, you are trying to convince your instructor that you understand how the diagnosis was made by supporting that diagnosis with data.

Be sure to handle all the bacterial cultures you are using in lab today as if they are pathogens!  Be sure to wash and sanitize your hands well at the completion of today’s lab.

Also, make sure you observe the results of someone in your lab who had an unknown different from yours . The Performance Objectives for Lab 15 tell you what you are expected to be able to do on the practical.

Rubric used for grading your Case Study Lab Report.

You must print two copies of the rubric for the case study (one for unknown #1 and one for unknown #2) from the link above and staple them to your Lab 15 Lab Report before you submit the report to your instructor!

 

For more information on writing your Lab Report, see Course evaluation for Lab (Core labs, case studies, lab quizzes) under Course Info in the menu of the BIOL 230 website.

Click here to print a PDF copy of this lab report.

 

Your Name:

Others in your group:

 

Lab section:

Date:

 

A. Case Study from Lab 15: Unknown #1

You must print two copies of the rubric for the case study (one for unknown #1 and one for unknown #2) from the link above and staple them to your Lab 15 Lab Report before you submit the report to your instructor!

A 57 year old female who is diabetic, a smoker, and who 30 days ago had hip replacement surgery presents with pain and signs of inflammation at the surgical site. Examination shows she has a fever of 101 °F and an increased total white blood cell count with a left shift. Radiologic examination shows a deep pelvic abcess. A culture of the implant site was taken.

 1. Patient’s history

Read the case study. Explain how any relevant parts of the patient’s history contributed to your diagnosis. You are urged to use the computers in lab to search reliable medically oriented Internet sources to support this. Reliable sources you might consider are Medscape (http://emedicine.medscape.com/infectious_diseases)
and The Centers for Disease Control and Prevention (CDC) at http://www.cdc.gov/. Cite any sources you use at the end of this Patient's History section in APA style (http://www.apastyle.org/). The patient's history and patient's symptoms should suggest a general type of infection, such as a urinary tract infection, a wound infection, gastroenteritis, strep throat, pneumonia, septicemia, etc. Search at least one medically-oriented reference article from a reliable site such as Medscape and use this article to support your diagnosis of the type of infection. Don't forget to cite any sources you used in APA style under the Patient's History and Patient's Symptoms sections of your Lab Report.

 

 

 

 

2. Patient’s symptoms

Read the case study. Explain how the patient’s symptoms contributed to your diagnosis. You are urged to use the computers in lab to search reliable medically oriented Internet sources to support this. Reliable sources you might consider are Medscape (http://emedicine.medscape.com/infectious_diseases)
and The Centers for Disease Control and Prevention (CDC) at http://www.cdc.gov/. Cite any sources you use at the end of this Patient's Symptoms section in APA style (http://www.apastyle.org/). The patient's history and patient's symptoms should suggest a general type of infection, such as a urinary tract infection, a wound infection, gastroenteritis, strep throat, pneumonia, septicemia, etc. Search at least one medically-oriented reference article from a reliable site such as Medscape and use this article to support your diagnosis of the type of infection. Don't forget to cite any sources you used in APA style under the Patient's History and Patient's Symptoms sections of your Lab Report.

 

 

 

 

 

 

 

3. Results of laboratory test given in the case study

List each lab test given and explain how the results of that test helps to contribute to your diagnosis.  The CBC test is described in Appendix C of this lab manual.

 

 

4. Microbiological lab tests you performed in Lab 15

a. Gram stain

Give the Gram reaction (Gram-positive or Gram negative and how you reached this conclusion) and the shape and arrangement of the unknown you were given. State how this contributed to your diagnosis and choice of microbiological tests and/or media to perform next. The Gram stain is discussed in Lab 6.

 

 

 

 

b. Blood agar with novobiocin (NB) disc

Give the results of the Blood agar with Taxo NB disc you performed on the unknown you were given, and how you reached this conclusion. State how this contributed to your final diagnosis. The possible results for Blood agar and NB disc were discussed in the beginning pages of this lab.

 

 

 

 

 

c. Mannitol Salt agar

Give the results of the Mannitol Salt agar you performed on the unknown you were given, and how you reached this conclusion. State how this contributed to your final diagnosis. The possible results for Mannitol Salt agar were discussed in the beginning pages of this lab.

 

 

 

 

 

d. DNase agar

Flood the surface of your DNase agar plate with 1N HCl. Give the results of the DNase agar you performed on the unknown you were given, and how you reached this conclusion. State how this contributed to your final diagnosis. The possible results for DNase agar were discussed in the beginning pages of this lab.

 

 

 

 

 

e. Coagulase test

Give the results of the Coagulase test you performed on the unknown you were given, and how you reached this conclusion. State how this contributed to your final diagnosis. The possible results for the Coagulase test were discussed in the beginning pages of this lab.

 

 

 

 

 

 

 

 

 

 

Final Diagnosis

Genus and species of unknown #1 = ________________________________

 

Infection: _______________________________

 

B. Case Study from Lab 15: Unknown #2

You must print two copies of the rubric for the case study (one for unknown #1 and one for unknown #2) from the link above and staple them to your Lab 15 Lab Report before you submit the report to your instructor!

A 57 year old female who is diabetic, a smoker, and who 30 days ago had hip replacement surgery presents with pain and signs of inflammation at the surgical site. Examination shows she has a fever of 101 °F and an increased total white blood cell count with a left shift. Radiologic examination shows a deep pelvic abcess. A culture of the implant site was taken.

 1. Patient’s history

Read the case study. Explain how any relevant parts of the patient’s history contributed to your diagnosis. You are urged to use the computers in lab to search reliable medically oriented Internet sources to support this. Reliable sources you might consider are Medscape (http://emedicine.medscape.com/infectious_diseases)
and The Centers for Disease Control and Prevention (CDC) at http://www.cdc.gov/. Cite any sources you use at the end of this Patient's History section in APA style (http://www.apastyle.org/). The patient's history and patient's symptoms should suggest a general type of infection, such as a urinary tract infection, a wound infection, gastroenteritis, strep throat, pneumonia, septicemia, etc. Search at least one medically-oriented reference article from a reliable site such as Medscape and use this article to support your diagnosis of the type of infection. Don't forget to cite any sources you used in APA style under the Patient's History and Patient's Symptoms sections of your Lab Report.

 

 

 

 

2. Patient’s symptoms

Read the case study. Explain how the patient’s symptoms contributed to your diagnosis. You are urged to use the computers in lab to search reliable medically oriented Internet sources to support this. Reliable sources you might consider are Medscape (http://emedicine.medscape.com/infectious_diseases)
and The Centers for Disease Control and Prevention (CDC) at http://www.cdc.gov/. Cite any sources you use at the end of this Patient's Symptoms section in APA style (http://www.apastyle.org/). The patient's history and patient's symptoms should suggest a general type of infection, such as a urinary tract infection, a wound infection, gastroenteritis, strep throat, pneumonia, septicemia, etc. Search at least one medically-oriented reference article from a reliable site such as Medscape and use this article to support your diagnosis of the type of infection. Don't forget to cite any sources you used in APA style under the Patient's History and Patient's Symptoms sections of your Lab Report.

 

 

 

 

 

3. Results of laboratory test given in the case study

List each lab test given and explain how the results of that test helps to contribute to your diagnosis.  The CBC test is described in Appendix C of this lab manual.

 

 

 

4. Microbiological lab tests you performed in Lab 15

a. Gram stain

Give the Gram reaction (Gram-positive or Gram negative and how you reached this conclusion) and the shape and arrangement of the unknown you were given. State how this contributed to your diagnosis and choice of microbiological tests and/or media to perform next. The Gram stain is discussed in Lab 6.

 

 

 

 

b. Blood agar with novobiocin (NB) disc

Give the results of the Blood agar with Taxo NB disc you performed on the unknown you were given, and how you reached this conclusion. State how this contributed to your final diagnosis. The possible results for Blood agar and NB disc were discussed in the beginning pages of this lab.

 

 

 

 

 

c. Mannitol Salt agar

Give the results of the Mannitol Salt agar you performed on the unknown you were given, and how you reached this conclusion. State how this contributed to your final diagnosis. The possible results for Mannitol Salt agar were discussed in the beginning pages of this lab.

 

 

 

 

 

d. DNase agar

Flood the surface of your DNase agar plate with 1N HCl. Give the results of the DNase agar you performed on the unknown you were given, and how you reached this conclusion. State how this contributed to your final diagnosis. The possible results for DNase agar were discussed in the beginning pages of this lab.

 

 

 

 

 

e. Coagulase test

Give the results of the Coagulase test you performed on the unknown you were given, and how you reached this conclusion. State how this contributed to your final diagnosis. The possible results for the Coagulase test were discussed in the beginning pages of this lab.

 

 

 

 

 

 

 

 

 

Final Diagnosis

Genus and species of unknown #2 = ________________________________

 

Infection: _________________________________

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PERFORMANCE OBJECTIVES FOR LAB 15

After completing this lab, the student will be able to perform the following objectives:

DISCUSSION

1. Name three common clinically important species of Staphylococcus and state which species is most pathogenic.

2. State the sources and the portal of entry for most Staphylococcus aureus infections.

3. Name and describe three types of abscesses caused by Staphylococcus aureus.

4. Name four systemic Staphylococcus aureus infections.

5. State the significance of Staphylococcus aureus enterotoxin, the exotoxin TSST-1, and the exotoxin exfoliatin.

6. Name the infection normally caused by Staphylococcus saprophyticus.

7. Name the types of infections most commonly caused by coagulase-negative staphylococci other than Staphylococcus saprophyticus.

 

ISOLATION AND IDENTIFICATION OF STAPHYLOCOCCI

1. State the Gram reaction and morphology of all staphylococci.

2. Describe the typical reactions of S. aureus, S. epidermidis, and S. saprophyticus on each of the following media:

a. Blood agar (pigment, hemolysis, novobiocin resistance)

b. Mannitol Salt agar (for mannitol fermentation)

c. DNase agar (for the enzyme DNase)

d. coagulase test with citrated rabbit plasma

e. Staphyloslide® test for bound coagulase and/or Protein A

RESULTS

1. Recognize staphylococci in a Gram stain preparation.

2. Recognize an organism as Staphylococcus aureus and state the reasons why after seeing the results of the following:

a. a Blood agar plate with a novobiocin disc

b. a Mannitol Salt agar plate

c. a DNase agar plate

d. a tube of citrated rabbit plasma

e. a Staphyloslide® test

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SELF-QUIZ

Self-quiz

Answers

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Lab Manual Table of Contents


Copyright © Gary E. Kaiser
All Rights Reserved
Updated: April, 2012
Please send comments and inquiries to Dr. Gary Kaiser