DISCUSSION

Staphylococci (see Fig. 1) 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. Fig. 2 shows a scanning electron micrograph of methicillin-resistant Staphylococcus aureus (MRSA), and Fig. 3 shows an electron micrograph of Staphylococcus aureus (MRSA) destroying an immune cell.

 

Fig. 1: Gram stain of Staphylococcus aureus	Fig. 2: Scanning Electron Micrograph of Methicillin-Resistant Staphylococcus aureus (MRSA)	Fig. 3: Scanning Electron Micrograph of Methicillin-Resistant Staphylococcus aureus (red) Killing an Immune Cell (Yellow Cell Debris)
Note Gram-positive (purple) cocci in clusters.	Note staphylococcus arrangement (cocci in irregular, often grape-like clusters).	Note staphylococcus arrangement (cocci in irregular, often grape-like clusters).
Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License	By Content Providers(s): CDC/ Janice Ayers [Public domain]. Courtesy of the Centers for Disease Control and Prevention.	By Content Providers(s): NAID/ Frank Deleo [Public domain]. Courtesy of National Institute of Allergy and Infectious Diseases.

 

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:

• A healthy nasal carrier, or
• Contact with an abscess from an infected individual.

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.Pustules
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. Furuncles or boils
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. Carbuncles
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 occurring 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.

In the past 20 years, both community-associated and hospital-acquired infections with Staphylococcus aureus have increased. This infection rate has been accompanied by a rise in antibiotic-resistant strains - most significantly, methicillin-resistant S. aureus (MRSA) and, more recently, vancomycin-resistant S. aureus.

For further information on virulence factors associated with S. aureus, see the following Softchalk lessons:

• Gram-Positive PAMPs
• The Ability to Adhere to Host Cells
• The Ability to Resist Phagocytic Engulfment
• The Ability to Resist Phagocytic Destruction
• The Ability to Evade Adaptive Immune Defenses
• Type I Toxins
• Type II Toxins that Damage Cell Membranes
• The Ability to Induce Autoimmune Responses
• Type III Toxins

 

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

Fig. 5 shows a scanning electron micrograph of Staphylococcus aureus forming a biofilm in an indwelling catheter.

 

Fig. 5: Scanning Electron Micrograph Showing Biofilm Formation by Staphylococcus aureus

Staphylococcus aureus found on the luminal surface of an indwelling catheter. Note the polysaccharide biofilm (B) woven around the cocci.

By Content Providers(s): CDC/ Janice Haney Carr [Public domain] Courtesy of the Centers for Disease Control and Prevention.

 

• Diseases and Organisms in Healthcare Settings; from CDC

 

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.

• Diseases and Organisms in Healthcare Settings; from CDC

 

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 several 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. Staphylococcus infections Staphylococcus aureus

 

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	Fig. 6A: Staphylococcus aureus (pigmented strain) on Blood Agar Fig. 6B: Staphylococcus aureus (non-pigmented strain) on Blood Agar	Fig. 6C: Staphylococcus epidermidis on Blood Agar	Fig. 6D: Staphylococcus saprophyticuson Blood Agar
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

 

Fig. 6A: Staphylococcus aureus  (Pigmented Strain) Growing on Blood Agar	Fig. 6B: Staphylococcus aureus (Non-pigmented Strain) Growing on Blood Agar	Fig. 6C: Staphylococcus epidermidis Growing on Blood Agar	Fig. 6D: Staphylococcus saprophyticus Growing on Blood Agar
Note beta hemolysis (complete lysis of the red blood cells around the colonies; see arrows) on the blood agar and the organism is sensitive to the antibiotic novobiocin in the Taxo NB® disk.	Note the non-pigmented colonies surrounded by a zone of beta hemolysis (complete lysis of the red blood cells around the colonies) on the blood agar.	Note there is no hemolysis (gamma reaction) on the blood agar and the organism is sensitive to the antibiotic novobiocin as shown by the zone of inhibition around the Taxo NB® disk.	Note there is no hemolysis (gamma reaction) on the blood agar and the organism is resistant to the antibiotic novobiocin in the Taxo NB® disk.
Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License	Image: Blood agar culture of Staphylococcus aureus © Rebecca Buxton, author. Licensed for use, ASM MicrobeLibrary.	Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License	Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License

 

2. Gram stain

All staphylococci appear as Gram-positive cocci, usually in irregular, often grape-like clusters (see Fig. 1 above).

 

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	Fig. 7A: Staphylococcus aureus Growing on Mannitol Salt Agar	Fig. 7B: Staphylococcus epidermidis Growing on Mannitol Salt Agar	Fig. 7C: Staphylococcus saprophyticus Growing on Mannitol Salt Agar
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

 

Fig. 7A: Staphylooccus aureus Growing on Mannitol Salt Agar.	Fig. 7B: Staphylooccus epidermidis Growing on Mannitol Salt Agar.	Fig. 7C: Staphylooccus saprophyticus Growing on Mannitol Salt Agar.
Mannitol salt agar is selective for staphylococci because of the high salt concentration. Acid from mannitol fermentation causes the pH indicator phenol red to turn from red (alkaline) to yellow (acid).	Mannitol salt agar is selective for staphylococci because of the high salt concentration. There is no mannitol fermentation and, therefore, no acid production. The pH indicator phenol red remains red (alkaline).	Mannitol salt agar is selective for staphylococci because of the high salt concentration. Acid from mannitol fermentation causes the pH indicator phenol red to turn from red (alkaline) to yellow (acid).
Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License	Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License	Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License

 

4. 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	Fig. 8A: Staphylococcus aureusCoagulase Test	Fig. 8B: Staphylococcus epidermidisCoagulase Test	Fig. 8C: Staphylococcus saprophyticusCoagulase Test
Coagulase production	Positive	Negative	Negative

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

 

Fig. 8A: Positive Coagulase Test for Staphylococcus aureus	Fig. 8B: Negative Coagulase Test for Staphylococcus epidermidis	Fig. 8C: Negative Coagulase Test for Staphylococcus saprophyticus
Rabbit plasma has gelled indicating the bacterium is coagulase positive.	Rabbit plasma has not gelled indicating the bacterium is coagulase negative.	Rabbit plasma has not gelled indicating the bacterium is coagulase negative.
Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License	Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License	Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License

 

5. 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	Fig. 9A: Staphyloside® Latex Test on Staphylococcus aureus	Fig. 9B: Staphyloside® Latex Test on Staphylococcus epidermidis	Fig. 9C: Staphyloside® Latex Test on 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

 

 

Fig. 9A: Staphyloslide® Test on Staphylococcus aureus	Fig. 9B: Staphyloslide® Test on Staphylococcus epidermidis	Fig. 9C: Staphyloslide® Test on Staphylococcus saprophyticus
The blue latex particles coated with human fibrinogen and human IgG have clumped together indicating the bacterium is positive for bound coagulase (clumping factor) and/or Protein A.	The blue latex particles coated with human fibrinogen and human IgG have not clumped together indicating the bacterium is negative for bound coagulase (clumping factor) and/or Protein A.	The blue latex particles coated with human fibrinogen and human IgG have not clumped together indicating the bacterium is negative for bound coagulase (clumping factor) and/or Protein A.
Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License	Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License	Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License

 

Concept map for Lab 15 - Organisms and Infections

 

For further information on coagulase and Protein A associated with S. aureus, see the following CourseArc lessons:

• The Ability to Resist Phagocytic Engulfment
• The Ability to Evade Adaptive Immune Defenses

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.

Return to Menu for Lab 15

 

SCENERIO FOR TODAY'S LAB

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

Case Study

A 57-year- old female who is diabetic, a long-time smoker, and who 28 days ago had hip replacement surgery presents with swelling, pain, inflammation, and erythema at the surgical site. Examination shows she has a fever of 101°F, is exhibiting malaise, and has an increased total white blood cell count with a left shift. Ultrasonography examination indicates a deep abscess. A culture from an aspiration of the infected surgical 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 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.

Videos reviewing techniques used in this lab:

How to Chemically Fix a Microscope Slide with Methanol

How to Prepare a Slide for Staining when using Bacteria from an Agar Culture

How to Make a Gram Stain

A Review of the Critical Decolorization Step of the Gram Stain

Preliminary Tips for Using a Microscope

Focusing Using Oil Immersion (1000X) Microscopy

How to Inoculate a Blood Agar Plate and Add a NB (Novobiocin) Disc

 

1. Do a Gram stain on the unknown (see Lab 6). Make sure you review the instructions before you do the Gram stain. Because Enterococci and Staphylococci can sometimes look similar in Gram stains done from a plate culture, perform a catalase test on your unknown to help differentiate an Enterococcus from a Staphylococcus (see Lab 8).

• Click here for a summary of the Gram stain procedure without pictures.

Instructions for the Gram Stain from Lab 6.

Instructions for Focusing a Microscope from Lab 1.

 

2. On the bottom of the petri plate, divide the plate into thirds with your wax marker and label as shown below. Before you streak your plate draw an "X" on the bottom of the blood agar plate in sector 2 to indicate where you will eventually place the Taxo NB disk as shown in Fig. 10, Step 1.

 

Fig. 10: Inoculating a Blood Agar Plate with your Unknown, Step 1

Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License

 

3. 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. 10, step 2, Fig. 10, step 3, and Fig. 10, step 4).

 

Fig. 10: Inoculating a Blood Agar Plate with your Unknown, Step 2	Fig. 10: Inoculating a Blood Agar Plate with your Unknown, Step 3	Fig. 10: Inoculating a Blood Agar Plate with your Unknown, Step 4
Using a sterile inoculating loop, streak one-third of the blood agar plate with your unknown. Flame the loop and let it cool.	Rotate the plate counterclockwise so sector 1 is at 9:00. Using a sterile inoculating loop, spread out some of the bacteria in area 1 over area 2. Flame the loop and let it cool.	Rotate the plate counterclockwise so sector 1 is at 9:00. Using a sterile inoculating loop, spread out some of the bacteria in area 2 over area 3.
Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License	Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License	Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License

 

html5 animation showing how to streak an agar plate for isolation: 3 sector method.

 

b. Using your inoculating loop,  stab the agar 2-3 times in each of the 3 growth areas in order to detect oxygen-sensitive hemolysins (Fig. 10, step 5).

 

Fig. 10: Inoculating a Blood Agar Plate with your Unknown, Step 5

Stab the agar 2-3 times in each if the growth areas.

Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License

 

c. Place a novobiocin antibiotic disk in the area where you drew the "X" in sector 2 (Fig. 10, step 6). Tap it lightly with your loop so that the disk sticks to the agar.

 

Fig. 10: Inoculating a Blood Agar Plate with your Unknown, Step 6

Place a Taxo NBdisk over the "X" in sector 2 and tap it lightly with your loop so that it sticks to the agar.

Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License

 

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.

 

4. Streak your unknown for isolation on a plate of Mannitol Salt agar (MSA) as shown in Fig. 11. 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.

 

Fig. 1: Inoculating a mannitol salt agar plate with Staphylococcus

Gary E. Kaiser, Ph.D. Professor of Microbiology The Community College of Baltimore County, Catonsville Campus This work is licensed under a Creative Commons Attribution 3.0 Unported License

 

html5 animation showing how to streak an agar plate for isolation: 3 sector method.

 

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

 

Return to Menu for Lab 15

 

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 type of infectious disease seen in your case study and identify the bacterium causing that infection.

2. Support your group’s diagnose based on:

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

The due date for this report can be found on the class calendar. 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. Remember, you are trying to convince your instructor that you understand how the diagnosis was made by supporting that diagnosis with data.

Grading:

The lab 15 Lab Report is worth 33 points each.

These case studies are based in part on your in-class participation as part of your group. Therefore:

a. If you were not in lab when the inoculations with your unknown were performed, 3 points will be deducted from your Lab Report score for labs 12, 14, and 15; 6 points from your Lab Report score for the Final Project.

b. If you were not in lab when the results of your lab tests were observed, 3 points will be deducted from your Lab Report score for labs 12, 14, and 15; 6 points from your Lab Report score for the Final Project..

c. For each day your Lab Report is late, 2 points will be deducted from your Lab Report score for labs 12, 14, and 15; 4 points from your Lab Report score for the Final Project.

 

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 Lab 15 Case Study Lab Report.

 

Click here to print a Word Document copy of this lab report.

 

Your Name:

Others in your group:

 

Lab section:

Date:

 

A. Case Study from Lab 15: Unknown #1

Case Study

A 57-year old female who is diabetic, a long-time smoker, and who 28 days ago had hip replacement surgery presents with swelling, pain, inflammation, and erythema at the surgical site. Examination shows she has a fever of 101°F, is exhibiting malaise, and has an increased total white blood cell count with a left shift. Ultrasonography examination indicates a deep abscess. A culture from an aspiration of the infected surgical site was taken.

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

 1. Patient’s history and predisposing factors

Read the case study. Explain how any relevant parts of the patient’s history contributed to your diagnosis as to the type of infectious disease seen here. The patient's history refers to anything given in the case study prior to that patient seeking medical attention for the current medical condition. 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 (https://www.scribbr.com/apa-examples/website/).

The patient's history should suggest a general type of infectious disease that is present, such as a urinary tract infection, a wound infection, gastroenteritis, pharyngitis, pneumonia, septicemia, etc. Do not look up the bacterium you eventually identify as the cause of this infectious disease. You do not know the causative bacterium at this point. You need to determine the general type of infection in order to determine what microbiological tests to perform to identify the bacterium causing the infection. 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 infectious disease seen here. Don't forget to cite any sources you used in APA style directly under this Patient's History and Patient's Symptoms sections of this Lab Report.

 

 

 

 

2. Patient’s signs and symptoms

Read the case study. Explain how the patient’s signs and symptoms contributed to your diagnosis as to the type of infectious disease seen here. Signs refer to anything being measured by a medical professional during a physical exam such as blood pressure, respiration rate, heart rate, oxygen saturation, and temperature. Symptoms refer to symptoms being reported by the patient. 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 (https://www.scribbr.com/apa-examples/website/) Also see appendix F (SIRS and Sepsis) in your lab manual for an indication of whether or not the patient has SIRS.

The patient's signs and symptoms should suggest a general type of infectious disease that is present, such as a urinary tract infection, a wound infection, gastroenteritis, pharyngitis, pneumonia, septicemia, etc. Do not look up the bacterium you eventually identify as the cause of this infectious disease. You do not know the causative bacterium at this point. You need to determine the general type of infection in order to determine what microbiological tests to perform to identify the bacterium causing the infection. 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 infectious disease seen here. Don't forget to cite any sources you used in APA style directly under this Patient's History and Patient's Symptoms sections of this Lab Report.

 

 

 

 

 

 3. Vocabulary list for medical terms used in the case study under signs and symptoms

List and define any medical terms used in your case study that describe the patients’s signs and symptoms that the average person not in the medical profession might not know.

 

 

 

 

 

 

4. Results of laboratory test given in the case study

List each lab test given in the case study that are done in a lab, such as total white blood count, differential white blood cell count, urinalysis, and X-ray, 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 and catalase test results

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. Because Enterococci and Staphylococci can sometimes look similar in Gram stains done from a plate culture, perform a catalase test on your unknown to help differentiate an Enterococcus from a Staphylococcus. State how this contributed to your decision as to which microbiological tests and/or media to use next. The Gram stain is discussed in Lab 6; the catalase test in Lab 8.

• Click here for a summary of the Gram stain procedure without pictures.

 

 

 

 

b. Blood agar with novobiocin (NB) disc

Video review - Staphylococcus aureus on Blood Agar with a NB Disc

Video review -Staphylococcus epidermidis on Blood Agar with a NB Disc

Video review - Staphylococcus saprophyticus on Blood Agar with a 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 your decision as to what bacterium is causing the infection. The possible results for Blood agar and NB disc were discussed in the beginning pages of this lab.

 

 

 

 

 

c. Mannitol Salt agar

Video review - How to Interpret 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 your decision as to what bacterium is causing the infection. The possible results for Mannitol Salt agar were discussed in the beginning pages of this lab.

 

 

 

 

 

 

 

 

d. Coagulase test

Video review - How to Interpret the 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 your decision as to what bacterium is causing the infection. 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

Case Study

A 57-year old female who is diabetic, a long-time smoker, and who 28 days ago had hip replacement surgery presents with swelling, pain, inflammation, and erythema at the surgical site. Examination shows she has a fever of 101°F, is exhibiting malaise, and has an increased total white blood cell count with a left shift. Ultrasonography examination indicates a deep abscess. A culture from an aspiration of the infected surgical site was taken.

 1. Patient’s history and predisposing factors

Read the case study. Explain how any relevant parts of the patient’s history contributed to your diagnosis as to the type of infectious disease seen here. The patient's history refers to anything given in the case study prior to that patient seeking medical attention for the current medical condition. 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 (https://www.scribbr.com/apa-examples/website/). Also see appendix F (SIRS and Sepsis) in your lab manual for an indication of whether or not the patient has SIRS.

The patient's history should suggest a general type of infectious disease that is present, such as a urinary tract infection, a wound infection, gastroenteritis, pharyngitis, pneumonia, septicemia, etc. Do not look up the bacterium you eventually identify as the cause of this infectious disease. You do not know the causative bacterium at this point. You need to determine the general type of infection in order to determine what microbiological tests to perform to identify the bacterium causing the infection. 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 infectious disease seen here. Don't forget to cite any sources you used in APA style directly under this Patient's History and Patient's Symptoms sections of this Lab Report.

 

 

 

 

2. Patient’s signs and symptoms

Read the case study. Explain how the patient’s symptoms contributed to your diagnosis as to the type of infectious disease seen here. Signs refer to anything being measured by a medical professional during a physical exam such as blood pressure, respiration rate, heart rate, oxygen saturation, and temperature. Symptoms refer to symptoms being reported by the patient. 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 (https://www.scribbr.com/apa-examples/website/).

The patient's signs and symptoms should suggest a general type of infectious disease that is present, such as a urinary tract infection, a wound infection, gastroenteritis, pharyngitis, pneumonia, septicemia, etc. Do not look up the bacterium you eventually identify as the cause of this infectious disease. You do not know the causative bacterium at this point. You need to determine the general type of infection in order to determine what microbiological tests to perform to identify the bacterium causing the infection. 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 infectious disease seen here. Don't forget to cite any sources you used in APA style directly under this Patient's History and Patient's Symptoms sections of this Lab Report.

 

 

 

 

3. Vocabulary list for medical terms used in the case study under signs and symptoms

List and define any medical terms used in your case study that describe the patients’s signs and symptoms that the average person not in the medical profession might not know.

 

 

 

 

 

 

4. Results of laboratory test given in the case study

List each lab test given in the case study that are done in a lab, such as total white blood count, differential white blood cell count, urinalysis, and X-ray, 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.

 

 

 

5. Microbiological lab tests you performed in Lab 15

a. Gram stain and catalase test

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. Because Enterococci and Staphylococci can sometimes look similar in Gram stains done from a plate culture, perform a catalase test on your unknown to help differentiate an Enterococcus from a Staphylococcus. State how this contributed to your decision as to which microbiological tests and/or media to use next. The Gram stain is discussed in Lab 6; the catalase test in Lab 8.

• Click here for a summary of the Gram stain procedure without pictures.

 

 

 

 

b. Blood agar with novobiocin (NB) disc

Video review - Staphylococcus aureus on Blood Agar with a NB Disc

Video review -Staphylococcus epidermidis on Blood Agar with a NB Disc

Video review - Staphylococcus saprophyticus on Blood Agar with a 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 your decision as to what bacterium is causing the infection. The possible results for Blood agar and NB disc were discussed in the beginning pages of this lab.

 

 

 

 

 

c. Mannitol Salt agar

Video review - How to Interpret 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 your decision as to what bacterium is causing the infection. The possible results for Mannitol Salt agar were discussed in the beginning pages of this lab.

 

 

 

 

 

 

 

d. Coagulase test

Video review - How to Interpret the 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 your decision as to what bacterium is causing the infection. 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. coagulase test with citrated rabbit plasma

d. 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 tube of citrated rabbit plasma

 

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SELF-QUIZ

Self-quiz

Answers

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Lab Manual Table of Contents

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Microbiology Laboratory Manual by Gary E. Kaiser, PhD, Professor of Microbiology
is licensed under a Creative Commons Attribution 4.0 International License.
Last updated: May, 2023