1. health and fitness
2. disease

CLS419 – Clinical Microbiology II
University of Nebraska Medical Center
Clinical Laboratory Science Program
Section Six:
Stool Cultures
CLS 419 Clinical Microbiology II
Page 89
Stool Cultures II – Task List
1. Review objectives for this unit
Objectives for all CLS 419 Bacteriology Units – Rotation II
Stool Cultures II
2. Read:
Textbook:
• Chapter 34 – Gastrointestinal Infections and Food Poisoning, pages 957-978
• Chapter 28 – Diagnostic Parasitology, pages 762-766, Amoeba 769-777,
Flagellates 778-782, and Cryptosporidium 795-797
Procedure:
• Stool Culture General Procedure (found in Rotation I Manual)
3. Review Parasitology Lecture Information from Student Lab:
• Stool Specimens
• Amoeba – intestinal and free-living
• Flagellates – intestinal and vaginal
• Cryptosporidium
4. Complete Study Questions
Stool Cultures II
5. Complete Worksheet
Parasitology Case Study Review
6. Complete bench time at your clinical site as assigned by your instructor(s)
7. Complete Stool Culture II Exam
CLS 419 Clinical Microbiology II
Rotation 2
Stool Cultures
Task List and Objectives
Page 90
STOOL CULTURES
NOTE:
All objectives in bold face are new objectives for CLS 419 and were not part of CLS 418.
Objectives:
After review of the Clinical Microbiology Study Manual and completion of the clinical
rotation for CLS 419 Clinical Laboratory Microbiology II, the Clinical Laboratory Science
student will be able to:
1. Discuss appropriate transport and handling of stool specimens for culture.
2. Determine the appropriateness of the following direct tests when processing stool specimens:
a. Detection of leukocytes
b. Gram stain
3. Describe the procedures for setting up a culture for the following potential stool pathogens, including
the types of media used and incubation requirements:
a. Salmonella/Shigella species
b. Campylobacter jejuni
c. Escherichia coli O157:H7
d. Yersinia enterocolitica
e. Vibrio species
f. Aeromonas/Plesiomonas species
g. Staphylococcus aureus
4. Evaluate stool culture findings based upon:
a. Normal flora
b. Potential pathogens
5. Recognize the following three stool pathogens that should always be screened for when a stool
specimen is submitted for bacterial culture regardless of patient age or geographic location:
a. Salmonella species
b. Shigella species
c. Campylobacter species
6. Explain the clinical significance of detecting Shiga-like toxin antigen in stool specimens.
7. Recognize the following laboratory methods for detection of Shiga-like toxin antigen in stool
specimens:
a. EIA
b. Molecular
8. Explain the advantages and disadvantages of the following methods in the diagnosis of hemolytic
uremic syndrome (HUS):
a. Antigen testing
b. Culture
ENTEROBACTERIACEAE
1. Describe the following characteristics common to the Enterobacteriaceae:
a. Gram stain morphology
b. Oxidase reaction
c. Glucose fermentation
d. Nitrate reduction
e. Growth on MacConkey agar
f. Flagellar arrangement
CLS 419 Clinical Microbiology II
Rotation 2
Stool Cultures
Task List and Objectives
Page 91
2. Evaluate the following media, including purpose, proper use, significant ingredients, inhibitory or
selective properties and colonial appearance:
a. MacConkey agar (MAC)
b. Hektoen enteric agar (HE)
c. Salmonella-Shigella agar (SS)
d. Xylose-lysine-deoxycholate agar (XLD)
e. Eosin-methylene blue agar (EMB)
f. Cefsulodin-irgasan-novobiocin agar (CIN) or Yersinia selective agar
g. Phosphate buffered saline
h. Gram-negative broth (GN)
i. MacSorbitol agar
3. Explain the principle of the following biochemical reactions and other characteristics,
including the interpretation of results:
a. Indole
b. Kliger’s iron agar (KIA)
c. Lysine iron agar (LIA)
d. Triple sugar iron agar (TSI)
e. Ornithine decarboxylase (ODC)
f. Oxidation-fermentation medium (OF)
g. Urease production
h. Gelatin liquefaction
i. Motility
j. Nitrate reduction
k. Citrate utilization
l. Voges-Proskauer
m. Deaminase reaction (tryptophan, lysine, phenylalanine)
n. Ortho-nitro-phenyl-beta-D-galactopyranoside (ONPG)
4. Evaluate the acceptability of results for tests listed in Objective #3, including appropriate
corrective action and/or further testing if necessary.
5. Differentiate between the following Enterobacteriaceae based on colonial morphology and the tests
listed in Objective #3.
a. Escherichia coli
k. Enterobacter cloacae
b. Shigella sonnei
l. Enterobacter aerogenes
c. Shigella species
m. Edwardsiella tarda
d. Salmonella species
n. Proteus vulgaris
e. Salmonella typhi
o. Proteus mirabilis
f. Serratia species
p. Morganella morganii
g. Citrobacter freundii
q. Providencia species
h. Citrobacter koseri
r. Providencia rettgeri
i. Klebsiella pneumoniae
s. Yersinia enterocolitica
j. Klebsiella oxytoca
6. Define the following terms:
a. Capsular antigen (K)
b. Somatic antigen (O)
c. Flagellar antigen (H)
d. Vi antigen
7. Evaluate the use of the antigens listed in objective #6 when performing serologic typing of organisms.
8. Discuss the following confirmatory methods used to identify Escherichia coli O157:H7:
a. Serogrouping / serotyping
b. Verotoxin detection
CLS 419 Clinical Microbiology II
Rotation 2
Stool Cultures
Task List and Objectives
Page 92
9. Correlate the clinical, epidemiological and laboratory findings associated with Enterobacteriaceae
related to the following infections:
a. Gastrointestinal infections
b. Hemolytic uremic syndrome (HUS)
d. Typhoid fever
f. Opportunistic/nosocomial infections
CAMPYLOBACTER AND HELICOBACTER
1. Evaluate Campylobacter blood agar (CVA) or Skirrow blood agar, including purpose, proper use,
significant ingredients, inhibitory or selective properties and colonial appearance.
2. Identify Campylobacter jejuni based on the following characteristics:
a. Gram stain reaction and morphology
b. Growth at 25ºC, 37ºC and 42ºC
c. Colony morphology
d. Catalase reaction
e. Oxidase reaction
f. Sodium hippurate hydrolysis
g. Nalidixic acid and Cephalothin susceptibilities
h. Motility
3. Identify Helicobacter pylori based on the following characteristics:
a. Gram stain reaction and morphology
b. Growth requirements
c. Direct specimen testing (i.e. rapid urea or antigen testing)
d. Oxidase reaction
e. Urease reaction
4. Discuss the use of serological testing in the diagnosis of Helicobacter pylori infections.
5. Correlate the clinical, epidemiological and laboratory findings associated with the following infections:
a. Campylobacter jejuni
1) Gastroenteritis
b. Helicobacter pylori
1) Gastritis
2) Peptic ulcer disease
ANAEROBES
8. Describe the Gram stain reaction and morphology of each of the following anaerobic species:
f. Clostridium
13. Correlate the clinical, epidemiological and laboratory finding associated with the following anaerobic
infections:
d. Clostridium difficile
1) Antibiotic-associated pseudomembranous colitis
GRAM-POSITIVE BACILLI
1. Describe the Gram stain morphology, oxygen requirements and catalase reaction for each of the
following gram-positive bacilli:
a. Spore-forming
1) Bacillus species
9. Correlate the clinical, epidemiological and laboratory findings associated with the following
infections:
b. Bacillus cereus
1) Food poisoning
CLS 419 Clinical Microbiology II
Rotation 2
Stool Cultures
Task List and Objectives
Page 93
11. Correlate the patient immune status with the pathogenicity of the following organisms:
b. Bacillus cereus
GRAM-NEGATIVE RODS - OXIDASE POSITIVE FERMENTERS
1. Discuss the following characteristics common to the Vibrionaceae:
a. Gram stain morphology
b. Glucose fermentation
c. Oxidase reaction
d. Growth on MacConkey agar
2. Evaluate the following media, including purpose, proper use, significant ingredients,
inhibitory or selective properties and colonial appearance:
a. Thiosulfate-citrate-bile-sucrose agar (TCBS)
b. Alkaline peptone broth
3. Distinguish Vibrio species from Aeromonas species and Plesiomonas species based on the
following characteristics:
a. Gram stain morphology
b. Colonial morphology
c. Growth on TCBS agar
4. Differentiate Aeromonas species from Plesiomonas species based on the following
characteristics:
a. Colonial morphology
b. Ornithine decarboxylase
6. Correlate the clinical, epidemiological and laboratory findings associated with the following
infections:
a. Vibrio cholerae
1) Cholera
b. Vibrio parahaemolyticus
1) Gastroenteritis
e. Plesiomonas shigelloides
1) Gastroenteritis
f. Aeromonas hydrophila
1) Gastroenteritis
STAPHYLOCOCCUS
4. Correlate clinical, epidemiological and laboratory findings associated with the following infections:
a. Staphylococcus aureus
8) Pseudomembranous enterocolitis
9) Food poisoning
INTRODUCTION TO PARASITOLOGY
1. Define the following terms:
b. Parasite
e. Protozoa
1) Pathogenic
1) Amoeba
2) Nonpathogenic (commensal)
2) Flagellates
3) Opportunistic
4) Sporozoa
3. Discuss the advantages and disadvantages of the following preservatives used to transport feces for
ova and parasite examinations:
a. Polyvinyl Alcohol (PVA)
b. 10% Formalin
c. One-vial fixatives (do not contain mercury)
CLS 419 Clinical Microbiology II
Rotation 2
Stool Cultures
Task List and Objectives
Page 94
4. Evaluate the acceptability of the following specimens for parasitic examination, including specimen
collection and transportation:
a. Feces
c. Scotch tape prep
b. Duodenal aspirate
i. Vaginal secretions
5. Evaluate the following methods used to recover parasites from clinical specimens:
a. Direct methods
1) Macroscopic examination
2) Saline preparations
3) Iodine wet preparations
b. Concentration methods
1) Formalin-ethyl acetate (sedimentation)
c. Permanent stained smears
1) Trichrome stain
3) Modified acid-fast
PROTOZOA – AMOEBA
1. Recognize the role (diagnostic or infective) of the following life cycle stages of the intestinal amoeba:
a. Cyst
b. Trophozoite
2. Recognize the following structures of the intestinal amoeba as they pertain to the identification of the
parasite:
a. Nucleus
d. Vacuoles
b. Karyosome
e. Chromatoid bars
c. Peripheral chromatin
3. Discuss the parasites listed below, emphasizing:
a. Infective form(s) for man
b. Identification of the diagnostic form(s)
c. Transmission, including significant vectors or reservoirs
1) Intestinal
a) Entamoeba histolytica
b) Entamoeba hartmanni
c) Entamoeba coli
d) Endolimax nana
e) Iodamoeba butschlii
f) Blastocystis hominis
2) Extraintestinal
a) Naegleria fowleri
b) Acanthamoeba species
4. Correlate clinical, epidemiological and laboratory findings associated with the following infections:
a. Entamoeba histolytica
1) Amebic dysentery
b. Naegleria fowleri
1) Primary amebic meningoencephalitis
c. Acanthamoeba species
1) Granulomatous amebic encephalitis
2) Acanthamoeba keratitis
PROTOZOA – FLAGELLATES
1. Recognize the role (diagnostic or infective) of the following life cycle stages for the flagellates:
a. Intestinal:
1) Cyst
2) Trophozoite
CLS 419 Clinical Microbiology II
Rotation 2
Stool Cultures
Task List and Objectives
Page 95
2. Recognize the following structures of the intestinal flagellates as they pertain to the identification of
the parasite:
a. Nucleus
d. Cytostome
b. Axoneme
e. Axostyle
c. Flagella
f. Undulating membrane
3. Discuss the parasites listed below, emphasizing:
a. Infective form(s) for man
1) Giardia lamblia
2) Trichomonas vaginalis
b. Identification of the diagnostic form(s):
1) Intestinal
2) Extraintestinal
a) Giardia lamblia
a) Trichomonas vaginalis
b) Chilomastix mesnili
c) Trichomonas hominis
d) Dientamoeba fragilis
c.
Transmission, including significant vectors or reservoirs:
1) Giardia lamblia
2) Trichomonas vaginalis
4. Correlate clinical, epidemiological and laboratory findings associated with the following infections:
a. Giardia lamblia
1) Diarrhea
2) Malabsorption syndrome
b. Trichomonas vaginalis
1) Urethritis / vaginitis
PROTOZOA - SPOROZOA
1. Recognize the role (diagnostic or infective) of the following life cycle stages for the sporozoa:
a. Intestinal:
1) Oocyst
2. Identify the diagnostic form(s) for the parasites listed below:
a. Intestinal
1) Cryptosporidium parvuum
3. Differentiate Cryptosporidium parvuum and Cyclospora species based on their oocyst size.
4. Recognize the clinical significance of the following pathogenic parasites:
a. Cryptosporidium parvuum
1) Diarrhea
CLS 419 Clinical Microbiology II
Rotation 2
Stool Cultures
Task List and Objectives
Page 96
Study Questions – Stool Cultures Rotation II
Utilize your Stool Culture Rotation II objectives as a guide for answering the following questions.
Suggested references in completing the study questions:
• Mahon & Manuselis, Textbook of Diagnostic Microbiology, 3rd Ed., Saunders, 2007. (Your textbook)
• Student Manuals
When indicated: Clinical Microbiology Blackboard course components are found under
<Assignments><Clinical Rotations - Bacteriology<Stool Culture II Unit><Study Questions> folder.
1. A stool submitted for a Yersinia Culture is best inoculated to _____________ and incubated at
______________________________.
a.
b.
c.
d.
Cefsulodin-irgasan-novobiocin (CIN) agar, 25°C in ambient air
Cefsulodin-irgasan-novobiocin (CIN) agar, 37°C in ambient air
Cycloserine-Cefoxitin Fructose (CCFA) agar, 25°C in ambient air
Cycloserine-Cefoxitin Fructose (CCFA) agar, 37°C in anaerobic conditions
What are the growth characteristics of Yersinia enterocolitica under these incubation conditions (i.e.,
rate of growth, colony morphology)?
What are the glucose and oxidase results of Yersinia enterocolitica?
2. A stool fecal leukocyte test performed on the stool of 24-year-old male was reported as having
moderate PMNs. After 24 hours of incubation at 35°C in ambient air, the routine stool culture is
growing:
MacConkey agar (Mac): Many clear colonies,
Hektoen Enteric agar (HE): Many clear colonies with black centers
What is your next step?
After recording your answer, go to the Clinical Microbiology Blackboard course component
<Assignments><Clinical Rotations - Bacteriology<Stool Culture II Unit><Study
Questions><Question 2> to continue with this question. Record further results here:
CLS 419 Clinical Microbiology II
Rotation 2
Stool Cultures
Study Questions
Page 97
3. A liquid stool from a 7-year-old girl is submitted for a stool culture. After 24 hours of incubation at
35°C in ambient air, the routine stool culture is growing a pure culture:
Blood agar: Many beta-hemolytic, large, opaque colonies
MacConkey agar (Mac): Many pink colonies
Hektoen Enteric agar (HE): Many yellow colonies
The organism is an oxidase positive, gram-negative rod.
What is your next step?
After recording your answer, go to the Clinical Microbiology Blackboard course component
<Assignments><Clinical Rotations - Bacteriology<Stool Culture II Unit><Study
Questions><Question 3> to continue with this question. Record further results here:
4. A liquid stool specimen from a 37-year-old male hospitalized for dehydration and hypotension was
sent for routine stool culture as well as a culture for cholera. The previous day the man had returned
from a South American trip.
What medium/media should be added to recover the etiologic agent of cholera?
After recording your answer, go to the Clinical Microbiology Blackboard course component
<Assignments><Clinical Rotations - Bacteriology<Stool Culture II Unit><Study
Questions><Question 4> to continue with this question. Record further results here:
CLS 419 Clinical Microbiology II
Rotation 2
Stool Cultures
Study Questions
Page 98
5. The laboratory received a gastric biopsy from a 35-year-old male diagnosed with peptic ulcer disease.
A rapid urease test (i.e., CLOtest) performed on a portion of the biopsy was negative. Due to the
negative rapid urease test, the physician ordered a culture looking specifically for an etiologic agent of
peptic ulcer disease.
What organism is he suspecting?
After recording your answer, go to the Clinical Microbiology Blackboard course component
<Assignments><Clinical Rotations - Bacteriology<Stool Culture II Unit><Study
Questions><Question 5> to continue with this question. Record further results here:
6. If a delay longer than 2 hours is anticipated for a stool submitted for bacterial culture, the specimen
should be:
a.
b.
c.
d.
Frozen at -60°C
Kept at room temperature up to 12 hours
Placed in a transport medium such as Cary-Blair
Refrigerated up to 24 hours
7. Routine stool culture should include testing for the presence of which of the following stool
pathogens:
a.
b.
c.
d.
e.
Routine culture media for the detection of Salmonella and Shigella
Routine culture media for the detection of Campylobacter species
Routine culture media for Yersinia enterocolitica
Thioglycollate broth to detect the presence of Clostridium botulinum
Both A and B
CLS 419 Clinical Microbiology II
Rotation 2
Stool Cultures
Study Questions
Page 99
8. Please match the following with its reservoir.
____a.
____b.
____c.
Salmonella species
Salmonella typhi
Shigella species
1. Only found in humans, not normal flora
2. Humans, and animal reservoirs
9. A 5-year-old patient was admitted to the hospital with acute enteritis. A routine stool culture and
screen for Yersinia are ordered. MacConkey Agar shows colorless colonies. Oxidase testing is
negative. Urease testing is positive. A red-centered colony with a clear periphery was isolated on
the CIN agar. Which of the following tests would be helpful to identify the suspected pathogen?
a.
b.
c.
d.
e.
Glucose Fermentation
Motility at 22 degrees Celsius
Motility at 35 degrees Celsius
All of the above
None of the above
What results would you expect on the suspected pathogen?
Which pathogen is suspected?
10. Which of the following organisms can grow in the small bowel and cause diarrhea in children,
traveler’s diarrhea, or a severe cholera-like syndrome through production of enterotoxins?
a.
b.
c.
d.
Escherichia coli
Salmonella typhi
Shigella dysenteriae
Yersinia enterocolitica
11. A. Which of the following organisms is most commonly associated with antibiotic-associated
diarrhea and pseudomembranous colitis?
a.
b.
c.
d.
Staphylococcus aureus
Clostridium difficile
Shigella sonnei
Clostridium perfringens
B. Which test on stool would be the most beneficial in diagnosis of this organism?
a.
b.
c.
d.
Routine culture and sensitivity
Gram stain
Antigen detection for toxin(s) by EIA or latex agglutination
Ova and parasite examination
CLS 419 Clinical Microbiology II
Rotation 2
Stool Cultures
Study Questions
Page 100
12. In addition to routine culture media it is suggested that media be set routinely to screen for what
organism when a bloody stool is received in the lab especially on small children?
a.
b.
c.
d.
Yersinia enterocolitica
E. coli 0157:H7
Vibrio cholera
All of the above
What additional media should be set up for this culture and how would you incubate the media
(temperature, atmosphere, and how long)?
13. Please list media you would set for routine stool culture and how you would incubate the media
(temperature, atmosphere and how long).
14. At 48 hours of incubation in 10% CO2 at 42˚C, the Campylobacter blood agar plate of a stool culture
was growing a few gray, flat colonies. Gram stain of culture plate shows small gram-negative
coccobacilli. Oxidase reaction is positive. It is the end of the day and the other microbiologists have
gone for the day. You are not sure of your gram stain results. What test could be performed to
confirm the presence of Campylobacter?
a. Subculture to two CVA plates. Incubate one in Microaerophilic conditions at 42˚C and one plate
at 35˚C in 5-7 % CO2.
b. Perform Sodium Hippurate testing and release results as Campylobacter jejuni if positive.
c. Perform Nalidixic Acid and Cephalothin disc testing.
d. Nothing. Release results as Campylobacter species
CLS 419 Clinical Microbiology II
Rotation 2
Stool Cultures
Study Questions
Page 101
University of Nebraska Medical Center
CLS419 Clinical Microbiology II: Parasitology
Parasitology Case Study Review Worksheet
Directions - to be used with the Blackboard Course component: Parasitology Case Study Review in the
Stool Culture Unit folder. Go to the corresponding case study to view the microscopic examples for each
case. Click on an image to see an enlarged view. Answer the following short answer or single answer
multiple choice questions for each case.
Case 1 – This organism was detected in a loose stool collected from a 4-year-old male that attends a
daycare center. The child’s mother indicated the boy has experienced diarrhea and abdominal pain for
approximately one week.
1. Identify the organism.
2. Besides stool, other specimen(s) acceptable for recovery of this organism include:
a.
b.
c.
d.
Anticoagulated blood
Duodenal aspirate, biopsy or contents as obtained with the string test
Genital specimens, urine
Liver aspirate or biopsy
3. Chronic infections with this organism often exhibit:
a.
b.
c.
d.
Extraintestinal development
Inflammatory reaction secondary to deposition of organism as it invades intestinal mucosa
Malabsorption
Vitamin B12 deficiency
Case 2 – This organism was detected in the stool of a 48-year-old AIDS patient. The organism was
detected with a modified-acid fast stain.
4. Identify this organism.
5. Humans acquire this infection via:
a.
b.
c.
d.
Contact with cat feces
Fecal/oral route
Ingesting undercooked, infected meat
Penetration of larval form
6. In immunocompromised hosts, this organism clinically exhibits as a/an:
a.
b.
c.
d.
Extraintestinal tissue infection
Parasitemia
Prolonged diarrhea that worsens progressively
Vitamin B12 deficiency
Case 3 – This organism was detected in stool submitted from a 64-year-old male farmer.
7. Identify this organism.
CLS 419 Clinical Microbiology II
Rotation 2
Stool Cultures
Study Questions
Page 102
8. Clinically, this organism is considered to be a/an:
a.
b.
c.
d.
Commensal, non-pathogenic organism
Contaminant, possible association with allergies
Etiologic agent of chronic diarrhea
Primary cause of intestinal ulceration & colitis
Case 4 – This organism was detected in stool of 45-year-old male complaining of a low-grade fever and
abdominal pain. The stool was described as loose and blood-tinged.
9. Identify this organism.
10. Clinically, this organism can present as a(an)
a.
b.
c.
d.
Asymptomatic, dysentery, or extraintestinal infection
Infection with localized intestinal lesions that often spread to the respiratory tract
Nonpathogenic, commensal organism
Vitamin B12 deficiency
Case 5 – These structures were detected in the stool of a 4-year-old boy. The stool was described as
loose, with areas of mucus, pus and blood flecks.
11. Based on the given information and microscopic images, the structures seen are:
a.
b.
c.
d.
Entamoeba coli cysts
Entamoeba coli trophozoites
Entamoeba histolytica cysts
Polymorphonuclear WBCs
Case 6 – These structures were detected in the stool of a 25-year-old male complaining of diarrhea and
abdominal pain.
12. Based on the given information and microscopic images, the structures seen are:
a.
b.
c.
d.
Artifact
Dientamoeba fragilis trophozoites
Endolimax nana trophozoites
Iodamoeba butschlii trophozoites
References:
Garcia, Lynne, Bruckner, D., 1997, 3rd ed. Diagnostic medical parasitology. Washington, D.C.: ASM
Press.
Howard, Barbara J., Klaas II, J., Rubin, S., Weissfeld, Al, Tilton, Rl, 1994. Clinical and pathogenic
microbiology. St. Louis: C.V. Mosby.
Koneman, E., Allen, S., Janda, W., Schrechenberger, P., Winn, W., 1997. Color atlas and textbook of
diagnostic microbiology., 5th ed. Philadelphia: Lippincott-Raven.
Mahon, C., & Manuselis G., 2000, Textbook of diagnostic microbiology, 2nd ed., Philadelphia: WB
Saunders.
CLS 419 Clinical Microbiology II
Rotation 2
Stool Cultures
Study Questions
Page 103