Solution Manual For Basic Clinical Laboratory Techniques 6th Edition

Solution Manual for Basic Clinical Laboratory
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Solution Manual for Basic Clinical Laboratory Techniques, 6th Edition
UNIT 7
Basic Clinical
Microbiology
UNIT OBJECTIVES
After studying this unit, the student will:

• Discuss the fields of study included in microbiology.
• Discuss the types of diseases caused by different groups of microorganisms.
• Explain why organisms are classified as normal flora, pathogens, or opportunistic
pathogens.
• Explain procedures for collecting and processing specimens for bacteriology.
• Discuss basic culture techniques and culture media used in bacteriology.
• Prepare and Gram stain bacterial smears.
• Describe three basic types of bacterial morphology and identify them microscopically.
• Perform a throat culture and a rapid test for group A Streptococcus.
• Perform a urine culture and colony count.
• Explain methods of bacterial identification and antibiotic susceptibility testing.
• Discuss laboratory test methods for the detection of sexually transmitted diseases.
• Discuss the role of transmission-based precautions in preventing infections in healthcare
settings.
• List five emerging infectious diseases and explain why they can be a threat to public health.
• List five potential agents of bioterrorism, the diseases they cause, and the role of the
clinical laboratory in surveillance and detection.
UNIT OVERVIEW
Unit 7 is an introduction to clinical microbiology, the study of the microorganisms that cause
disease and some of the laboratory tests to detect these organisms. The clinical laboratory’s
microbiology department isolates and identifies medically important bacteria, viruses, fungi, and
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parasites. The test methods presented in this unit emphasize bacteriological tests suitable for the
smaller laboratory or physician’s office. Advances in technology are providing an increased
number of rapid, easy-to-use tests for bacteriology, virology, and parasitology.
Lesson 7-1 concentrates on history, background information, terminology, and knowledge
about the different groups of microorganisms in microbiology and the diseases they cause.
Procedures for collecting specimens for culture in the bacteriology laboratory are discussed in
Lesson 7-2, along with explanations of the use of transport media.
Growth requirements of various bacteria and general culture techniques are presented in
Lesson 7-3. The differences in primary, selective, and indicator media are described, and
explanations and tables are provided to explain the use of each type. Safety in the bacteriology
laboratory and use of aseptic technique are emphasized.
The stained bacterial smear is an important tool for identifying bacteria. Lesson 7-4 details
how to prepare a smear, perform a Gram stain, and examine the smear microscopically for Gram
stain reaction and bacterial morphology.
Rapid tests for group A Streptococcus, the cause of strep throat, are performed many times a
day in most laboratories. The procedures for rapid strep tests and throat culture for group A
Streptococcus are given in Lesson 7-5. Lesson 7-6 describes how to perform a urine culture and
colony count. Urine culture is another of the most frequently performed laboratory tests. Urine
culture can be performed in the small laboratory as long as quality assessment measures are
followed and qualified personnel are available. Many smaller facilities send urines to be cultured
to a hospital or reference laboratory. Lesson 7-7 describes bacterial identification methods and
antibiotic susceptibility testing.
Sexually transmitted diseases (STDs) and the types of STD diagnostic tests available,
including those suitable for the small laboratory, are discussed in Lesson 7-8. Methods of
preventing healthcare-associated infections (HAIs) and the Centers for Disease Control and
Prevention (CDC) categories of transmission-based precautions are discussed in Lesson 7-9.
Lessons 7-10 and 7-11 address two microbiology topics important to public health.
Lesson 7-10 reviews several emerging infectious diseases and discusses why these diseases
are on the increase and how they pose a threat to public health. Lesson 7-11 addresses the
topics of bioterrorism, biological weapons, and the role of clinical laboratories in preparing for
and investigating possible threat incidents.
LESSON 7-1
Introduction to Clinical
Microbiology
LESSON OBJECTIVES
After studying this lesson, the student will:
• List the fields of study included in microbiology.
• Describe the microbiology department’s organization and function in small and
large laboratories.
• Discuss the differences among normal flora, pathogens, and opportunistic
pathogens.
• Discuss the three basic shapes of bacteria.
• Explain the importance of microbiology specimen collection and processing
techniques.
• Discuss methods used to identify bacteria.
• Explain the functions of the parasitology laboratory.
• Explain how virology diagnostic procedures differ from bacteriology procedures.
• Discuss diagnostic methods used in mycology.
• Define the glossary terms.
GLOSSARY
aerobic / requiring oxygen for growth

anaerobic / growing in the absence of oxygen
antibiotic susceptibility testing / determining the susceptibility of bacteria to specific antibiotics
bacillus / a rod-shaped bacterium
coccus / a spherical bacterium
colony / a defined mass of bacteria assumed to have grown from a single organism
communicable / able to be transmitted directly or indirectly from one individual to another
350 Lesson 7-1 ● Introduction to Clinical Microbiology
culture / growth of microorganisms in a special medium; the process of growing

microorganisms in the laboratory
deoxyribonucleic acid (DNA) / the nucleic acid that carries genetic information and that is
found primarily in the nucleus of all living cells
fastidious bacteria / bacteria that require special nutritional factors to survive
fission / reproductive process in which the parent cell divides into two identical independent
cells
gram negative / designation for bacteria that lose the crystal violet (purple) stain and retain the
safranin (red ) stain in the Gram stain procedure
gram positive / designation for bacteria that retain the crystal violet (purple) stain in the Gram
stain procedure
Gram stain / a differential stain used to classify bacteria
host / the organism from which a parasite obtains nutrients and in which some or part of the
parasite’s life cycle is completed
hyphae / filaments of a fungus that make up the mycelium
immunoassay / a diagnostic method using antigen–antibody reactions
infection / a pathological condition caused by growth of microorganisms in the host
medium / a substance used to provide nutrients for growing microorganisms
minimum inhibitory concentration (MIC) / minimum concentration of an antibiotic required
to inhibit the growth of a microorganism
mycelium / mass of hyphae that makes up the vegetative body of a fungus
mycosis / infection caused by fungi
normal flora / microorganisms normally present at a specific body site and that do not cause
disease in healthy individuals
opportunistic pathogen / a microorganism that causes disease only when the host’s normal
defense mechanisms are impaired or absent
pathogen / an organism or agent capable of causing disease in a host
progeny / offspring or descendants
ribonucleic acid (RNA) / the nucleic acid that is important in protein synthesis and is found in
all living cells
spiral bacteria / motile bacteria having a helical or spiral shape
TEACHING AIDS AND RESOURCES

• Newspaper or magazine articles describing a recent infectious disease outbreak
• Guest speaker—a clinical laboratory professional employed in a microbiology laboratory,
an epidemiologist, or a microbiology professor from a nearby college or university
• Examples of bacterial, fungal, viral, or parasitic test kits, cultures, identification kits,
specimens, etc.
Lesson 7-1 ● Introduction to Clinical Microbiology 351
• Images (digital, transparencies, or overheads) of Figures 7-1 through 7-4

• Images (digital, transparencies, or overheads) of Tables 7-1 through 7-9
• Instructor’s Resources CD accompanying Basic Clinical Laboratory Techniques,
6th edition, including computerized test bank and PowerPoint
LESSON CONTENT
I. Introduction
II. Rules of Nomenclature
III. Clinical Bacteriology
A. General Characteristics of Bacteria
1. Bacterial morphology
a. Multiply by fission
b. Form colonies
c. Coccus (round)
d. Bacillus (rod)
e. Spiral bacteria
2. Gram stain reactions
a. Gram positive
b. Gram negative
3. Growth requirements
a. Aerobic
b. Anaerobic
c. Fastidious
d. Enhanced growth in 5% to 10% carbon dioxide
B. Pathogenic Bacteria
1. Normal flora
2. Opportunistic pathogens
3. Pathogens
C. Bacteriological Procedures in the Small Laboratory
1. Specimen collection
2. Identifying bacteria
3. Antibiotic susceptibility testing
IV. Parasitology
A. Intestinal Parasites
1. Stool examination for parasites
2. Preserving and transporting specimens
B. Blood and Tissue Parasites
V. Virology
A. Characteristics of Viruses
B. Diagnostic Testing in Virology
1. Viral culture
2. Immunoassays
3. Rapid tests, CLIA-waived
VI. Mycology
A. Characteristics of Molds
1. Hyphae
2. Mycelium
B. Characteristics of Yeasts
1. Eukaryotic
2. Reproduce by budding
C. Fungal Diseases—Mycoses
1. Dimorphic fungi
2. Opportunistic fungi
a. Candida
b. Aspergillus
c. Pneumocystis
3. Dermatophytes
D. Identifying Molds and Yeasts
VII. Summary
STUDENT ACTIVITIES
1. Complete the written examination for this lesson.

2. Visit a microbiology laboratory in the community. Find out what types of tests are performed
in the laboratory and which tests are sent to reference laboratories.
3. Interview an employee of a hospital microbiology laboratory and report on his or her job
functions and responsibilities.
Web Activity
Use the Internet to find information on a disease caused by an agent from each microbiology
category (bacteria, viruses, fungi, and parasites). For each disease, report on the causative agent,
disease symptoms, method of diagnosis, and treatment.
Lesson 7-1 ● Introduction to Clinical Microbiology 353
REVIEW QUESTIONS AND ANSWERS

1. When was the term microbiology first used?
Louis Pasteur first used the term microbiology in the 1860s.
2. What four areas of study are encompassed by clinical microbiology?

Bacteriology, virology, mycology, and parasitology
3. What are the functional differences between a small and large microbiology laboratory?
A large laboratory may have individual departments that are responsible for each type
of microorganism—virology, parasitology, mycology, and bacteriology. In a small
laboratory, one department may perform the less complex procedures for all of these
areas, and some small laboratories send many samples to a reference laboratory.
4. What is the difference between a pathogen and an opportunistic pathogen?

A pathogen is an organism that can cause disease in most hosts; an opportunistic
pathogen is usually only able to cause disease in immunocompromised hosts.
5. How do bacterial pathogens cause host damage?

Bacterial pathogens cause host damage by invading tissues, growing, and often
producing toxic products.
6. What are the three morphological types of bacteria?

a. Cocci—round or spherical bacteria
b. Bacilli—rod-shaped bacteria
c. Spiral—helical or spiral shaped bacteria
7. What is a Gram stain?

The Gram stain is a staining procedure that differentiates bacteria according to
structural differences in their cell walls.
8. What are two morphological forms of fungi?

Molds and yeasts
9. What is the difference between aerobic and anaerobic bacteria?

Aerobic bacteria require oxygen for growth; anaerobic bacteria grow in the absence of
oxygen.
10. How are viruses different from microorganisms? Name three viral diseases.
Viruses are not living cells and can only replicate by invading a cell; they have only
one type of nucleic acid and are too small to be seen with the light microscope.
Microorganisms have both DNA and RNA, can grow on culture media, and can be
seen with the light microscope. Any three of the following examples of viral diseases
are acceptable: (1) mumps, (2) AIDS, (3) common cold, (4) influenza, (5) rubella,
(6) rubeola, (7) hepatitis, (8) herpes, and (9) infectious mononucleosis (Table 7-6).
11. What type of specimen is required to detect malaria? To detect Giardia?

Malaria is detected by examination of a stained blood smear. Giardia is detected by
examination of stool specimens.
12. List five methods used to help identify bacteria.

Any five of the following are acceptable:
a. Microscopic appearance
b. Colonial morphology
c. Selective or indicator media
d. Gram and other stains
e. Biochemical reactions
f. Gene probes
g. Antibody reactions
13. Define aerobic, anaerobic, antibiotic susceptibility testing, bacillus, coccus, colony,
communicable, culture, deoxyribonucleic acid, fastidious bacteria, fission, gram negative,
gram positive, Gram stain, host, hyphae, immunoassay, infection, medium, minimum
inhibitory concentration, mycelium, mycosis, normal flora, opportunistic pathogen,
pathogen, progeny, ribonucleic acid, and spiral bacteria.
See Glossary.
LESSON 7-2
Bacteriology Specimen
Collection and Processing
LESSON OBJECTIVES
After studying this lesson, the student will
• List seven specimens that are frequently cultured in the microbiology laboratory.
• List five reasons a culture might be ordered.
• Discuss the collection and processing of a throat culture.
• Explain why a nasal culture would be required.
• Explain the procedures for the collection and processing of urine for culture.
• List three methods for collecting specimens from a wound.
• Discuss the collection and processing of a sputum specimen.
• Explain how specimens are collected for a stool culture.
• Describe the procedure for collecting blood cultures.
• Explain the function and importance of transport media.
• Explain how the type of specimen must be considered in the choice of transport media.
• Discuss safety precautions that must be observed when collecting and processing
specimens for bacteriology testing.
• Discuss how quality assessment procedures are used in the collection and processing
of specimens for bacteriology testing.
• Define the glossary words.
GLOSSARY
carrier / an individual who harbors an organism and is capable of spreading the organism to
others, but has no symptoms or signs of disease
culture / growth of microorganisms in a special medium; the process of growing
microorganisms in the laboratory
356 Lesson 7-2 ● Bacteriology Specimen Collection and Processing
culture medium /a substance used to provide nutrients for growing microorganisms

Escherichia coli (E. coli) / a bacterium that is part of the normal flora of the intestines
sepsis / the presence of microorganisms and/or their toxic products in the blood and other tissues
septicemia / the presence and growth of pathogenic microorganisms in the blood
Streptococcus pyogenes / bacterium that causes a common type of streptococcal infection, most
notably “strep” throat
transport medium / containers specially designed to provide nutrients and an environment that
preserves the viability of microorganisms during transport
virulent / highly infectious
wound / a break in the continuity of soft parts of the body structure; trauma to tissues

• Examples of specimen collection and transport materials for various types of
bacteriological cultures
LESSON CONTENT
I. Introduction
II. Reasons for Ordering Cultures
III. Specimen Collection Manual
A. Contains Collection Instructions
B. Provided by Reference Laboratory
IV. Collection and Transport Supplies
A. Supplies for a Variety of Specimen Types
B. Support Aerobic and Anaerobic Microorganisms
C. Requirements for Transport Containers
V. Safety Precautions
A. Standard Precautions
B. Personal Protective Equipment (PPE)
C. Disinfect Setup Area
VI. Quality Assessment
A. Follow SOP Manual
Lesson 7-2 ● Bacteriology Specimen Collection and Processing 357
B. Label Correctly
C. Do Not Use Cotton Swabs
D. Do Not Use Expired Media
VII. Collecting the Specimen
A. Throat Culture
1. Swab tonsils
2. Immediately inoculate blood agar plate or place swab in transport medium
B. Nasal Cultures
1. MRSA carriers
2. Bordetella pertussis
3. Corynebacterium diptheriae
4. Other
C. Urine for Culture
1. Midstream, clean-catch
2. Label container appropriately
3. Use transport system if testing is delayed
D. Wound Specimens
1. Aspirates are the preferred specimen
2. Inoculate transport medium immediately
3. Use transport system that supports anaerobes and anaerobes
E. Respiratory Specimens
1. Sputum
2. Rinse mouth before collecting
3. Streptococcus pneumoniae and Klebsiella pneumoniae
F. Stool Specimens
1. Stool not contaminated with water or urine
2. Do not collect from disposable diaper
3. Use transport medium that supports enteric pathogens
4. Enteric pathogens include: Salmonella, Shigella, Vibrio, Campylobacter,
Clostridium difficile, E. coli 0157:H7
G. Blood Cultures
1. Septicemia or sepsis
2. Phlebotomist wears sterile gloves
3. Puncture site cleansed with alcohol followed by iodine
4. Various types of collection bottles
VIII. Case Studies
IX. Summary
Case Studies and Answers
Case Study 1
Carol was working in a point-of-care testing (POCT) site. A rapid test for group A strep was
negative on a 10-year-old boy. The provider ordered a confirmatory throat culture for
S. pyogenes. Carol collected another throat specimen with a sterile swab. Several patients were
waiting for laboratory tests. Carol had to decide whether to set up the culture immediately or
wait until after she collected blood samples for the other patients.
1. Carol should:
a. Place the swab back into the original package until she has time to set up the culture.
b. Place the swab into an open test tube on the counter until she can set up the culture.
c. Let the patients wait while she sets up the culture from the swab.
2. How could Carol have avoided her dilemma?

Carol could have collected the specimen using a swab and transport medium system
that would protect the organisms until the specimen could be inoculated to culture
medium.
Case Study 2
Rick had performed a rapid test for group A strep on a young child. The result was negative.
He knew the laboratory protocol required that a confirmatory culture be performed; however, the
child did not seem very ill. His thinking was “Why waste time processing a throat culture when it
would probably be negative?”
A. Evaluate the following statements for correctness:
TF 1. S. pyogenes infections have no risk of complications.
T F2. Rick must follow the laboratory protocol.
TF 3. A throat culture swab should be inoculated to a blood agar plate.
B. Have a class discussion about Rick’s attitude. Include topics such as professionalism, ethics,
best practices for patient care, job responsibility.
Guide student discussions emphasizing that personnel must follow laboratory protocol.
Rick has a poor attitude; it is not Rick’s responsibility to make an independent decision
about the severity of the patient’s condition; the goal should be to provide the best
patient care possible, etc.
Case Study 3
Two different organisms normally found on skin were isolated from a blood culture. No
pathogenic organisms were isolated. Choose from the answers below for the most likely reason(s)
for the culture results.
A. The phlebotomist used sterile gloves instead of plain vinyl gloves.
B. The phlebotomist cleaned the septum of the culture bottle with alcohol followed by iodine.
C. The venipuncture site was cleansed using an alcohol swab.
D. All of the above.
Lesson 7-2 ● Bacteriology Specimen Collection and Processing 359
STUDENT ACTIVITIES

2. Design a patient instruction card explaining how to collect a stool specimen for culture.
Web Activities
1. Search the Internet for infections and complications (other than mentioned in this lesson)
caused by S. pyogenes and prepare a brief report on them.
2. Search the Internet for sputum collection systems for mycobacteria. Choose two and report
on their use.

1. What can cause contamination of a throat swab during collection?
The swab can become contaminated with normal flora of the mouth if the swab touches
the tongue or the inside of the mouth.
2. What mistakes can occur in the collection of urine for culture?

Failure to correctly collect urine for culture (midstream and clean-catch specimen) can
introduce mucus, epithelial cells, and bacterial contaminants from skin into the urine. If
the label is placed on the lid (instead of the side) of the urine container, the lid could be
accidentally discarded or switched to another container.
3. Explain why stool specimens collected in an infant’s disposable diaper may not grow
microorganisms.
Disposable diapers can contain bacteriostatic chemicals that inhibit recovery of
microorganisms from the specimen.
4. Why should the patient rinse the mouth with water before collecting a sputum specimen?
Rinsing the mouth with water reduces the normal flora of the mouth that could
contaminate the sputum sample.
5. Why is an aspirate of a wound preferred over a swab?

Material aspirated from the wound is preferred to a swab of the wound because the
swab is more prone to drying and may not pick up sufficient bacteria for culture.
6. Why are both alcohol and an iodine preparation used in the collection of blood cultures?
Alcohol is used first to remove dirt and skin oils. Iodine is used to sterilize the site as
much as possible to prevent contamination of the blood culture with skin bacteria.
7. Discuss why wound specimens are usually sent to regional reference or hospital
laboratories.
Most small laboratories send wound specimens to regional or reference laboratories
instead of processing the specimens because a wide variety of microorganism(s) can
cause wound infections, requiring several types of media to be kept on hand as well as
having varying growth environments available to incubate cultures.
8. List three important properties of transport systems for bacteriology specimens.

Any three are acceptable:
Transport systems must:
a. Enclose the specimen to protect it from contamination
b. Protect the transporter and receiving personnel from contamination
c. Be labeled as containing biohazard material
d. Enclose the inner specimen container in an outer container
e. Be of a design approved for transport by the carrier
f. Contain media, nutrients and an environment to support viability of
microorganisms until the specimen can be transferred to culture media
9. What culture can be performed to determine if a person is a carrier of methicillin-resistant

Staphylococcus aureus (MRSA)?
Nasal culture
10. Define carrier, culture, culture medium, Escherichia coli, sepsis, septicemia, Streptococcus
pyogenes, transport medium, virulent, and wound.
See Glossary.
LESSON 7-3
Culture Techniques for
Bacteriology
LESSON OBJECTIVES
• Explain the use of aseptic technique in bacteriology.
• Explain the differences between antiseptics and disinfectants and discuss how each
is used.
• Describe the different types of biological safety cabinets.
• Explain the differences in primary, selective, and indicator media.
• Describe how to inoculate different forms of media.
• Demonstrate the method for inoculating an agar plate and streaking for isolated
colonies.
• Discuss why different incubation conditions are required for growing cultures of
bacterial pathogens.
• Discuss safety precautions that must be observed when performing bacterial culture
techniques.
• Explain the quality assessment procedures involved in culturing bacteria.
GLOSSARY
agar / a seaweed derivative used to solidify microbiological media
antiseptic / a chemical used on living tissues to control the growth of infectious agents
aseptic technique / work practices used to prevent contamination when working with
microorganisms
disinfectant / a chemical used on inanimate objects to kill or inactivate microbes
enriched medium/ a medium that contains nutrients to support a wide variety of organisms,
including fastidious organisms
hemolysis / the rupture or destruction of red blood cells resulting in the release of hemoglobin
HEPA filter / high-efficiency particulate air filter used in biological safety cabinets
362 Lesson 7-3 ● Culture Techniques for Bacteriology
indicator medium / a bacteriological medium that differentiates bacteria on the basis of certain
chemical reactions; differential medium
inoculating loop / an instrument used to transfer bacteria
inoculation / the process of transferring microorganisms to a growth medium
inoculum / the microorganisms transferred from one medium to another
isolated colonies / bacterial colonies growing on an agar surface and not touched by other
colonies
primary medium / a medium that provides nutritional requirements for an microorganism and is
used to recover the microorganism from a specimen
quadrant / one-fourth of a circle; one-fourth of an agar plate
selective medium / a bacteriological medium that contains chemicals or substances that allow
growth of some organisms while inhibiting growth of others
sterilization / the act of eliminating all living microorganisms from an article or area

• Examples of media in culture plates and agar slant tubes
• Examples of reusable and disposable inoculating loops
• Electric incinerator for sterilizing loops
• Bacterial cultures growing on agar
• Student Performance Guide
LESSON CONTENT
I. Introduction
A. Basic Techniques Must Be Mastered
B. Safe Work Practices
C. Selection and Use of Media
D. Knowledge of Culture Techniques
II. Aseptic Technique
A. Physical Barriers
1. Protective clothing
2. Safe use of equipment
a. Sterilizing loops
b. Avoiding creation of aerosols
Lesson 7-3 ● Culture Techniques for Bacteriology 363
3. Microbiological safety cabinets

a. Class II
b. HEPA filter
B. Disinfectants and Antiseptics
1. Disinfectants—surfaces
2. Antiseptics—skin
C. Sterilization
1. Autoclave
2. Chemical or gas sterilization
III. Growth Media for Clinical Bacteriology
A. Primary Media
1. Blood agar
2. Enriched medium
B. Selective Media
1. EMB
2. MacConkey’s
3. Hektoen
C. Indicator Media
1. EMB, selective and indicator
2. MAC, selective and indicator
D. Anaerobic Media
IV. Culture Techniques
A. Safety Precautions
1. Standard Precautions
2. Personal protective equipment
3. Aseptic technique
4. Frequent handwashing
5. Surface disinfection
6. Autoclave
B. Quality Assessment
1. Internal quality assessment program
2. External quality assessment program
C. Collecting and Transporting Specimens
D. Inoculating the Media
1. Agar plates
a. Inoculating the plate
b. Streaking for isolated colonies
2. Agar slant
3. Indicator or selective medium
E. Incubating the Inoculated Media
1. Temperature
2. Oxygen/carbon dioxide requirements
a. Aerobic
b. Anaerobic
c. Microaerophilic
d. Increased CO2
F. Observing the Culture after 24 Hours
1. Isolated colonies
2. Colony characteristics
3. Hemolysis
V. Safety Reminders
VI. Procedural Reminders
VII. Critical Thinking Problem
VIII. Summary
Critical Thinking Problem and Answers

Bill’s work assignments in bacteriology included wiping down work surfaces and disposing of
all contaminated materials and cultures appropriately. He used a surface disinfectant to wipe the
counters every hour during the work day. Several times during each shift he gathered all the used
culture plates and other contaminated supplies into biohazard bags. After closing the bags
securely, he placed them alongside the other trash to be picked up by housekeeping services.
1. Should Bill be commended for ensuring the safety of everyone working in the laboratory?
No, Bill’s safety practices were only partially acceptable.
2. Explain your answer.

Bill has done a good job of disinfecting the work counters and providing an uncluttered
work environment by keeping the used culture plates and supplies picked up and out of
the way of other workers. However, he should not have placed the filled biohazard bags
with the other trash. Contaminated materials, including culture materials, must never
be included with general trash. The materials must be decontaminated and disposed of
according to the facility’s protocol. This might be done by autoclaving before disposal,
by incineration, or by using a medical waste disposal service.
STUDENT ACTIVITIES

2. Inquire about the types of disinfectants used in local POLs.
3. Practice using aseptic technique to inoculate and streak an agar plate, as outlined in the
Student Performance Guide.
Web Activities
1. Search the Internet for disinfectants appropriate for the microbiology laboratory. Select three
and make a table including characteristics, ingredients, and classes of microbes for which
they are effective.
2. Search the Internet for information about vancomycin-resistant Enterococcus (VRE). Report
on the Gram stain reaction and one condition caused by VRE.
3. Examine the labels of two household disinfectants. Use the Internet to compare their active
ingredients with those of disinfectants used in healthcare settings. Find out if household
disinfectants are bactericidal or bacteriostatic.

1. Explain why the use of aseptic technique is important in the bacteriology laboratory.
In the bacteriology laboratory, aseptic technique is a set of work practices that ensures
safety and quality of work. Aseptic technique is used to prevent (1) workers from
becoming infected with bacteria from patient specimens or cultures; (2) contamination
of work surfaces; (3) cross-contamination of cultures; and (4) contamination of
cultures with environmental bacteria.
2. What are the differences between disinfectants and antiseptics?

Disinfectants kill or control growth of microorganisms on inanimate objects or
surfaces; antiseptics control growth of microorganisms on living tissue.
3. What are two types of safety cabinets used in microbiology?

Two types of safety cabinets used in microbiology are Class I and Class II. Class I
protects the worker and environment from contamination; Class II incorporates a
HEPA filter to protect both the worker and the culture from contamination.
4. What are the purposes of primary, selective, and indicator media?

The purpose of a primary medium is to provide necessary growth requirements of the
specimen; the primary medium is chosen according to the type of specimen being
cultured. Selective media inhibit the growth of certain bacteria and allow the growth
of others. Indicator media visually indicate biochemical reactions of bacteria. Some
media combine selective and indicator functions. See Table 7-17.
5. Describe how to inoculate an agar plate using a swab.

Obtain a specimen swab; open the agar plate just enough to admit the swab; gently
roll the swab over the surface of one quadrant of the plate (return specimen swab to
transport container); and then streak for isolation using a sterile loop.
(See Figure 7-19A.)
6. Describe how to streak an agar plate for isolated colonies.

The inoculum is applied to the first quadrant of an agar plate using a swab or loop; a
sterile loop is then used to make six to eight streaks from the first quadrant into the
second quadrant, crossing the inoculated area in the first quadrant each time; the third
quadrant is streaked in the same manner; to streak the fourth quadrant, the loop is
used to make one streak from inside the third quadrant into the fourth, and this streak
is continued in a pattern to produce isolated colonies. The loop is sterilized and cooled
after the streaking of each quadrant. (See Figure 7-20.)
7. Describe how to transfer an inoculum from an agar plate to an agar slant.

The tube containing the slant is held in one hand while the fourth and fifth fingers of
the hand holding the inoculating loop are used to remove and hold the cap from the
tube; the sterile loop is used to pick up an isolated colony from the agar plate; the loop
is then inserted into the tube and used to make a zigzag pattern on the agar slant; the
loop is withdrawn; the rim of the tube is sterilized and the cap is replaced on the tube.
(See Figure 7-21.)
8. What are important colony characteristics that can be observed?

Important colony characteristics include colony size, shape, and color; some bacteria
also produce a distinctive odor.
9. What hemolytic reactions of bacteria can be used to identify the organism?

Three types of hemolysis are alpha (incomplete hemolysis giving agar a greenish
color), beta (complete hemolysis), and gamma (none). Because only certain bacteria
are hemolytic, this can be an important factor in identification. (See Figure 7-23.)
10. What medium is used to indicate hemolysis?

Blood agar
11. Why are transport media used?

A transport medium provides the nutrients and environment necessary for
microorganisms in a patient specimen to remain viable until the specimen can be
inoculated onto growth media. Transport supplies are sterile and include ingredients
that will protect microorganisms from drying for several hours. Transport media are
available to meet the growth requirements of different types of microorganisms,
including aerobic and anaerobic.
12. Discuss quality assessment programs for the bacteriology laboratory.

A QA program is important in the bacteriology laboratory to help ensure reliable
culture results. The QA program should include checking the quality of all new lots of
media and reagents, subscribing to a proficiency program, and keeping performance
records of equipment, such as incubator and refrigerators.
13. What safety techniques must be used in the bacteriology laboratory?

Technicians in the bacteriology laboratory must observe Standard Precautions,
keeping in mind that the specimens they handle can contain not only bacteria, but also
other potentially infectious material (OPIM). Frequent handwashing and donning
clean gloves are important when processing specimens. Other PPE should be used as
deemed necessary. Aseptic technique, physical methods of preventing contamination,
and chemical disinfection methods must be combined to ensure the safety of everyone
working in the bacteriology laboratory.
14. Define agar, antiseptic, aseptic technique, disinfectant, enriched medium, hemolysis, HEPA
filter, indicator medium, inoculating loop, inoculation, inoculum, isolated colonies, primary
medium, quadrant, selective medium, and sterilization.
See Glossary.
LESSON 7-4
The Gram Stain
LESSON OBJECTIVES
• Explain the principle of the Gram stain.
• Prepare smears of bacteria from a swab and from a bacterial culture.
• Perform the Gram stain procedure.
• Identify gram-positive organisms on a smear.
• Identify gram-negative organisms on a smear.
• List the safety precautions to be observed when performing the Gram stain.
• Discuss quality assessment procedures that must be a part of preparing smears and
performing Gram stains.
GLOSSARY
bibulous paper / a special absorbent paper used to dry slides; blotting paper
counterstain / a dye that adds a contrasting color
mordant / a substance that fixes a dye or stain to an object

• Gram-stained bacterial smears, bacteriology atlas, and digital images or other visuals
showing gram-positive and gram-negative bacteria
• Gram stain kit
• Images (digital, transparencies or overheads) of Figures 7-24 through 7-29
• Image (digital, transparency or overhead) of Table 7-18
Lesson 7-4 ● The Gram Stain 369
LESSON CONTENT
I. Introduction
A. Prepare Smear from Culture or Specimen Swab
B. Gram Stain Reveals Bacterial Morphology and Gram Reaction
C. Important to Bacterial Identification and Antibiotic Susceptibility
II. Preparing the Bacterial Smear
1. Use aseptic technique
2. Wear appropriate PPE
3. Clean work area frequently with disinfectant
4. Decontaminate materials before disposal
1. Use control slides with Gram stain
2. Heat-fix slides carefully
3. Time Gram stain procedure carefully
C. Preparing a Smear from a Swab
1. Inoculate media before making smear
2. Use clean slide (95% ethanol)
3. Roll swab gently across slide
D. Preparing a Smear from a Culture
1. Using an agar slant
2. Using an agar plate
E. Heat-Fixing the Smear
1. Smear must be dry
2. Affixes bacteria to slide
3. Avoid excessive heating
III. The Gram Stain
A. Performing the Gram Stain
1. Primary stain
a. Stain 1 minute in crystal violet
b. Rinse with water
2. Gram’s iodine
a. Iodine is a mordant
b. Apply for 1 minute and rinse
370 Lesson 7-4 ● The Gram Stain
3. Decolorizer
a. Alcohol or acetone-alcohol mixture
b. Crystal violet washed out of gram-negative cells
c. Apply for only a few seconds and rinse immediately
4. Counterstain
a. Stain 1 minute in safranin
b. Rinse slide and blot dry
IV. Observing the Stained Bacteriological Smear
A. Oil-immersion objective
B. Observe Gram reaction
1. Gram-positives are purple
2. Gram-negatives are pink
C. Observe morphology
1. Rod, coccus, or coccobacillus
2. Growth patterns (clusters, chains, etc.)
V. Safety Reminders
VI. Procedural Reminders
VII. Critical Thinking Problem
VIII. Summary
Critical Thinking Problem and Answers

Marion made a smear for a Gram stain and left it to dry while she went to lunch. While she was
gone Christine had extra time, so she stained it. When she examined it under the microscope
bacteria were scarce and large clear areas were present on the slide. Christine prepared another
smear, let it dry, heat-fixed it, and stained it. She had no problem observing the morphology of
the bacteria present on the second slide.
1. Which of the following is the most likely cause of the problem with the first smear/stain?
a. Failing to heat-fix the smear
b. Rubbing the swab across the slide
c. Leaving decolorizer on too long
d. Failing to sterilize the loop
2. Which is most likely the problem—Marion’s smear technique or Christine’s processing of

the slide? Explain.
Christine’s processing of the slide is most likely the problem. The first slide was not
heat-fixed before Christine stained it, so the majority of the bacteria were washed away
in the staining procedure. Christine should have asked if the slide had been heat-fixed
before proceeding with the Gram stain.
Lesson 7-4 ● The Gram Stain 371
STUDENT ACTIVITIES
2. Obtain Gram-stained bacterial smears and practice identifying bacterial morphology and
Gram-stain reactions using the microscope.
3. Practice preparing bacteriological smears, performing the Gram stain, and identifying gram-
negative and gram-positive organisms as outlined in the Student Performance Guide.
4. Prepare duplicate smears from a culture of gram-positive organisms. Stain the smears
following the Gram stain procedure. Decolorize one smear for the normal time and the other
for 30 seconds. Examine the smears microscopically, compare the staining results, and
discuss your findings.
Web Activities
1. Use the Internet to find tutorials demonstrating how to perform a Gram stain.
2. Use the Internet to find the morphology and Gram reactions of Clostridium difficile and
Haemophilus.

1. Discuss the use of Standard Precautions and aseptic technique in the bacteriology laboratory.
The goal of Standard Precautions is to protect laboratory personnel from exposure to
microbes that might be present in a body fluid, tissue, or other specimen that is being
cultured. The use of aseptic technique protects the worker, the environment, and
coworkers from exposure to bacteria in a patient specimen or a culture; it also protects
the culture or specimen from contaminants in the environment.
2. Why is it necessary to wear a laboratory coat when working with bacteria?

A laboratory coat can prevent contamination of skin and clothing from spills and
splashes of culture material; it can also protect cultures from being contaminated by
bacteria and cells from skin of workers.
3. Why is it important to gently roll the swab across the slide when preparing a smear?
If the specimen swab is dragged across the slide or rubbed on the slide, bacterial
morphology can be destroyed.
4. Why is the swab used to inoculate the culture medium before it is used to make the smear?
The culture medium is inoculated first to prevent possible contamination of the swab
with microorganisms that might be present on the nonsterile slide and subsequent
transfer of these contaminants to the culture medium.
5. Explain the differences in the procedures for preparing smears from tube cultures and from
agar plate cultures.
The tube culture is not as easily contaminated as the agar plate culture; the mouth of
the tube is sterilized, but no similar procedure can be performed on the agar plate. It is
easier to pick isolated colonies from the surface of the agar plate.
372 Lesson 7-4 ● The Gram Stain
6. Why must the smear for Gram stain be heat-fixed before it is stained?
Heat-fixing causes the material in the smear to adhere to the glass slide, preventing the
material from being washed off in the staining procedure.
7. Explain how to perform a Gram stain.

An air-dried and heat-fixed smear is placed on a staining rack; crystal violet is poured
onto the slide for the prescribed time and then rinsed off with water; Gram’s iodine is
then poured onto the slide for a specific time and rinsed off with water; decolorizer is
applied for a few seconds and the slide is then rinsed with water; the counterstain
(safranin) is applied for a specific time and rinsed off with water; the slide is then
blotted dry with bibulous paper. (See Figure 7-28.)
8. Why are some bacteria gram-positive and others gram-negative?

Gram stain reactions are based on chemical differences in the structures of bacterial cell
walls. The walls of gram-negative cells are chemically more complex than the walls of
gram-positive cells. Both types of bacteria contain peptidoglycan in their cell walls, but
the Gram-negative cell wall contains more lipid, polysaccharide, amino acids, and
lipoprotein complexes than gram-positive cell walls. Both types of cell walls take up the
primary stain, which is crystal violet. The chemical composition of gram-positive cell
walls causes them to retain the crystal violet and resist decolorization. However, the
components in the cell walls of gram-negative bacteria allow them to be decolorized
(lose the crystal violet stain) and be counterstained by taking up the safranin stain.
9. What is the appearance of gram-positive cocci? Gram-negative rods?

Gram-positive cocci are round or spherical and stain a dark blue-purple; gram-negative
rods are rod-shaped, are longer than they are wide, and stain pink-red.
10. Why is it necessary to wipe countertops often with surface disinfectant?

Frequent countertop disinfection eliminates bacteria that might be present from
accidental spills and splashes of culture material or specimens; this prevents
contamination of clothing, skin, and equipment.
11. What QA procedures must be followed when making and staining smears?
Make smears thin enough to be just barely visible before staining; use gentle heat for
heat-fixing to avoid damaging the cells; do not use stains after their expiration date;
follow instructions and timing exactly; use control slides to check that stains are
working properly and to check technician stain technique; keep microscopes in good
working order and lenses clean.
12. Define bibulous paper, counterstain, and mordant.

See Glossary.
LESSON 7-5
Tests for Group A
Streptococcus
LESSON OBJECTIVES
• Discuss the importance of identifying infections caused by group A Streptococcus.
• Collect a pharyngeal (throat) swab.
• Perform a throat culture and interpret the results.
• Use a bacitracin disk to identify group A Streptococcus.
• List two major types of technology used in rapid tests for group A Streptococcus.
• Perform a rapid test for group A Streptococcus.
• Discuss safety precautions that must be observed when performing throat cultures
and rapid tests for group A Streptococcus.
• Discuss quality assessment procedures essential to the performance of throat
cultures and rapid tests for group A Streptococcus.
GLOSSARY
fossae / in the throat, shallow depressions where the tonsils were located before surgical removal
hemolysis / the rupture or destruction of red blood cells resulting in the release of hemoglobin
pharyngeal / having to do with the back of the throat or pharynx

• Recent magazine, newspaper, or journal articles about invasive group A Streptococcus
• Group A Streptococcus growing on a blood agar plate, showing beta-hemolysis
374 Lesson 7-5 ● Tests for Group A Streptococcus
• Gram-stained smears of streptococci

• Examples of rapid tests for group A Streptococcus
• Images (digital, transparencies or overheads) of Tables 7-19 and 7-20
LESSON CONTENT
I. Introduction
A. Frequently Performed
B. Identifies Strep Throat
1. Caused by Streptococcus pyogenes
2. Referred to as Group A Streptococcus
3. Complications can develop if untreated
4. Other Lancefield groups can cause tonsillitis
II. Detecting Group A Streptococcus
A. Confirm by Culture and Rapid Test
B. Safety Precautions
1. Wear appropriate PPE when collecting throat swab
2. Disinfect surfaces frequently
3. Autoclave waste before disposal
4. Wear mask when collecting throat culture swab
C. Quality Assessment
1. Follow SOP manual
2. Check blood agar for ability to demonstrate hemolysis
3. Use appropriate controls with test kits
D. Performing the Throat Culture
1. Collecting the specimen
a. Sterile Dacron swab
b. Swab tonsils or fossae
c. Do not touch tongue or other mouth surfaces
2. Inoculating the media
a. Blood agar
b. Stabs
c. Bacitracin disk (A disk)
Lesson 7-5 ● Tests for Group A Streptococcus 375
3. Incubating the culture

a. Reduced oxygen (5% to 10% CO2) environment
b. CO2 incubator, candle jar, or gas-generating system
4. Reading the throat culture plate
a. Beta-hemolysis
b. Inhibition of growth by bacitracin
c. Antibody tests
E. Performing a Rapid Test for Group A Streptococcus
1. Immunoassays for group A Streptococcus
2. ICON Strep A tests
a. ICON SC Strep A
b. ICON DS Strep A
c. CLIA-waived
d. Specimen used is throat swab
e. Antigen extraction
f. Color end-point
3. Latex agglutination tests
4. Reliability of rapid tests for group A Streptococcus
a. Sensitive and specific for moderate to heavy infections
b. Results depend on thorough swabbing of throat
c. Patient should not eat, drink, or gargle 30 minutes before throat swab
d. Negative test should be confirmed by culture
III. Invasive Group A Streptococcus
A. Necrotizing Fasciitis
B. Caused by Subgroup of Group A Streptococcus
IV. Safety Reminders
V. Procedural Reminders
VI. Critical Thinking Problems
VII. Summary
Critical Thinking Problems and Answers
Critical Thinking 1
Sue was working in the campus health clinic when Tony came in to have a throat swab
performed. His healthcare provider had ordered a rapid test for GAS because Tony had a fever
and sore throat. The result from the rapid test was negative, so a throat culture for strep was
ordered. Sue swabbed Tony’s throat, and the healthcare provider gave him a prescription for an
antibiotic. Sue inoculated the first quadrant of the plate using the specimen on the swab. She
disposed of the swab in the appropriate biohazard container, sterilized the loop, and began to
streak the inoculum into the other quadrants. A fellow worker interrupted her, and she laid the
loop down momentarily and then continued streaking the plate. After overnight incubation of the
plate at 35° to 37°C in a CO2 incubator, Tom, a coworker, examined the plate and observed
atypical colonies that did not look like strep. He performed a Gram stain and saw some gram-
positive bacilli, similar in appearance to those commonly found on environmental surfaces.
1. What could be the source of gram-positive bacilli?
The loop probably became contaminated with environmental bacteria from the
countertop when Sue laid down the loop. These environmental bacteria can grow on
blood agar.
2. Were mistakes made in the laboratory procedures? What were they?

Yes, the inoculating loop should never be laid down on a counter but should be placed
in a loop holder. The loop should have been sterilized again before Sue continued
streaking the plate.
3. Which laboratory technician needs a refresher course in culture techniques?

Sue
4. Since the rapid strep test was negative, was the throat culture necessary?
Yes, negative results in a rapid GAS test should be confirmed by throat culture.
Critical Thinking 2
Maria worked the day shift in bacteriology. One of her first responsibilities each day was to
examine cultures that had been incubating overnight. She saw that both throat and urine cultures
had been set up on specimens from Mrs. Cheng. Maria pulled the plates to read the two cultures
and found that the blood agar plate for the throat culture and the blood agar and MAC urine
culture plates were all in the 37°C aerobic incubator. The blood agar throat culture plate had
scant growth.
Should the results be reported? Explain your answer.
The urine culture results can be reported because the culture plates were incubated
correctly. The results of the throat culture cannot be reported because the blood agar plate
was incubated incorrectly. The throat culture plate should have been incubated in 5% to
10% CO2 to enhance growth of any group A Streptococcus that have been present in the
throat specimen.
STUDENT ACTIVITIES

2. Survey local clinics or physician office laboratories (POLs) to find out which rapid tests for
GAS are used and what procedure is followed when a rapid test is negative.
3. Practice performing a throat culture, as outlined in the Student Performance Guide.
4. Practice performing rapid tests for GAS, as outlined in the Student Performance Guide.
Lesson 7-5 ● Tests for Group A Streptococcus 377
Web Activities
1. Use the Internet to find two additional rapid strep tests not mentioned in this lesson. Report
on your findings, including information such as test principle, test sensitivity and specificity,
and CLIA complexity level.
2. Use the Internet to research a complication that can result from an untreated strep infection.
3. Search the Internet for a video demonstrating the procedures for collecting a throat swab and
streaking a throat culture plate.

1. Why is early diagnosis of group A Streptococcus (GAS) infection important?
Early diagnosis is important because untreated strep infections can have serious
complications.
2. Explain why the tongue and mouth should not be touched when collecting a throat swab for
strep.
The tongue and mouth surfaces are inhabited by a variety of normal flora that can
grow on the medium and contaminate the culture, interfering with culture results and
test interpretation.
3. Why is a throat culture inoculated to blood agar?

Blood agar provides the nutrients required by most throat pathogens and also
demonstrates the presence of hemolysis, a characteristic of GAS.
4. What is the purpose of the bacitracin disk?

The bacitracin disk is used to help identify GAS. Bacitracin inhibits growth of GAS;
this is demonstrated by the presence of a zone of inhibition (no bacterial growth)
around the disk.
5. What is the reason for making stabs in the agar?

Oxygen concentration is reduced below the surface of the agar, a condition favorable to
growth of GAS, and to demonstration of beta-hemolysis.
6. Explain the different types of hemolysis.

In beta-hemolysis, the red cells are completely lysed and the agar is almost clear; alpha-
hemolysis produces a green discoloration in the agar; gamma hemolysis refers to
absence of hemolysis.
7. Explain the principles of agglutination tests and rapid immunoassay tests for GAS.
Agglutination tests are performed by adding latex beads coated with anti-strep A
antibodies to a mixture containing the organism to be tested (antigen); a positive result
is demonstrated by agglutination. In the rapid immunoassay tests, the anti-strep A
antibody is coated on a test membrane contained in a test cassette. Antigen present on
the throat swab is extracted, and the extract is added to the membrane. Any strep A
antigen present in the extract binds to the antibody on the membrane and produces a
positive reaction, usually a colored line.
8. Explain the possible consequence of a false-negative result in a rapid strep A test.

A possible consequence of a false-negative result in a rapid strep A test is that the
patient might not receive treatment for the infection and can possibly develop
complications of GAS infection. For this reason, laboratory policy should be to follow
up all negative rapid strep A tests with a throat culture.
9. List four safety procedures that must be observed when collecting or processing throat
culture swabs.
Any four of the following are acceptable:
a. Observe Standard Precautions.
b. Wear appropriate PPE.
c. Treat used swabs as if infectious.
d. Wipe work area frequently with disinfectant.
e. Wear mask and face protection when collecting the throat swab if patient is
coughing excessively.
f. Autoclave all specimens and culture materials before disposal
10. What QA procedures are important when testing for GAS?

Quality assessment (QA) programs must be in place to ensure reliability of media and
test kits. Each new lot of swabs should be tested with stock cultures and rapid strep
kits to be sure the swabs support viability of group A strep and do not interfere with
the rapid tests, causing false reactions. Each lot of blood agar media should be
inoculated with a known culture of GAS to check for support of growth and
demonstration of hemolysis.
11. Define fossae, hemolysis, and pharyngeal.

See Glossary.
LESSON 7-6
Urine Culture and Colony
Count
LESSON OBJECTIVES
• Discuss the reasons why a urine culture and colony count might be requested.
• Select the correct primary medium and indicator medium for a urine culture.
• Demonstrate the streaking technique for urine colony count.
• Perform a colony count on a urine culture.
• Discuss the medical significance of urine culture results.
• Name four bacteria that are common causes of urinary tract infections.
• Discuss aspects of quality assessment that must be considered when performing
urine culture and colony count.
• Describe the safety precautions to observe when performing urine culture and
colony count.
GLOSSARY
colony count / an estimation of the number of bacteria in 1 mL of urine made by counting the
colonies on a urine culture plate

• Calibrated loops, disposable and reusable
• Urine culture plates with bacterial colonies
• Images (digital, transparencies or overheads) of Figures 7-35 and 7-36
380 Lesson 7-6 ● Urine Culture and Colony Count
LESSON CONTENT
I. Introduction
A. Detects Urinary Tract Infection (UTI)
B. Symptoms of UTI
1. Frequent urination
2. Pain and burning during urination
3. Blood in urine
4. Backache
C. Gram-Negative Bacteria
1. Escherichia coli most common
2. Pseudomonas, Proteus, and Klebsiella also cause UTIs
D. Gram-Positive Coccus—Staphylococcus saprophyticus
II. Performing the Urine Culture
C. Collecting the Specimen
1. Clean-catch
2. Label container, not lid
3. Inoculate to culture media within 1 hour of collection
D. Streaking the Plates
1. Inoculate blood agar
a. Primary medium
b. Supports growth of most urinary tract pathogens
2. Inoculate indicator/selective medium such as MAC or EMB
a. Inhibit growth of gram-positive bacteria
b. Indicate chemical reactions of gram-negative bacteria
3. Use calibrated loop, 0.01mL or 0.001 mL
4. Make vertical and horizontal streaks
5. Incubate overnight
E. Colony Count and Interpretation
1. Colony count
a. Observe plates for growth
b. Note morphology and hemolysis, if any
c. Count colonies on blood agar plate
d. Calculate number of colonies per milliliter of urine
2. Interpretation
Lesson 7-6 ● Urine Culture and Colony Count 381
III. Safety Reminders

IV. Procedural Reminders
V. Case Study
VI. Critical Thinking
VII. Summary
Case Studies and Critical Thinking Problem and Answers
Case Study 1
Shawna was working in a physician office laboratory (POL) when a urine dipstick result
indicated presence of blood and WBCs in the urine she was testing. Laboratory protocol
specified that a urine culture and sensitivity (C & S) should be set up. Shawna obtained a blood
agar plate from stock. On the bottom of the plate she placed the patient's barcode label and
streaked the plate for colony count. She then placed the culture plate into the incubator to be read
the next morning.
1. Was the urine culture set up correctly?
No, (1) the urine culture should have been on a clean-catch urine; (2) culture should be
set up from clean-catch urine before non-sterile tests are performed which could
introduce contaminants; and (3) an indicator plate such as EMB or MAC should also
have been inoculated.
2. Does blood agar support the growth of both gram-positive and gram-negative organisms?
Yes, BA supports growth of both.
Case Study 2
Tina is a technician in a small hospital laboratory. She works in the hematology, chemistry, and
microbiology sections. On this morning, she was in microbiology examining culture plates that
had been set up the previous day. She examined BA and MAC plates set up for a urine culture.
Both plates had many colonies growing, and the colonies on each plate appeared to have the
same morphology. Tina counted 132 colonies on the MAC plate. From the requisition slip, she
saw that the person who set the culture up had used a 0.001-mL loop. Tina reported a colony
count of 13,200/mL for the culture.
1. Evaluate Tina’s performance in evaluating the urine culture.
Tina made some mistakes. First, she should have counted colonies from the blood agar
plate rather than from the MAC plate. Second, if 132 colonies were counted and the
culture was set up using a 0.001 mL loop, the count should have been calculated to be
132,000/mL.
2. Does the patient have a urinary tract infection?

Colony counts of 100,000/mL or greater are evidence of UTI, even in the absence of
symptoms.
382 Lesson 7-6 ● Urine Culture and Colony Count
Critical Thinking Problem

Mrs. Miller went to her healthcare provider complaining of frequent, painful urination
accompanied by a burning sensation, symptoms she had never had before. The provider ordered
a routine urinalysis and a urine culture and colony count. In the laboratory, Mrs. Miller gave the
laboratory order to John, who handed her a urine collection cup just as the phone rang. Mrs.
Miller took the cup to the restroom and brought back a urine specimen, with her ID label on the
cup. John set up the urine culture and gave the urine specimen to Timothy, the other technician.
Timothy performed a routine urinalysis including microscopic analysis of the urine sediment and
observed 5 to 10 epithelial cells/low power field, mucus threads, and many bacteria. The next
morning the BA culture plate had at least four different colony types growing on it.
1. Should the laboratory try to identify all four isolates?
No.
2. What mistake(s) affected the routine urinalysis and culture?

Mrs. Miller should have been given a urine culture collection kit and instructions on
how to collect a midstream clean-catch urine for culture.
STUDENT ACTIVITIES

2. Practice performing the urine culture and colony count, as outlined in the Student
Performance Guide.
Web Activities
1. Use the Internet to find information on cystitis. Report on the complications that can occur if
cystitis is untreated.
2. Search the Internet for tutorials or videos demonstrating how to streak urine culture plates.

1. Why must clean-catch urine be used for urine culture?
The clean-catch urine must be used for culture to ensure that only the bacteria
responsible for the infection grow in the culture. (The clean-catch method cleanses the
urethral opening to remove epithelial cells, mucus, and skin bacteria.)
2. What types of media are used for urine culture?

A primary medium—blood agar--and indicator/selective medium such as EMB or
MAC are used for urine culture.
3. Which culture plate is used to make the colony count?

The blood agar plate
Lesson 7-6 ● Urine Culture and Colony Count 383
4. Describe how to streak the BA plate for a colony count.

A calibrated, sterile loop (disposable or reusable) is dipped into the well-mixed urine;
the loop is inspected to be sure it is filled with urine, and then one continuous vertical
streak is made down the center of the surface of the BA; the loop is then used to make
15 to 20 horizontal streaks, crossing the vertical streak each time. The plate is turned
90 degrees and another set of 15 to 20 horizontal streaks is made (see Figure 7-35).
5. How does the loop used for streaking affect the colony count?
The size of loop used must be known in order to calculate the colony count, which is
reported as colonies per milliliter. If the loop capacity is 0.01 mL, the number of
colonies counted is multiplied by 100; if the loop capacity is 0.001 mL, the colony
number is multiplied by 1,000.
6. What measure(s) should be used in the urine culture procedure to increase safety?
Standard Precautions must be observed and appropriate PPE should be worn.
Formation of aerosols must be avoided when sterilizing the loop or mixing or
disposing of urine samples. All surfaces must be wiped frequently with surface
disinfectant. Hands must be washed with antiseptic after removing gloves and anytime
hands become contaminated. All contaminated materials must be disposed of
appropriately.
7. Why is it important to perform quality assessment procedures on new shipments of media?

How is this done?
A plate or tube from each new lot of media must be checked for sterility and for ability
to support bacterial growth. This is done by incubating one item from each lot at 37°C
and examining for growth of contaminants and inoculating one item from each lot
with a known organism to be sure the medium can support growth of the
microorganisms that cause UTIs.
8. What are the symptoms of UTIs?

Symptoms include frequent urination, pain and burning during urination, blood in the
urine, and sometimes fever and backache.
9. What bacterial species is the most common cause of UTIs?

The most common microorganism responsible for UTI is Escherichia coli, a gram-
negative rod that is part of the normal flora of the intestinal tract.
10. Define colony count.

See Glossary.
LESSON 7-7
Bacterial Identification
and Antibiotic
Susceptibility Testing
LESSON OBJECTIVES
• Describe general steps followed to identify gram-positive or gram-negative bacteria
from culture.
• Use the microscope to identify Gram stain reactions of bacteria.
• Explain the purpose of the catalase and coagulase tests.
• Perform the catalase test and interpret the results.
• Perform the coagulase test and interpret the results.
• Explain how to report the presumptive identification of gram-positive and gram-
negative bacteria.
• Discuss the use of manual and automated identification systems for bacteria.
• Perform the disk antibiotic susceptibility test and interpret the results.
• Discuss the safety precautions that must be observed when performing bacterial
identification and antibiotic susceptibility tests.
• Discuss quality assessment procedures that must be followed when performing
bacterial identification and antibiotic susceptibility tests.
GLOSSARY
catalase test / a test to differentiate between Streptococcus and Staphylococcus species

coagulase test / a test to differentiate between Staphylococcus aureus and other Staphylococcus
species
Lesson 7-7 ● Bacterial Identification and Antibiotic Susceptibility Testing 385
coliform / referring to certain fermentative gram-negative enteric bacteria including Escherichia

coli, Enterobacter, and Klebsiella
minimum inhibitory concentration (MIC) / minimum concentration of an antibiotic required
to inhibit the growth of a microorganism
pleomorphic / having varied shapes
zone of inhibition / in the Bauer-Kirby antibiotic susceptibility test, the area around an antibiotic
disk in which bacterial growth is inhibited

• Examples of bacterial identification systems
• Examples of antibiotic disks and disk dispensers; MIC plate; Mueller-Hinton plate;
McFarland’s standards; ruler or template for measuring zones of inhibition
• Atlases containing images of bacterial colonial morphology and fermentation reactions
LESSON CONTENT
I. Introduction
II. Bacterial Identification
C. Gram Stain and Primary Media Results
1. Gram stain reveals morphology and gram reaction
2. Pleomorphic bacteria
3. Gram-positives do not grow on selective/indicator medium
D. Identification of Gram-Positive Bacteria
1. Catalase test—differentiates Staphylococcus from Streptococcus
2. Coagulase test—differentiates pathogenic Staphylococcus from other
Staphylococcus species
3. Latex agglutination tests
E. Methicillin-Resistant Staphylococcus aureus (MRSA)
1. Penicillin-resistant strains appeared in 1940s
2. Rapid resistance developed to new antibiotics
a. Methicillin and cloxacillin—new forms of penicillin
386 Lesson 7-7 ● Bacterial Identification and Antibiotic Susceptibility Testing
b. MRSA reported
c. Unexplained drop in MRSA in 1970s
3. 1980s—Re-emergence of resistant strain
4. Some strains resistant to vancomycin
5. Tests for MRSA
6. Increased incidence in long-term care facilities
F. Identification of Gram-Negative Bacteria
G. Manual Bacterial Identification Systems
1. bioMerieux ChromID media
2. API Microbial Identification Strips
3. Enterotube II
H. Automated Bacterial Identification Systems
1. VITEK-2
2. MicroScan Walk-Away
III. Antibiotic Susceptibility Tests
A. Automated and Semi-Automated Methods
B. Manual Methods of Antibiotic Susceptibility
1. Bauer-Kirby Susceptibility Test
a. Performing the Bauer-Kirby Test
b. Interpreting the Bauer-Kirby Test
2. AB Biodisk E (E test)
IV. Safety Reminders
V. Procedural Reminders
VI. Case Study
VII. Summary
Case Study and Answers

It was Natasha’s day to read and interpret the urine cultures in the microbiology laboratory.
When she examined Ms. Gonzalez’s BA and EMB plates, she saw two distinct colony types on
the BA plate and only one type on the EMB plate. Natalie counted 74 colonies total on the BA
plate which had been streaked using a 0.001 mL loop.
1. What is the colony count on this patient’s urine? Explain your answer.
The colony count is 74,000 /mL. The 0.001 mL loop was used so the number of colonies
counted must be multiplied by 1,000 to find the number of colonies in 1 mL of urine.
2. What is the probable gram stain reaction of the colony(ies) growing on the EMB plate?
Explain your answer.
The colonies on the EMB plate are most likely gram-negative, because EMB inhibits
growth of most gram-positive bacteria.
3. Which colonies should be tested for the catalase reaction? Explain.

Natalie saw two different colony types on the blood agar but only one type on the EMB.
One of the two types of colonies on the BA plate is likely a gram-positive organism.
Natalie should perform a Gram stain on one colony of each type. If the colony type that
gives a gram-positive reaction is also a coccus (morphology revealed by the Gram
stain), then the catalase test can be performed on one of those colonies to differentiate
Staphylococcus from Streptococcus.
STUDENT ACTIVITIES

2. Practice performing bacterial identification and antibiotic susceptibility tests as outlined in
the Student Performance Guide.
Web Activities
1. Use the Internet to find recent information on microbial resistance to commonly prescribed
antibiotics such as the Zithromax Z Pack, penicillin, or amoxicillin.
2. Search the Internet for information about recent revelations of resistant bacteria, including
VRE.
3. Use the Internet to search a web site such as WebMD or Lab Tests Online; find the names of
two antibiotics useful for treatment of gram-negative infections with E. coli and
Pseudomonas aeruginosa.
4. Use the Internet to find two antibiotics used in antibiotic susceptibility tests for both gram-
negative rods and gram-positive cocci.

1. What is known about a culture that grows on both blood agar and EMB?
A culture that grows on both BA and EMB is most likely a gram-negative rod.
2. Why is a correctly performed Gram stain important to the process of identifying bacteria?
Microscopic examination of a Gram-stained colony reveals the gram reaction and the
bacterial morphology. Growth characteristics can also be observed. Gram stain results
help determine which biochemical tests are needed to aid in identification of the
bacteria, because different test methods are used to identify gram-positive and gram-
negative bacteria.
388 Lesson 7-7 ● Bacterial Identification and Antibiotic Susceptibility Testing
3. How is the MIC determined?

The minimum inhibitory concentration (MIC) is determined by exposing a
standardized suspension of bacteria to varying (decreasing) concentrations of an
antibiotic in a microtiter plate. The lowest concentration of antibiotic that inhibits
growth of the bacteria is the MIC.
4. In the Bauer-Kirby susceptibility test, what is indicated by the presence of a large clear area
around an antibiotic disk?
A large clear area (no bacterial growth) around an antibiotic disk is called a zone of
inhibition and indicates that the particular antibiotic can inhibit growth of the bacteria.
5. Discuss how to presumptively identify bacteria.

Presumptive identification of bacteria can be made by (1) observing growth and colony
characteristics on the BA and the selective or indicator (EMB or MAC) medium and
(2) observing bacterial morphology and Gram stain reaction from a stained smear.
Definitive identification of bacteria is made using more specific methods such as
biochemical reactions.
6. How is the catalase test performed?

Catalase is an enzyme possessed by some bacteria; it breaks down hydrogen peroxide
into H2O and O2, causing bubbles to form as the O2 is released. The catalase test is
performed by placing a small portion of a colony in a drop of hydrogen peroxide on a
slide and observing for the development of bubbles, a positive test. The catalase test is
used to distinguish between streptococci (catalase-negative) and staphylococci
(catalase-positive).
7. How is the coagulase test performed?

The coagulase test detects the ability of certain organisms to cause clumping or clotting
of plasma. Pathogenic staphyococci produce a positive coagulase test. A portion of a
colony is inoculated into a tube of coagulase plasma, incubated, and observed for
thickening of the plasma. A positive coagulase result indicates that the organism is a
pathogenic Staphylococcus and is presumptive identification of Staphylococcus aureus.
8. List two automated methods used to identify bacteria.

Any two of the following are acceptable:
a. VITEK-2
b. MicroScan Walk-Away
c. Phoenix
9. Give an example of a manual method of identifying bacteria using biochemical reactions.

Manual identification methods include API strips, bioMérieux ChromID Media, and
Enterotube II.
10. Explain how to set up a disk antibiotic susceptibility test.

A Mueller-Hinton agar plate is labeled on the bottom with the patient identification. A
standardized suspension of the bacterial culture is streaked onto the surface of the
agar with a swab: Starting at the 12 o’clock position, the plate is streaked continuously
in horizontal streaks from the top to bottom of the plate. The plate is turned 90
degrees and is again streaked continuously from top to bottom, crossing the original
streaks. Antibiotic disks are then applied to the surface of the agar and tamped down
to be sure they adhere to the agar surface. The plate is incubated upside down
overnight.
11. How are the zones of inhibition around the disks measured in the Bauer-Kirby test?
After overnight incubation, the Mueller-Hinton plate is placed upside down on the
work surface. The zones are measured through the bottom of the plate using calipers,
a ruler, or a transparent template made especially for this purpose. The zone size is
compared to information on the template or in the package insert to determine if the
organism is resistant or sensitive to the antibiotic.
12. Define catalase test, coagulase test, coliform, minimum inhibitory concentration,
pleomorphic, and zone of inhibition.
See Glossary.
LESSON 7-8
Tests for Sexually
Transmitted Diseases
LESSON OBJECTIVES
• Explain the importance of detecting sexually transmitted diseases (STDs).
• Discuss the incidence of certain STDs in the United States.
• Discuss four basic types of tests used to detect STDs.
• List five STDs common in the United States.
• Name one method of detection for each of the five common STDs.
• Explain the three-slide test for females and list the types of microorganisms that can
be detected.
• Explain the special culture conditions for the growth of Neisseria gonorrhoeae.
• Explain the safety precautions that must be followed when performing tests for
STDs.
• Discuss the quality assessment procedures involved in testing for STDs.
GLOSSARY
Candida albicans / yeast that causes vaginitis and other infections, especially following
antibiotic therapy
Chlamydia trachomatis / species of gram-negative intracellular bacteria that is a cause of
sexually transmitted diseases (STDs)
clue cells / vaginal epithelial cells covered with tiny, gram-variable bacteria and seen in vaginal
secretions of patients with bacterial vaginosis
gonorrhea / contagious infection spread by sexual contact and caused by Neisseria gonorrhoeae
herpes simplex virus, type 1 (HSV-1) / the virus causing oral herpes
herpes simplex virus, type 2 (HSV-2) / the virus causing genital herpes
Lesson 7-8 ● Tests for Sexually Transmitted Diseases 391
human immunodeficiency virus (HIV) / the retrovirus that has been identified as the cause of
acquired immunodeficiency syndrome (AIDS)
human papillomavirus (HPV) / a group of DNA viruses, some of which are sexually
transmitted
nongonococcal urethritis / gonorrhea-like STD caused by microorganisms other than gonococci
oxidase test / an enzyme test used to identify certain bacteria such as Neisseria
sexually transmitted disease (STD) / a disease transmitted by sexual contact; sexually
transmitted infection (STI)
sexually transmitted infection (STI) / an infection transmitted by sexual contact; sexually
transmitted disease (STD)
spirochetes / motile, helical or spiral bacteria of the family Spirochaeta
syphilis / an infectious, chronic, sexually transmitted disease caused by a spirochete,
Treponema pallidum
trichomoniasis / a sexually transmitted genitourinary tract infection caused by the parasitic
protozoan, Trichomonas vaginalis
urethritis / infection or inflammation of the urethra
vaginitis / infection or inflammation of the vagina
venereal / having to do with, or transmitted by, sexual contact

• Recent newspaper, magazine, or journal articles discussing the incidence of STDs
• Recent data on STD case numbers from the CDC, state, or county public health department
• Guest speaker from a local public health laboratory, a health educator from a nearby college
or university, or an employee of a facility that specializes in treating STDs
• Examples of STD screening tests such as RPR, VDRL, rapid HIV tests, etc.
LESSON CONTENT
I. Introduction
A. Caused by Bacteria, Viruses, Fungi, or Protozoa
B. Can Have Serious Consequences
C. Common STDs in United States
1. Chlamydial infections
2. Gonorrhea
3. Herpes
392 Lesson 7-8 ● Tests for Sexually Transmitted Diseases
4. Human papillomavirus (HPV)

5. Hepatitis B
6. Trichomoniasis
7. Syphilis
8. Candidiasis
9. Human immunodeficiency virus (HIV)
D. Symptoms of Sexually Transmitted Diseases
II. Detection of STDs in Females
A. Vaginitis—caused by bacteria, fungi, or protozoa
B. The Three-Slide Test for Vaginitis
1. Saline wet preparation
a. Trichomonas
b. Gardnerella vaginalis, clue cells
2. KOH preparation for yeasts (fungi)
3. Gram stain for bacteria and yeasts
a. Smear for Gram stain
b. Neisseria gonorrhoeae, gram-negative intracellular diplococci
c. Gardnerella vaginalis, gram-variable
d. Lactobacillus, normal flora, gram-positive rods
C. Neisseria gonorrhoeae Culture
1. Modified Thayer-Martin agar
2. Incubate in 5% to 10% CO2
D. Rapid Tests for Vaginitis and Vaginosis
III. Detection of STDs in Males
A. Symptoms
1. Urethritis
2. Penile discharge
B. Urinalysis and Urine Culture
C. Urethral Culture
1. For Neisseria gonorrhoeae
2. Modified Thayer-Martin agar
3. Incubate in 5% to 10% CO2
D. Urethral Gram Stain
IV. Identification of Neisseria
A. Appearance of Colonies
B. Confirmatory Tests from Culture

1. Oxidase
2. Gram stain
3. Agglutination
C. Nucleic Acid Methods for Detection of N. gonorrhoeae
V. Human Papillomavirus
VI. Tests for Other STDs
A. Tests for Herpes Infection
B. Tests for Chlamydia Infection
C. Tests for Syphilis
1. The Venereal Disease Research Laboratory (VDRL) test
2. The Rapid Plasma Reagin (RPR) test
3. Confirmation of a reactive syphilis screening test
D. Tests for HIV
E. Tests for Hepatitis B Infections
VII. Summary
STUDENT ACTIVITIES

2. Survey POLs in your community to find out the extent of STD testing performed.
3. Determine how a nearby reference laboratory confirms positive HIV tests and reactive RPR
tests.
4. Tour your state’s public health laboratory. Ask for copies of their epidemiology reports on
STD incidence in your state.
Web Activities
1. Use the Internet to search for the Morbidity and Mortality Weekly Reports (MMWR).
Find statistics for the incidence of Chlamydia cases in the United States for the last 2 years
for which reports are available.
2. Use the Internet to find information about PCR or DNA and RNA probe technology.
Report on the use of one of these technologies in detecting an STD.
3. Search the Internet for molecular diagnostic tests for an STD.
394 Lesson 7-8 ● Tests for Sexually Transmitted Diseases

1. What is the importance of detecting STDs?
Early detection of STDs leads to treatment, helps prevent the spread of disease to
others, increases the life span or survival of patients, and reduces patients’ chances of
complications or permanent damage (such as infertility).
2. When is modified Thayer-Martin (MTM) medium used?

MTM medium is used to isolate and grow Neisseria gonorrhoeae.
3. Why should personnel in POLs and other small laboratories be knowledgeable about STDs
and the methods used to detect them?
Because of the development of rapid test methods, STD testing is no longer limited to
hospital, reference, or public health laboratories; the availability of rapid test methods
to detect STDs means that more patients can be tested in the office of their personal
physician.
4. What are five common STDs in the United States?

Any five of the following STDs are acceptable:
a. Syphilis
b. Gonorrhea
c. Chlamydial infection
d. Herpes infections
e. Hepatitis B infection
f. HIV infection
g. Trichomoniasis
h. Candidiasis
i. HPV infection
5. Name one method of detection for each of five STDs.

Any five of the following are acceptable:
a. Chlamydial infection—EIA, DNA probe, cell culture, fluorescent antibody
b. Gonorrhea—culture, RNA and DNA probes, PCR, agglutination tests
c. Herpes—cell culture, serum antibodies, rapid tests
d. Hepatitis B—test for serum antibodies or viral antigen
e. Trichomoniasis—wet prep
f. Syphilis—VDRL, RPR, fluorescent antibody, ELISA
g. HIV infection—serum antibody, viral antigen
h. HPV infection––PAP smear, tissue biopsy, RNA probe
i. Candidiasis––KOH prep, Gram stain
6. What does the presence of HBsAg indicate about a patient?

A positive result for hepatitis B surface antigen (HBsAg) indicates that the patient now
has, or has had, an HBV infection. A positive test several months after infection
indicates that the person is a chronic HBV carrier.
7. What is the danger of chronic HBV infection?

Chronic HBV infection can cause hepatocarcinoma (liver cancer).
8. Why should patients be tested for both HSV-1 and HSV-2?

HSV-1 was formerly considered to be confined to oral mucous membranes (cold sores)
and HSV-2 was considered to be a genital pathogen; it is now recognized that both can
be transmitted sexually and isolated from oral or genital sites.
9. Define Candida albicans, Chlamydia trachomatis, clue cells, gonorrhea, herpes simplex
virus type 1, herpes simplex virus type 2, human immunodeficiency virus, human papilloma
virus, nongonococcal urethritis, oxidase test, sexually transmitted disease, sexually
transmitted infection, spirochetes, syphilis, trichomoniasis, urethritis, vaginitis, and venereal.
See Glossary
LESSON 7-9
Infection Prevention in
Healthcare Settings
LESSON OBJECTIVES
• Discuss the role of the institution’s infection prevention department.
• List five exposure control methods that are included in Standard Precautions.
• Explain why isolation techniques are used.
• List the three types of transmission-based precautions and explain the basis for each
classification.
• Demonstrate handwashing technique.
• Demonstrate gowning technique.
• Demonstrate the method of putting on a mask.
• Demonstrate the technique for donning sterile gloves.
• Demonstrate the techniques for removal and disposal of mask, gown, and gloves.
GLOSSARY
Airborne Precautions / a Centers for Disease Control and Prevention (CDC) isolation category
designed to prevent transmission of infectious diseases, such as measles, that are spread by
the airborne route
APIC / Association for Professionals in Infection Control and Epidemiology, an organization
working across a spectrum of professionals, organizations, and institutions to prevent
healthcare-associated infections
carrier / a individual who harbors an organism and is capable of spreading the organism to
others, but has no symptoms or signs of disease
Contact Precautions / a CDC isolation category designed to prevent transmission of diseases
spread by close or direct contact
Droplet Precautions / a CDC isolation category designed to prevent transmission of diseases
spread through the air over short distances
Lesson 7-9 ● Infection Prevention in Healthcare Settings 397
fomites / inanimate objects, such as bed rails, linens, or eating utensils, which can be
contaminated with infectious organisms and serve as a means of their transmission
healthcare-associated infection (HAI) / infection acquired while being treated for another
condition in a healthcare setting; synonym for healthcare-acquired infection; formerly called
nosocomial infection
infection / a pathological condition caused by growth of microorganisms in the host
isolation / the practice of limiting the movement and social contact of a patient who is
potentially infectious or who must be protected from exposure to infectious agents
nonpathogenic / not normally causing disease in a healthy individual
nosocomial infection / an infection acquired in a hospital or healthcare facility; healthcare-
associated infection
protective isolation / an isolation category designed to protect highly susceptible patients from
exposure to infectious agents; reverse isolation
Standard Precautions / a set of comprehensive safety guidelines designed to protect patients
and healthcare workers by requiring that all patients and all body fluids, body substances,
organs, and unfixed tissues be regarded as potentially infectious

• Examples of personal protective equipment such as gloves, gowns, face shields, masks,
N95 respirator
• Examples of precautions signs used on patient room doors
• Video demonstrating the use of PPE for transmission-based precautions
• Images (digital, transparencies or overheads) of Tables 7-25 through 7-27
LESSON CONTENT
I. Introduction
A. Infection Prevention Department
B. Healthcare-Associated Infections
C. Guidelines for Managing Contagious Patients
II. Causes of Infection
A. Source of Microorganisms
B. Susceptible Host
C. Method of Transmission
398 Lesson 7-9 ● Infection Prevention in Healthcare Settings
III. Healthcare-Associated Infections

A. Incidence
B. Put patients at greater risk
C. Increases cost of care
D. Multidrug-resistant bacteria
1. MRSA
2. Clostridium difficile
3. VRE
IV. Clostridium difficile
A. Produces Endotoxin and Cytotoxin
B. Associated with Long-term Stays in Healthcare Settings
C. Pathology
D. Treatment
E. Preventive Measures
V. Transmission-Based Precautions
A. Used in Addition to Standard Precautions
B. Contact Precautions
C. Droplet Precautions
D. Airborne Precautions
E. Reverse or Protective Isolation
VI. Complying with the Exposure Control Plan
VII. Exposure Control Practices
A. Hand Hygiene
1. Waterless handrubs
2. Handwashing technique
B. Using Personal Protective Equipment
1. Masks
2. Gowns
3. Gloves
C. Entering and Exiting Precaution Room
VIII. Summary
STUDENT ACTIVITIES
2. Practice the procedures for handwashing and donning and removing mask, gown, and gloves, as
outlined in the Student Performance Guide.
Web Activities
1. Explore the web sites of infection prevention organizations to see what publications, alerts, or regulatory
information is available. One such site is www.apic.org.
Lesson 7-9 ● Infection Prevention in Healthcare Settings 399
2. Select a disease or condition from the list at the end of this section. Use the Internet to
determine which (if any) of the transmission-based precautions should be used for the
condition selected. If a disease was selected, use the Internet to research how the disease is
transmitted. Outline a scheme, listing the precautions to use for a hospitalized patient with
that condition. Include the PPE that must be used, where the PPE should be located, and
where and how used materials should be discarded.
Select a topic: Colostomy, chickenpox, tetanus, whooping cough, rubeola, HIV infection, West
Nile virus, filovirus

1. What is the function of the institution’s infection prevention department?
The infection prevention department monitors contagious disease within the hospital or
other healthcare facility, sets guidelines for managing contagious patients and patients
highly susceptible to infection (have little resistance or immunity), and sets standards to
ensure that patients do not acquire healthcare-associated infections (HAI) and
infections do not spread to visitors and healthcare personnel.
2. Why are transmission-based precautions used?

Transmission-based precautions are used in addition to Standard Precautions to
provide patients, workers, and visitors additional protection from contagious patients.
Transmission-based precautions specify methods to prevent or interrupt disease
transmission in healthcare facilities.
3. What are the three categories of transmission-based precautions? Give an example of a

condition or disease requiring each type of precaution.
See TABLES 7-26 and 7-27.
a. Contact Precautions—used when a patient has a disease spread by close or direct
contact, such as infective material from a wound or fecal matter, skin infections, or
colonization with multidrug-resistant pathogens
b. Droplet Precautions—used when patients are infected with pathogens easily
transmitted through the air over short distances by large-particle droplets, such as
mumps, rubella, pertussis (whooping cough), and certain pneumonias
c. Airborne Precautions-–used when a patient is known or suspected of being infected
with pathogens transmitted by the airborne route, such as measles (rubeola),
chickenpox (varicella), and infectious pulmonary tuberculosis.
4. Describe correct handwashing technique. When must handwashing be performed?

See Figure 7-60. Wet hands with warm or room temperature water; apply antiseptic soap
to hands; lather hands and wrists, rubbing hands together vigorously for at least 15 to 30
seconds, covering all surfaces of the hands and fingers. Hold hands in downward position,
rinse from the arm or wrist toward the tips of the fingers. Dry hands thoroughly with a
disposable towel. Use clean disposable towel to turn off hand-operated faucets. Hands
should be washed (decontaminated) before and after all procedures.
400 Lesson 7-9 ● Infection Prevention in Healthcare Settings
5. What is the correct technique for putting on and removing a gown?

See Figure 7-61. Remove laboratory coat before donning gown; touch gown only on
the inside surface; place hands inside the gown shoulders; slip fingers inside the neck-
band and tie gown at neck, overlapping the back edges of the gown to cover all
clothing and secure the waist ties. Remove gown by turning the inside of the gown to
the outside and folding the contaminated outer side inward.
6. Explain how to put on sterile gloves. How should used gloves be removed to prevent
exposure to possible contaminants on glove surfaces?
See Figures 7-62 and 7-63. Sterile gloves are used to handle sterile equipment or
instruments and can be used for certain other techniques such as collecting a blood
culture. Open sterile pack being careful not to touch outer surface of gloves; pick up
first glove by the cuff to insert hand, taking care not to touch outside of glove; use the
sterile gloved hand to don second glove; unfold cuffs to cover exposed skin by sliding
fingers under cuffs. Gloves are removed by pulling one glove down over the hand
(inside out), enclosing the removed glove in the palm of the remaining gloved hand;
inserting bare fingers inside remaining glove cuff and pulling the glove inside out
down over the hand, taking care not to the touch outside of glove. Discard gloves into
biohazard container.
7. What are five exposure-control methods used to prevent exposure to blood and body fluids?
a. Use correct hand hygiene techniques
b. Wear appropriate PPE
c. Wear eye or face protection if aerosols or splashes are likely
d. Discard used sharps into rigid, puncture-proof sharps containers
e. Clean up spills immediately with appropriate disinfectant
8. How do Standard Precautions and transmission-based precautions differ? When are

Standard Precautions used?
Standard Precautions are used for all patients and all patient specimens.
Transmission-based precautions are used in addition to Standard Precautions when
patients have, or are suspected of having, a contagious disease.
9. What exposure-control methods are used in each of the three categories of transmission-
based precautions?
See Figures 7-57, 7-58, and 7-59. Standard Precautions are always used. In addition
the following exposure control methods are used with transmission-based precautions:
Contact Precautions—wear gloves and gown; use disposable or patient-dedicated
equipment.
Droplet Precautions—wear mask in patient room
Airborne Precautions—wear N95 respirator, place patient in airborne infection
isolation room, keep patient room door closed
10. Define Airborne Precautions, APIC, carrier, Contact Precautions, Droplet Precautions,
fomites, healthcare-associated infection (HAI), infection, isolation, nonpathogenic,
nosocomial infection, protective isolation, and Standard Precautions.
See Glossary.
LESSON 7-10
Emerging Infectious
Diseases
LESSON OBJECTIVES
• Explain what is meant by emerging infectious disease.
• List five factors that influence the emergence or re-emergence of disease.
• Discuss how emerging infectious diseases present a threat to public health.
• Name four emerging diseases and explain transmission, symptoms, precautions for
caregivers, and treatment for each disease.
• Explain the role of the Laboratory Response Network in responding to emerging
diseases.
• Explain how natural disasters can create a public health emergency.
GLOSSARY
avian influenza / an infection of birds with one of the influenza A viruses; bird flu
biosafety level 4 (BSL-4) / a designation requiring use of a combination of work practices,
equipment, and facilities to prevent exposure of individuals or the environment to pathogens
that can be transmitted by aerosol and that pose a high risk for life-threatening disease for
which treatment or vaccine is not generally available
bovine spongiform encephalopathy (BSE) / a fatal, neurological disease of cattle caused by an
unconventional transmissible agent called a prion; commonly called mad cow disease
Ebola virus / a highly infectious filovirus that causes a hemorrhagic fever
epidemic / disease affecting many persons at the same time, spread from person to person, and
occurring in an area where the disease is not prevalent
epizootic / an outbreak of disease in an animal population
402 Lesson 7-10 ● Emerging Infectious Diseases
Laboratory Response Network (LRN) / a national network of laboratories coordinated by the

Centers for Disease Control and Prevention with the ability for rapid response to threats to
public health
Marburg virus / a filovirus that causes a hemorrhagic fever
Mycobacterium tuberculosis / an acid-fast bacillus that causes tuberculosis
pandemic / widespread disease transmitted person to person and occurring over an entire
country, continent, or even worldwide
SARS / the acronym for severe acute respiratory syndrome, a condition caused by a coronavirus
zoonotic / infection or disease that can be transmitted from vertebrate animals to humans

• Newspaper or magazine articles about emerging infectious diseases
• Images (digital, transparencies or overheads) of Figures 7-65 and 7-73
LESSON CONTENT
I. Introduction
A. Infectious Diseases a Major Cause of Death Worldwide
B. World Health Organization (WHO)
II. Emerging Infectious Diseases
A. Cause for Concern
1. High morbidity and mortality
2. Vaccines generally not available
3. Animal disease spreading to humans
4. Potential for pandemic
B. Contributing Factors
1. Environmental changes, climate change
2. Population concentrations
3. Global travel
4. Public health, public policy, and disease surveillance failures
5. Mutation or adaptation of pathogens
6. Natural disasters
C. West Nile Virus
D. Viral Hemorrhagic Fevers
Lesson 7-10 ● Emerging Infectious Diseases 403
1. Some are BSL-4 pathogens

2. Rift Valley fever
3. Ebola and Marburg viruses
E. Severe Acute Respiratory Syndrome (SARS)
F. Mycobacterium tuberculosis
1. 3 billion infected worldwide
2. Multidrug-resistant forms increasing
3. XDR-TB
G. Avian Influenza—H5N1
1. Bird flu outbreaks
2. Prevention
III. Pandemic Influenza
A. 2009–2010
B. H1N1— “swine” virus
C. Vaccine successful
D. Other Pandemics
IV. Role of the Clinical Laboratory in Emerging Diseases
A. Laboratory Response Network
1. National laboratories
2. Reference laboratories
3. Sentinel laboratories
B. Rapid response capability
C. Surveillance
V. Summary
STUDENT ACTIVITIES

2. Find articles about emerging diseases in news magazines, newspapers, and other periodicals.
Report on your findings.
Web Activities
1. Use the Internet to look up information on emerging infectious diseases on web sites of the
CDC, WHO, FDA, or USDA. Note how many diseases the CDC categorizes as emerging
infectious diseases. Report on an emerging infectious disease that is not discussed in this
lesson. Include information about where it is endemic, how it is transmitted, and symptoms,
treatment, and prevention.
2. Search the CDC web sites to find current information on tuberculosis in the United States.
Get information on which states report the highest numbers of infections.
3. Polio has not been a problem in the United States since the development of effective vaccines
and institution of childhood vaccinations. Use the Internet to search for information on recent
outbreaks of polio around the world. Report on the cause(s) of these outbreaks.
4. Search the Internet for information on recent contagious disease outbreaks associated with
natural disasters, such as the 2010 Haiti earthquake or Pakistan floods. Report on what
diseases created problems, or what diseases health agencies were most concerned about.

1. What is the difference between an emerging infectious disease and a re-emerging infectious
disease?
Emerging diseases are newly recognized diseases or diseases that have increased in
humans or threaten to increase in the near future; re-emerging diseases are diseases
that were on the decrease for a time but have again become major health threats. These
diseases gain attention because they have high morbidity and mortality rates and no
reliable method of prevention.
2. What five factors can influence the emergence of a disease?

See Table 7-28. Any five of the following are acceptable:
a. Environmental changes
b. Global climate changes
c. Natural disasters
d. Changes in population concentrations, more crowded living conditions
e. Increase in international travel and trade
f. Failure of public health policies or disease surveillance
g. Mutations or adaptations of pathogens
3. The most serious emerging infectious disease pathogens are currently not a problem in the
United States. What conditions could cause the spread of these pathogens to the United
States?
Emerging infectious disease pathogens could be spread to the United States by infected
travelers, global spread of disease, or because of natural disasters or manmade disasters
such as bioterrorism.
4. What characteristics do most emerging infectious diseases have in common?

Emerging infectious diseases characteristically have high morbidity, high mortality,
and no reliable preventive measures such as vaccines.
5. Why is avian influenza, or bird flu, a problem for humans?

Humans have little or no immunity against avian influenza viruses. Some avian
influenza viruses have been shown to cause high morbidity and mortality in humans.
Lesson 7-10 ● Emerging Infectious Diseases 405
6. What must happen to an animal virus in order for it to cause a pandemic?

The virus would have to change abruptly and develop the ability to pass easily from
person to person, before the general public has time to develop immunity.
7. What are three viral hemorrhagic fevers? Why are they given this designation?
Ebola, Marburg, and Rift Valley fevers are called hemorrhagic fevers because their
symptoms include hemorrhaging (bleeding) under the skin, internally, and from body
orifices.
8. Why are insect control programs important in developing nations?

Insects are vectors for many of the emerging diseases.
9. Why has tuberculosis resurfaced as a disease on the increase?

It is believed that prolonged exposure to antibiotics has contributed to the
re-emergence of tuberculosis, particularly drug-resistant strains; in addition, the
tuberculosis has increased with the increased incidence of immunosuppressive diseases
such as AIDS.
10. Explain the role of the LRN in responding to emerging diseases.

The LRN is a national network of laboratories with the ability to respond rapidly to
public health threats such as emerging diseases and acts of bioterrorism. Many
laboratories in the network have BSL-3 facilities and can perform confirmatory tests
on BSL-3 agents. The laboratories are provided with personnel training, vaccinations
for workers, and reagents and controls needed to identify diseases.
11 Explain how natural disasters can create a public health emergency.

Natural disasters can cause several public health emergencies. Flood waters can
contain raw sewage and also create environments where insects and pathogens can
grow and spread disease. Increases in foodborne illnesses such as cholera can result
from contamination of water or food supplies.
12. Name five emerging diseases and explain transmission, symptoms, precautions for
caregivers, and treatment.
See Table 7-28.
a. West Nile virus—transmitted by mosquitoes; mild febrile illness to severe
encephalitis or meningitis; not transmitted human to human so no special
precautions other than Standard Precautions; no specific treatment or human
vaccine available
b. SARS—person-to person transmission; fever, headache, severe respiratory
symptoms; Standard Precautions, Droplet and Airborne Precautions, and N95
respirator; no specific treatment
c. Mycobacterium tuberculosis—person-to-person transmission; lung disease;
Standard Precautions and Airborne Precautions; long-term drug treatment
d. Avian influenza H5N1—transmitted by wild birds or poultry; flu symptoms;

Standard Precautions, Droplet and Airborne Precautions; no specific treatment
e. Viral hemorrhagic fevers—transmitted by animals or arthropods (some unknown)
and person-to-person; fever, fatigue, bleeding, multiple organ damage, and
hemorrhages; Standard Precautions, Contact, and Airborne Precautions; no
specific treatment
13. Define avian influenza, biosafety level 4, bovine spongiform encephalopathy, Ebola virus,
epidemic, epizootic, Laboratory Response Network, Marburg virus, Mycobacterium
tuberculosis, pandemic, SARS, and zoonotic.
See Glossary.
LESSON 7-11
Biological Threat Agents
LESSON OBJECTIVES
• List five pathogens that have potential use in bioterrorism or as biological weapons
and explain how they are transmitted.
• Name six characteristics that make an agent useful as a biological weapon.
• Explain how the threat of bioterrorism affects the agricultural industry.
• Explain the role of laboratories and primary healthcare providers in recognizing
and responding to potential bioterrorism agents.
GLOSSARY
agroterrorism / acts of terrorism involving threats to agricultural products, including food

animals and crops
botulinum intoxication / a condition in which body tissues are affected by the botulinum toxin
botulinum toxin / neurotoxin produced by Clostridium botulinum
Department of Homeland Security / a federal agency whose primary mission is to prevent,
protect against, and respond to acts of terrorism on U.S. soil
virion / the infectious form of a virus
virulent / highly infectious or highly pathogenic

• Articles from newspapers, magazines, or web sites about anthrax scares, current state of
smallpox virus stores, biological weapons, actions to prevent agroterrorism, etc.
408 Lesson 7-11 ● Biological Threat Agents

LESSON CONTENT
I. Introduction
A. Department of Homeland Security
B. Emergency Preparedness
II. Biological Weapon Agents
A. Characteristics of Potential Biological Weapon Agents
1. Ease of person-to-person transmission
2. Easily disseminated
3. High mortality
4. Major public health impact
5. Difficult to reach state of preparedness
6. Potential to cause panic
7. Classified as Class A agents
B. Anthrax
1. Bacillus anthracis, spore-forming gram-positive bacterium
2. Occurs in wild and domestic animals
C. Smallpox
1. Variola virus
2. Virulent and highly contagious
3. Discontinued routine smallpox vaccinations in 1972
D. Plague
1. Yersinia pestis, gram-negative bacillus
2. Rodents are reservoir host
3. Bubonic, septicemic, and pneumonic forms
E. Tularemia
1. Francisella tularensis, gram-negative coccobacillus
2. Rabbit fever
F. Botulism
1. Clostridium botulinum, gram-positive spore-forming bacterium
2. Neurotoxin
G. Hemorrhagic Fever Viruses
Lesson 7-11 ● Biological Threat Agents 409
III. Threats to Agriculture

A. Agroterrorism
B. Plant and Animal Diseases
C. Foot-and-Mouth Disease
IV. Food and Water Safety
V. Laboratory Role in Bioterrorism Response
A. Laboratory Response Network in Action
1. 2001 Anthrax Attack
2. SARS
3. BioWatch
B. Laboratory Preparedness
VI. Summary
STUDENT ACTIVITIES
2. Visit a regional hospital, reference laboratory, or state public health laboratory. Find out what
procedures they would follow if they suspected the presence of a threat agent. Ask if the
laboratory is a sentinel or reference laboratory.
3. Look in newspapers, news magazines, or periodicals for information on recent natural
outbreaks of agents such as the anthrax or plague bacterium. Find out what kind of disease
was caused and what the mortality was.
Web Activities
1. Use the Internet to explore the Homeland Security web site or another web site with
information about bioterrorism response. List six biological threat agents. Report which, if
any, of these agents have caused animal or human illness in the last 5 years.
2. Use the Internet to search for outbreak information. Use reliable web sources such as the
CDC’s Morbidity and Mortality Weekly Reports or ProMed (Program for Monitoring
Emerging Diseases) at www.promedmail.org.

1. What is meant by agroterrorism? What is one example of an agent that might be used in this
way?
Agroterrorism includes acts of terrorism involving threats to agricultural products such
as food crops and animals used for food. Threats to animals might include foot-and-
mouth disease, avian influenza or imported insect pests. Threats to crops might include
non-native insect pests.
Solution Manual for Basic Clinical Laboratory Techniques, 6th Edition
410 Lesson 7-11 ● Biological Threat Agents
2. What is the causative agent and reservoir for each of the following disease threats: plague,
tularemia, and smallpox?
See Table 7-30
a. Plague—Yersinia pestis; reservoir is rodents.
b. Tularemia—Francisella tularensis; reservoirs are wild and domestic animals.
c. Smallpox—variola virus; reservoirs are laboratory stock cultures; smallpox was
considered eradicated in 1972.
3. What are six characteristics that make an agent useful as a threat agent?
a. Ease of person-to-person transmission
b. Easily disseminated or dispersed
c. Causes high mortality
d. Outbreak would have potential for major public health impact
e. Special measures required to reach a state of preparedness
f. Outbreak might cause panic or disrupt society
4. Why is bioterrorism a hot topic?

Bioterrorism is a hot topic because of the rise of terrorist activities around the world;
these include the 9/11 attack and various attacks involving anthrax in the United States.
5. What was the role of the LRN in the 2001 anthrax outbreak in the United States?
When 22 people became infected with anthrax shortly after the 9/11 attacks, the LRN
and CDC worked to identify the agent involved in the first case. A Florida public health
laboratory, an LRN member, confirmed the identification. LRN laboratories performed
follow-up testing on environmental and clinical samples, ultimately testing more than
125,000 specimens related to the one incident. Because of rapid response treatment was
able to be instituted and more patients survived the infection than would have normally
been expected.
6. What is BioWatch?
BioWatch is an around-the-clock environmental surveillance program that samples air
in certain densely populated cities. The filters are removed daily and analyzed using
polymerase chain reaction (PCR) technology. If a harmful agent were found, it would
trigger a set of emergency responses.
7. What two threat agents have an ancient history of causing disease?

Anthrax and smallpox
8. Define agroterrorism, botulinum intoxication, botulinum toxin, Department of Homeland

Security, virion, and virulent.
See Glossary.
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aerobic / requiring oxygen for growth

culture / growth of microorganisms in a special medium; the process of growing

TEACHING AIDS AND RESOURCES

• Images (digital, transparencies, or overheads) of Figures 7-1 through 7-4

1. Complete the written examination for this lesson.

REVIEW QUESTIONS AND ANSWERS

2. What four areas of study are encompassed by clinical microbiology?

4. What is the difference between a pathogen and an opportunistic pathogen?

5. How do bacterial pathogens cause host damage?

6. What are the three morphological types of bacteria?

7. What is a Gram stain?

8. What are two morphological forms of fungi?

9. What is the difference between aerobic and anaerobic bacteria?

11. What type of specimen is required to detect malaria? To detect Giardia?

12. List five methods used to help identify bacteria.

culture medium /a substance used to provide nutrients for growing microorganisms

TEACHING AIDS AND RESOURCES

Case Studies and Answers

2. How could Carol have avoided her dilemma?

1. Complete the written examination for this lesson.

REVIEW QUESTIONS AND ANSWERS

2. What mistakes can occur in the collection of urine for culture?

5. Why is an aspirate of a wound preferred over a swab?

8. List three important properties of transport systems for bacteriology specimens.

9. What culture can be performed to determine if a person is a carrier of methicillin-resistant

TEACHING AIDS AND RESOURCES

3. Microbiological safety cabinets

Critical Thinking Problem and Answers

2. Explain your answer.

1. Complete the written examination for this lesson.

REVIEW QUESTIONS AND ANSWERS

2. What are the differences between disinfectants and antiseptics?

3. What are two types of safety cabinets used in microbiology?

4. What are the purposes of primary, selective, and indicator media?

5. Describe how to inoculate an agar plate using a swab.

6. Describe how to streak an agar plate for isolated colonies.

7. Describe how to transfer an inoculum from an agar plate to an agar slant.

8. What are important colony characteristics that can be observed?

9. What hemolytic reactions of bacteria can be used to identify the organism?

10. What medium is used to indicate hemolysis?

11. Why are transport media used?

12. Discuss quality assessment programs for the bacteriology laboratory.

13. What safety techniques must be used in the bacteriology laboratory?

TEACHING AIDS AND RESOURCES

Critical Thinking Problem and Answers

2. Which is most likely the problem—Marion’s smear technique or Christine’s processing of

REVIEW QUESTIONS AND ANSWERS

2. Why is it necessary to wear a laboratory coat when working with bacteria?

7. Explain how to perform a Gram stain.

8. Why are some bacteria gram-positive and others gram-negative?

9. What is the appearance of gram-positive cocci? Gram-negative rods?

10. Why is it necessary to wipe countertops often with surface disinfectant?

12. Define bibulous paper, counterstain, and mordant.

TEACHING AIDS AND RESOURCES

• Gram-stained smears of streptococci

3. Incubating the culture

Critical Thinking Problems and Answers

2. Were mistakes made in the laboratory procedures? What were they?