Download Microbiology Lab 156 - BioLab156

Bio Lab 156
Lucia Testa
Rev 1/2011
Instructor: Lucia Testa
Contact Info: E-mail: [email protected]
Location: Science Building Room 200
Website for Class Info : BioLab156.wikispaces.com
Microbiology Lab 156
SyllabuS…………………………………………………
2
Study tipS …………………………………………..…
9
CaSe StudieS/aSSignmentS……………………… 10
Things to know/ Study Guides…………..… 18
miCroorganiSm abbreviationS…………..22
Journal writing Protocol………………… 23
SELECTIVE & DIFFERENTIAL MEDia guide….. 25
Unknown Microorganism Project……...29
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Bio Lab 156
Lucia Testa
Syllabus
Course Objective:
This course explores the biology of microorganisms and is intended for pre-Allied Health students.
Laboratory assignments will serve as an active means of learning and will cover aseptic techniques as well as
methodology for culturing and identifying certain microorganisms.
Grade Points:
7 Homework Assignments
8 Quizzes
Journal 1 check
Journal 2 check
Lab Practical 1- Technique
Lab Practical 2- Technique
Unknown Project
14%
40% (the lowest grade from the 9 quizzes is dropped)
10%
5%
5%
10%
_16%
100%
Prerequisite or Co requisite: BIO 155
Required:
- Lab coat, long sleeve, ¾ length
- Black permanent marker (like a Sharpie)
- Bound notebook
- Plastic bag (for lab coat)
- Colored pencils
- Optional (but recommended):
1 inch ring binder
Required Lab Manual:
Cappuccino, J. and Sherman, N. 2011. Microbiology: A laboratory manual. 9th Ed.
Benjamin Cummings Press, NY, NY.
WEEK
1
B
LAB ACTIVITY
ASSIGNMENTS DUE AND
IN CLASS QUIZZES
Lab Safety, Course Introduction
Ex. 4 Microscopy
Ex. 1 Culture Transfer techniques (no organisms)
Homework:
- Prepare journal for the semester (label/ journal for identification,
number all pages, start table of contents, write Lab Manual reference in back.)
- Read “Journal Writing Protocol” from this packet.
- Review online tutorial for journal writing .
- Write in your journal the Purpose, Materials, and Procedure sections for Ex 4.
- Prepare Case Study/ Assignment 1
(typewrite- no handwritten assignment)
- Read Ex. 4 Microscopy pg 31-37
- Answer Review Question 2
- Read Ex. Part 1 and Culture Transfer Techniques pg 1 – 11
- Answer Review Questions 1 - 3
- Read Ex. 2A Isolation of pure culture pg 13 -15, 19
- Consult the Study Guide for things to know on the experiments listed above.
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WEEK
LAB ACTIVITY
2
Read results
Ex. 1 Culture transfer with organisms
Ex. 2A Isolation of pure culture
Ex. 4 Microscopy continued
W
ASSIGNMENTS DUE AND
IN CLASS QUIZZES
- Assignment 1 Due
Homework:
- Complete journal entry for Exp 4, 1, 2A
- Prepare Case Study/ Assignment 2
- Study for Quiz #1 (safety, Ex 1, 4, 2A & intro to 2B, 3; definitions from Assignment 2)
- Ex. 2A Isolation of pure culture- Answer Review Questions 1 & 2
- Read Ex. 3 Cultural Characteristics pg 23-27
- Read Ex. 2B Isolation of Pure culture pg 15-17, 20
- Consult the Study Guide for things to know on the experiments listed above.
3
B
Read results
Ex. 3 Cultural Characteristics
Ex. 2B Isolation of Pure culture
- Assignment 2 Due
- Quiz #1
Homework:
- Complete journal entries from today’s class.
- Prepare Case Study/ Assignment 3.
- Study for Quiz 2 (Ex 2B, 3 & intro 7, 8, 10; definitions from Assignment 3)
- Ex. 2B Isolation of Pure culture - Answer Review Questions 3 & 4
- Read Ex. 7 Prep of Smears pg 53-57
- Read Ex. 9 Simple Stain pg 59-63
- Read Ex.11 Gram Stain pg 69-73
- Consult the Study Guide for things to know on the experiments listed above.
4
W
Read results
Ex. 7 Prep of Smears
Ex. 8 Simple Stain
Ex.10 Gram Stain
- Assignment 3 Due
- Quiz #2
Homework:
- Complete journal entries from today’s class.
- Study for Quiz #3 (Ex 7, 8, 10 & intro 14)
- Ex. 7 Prep of Smears- Answer Review Questions 1 - 4
- Ex. 8 Simple Stain- Answer Review Questions 2 & 3
- Ex.10 Gram Stain - Answer Review Questions 1 – 5
- Read Ex.14 Selective and Differential media pg 99-106
- Consult the Study Guide for things to know on the experiments listed above.
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WEEK
LAB ACTIVITY
5
Read results
Ex.14 Selective and Differential media
Ex.10 Gram Stain continued
B
ASSIGNMENTS DUE AND
IN CLASS QUIZZES
- Quiz #3
Homework:
- Complete journal entries from today’s class.
- Prepare Case Study/ Assignment 4
- Study for Quiz 4 (Ex 14 & intro 21)
- Study “Selective & Differential Media Guide” from this packet.
- Study “Chart of Actions of Different Types of Media”
- Ex. 14 Selective and Differential media
- Answer questions Observation and Results Section on pg 106-107
- Answer Review Question 1
- Review Journal for completeness
- Review Gram Stain & Streak plate Technique
- Read Biochemical Activities Part 5 Introduction pg 147 – 148
- Read Ex. 21 Hydrolysis (Starch and Gelatin) pg 149 – 153
(Omit lipid and casein sections)
- Consult the Study Guide for things to know on the experiments listed above.
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W
Read results
Ex. 21 Hydrolysis (Starch and Gelatin)
Lab Practical 1- Gram Stain & Streak plate technique
- Assignment 4 Due
- Quiz #4
- Journal Check
Homework:
- Prepare Case Study/ Assignment 5
- Read Ex. 22 Carbohydrate Fermentation pg 155 - 159
- Read Ex. 23 Triple Sugar Iron Agar pg 161 - 163
- Consult the Study Guide for things to know on the experiments listed above.
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B
Read results
Ex. 22 Carbohydrate Fermentation
Ex. 23 Triple Sugar Iron Agar
- Assignment 5 Due
Homework:
- Complete journal entries from today’s and last week’s class.
- Study for Quiz 5 (Ex 21, 22, 23 & intro 24, 25, 26, 30)
- Ex. 22 Carbohydrate Fermentation - Answer Review Questions 1 – 4
- Ex. 23 Triple Sugar Iron Agar- Answer Review Questions 1 – 4
- Read Ex. 24 IMViC Part A, Part D pg 165 - 166, 169 – 171, 173, 175
- Read Ex. 25 Hydrogen Sulfide Test pg 177- 179
- Read Ex. 26 Urease Test pg 181- 183
- Read Ex. 30 Oxidase Test pg 199- 201
- Consult the Study Guide for things to know on the experiments listed above.
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WEEK
LAB ACTIVITY
8
Read results
Ex. 24 IMViC Part A Indole, Part D Citrate Test
Ex. 25 Hydrogen Sulfide Test
Ex. 26 Urease Test
Ex. 30 Oxidase Test
W
ASSIGNMENTS DUE AND
IN CLASS QUIZZES
- Quiz #5
Homework:
- Complete journal entries from today’s class.
- Study for Quiz #6 (Ex 24, 25, 26, 30 & intro 29, 62, 63)
- Ex. 24 IMViC Part A, Part D- Answer Review Questions 1- 3
- Ex. 25 Hydrogen Sulfide Test - Answer Review Questions 2 & 3
- Ex. 26 Urease Test - Answer Review Questions 1 – 4
- Ex. 30 Oxidase Test - Answer Review Questions 1 – 4
- Read Ex. 29 Catalase pg 195 - 197
- Read Ex. 62 pg 425 & Coagulase Section pg 426-431
- Read Ex. 63 (Only the Introduction) pg 433-435 (no journal entry)
(Hemolytic activity and Bacitracin test only)
- Consult the Study Guide for things to know on the experiments listed above.
9
B
Read results
Ex. 29 Catalase
Ex. 62 Coagulase Section
Blood and MSA review for Strep and Staph
- Quiz #6
Homework:
- Complete journal entries from today’s class.
- Study for Quiz 7 (Ex 29, 62, 63 & intro 15, 16, 17, 18)
- Ex. 29 Catalase - Answer Review Questions 1 & 2
- Review Blood and MSA for Strep and Staph
- Read Ex.15 Temperature pg 109-113
- Read Ex.16 pH pg 115-117
- Read Ex.17 Oxygen requirements pg 119-121
- Read Ex. 18 Anaerobic Microorganisms pg 125-128
- Consult the Study Guide for things to know on the experiments listed above.
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WEEK
LAB ACTIVITY
10
Read results
Ex.15Temperature
Ex.16 pH
Ex.17 Oxygen requirements
Review Standard Tests for G+ and G- organisms
W
ASSIGNMENTS DUE AND
IN CLASS QUIZZES
- Quiz #7
Homework:
- Complete journal entries from today’s class.
- Study for Quiz #8 (Ex 15, 16, 17, 18 & intro 43)
- Ex.15 Temperature - Answer Review Questions 1-3
- Ex.16pH- Answer Review Questions 1 – 5
- Ex.17 Oxygen requirements- Answer Review Questions 2 – 5
- Ex 18 Answer Review Question 1 (for thioglycollate only) and 2
- Review Standard Tests for G+ and G- organisms
- Read Ex. 43 Chemotherapeutic Agents Part A pg 287-290, 295
- Read Unknown/Project Report Example.
11
B
Read results
Ex. 43 Chemotherapeutic Agents Part A
Ex. 69 Identification of an Unknown Organism
Part 1(Gram Stain and Isolation Technique)
- Quiz #8
Homework:
- Complete journal entries from today’s class.
- Prepare Case Study/ Assignment 6.
- Read Ex. 72 Identification of an Unknown Organism pg 473-477
- Read Ex. 45 Disinfectants and Antiseptics pg 303 & 306 (not responsible for Table 45.1)
- Consult the Study Guide for things to know on the experiments listed above.
12
W
Read results
Ex. 45 Disinfectants and Antiseptics
Ex. 69 Unknown Part 2 continued
- Assignment 6 Due
Homework:
- Complete journal entries from today’s class.
- Study for Quiz 9 - (Ex 45, 43 & intro 65; definitions from Assignment 7)
- Prepare Case Study/Homework Assignment 7
- Read Ex. 65 Enterotube pg 447 - 448 (Demo)
- Ex. 65 Enterotube - Answer Review Questions 1 , 2, 3
- Consult the Study Guide for things to know on the experiments listed above.
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WEEK
LAB ACTIVITY
13
Read results
Ex. 69 Unknown Part 3 continued
Ex. 65 Enterotube- Demonstration
B
ASSIGNMENTS DUE AND
IN CLASS QUIZZES
- Assignment 7 Due
- Quiz #9
Homework:
- Complete journal entries from today’s class.
- Prepare Unknown Organism Report
- Review Journal for completeness
- Review Gram + and Gram – Chart for completeness.
- Review lab techniques learned this semester.
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W
Lab Practical 2- Technique
- Unknown Written Report Due
- Journal Due
- Gram pos & Gram neg Charts Due
Conduct:
Please turn off your cell phone, pager or any other device that rings, beeps, or otherwise makes noise while in
class. Additionally, lab is an interactive environment, but it is not a social hour. Please be professional in your
conduct as the laboratory setting contains expensive equipment and potentially hazardous substances.
Absolutely no food (including chewing gum) or beverage consumption in the lab. Smoking is
prohibited in the lab.
It is important to come prepared to lab by reading the lab manual and complete the homework each
week. You are required to wear a lab coat and closed shoes for this lab to ensure your safety. Be
prepared to start lab on time, lateness and not being prepared can result in grade reductions. Lab
attendance is required; missing lab can result in significant grade reduction.
Additionally, you are expected to clean up after yourself in the lab and to observe all safety measures. Lab
benches must be wiped down with disinfectant after each lab exercise and, for your safety, you must wash
your hands prior to leaving the lab.
Turning in assignments:
Late assignments will not be accepted. You may turn in the assignment early or send it with someone on or
before the deadline.
Journals will be evaluated for protocol, format (Exp#, headers, dates, etc), weekly entry protocol, charts,
procedures, citations, results, and content.
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Labs and quizzes:
There are no make up labs, it is your responsibility to obtain missed information from your classmates.
Also use your lab syllabus, Study Guide, BioLab156.wikispaces.com website and lab manual as resources.
Failure to follow instructor’s directions and/or conducting unauthorized experiments and/or not following
safety guidelines may result in grade reduction.
Except for the last quiz, quizzes will be administered at the beginning of the class. See above listing for week
the quizzes will be administered. Be on time. If you are late, you will reduce the time you have to take the
quiz. There will be no make up quizzes…so don’t miss class.
Academic Integrity:
Academic dishonesty is considered a serious offense and is expressly prohibited. This policy applies to all
course assessment and related assignments. Cheating is defined by the college as "the giving or taking of
any information or material with the intent of wrongfully aiding oneself or another in academic work
considered in the determination of a course grade or the outcome of a standardized assessment." Students
found to have cheated in a course will face several possible consequences ranging from receiving an "F" on
the assignment(s) to expulsion from the college. Please refer to the course catalog section on academic
integrity for additional definitions of ‘cheating’ or for more detail.
Plagiarism is also unacceptable. Plagiarized work will result in zero credit for the plagiarized assignment.
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Because of the many contributing areas of science, microbiology is often perceived as a
complex and difficult subject. However, if you study and focus on microbiology, assisted
by other sciences, you will gain insight into the science of microbiology and how it has
impacted human culture.
S
t
u Here are some study tips to help you:
d Come to class ready to participate. Read assigned material before each respective lecture.
The material will make better sense after you have read about it at least once. Reading the
y material before class, coming to class with questions or answers, and showing up with an
T open mind is an excellent way to learn.
assistance with difficult concepts early. See your instructor for an explanation of
i Seek
terms, principles or techniques, which you don’t understand.
p Remember, microbiology is a comprehensive biology course. It is not a course that you
pass by just memorizing the facts. Think through the reasons why experiments were
s can
performed. Analyze data or relationships to understand conclusions drawn from them.
Regularly attend classes. Information will be presented and concepts will be developed in
class that will not be effectively communicated by reading the notes of another. You need
to experience each class yourself.
Don’t wait until the day before a quiz to prepare for it. Learning is an ongoing, full-time
job. Read before class. Re-read after class. Review your notes frequently. Ask questions.
Get feedback. Don’t wait until it’s too late to get help.
Use study aids. (1) Use charts and tables distributed in class. (2) Keep class notes and
corresponding manual notes next to the lab in your journal. (2) After each lab, write down
3-5 questions that reflect the essence of the lab material. (3) Find other members of the
class who will agree to write questions for each lab, and share them. (4) Explore related
topics on the World Wide Web.
Like everything else, "practice makes perfect"!
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Case Studies/Assignments
Evaluating cause and effect relationships, such as microbial causes of disease, by the scientific
method are good exercises for developing critical thinking skills. Following are some case
studies for you to investigate.
When preparing the case studies/homework follow the directions below:
1. Read the assignment. Underline the significant facts.
2. Research the case study/subject by using text references, and Internet resources (some
are included in the assignment as a starting point). Additional resources should also be
documented and footnoted at the end of your report.
3. Write your report using the following procedure.
a. Header of report should include your Name, Assignment #, Title(s) of
Assignment Section, Date, Complete Section # (156- your section number)
b. Write each question. Then write the answer afterwards.
c. Write the answer in complete sentences with correct grammar and spelling.
d. The report needs to be typed.
e. The report should be no longer than two (2) pages long.
4. The answer should reflect scientific and medical accuracy.
5. Answers should be thoughtful and reflect comprehension of the type of bacterial
infection. Explain in thorough detail the thought process of how you arrived to your
answer.
6. Names of bacteria should be written correctly [Example- in italics with Genus
capitalized and species in lower case Staphylococcus aureus.)
7. Properly write your references (end noting) with your report!
8. Grades will be based on the criteria stated above.
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Assignment #1
Case Study
A 24-year old female automotive technician presents herself at the doctor’s office. She complains of
fever and of pain in her left hand.
On physical examination, the patient had a deep wound on her left palm that was oozing pus. She
had purplish, red streaks running up her left arm. She had enlarged lymph nodes at the elbow and
under her arm. The patient’s skin was warm and dry.
In her history, the patient had punctured her left palm with sharp metal from the undercarriage of a
"real cherry" 1977 Malibu about a week earlier. She said the wound had bled for a few minutes and
she thought that she had washed it "real good" with soap and water. She had covered the wound
with a large "band-aid" and gone back to work. She developed a fever about three days later. For
the past couple of days, she "did not feel so good" and had vomiting and diarrhea.
1. What type of infection do you believe she has in this hand?
a. Streptococcus pyogenes
b. Staphylococcus aureus
c. Clostridium
d. Pseudomonas
e. Pasteurella multocida
2. From complaint and physical examination, which of the symptoms lead you to your choice of
agent?
a. the fever and pain in the hand
b. the fever, wound with oozing pus and enlarged lymph nodes
c. the lymph nodes and red streaks
d. the warm dry skin
e. the pain, red streaks and enlarged lymph nodes
3. From the history, which of the information confirmed your choice?
a. The wound had bled for a while.
b. The wound was cleaned with soap and water.
c. The wound had been covered and had perhaps become anaerobic.
d. She had diarrhea and vomiting.
e. The cut was from a rusting car frame.
4. Which of the following is most likely to follow this infection?
a. gangrene and amputation
b. toxic shock syndrome
c. muscle spasms
d. neurological dysfunction of the hand
e. arthritis
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Assignment #2
Infections
1.
2.
3.
4.
What steps can one take to control infections (list five)? Which is the most important?
Define the word pathogens.
Define the word etiology.
Explain the difference between a primary and secondary infection.
http://www.nlm.nih.gov/medlineplus/infectioncontrol.html
Case Study
A 27-year old white female presented at the walking clinic of her local physician on August 15. On
physical exam, the patient had a fever of 38.5C. She appeared fatigued, had tender joints, and
complained of a headache, a stiff neck and a backache. The physician noticed a circular "rash"
about 5 inches in diameter, with a bright red leading edge and a dim center in the form of a "bull’s
eye". The physician noted an irregular heart beat. The patient complained of lack of ability to
concentrate.
The patient gave the following history: She is a graduate student in the wildlife program at the
university in town. She was in the field for three weeks in Wisconsin during the months of May and
June. She tracks small mammals in the field and studies their behavior. It had been a warm, wet
spring and she complained of a large number of biting flies, mosquitoes and ticks in the area. She
felt well until about 2 weeks after returning to her home. Since that time, many of her symptoms had
progressed. She finally found that she could take it no more.
1. What is your best diagnosis of this case?
2. What features are critical to your diagnosis?
3. What further steps should be taken to clear up the problem?
http://en.wikipedia.org/wiki/Lyme_disease
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Assignment #3
Nosocomial Diseases
1.
2.
3.
4.
5.
Define nosocomial infections.
How are nosocomial diseases transmitted?
What factors cause patients to be more predisposed to acquire a nosocomial disease?
Describe two bacteria which have grown resistant to antibiotics.
How can nosocomial diseases be prevented?
http://en.wikipedia.org/wiki/Nosocomial_infection
Case Study
A lethargic 22-month old black female was presented by her mother to the emergency room at
2:15am on a Sunday. The child had a history of a runny nose, hoarse cough and low-grade fever
(~99F) for the past 48 hours. The mother was concerned about the forced and noisy breathing of
the child. The pediatrician examined the child and found cloudy eyes and mild inflammation of the
ears, but no overt signs of bacterial infection (no significant changes in the eardrums). The throat of
the child was red and coated with mucus. The larynx was swollen and raw.
The physician performed a rapid Strep test and found it was negative. Throat swabs were taken for
culture. The physician placed the child in a room with a warm vaporizer for about 30 minutes. This
dramatically improved the breathing of the child.
1. What is the presumptive diagnosis for this case?
2. Will the throat cultures likely show evidence of Streptococcus pyogenes? How about
Staphylococcus aureus?
3. Do you believe that this is a bacterial or viral disease? Why?
4. What further treatment is indicated for this case?
http://kidshealth.org/parent/infections/bacterial_viral/whooping_cough.html
http://www.medicinenet.com/staph_infection/article.htm
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Assignment #4
Case Study
A 4-year old girl presents at the emergency room with bloody diarrhea, fever and vomiting. The
child’s mother reports that the child has had these symptoms for about 24 hours and she has not
passed any urine for about 12 hours.
The child is enrolled in a day care center and the group had recently made a field trip to a fast food
place to learn about different jobs. The children had a lunch of ground beef, fries and cola after
meeting with different workers. This field trip was 4 days earlier on Friday. The child had a
temperature of 39C and showed physical signs of dehydration. Blood samples drawn showed
evidence of greatly reduced kidney function and lysed red blood cells.
1. What is your diagnosis here? What is organism is responsible?
2. What pathogenic feature of this organism caused the severity of this problem?
3. What were the critical features to your diagnosis?
http://www.emedicinehealth.com/food_poisoning/page3_em.htm#Food%20Poisoning%20Symptoms
http://www.bernco.gov/live/standalone.asp?dept_id=2330&link_id=4181
Assignment #5
Case Study
A 22-year old woman presents at the walk-in health clinic with slight fever (38.5C), a complaint of
frequent urination, burning on urination, vaginal discharge and a small lesion on the labia.
The woman reported that she was moderately sexually active and had three sexual partners in the
past six months. Her last sexual contacts were about 7 days earlier. She had developed mild
symptoms about 5 days earlier, beginning with a discharge from the vagina. She began having pain
on urination about 3 days earlier.
Urine analysis revealed a pH of 8.2, some white cells and a few red blood cells. There was protein
in the urine. A smear of the vaginal secretion showed a number of Gram-negative cocci.
1. What is your diagnosis here?
2. What clinical features are critical to your diagnosis?
3. What further actions must be taken?
http://www.avert.org/std.htm
http://www.emedicinehealth.com/vaginal_infections/article_em.htm
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Assignment #6
Lucia Testa
(CASE Study A & B)
Case Study A
A white male, 17 years of age presented at the emergency room with a severe headache, vomiting,
and a stiff neck with pain running up his back. On admission, his temperature was 101F. The young
man appeared to have trouble hearing during the nurse’s interview and also seemed to have trouble
concentrating.
The history revealed that the young man is a wrestler for the local high school team. He had felt as
though he were getting a cold the past few days, since his last meet in Hicksville. He did not smoke
or drink, but he had attended a party two days earlier thrown by his girlfriend and the other
cheerleaders to celebrate his victory in the sectionals. He had been holding his weight at 162 for the
season, so he ate little and did not drink on the day of meets (today is a day of the meet).
On physical exam, the physician noticed several areas of small purplish spots on the skin of the
back, thigh and arm. The boy thought those were from wrestling.
1. Which of the following is the most likely diagnosis?
a. Neisseria gonorrhoae induced meningitis
b. E. coli induced meningitis
c. Neisseria meningitidis induced meningitis
d. Streptococcus pneumonae induced meningitis
e. LaCrosse encephalitis
2. Which of the following was a critical factor in your choice of diagnosis?
a. The fact the he is a wrestler and he does not eat or drink much.
b. The headache, vomiting, stiff neck, fever and purplish spots.
c. The party he went to with the cheerleaders.
d. His trouble hearing.
e. The cold symptoms.
3. Which of the following will likely happen if he is not treated?
a. He will continue to have a fever and stiff neck for a few days and recover.
b. His symptoms will progressively worsen until he develops shock and dies.
c. He may get better in a few days, but will likely have permanent hearing loss.
d. It is impossible to tell, because not enough data is available for an assessment of his
condition.
e. He will likely recover from the acute illness, but develop chronic arthritis later.
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Case Study B
A missionary couple, living in West Africa, brings their 4-year old son to the office of their physician
on the second day of a visit home to Minnesota. The boy had a mild episode of diarrhea about
seven days earlier and would not eat. He seemed to recover, but the mother noticed that the boy
was having trouble walking the previous night and had seemed to have trouble dressing himself and
walking that day.
On examination, the patient had no significant fever (98.9) and normal bowel sounds. His chest,
ears and eyes were clear. He had no rebound tenderness in the abdomen. The physician noted that
the child had poor muscular reflexes in his arms and legs. The child also was a bit lethargic and
seemed confused.
The family lives in an isolated village in Africa. The mother opposes vaccinations on personal
grounds, so the child has only had the initial series of DPT shots and no other typical vaccines. The
village where they live has many problems with parasites and insect borne fevers (including
Dengue).
Urine, stool and blood samples were collected for analysis.
1. This disease is most likely:
a. an intestinal infection or parasite.
b. a respiratory infection or parasite.
c. a neurological infection or parasite.
d. a urinary tract infection or parasite.
e. Not enough information is given to determine this.
2. My best guess at a diagnosis is:
a. Hib meningitis
b. sleeping sickness
c. polio
d. a parasitic worm
e. malaria
3. Which of the following samples could easily provide confirmation of your diagnosis?
a. The stool would show the presence of the worms.
b. The serum would have antibody against polio.
c. The urine would contain H. influenzae organisms.
d. The blood would contain red blood cells with malarial parasites.
e. Only CSF will show the answer.
4. Would receiving the childhood vaccine series have prevented this problem?
a. yes, without doubt
b. yes, most likely
c. possibly, but living in Africa the pathogen load might be too high
d. no, no vaccine is normally given
e. no way, no vaccine exists
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Assignment #7
Epidemiology
1. Define epidemiology.
2. What is the function of epidemiological studies and how are they applied?
3. Describe an example of an epidemiologic investigation.
http://www.epidemiolog.net/evolving/Introduction.pdf
Case Study
A 58-year old lawyer presents in the emergency room with headache, irritability, generalized muscle
pain and uncontrollable back spasms. He has become very restless and worried because he has
had the back spasms all through his court case that afternoon and they became extremely painful.
In his history, the lawyer states that he has a very busy practice. He is on medication for high blood
pressure (beta blocker) and has mild asthma. He injured himself about 10 days earlier, puncturing
his left arm with a nail from an old barn he is tearing. The wound has produced moderate quantities
of pus, but he has been keeping it clean. When asked, he did not remember having a tetanus shot
since he was a kid.
The wound was sampled for microscopic examination and culture. The back appears to have very
tight contractions and spasms. The patient is in obvious agonizing pain.
1.
2.
3.
4.
What is your diagnosis here?
What is the proper treatment of this problem?
How could this have been prevented?
If left untreated what are the possible consequences?
http://en.wikipedia.org/wiki/Tetanus
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Things to know / Study Guides
Resources to study from: Use your lab journal, homework assignments, handouts, Power Point
Presentations, and the Microbiology Lab Manual.
Note: - You will be expected to understand the concepts/topics covered in your homework
assignments for the quizzes.
- You will not be expected to know/memorize any chemical structure unless noted.

Safety and Routines - Know routines for best practices for labs.

Ex. 4 Microscopy
o Know answer to assigned Review Question from homework.
o Know the meaning of the bold words.
o Know parts of microscope, their location (Fig 4.1), and function.
o Know rules of microscope use.
o Know routine procedures for microscope use.

Ex. 1 Culture Transfer techniques
o Know answer to assigned Review Questions from homework.
o Know the complete procedure for subculturing (Fig 1.1).

Ex. 2A Isolation of pure culture
o Know answer to assigned Review Question from homework.
o Know the complete procedure for the streak-plate inoculation (Fig 2.1).

Ex. 3 Cultural Characteristics
o Know meaning of cultural characteristics.
o (Do not memorize each description- a chart will be provided for you.)

Ex. 2B Isolation of Pure culture
o Know answer to assigned Review Questions from homework.
o Know the complete procedure for preparation of pure culture (Fig 2.4).

Ex. 7 Prep of Smears
o Know rules and procedure (Fig 7.2) for preparing bacterial smears.
o Know answer to assigned Review Questions from homework.

Ex. 8 Simple Stain
o Know purpose for simple stain.
o Know terms for morphology and arrangements for bacterial cells (Fig 8.1)
o Know answer to assigned Review Questions from homework.
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
Ex.10 Gram Stain
o Use Color Plates 6 -11 from the middle of the manual as references.
o Know meaning and functions of words in bold.
o Know mechanisms of action of each reagent used.
o Know precautions for adequately preparing stained smears.
o Know Gram stain procedure steps.
o Know answer to assigned Review Questions from homework.

Ex.14 Selective and Differential media
o Understand the differences amongst the use and functions of each type of specialized media
(selective, differential, and enriched).
o Know chemicals and compounds added to the following media (and their functions): PEA,
mannitol salt, MacConkey, eosin-methylene blue, and blood agar.
o Know the results produced on each of the above media and how to interpret them.
o Know answers to assigned Observation and Results questions from this section.
o Know answer to assigned Review Question from homework.

Introduction to Part 5
o Understand the differences between exoenzymes and endoenzymes.
o Know the various purposes of conducting biochemical assays on microorganisms.

Ex. 21 Hydrolysis (Starch and Gelatin)
(omit lipid and casein sections)
o Understand the function of the microbial extracellular enzymes for starch and gelatin
hydrolysis.
o Know meaning and functions of words in bold in the sections above.
o Know chemicals and compounds in the media (and their functions) to the test for the above
assays.
o Know the results produced for each test and how to interpret them.

Ex. 22 Carbohydrate Fermentation
o Understand the difference between cellular respiration and fermentation.
o Understand the difference between aerobic, anaerobic, and facultative aerobic organisms.
o Understand (in general) the glycolic pathway.
o Know chemicals and compounds in the media (and their functions) to the test for the assay
for the various carbohydrates.
o Know the results produced for each test and how to interpret them.
o Know answer to assigned Review Questions from homework.
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
Ex. 23 Triple Sugar Iron Agar
o Understand the purpose of the TSI test.
o Know chemicals and compounds in the media (and their functions) to the test for these
assays.
o Know the procedure/technique used for this test.
o Know the results produced for each test and how to interpret them.
o Know answer to assigned Review Questions from homework.

Ex. 24Part A, Part D
o Be able to classify the organisms we are working with as enteric or non enteric.
o Know compound in the SIM agar (and its function) to test for indole production.
o Understand the role the role of Kovac’s Reagent to test for indole production.
o Understand the role of tryptophanase in the indole production assay.
o Know the compound in the media (and its function) to conduct the Citrate Utilization Test.
o Know the results produced for both the Indole Production and Citrate Utilization Test and
how to interpret them.
o Know answer to assigned Review Questions from homework.

Ex. 25 Hydrogen Sulfide Test
o Know the results produced for the Hydrogen Sulfide Test and how to interpret it.
o Know answer to assigned Review Questions from homework.

Ex. 26 Urease Test
o Know purpose for conducting this test.
o Know the result produced for this test and how to interpret it.
o Know answer to assigned Review Questions from homework.

Ex. 30 Oxidase Test
o Know the procedure and chemical used for this test.
o Know the result produced for this test and how to interpret it.
o Know answer to assigned Review Questions from homework.

Ex. 29 Catalase
o Know the function of catalase.
o Know the procedure and chemical used for this test.
o Know the result produced for this test and how to interpret it.
o Know answer to assigned Review Questions from homework.

Ex. 62 Coagulase
o Know the function of coagulase in microorganisms.
o Understand mechanism/principle, technique, and test interpretations for the following : MSA,
coagulase.
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Bio Lab 156
 Ex. 63 Streptococcal Pathogens
o Be familiar with the following terms only: hemolytic activity, Alpha-hemolysis,
Beta-hemolysis, Gamma-hemolysis, Bacitracin test
Lucia Testa

Ex.15 Temperature
o Understand the diverse growth temperature requirements of bacteria including cardinal
temperature points and classification groups.
o Know answers to assigned Review Questions from homework.

Ex.16 pH
o Understand the diverse pH requirements of microorganisms including the classification
groups.
o Know answer to assigned Review Questions from homework.

Ex.17 Oxygen requirements and Ex. 18 Anaerobic Microorganisms
o Understand the diverse atmospheric oxygen requirements of microorganisms especially
the five classification groups.
o Understand the purpose and function of the GasPack and thioglycollate media.
o Know answers to assigned Review Questions from homework.

Ex. 43 Chemotherapeutic Agents
o Understand the mode of action/mechanism of chemotherapeutic agents.
o Know the difference between antibiotics and synthetic drugs.
o Know the variables that determine the zone of inhibition.
o Understand the following terms: competitive inhibition and zone of inhibition.

Ex. 45 Disinfectants and Antiseptics
o (Not responsible for information on Table 45.1)
o Know what are antiseptics and disinfectants.
o Know the factors which influence the efficacy of antiseptics and disinfectants.

Ex. 65 Enterotube
o Know general procedure and interpretation involved in conducting the Enterotube II
Multitest System and ENCISE described
o Know answer to assigned Review Questions from homework.
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Microorganism Abbreviations












Bacillus subtilis
Bacillus stearothermophilus
Clostriduim sporogenes
Enterobacter aerogenes
Escherichia coli
Micrococcus luteus
Proteus vulgaris
Pseudomonas aeruginosa
Serratia marcescens
Staphylococcus aureus
Staphylococcus epidermidis
Streptococcus salivarius
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BS
BST
CS
EA
EC
ML
PV
PA
SM
SA
SE
SS
Bio Lab 156
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Journal entry and Notebook
Protocol for Microbiology
laboratory Experiments
Journal entry Protocol
Header: Exp. #, Title, Date.
•
•
Every page shall have the Header with: Exp. #. Title, date
Example: Header: Exp. 2 Handling & Examining Cultures
June 5, 2002
Purpose: The purpose and/or objective of the experimental activity, hypothesis, and premise. (Often
found at beginning of each experiment in lab manual.)
•
Example: Purpose: To make aseptic transfers of pure cultures and to examine them for gross features.
Materials: List cultures, media, & equipment for activities.
•
Example: Material: E. coli broth culture, B. cereus slant culture, inoculation loop, (2) TSA broth, (2)
TSA plates.
Procedures/Activity: briefly describe procedures, reference sections and page numbers of the lab
manual for specifics and details.
•
Example: Procedure: Obtained cultures from instructor, aseptically transferred cultures per
instructor’s inoculation scheme and Exp. 2. (Cappuccino and Sherman, pages 7-10) to TSA broth,
TSA plate, incubated at 37oC for 24 hrs, refrigerated until next period. Reviewed and recorded results.
Result: data, drawing, observations, tables of results of tests, details of results.
•
•
•
All diagrams of microscopic field should be 3 inch (5 cm) in diameter and labeled with color and
magnification.
Make drawing of cultures and/or slides. Make a table of results of tests.
Example: Results: record observation of cultures: colors, consistency, odors, appearance.
Conclusion: Restate objective, then make statements of fact about the data to support objective,
hypothesis, and premise.
• Example: Summary of results.
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Bio Lab 156
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Journal Notebook Protocol
1. Preparing the Journal:
a. On front cover and/or inside cover write you name, class, and information to find
you if journal is lost.
b. Number every page of book on the top right side only.
c. Label the top of the first six pages (on the right) as: “Table of Content”.
2. Table of Contents Section includes the following for each entry:
a. Experiment #
b. Experiment name
c. Page numbers
d. Dates of the work
3. Each Experiment Entry is written using the following protocol:
a. Entries are in written black ink pen.
b. Entries are made on right hand pages only. (Left hand pages are for class
notes, calculations, and instructions.)
c. Entries are in chronological order by date.
d. Entries are made directly to book (no scrap paper or other notebooks).
e. Each page header includes the following:
i. Experiment #
ii. Experiment name
iii. Date when the entry is made.
f. State sources for all literature or outside sources of information.
g. End of each page includes the following on the lower right hand corner:
i. Denote whether continuing (cont. on page #) or ending (end) experiment
entry.
ii. Your initials.
4. Blank space and mistakes protocol:
a. Do not leave blank page or blank areas on a page; draw a diagonal line through
the unused portion or pages. (Never rip out any pages.)
b. Cross out, do not erase or white out mistakes. Use a single strike through line
and initial the cross out.
5. Tables and handouts protocol:
a. Tables may be drawn in or taped in from a lab manual or spread sheet.
b. Hand outs with procedures, charts, reference data, etc. may be taped or stapled in
with the referenced author, book, etc. and date of entry.
c. Absolutely no loose or hanging out papers/tables/handouts/copies are allowed
in the journal.
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SELECTIVE & DIFFERENTIAL MEDIA
Study guide
TYPES OF MEDIA
All-Purpose Media: Media which are designed to support the growth of a wide
range of microorganisms. Examples of these include Tryptic Soy agar, Nutrient
agar, Brain Heart Infusion Agar, and Sabouraud Dextrose Agar which is used to
grow fungi.
Selective Media: Media which are formulated to allow some microorganisms to
grow, while inhibiting others from growing. This is accomplished
by including chemicals which will inhibit the growth of some
organisms while selecting for the growth of others.
Differential Media: Media formulated to allow the differentiation of microbes
based on differences in appearance of colonies or changes in color or opacity of the
medium. This is in response to the production of/ or inability to produce certain
enzymes which allows them to use a certain nutritive component (usually a sugar) in
the media.
Enriched Media: Media formulated with highly nutritious materials for the purpose
of growing certain organisms.
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MEDIA
Phenylethyl Alcohol Agar [PEA]: Selective only. For isolation of Gram positive
bacteria from mixed cultures.
1. Selects for Gram positive bacteria.
2. Components:
a. Phenylethyl alcohol – inhibits the growth of Gram negatives, thus selecting for Gram
positives
Mannitol Salt Agar [MSA]: Both selective and differential.
For isolation of Staphylococci from mixed cultures.
1. Selects for species of the genus Staphylococcus.
2. Differentiates between species which ferment mannitol and those which do not.
3. Components:
a. 7.5% NaCl – selects for species of Staphylococcus. This concentration of salt is too high
for most other bacteria and, therefore, inhibits their growth.
b. Mannitol – alcohol of the carbohydrate mannose. Mannitol fermentation produces acid end
products which turn the medium yellow. Yellow indicates mannitol+ and no color change
indicates mannitol-.
c. Phenol red pH indicator – yellow in acid pH (same indicator used in phenol red
carbohydrate fermentation broths).
4. Staphylococcus epidermidis produces colorless colonies with no change in the media because it
does not ferment mannitol.
Staphylococcus aureus produces yellow colonies with a yellow zone in the media surrounding
them because it ferments mannitol.
(Media continued on next page.)
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Bio Lab 156
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Media: Continued
MacConkey Agar [MAC]: Both selective & differential. For isolation of
Gram negative bacteria from mixed cultures.
1. Selects for Gram negative bacteria
2. Differentiates lactose fermenting from non-lactose fermenting bacteria.
3. Components:
a. Bile salts and Crystal Violet – these inhibit Gram positive bacteria, thus selecting for Gram
negatives
b. Lactose – some bacteria can ferment lactose to acid-end products, others cannot.
c. Neutral red pH indicator - * hot pink in acid pH
* rose in neutral pH
* tan in alkaline pH
4. Fermentation of lactose to acid end-products causes a drop in pH resulting in
hot pink colonies (lactose+). Organisms that do not ferment lactose will not
produce the hot pink color (lactose-).
5. Escherichia coli and Enterobacter aerogenes produce pink/red colonies with
pink/red
media surrounding it because they ferment lactose.
Pseudonomas aeruginosa and Proteus vulgaris produce colorless colonies because they do not
ferment lactose.
Eosin-methylene blue Agar [EMB] (Levine): Both selective and differential.
For isolation
of Gram negative bacteria from mixed cultures.
1. Selects for Gram negative bacteria.
2. Differentiates lactose fermenting bacteria from one another and from non-lactose fermenting
bacteria.
3. Components:
a. Lactose – a disaccharide which can be fermented by some bacteria to acid end products.
b. Eosin and Methylene Blue – dyes which inhibit the growth of most Gram positive bacteria.
They also react with any acid produced from lactose fermentation to color the colonies.
4. Acid production from lactose fermentation causes precipitation of the dyes on the surface of the
colony resulting in different colors.
a. Large amounts of acid  green metallic sheen
b. Small amounts of acid  pink
c. No fermentation  colorless
5. Enterobacter aerogenes produces colonies which are pink-to-buff around dark centers.
Escherichia coli produces dark colonies with a green metallic sheen.
Pseudonomas aeruginosa and Proteus vulgaris produce colorless colonies because they do not
ferment lactose.
Salmonella and Shigella are both pathogens which produce colorless colonies.
(Media continued on next page.)
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Media: Continued
Blood Agar: Both differential and all-purpose.
1. An enriched all-purpose medium which supports the growth of many types of
bacteria, while allowing for hemolytic differentiation.
2. Components:
a. 5% to 10% animal blood
3. Hemolysis:
a. Beta hemolysis results in complete lysis of erythrocytes in the medium resulting in a clear
area around the colonies.
b. Alpha hemolysis results in incomplete lysis of erythrocytes in the medium resulting in a
greenish area around the colonies.
c. Gamma hemolysis is a term used for nonhemolytic bacteria.
4. Staphylococcus epidermidis produces greenish halo around the colonies due to incomplete lysis of
red blood cells has occurred, which reduced hemoglobin to methemoglobin.
Staphylococcus aureus produces clear zone around the colonies due to complete lysis of red blood
cells.
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Unknown Microorganism Project
Grading Rubric:
Gram stains
SPD (technique & isolation in 3rd and 4th quadrant)
Tests/Choice of tests
Written Report (complete and typed)
4 points
4 points
4 points
4 points
16 points
EXAMPLE OF UNKNOWN REPORT:
IDENTIFICATION OF UNKNOWN MICROORGANISM # ________
Name:
Date:
Course number & section:
Abstract: Include short statements (kind of like a summary) of the objective, procedure, data, and
conclusion. Last sentence should say: Unknown #_______ is: Gram positive cocci (or whatever color and
shape/arrangement); Genus family. (Name of bacteria should be written correctly [Example- in italics with
Genus capitalized and species in lower case Staphylococcus aureus.)
Introduction: State the objective of the experiment.
Procedures:
1. Used Streak Plate Technique to isolate organism (*Cappuccino & Sherman, pg ?).
2. Gram stained organism (*Cappuccino & Sherman, pg ?).
3. Selected and performed the following Media & Biochemical Tests:
List in a bulleted format performed- make sure to reference them to the journal.
4. Draw a the logic flow (tree-style) chart of how you selected the media & tests you conducted.
(can be hand drawn)
Data:
1. Gram stain was (positive or negative) and the shape was (cocci or bacillus).
2. Drawing of organism (1000X) in color. (can be hand drawn)
3. Results of above tests. List in a bulleted format.
Discussion and Conclusions:
Based on the procedures x, y, and z (name what you did), unknown #___ is: Gram positive cocci; Alpha beta.
(This is the section where you may explain your choice of media and tests, and/or explain your thought
process for deciding on the organism that you did and any other information that may pertain to this report.)
Name
Signature
Date
References: * Full references
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