Download UNIT 2 SEMINAR: Observation of Microorganisms Evelyn I. Milian

Fundamentals of Microbiology
UNIT 2 SEMINAR:
Observation of Microorganisms
Evelyn I. Milian
Instructor
2011
Microbiology: Unit 2 Seminar – Observation of Microorganisms
Agenda
1.
Discussion of unit 2 seminar topic:
 Observation of microorganisms
2.
Question and answer session (~last 5 minutes):
 Course syllabus, assignments, grading, requirements.
 * Students in sections with other instructors are also
invited to stay for this session; however, specific
questions about assignments or grading should
be addressed to your instructor.
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Questions Assigned for Discussion (Unit 2 Seminar Page)
1.
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Give 3 examples of safety procedures you should always follow in the
microbiology lab.
Why do you stain cells before observing them under the microscope?
 Name some common shapes and arrangements observed when viewing
bacteria under a microscope.
In a Gram stain, one step could be omitted and still allow differentiation
between gram positive and gram negative cells. What is that one step?
Assume that you are viewing a Gram-stained sample of vaginal discharge.
Large (10um) nucleated red cells are coated with small (0.5 um X 1.5 um) bluepurple cells on their surfaces. What is the most likely explanation for the red
cells and blue cells?
A sputum sample from Calle, a 30-year-old Asian elephant, was smeared onto
a slide and air dried. The smear was fixed, covered with carbolfuchsin, and
heated for 5 minutes. After washing with water, acid alcohol was placed on the
smear for 30 seconds. Finally, the smear was stained with methylene blue for
30 seconds, washed with water, and dried. On examination at 1000X, the zoo
veterinarian saw red rods on the slide. What infection do the results
suggest? (Calle was treated and recovered.)
 Discuss 2 other examples of staining techniques, and when they are used.
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Basic Safety Procedures in the Microbiology Lab

The microbiology laboratory is unique in many ways
compared to other biological laboratories. While safety is
paramount in any lab, the added complication of unseen
contaminants and potential hazards must be addressed.

Microorganisms are normally present in the environment,
and many kinds are also found on and in the human body.

Improper handling of chemicals, equipment and/or microbial
cultures is dangerous and can result in injury or infection.

Avoiding contamination and possible infection warrants
stringent safety practices at all times.
1. Give 3 examples of safety procedures you should
always follow in the microbiology lab.
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Safety in the Microbiology Lab is Priority!
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Basic Safety Procedures in the Microbiology Lab

Do not eat, drink, smoke, or bring food or drinks into the lab room.
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Do not apply cosmetics or handle contact lenses in the lab.

Wear protective clothing (i.e., a lab coat) and closed-toed shoes in lab.

Tie back long hair, as it is a potential source of contamination as well as a
likely target for fire.

Do not work with an uncovered open cut or wound. Protect it with a
bandage and wear plastic gloves.

Wipe your work area or bench with disinfectant before and after work.

Keep all sources of possible contamination out of your mouth and hands:
pencils, laboratory ware and utensils, and other items.
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Discard contaminated items—pipettes, Petri dishes, test tubes, and other
items—in the designated containers (many times labeled “biohazard”).

Practice aseptic technique at all times when dealing with microbial cultures.
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Wash your hands thoroughly for 20 seconds with soap and water after
handling living microbes and before leaving the laboratory.
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Laboratory Safety Items and Equipment
2010
Biology I - Lab Te st 1 - Prof. Eve lyn I . Milian
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Staining Techniques to Observe Microorganisms

Observing microorganisms also presents a special challenge
to microbiologists, and to answer that challenge there are a
vast array of staining techniques available to enhance
observations. There are special staining techniques that take
the physiology of the target cells into account.
1. Why do you stain cells before observing them under the microscope?

Name some common shapes and arrangements observed when
viewing bacteria under a microscope.
2. In a Gram stain, one step could be omitted and still allow differentiation
between gram positive and gram negative cells. What is that one step?
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Staining Techniques to Observe Microorganisms

Staining increases contrast.
 Contrast is the effect of a striking difference, as in color
or tone, of adjacent parts (for example, in a photograph or
image). It is based on the differential absorption of light by
parts of the specimen.
 Microscopists improve contrast by coloring specimens
with stains (dyes) that bind to cellular structures and
absorb light to provide contrast.
 Live or unstained cells have little contrast with the
surrounding medium. However, researchers do make
discoveries about cell behavior looking at live specimens.
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Bacterial Shapes and Arrangements

Most common shapes
1) Bacillus (rod-shaped; bacilli in plural)
2) Coccus (spherical; cocci in plural)
3) Spiral (corkscrew or curved)

There are variations to these basic
shapes.
 Coccobacillus (oval-shaped)

Other less common shapes:
 Square
 Star-shaped

Cell arrangements: The cells may
form groups.
 Pairs
 Chains
 Clusters

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Average size: 0.2-2.0 µm wide  2-8
µm long
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Prokaryotic Cells: Bacterial Shapes

Bacillus (plural: bacilli)
 Rod-shaped cells.
 Arrangements: pairs, chains.
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Prokaryotic Cells: Bacterial Shapes

Coccus (plural: cocci)
 Spherical cells
 Groups arrangements:
 Diplococci (pairs)
 Streptococci (chains)
 Tetrad (4 cells in cube)
 Sarcinae (8 cells in cube)
 Staphylococci (grapelike
clusters of many cells)
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
The Gram Stain: A Differential Stain

2011
Eve lyn I . Milian - I nstructor
In a Gram stain,
one step could be
omitted and still
allow differentiation
between gram
positive and gram
negative cells.
What is that one
step?
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Differential Stains: Gram Stain

In a Gram stain, one step
could be omitted and still
allow differentiation
between gram positive
and gram negative cells.
What is that one step?

Answer:
 The counterstain (or
secondary stain) =
safranin.
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
The Gram Stain and the Bacterial Cell Wall

In the Gram staining technique, samples are first stained with crystal violet dye
and iodine (a mordant that helps retain the stain), then rinsed with alcohol, and
finally counterstained with a red dye such as safranin. * The structure of a
bacterium’s cell wall determines the staining response. *
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
The Prokaryotic Cell Envelope: Bacterial Classification According to
Their Cell Wall Structure and Gram Stain Properties
GRAM-POSITIVE BACTERIA
GRAM-NEGATIVE BACTERIA
Envelope with two layers: cell wall and cell Envelope with three layers: outer
(cytoplasmic) membrane.
membrane, cell wall and cell membrane.
Retain the Gram stain (crystal violet) due
to their cell wall structure; purple-colored
(which is applied first in the procedure).
Do not retain the Gram stain (crystal
violet) and will appear pink-red after the
counterstain (safranin) is applied.
Cell wall with thick (multilayered)
peptidoglycan, containing teichoic acids.
Cell wall with thin (single-layered)
peptidoglycan layer. No teichoic acids.
No outer membrane; no LPS, low lipid and
lipoprotein content.
Protective outer membrane with LPS
(lipopolysaccharides), lipoproteins, and
phospholipids.
Their cell wall is almost completely
destroyed by lysozyme (a digestive
enzyme in eukaryotic cells, found in
mucus, saliva and tears).
Their cell wall usually is not destroyed by
lysozyme to the same extent as in grampositive cells; some of the outer membrane
also remains.
Highly susceptible to penicillin and
sulfonamide (antimicrobial agents).
Low susceptibility to penicillin and
sulfonamide.
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Staining Techniques: Clinical Applications (Chapter 3)

Assume that you are
viewing a Gram-stained
sample of vaginal
discharge. Large (10um)
nucleated red cells are
coated with small (0.5 um
x 1.5 um) blue-purple
cells on their surfaces.

What is the most likely
explanation for the red
cells and blue cells?
2011
Eve lyn I . Milian - I nstructor
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Staining Techniques: Clinical Applications (Chapter 3)

The large red cell is an epithelial
cell from the vaginal tissue.

Blue-purple cells are grampositive bacteria; there are also
some gram-variable bacilli
(Gardnerella vaginalis).

This is bacterial vaginosis (BV),
an infection caused by
Gardnerella vaginalis.

Vaginosis is most likely a result
of a shift from a predominance of
“good bacteria” (lactobacilli) in
the vagina to a predominance of
“bad bacteria”.
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

Gram stain of sample of
vaginal discharge
Note that there are 2 white
blood cells (left bottom).
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Staining Techniques: Clinical Applications (Chapter 3)

A sputum sample from Calle, a 30-year-old Asian elephant, was smeared
onto a slide and air dried. The smear was fixed, covered with carbolfuchsin,
and heated for 5 minutes. After washing with water, acid alcohol was
placed on the smear for 30 seconds. Finally, the smear was stained with
methylene blue for 30 seconds, washed with water, and dried. On
examination at 1000X, the zoo veterinarian saw red rods on the slide.
 What infection do the results suggest? (Calle was treated and recovered.)
2011
Eve lyn I . Milian - I nstructor
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Staining Techniques: Clinical Applications (Chapter 3)


Figure 3.13 Acid-fast bacteria.
The Mycobacterium bacteria that have
infected this tissue have been stained red with
an acid-fast stain. Non–acid-fast cells are
stained with the methylene blue counterstain.
2011
Eve lyn I . Milian - I nstructor
The acid-fast stain binds
strongly only to bacteria that
have mycolic acid in their
cell walls, a waxy material.
This stain is used to identify
all bacteria in the genus
Mycobacterium, including
Mycobacterium tuberculosis,
the causative agent of
tuberculosis, and
Mycobacterium leprae,
cause of leprosy. This stain
is also used to identify the
pathogenic strains of the
genus Nocardia. Bacteria in
the genera Mycobacterium
and Nocardia are acid-fast.
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Special Staining Techniques
2011

Negative staining – capsules:
using India ink or nigrosin for
background and safranin; capsules
appear as halos surrounding each
stained bacterial cell (red).

Endospore staining: SchaefferFulton: malachite green and heat
to help stain penetrate endospore;
safranin for other cellular parts.

Flagella staining: carbolfuchsin
and a mordant to make flagella
wide enough to be seen.
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Questions???
INSTRUCTOR INFORMATION
Instructor and Credentials
Evelyn I. Milian, M.S.; Microbiology
Kaplan Email Address
[email protected]
AIM (AOL Instant
Messenger) Name
milianevelyn
AIM Office Hours
By appointment. Contact me by email or
AIM at the above addresses to set one up.
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2011
Please keep your Course Syllabus handy and use it as your
guide throughout the entire term.
Read all sections of the syllabus and make sure that you
understand them.
Do not hesitate to contact me by e-mail or AIM as soon as
you have a question.
* Students in other sections: Please contact your instructor
for specific questions about assignments or grading.
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
Some Recommendations to be Successful in the Course
1.
2.
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6.
7.
8.
9.
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Read your e-mails and course announcements every day.
Work consistently and stay caught up; make a plan and stay organized.
Post early and often to the Discussion Board.
Keep up with the assignments and projects.
Come prepared to the weekly seminars and participate meaningfully
throughout the entire hour.
Always support all your work with complete APA style references and
in-text citations and rely on quality literature resources.
Avoid copying and pasting anything longer than a line or two from any
given source; posts and projects must always be made up of your own
words, supported by literature sources properly cited.
Make sure that you meet the learning activity submission deadlines in
each Unit. Units start on Wednesdays each week and end at 11:59 PM
Eastern Time the following Tuesday.
Use all Kaplan resources available to help you in your courses.
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Microbiology: Unit 2 Seminar – Observation of Microorganisms
References

Alters, Sandra & Alters, Brian. (2006). Biology, Understanding Life. John Wiley & Sons,
Inc. NJ, USA.

Audesirk, Teresa; Audesirk, Gerald & Byers, Bruce E. (2005). Biology: Life on Earth.
Seventh Edition. Pearson Education, Inc.-Prentice Hall. NJ, USA.

Black, Jacquelyn G. (2005). Microbiology, Principles and Explorations. Sixth Edition.
John Wiley & Sons, Inc. NJ, USA. www.wiley.com/college/black.

Campbell, Neil A.; Reece, Jane B., et al. (2008). Biology. Eighth Edition. Pearson
Education, Inc.-Pearson Benjamin Cummings. CA, USA.

Cowan, Marjorie Kelly; Talaro, Kathleen Park. (2009). Microbiology A Systems Approach.
Second Edition. The McGraw-Hill Companies, Inc. NY, USA. www.mhhe.com/cowan2e

Dennis Kunkel Microscopy, Inc. (2010). http://www.denniskunkel.com

Mader, Sylvia S. (2010). Biology. Tenth Edition. The McGraw-Hill Companies, Inc. NY, USA.

Tortora, Gerard J.; Funke, Berdell R.; Case, Christine L. (2010). Microbiology An
Introduction. Tenth Edition. Pearson Education, Inc.-Benjamin Cummings; CA, USA.
www.microbiologyplace.com.
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