UNIT 2 SEMINAR: Observation of Microorganisms Evelyn I. Milian Instructor 2011 Fundamentals of Microbiology

UNIT 2 SEMINAR:Observation of Microorganisms

Evelyn I. MilianInstructor

2011

Fundamentals of Microbiology

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.

2011 Evelyn I. Milian - Instructor 2


Questions Assigned for Discussion (Unit 2 Seminar Page)

1. Give 3 examples of safety procedures you should always follow in the microbiology lab.

2. Why do you stain cells before observing them under the microscope? Name some common shapes and arrangements observed when viewing

bacteria under a microscope.3. 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?4. 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?

5. 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.



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.



Safety in the Microbiology Lab is Priority!



Basic Safety Procedures in the Microbiology Lab

Do not eat, drink, smoke, or bring food or drinks into the lab room. 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. 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. Wash your hands thoroughly for 20 seconds with soap and water after

handling living microbes and before leaving the laboratory.



2010 Biology I - Lab Test 1 - Prof. Evelyn I. Milian 7

Laboratory Safety Items and Equipment


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?



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.



Bacterial Shapes and Arrangements

Most common shapes1) 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

Average size: 0.2-2.0 µm wide 2-8 µm long

102011 Evelyn I. Milian - Instructor


Prokaryotic Cells: Bacterial Shapes


Bacillus (plural: bacilli) Rod-shaped cells.

Arrangements: pairs, chains.


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)

12Evelyn I. Milian - Instructor


2011 13Evelyn I. Milian - Instructor


The Gram Stain: A Differential 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?



2011 15Evelyn I. Milian - Instructor


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.



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. *



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 (cytoplasmic) membrane.

Envelope with three layers: outer 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 gram-positive cells; some of the outer membrane also remains.

Highly susceptible to penicillin and sulfonamide (antimicrobial agents).

Low susceptibility to penicillin and sulfonamide.



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?



Gram stain of sample of vaginal discharge

Note that there are 2 white blood cells (left bottom).


The large red cell is an epithelial cell from the vaginal tissue.

Blue-purple cells are gram-positive 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”.



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.)




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.


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.


Special Staining Techniques

Negative staining – capsules:

using India ink or nigrosin for

background and safranin; capsules

appear as halos surrounding each

stained bacterial cell (red).

Endospore staining: Schaeffer-

Fulton: 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.



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 HoursBy appointment. Contact me by email or AIM at the above addresses to set one up.

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.



Some Recommendations to be Successful in the Course

1. Read your e-mails and course announcements every day.

2. Work consistently and stay caught up; make a plan and stay organized.

3. Post early and often to the Discussion Board.

4. Keep up with the assignments and projects.

5. Come prepared to the weekly seminars and participate meaningfully throughout the entire hour.

6. Always support all your work with complete APA style references and in-text citations and rely on quality literature resources.

7. 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.

8. 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.

9. Use all Kaplan resources available to help you in your courses.



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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UNIT 2 SEMINAR: Observation of Microorganisms Evelyn I. Milian Instructor 2011 Fundamentals of Microbiology