A Detailed Lesson Plan I.

OBJECTIVES: Given a set of activities, the students should be able to attain at least 75% proficiency and should be able to: A. describe staining; B. enumerate the common stains; C. differentiate between an acidic dye and a basic dye; D. compare simple, differential and special stains; and E. list the steps in preparing a Gram stain and describe the appearance of a gram-positive and gram- negative cells after each step.\ II. SUBJECT MATTER: Biological Techniques Topic: Staining A. Materials: cartolina- used during the activity in lesson proper(flashcards) iodine- used in the activity in application glass slide- used in the activity in application(wet mount) cover glass- used in the activity in application(wet mount) ball of cloth- used in the activity in motivation manila paper- used in the activity in lesson proper(table) improvised canal- used in the activity in motivation mud- used in the activity in motivation B. References: Tortora, Geral J., Funke, Berdell R., and Case, Christine L., Microbiology (An Introduction) fifth edition, the Benjamin/ Cummings Publishing Company, Inc., pages 61-66. Internet websites: Bruckner, Monica Z., Basic Cellular Staining. serc.carleton.edu, April 14, 2013 http://serc.carleton.edu/microbelife/research_methods/microscopy/cellstain.html ANTRANIK, Stain uses, types and applications.antranik.org, March 11, 2012 http://antranik.org/stain-uses-types-and-applications/ Staining Techniques, from http://www.cliffsnotes.com/study_guide/StainingTechniques.topicArticleId8524,articleId-8413.html Bismarck Brown,May 2005, from http://stainsfile.info/StainsFile/dyes/21000.htm Mifflin,Houghton.,Papanicolaoustain.http://medicaldictionary.thefreedictionary. com,2007 from http://medical-dictionary.thefreedictionary.com/Papanicolaou+stain

Abedon,StephenT.,MicroscopyandStaining.mansfield.ohiostate.edu,3/28/2003 from http://www.mansfield.ohiostate.edu/~sabedon/black03.htm III. PROCEDURE A. Motivation Teachers Activity

Students Activity Good afternoon sir!

Good afternoon class! We will play a game .I will give you a ball of cloth. All you have to do is to roll the ball of cloth into the improvised canal with mud. Who wants to volunteer? What did you observe when you rolled the ball of cloth into the improvised canal with mud? Yes Mark. Thats right! After you observed that the ball of cloth got stained, what is staining then? Yes Abegail. So what do you think is the purpose of staining? Yes Sharmaine. Very good! Staining is the application of stain to a sample to color cells, tissues components or metabolic processes. This process may involve immersing the sample (before or after fixation or mounting) in a dye solution and then rinsing and observing the sample under a microscope. B. Lesson Proper There are several types of staining media, each can be used for different purpose. All these stains may be used on fixed, or non-living cells, and those that can be used on living cells.

(students will raise their hands)

The ball of cloth got stained. Staining simply means coloring the microorganisms with a dye. It emphasizes certain structures sir.

I have prepared an activity; on the board are functions of common stains. Your job

is to determine what stain the function belongs to. You have to plot/paste the common stains. I have prepared strips (common stains). You get three guesses, if you guess correctly you get five extra points. You can ask for a clue but fore very clue, you give up one extra point.

Functions/uses 1. Used in gel electrophoresis. 2. Used in Gram Staining. 3.Stains animal cells to make nuclei more visible 4. Used as a starch indicator. 5. A protein-specific fluorescent stain used in fluorescence microscopy. 6. A nuclear stain used as a counterstain or to color collagen yellow. 7. Used to detect proteins after electrophoretic separation on polyacrylamide gels. 8. It is frequently used method for examining cell samples from various bodily secretions. 9. Colors acid mucins, a type of protein yellow and maybe used to stain live cells. 10. A nuclear stain that with a mordant stains nuclei blue-violet or brown. Will you read function number 1? What do you think is that stain? (clue: this stain sounds like the word Coco Martin on the first word and the second word is a color) Very good! Its Coomasie blue. This

(the student then will read the function no.1) (the student then will answer number 1) Coomasie blue

stains proteins a brilliant blue. Will you read function number 2? What do you think is that stain? (clue: this stain is frequently used by the fortune tellers and the second word is a dark color) Thats right! Its Crystal violet. This it stains cell walls purple when combined with a mordant.

(the student then will read the function no.2) (the student then will answer number 2) Crystal violet

Will you read function number 3? What do you think is that stain? (clue: in the first word of this stain, it begins with letter M and the second word is also color) Very good! Its Methylene blue. It is a dark green dye used as a stain, an antiseptic, a chemical indicator, and an antidote in cyanide poisoning. Will you read function number 4? What do you think is that stain? (clue: this stain is used to stain cheek cells in zoology) Thats right! Its Iodine. When in solution starch and iodine form a dark blue color. Will you read function number 5? What do you think is that stain? (clue: this stain is only one word and the first letter begins with letter R) Very good! Its Rhodamine. This stain used especially in coloring paper and as a biological stain. Will you read function number 6? the student then will read the function no.5) (the student then will answer number 5) Rhodamine (the student then will read the function no.4) (the student then will answer number 4) Iodine (the student then will read the function no.3) (the student then will answer number 3) Mehtylene blue

What do you think is that stain? (clue: this stain is only one word and the last letter is S) Yes. Its Safranin. This stain is any of various usually red synthetic dyes that are amino derivatives of bases. Will you read function number 7? What do you think is that stain? (clue: the first word is a shining shimmering color) Very good! Its Silver staining. This is used to show both substances inside and outside cells and use of silver to stains histologic sections Will you read function number 8? What do you think is that stain? (clue: the first word is sounds like the brand for ketchup) Thats right! Its Papanicolaou staining. It is frequently need to stain Pap smear specimen. Will you read function number 9? What do you think is that stain? (clue: the first word is sounds like biscuit and the second word is like the color of a tree) Very good! Its Bismarck Brown. It is a brown basic diazo dye that is used as a biological stain. And lastly no. 10. Will you read the function number 10? What do you think is that stain? (clue: this stain begins with letter H) Very Good! Its Hematoxylin. It is used chiefly as a biological stain because of its ready oxidation to hematein.

the student then will read the function no.6) (the student then will answer number 6) Safranin

the student then will read the function no.7) (the student then will answer number 7) Silver staining

the student then will read the function no.8) (the student then will answer number 8) Papanicolaou staining

the student then will read the function no.9) (the student then will answer number 9) Bismarck Brown

(the student then will read the function no.10) (the student then will answer number 10) Hematoxylin

I want you to be divided to form two groups. Please count off 1 and 2. Now all 1s please stand up and go to this right side. All 2s please stand up and go to this left side. Select a leader and secretary in your group. The secretary will list the names of the members of each group. You and your groupmates will be playing a game. This game is called arrange me seconds to win it! You only have 20 seconds to arrange the jumbled letters. I will read the questions twice. If you got the answers, you will receive prices and points for every stage. And now, are you ready to play the game! This is your first question: 1. Stains are salts composed of a positive and a negative ion one which is colored and is known as the _________. (The game begins in 3, 2, 1) go. Very good! Next question. 2. It is a positive ion that is attracted to the negatively charged bacterial cell. (The game begins in 3, 2, 1) go. Thats right! Were going down to third question. 3. This is not attracted to most types of bacteria because the dyes negative ions are repelled by the negatively charged bacterial surface. (The game begins in 3, 2, 1) go. Very good! And now were going down to our last question. This is a safety level. The group who will be the first to arrange the jumbled letters and give the correct answer will be given 6 pieces of glass slide and cover glass plus 10 points and Yes sir! (The students then will arrange the jumbled letters for 20 seconds) answer would be Chromophore. (1,2, 1, 2 up to the last student)

(The students then will arrange the jumbled letters for 20 seconds) answer would be Basic dyes.

(The students then will arrange the jumbled letters for 20 seconds) answer would be Acidic dyes.

the remaining group will be given at least a price for their efforts. 4. It is a method of demonstrating the form of small objects (as bacteria) by surrounding them with a stain that they do (The students then will arrange the not take up so that they appear as jumbled letters for 20 seconds) answer sharply outlined unstained bright bodies would be Negative staining. on a colored ground. (The game begins in 3, 2, 1) go. Very Good! Thank you. Please go back to your seats. Did you enjoy the game? What are the three kinds of staining techniques? Yes. Jarolyne? Will you describe simple stains? Yes. Cristina? Good. And what is its purpose? Yes.Geralden? Yes Sir! Simple, differential and special stains sir. It is an aqueous or alcohol solution of a single basic dye. The primary purpose of a simple stain is to highlight the entire microorganisms so that the cellular shapes and basic structures are visible. Mordant sir. One function of a mordant is to increase the affinity of a stain for biological specimens.

Very good! And what chemical is added to the solution to intensify the stain? Yes. Jonalyn? Yes. And what is the function of mordant? Evelyn? Very good! And another function of a mordant is to coat a structure to make it thicker and easier to see after it is stained with dye. Next staining techniques are the differential techniques. This technique distinguishes two kinds of organisms. What are they? Yes. Sharmaine?

Gram stain and acid-fast stain sir. Very good! By the year 1884, who discovered the Gram stain? Yes. Cherry anne? Hes a Danish bacteriologist Hans Christian Gram sir.

Yes. Thats right! Why is it considered to be one of the most useful staining procedures? Yes. Glaiza? It is one of the most useful staining procedures because it divides bacteria into 2 large groups, gram-positive and gramnegative. It is used to differentiate species of mycobacterium from the other bacteria. It is used to color and isolate specific parts of microorganisms such an endospores and flagella and to reveal the presence of capsules The application of crystal violet sir.

The dye is washed off. Sir. The application of iodine sir.

Both are purple. The slide is washed with ethanol or an alcohol-acetone solution and then washed with water. The gram-positive cells are purple and gram-negative cells are colorless. Safranin is added as a counterstain and the slide is washed, dried and examined microscopically. Gram-positive retain the purple dye even though the alcohol wash. Gram-negative

appear pink. Very good! And what is an acidic fast stain? Yes. Jinky? Very good! And lastly is the special stains. What is the use of special stains? Yes. Lovelyn? Gram-negative appear pink because the pick up the safranin (counterstain).

What are the steps in preparing a Gram stain? Yes. Mark? Very good! A fixed smear of cocci and rods is first covered with a basic dye such as the crystal violet. And what will happen next? Yes. Chideth? Yes. And what is the next step? Abegail? Thats right! The smear is covered with iodine and washed off. What would be the appearance of iodine(mordant) in a grampositive and gram-negative cells? Yes. Jonabelle? Very good! What will happen next? Yes. Glaiza? Yes thats right! What would be the appearance of decolorization of grampositive and gram-negative cells? Yes. Sharmaine? Very good! And lastly, what is the final step? Yes. Jarolyne? Yes. What would be the appearance then? Cristina? Why does the Gram-negative appear pink? Yes. An-an? C. Generalization

Okay, Before going on to our next activity, None sir. does anyone have any questions? Okay. Please summarize then what we tackled today. We were introduced to staining which is Staining is the application of stain to a sample to color cells, tissues components or metabolic processes. This process may involve immersing the sample (before or after fixation or mounting) in a dye solution and then rinsing and observing the sample under a microscope. We came to answer in the table from which we will paste the common stains beside the description or their uses. And we came to differentiate between acidic dyes and basic dyes by playing the game. The color of so-called basic dyes is in the positive ion; in acidic dyes, it is in negative ion. We came also to compare the simple, differential, and simple stains. In the steps in preparing Gram stain: first is the application of crystal violet then the appearance would be both purple. 2ndstep is the application of iodine (mordant) and the appearance of gram-positive and gram-negative are both purple. Next step that the slide is washed with ethanol or an alcohol-acetone solution and then washed with water. The gram-positive cells are purple and gram-negative cells are colorless. And lastly the application of safranin. Gram-positive retain the purple dye even though the alcohol wash. Gramnegative appear pink.

Anyone else?

Very good class. D. Application Please get into the same group again. You and your groupmates will be creating your own wet mount slide. I will give you 20 minutes to finish the activity work wisely and every group should have a final product by the end of the 20 minutes. If you have any questions, you

may approach me anytime. I will be going around as you do your work. Your output will be counted as a test.( see attached activity) Did you understand the procedure?

(The students will do then the wet mount for 20 minutes)

IV. Evaluation Matching type. Match the descriptions in column A with the stain in column B. Write the letter of your answer on the spaces provided before the items. Column A ____1. It is the application of stains to a sample to color cells, tissues, components or metabolic process. ____2. Used to color and isolate specific parts of microorganisms. ____3. Its purpose is to highlight the entire microorganisms so that the cellular shapes and basic structures are visible. ____4. It was developed in 1884 and one of the most useful staining procedures. ____5. It is commonly used as a starch indicator. ____6. It is used to show both substances inside and outside cells and use of silver to stains histologic sections. ____7. Used chiefly as a biological stain because of its ready oxidation to hematein. ____8. A nuclear stain used as a counterstain or to color collagen yellow. ____9. It is frequently need to stain Pap smear specimen. ____10. Used especially in coloring paper and as a biological stain V. Assignment Please do a research on the following: 1. What is fixation? 2. What are the fixation techniques? 3. Enumerate the preparation for fixation. Column B

A. Gram Stain B. Hematoxylin C. Iodine D. Papanicolaou staining E. Rhodamine F. Safranin G. Silver staining H. Simple stain I. Special stain J. Staining

Preparing a Wet Mount Slide

A wet mount slide will allow you to observe preserved or live material under the microscope. Follow the instructions below to prepare a wet mount for the specimen you wish to observe. Materials: Clean, dry microscope slide Water (or liquid) Specimen to be observed Cover Slip Stain (if required) 1. Using a pipette or dropper bottle, place a drop or two of water on a clean, dry slide.(If the specimen you wish to observe is in a liquid environment, you do not need to do this step.) 2. Place your specimen in the drop of water. 3. Place a cover slip over the water and your specimen by holding the cover slip at a 45 degree angle and slowly lowering the cover slip on to the specimen. (Applying the cover slip in this way should prevent air bubbles from forming under your cover slip.) Assessment Rubric: Criteria Excellent (109) -Successfully created a wet mount slide without any help Satisfactory (8-7) -Created a wet mount slide with minimal amount of help Limited (6-5) Unsatisfactory (<5 ) -Was not successful at creating a wet mount slide

Context

-Created a wet mount slide with a considerable amount of help

http://www.scs.sk.ca/cyber/elem/learningcommunity/sciences/biology20/curr_content/bi ology20/wet_mount_slide.htm

Mariano Marcos State University COLLEGE OF TEACHER EDUCATION Laoag City

A Detailed Lesson Plan In Biotechniques

Topic: Staining

Prepared by: Ronel M. Batangan BSE-IIIC

Submitted to: Prof. Elsie R. Marcelino