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Gram Staining

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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.

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, 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. topicArticleId-8524,articleId-8413. html Bismarck Brown,May 2005, from http://stainsfile. info/StainsFile/dyes/21000. htm Mifflin,Houghton. ”Papanicolaoustain. ”http://medicaldictionary. thefreedictionary. cHYPERLINK “http://medical-dictionary. thefreedictionary. com/”om,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 |Teacher’s Activity |Student’s Activity | |Good afternoon class! Good afternoon sir! | | | | |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? | | |(students will raise their hands) | |What did you observe when you rolled the ball of cloth into the | | |improvised canal with mud? Yes Mark. | | | | | |That’s right!

After you observed that the ball of cloth got stained, |The ball of cloth got stained. | |what is staining then? Yes Abegail. | | | | | |So what do you think is the purpose of staining? Yes Sharmaine. |Staining simply means coloring the microorganisms with a dye. | | | | |Very good!

Staining is the application of stain to a sample to color | | |cells, tissues components or metabolic processes. This process may |It emphasizes certain structures sir. | |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. | | | | | |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. |(the student then will read the function no. ) | | |(the student then will answer number 1) Coomasie blue | |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. |(the student then will read the function no. 2) | | |(the student then will answer number 2) Crystal violet | |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 |(the student then will read the function no. ) | |and the second word is a color) |(the student then will answer number 3) | | |Mehtylene blue | |Very good! It’s Coomasie blue. This 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) |(the student then will read the function no. ) | | |(the student then will answer number 4) | |That’s right! It’s Crystal violet. This it stains cell walls purple |Iodine | |when combined with a mordant. | | | | | |Will you read function number 3? | | | | |What do you think is that stain? |the student then will read the function no. 5) | |(clue: in the first word of this stain, it begins with letter M and |(the student then will answer number 5) | |the second word is also color) |Rhodamine | Very good! It’s 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? | | |the student then will read the function no. 6) | |What do you think is that stain? |(the student then will answer number 6) | |(clue: this stain is used to stain cheek cells in zoology) |Safranin | | | | |That’s right!

It’s Iodine. When in solution starch and iodine form a | | |dark blue color. | | |Will you read function number 5? | | | |the student then will read the function no. 7) | |What do you think is that stain? (the student then will answer number 7) | |(clue: this stain is only one word and the first letter begins with |Silver staining | |letter R) | | |Very good! It’s 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. 8) | | |(the student then will answer number 8) | |What do you think is that stain? Papanicolaou staining | |(clue: this stain is only one word and the last letter is S) | | |Yes. It’s 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? |the student then will read the function no. 9) | |(clue: the first word is a shining shimmering color) |(the student then will answer number 9) | |Very good!

It’s Silver staining. This is used to show both substances|Bismarck Brown | |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) |(the student then will read the function no. 0) | | |(the student then will answer number 10) | |That’s right! It’s Papanicolaou staining. It is frequently need to |Hematoxylin | |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! It’s Bismarck Brown. It is a brown basic diazo dye that is|(1,2, 1, 2… up to the last student) | |used as a biological stain. | | |And lastly no. 0. Will you read the function number 10? | | |What do you think is that stain? | | |(clue: this stain begins with letter H) | | |Very Good! It’s Hematoxylin. It is used chiefly as a biological stain| | |because of its ready oxidation to hematein. | | | | |I want you to be divided to form two groups. Please count off 1 and | | |2. | | |Now all 1’s please stand up and go to this right side.

All 2’s please| | |stand up and go to this left side. Select a leader and secretary in |Yes sir! | |your group. The secretary will list the names of the members of each| | |group. |(The students then will arrange the jumbled letters for 20 seconds) | | |answer would be Chromophore. | |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,|(The students then will arrange the jumbled letters for 20 seconds) | |are you ready to play the game! answer would be Basic dyes. | | | | |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 students then will arrange the jumbled letters for 20 seconds) | |(The game begins in 3, 2, 1) go. |answer would be Acidic dyes. | | | | |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. | | | | | |That’s right! We’re 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. | | | |(The students then will arrange the jumbled letters for 20 seconds) | |Very good! And now we’re going down to our last question.

This is a |answer would be Negative staining. | |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 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 not take up | | |so that they appear as sharply outlined unstained bright bodies on a |Yes Sir! | |colored ground. | | |(The game begins in 3, 2, 1) go. | | |Simple, differential and special stains sir. | |Very Good! Thank you. Please go back to your seats. Did you enjoy | | |the game? | | | |It is an aqueous or alcohol solution of a single basic dye. | |What are the three kinds of staining techniques?

Yes. Jarolyne? | | | | | |Will you describe simple stains? Yes. Cristina? |The primary purpose of a simple stain is to highlight the entire | | |microorganisms so that the cellular shapes and basic structures are | | |visible. |Good. And what is its purpose? Yes. Geralden? | | | | | | |Mordant sir. | | | | |Very good!

And what chemical is added to the solution to intensify |One function of a mordant is to increase the affinity of a stain for | |the stain? Yes. Jonalyn? |biological specimens. | | | | |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. Gram stain and acid-fast stain sir. | |Sharmaine? | | | | | |Very good! By the year 1884, who discovered the Gram stain? Yes. |He’s a Danish bacteriologist Hans Christian Gram sir. | |Cherry anne? | | | | | |Yes.

That’s 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 gram-negative. | | | |Very good! And what is an acidic fast stain? Yes. Jinky? |It is used to differentiate species of mycobacterium from the other | | |bacteria. | |Very good! And lastly is the special stains. What is the use of | | |special stains? Yes. Lovelyn? It is used to color and isolate specific parts of microorganisms such | | |an endospores and flagella and to reveal the presence of capsules | | | | | | | |What are the steps in preparing a Gram stain? Yes. Mark? |The application of crystal violet sir. | | | |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? |The dye is washed off. Sir. | |Yes. And what is the next step? Abegail? | | | |The application of iodine sir. |That’s right! The smear is covered with iodine and washed off. What | | |would be the appearance of iodine(mordant) in a gram-positive and | | |gram-negative cells? Yes. Jonabelle? | | | | | |Very good!

What will happen next? Yes. Glaiza? |Both are purple. | | | | |Yes that’s right! What would be the appearance of decolorization of | | |gram-positive and gram-negative cells? Yes. Sharmaine? |The slide is washed with ethanol or an alcohol-acetone solution and | | |then washed with water. |Very good! And lastly, what is the final step? Yes. Jarolyne? | | | |The gram-positive cells are purple and gram-negative cells are | | |colorless. | |Yes. What would be the appearance then? Cristina? | | | | | |Safranin is added as a counterstain and the slide is washed, dried and| |Why does the Gram-negative appear pink? Yes. An-an? |examined microscopically. | | | | | |Gram-positive retain the purple dye even though the alcohol wash. | |Gram-negative appear pink. | | | | | |Gram-negative appear pink because the pick up the safranin | | |(counterstain). | C. Generalization |Okay, Before going on to our next activity, does anyone have any |None sir. |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. ndstep 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 | |Anyone else? |gram-negative cells are colorless. And lastly the application of | | |safranin. Gram-positive retain the purple dye even though the alcohol | | |wash. Gram-negative appear pink. | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |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 |(The students will do then the wet mount for 20 minutes) | |activity) | | | | | |Did you understand the procedure? | | 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 AColumn B ____1. It is the application of stains to a sample A. Gram Stain to color cells, tissues, components or metabolic process. ____2. Used to color and isolate specific parts B. Hematoxylin of microorganisms. ____3. Its purpose is to highlight the entire micro-C. Iodine organisms so that the cellular shapes and basic structures are visible. ____4. It was developed in 1884 and one of the D. Papanicolaou staining most useful staining procedures. ____5. It is commonly used as a starch indicator. E. Rhodamine ____6. It is used to show both substances inside andF. Safranin outside cells and use of silver to stains istologic sections. ____7. Used chiefly as a biological stain because G. Silver staining of its ready oxidation to hematein. ____8. A nuclear stain used as a counterstain H. Simple stain or to color collagen yellow. ____9. It is frequently need to stain Pap smear specimen. I. Special stain ____10. Used especially in coloring paper andJ. Staining 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. 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 (10-9) |Satisfactory (8-7) |Limited (6-5) |Unsatisfactory (<5 ) | | | | | | | | | | | | | | |-Successfully created a |-Created a wet mount slide |-Created a wet mount |-Was not successful at creating a| | |wet mount slide without |with minimal amount of help |slide with a |wet mount slide | |Context |any help | |considerable amount of | | | | | |help | | | | | | | | http://www. scs. sk. ca/cyber/elem/learningcommunity/sciences/biology20/curr_content/biology20/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

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Gram Staining. (2016, Oct 24). Retrieved from https://graduateway.com/gram-staining-2/

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