Bacterial Smears and Staining Techniques
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Questions and Answers

What is the shape of cocci bacteria?

  • Comma-shaped
  • Round (correct)
  • Spiral
  • Rod-shaped

Which of the following is a common type of bacteria?

  • Amoeba proteus
  • Euglena gracilis
  • Paramecium caudatum
  • Bacillus subtilis (correct)

Before heat-fixing a slide with a bacterial smear, what must be done?

  • Cover with immersion oil
  • Rinse with ethanol
  • Apply stain
  • Air-dry the slide (correct)

What happens if a bacterial smear is too thick?

<p>The smear will stain unevenly. (D)</p> Signup and view all the answers

What is the purpose of heat-fixing a slide?

<p>To make the bacteria stick to the slide (A)</p> Signup and view all the answers

What should you add to the slide when making a smear from a culture growing on a solid medium?

<p>One loopful of water (B)</p> Signup and view all the answers

What is the first step in preparing a bacterial smear?

<p>Prepare smear and let it dry (C)</p> Signup and view all the answers

When using broth, what should you NOT add when preparing a slide?

<p>Water (B)</p> Signup and view all the answers

Where should the circle be drawn on the slide for bacterial preparation?

<p>Below the slide, opposite where bacteria will be added (B)</p> Signup and view all the answers

What should be observed in slants, plates, and broths?

<p>Growth (C)</p> Signup and view all the answers

What is the term for bacterial growth that is uniformly cloudy in broth tubes?

<p>Turbidity (A)</p> Signup and view all the answers

What is the growth called when it settles on the bottom of a broth tube?

<p>Sediment (B)</p> Signup and view all the answers

What is the purpose of heat fixing a bacterial smear?

<p>To kill and adhere the bacteria to the slide (D)</p> Signup and view all the answers

What techniques are used to transfer an inoculum?

<p>Aseptic techniques (A)</p> Signup and view all the answers

What follows inoculation and transfer techniques in bacterial characterization?

<p>Preparation and staining slides (B)</p> Signup and view all the answers

Most stains used in microbiology are what type?

<p>Basic (A)</p> Signup and view all the answers

Methylene blue is an example of what kind of stain?

<p>Basic stain (C)</p> Signup and view all the answers

Which of the following is a morphological feature of bacteria?

<p>All of the above (D)</p> Signup and view all the answers

What is the purpose of a simple stain?

<p>To highlight the entire microorganism for basic structure visualization (D)</p> Signup and view all the answers

What might be added to a stain to increase its affinity for a cell structure?

<p>A mordant (C)</p> Signup and view all the answers

Which of the following describes cocci bacteria?

<p>Round shaped (D)</p> Signup and view all the answers

What term describes bacteria that have different shapes?

<p>Pleomorphism (C)</p> Signup and view all the answers

When preparing a smear from solid media, what should be added to the slide first?

<p>A drop of water (B)</p> Signup and view all the answers

After allowing a bacterial smear to air dry, what is the next step?

<p>Fixing the smear (C)</p> Signup and view all the answers

What is the primary purpose of simple staining?

<p>To observe bacterial morphology and arrangement (D)</p> Signup and view all the answers

Which of the following is a basic bacterial shape?

<p>Coccus (B)</p> Signup and view all the answers

What is a smear in the context of bacterial staining?

<p>A thin film of bacteria applied to a slide (B)</p> Signup and view all the answers

Why is heat fixing important in preparing a bacterial smear?

<p>To denature bacterial enzymes and adhere cells to the slide (B)</p> Signup and view all the answers

What should be added to a slide when preparing a smear from a solid culture?

<p>A loopful of water (B)</p> Signup and view all the answers

Which of the following is a purpose of staining microorganisms?

<p>To add contrast and make them easier to see (B)</p> Signup and view all the answers

What is the first step in preparing a smear from a broth culture?

<p>Add bacteria to the slide (D)</p> Signup and view all the answers

What is the correct order for preparing a bacterial smear?

<p>Add bacteria, air dry, heat fix (C)</p> Signup and view all the answers

Besides using a Bunsen burner, what is another method of fixing a smear?

<p>Incubating at 60°C (C)</p> Signup and view all the answers

Which of the following is NOT a common shape of bacteria?

<p>Pyramid (B)</p> Signup and view all the answers

What is the first step in preparing a bacterial smear for simple staining?

<p>Add a loopful of water to the slide (C)</p> Signup and view all the answers

Why is a bacterial suspension air-dried before heat-fixing?

<p>To ensure the bacteria adhere to the slide (C)</p> Signup and view all the answers

Which of the following is used to apply the bacterial sample to the slide?

<p>Loop (B)</p> Signup and view all the answers

Which of the following is a basic dye used in simple staining?

<p>Safranin (A)</p> Signup and view all the answers

What should you do if you are using methylene blue as a stain?

<p>Stain the smear for 4 to 5 minutes (A)</p> Signup and view all the answers

After staining, what is used to remove excess stain from the slide?

<p>Running water (B)</p> Signup and view all the answers

How should a stained slide be dried?

<p>By placing it between pieces of blotting paper (A)</p> Signup and view all the answers

What objective is used to get the highest magnification?

<p>Oil immersion (C)</p> Signup and view all the answers

Which of the following is an example of a bacterial morphology that can be identified with staining?

<p>Cell shape (D)</p> Signup and view all the answers

Flashcards

Simple Stain

Using one stain to observe bacterial morphology

Basic Stain Uses

Stains all cells the same color, useful for observing cell shape and arrangement.

Bacterial Shapes

Bacillus (rod), coccus (sphere), and spirillum (spiral).

Smear

A thin film of bacteria applied to a slide.

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Smear purpose

Attaches microbes to the slide, necessary before many bacterial stains are done.

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How to make a Smear

Spreading a bacterial suspension on a clean slide and allowing it to air dry.

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Heat Fixing

Passing the dry smear through a Bunsen burner flame to kill the bacteria.

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Fixing purpose

Denatures bacterial enzymes, preventing them from digesting cell parts and enhances adherence to the slide.

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Smear from solid medium

Add water to the slide, then add your inoculum to create a suspension.

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Smear preparation from broth

Add bacteria (several loopful) to the slide.

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Heat Fixation

Heating a slide to adhere a specimen.

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Stains

Synthetic dyes used to color microorganisms for better viewing.

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Basic Stains

Stains with positive ions (cationic).

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Acidic Stains

Stains with negative ions (anionic).

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Differential Staining

Using two or more stains to differentiate structures.

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Mordant

A substance added to a stain to increase its affinity for a cell structure.

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Cocci

Spherical or round-shaped bacteria.

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Bacilli

Rod-shaped bacteria.

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Spirilla/Spirochetes

Spiral or curved-shaped bacteria.

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Prepare Smear & Dry

First step in slide preparation; apply specimen to slide and allow to air dry completely before staining or heat-fixing.

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Broth Smear

Adding bacteria directly without water if broth is used as the source of the bacteria.

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Slide Circle

Draw a circle on the underside of the slide to mark where the sample will be placed on the opposite side.

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Colony Margin

The edge characteristics or shape of a colony's margin when grown on a solid medium, such as an agar plate or slant.

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Entire Margin

Smooth, continuous edge.

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Undulate Margin

Wavy edge.

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Turbidity

A broth in which the liquid appears uniformly cloudy throughout.

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Flocculent Broth Growth

Visible chunks or flecks of growth are scattered throughout the broth

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Pellicle

A film or layer of bacteria covering the surface of the broth.

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Bacterial Morphology and Arrangement

The shape and arrangement of bacterial cells.

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Purpose of Heat-Fixing

To kill bacteria, adhere them to the slide, and prepare them for staining.

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Chemical Basis for Staining

Dyes with charged groups bind to oppositely charged cellular components.

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Loop

A tool used to transfer small amounts of liquid culture.

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Purpose of Suspension

To create a thin layer of bacteria on the slide for better viewing.

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Air Drying Smear

Allows the bacteria to dry completely before heat-fixing.

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Staining Time Differences

Methylene blue stains for 4-5 minutes, while crystal violet and safranin stain for 30 seconds to 1 minute.

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Drying the Slide

Blotting paper gently removes excess water without disrupting the smear.

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Microscope Objectives

Low, high-dry, and oil immersion objectives are used to view the slide at increasing magnifications.

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Bacillus subtilis

A common lab bacterium; bacillus (rod shaped).

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Staphylococcus aureus

Common bacterium, often appears as clustered cocci (spherical shaped).

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Escherichia coli

Common Gram-negative bacillus (rod shaped) bacterium, frequently used in lab studies.

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Study Notes

  • The first step is to prepare a smear and let it dry.

Preparing a Smear

  • If using broth, add it directly to the slide without water.
  • When adding a circle to the slide, place it below, opposite from where the bacteria will be added.
  • Add water first when dealing with a solid.
  • Place the liquid on the slide.
  • Add microbes to the liquid, spreading over a 1 cm area.
  • Air dry or heat gently, then briefly heat fix the cells to the slide when dry.

Background

  • Simple stains use one stain to observe bacterial morphology
  • Basic stains, such as safranin or methylene blue, stain all cells on the slide the same color.
  • Simple staining observes the morphology or shape and the arrangement of cells.
  • The three most common bacteria shapes include bacillus (oblong, rod), coccus (sphere), and spirillum (comma-shaped, spiral).
  • To stain bacteria, a smear is required, which involves a thin film of bacteria applied to a slide.

Smears

  • Staining adds contrast, making it easier to see microorganisms
  • Staining allows for simple viewing
  • A smear is required to begin a simple stain.
  • A smear attaches the microbes to the slide
  • A smear involves a thin film of a solution of microbes on a slide.
  • Smears are performed before staining

Smear Preparation Steps

  • Bacteria attach to a slide and are then stained.
  • Spread a bacterial suspension on a clean slide and let air dry.
  • Add a drop of water before the bacteria if collected from solid culture to create a suspension for spreading.
  • Pass the dry smear through a Bunsen burner flame to "heat fix" the bacteria.
  • Heat fixing denatures bacterial enzymes.
  • Heat fixing enhances adherence of bacterial cells to the microscope slide
  • Fixing can be done by incubating at 60 degrees Celsius or with chemicals.

Preparing Smears from Liquid and Solid Cultures

  • For a broth sample, add bacteria to the slide (several loopfuls)
  • Let it air dry to help attach the bacteria.
  • Heat fix after the smear is air dried by passing the slide through the flame about three times.
  • For a solid medium sample, add a loopful of water to the slide to create a suspension for spreading the bacteria.
  • Add some inoculum to the water and create a suspension.
  • Let it air dry.
  • Heat fix by passing the slide through the flame about three times after the smear is air dried.

Stains

  • Stains are synthetic dyes composed of charged colored ions or chromophores
  • Ions can be basic/cationic, if positive
  • Ions can also be acidic/anionic, if negative
  • Most dyes used will be basic, like methylene blue, crystal violet, and safranin.
  • Basic dyes readily stain nucleic acid (being net negative charge)
  • Acidic stains will readily stain proteins (being net positive charge)

Simple Stains

  • Simple stains use an aqueous or alcohol solution of a single basic dye.
  • The cells are fixed ,then stained.
  • Simple stains highlight the entire microorganism, showing its structure/arrangement.
  • Mordants may be added to increase a stain’s affinity to cell structure.
  • Simple stains help in viewing the size, shape and arrangement of bacteria stained
  • The basic shapes of bacteria: bacilli (rod shape), cocci (round), spirochete or spiral (curves)

To Prepare Smears during Today's Activities

  • Aseptically transfer a loop full of specimen to a slide
  • Add 3–5 loop fulls if from a broth.
  • Place a drop of water on the slide first if from solid media (slants, plates, or deeps) before transferring the specimen.
  • Spread the broth or specimen over a small area and let dry.
  • Draw a circle where the specimen is located prior to adding the bacteria
  • Flame the inoculating instrument.
  • Allow the slides to air dry
  • Heat fix the smear by passing it through the flame and it is now ready for staining.

Todays Exercise Materials

  • Slides
  • Staining tray
  • Slide holder or clothespin
  • Bunsen burner and striker
  • Methylene blue
  • Crystal violet
  • Safranin
  • Water bottle
  • Bibulous paper or paper towel
  • Loop

Simple Staining Procedure

  • Create a suspension to spread the bacteria by adding a loopful of water to the slide.
  • Add your inoculum to the drop of water
  • Then let it air dry.
  • Heat fix the slide through the flame about three times after it's air dried
  • Cover the smear with the stain: methylene blue
  • Place on a staining rack.
  • Then let the smear stain, pour off the stain, and rinse with slowly running water.
  • Dry the slide by placing it between pieces of blotting paper
  • Don't rub
  • Examine the slide using low, high-dry, and oil immersion objectives.
  • Use safranin or crystal violet to stain the smear from 30 seconds to 1 minute.
  • Use methylene blue to stain the smear for 4 to 5 minutes.

Bacterial Shapes and Arrangements

  • Cocci are sphere-shaped and can be arranged into coccus, diplococci, streptococci, tetrad and staphylococci
  • Bacilli are rod-shaped and can be arranged into coccobacillus, bacillus, diplobacilli, streptobacilli, and pallisades.
  • Spirals are spiral-shaped and can be arranged into vibrio, spirilla, and spirochetes.

Tips for Success

  • When making a smear from a culture growing on a solid medium, add only one loopful of water to the slide.
  • Slides must air-dry before being heat-fixed.
  • Too much added water will increase evaporation time.
  • Add a small amount of inoculum to the water drop.
  • Too many bacteria can make the sample too thick and difficult to view.
  • It can also make the smear stain unevenly and prevent proper heat-fixing.
  • Spread the smear adequately.
  • Should be a very thin film, not big chunks of bacteria.
  • Bacteria will wash off non heat-fixed slides.
  • Check your slide is positioned with the smear facing up.

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Description

Explore bacterial shapes, smear preparation, and staining. Learn about heat-fixing, proper smear thickness, and inoculation techniques. Understand bacterial growth patterns in different media and the characteristics of microbial stains.

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