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Questions and Answers
What should be used to prepare a smear when working with broth cultures?
What should be used to prepare a smear when working with broth cultures?
- Water
- Saline solution
- Alcohol
- Broth directly (correct)
Where should the circle be made on the slide for a bacterial smear?
Where should the circle be made on the slide for a bacterial smear?
- On the edge of the slide
- Below where bacteria will be added (correct)
- Above where bacteria will be added
- In the center of the slide
What is the first step in preparing a bacterial smear?
What is the first step in preparing a bacterial smear?
- Heat-fixing the slide
- Applying stain
- Preparing the smear and letting it dry (correct)
- Adding water
Which of these is a differential stain?
Which of these is a differential stain?
What is one characteristic used to classify bacteria?
What is one characteristic used to classify bacteria?
In bacterial characterization, what follows inoculation and aseptic transfer techniques?
In bacterial characterization, what follows inoculation and aseptic transfer techniques?
What is the purpose of using aseptic techniques during inoculation and transfer?
What is the purpose of using aseptic techniques during inoculation and transfer?
Which session focused on microscopy?
Which session focused on microscopy?
What information should be mentioned when observing bacteria under a microscope?
What information should be mentioned when observing bacteria under a microscope?
Which of the following is NOT a further characterization technique for bacteria?
Which of the following is NOT a further characterization technique for bacteria?
What is a component of all bacterial cell walls?
What is a component of all bacterial cell walls?
Which of the following can cause errors in Gram staining?
Which of the following can cause errors in Gram staining?
What is the primary function of the bacterial cell wall?
What is the primary function of the bacterial cell wall?
Which type of bacteria has an outer membrane?
Which type of bacteria has an outer membrane?
What color are Gram-positive cells after the primary stain (crystal violet) is applied?
What color are Gram-positive cells after the primary stain (crystal violet) is applied?
During Gram staining, what is the effect of the decolorizing agent on Gram-negative cells?
During Gram staining, what is the effect of the decolorizing agent on Gram-negative cells?
Which component is unique to Gram-positive cell walls?
Which component is unique to Gram-positive cell walls?
What is the effect of excessive rinsing during Gram staining?
What is the effect of excessive rinsing during Gram staining?
Which of the following is a characteristic of Gram-negative bacteria?
Which of the following is a characteristic of Gram-negative bacteria?
What can excessive staining cause?
What can excessive staining cause?
What is the primary purpose of a Gram stain?
What is the primary purpose of a Gram stain?
What color do Gram-positive bacteria appear after undergoing a Gram stain?
What color do Gram-positive bacteria appear after undergoing a Gram stain?
Which of the following is the primary stain used in the Gram stain procedure?
Which of the following is the primary stain used in the Gram stain procedure?
What is the function of Gram's iodine in the Gram stain procedure?
What is the function of Gram's iodine in the Gram stain procedure?
Which reagent is used as a decolorizer in the Gram stain?
Which reagent is used as a decolorizer in the Gram stain?
What color do Gram-negative bacteria appear after the addition of safranin?
What color do Gram-negative bacteria appear after the addition of safranin?
Which bacterial cell wall component is associated with Gram-positive bacteria?
Which bacterial cell wall component is associated with Gram-positive bacteria?
Which of the following is true of Gram-negative bacteria compared to Gram-positive bacteria?
Which of the following is true of Gram-negative bacteria compared to Gram-positive bacteria?
Who discovered the Gram stain technique?
Who discovered the Gram stain technique?
The acid-fast stain is used to identify bacteria in which genus?
The acid-fast stain is used to identify bacteria in which genus?
What color does safranin stain cells?
What color does safranin stain cells?
Which step in the Gram staining procedure is the most critical for differentiating Gram-negative bacteria?
Which step in the Gram staining procedure is the most critical for differentiating Gram-negative bacteria?
Which of the following is an example of a Gram-positive bacteria?
Which of the following is an example of a Gram-positive bacteria?
Which step comes first when preparing a bacterial smear?
Which step comes first when preparing a bacterial smear?
What is the purpose of heat-fixing a bacterial smear?
What is the purpose of heat-fixing a bacterial smear?
In a mixed bacterial sample containing E. coli and S. aureus, what colors would you expect to see after Gram staining?
In a mixed bacterial sample containing E. coli and S. aureus, what colors would you expect to see after Gram staining?
What is initially added to the slide when preparing a smear from a bacterial culture?
What is initially added to the slide when preparing a smear from a bacterial culture?
What should be done with the bacterial smear after it has been created?
What should be done with the bacterial smear after it has been created?
In Gram staining, if the decolorization step is skipped, what color would all bacteria appear under the micropscope?
In Gram staining, if the decolorization step is skipped, what color would all bacteria appear under the micropscope?
Flashcards
Bacterial Smear
Bacterial Smear
A technique used to prepare a bacterial sample for staining. It involves spreading a thin layer of bacteria on a slide and allowing it to dry.
Broth use
Broth use
Instead of water use broth to prepare a bacterial smear.
Gram Stain
Gram Stain
Gram staining is a differential staining technique used to classify bacteria based on their cell wall structure.
Aseptic Technique
Aseptic Technique
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Bacterial Pigment
Bacterial Pigment
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Inoculum
Inoculum
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Inoculation
Inoculation
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Differential Stain
Differential Stain
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Microscopy
Microscopy
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Broth
Broth
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Gram Stain: Primary Stain
Gram Stain: Primary Stain
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Mordant
Mordant
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Gram Stain: Decolorizer
Gram Stain: Decolorizer
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Gram Stain: Counterstain
Gram Stain: Counterstain
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Gram-Positive Bacteria
Gram-Positive Bacteria
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Gram-Negative Bacteria
Gram-Negative Bacteria
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Acid-Fast Stain
Acid-Fast Stain
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Hans Christian Gram
Hans Christian Gram
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Autolysin Levels
Autolysin Levels
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Growth Conditions
Growth Conditions
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Dirty Slides
Dirty Slides
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Faulty Reagents
Faulty Reagents
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Excessive Staining
Excessive Staining
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Excessive Rinsing
Excessive Rinsing
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Bacterial Cell Envelope
Bacterial Cell Envelope
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Cell (Plasma) Membrane
Cell (Plasma) Membrane
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Cell Wall
Cell Wall
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Outer Membrane
Outer Membrane
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Safranin
Safranin
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Decolorization
Decolorization
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Gram-Positive
Gram-Positive
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Gram-Negative
Gram-Negative
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Smear Preparation
Smear Preparation
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Bacterial Suspension
Bacterial Suspension
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Air Drying
Air Drying
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Staphylococcus
Staphylococcus
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Streptococcus
Streptococcus
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Study Notes
- To prepare a smear, first prepare the smear and let it dry.
- In this process, broth will be used, so no water is needed; just add directly.
- The circle will be done below the slide, on the opposite side from where the bacteria will be added.
- To prepare the smear:
- Place the liquid on the slide.
- Add the microbes to the liquid and spread over a 1 cm area.
- Air dry or heat gently.
- When dry, briefly heat-fix the cells to the slide.
Bacterial Shapes and Arrangements
- Bacteria can be classified based on:
- Color (some produce pigment)
- Size
- Shape (rod/bacilli, spherical/cocci, spiral)
- Behavior/Reactivity
- DNA
- Protein content
- Internal and external structures
- Bacterial shapes include Cocci, Bacilli, and Spiral.
- Arrangements of Cocci include:
- Coccus
- Diplococci
- Tetrad
- Sarcina
- Staphylococci
- Streptococci
- Arrangements of Bacilli include:
- Bacillus
- Diplobacilli
- Streptobacilli
- Palisades
- Coccobacilli
- Arrangements of Spiral bacteria:
- Spirochetes
- Spirilla (Helical-shaped/Corkscrew form)
- Vibrio
Gram Staining
- Used for preparation and staining slides, to study characteristics.
- Gram staining is a differential stain technique.
- Gram + bacteria color is purple.
- Gram - bacteria color is pink.
- Hans Christian Gram (1853 – 1938) developed the technique in 1884.
- The Gram stain is useful to discriminate between two types of bacteria (Gram-positive and Gram-negative) with similar clinical symptoms.
- Gram staining can help identify Streptococcus pneumoniae (also known as the pneumococcus) and Klebsiella pneumoniae.
- The Gram stain method is based on the chemical and physical properties of bacterial cell walls.
- Differential stains use two or more stains or dyes.
- Differential stains react uniquely with different bacteria/ bacterial structures.
- Gram stain is used to identify Gram-positive or Gram-negative bacteria
- A primary stain like crystal violet is used.
- A mordant like Gram’s iodine is applied to help the stain bind more strongly by forming complexes with the crystal violet, trapping the dye.
- Decolorizing acetone alcohol is important to identifying Gram-negative bacteria because it removes the crystal violet from the cells.
- A counter stain like safranin is utilized.
- Gram-negative stains pink.
- Gram-positive stains and appears purple.
- The Gram stain classifies bacteria into Gram-positive or Gram-negative, the two largest groups of bacteria.
- Gram-positive bacteria are often killed by penicillin and detergents.
- Gram-positive bacteria are more susceptible due to their thick cell wall (peptidoglycan).
- Gram-negative bacteria are more resistant to antibiotics because thin cell wall (peptidoglycan).
- Acid-fast stain can identify all bacteria in the genus Mycobacterium that have a special cell wall with mycolic acid.
- Capsule and endospore stain can also be performed.
Factors Affecting Gram Stain Results
- Factors affecting Gram stain:
- Cell age
- Autolysin levels
- Growth conditions
- Dirty slides
- Faulty reagents
- Excessive staining
- Excessive rinsing
- Too much or too little bacteria
Bacterial Cell Envelopes
- Consist of:
- Cell membrane or plasma membrane (all bacteria)
- Lipid bilayer
- Cell wall (most bacteria) All bacterial cell walls are made of peptidoglycan
- The function of peptidoglycan is to protect the cell from internal pressure.
- Few bacteria have no cell wall, like Mycoplasma.
- Outer membrane only exists if present (only Gram negatives).
Gram + vs Gram -
- Gram-positive has a thick layer of peptidoglycan.
- They have 2 layers as the cell envelope: the inner membrane and cell wall.
- Gram-positive cells do not have an outer membrane.
- Lipoteichoic acid and teichoic acid are attached to the cell wall in Gram-positive bacteria.
- Gram-negative has a mono or thin layer of peptidoglycan.
- They have 3 layers: inner and outer membranes and a cell wall.
- Gram-negative cells have an outer membrane.
- Lipopolysaccharides and porins are found in Gram-Negative bacteria
General Grouping of Bacteria
- Bacterial groupings include:
- gram positive, which have a thick layer of peptidoglycan
- gram negative, which have a mono or thin layer of peptidoglycan
- gram-variables, may appear gram positive or gram negative, depending on other factors
- gram-nonreactive, have no reaction to gram stain like Mycobacterium, M. tuberculosis
Smear Preparation
- Add a loopful of water to the slide to create a suspension and spread the bacteria onto the slide.
- Add the inoculum to the drop of water to create a suspension on the slide.
- Let is air dry in less than 10 minutes.
- Once the smear is air dried, heat fix by passing the slide through the flame about three times.
Gram Staining Procedure
- Prepare a smear like in a simple stain.
- Fix the smear like in a simple stain.
- Steps for Gram staining include:
- Stain with crystal violet for 1 min.
- Rinse the slide in slowly running water for 5 seconds.
- Cover the smear with iodine and allow it to remain for 1 minute.
- Rinse the smear with running water like in step 2.
- Apply the alcohol decolorizer dropwise and slowly, continuing until the dye no longer runs off.
- This is important for identifying Gram-negative bacteria.
- Rinse with running water again like in step 2 to prevent additional decolorization.
- Cover smear with safranin for 1 minute.
- Rinse again with water like in step 2.
- Blot dry, but do not rub.
- Observe from low objective to oil immersion.
- Characterize morphology of cells.
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Description
Questions cover bacterial smear preparation, staining techniques like Gram staining, and characteristics used to classify bacteria. Also covers aseptic techniques, microscopy, and cell wall components. Key steps in bacterial characterization are highlighted.
