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Questions and Answers
What is the primary purpose of creating a smear in the context of bacterial staining?
What is the primary purpose of creating a smear in the context of bacterial staining?
- To obtain a thin film of bacterial cells for better visualization under a microscope. (correct)
- To increase the size of individual bacterial cells for easier viewing.
- To kill all bacteria present in the sample.
- To change the genetic make-up of the bactera.
How does the process of fixing a bacterial sample typically work to preserve it for staining?
How does the process of fixing a bacterial sample typically work to preserve it for staining?
- By denaturing bacterial enzymes (autolysis) and preventing digestion of cell parts. (correct)
- By dissolving the cell walls to enhance stain penetration.
- By precipitating the DNA, making it more visible.
- By cooling cell to stop activity.
In the context of staining, what is a chromophore?
In the context of staining, what is a chromophore?
- The specific structure used to stick to the structure.
- The buffer applied to balance the pH.
- The dissolving agent.
- The ion that gives a stain its color. (correct)
If a bacterium is stained by a basic stain, what does this indicate about the charge of the chromophore and its interaction with the cell?
If a bacterium is stained by a basic stain, what does this indicate about the charge of the chromophore and its interaction with the cell?
Why are most bacteria readily stained by basic stains?
Why are most bacteria readily stained by basic stains?
What is a key characteristic of a simple stain?
What is a key characteristic of a simple stain?
What cellular characteristic can be determined using simple stains?
What cellular characteristic can be determined using simple stains?
Why are strong chemicals avoided during the process of negative staining?
Why are strong chemicals avoided during the process of negative staining?
Under what circumstances is negative staining particularly useful?
Under what circumstances is negative staining particularly useful?
What physical change is typically done after crystal violet is applied but before Gram's iodine?
What physical change is typically done after crystal violet is applied but before Gram's iodine?
Flashcards
What is a smear?
What is a smear?
To obtain a thin film of bacterial cells on a slide for staining and observation.
What is fixing in microscopy?
What is fixing in microscopy?
Denatures bacterial enzymes, preventing autolysis and digestion of cell parts. Methods include heat fixing and chemical fixing (e.g., methanol).
What is a chromophore?
What is a chromophore?
The ion that gives a stain its color; can be positive (basic stain) or negative (acidic stain).
What are simple stains?
What are simple stains?
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What is a negative stain?
What is a negative stain?
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Why not use fixing method?
Why not use fixing method?
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What is Gram stain?
What is Gram stain?
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What are the steps of Gram stain?
What are the steps of Gram stain?
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What does decolorizing agent do?
What does decolorizing agent do?
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What are gram-negative bacteria?
What are gram-negative bacteria?
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Study Notes
- A smear is used to obtain a thin film of bacterial cells
Fixing
- Fixing denatures bacterial enzymes (autolysis) and prevents cells from digesting.
- Fixing methods include heat fixing or chemically fixing with methanol for 1 minute.
Components of Staining
- A chromophore is the ion that is colored.
- Basic stains have a positive chromophore.
- Acidic stains have a negative chromophore.
Bacterial Staining
- Most bacteria are stained by a basic stain which permeates the cell wall, since the cell wall is negatively charged.
Simple Stains
- A simple stain uses one stain.
- It is a direct stain.
- A negative stain (unstained bacteria) is an example of a simple stain on bacteria.
- An example of a simple stain is for the background.
- Simple stains can test for morphology, cell arrangement, and size.
- Methylene blue is a dye name.
- After fixing, use 95% methanol.
Negative Stains
- Background is stained, not the bacteria.
- It is repelled by charge.
- Colloidal stains like India ink can be used.
- Nigrosin (doesn't enter cell, negatively charged and pH7) is a colloidal suspension used in negative stain
- Fixing or strong chemicals aren't used in negative staining to prevent distortion of bacteria.
- Use when other staining techniques don't clearly show cell morphology/size.
- Especially useful for coccobacilli (short and oval bacilli).
- Rod-shaped bacteria will look like short rods with small cocci.
Gram Staining
- Gram staining is used to classify gram-negative vs. gram-positive bacteria.
- Hanns saw colored cells losing color when washed off.
Steps of Gram Staining
- Primary stain with crystal violet basic dye.
- Mordant, using Gram's iodine.
- Decolorizing agent, such as ethanol/acetone.
- Secondary stain/counterstain, with safranin.
- Crystal violet picked up by peptidoglycan.
- Mordant combines with crystal violet to form crystal violet iodine complex.
- Decolorizing agent removes primary stain from some bacteria.
- Secondary stain is a basic dye that stains decolorized bacteria red.
- Gram-negative bacteria decolorize easily.
- Gram-positive bacteria decolorize slowly and retain primary stain.
- Peptidoglycan in the cell wall causes different staining results. Positive bacteria have multiple layers, while negative bacteria have a thin layer with an outer layer of lipoproteins, phospholipids, and lipopolysaccharides.
- Crystal violet is picked up by peptidoglycan to form crystal violet competition (cv-I) that is insoluble in water.
- CV-I is not washed out by alcohol.
- Stain washes out of gram-negative bacteria because the decolorizing agent dissolves the outer lipopolysaccharide layer and CV-I, washing it out of this Pepti layer.
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