3.2: Sample Detection: Staining Flashcards
True or False: Most cells are relatively transparent
True
Stains can be used to examine ___1___, ___2___or even ___3___.
- tissues (muscle or connective)
- specific types of cells (blood, bacterial, etc.)
- organelles within an individual cell (nucleus, ER, etc.)
Gram staining was first developed by ___1___ in the year ___2___ and is still used to this day.
- Hans Christian Gram
- 1884
In Gram Staining the differentiation, based on color, divides bacteria into two categories: ___1___ or ___2___.
- Gram-positive
- Gram-negative
______bacteria have a thick cell wall with many overlapping strands of peptidoglycan.
Gram-positive
Gram-positive bacteria have a ___1___ cell wall with many overlapping strands of ___2___.
- thick
- peptidoglycan
A thick peptidoglycan layer provides a ______ to the surrounding environment.
vital protective barrier
True or False: The thick peptidoglycan layer is impermeable, preventing any molecules from passing through to the intracellular space.
False. The thick peptidoglycan layer is not impermeable, meaning select molecules (water, nutrients, etc.) can still pass through to the intracellular space.
The Gram stain exploits this characteristic of the thick peptidoglycan layer by using the dye combinations of ___1___ and ___2___.
- Crystal Violet
- Iodine
Crystal violet is ______ by the thick peptidoglycan cell wall and forms a stable complex with ___2___ (upon its addition) effectively trapping the dyes in the cell. The resulting mixture is a ___3___ coloration of the cell. Thus, Gram-positive cells appear ___3___.
- retained
- iodine
- purple
___1___ bacteria have a relatively thin peptidoglycan layer followed by an outer membrane composed of lipopolysaccharides (LPS).
Gram-negative
Gram-negative bacteria have a relatively ___1___ layer followed by an outer membrane composed of ___2___.
- thin peptidoglycan
- lipopolysaccharides (LPS)
During the Gram staining procedure, Gram-negative bacteria will initially retain the crystal violet dye, however, by next washing the cells with alcohol, a step referred to as the ___1___, the LPS and thin peptidoglycan layers of Gram-negative bacteria are unable to retain the dye, and the outer membrane is depleted of its color.
decolorization wash
True or False: Gram-positive bacteria remain unaffected by the decolorization step.
True
True or False: Gram-negative bacteria remain unaffected by the decolorization step.
False. By washing the cells with alcohol, a step referred to as the decolorization wash, the LPS and thin peptidoglycan layers of Gram-negative bacteria are unable to retain the dye, and the outer membrane is depleted of its color.
To visualize unstained gram-negative bacteria after it has undergone the decolorization wash, a secondary (counterstain) dye called ______ is added.
safranin
By counterstaining with the ___1___ Safranin dye, Gram-negative bacteria now retain a ___2___ color.
- positively charged
- pink
Identify each type of gram staining in the following pictures.
(A) Gram-positive bacillus
(B) Gram-negative bacillus
(C) a mixed culture of Gram-positive coccus and Gram-negative bacillus are shown.
Gram stain distinguishes between bacteria with a thick peptidoglycan wall and those without (or very thin), this is referred to as a ______.
differential stain
______ is a generalized term used for any staining technique that separates specimens into further subgroups. This process most often utilizes at least two dyes.
Differential staining
One of the disadvantages of the Gram stain is the requirement of the cells to be ______ to a glass slide.
fixed (attached)
The most common method to fix a sample is via ______.
heat fixation
In the ______ process, samples are added to a glass slide and then passed through a flame until all liquid in the sample has been removed.
Heat Fixation
What is the Heat Fixation process?
A process for cells to be fixed (attached) to a glass slide for the Gram Staining Process. The samples are added to a glass slide and then passed through a flame until all liquid in the sample has been removed.
______ strategies Include the use of paraformaldehyde, ethanol, or methanol to fix cells.
chemical fixation
What 3 chemicals are used in the process of chemical fixation?
- paraformaldehyde
- ethanol
- methanol
What is the downside of using the Chemical Fixation process?
It kills the microorganism and characteristics related to motility (movement) are not possible.
______ is a basic form of sample preparation for viewing live samples. A small liquid culture (usually just a drop) containing a microorganism of interest is prepared, added to a slide, and then covered with a glass coverslip. The coverslip is present to both protect the objective and the specimen while also holding the microorganism in place. Note: Heat fixing is not performed. Wet mounts are used to observe the motility and behavior of an organism.
Wet mount
What is Wet Mounting?
a basic form of sample preparation for viewing live samples. A small liquid culture (usually just a drop) containing a microorganism of interest is prepared, added to a slide, and then covered with a glass coverslip. The coverslip is present to both protect the objective and the specimen while also holding the microorganism in place. Note: Heat fixing is not performed. Wet mounts are used to observe the motility and behavior of an organism.
Wet Mounting is used to observe what?
Wet mounts are used to observe the motility and behavior of an organism.
True or False: Heat fixing is performed during the process of wet mounting.
False. Heat fixing is not performed.
______ uses a solution of a positively charged dye, such as methylene blue, crystal violet, safranin, or fuchsin, to bind to and stain the negatively charged membrane of the microorganism. This simple technique is often used to quickly observe the size, shape, and arrangement of cells.
Simple staining
Simple staining uses a solution of a ___1___, such as ___2___, ___3___, ___4___, or ___5___, to bind to and stain the ___6___ membrane of the microorganism.
- positively charged dye
- methylene blue
- crystal violet
- safranin
- fuchsin
- negatively charged
______ is a simple technique often used to quickly observe the size, shape, and arrangement of cells.
Simple Staining
Simple Staining is a technique used to observe what about a cell?
- Size
- Shape
- Arrangement
______ is an alternative to the positively charged simple staining dyes.
Negative staining
True or False: A negative stain can be thought of the inverse of a simple stain.
True
In Negative Staining, instead of staining the microorganism, you stain……
……everything except the microorganism.
In which process of staining do you stain everything except for the Microorganism?
Negative Staining
In Negative Staining, applying the dark stain ___1___to a sample, its negative charge is repelled by the ___2___, resulting in a sharp contrast between the unstained specimen and the now dark background.
- nigrosin (or India ink)
- negatively charged membrane
What is the name of the dark ink used in Negative Staining?
nigrosin (or India ink)
A negative stain is only mildly ___1___ and may not kill the microorganism. As such, a negative stain is contraindicated for ___2___samples.
- invasive
- pathogenic
______ is a differential stain used to identify bacterial stains showing a high degree of resistance to decolorization.
Acid-fast staining (also known as the Ziehl-Neelsen stain)
What is Acid-fast staining (also known as the Ziehl-Neelsen stain) used for?
Is a differential stain used to identify bacterial stains showing a high degree of resistance to decolorization.
______ is the most common use for an acid-fast stain as it is the causative agent of tuberculosis.
Mycobacterium tuberculosis
The Acid-fast staining procedure uses the red dye___1___, initially staining all cells red. However, following the decolorization wash step, only cells with a ___2___ remain red.
- carbolfuchsin
- thick lipid-based (acid-fast) protective membrane
After samples undergoing the Acid-fast staining procedure complete the decolorization wash, they are then counterstained with ___1___ to stain ___2___. This staining procedure is vital in the diagnosis of ___3___ and is most often performed on ___4___ samples obtained from patients.
- methylene blue
- non-acid-fast bacterium
- tuberculosis
- sputum
Describe what is being shown in this picture as well as which Staining Process is being used.
Acid-fast stain. A representative image of Mycobacterium tuberculosis is shown. The causative agent of TB is shown in red, while non-acid fast cells only retain the counterstain and are shown in blue.
______ is also a differential stain often used in clinical settings.
Giemsa
Giemsa combined with Wright’s stain (stains blood cells), the resulting combinatorial stain can be applied to ___1___ to determine the presence (or absence) of ___2___—human (blood) cells appear ___3___, and bacterial cells appear as ___4___.
- blood smears
- pathogenic bacteria
- Purple
- Pink
___1___ combined with ___2___, the resulting combinatorial stain can be applied to blood smears to determine the presence (or absence) of pathogenic bacteria—human (blood) cells appear purple, and bacterial cells appear as pink.
- Giemsa
- Wright’s stain (stains blood cells)
Which staining is most commonly used for the diagnosing of Malaria and other blood parasites?
Giemsa in combination with Wright’s stain.
