Staining Flashcards
What is a smear prep?
a technique used to produce a sample of microorganisms, typically for microscopy or staining.
From what sources are bacterial smears most often created?
1) bacteria in liquid broth culture,
2) bacteria on agar media.
Describe the basic concepts of smear preparation.
- Growing a bacterial culture in liquid nutrient broth or on an agar plate
- Acquiring a portion of the sample for smear creation using aseptic technique
- Applying the sample to a slide to distribute the organisms
- Heat fixation of the smear to adhere the sample to the slide
- Evaluation of the quality of the smear right away using a simple stain called methylene blue
How long is a properly performed bacterial smear good for?
can be stored in a dry place for months before further use.
Name some fields of study where smear preparation and staining are necessary tools.
+ Microbiology
+ Cytology
+ Histology
+ Pathology
What is the purpose of using stains?
a colored chemical dye, known as a stain, is often added to provide additional contrast to help visualize the microbial cells and their external structures such as capsules, endospores, or flagella.
Briefly define:
Bacterial cellular arrangement
Description of overarching patterns and organization of bacterial cells seen across a microscopic field. Examples include descriptors such as “clusters” or “in pairs.”
Briefly define:
Bacterial cellular morphology
Description of shapes and structure of bacterial cells seen across a microscopic field. Examples include descriptors such as cocci or bacilli.
Briefly define:
Heat fixation
The process of applying gentle heat to a sample.
Explain the benefits of heat fixation.
- preserves various cellular components in a natural state with minimal distortion,
- while simultaneously killing the specimen and provides a measure of safety
- securing it to the slide.
- Makes cells more permeable to stains.
Briefly define:
Methylene blue
A blue dye used to visualize cells in bacteriological and biological staining.
Briefly define:
Smear
- An isolated and prepared sample of microorganisms for use in microscopy and staining.
- A thin layer spread across a glass slide.
Briefly define:
Staining
A technique that utilizes dye(s) to add color for visualizing and distinguishing cells on a smear by increasing contrast.
Briefly describe the steps for slide prep with heat fixation.
- Obtain and label a slide.
2a. Two loopfuls of liquid.
2b. Disperse culture over 1/3 of slide. - Allow to air dry.
- Heat source applied to slide.
Briefly describe the bacterial capsule.
Some species of bacteria produce a thick extracellular layer that provides several advantages to the bacterial cell, including adherence and protection from immune responses.
Describe the major categories of microbial cell staining.
There are three
* Simple staining uses only a single dye to reveal morphology and arrangement.
* Differential uses multiple dyes to discriminate between cell types or structures.
* Structural staining assists in the observation of structures external to the cell wall.
Name some of the common staining methodologies.
There are many common staining methodologies used in microbiology, including acid-fast staining, capsule staining, endospore staining, Gram staining, negative staining, simple staining.
name several common terms for capsule
several common terms all refer to similar structures. These terms include capsule, glycocalyx, and slime layer.
Briefly describe the difficuties staining capsules.
Because of their molecular architecture and permeability properties, capsules are not always easily penetrated by stains and may require unique steps.
Name some uses for capsule staining.
- Recognizing bacterial genera that produce capsules.
- Visualizing the capsule.
- Differentiating the morphological characteristics of the specimen.
The protocol for performing a capsule stain is as follows:
- Stain the background of a clean slide with the dark primary stain
- Mix bacterial sample into primary stain
- Create a thin smear across the sample slide using the edge of another clean slide
- Air dry sample to fix to slide, but do not use heat
- Apply secondary stain to visualize bacterial cells
- Rinse briefly with water to ready slide for microscope viewing
Name the major category of staining used for capsules.
all capsule stains are structural, where the structures are differently colored from the background
What is the name given to the method of using a dark dye to stain the background?
Negative staining.
What dyes are often used to stain the background?
often a dark-colored compound, such as india ink or nigrosin.
Explain why heat fixation should be avoided.
Heat fixation should be avoided if possible, as the heat may destroy bacteria, capsule structure, or overall morphology.
What is the fancy term that describes bacteria with capsules?
encapsulated
Name two clinically significant genera of encapsulated bacteria.
Streptococcus and Pseudomonas.
Describe the notable outcomes of capsule staining.
Capsule stains have several notable outcomes.
* Backgrounds are colorized black or dark blue by the primary stain.
* Capsules retain no color.
* Bacterial cells and cytoplasm may stain purple or pink, based on the secondary stain used.
Briefly define:
Bacillus
(as related to morphology)
Bacterial cell shape that is cylindrical or rod-like (longer than it is wide).
Describe the capsule as if it were an organelle.
an organized glycocalyx structure composed of repeating polysaccharide (sugar) units, of protein, or of both
Compare the capsule against a slime layer.
Capsule is bound more tightly to the cell than a slime layer and has a thicker, gummy consistency that gives a sticky or mucoid character grown on agar plates.
Describe the clinical significance of the presence of a capsule.
This layer is protective and can be associated with virulence.
Briefly define:
Counterstain
A dye that is visibly contrasting to the principal stain.
Name the most common counterstain used with capsule staining.
In capsule staining, it is most commonly the purple dye known as crystal violet.
Briefly define:
Crystal violet
A purple dye that interacts with components of the bacterial cell wall and cytoplasm
Briefly define:
Structure of the Glycocalyx
A filamentous network of carbohydrate-rich molecules that coats cells.
Name two common terms used to describe types of glycocalyx.
Highly organized glycocalyx structure is also termed as “capsule”, while unorganized glycocalyx formation is termed as “slime layer”.
Name two common terms used to describe types of glycocalyx.
Highly organized glycocalyx structure is also termed as “capsule”, while unorganized glycocalyx formation is termed as “slime layer”.
Briefly define:
Gram stain
A differential stain for bacteria based on cell wall composition, useful in identification and taxonomy.
Briefly describe:
India ink
A suspension of carbon black particles in a medium (such as ethylene glycol)
Name two common uses of india ink.
commonly used as a staining dye or anatomic marker for surgery and radiotherapy.
Briefly describe:
Nigrosin
A mixture of black synthetic dyes often used in negative staining and capsule staining of bacteria
Briefly define:
Primary stain
The first dye applied in a differential staining technique.
Briefly describe:
Safranin
A red dye that is readily absorbed into most bacteria, visibly contrasting to the principal stain.
Briefly define:
Slime layer
A loosely adhered glycocalyx formation, lacking specific organizational units.
Briefly describe the benefits of a slime layer to bacteria.
Slime layers cover bacterial cells with a loose shield that protects from dehydration, loss of nutrients, and provides simple adhesion capability.
Briefly define:
Staphylococcus
Round-shaped bacterial cells arranged in irregular clusters.
Briefly define:
Streptococcus
Round-shaped bacterial cells arranged in chains.
Briefly define:
Vegetative cell
A metabolically active and growing cell.
Name some reasons for using the Gram stain.
- Visualization of bacterial cells
- Differentiation of bacterial cell shapes and arrangements
- Identification of bacterial cell wall structures
- Differentiation between gram-positive and gram-negative cells
List the steps of the Gram stain.
The procedure involves four steps:
1. Primary staining with crystal violet
2. Fixation with Gram’s iodine
3. Decolorization with ethanol
4. Secondary staining with safranin
Describe the consequences of a bad Gram stain.
Skipping steps or using a flawed order prevents the stains from binding properly to bacterial cell structures and produces inaccurate identification characteristics on the microscope.
A correctly performed Gram staining separates bacteria into what broad groups?
there are two, known as gram-positive and gram-negative
What cell structure is the reason for the differences in Gram staining.
based on the differences in the cell wall structure of the stained bacterium.
Describe the colors resulting from Gram staining.
- Gram-negative cells are decolorized and stained by the red secondary stain.
- Gram-positive cells retain the purple dye-mordant complex.
Briefly define:
Coccus
Bacterial cell shape that is spherical or rounded in appearance
Define and describe:
Decolorizer
- A chemical, usually ethanol or acetone, that dehydrates the peptidoglycan layer, shrinking and tightening it.
- With the thinner peptidoglycan layer of Gram negative cells lose their color because they are unable to retain the crystal violet-iodine complex.
Describe the reason gram-positive bacteria retain their color.
The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in gram-positive bacteria.
Describe the consequences if the decolorizing agent is left on too long.
Decolorization is critical and must be timed correctly, as the crystal violet stain is removed from both gram-positive and gram-negative cells if the decolorizing agent is left on too long.
Briefly define:
Fixative
A chemical that fixes dye in or on cells by forming an insoluble compound. Also referred to as a “mordant.”
Describe:
Gram’s iodine
A chemical that binds to crystal violet in peptidoglycan, forming a complex. This complex is a larger molecule than the original crystal violet stain and is insoluble in water.
Briefly describe:
Gram-negative bacteria
Refers to a bacterial cell wall with 2 membranes (an inner and an outer) and a thin layer of peptidoglycan.
Briefly describe the appearance of gram-negative bacteria.
Gram-negative organisms appear red after loss of crystal violet and absorbance of the safranin counterstain.
Briefly describe:
Gram-positive bacteria
Refers to a bacterial cell wall with 1 membrane and a thick layer of peptidoglycan.
Briefly describe the appearance of gram-positive bacteria.
Gram-positive organisms appear purple from the retention of the crystal violet-iodine complexes.
Briefly define:
Mordant
Also known as a fixative, this is another term for the iodine staining step.
Briefly describe:
Peptidoglycan
A network of polysaccharide chains cross-linked by short peptides that form the rigid part of bacterial cell walls.
Contrast the quantity of peptidoglycan in gram-positive and gram-negative bacteria.
Gram-negative bacteria have a smaller amount of peptidoglycan than do gram-positive bacteria.
Name the morphologies:
bacterial cell with a helical or corkscrew appearance.
- Spirillum
- Spirochete
Name the morphologies:
bacterial cell with a curved rod
or comma-shaped.
Vibrio
name the reagents for an acid-fast staining procedure
Carbolfuchsin primary stain
Decolorization with acid alcohol
Methylene blue counterstain
Describe the bacterial structure suited for acid-fast staining.
used for cell walls with an unusual composition of waxy lipid components, such as mycolic acid, that significantly changes their staining properties.
Describe the feature of acid-fast cell walls that make it resistant to some staining steps.
An acid-fast cell wall composition is less permeable to chemicals, making bacteria resistant to some staining steps, such as decolorization, and makes classic Gram staining ineffective.
Name some diseases that can be from organisms identified with acid-fast staining.
tuberculosis, leprosy, pneumonias, and diarrheas
Name the two methodologies for performing an acid-fast stain and identify their one key difference.
- The Ziehl-Neelsen method, which uses heated stain,
- The Kinyoun method, which does not use heat.
What color results from acid-fast staining?
Acid-fast bacterial cells are stained red
Everything else is not stained.
Briefly define:
Acid-fast
A term referring to the property of bacteria to retain carbolfuchsin even in the presence of acid alcohol.
Briefly define:
Carbolfuschin
A red-colored solution of dye that, when bound to lipids in the cell wall of some bacterial species, cannot be removed with an acid wash.
Briefly define:
Cell wall
In bacteria, a rigid structure made of peptidoglycan or lipids that lies just outside the cytoplasmic membrane.
Briefly describe the imporance of the cell wall to staining techniques.
Architecture of the cell wall affects what stains are held and seen.
Describe the features of spores that make it resistant to some staining steps.
Because of their thick spore coat and resistant properties, spores are not easily penetrated by stains and require unique chemicals and steps in the staining protocol to be visualized.
Name some uses for spore staining.
- Recognizing a bacterial genera that produce spores
- Describing the spores
- Differentiating spores from metabolically active cells
Name the key steps of spore staining.
There are three.
1. Primary staining with malachite green
2. Using heat to help permeabilize spores, allowing for dye entry
3. Secondary staining with safranin
Name the spore staining method as we performed in lab with malachite green.
Schaeffer-Fulton method
Name the spore staining method that uses carbol fuchsin and methylene blue.
Moeller method
Correctly performed spore staining separates bacteria into what broad groups?
sporulators and non-sporulators, based on the presence or absence of spore structures on the sample.
Describe the spore staining colors and what they identify.
Spores are colorized green by the primary stain.
Vegetative cells retain red color.
Briefly define:
Endospore
A descriptive term used to describe a spore that is contained in the cytoplasm of a bacterium.
Briefly define:
Exospore
endospore released (or lysed) is then re-termed an “exospore” or “spore”.
Briefly describe:
Malachite green
A green dye that is able to penetrate through the bacterial spore coat and is retained in the spore for observation
Define:
Spore
A highly resistant, dormant structure produced by some bacterial species in order to withstand hostile conditions that would kill a vegetative (growing) cell.
How would the gram stain appear if you didn’t decolorize enough?
crystal violet would not be washed out of the Gram negative cells. you would have false positives
Describe the appearance of a cell without a capsule when a capsule stain is used.
When a capsule is not present, the background stain will be up against the cell. There will be no “halo.”
Briefly describe the appearance of the endospores and vegetative cells when the endospore stain is used on sporulating bacillus.
Endospores will appear as green ovals inside of the red bacterial cells. Vegetative bacterial cells will appear red.
The prefix “diplo-” refers to what?
a pair of cells.
The prefix “Strepto-” refers to
the cells are arranged in chains.
The prefix “Staphylo-” refers to
a cluster arrangement.
What are the things to consider when making a smear from a bacterial culture on solid media.
There are two.
* First, select a well-isolated colony to be sure that you are only sampling one type of organism.
* Also, use only a tiny amount of a bacterial colony. Otherwise, your smear may be too thick to stain well.
Consequences of Inadequate Heat Fixation
An unfixed or inadequately fixed smear is likely to wash off the slide during staining and rinsing
Consequence of Excessive Heat Fixation
Excessive heat fixation may rupture bacterial cells or distort their size and shape and lead to poor staining results.
List the types of reagents used with differential stains.
- Primary Stain
- Decolorizing Agent
- Secondary Stain (Counterstain)
(also, the Mordant with Gram)
