BIO 261L - lab 3 ex 14,16

Mordant

A mordant intensifies a stain or can be used to coat structures such as flagella for viewing.

mycobacterium

Mycobacterium and some members of the genus Nocardia have a layer of mycolic acid that prevents them from being properly stained. • Mycolic acid is a waxy material in their cell wall.

gram-variable

Some bacteria are considered gram-variable be- cause some cells will retain the crystal violet stain, while others will not and appear red from the counterstain.

term acid-fast

Subsequent treatment of the cells with acid-alcohol, a decolorizer, does not remove the entrapped stain from the cells. Hence, these bacte- ria are termed acid-fast

Kinyoun acid-fast method

The Kinyoun acid-fast method is a modification in which the concentrations of primary stain, basic fuchsin (substituted for carbolfuchsin), and phenol are increased, making it unnecessary to heat the cells during the staining procedure. he in- creased concentrations of basic fuchsin and phenol are sufficient to allow the penetration of the stain into acid-fast cells, and the basic fuchsin is not removed during destaining with acid-alcohol. This procedure is safer because phenol fumes are not generated during staining of the cells.

What is the primary stain in the Kinyoun acid-fast stain? How does this differ from the primary stain in the Ziehl-Neelsen method?

The basic fuchsin is the primary stain in the Kinyoun. Ziehl-Neelson is the carbol fuchsin. They're different because the basic makes heat unnecessary

If you conducted an acid-fast stain on a sputum sample from a person with tuberculosis, what would you expect to see on the slide? color

Will be stained pink to red

acid-fast bacteria

ave a unique cell wall made of "waxy" lipids. Mycobacteria, the caus- ative agents of tuberculosis and Hansen's disease (lep- rosy), are acid-fast bacteria. These cells may appear as either nonreactive or gram-positive after the Gram stain technique, but a special acid-fast staining tech- nique (see Exercise 16) can be used to identify bacteria with this type of cell wall.

Escherichia coli under the microscope if you forgot to apply safranin

colorless

Moraxella catarrhalis after decolorization

colorless

Pseudomonas aeruginosa after decolorization

colorless

Staphylococcus aureus before the primary stain

colorless

mycolic acid

complex lipid that is composed of fatty acids and fatty alcohols that have hydrocarbon chains up to 80 carbons in length. It significantly affects the staining properties of these bacteria and prevents them from being stained by many of the stains routinely used in microbiology. e acid-fast stain is an important diagnostic tool in the identification of Mycobacterium tuberculosis, the causative agent of tuberculosis, and Mycobacterium leprae, the bacterium that causes Han- sen's disease (leprosy) in humans.

Hans Christian Gram

e discovered that certain stains were retained by some types of bacterial cells but removed from others during the staining pro- cess.

What other bacterial genus is acid-fast?

mycobacterium, nocaria

acid fast genuses example

mycoplasmas, norcardias

gram stain colors, +/-

n the Gram stain, two kinds of cells, gram-positive and gram-negative, are differentiated based on their cell wall structure and composition. These types of cells can be identified by their respective colors, purple and pink or red, after performing the staining method.

Why is the gram stain considered a differential stain?

n the Gram stain, two kinds of cells, gram-positive and gram-negative, are differentiated based on their cell wall structure and composition. These types of cells can be identified by their respective colors, purple and pink or red, after performing the staining method. The procedure is based on the fact that gram-positive bacteria retain a purple dye complex, whereas gram-negative bacteria are decolorized and must be counterstained with a red dye in order to be visualized by microscopy

bacillus megaterium under the microscope if you forgot to apply iodine

pink color

Pseudomonas aeruginosa after the primary stain

purple

bacillus megaterium after the addition of the mordant

purple

Escherichia coli under the microscope if you forgot to apply decolorize

purple color

Staphylococcus aureus after decolorization

purplee

acid fast stain steps lecture

rinse with water blot dry look under microscope

safety concerns of staining with phenol

the application of heat to cells during staining with carbolfuchsin and phenol is not without safety concerns. Phenol can vaporize when heated, giving rise to noxious fumes that are toxic to the eyes and mucous membranes.

methylene blue, color

the counterstain that can show if cells are non acid fast The acid fast cells tend to appear red or pink and the non acid fast cells appear blue.

how does alcohol wash off dye in gram negative cells

the decolorizer dissolves the lipids in the outer membrane of gram-negative bacteria, al- lowing the dye-mordant complexes to escape through the thin peptidoglycan layer.

Ziehl-Neelsen staining method

the primary stain, carbolfuchsin, contains phenol, and the cells are heated for 5 minutes during the staining procedure. Phenol and heat facilitate the penetration of the car- bolfuchsin into the cell.

purple

Bacillus megaterium after adding the counterstain

gram stain procedure m

Cover a heat-fixed smear with crystal violet and let stand for 30 seconds (see figure 14.3). Briefly wash off the stain, using a wash bottle of distilled water. Drain off excess water. Cover the smear with Gram's iodine solution and let it stand for 1 minute. (Your instructor may prefer only 30 seconds for this step.) Wash off the Gram's iodine. Hold the slide at a 45-degree angle and apply the decolorizer, allowing it to flow down the surface of the slide. Do this until the decol- orizer is colorless as it flows from the smear down the surface of the slide. This should take no more than 15 seconds for properly prepared smears. Note: Thick smears can take longer for decolorization. Stop decolorization by washing the slide with a gentle stream of water. Cover the smear with safranin for 1 minute. 7. Wash gently for a few seconds and blot dry with bibulous paper. 8. Examine the slide under oil immersion.

diferrential staining

Differential staining reactions take advantage of the fact that cells or structures within cells display dissimilar staining reactions that can be distinguished by the use of different dyes.

How do gram-positive and gram-negative bacteria differ in cellular structure, and how does this con- tribute to their differential staining properties?

Gram (-) cells have a greater lipid content in their cell walls than gram (+) cells. Lipids are more soluble in alcohol and acetone that causes more rapid decolorization of gram (-) cell Gram-positive bacteria have a thicker layer of peptidoglycan that retains the crystal violet-iodine complex.

what is grams staining

Gram staining is a valuable diagnostic tool used in the clinical and research setting

In what type of cell, gram-positive or gram-negative, would you find lipopolysaccharide in its cell wall?

Gram-negative bacteria are surrounded by a thin peptidoglycan cell wall, which itself is surrounded by an outer membrane containing lipopolysaccharide. Gram-positive bacteria lack an outer membrane but are surrounded by layers of peptidoglycan many times thicker than is found in the Gram-negatives.

when doing acid fast, what is the mordant ? what does this mean for the decolorizer?

Heat is acting as a mordant to make the mycolic acid and cell wall lipids more permeable to the stain. acid-alcohol, a decolorizer, does not remove the entrapped stain from the cells.

Which step in the Gram stain procedure is most prone to error? If done incorrectly, how might that step affect the end result?

If done too long, gram-positive can look gram-negative and if done too short, gram-negative and look gram-positive. decolorization

colors of fast staining Staphylococcus as an example

In the acid-fast staining method, acid-fast bacteria such as Mycobacterium are not decolorized by acid- alcohol and are therefore stained pink to red by the basic fuchsin. Because non-acid-fast bacteria such as Staphylococcus are decolorized by the acid-alcohol, a secondary stain, methylene blue, must be applied to visualize these cells in stained preparations. appears blue after staining

steps of gram, colors

Initially, both gram-positive and gram-negative cells are stained by the primary stain, crystal violet. In the second step of the procedure, Gram's iodine is added to the smear. Iodine is a mordant that combines with the crystal violet and forms an insoluble complex in gram-positive cells. At this point, both types of cells will still appear as purple. During decolorization with alcohol and/or acetone, gram-positive cells retain the crystal violet- iodine complex, and therefore these cells will ap- pear purple under the microscope. Alternatively, the dye-mordant complex is removed from gram-negative cells, leaving them colorless. Safranin is applied as a counterstain, coloring the gram-negative cells pink or red. The safranin also sticks to the gram-positive cells, but their appearance is unchanged because the crystal violet is a much more intense stain than safranin.

Several factors can affect the outcome of the procedure:

It is important to use cultures that are 16-18 hours old. Gram-positive cultures older than this can convert to gram-variable or gram-negative and give erroneous results. gram-negative bacteria never convert to gram-positive It is critical to prepare thin smears. Thin smears allow the observation of individual cells and any arrangement in which the cells occur. However, thick smears can entrap the primary stain, pre- venting decolorization. Cells that occur in the entrapped stain may falsely appear gram-positive. Decolorization is the most critical step in the Gram stain procedure. If the decolorizer is overapplied, the dye-mordant complex may be removed from gram-positive cells, causing them to incorrectly appear as gram-negative cells.

gram + bacteria

have a thick layer of peptidoglycan an will retain initial dye even if decolorization with alcohol or acetone is done

gram - bacteria

have a thin layer of peptidoglycan in addition to an outer membrane , will appear red after a counterstain with safarin dye post being decolorized by with alcohol or acetone

What is the function of a mordant, and which reagent serves this purpose in the Gram stain procedure?

iodine A mordant intensifies a stain or can be used to coat structures such as flagella for viewing.

facilitate the staining of acid-fast bacteria

it is necessary to use methods that make the cells more permeable to stains because the mycolic acid in their cell walls prevents the penetration of most stains