Gram stain/ Acid-fast / Endospores

-pairs

-Diplococci

___ is a chemical used as a decolorizer in the ACID-FAST stain > safranin > alcohol >differential > acid-alcohol

> acid-alcohol

-organisms' oxygen requirements.

Growth layered at the top, center, or bottom of a broth tube reveals?

1) - stains make microbes stand out against the background. 2) - helps investigators group major categories 3) - helps to distinguish parts of a cell.

What reason do we need to stain bacteria?

___ is the color of ACID-FAST staining reaction under the microscope. [Note that brilliant green dye is the *alternative*counterstain for the dye used to produce this color reaction] > green (from brilliant green dye counterstain) > blue (from methylene blue dye counterstain) > red (from safranin dye counterstain) > violet (from crystal violet dye primary stain)

> blue (from methylene blue dye counterstain)

___ is the type of stain where various types of microbes can be identified based on color > simple > differential

> differential

___ is the color of Gram-negative staining reaction under the microscope > green (from brilliant green dye counterstain) > blue (from methylene blue dye counterstain) > red (from safranin dye counterstain) > violet (from crystal violet dye primary stain)

> red (from safranin dye counterstain)

___ is the color of Gram-positive staining reaction under the microscope. > green (from brilliant green dye counterstain) > blue (from methylene blue dye counterstain) > red (from safranin dye counterstain) > violet (from crystal violet dye primary stain)

> violet (from crystal violet dye primary stain)

-culture medium (plural, media). it may be prepared in liquid form, as a broth or solidified with agar.

A mixture of nutrients such as proteins, carbs, minerals, vitamins, and moisture in the right composition is called?

a glass slide, stained, and heated. The cells in the sample hold onto the dye. The lab team member washes the slide with an acid solution and applies a different stain. The bacteria that hold onto the first dye are considered "acid-fast" because they resist the acid wash. This type of bacteria is associated with tuberculosis and other infections.

Acid fast stain

chemical used to decolorize the Gram stain

Alcohol

-an intact cell wall.

An organism's appropriate Gram-stain reaction depends on the presence of?

When you have an infection, you should request an acid-fast strain. The test can tell if you have come into contact with the bacteria that causes tuberculosis and related infections.

When you have an infection, you should request an acid-fast strain. The test can tell if you have come into contact with the bacteria that causes tuberculosis and related infections.

-rich in RNA(contained in their abundant ribosomes) and stain well in basic.

Bacterial cells are rich in what? and stain very well in what dyes?

-small particles of any kind, including bacteria (whether motile or not), are constantly bombarded by the vibration of the fluid molecules, and so are bobbed up and down, back and forth.

Brownian movement?

- you CANNOT distinguish between Staphylococcus (positive/purple) and Streptococcus (positive/purple) - you CAN distinguish between Staphylococcus (positive/purple) and Neisseria (negative/red) - you CANNOT distinguish between Escherichia (negative/red) and Proteus (negative/red) - you CAN distinguish between Escherichia (negative/red) and Bacillus (positive/purple)

Can you distinguish between various organisms on the basis of color alone, not shape?

-stained preparation is good for studying morphology but not motility because bacteria are already killed. -wet mount is good for studying bacteria motility.

Compare wet mount and stained preparation?

Not always. Saprophytic Mycobacteria are acid-fast positive and don't cause disease. The kinds of acid-fast organisms that do cause disease, like Mycobacterium tuberculosis, are more difficult to treat with antibiotics because of the large amounts of lipids in their cell walls so they do cause serious disease.

Does the presence of acid-fast organisms in a clinical specimen always suggest?

-small, round hollow bodies in cell

Endospore-forming

A differential stain is used to distinguish between two different organisms. It is used to see a difference in the elements of a cell. A couple of examples of differential stains are acid fast and gram stain.

Examples of Differential Stains?

-lipids (fats) in their outer membranes. the lipids dissolve in the decolorizer (alcohol, acetone, or a mixture) and are washed away along with the crystal violet-iodine complex.

Gram negative bacterial walls have high concentration of

- Thick, tightly linked peptidoglycans (protein-sugar complexes) in which the crystal violet iodine complex becomes trapped, thereby enabling the cells to resist decolorization.

Gram positive cell walls are composed of:

-relative numbers and morphology of bacterial in the specimen. -this information is valuable to the physician in planning the patients treatment before the specimen.

Gram stained smears are read promptly to determine the

The Acid-fast stain method, to detect the bacteria Nocardia starts by obtaining a sample of sputum, fluid from the trachea or lungs,or a sample of tissue collected during surgery. Preparing a smear for staining involves applying a very small sample to the center of a carefully cleaned glass slide. Allow the smear to dry completely. Fix the smear at 80 degree Celsius.

How should the acid-fast stain of a sputum specimen from a patient with suspected pulmonary Nocardia infection be performed?

-single colony, pure cultures.

In order to study the properties of individual species without interference from other species, the microbiologist must work with-

-spirochetes

Long sinuous organisms with many loose or tight coils with flexible bodies are called?

Some organisms have complex nutritional requirements that is not easily mimicked with nutrient agar. Using blood agar will allow them to grow better.

Why is a blood agar plates used for the growing culture from the mouth rather than nutrient agar?

MacConkey agar 1. Selective media - bile salts to support the growth of Gram negative microbes - crystal violet to inhibit the growth of Gram positive microbes 2. Differential media - neutral red indicator as visual response

Why is macconkey agar selective as well as differential?

-complex cells of higher forms because they permit differentiation of interior structures, some which are basic and other are acidic.

Neutral stains are made by combining acidic and basic dyes which are most useful for staining?

-V, X, Y forms, clubbing

Palisades

-varying widths and lengths

Pleomorphism

-short, curved, or spiral forms with rigid bodies.

Spirilla

-measured for size and classified by their shapes and groupings.

Stained bacteria can be measured for what? and classified by their?

grapelike, clusters

Staphylo-

-chains

Strepto-

-packet of four

Tetrads

-either an acidic, basic, or neutral manner. acidic and basic are used primarily in bacteriologic work.

The aniline dyes react in

-cations(positively charged) that combine with and acid in the stained material to form a salt.

The basic dyes possess

-aniline dyes( synthetic organic dyes made from coal-tar products).

The best bacterial stains are?

-anions(negatively charged) that combines with cations of a base in the stained cell to form a salt.

The free ions of acidic dyes are

-is motile, but also permits an undistorted( untwisted) view of natural cell groupings and of individual cell size and shape.

The method of suspending bacteria with fluid is used to determine whether an organism

-the concentration of the dyes, length of staining time for each dye, and composition of the decolorizer.

The numerous modifications of Gram's orginal method are based on

-suspend them in a fluid( water, saline, or broth) and prepare a "wet mount."

The simplest method for examining living microorganisms is to

nutrients such as proteins, carbs, minerals, vitamins, and moisture in the right composition.

To cultivate microorganisms we must provide them with

-movement caused by currents in liquid. if a wet mount contains bubbles, air currents set up reacting fluid currents.

True motility

1. bad staining technique 2. bacteria with cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of Gram-positive cells

What 2 conditions may cause gram variable staining (both positive and negative reactions)?

-stain can penetrate the lipid cell wall and reach the cell cytoplasm. once the cytoplasm is stained, it resist decolorization, even with harsh agents such as acid-alcohol.

What happen to cell wall when applying basic dye such as carbolfuchsin with heat or in concentration solution.

-contain large amount of lipid substances within their cell walls. These fatty waxes also known as mycolic acids, resist staining by ordinary methods.

What is special about Mycobacterium and staining

Chocolate agar is essentially blood agar that contains lysed red cells and a couple of growth-promoting agents to grow organisms of Hemophilus and Neisseria. MTM is essentially chocolate agar, but it contains inhibitory agents that are more selective for STDs. MTM is only used when a genital culture is ordered. Chocolate is used in a variety of culture types

What is the difference between Modified Thayer-Martin MTM agar and Chocolate Agar? When would you use MTM?

-ethyl alcohol slowest which permit the student to gain experience with decolorization. -Acetone is the fastest with an equal mixture of 95% ethyl alcohol. -the most popular is the combination of acetone-alcohol.

What is the lowest and fastest decolorizers?

-Cervical smear - test for cancer -a thick blood smear for malaria disease, viability of sperm cells from semen

What kind of clinical situation would a direct smear report from the laboratory be of urgent importance?

-when bacteria culture age and wall begins to disintegrate.

What makes the gram-positive of crystal violet-iodine complex appear falsely gram-negative or mixture?

Acid fast bacteria are not classified as either Gram-positive or Gram-negative because they do not have the chemical characteristics of either, although the bacteria do contain peptidoglycan (murein) in their cell wall. If a Gram stain is performed on Mycobacterium tubersulosis, for instance, it stains very weakly Gram-positive or not at all (referred to as "ghosts"). Mycobacterium species, along with members of a related genus Nocardia, are classified as acid-fast bacteria due to their impermeability by certain dyes and stains ( thus explaining why the gram stain isn't useful). Despite this, once stained, acid-fast bacteria will retain dyes when heated and treated with acidified organic compounds. One acid-fast staining method for Mycobacterium tuberculosis is the Ziehl-Neelsen stain. When this method is used, the M.TB. smear is fixed, stained with carbol-fuchsin (a pink dye), and decolorized with acid-alcohol. The smear is counterstained with methylene-blue or certain other dyes. Acid-fast bacilli appear pink in a contrasting background. Because their cell walls do not absorb the dyes used in the Gram stain

Why isn't the Gram stain used on acid-fast bacteria?

-mordant (a chemical that fixes a dye in or on a substance by combining with the dye to form an isoluble compound) for the first crystal violet stain.

an iodine solution is used as a

In differential staining, the cells retaining the first or primary stain are usually termed: a) fast b) positive c) resistant d) simple

b) positive

Which stain is used as the counterstain for the Gram stain? a) iodine b) safranin c) cystal violet d) methylene blue

b) safranin

The chemical found in Gram-positive cell walls not in Gram-negative cell walls that reacts w/the crystal violet-iodine complex is: a) lipopolysaccharides b) teichoic acid c) protein coat d) peptidoglycan

b) teichoic acid

The reason why certain bacteria are termed "acid-fast" is: a) they release HCI from their cell walls b) they resist penetration of an alcohol/HCI solution into their cells c) they require a low pH to grow d) they use acid to dissolve needed nutrients

b) they resist penetration of an alcohol/HCI solution into their cells

What color would Gram-positive cells show under the microscope? a) pink b) violet c) blue d) green

b) violet

-basis for their classification.

bacteria have rigid cell walls and maintain a constant shape that is used as

A cell is first stained with carbol fuchsin, decolorized, and then exposed to methylene blue. When observed under the microscope, the cells exhibit a blue color. This indicates that these cells are: a) Gram-positive b) Gram-negative c) acid-fast d) nonacid-fast

c) acid-fast

The stain used to visualize Mycobacteria is: a) simple stain b) Gram stain c) acid-fast stain d) endospore stain

c) acid-fast stain

Peptidoglycan and teichoic acid form a molecular complex with: a) safranin b) carbol fushin c) crystal voilet/iodine d) methylene blue

c) crystal voilet/iodine

appearance of colonial growth on agar media such as color, density, consistency, surface texture, shape, and size.

colonial morphology

a visible aggregate of cells that is formed from a single bacteria cell deposited of a certain area on an agar plate that multiplies at the site.

colony

A normally Gram-positive cell shows up as Gram-variable. Which explanation reveals why this occurred? a) the smear was too thick b) the smear was exposed to the decolorizer too long c) a 48-hour culture was used d) all of these

d) all of these

Differential staining allows one to: a) identify pathogenic vs nonpathogenic bacteria b) distinguish b/w eukaryotic and prokaryotic cells c) identify various bacterial types based on color d) all of these

d) all of these

The iodine in the Gram stain is used: a) to remove safranin from the cell wall b) to decolorize c) as a primary stain d) as a fixative or mordant

d) as a fixative or mordant

A microbial cell that shows up positive in a differential stain: a) is impermeable to water b) has a thick cell wall c) has been decolorized d) retains the color of the primary stain

d) retains the color of the primary stain

The iodine in Gram stain fixes the crystal violet to the bacterial cell wall of the Gram positive bacteria.

how does iodine has an affect on staining?

EMB agar - Inhibits other organisms due to the presence of methylene blue dye MacConkey agar - Inhibits other organisms due to the presence of crystal violet dye Mannitol salt agar (MSA) - High salt concentration does not allow other organisms to grow only S.aureus can grow in a high salt medium.

if you wanted to isolate s. aureus from a pus specimen containing a mixed flora, what medium would you choose to get the results most rapidly?

container growing two or more indentified species

mixed culture

-seaweed.

nonnutritive solidifying agent extracted from?

-Mycobacterium species are found in the environment and are rarely pathogenic or have not yet been associated with infection.

saprophytic mycobacteria

-isolating and separating bacteria growing together in a specimen collected from a patient; for example, urine or sputum.

solid media are essential for

-spherical(coccus, plural cocci). -rod(bacillus, plural bacilli) -spiraled(spirillum plural spirilla)

what are the bacteria three basic shapes?

-Other bacteria whose cell walls do not contain high concentrations of lipids are readily decolorized by acid-alcohol after staining with carbolfuchsin.

what is non-acid fast

-Gram positive, and gram negative. -gram pos look like a dark blue/purple colour, and gram neg look red

what is the purpose of gram stain

-for isolation colonies

what is the purpose of quadrant streaking

The alcohol serves to dehydrate the peptitoglycan layer of the cell wall. Because it is the outermost layer of the Gram positive bacterial cell wall, it is dehydrated by the alcohol. This causes pores to shut in the crystal violet, staining it purple. The cell wall of a Gram negative bacterial cell does not have its peptidoglycan on the outermost layer, so it does not dehydrate and retain the dye, and thus stains with the counterstain. If this step were omitted and every other step were carried out, all cells would stain with the counterstain, and it would be impossible to tell whether or not they are Gram positive or negative by looking at the colour.

what is the purpose of the alcohol solution in the gram stain?

-it allows the microbiologist to determine how many types of bacteria are present, which are causing the disease, and test which antimicrobial agents will be effective for treatment.

why streaking plates to obtain pure cultures is critical?