Microbiology Test 4 A

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Microscopic examination is an invaluable tool used to

detect and identify microorganisms in clinical specimens; and to provide information regarding the size, shape, arrangements, and staining reactions of microorganisms grown on artificial culture media.

The use of various dyes or stains enhances the process by

providing more distinct contrast of the organism and its structure against the background material, or by differentiating structures or organisms based on differing reactions to the particular stains used.

A specimen for microscopic examination is generally prepared by

mounting it on a glass microscope slide.

The actual preparation of the specimen depends on the

condition of the specimen (live or preserved); the intent of the examination (observe movement, observe overall structure, observe differential characteristics, etc.); and the type of microscopy to be used (bright-field, dark-field, phase-contrast, fluorescence, etc.).

Wet mounts of hanging drop mounts are used for

live specimens in order to observe the organism as near its natural state as possible.

In wet mounts, the specimen is

suspended in a suitable fluid (water, saline, broth). A drop or two of the suspension is placed on the microscope and overlaid with a cover glass.

the wet mount fluid provides a

suitable medium to maintain the viability of the organisms for a short period of time, and provides a medium for locomotion

The wet mount type of preperation is intended to

allow observation of the size, shape, arrangement, and motility of the organisms observed.

Certain dyes or stains may be added to the wet mount preparation to

enhance the contrast between the organisms and the background material. This is a short-term preparation that is not intended to be retained for later use.

More permanent preparation of specimens for longer-term use can be accomplished by preparing

fixed, stained specimens. This process is called heat fixation. The fixed specimen can then be stained according to the particular staining method.

With a fixed, stained specimen, a liquid suspension of the organisms to be observed (or the clinical specimen) is

spread as a thin smear on the microscope slide and allowed to air-dry. The air-dried smear is usually heated gently.

Gently air-drying the smear is called

heat fixation

Heat fixation does what

kills the organisms, adheres the material to the glass microscope slide, and preserves the cellular detail with minimal distortion.

Fixation may also be performed using (other than heat)

chemicals rather than heat.

The two basic types of staining techniques depend on

how the dye reacts with the specimen.

Staining methods in which the dye actually sticks to the specimen and gives it color are called

positive stains.

Staining methods in which the dye does not stick to the specimen but settles around its boundary, forming a silhouette, are called

negative stains.

The dyes most commonly used for negative staining are

nigrosin (blue-black) and India ink (black suspension of carbon particles).

In negative staining procedures, the cells themselves do not stain because the negatively charged (anionic) dyes are

repelled by the negatively charged surface of the cells.

In negative staining, the specimen is observed as a

light image silhouetted against a dark background. There is little shrinkage or distortion of the cells since the specimen is not heat fixed. Relatively simple assessments of the size, shape, and arrangement of organisms can be made.

Negative staining is frequently used to

accent the presence of a capsule that surrounds many bacteria and yeasts.

Positive staining methods are classified as

simple, differential, or special based on the intent of the staining procedure.

Simple stains require only

a single dye and an uncomplicated procedure.

Most simple stain procedures take advantage of the

attraction and binding of positively charged (cationic) dyes to the negatively charged surface of the cells.

Simple stains cause all cells in the smear to appear

about the same color, regardless of type;

Simple stains can reveal

characteristics such as size, shape, and arrangement.

Examples of cationic dyes used in simple staining procedures include

methylene blue, malachite green, crystal violet, basic fuchsin, and safranin.

Differential stains utilize

two different stains to distinguish between different types of cells or different parts of cells.

The two stains used in differential staining are referred to as

the primary stain and the counterstain

Properly performed differential stains provide

clear contrast between two different types of cells or cell parts. These differences are observable as color differences between the cell types or parts.

Common color combinations used in differential stains include

red and purple, red and green, or pink and blue.

In addition to observing size, shape, and arrangement of cells; differential stains may distinguish organisms based on

Gram reaction, acid-fastness, presence of spores, or other stain reactions.

Examples of differential staining procedure include

Gram stains, acid-fast stains, and spore stains.

Special stains are used to highlight or emphasize certain cell parts that may not be revealed by

conventional staining methods. These stains may be used to accent structures such as capsules and flagella. The presence or absence of these structures can be a useful feature for identifying the organisms.

The Gram stain was developed by

Christian Gram in the late 1800's. This differential staining procedure allows bacteria to be distinguished into two major categories termed Gram-positive and Gram-negative.

Differentiation of bacteria as gram-positive vs. gram-negative is based on the

different cell wall compositions of the two groups.

The relatively thick peptidoglycan layer of the cell wall of gram-positive bacteria resists

decolorization of the primary stain.

The primary stain is easily removed from the gram-negative bacteria which then are stained with

the counterstain.

Gram stain reaction serves as a major differentiation of bacteria and is the basis of several bacteriological issues including

bacterial taxonomy, identification and diagnosis of infections, and selection of appropriate antimicrobial therapy.

The Gram stain consists of

timed, sequential application of a primary stain, mordant, decolorizer, and counterstain.

The primary stain used in the Gram stain procedure is

crystal violet. After the smear has been prepared, air-dried, heat-fixed (or chemically fixed), and cooled; the smear is covered with crystal violet solution. The crystal violet is not specific for gram-positive cells, but stains all cells the same purple color.

The second step in the gram stain process is the application of an

iodine solution which serves as a mordant. Entrapment of the crystal violet crystals is far more extensive in the thick peptidoglycan layer of gram-positive cell walls than in the gram-negative cells.

A mordant is a

stabilizer that causes the crystal violet to form large crystals in the peptidoglycan layer of the cell wall.

The third step in the gram stain process is

application of an alcohol decolorizer which dissolves lipids in the outer membrane and removes the crystal violet dye from the peptidoglycan layer. The crystals of crystal violet that are tightly embedded in the thick peptidoglycan layer of the gram-positive cell walls are relatively inaccessible and resistant to removal.

This alcohol decolorization step results in

retention of the crystal violet (purple color) by the gram-positive organisms, but removal of the crystal violet from the gram-negative organisms leaving them colorless.

The last step in the gram staining process is

application of safranin which serves as the counterstain. Safranin imparts its red color to the gram-negative organisms.

After completion of the Gram stain procedure, bacteria observed can be differentiated into the two major categories of

Gram-positive and Gram-negative based on their resulting colors. Gram-positive organisms appear purple, and gram-negative organisms appear red-to-pink.

The Gram stain is an old procedure but remains the

universal basis for classification and identification of bacteria.

In addition to differentiating bacteria as gram-positive vs. gram-negative; this stain also allows observation of the

sizes, shapes, and arrangements of the bacteria involved.

The acid-fast stain is another differential stain that allows determination of bacteria that have the characteristic of

acid-fastness from those that lack this quality. The Ziehl-Neelsen acid-fast stain is a common example of this type stain.

Some organisms, particularly members of the genera ?, ?, and some ? have a high lipid content in their cell wall.

Mycobacterium, Nocardia, and some Actinomyces

The high lipid characteristic is responsible for some organiisms

resistance to drying, acids, and various germicides.

Organisms demonstrating this high lipid characteristic are called

acid-fast.

Acid-fast organisms resist

decolorization of the primary stain by acidic decolorizing agents. This characterisitic allows for differential staining that will distinguish acid-fast vs. non-acid-fast organisms.

One of several acid-fast stains is called

The Ziehl-Neelsen acid-fast stain

The primary stain for the Ziehl-Neelsen procedure is

carbol fuchsin. After the smear has been prepared, air-dried, heat-fixed (or chemically fixed), and cooled; the smear is covered with carbol fuchsin. Absorption of this stain is enhanced by heating or steaming.

The second step in the Ziehl-Neelsen acid-fast stain procedure, after the slide has cooled, is

application of an acid-alcohol decolorizer.

Organisms demonstrating the characteristic of acid-fastness will

resist decolorization and retain the red color associated with the carbol fuchsin. Non-acid-fast organisms will be decolorized and will be colorless after decolorization.

The last step in the Ziehl-Neelsen acid-fast procedure is application of

methylene blue as a counterstain. Methylene blue imparts its blue color to the non-acid-fast organisms.

After completion of the Ziehl-Neelsen acid-fast stain procedure, bacteria observed can be differentiated as acid-fast or non-acid-fast based on

their resulting colors.

Acid-fast organisms appear ?, and non-acid-fast organisms appear ?.

red to pink
blue

The Schaeffer-Fulton spore stain is a differential stain used for demonstrating the presence of

endospores. It is based on differential staining characteristics of the endospores and vegetative cells.

The formation of endospores is a significant characteristic of certain organisms, particularly those of the genera

Bacillus and Clostridium.

The Schaeffer-Fulton spore stain allows the endospores to be distinguished from

the cells that produce them (vegetative cells).

The primary stain for the Schaeffer-Fulton spore stain is

malachite green. After the smear has been prepared, air-dried, heat-fixed (or chemically fixed), and cooled; the smear is covered with malachite green. Absorption of this stain into the endospores is enhanced by heating or steaming.

After the slide has cooled, the excess stain is rinsed off with distilled water. The last step in the Schaeffer-Fulton spore stain is application of

safranin as a counterstain.

After completion of the Schaeffer-Fulton spore stain procedure, endospores can be differentiated from their vegetative cells based on

their resulting colors. Endospores retain the malachite green stain and appear green. The vegetative cells accept the safranin counterstain and appear red-to-pink.

Special stains are used to highlight or emphasize certain cell parts that may not be revealed by conventional staining methods. Examples of special stains include ones used to demonstrate

capsule formations and flagella.

Capsules are relatively unstructured protective layers surrounding the cells of some

bacteria and fungi.

Because capsules do not react with most stains, they are most often demonstrated by

negative staining procedure using India ink or nigrosin. These structures may also be demonstrated by special positive stains

The fact that not all organisms produce capsules provides a useful feature in

identifying the organisms.

A diagnostic example of the use of Schaeffer-Fulton endospore stain is associated with the serious fungal pathogen

Cryptococcus neoformans. Microscopic observation of an encapsulated yeast form in a clinical specimen (frequently cerebrospinal fluid) from a patient with compatible symptoms is highly suggestive of cryptococcal infection.

Flagellar staining is used to demonstrate

the presence, number, and arrangement of flagella on the organisms observed.

The width of bacterial flagella is smaller than the

resolving power of the light microscope. In order to be seen, the flagella must be enlarged by depositing a coating on the outside of the filament and then staining it. (like mascara)The presence, number, and arrangement of flagella is characteristic of the particular bacteria.

Direct microscopic examination of clinical specimens or microorganisms grown on artificial culture media provides the microbiologist with valuable information regarding the

size, shape, and arrangement of organisms as well as differential staining characteristics.

The use of various stains or dyes provides more distinct contrast between the ? and the?, or may provide differentiation among organisms based on?

organisms, background,
*differing reactions to the particular stains or staining procedures.

Staining procedures may involve

simple stains, differential stains, or special stains.

Simple stain procedures require only

a single dye and an uncomplicated procedure.

Most simple stains depend on the attraction of a ?dye to the ? cell surface.

positively charged (cationic) dye
negatively charged cell surface.

Simple stains cause all cells to appear about the same color, but can reveal

characteristics such as size, shape, and arrangement.

Simple stains do not provide any

differentiation among the organisms observed.

Examples of simple stains include

methylene blue, crystal violet, malachite green, safranin, etc.

Differential stains utilize two different

stains to distinguish between different types of organisms, cells or parts of cells.

The two different stains in differentail staining are generally referred to as the

primary stain and the counterstain (secondary stain)

Properly performed differential stains provide

clear contrast between different types of organisms, cells, or parts of cells.

The differences among the types of organisms or cells in differentail staining are typically observed as

color differences resulting from the differential stain procedure.

Examples of differential stains include the

Gram stain procedure, acid-fast stain procedures, spore stains, etc.

Other special stains may be used to highlight certain cell parts that may not be apparent with other conventional stain procedures. These special stains may be used to

highlight structures such as flagella and capsules.

Specimens must be properly prepared for staining, The quality of the resulting stained material is directly related to

the quality of the specimen subjected to the staining procedure.

The specimen to be stained must be prepared on the microscope slide. The actual method of slide preparation varies depending on

the source of the material to be stained.

Specimen material to be stained may be obtained from

broth cultures, solid media, or may be direct clinical specimens collected directly from a patient.

Smear preparation from liquid broth (11 steps)

1. Obtain a clean microscope slide and the liquid broth
2. Label the microscope slide
3. Sterilize an inoculating loop
4. remove the cap from the broth culture tube.
5. Flame the mouth of the tube
6. Insert the sterilized (but cool) inoculating loop into the broth culture and remove a loopful of the broth.
7. Reflame the mouth of the broth culture tube, replace the cap onto the tube.
8. Place the loopful of the broth culture directly onto the microscope slide. Spread the drop to about one centimeter diameter (about the size of a dime).
9. Resterilize the inoculating loop.
10. Allow the slide to air dry. The smear will turn opaque as it dries.
11. Once the smear is air-dried, it is ready to be stained. Heat-fixing may be required depending on the stain procedure to be used.

Smear preparation from solid culture media (12 steps)

1. Obtain a clean microscope slide, a small amount of distilled/sterile water, and the culture media (slant tube or plate) containing the culture material to be stained.
2. Label the microscope slide
3. Sterilize an inoculating loop
4. Use the inoculating loop to obtain a drop of distilled water and place it onto the microscope slide. Do not smear or disperse the drop of water at this time.
5. Resterilize the inoculating loop.
6. If the culture media is in the form of a slant tube, remove the cap and pass the mouth of the tube through the flame
7. If the culture media is in plate form, set the plate upside down on the bench and remove the bottom of the dish (containing the media) from the lid.
8. Use the sterilized (but cooled) inoculating loop to transfer a small sample (single colony if possible) of the bacteria to be stained to the microscope slide. Mix the bacteria into the drop of distilled water previously placed on the slide. Spread the liquid mixture to about one centimeter diameter (about the size of a dime).
9. Resterilize the inoculating loop.
10. Reflame the slant tube and replace the cap, or return the bottom of the petri dish to the lid.
11. Allow the slide to air dry. The smear will turn opaque as it dries.
12. Once the smear is air-dried, it is ready to be stained. Heat-fixing may be required depending on the stain procedure to be used.

Depending on the stain procedure to be used, the prepared smear may require

heat-fixation prior to staining. The smear should be subjected to moderate heat for a short exposure. Overheating the smear will result in morphological distortion of the organisms and render the smear unusable. Other fixation techniques are available that utilize certain chemicals rather than heat as the fixation agent.

Heat-fixation does what?

kills the organisms and adheres them to the slide. This process helps prevent the smear from being washed off the slide during the staining procedure.

Heat-fixation of prepared smear

1. Obtain the prepared, air-dried smear.
2. Using a slide holder (or other suitable device). Pass the slide through the Bunsen burner flame 3-4 times. The slide must not be heated too much. Overheating will cause morphological distortion of the organisms. Staining results will be useless.
3. After heat-fixing, allow the slide to cool.
4. After cooling, the heat-fixed smear is ready to be stained.

A simple stain using methylene blue (or some other cationic dye) is used to allow microscopic examination of the

size, shape, and arrangement of organisms and/or other cells. The simple stain colors the material to enhance the visual image, but does not provide any differential characteristics.

The gram stain is a differential stain procedure in which crystal violet is used as the primary stain and safranin is used as the counterstain (secondary stain). Iodine is used as a mordant. A mordant is a substance that helps chemically bind the basic dye to the cell wall material. The procedure allows differentiation of organisms into two major groups referred to as

gram positive and gram negative. Differentiation is based on variations in the cell wall structure of the two groups. Gram positive organisms resist decolorization and appear purple (or blue) due to retention of the crystal violet primary stain. Gram negative organisms are decolorized and appear red (or pink) due to the safranin counterstain.

The Ziehl-Neelsen stain is a differential stain procedure in which carbol fuchsin is used as the primary stain and methylene blue is used as the counterstain (secondary stain). This procedure allows differentiation of

acid fast organisms from non-acid fast organisms. Acid fast organisms resist decolorization with an acid alcohol decolorizer and appear red to pink due to the carbol fuchsin primary stain. Non-acid fast organisms are decolorized with the acid alcohol decolorizer and appear blue due to the methylene blue counterstain.

Staining procedures that use two different stains to distinguish between different types of organisms or cells are called _______________ stains.

differential

Organisms that appear purple as a result of the gram stain procedure are referred to as ____________________ organisms.

gram positive

The Ziehl-Neelsen staining procedure is used to detect _______________ organisms

acid fast

The primary stain used in the gram stain procedure is ____________________

crystal violet

A substance used in the staining process that chemically binds the basic dye to the cell wall material is called a _______________ .

mordant

The primary stain used in the Ziehl-Neelsen stain procedure is ____________________

carbol fuchsin

The gram stain procedure causes gram negative organisms to appear _______________

red/pink

Organisms that appear red as a result of the Ziehl-Neelsen stain procedure are called _______________ .

acid fast

The counterstain used in the gram stain procedure is _______________ .

safranin

The process in which a prepared smear is passed through a flame several times prior to staining is called ____________________ .

heat fixation

The process in which a prepared smear is passed through a flame several times prior to staining is called ____________________ .

kills organism
adheres the organism to the slide

The mordant used in the gram stain procedure is _______________

iodine

In order to adequately examine bacteria microscopically, __________ total magnification is required.

1000x

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