ACID FAST STAINING Flashcards by Kyra Catedral

A. Sputum Smear Preparation

Prepare a clean glass slide. Label specimen number at the frosted end of the slide.
Fish out ________of sputum.
Spread one loopful of the sputum evenly on a clean labeled glass slide, approximately________ in size.
Allow the smear to dry completely at________. Do not placed it _________.
After drying completely, fix it by passing through the ______about_____each. Flame at the back of smeared surface of the slide. Never scorch the smear. Blot dry with a paper towel or tissue.

one loopful of yellowish particle

2 cm x 3 cm

room temperature; under the heat of the sun or over a flame

flame 2 to 3 times

2-3 seconds

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The cell wall of some organisms, such as those belonging to the Genus_____, contains a substantial amount of lipids.

Mycobacterium

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_______ are very difficult to stain because it resists penetration of the primary dye into the cellular cytoplasm.

Because of this factor, application of_________ or _______ is needed.

Once stained, these organisms retain the stain so that even acid alcohol could not remove it. Hence, they are referred to as_______.

Lipids

heat or prolonging the exposure of the smear with the primary dye

acid fast

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Steps in Acid-Fast staining:

A._________ is applied to a fixed smear, and the slide is gently_____ for several minutes.

B. Slide is cooled and washed with water.

C. Smear is next treated with_______, a decolorizer, which removes the red stain from________

D. The smear is then stained with a________. _______appear blue after application of the counterstain.

Red dye carbolfuchsin; heated

acid alcohol; bacteria that are not acid-fast.

methylene blue counterstain; Nonacid-fast cells

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• The acid-fast microorganisms retain the red color because the______ is more soluble in the_______ than in the______

carbolfuchsin

cell wall lipids

acid-alcohol

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• In non-acid fast bacteria, whose _______lacks the_____ components, the carbolfuchsin is rapidly removed during______, leaving the cells colorless.

cell wall; lipid

decolorization

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MAIN CAUSES OF FALSE READINGS IN AFS SMEARS:

If smear is too big or too small

False negative

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MAIN CAUSES OF FALSE READINGS IN AFS SMEARS:

If smear is uneven or sloughed off

False negative

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MAIN CAUSES OF FALSE READINGS IN AFS SMEARS:

Is smear is too thick or too thin

False negative

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MAIN CAUSES OF FALSE READINGS IN AFS SMEARS:

If smear has dirt or an artifact

Dirt: false +/-

Artifact: false +

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MAIN CAUSES OF FALSE READINGS IN AFS SMEARS:

If sputum has saliva

False negative

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MAIN CAUSES OF FALSE READINGS IN AFS SMEARS:

Staining:

Overheating
Insufficient heating
Under decolorization
Over decolorization

Overheating - False +/-
Insufficient heating - False -
Under decolorization - False +/-
Over decolorization - False -

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The______ end of the applicator stick can be used instead of wire loop to take sputum to smear and the_____ end may be used to make the smear.

The used sticks are burnt after day’s work.

rough

pointed

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Acid Fast Staining procedure

Carbolfuchsin -> Heat till steam comes off -> do not boil and dry -> let sit for 10mins

Tilt slide -> wash gently -> tilt slide

Cover whole slide with Acid alcohol 25-30secs

Wash slide gently -> tilt slide

Cover whole slide with Methylene blue 10secs

Tilt slide -> wash gently -> tilt slide -> dry rack do not place under the sun

OIO -> immersion oil -> Acid fast bacilli appear red or pink color and the background is stained in blue color

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Evaluation of Acid-Fast Stained Smear
1. Read the slide________. Do not read the smear in a______ manner because this will increase the straining of the eyes.

Proceed from ________end to the other end. Come_____ a little and then proceed either from______ (or three vertical lines of the smear).
One horizontal line of a______ width of the smear corresponds to approximately _______of magnification x 1000 (100 x objective and 10 x eyepiece).

systematically; zigzag

left or right ; down; left to right

3 cm; 150 visual fields (VF)

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Evaluation of Acid-Fast Stained Smear

NOTE:
One may read along one imaginary horizontal line from one end to the other of the smear if the reading is

positive (+n, 1+, 2+, 3+)

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Evaluation of Acid-Fast Stained Smear

Read two horizontal lines which correspond to _______to report the slide as______.
Read at least______. When AFB is seen, count the number of bacilli present. But stop reading the smear when the average number of AFB in each field is________ in less than one horizontal line and the smear is reported as_____. When AFB is not observed on one horizontal line,_______
Report as _______and not such a comment as M. tuberculosis because the identification of Mycobacterial species is not possible by microscopic examination, but it is only possible after_______ of the bacilli.

300 visual fields ; negative

one horizontal line; 1or more; (+++), continue to read second horizontal line

Acid-Fast Bacilli; cultivation

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NATIONAL STANDARD REPORTING SCALE

No AFB seen in 300 visual fields

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NATIONAL STANDARD REPORTING SCALE

1-9 AFB / 100 visual fields

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NATIONAL STANDARD REPORTING SCALE

10-99 AFB / 100 visual fields

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NATIONAL STANDARD REPORTING SCALE

1-10 AFB / OIO in at least 50 visual fields

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NATIONAL STANDARD REPORTING SCALE

More than 10 AFB / OIO in at least 20 visual fields

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is considered a reasonably sensitive and rapid procedure for the presumptive identification of Mycobacterium spp. in clinical specimens.

Microscopy

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The cell walls of mycobacteria contain long-chain multiple cross-linked fatty acids called _______.

These contribute to the characteristic of acid-fastness that distinguishes mycobacteria from other bacteria.

mycolic acids

Mycobacteria are not the only group with this unique feature (mycolic acid). Species of _____ and _____are also partially acid fast

Nocardia and Rhodococcus

a causative agent in pneumonia, is partially acid fast in tissue. Gram negative

Legionella micdadei

Cysts of the genera______ and _____are distinctly acid fast.

Cryptosporidium Isospora

The ______ and ______ in the mycobacterial cell wall account for the unusual resistance of these organisms to the effects of drying and harsh decontaminating agents in addition to the property of acid fastness.

mycolic acids and lipids

When Gram stained, mycobacteria usually appear as slender, poorly stained and beaded gram-____bacilli; sometimes they appear as "gram neutral" or "gram ghosts" by failing to take up either crystal violet or safranin.

positive

Three types of staining procedures are used in the laboratory for the rapid detection and confirmation of AFB:

Auorochrome Ziehl-Neelsen Kinyoun

Visualization of AFB in sputum or other clinical material should be considered presumptive evidence of_____ because staining does not specifically identify_______.

tuberculosis M. tuberculosis

Fuchsin Acid-Fast Stains. The classic carbolfuchsin stain requires ***heating of the slide*** for better penetration of the stain into the mycobacterial cell wall; hence it is also known as the ***hot stain procedure***

Ziehl-Neelsen method

With Ziehl-Neelsen staining, Mycobacterium spp. appear_____ beaded appearance, whereas nonmycobacteria appear_____.

red or have a red-blue blue

The method is similar to Ziehl-Neelsen staining but no heat is used; this is known as the ***cold stain procedure.*** If present, typical AFB appear as______, slightly curved, short or long rods (2 to 8 um); they also may appear beaded or banded (Mycobacterium kansasii). For some nontuberculous species, such as MAC, they appear pleomorphic and usually coccoid.

Kinyoun acid-fast stain purple to red

In the______ technique, carbolfuchsin is heated to drive the stain into the cell wall of the myco-bacterium. In the_____ carbolfuchsin method, a detergent is used with the carbolfuchsin to enable the stain to penetrate the cell wall.

Ziehl-Neelsen Kinyoun

The acid-fast stain is an important tool in the preliminary identification of the_______.

mycobacteria

A positive smear provides a presumptive but vital clue for the clinician in the diagnosis of______ when combined with other clinical symptoms.

tuberculosis

the acid-fast stain method,______ binds to the______ in the cell wall of mycobacteria, using _____ or ____to drive the stain into the cell wall. The stain cannot be removed, even with acid alcohol, giving the mycobacteria the property of acid fastness.

carbolfuchsin mycolic acid heat or detergent

Mycobacteria are called "acid-fast" because

they retain their stain even after being treated with acid-alcohol, which is used to decolorize non-acid-fast cells.

is a required component in media like Lowenstein-Jensen medium, which is commonly used for the cultivation of Mycobacterium species. The egg provides essential nutrients and helps solidify the medium.

Whole egg

Purpose of heating primary stain - carbolfuchsin

Steam is used during acid-fast staining to soften the waxy, mycolic acid-rich cell walls of mycobacteria, allowing the carbolfuchsin dye to penetrate. This step ensures that once stained, the bacteria retain the dye even after treatment with acid-alcohol, classifying them as "acid-fast."

A sputum smear reveals an average of five acid-fast bacilli per 1,000% field. According to CDC guidelines, report this result as: a. Negative b. Rare c. 1+ or few d. 2+ or moderate e. 3+ or numerous

The correct answer is c According to CDC guidelines, finding an average of five acid-fast bacilli per 1,000 fields in a sputum smear is typically reported as 1+ or few.

Mechanism of carbolfuchsin Ngano di siya matanggal after acid alcohol?

Carbolfuchsin enters bacteria and binds strongly to the waxy cell walls of acid-fast bacteria. This causes the dye to stay in these bacteria even after washing with acid alcohol, which removes the dye from non-acid-fast bacteria.

Purpose of heat

Heat helps carbolfuchsin dye penetrate the thick, waxy cell walls of acid-fast bacteria, making the staining process more effective.

Purpose of acid alcohol

Acid alcohol removes the dye from non-acid-fast bacteria, but acid-fast bacteria keep their color because of their waxy cell walls.

Purpose of methylene blue

Methylene blue stains non-acid-fast bacteria and the background, providing contrast to the red color of acid-fast bacteria.

Purpose of cooling slide after heating with carbolfuchsin

Cooling helps to stabilize the dye within the bacterial cells, making the staining more effective and reliable.

Why is carbolfuchsin used for detecting acid fast bacteria?

It is lipid soluble and contains phenol which helps the stain to penetrate the cell wall

Acid alcohol

Strips the color from non acid fast organisms

Why must decolorizer be strong

The decolorizer must be strong to effectively remove the dye from non-acid-fast bacteria while leaving the dye intact in acid-fast bacteria. Acid-fast bacteria have a waxy, lipid-rich cell wall that resists decolorization, so a strong decolorizer is necessary to differentiate between acid-fast and non-acid-fast organisms.

A direct sputum smear involves

taking a sample of sputum (mucus from the respiratory tract) and spreading it onto a microscope slide

Why shouldn’t smears be fire dried?

It can produced aerosols which can spread harmful microorganisms

Why not boil?

This can lead to loss of bacterial morphology and make the staining process less effective, potentially resulting in inaccurate or unreliable diagnostic results. Heat fixing, applied gently, is sufficient to adhere bacteria to the slide without causing damage.

Underdecolorized…

Non acid fast remain stained False positive acid fast

Why is blotting not allowed?

Blotting is avoided because it can remove the dye from the specimen and may risk spreading infectious agents, such as those from a tuberculosis sample. Air drying preserves the stain and minimizes contamination risks.

Sunlight…

Fade acid fast bacilli Exposure to sunlight can cause fading or degradation of the dye on the slide, leading to inaccurate staining results and potentially obscuring the bacterial details.

When there is positive results, should we report presence of M. tuberculosis?

In reporting, "positive" should be avoided for *Mycobacterium tuberculosis* because it is not specific enough and does not provide detailed information about the amount or severity of the infection. Instead, reports should use descriptive terms such as "acid-fast bacilli present" with a quantitative description or a specific diagnostic interpretation to ensure clarity and accuracy in the results.

If microscopy is not specific for M. tuberculosis, what is?

tuberculosis culture