Gram Stain Differential stain (Hans Christian Gram, a Danish doctor ). He developed a - PowerPoint Presentation

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Gram Stain

Differential stain (Hans Christian Gram, a Danish doctor ). He developed a new method to stain bacteria so they can be visible in specimen samples.

Differentiate bacteria into two large groups (the Gram Positive and the Gram negative).

Gram status is important in medicine; the presence or absence of a cell wall will change the bacterium's susceptibility to some antibiotics.

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Almost all bacteria are described by their Gram stain characteristics.

Based on differences of Cell wall structuresSlide4

Gram Positive & Negative Bacteria

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Gram-negativeGram-positive

Thin (single-layered)

Thick (multilayered)Peptidoglycan layerAbsent

Present in manyTeichoic acids

Present

Absent/smallerPeriplasmic membrane

High

None

Lipopolysaccharaide

(LPS) content

High (outer

membrane)

low

Lipid and lipoprotein content

Present

Absent

Outer

membrane

Endotoxin

Exotoxin

Toxins produced

They don't retain the Gram stain when washed with absolute

alcohol

They

r

emain colored purple with gram stain when washed with absolute alcohol and water.

Gram reaction Slide7

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Reagents for Gram Stain

Crystal Violet (purple).Primary stain; positive stain

Stains cell wall purple

Iodine

Mordant

Combines with primary stain to form an insoluble complex that gets trapped in bacterial cell wallSlide9

Ethanol/acetone

Decolorizer

CV-I complex washed out of Gram negative organisms because it cannot be trapped by lippopolysaccharide layer; flows right through outer

membrane

Safranin (pink)

Counterstain

Simple positive stain that provides contrasting dye for decolorized cells (Gram negative)

Stains all cells, but only the negative ones actually appear pink.

Gram +

Gram –

Primary stain:

Crystal violet

Purple

Purple

Mordant:

Iodine

Purple

Purple

Decolorizing agent:

Alcohol-acetone

Purple

Colorless

Counterstain:

Safranin

Purple

PinkSlide10

Gram Staining Procedure

After

air drying and heat fixation.

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Errors During Staining

Never ever used old culture.

Never ever used sample for patient take antibiotic.Time of Decolorizer:

Over: G + see as G -.Low: G- see as G +.

Time of fixation:

Over: G + see as G -.

Low: no sample on slide.Slide15

Acid-fast stain (

Ziehl-Neelsen stain)

The acid-fast stain is another differential staining method.

In this case, the target cells are usually members of the genus Mycobacterium.

The cell walls of these bacteria contain an unusually high concentration of waxy lipids, thus making conventional simple stains and Gram stains useless.

The genus

Mycobacterium

contains two important human pathogens,

M. tuberculosis

and

M. leprae

,

which cause tuberculosis and leprosy, respectively. Slide16

The reagents used are

Ziehl

–

Neelsen

carbolfuchsin, acid alcohol, and methylene blue.

Acid-fast bacilli will be bright red after staining.Slide17

Acid-fast stains are useful in identification of acid-fast bacilli (AFB)and rapid, preliminary diagnosis of

tuberculosis (with greater than 90% predictive value from sputum samples).

It also can be performed on patient samples to track the progress of antibiotic therapy and determine their degree of contagiousness. Slide18

Acid Fast Reagents

Carbolfuchsin (red), a phenolic stain: is the primary stain in the acid-fast test. It is soluble in the lipids of the mycobacterial cell wall. It’s consist of

basic fuchsin(red) and

carbolic acid(phenol).Heating the specimen, or adding a wetting agent such as

Tergitol

, increases the penetration of the carbolfuchsin

.

Following application of the carbolfuchsin, the specimen is cooled and decolorized with a solution of 3% hydrochloric acid and 95% ethanol

(

acid-alcohol

). Slide19

Since carbolfuchsin is more soluble in waxy cell

lipids than in acid-alcohol, the acid-alcohol removes

the carbolfuchsin from non-acid-fast organisms, but not from acid-fast organisms.

Following decolorization, the sample is counterstained with methylene blue which Cannot penetrate mycolic acid; provides contrast to non acid fast cells.

Mycobacteria

 are 

difficult

to stain

, but once stained are

difficult to decolorize

even in the presence of strong alcohol.Slide20

Procedures

Prepare a smear organism and a on glass slides. Allow the slides to air dry, and then heat fix the organisms.

Apply enough of carbolfuchsin with Tergitol to cover the bacteria. Allow it to set for five minutes

.(Alternate) If Tergitol

is not available, apply enough carbolfuchsin to cover the bacteria. Place the slide on a pre-warmed hot plate set on low for 8 minutes.

Do not allow the stain to evaporate or Boil. Add additional stain, if necessary. Remove the slide and allow it to cool.Slide21

Apply

enough methylene blue to cover the bacteria.

Allow it to set for 30 sec. Gently rinse the slide with water.

Blot (don't wipe) the slide dry with bibulous paper.

Allow

the slide to air dry

.

Examine the slide under oil immersion.

Positive

organisms will appear pink or red;

Negative

organisms will appear blue.

Rinse the slide with acid-alcohol (15-20 sec), drop by drop, just until the alcohol runs clear.

Gently rinse the slide with water.Slide22
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Kinyoun stain (cold method)

Method of staining acid-fast microorganisms, specifically mycobacterium. Procedure is similar to Ziehl-Neelson stain, but does not involve heating the slides.Kinyoun

staining method uses carbol-fuchsin as a primary stain, followed by decolorization with an acid-alcohol solution and methylene blue as a

counterstain. Kinyoun carbol-fuschsin has a greater concentration of phenol and basic

fuchsin

and does not require heating in order to stain properly.When viewed under a microscope,

Kinyoun

stained slides will show acid-fast organisms as red and non acid-fast organisms as blue.Slide26

Auramine-Rhodamine stain

Auramine-Rhodamine Fluorochrome staining is used to visualize Acid fast bacilli (AFB) bacteria using fluorescence microscopy, notably species in the Mycobacterium

 genus. Acid-fast organisms display a reddish yellow fluorescence.Slide27

Note

The

reddish-pink

color and “cording” (sticking together in long ropy masses) of the

Mycobacterium

cells due to the excess lipids of the cell wallSlide28