EXERCISE 3: BACTERIAL SMEAR PREPARATION

Clinical specimen

Clinical - diseased state or infection of the patietn
specimen- anything taking out of the patient. blood, etc

importance of microscopic examination

Establish the presence of microorganism in the clinical specimen

The clkinical labarototry’s 3 phases of workflow

• Pre analytical phase - collection of the specimen, checking its integrity, instruction from the MT on how the patient should collect the sample

• analytical phase -

• post analytical phase -

why stain microorganism

identification of the microrganimsm causing disease to the patient. Identify the morphology of the mircoogranism. Identfiy if gram + or -

Color of gram positive

violet/ purple or blue

Cell wall has a thick peptidoglycan layer with teichoic acid

• insoluble in alcohol

• Disaccharides and amino acids

Color of gram negative

red or pink

composed of thin peptidoglycan layer with lipopolysaccharides

• Are soluble in alcohol

• Lipids and polysaccharides

Acid fast staining

staining technique that differentiates acid fast organisms from non-acid fast organisms

Preparation of samples

• Smears from swabs - Transfer swabs into a clean microscope slide. Line, left to it is the frosted end. Rest is non frosted.

  • Frosted end - details of the patient (name, birth date, sex, date and collection fo the sample, psecimen source)

  • Roll the cotton swab all over the glass

  • 2 different smears from the same patient and specimen source

    • For consistency of the result

• Smears from thick liquids/ semisolids (ex. stool)

  • cotton swabs absorb the material in the container

  • rolled over the surface of the glass

  • 2nd swab/ smear

• Smears from thick, granular or mucoid materials

  • Make smear from the granular material (abnormal)

  • Fish out the granules from the container and place it in the center of glass slide

  • Using a 2nd glass, place on top the 1st glass, then push down to crush the granules

  • Then rotate to completely crush

  • Now created 2 different smears

• Smears from thin fluid (low protein content and low cellular count)

  • Instead of spreading, place a drop in the center which was pre marked in the non-frosted side

  • Cytocentrifuge preparation - method of making bacterial smears for thin fluids ensuring the cells are concentrating the collected test tube

    • emulsify the thick material and make a thin smear by motion to the right

    • Do examination on the thin potion

Before stain, 2 prior processes

• Air drying the smears

  • depends on the thickness of the smear for time

• Heat fix

  • pass it to the heat of an alcohol lamp for several times. Adheres the smear to the glass slide. Avoid washing off.

4 theories of staining

1. Stains are composed of 2 ions - positive and negative ion

2. one of the 2 ions in the staining solutions successfully colors the microorganism (chromophore).

3. The positive ion is carrying the colors of the basic dye. The negative ion is carrying the colors of the acidic dye

4. Cell wall of microorganism at pH 7 is slightly negative

Simple stain

• Aquaious solution of a single dye

  • cannot be used because positive ion is the chromophore, and negative ion is the background. Cannot be seen

• adds a chemical known as mordant

• directed toward coloring the forms and shapes present

• methylene blue, crystal violet, carbolfuchsin, and safranin

Differential stain

• Employs more than one stain

  • Differentiate gram positive and negative organisms

• Reacts differently with any kinds of bacteria

• directed towards coloring specific components of the organism

• Gram stain and acid-fast chain

GRAM’S STAINING (Hans Christian Gram)

1. Gentian Violet (substitute if no crystal violet): The primary stain, a basic dye, forms a complex with the peptidoglycan and initially stains the bacterial cell wall.

2. Gram’s Iodine: The mordant chemically binds with the bacterial cell wall and increases the affinity for the crystal violet.

3. Acetone Alcohol: The decolorizing agent, critical for distinguishing between Gram (+) and Gram (-) bacteria. Gram (-) bacteria contain lipids that dissolve in alcohol, causing them to appear clear.

4. Safranin: A red dye that stains Gram (-) organisms pink/red.

   • Gram (+) → Blue/Violet

   • Gram (-) → Red/Pink

• Primary stain

  • Crystal violet (hexamethyl-para-rosaniline chloride)

• Mordant

  • Gram’s iodine - brown colored reagent

  • Intensify the color of the stain to the cell wall

• Decolorization

  • acetone alcohol

  • cell wall of the gram positive will remain intact. Violet color retained

  • cell wall of the gram negative will be destroyed. Decolorizes the cell wall.

• Counter staining

  • Safranin - red colored stain

  • gram positive remain purple

  • gram negative, cell wall already destroyed = colorless. Take red color of safranin = appear red

Hucker’s Method:

• Gram (+) organisms: Have thick peptidoglycan and lipoteichoic acid cross-links.

• Gram (-) organisms: Have thin peptidoglycan and lipids.

Gram Staining Issues:

• Gram (+) appears Gram (-):

  • Removal of mRNA via salt precipitation

  • Aging/old specimens

  • Over-drying

  • Use of acidic stain

  • Technical errors (e.g., over-decolorization)

• Gram (-) appears Gram (+):

  • Incomplete decolorization

  • Thick smear

Gram’s Rule:

• All cocci are Gram (+), except:

  • Neisseria, Moraxella, Veillonella, Branhamella

• All bacilli are Gram (-), except:

  • Mycobacterium, Corynebacterium, Bacillus, Erysipelothrix, Lactobacillus, Listeria, Nocardia

• All spirals are Gram (-).

ACID-FAST STAINING / ZIEHL-NEELSEN (Franz Ziehl and Friedrich Neelsen)

Acid-fast bacteria resist decolorization by acid once stained due to high lipid content (mycolic acid) in their cell walls, known as the "Lipid-Barrier Principle." Acid-fast organisms are hard to stain but once stained, they are difficult to decolorize.

2 methods used:

• Ziel-Neelsen method - hot method; invloves steaming or heating the smear

  1. Use of Carbol Fuchsin - red colored dye

  2. heating or steaming - mordant

  3. Decolorization - acid alcohol

  4. Counter staining - lamb Methylene Blue

• Kinyoun method - cold method; does not involve heating

  • Increases the concentration of the phenol so the acid-fast organism is stained effectively

General Rule: All bacteria are non-acid-fast except for Mycobacterium and Nocardia (slightly acid-fast).

1. Carbol Fuchsin: The primary stain contains phenol, which penetrates the cell wall of the Mycobacterium genus.

2. Steaming: Softens mycolic acid, allowing penetration of the stain (does not act as a mordant).

3. Acid Alcohol: The decolorizer contains 95% ethanol in concentrated HCl.

4. Loeffler’s Methylene Blue: The counterstain.

   • Mycobacterium → Red or Green background

   • Non-acid-fast → Blue or Green

   • Macrophage → Green/Blue

Alternative Techniques to Avoid Steaming:

• Increase the concentration of phenol or dye.

• Prolong dye application.

• Add a wetting agent, such as Tergitol.

ACID-FAST organisms

• Cell walls composed of mycolic acid

  • fatty acids; very slippery

  • also known as hydroxymethoxyl acid

  • Responsible for the fastness of acid fast

non ACID-FAST organisms

• Cell walls have no mycolic acid

Special stains

Used to stain specific structure of the organism

• Wirtz’s ,ethod for spore/endospore

• Maneval’s staining for capsule

• Leifson’s method for flagella

• Albert stain, Loeffler’s methylene blue or Gram stain for metachromic granules

C. MODIFIED ACID-FAST STAINING / KINYOUN METHOD (Joseph Kinyoun)

This method differentiates acid-fast organisms from non-acid-fast ones and is referred to as the "Cold Method" because no heating or steaming is required. Instead, it uses a higher concentration of phenol to enhance cell wall penetration.

1. Kinyoun Carbol Fuchsin: Primary stain, consisting of basic fuchsin in 95% ethanol with liquefied phenol. After application, wash with distilled water.

2. Acid Alcohol: The decolorizer, 95% ethanol in concentrated HCl. Wash again with distilled water.

3. Methylene Blue or Malachite Green: The counterstain. Wash with distilled water.