Bacterial Staining Techniques

Conclusions From Previous Lab

• The microscopic field decreases with Magnification
• Light intensity needs to be increased with magnification
• The working distance of the microscope decreases with magnification
• The resolving power of the microscope increases with magnification
• Bacteria can be either motile or non-motile
• Bacteria are minute and colorless, making them impossible to see with the naked eye and hard to see under a microscope

Why is it Hard to See Bacteria

• They are minute and colorless
• They are impossible to be seen by the naked eye
• They are hard to see under the microscope

Improving Visibility

• To increase visibility of bacteria under a microscope increase the contrast
• Contrast can be increased by staining

Staining Procedure Needs

• Materials, specifically stains
• Technique

Stain Components

• Stain is an inorganic compound composed of Chromogen + Auxochrome
• Stain (dye) = Chromogen + Auxochrome
• Chromogen = Benzene ring + chromophore which gives color to chromogen
• Auxochrome gives charge to chromogen

Chromogen Properties

• The chromogen may become positive or negative
• Benzene ring is the colorless part of a dye and the basic structural component of a dye
• Chromophore is the functional group of a dye that gives color to the stain
• Auxochrome is the group that gives ionic property to the stain

Types of Stain

• According to the charge of stain, there are three types of stain

Acidic Stains

• Acidic stains are anionic stains and have a negative charge
• Nigrosine, India ink, and Eosin are examples of acidic stains

Basic Stains

• Basic stains are cationic stains and have a positive charge
• Crystal violet, Methylene blue, Safranine, Carbolfuchsin, and Malachite green are examples of basic stains

Neutral Stains

• Neutral stains have no net charge

Acidic Stain Properties

• The chromogen of acidic stain is negatively charged and known as an Anionic stain
• Acidic stains are used to stain the positively charged components such as background staining
• The cell wall for bacteria is negatively charged

Basic Stain

• The chromogen, or colored part, of basic stain is positively charged, also known as a cationic stain
• Basic stains are used to stain negatively charged components, such as bacterial cells
• Methylene blue, safranin, malachite green, basic fuschin, crystal violet, and Carbolfuchsin are examples of basic dyes

Basic Dyes

• Chromophore is the positive ion dye attracted by bacteria, so cells of bacteria are stained

Acidic Dyes

• Chromophore is the negative ion dye rejected by the cell and stains the background of the slide

Bacteria and Dyes

• Bacteria are slightly negative and are attracted to the positive chromophore of the basic dye

Neutral Stain

• Neutral stains are a salt of acidic and basic stain
• Giemsa stain is one example

Staining Techniques

• According to the purposes, staining techniques divide into three types

Staining Types

• Simple staining
• Differential staining
• Structural staining

Simple Staining

• Uses only one dye, and this dye reacts similarly with all types of bacteria

Direct Simple Stain

• Stains bacteria itself, so a basic dye must be used

Indirect Simple Stain

• Stains negative or background
• Stains the background, so acidic dye must be used

Differential Staining

• Uses more than one dye; these dyes react differently with different types of bacteria

Differential Staining Types

• Gram staining is a type
  • It is used for gram-positive bacteria
  • It also used for gram-negative bacteria
• The acid-fast staining is also a type
  • It is used for acid-fast bacteria
  • It is also used for non-acid-fast bacteria

Structural Staining

• Uses one dye or more than one dye to stain certain structures within bacteria
• Bacterial structures that can be stained include capsule, cell wall, endospore, flagella, ribosomes, and genetic material

Smear Preparation

• All staining techniques need smear preparation, EXCEPT negative staining, also called background or indirect staining

During Smear Preparation

• Spread bacteria over a proper area of the slide so at least 70% of the cells arrange look like they would in nature

Cooling the Loop

• Check the cooling of the loop by inserting it into the agar of the culture from where the bacteria will be taken, and away from the bacteria
• This prevents scattering of the bacteria in the lab air, prevents infection, and prevents killing of bacteria

Heat Fixation

• Serves to kill the bacteria
• Serves to fix the bacteria to the slide so it will not wash out during staining

Direct Simple Stain

• It is used to stain bacteria itself, while the background remains colorless
• Cation (basic) dye should be used to stain bacteria itself

Direct Simple Stain

• The materials needed include a loop, slides, crystal violet or any basic dye, sterile distilled water, and bacteria
• Work should be done under aseptic conditions

Direct Simple Stain- Method

• Smear preparation is the first step
• Load the smear with basic dye (as crystal violet)
• Wash gently with tap water, but prevent water from dripping directly over the smear
• Blot with filter paper
• Observe under the Microscope

Indirect Simple Stain

• It stains the background and the bacteria remain colorless
• It is also called Negative or background stain

Negative, Background Staining Materials

• Includes bacterial culture (Bacillus), inoculating loop, two sterile cleaned microscopic slides, and dye (stain)

Indirect Simple Staining

• There are two types of stain (dye) that can be used indirect simple staining

Types of Stains for Indirect Simple Staining

• Acidic dyes: such as Nigrosine and India ink, the negative charge of this dye repelled by the negative charge of the bacteria
• The dye will not access the cell & will remain outside the cell to stain the background and not the bacterial cells
• Stain with large molecular size: Pelikan 4001 dye has large molecules, larger than the pores in the cell wall of bacteria
• They can’t penetrate bacterial cells and remains in the background to stain it

Why Use Negative Staining

• Needed to stain the bacteria that can’t be stained by other staining techniques besides negative staining, such as Spirilli bacteria
• Needed to study the natural shape and size of the bacteria because no stain enters it, so the cell will not enlarge and remains normal
• Furthermore, no heat applies to the preparation; most of the bacterial cell is water When applying heat to the cell, the water evaporates and the cell becomes smaller. In negative staining, there is no heat and the cell remains normal