Observing Microorganisms through a Microscope Dr. Bhavesh Patel Principal V.P. and R.P.T.P. Science College Vallabh Vidyanagar –

Basic techniques needed to study Bacteria 1. Grow Bacteria 1. Grow Bacteria 2. Isolate Bacteria 2. Isolate Bacteria 3. Grow Bacteria in pure culture 3. Grow Bacteria in pure culture 4. Observe Bacteria 4. Observe Bacteria 5. Identify Bacteria 5. Identify Bacteria

Preparing smears for staining 1. Bacteria on slide (preparation of smear) 1. Bacteria on slide (preparation of smear) 2. Air Dry 2. Air Dry 3. Bacteria are HEAT FIXED to the slide 3. Bacteria are HEAT FIXED to the slide 4. Stain is applied 4. Stain is applied

Staining Reaction Stains - salts composed of a positive and negative ion, one of which is colored (chromophore). Purified preparation (uneconomic) Stains - salts composed of a positive and negative ion, one of which is colored (chromophore). Purified preparation (uneconomic) Dyes – Coloring agent that are relatively impure preparation (economic) Dyes – Coloring agent that are relatively impure preparation (economic) Basic Dyes - positively charged Basic Dyes - positively charged Acid Dyes - negatively charged Acid Dyes - negatively charged

Basic Dye Bacteria have slight negative charge on surface, so are attracted to the positively charged stain/ dye Bacteria have slight negative charge on surface, so are attracted to the positively charged stain/ dye Common Basic Dyes Common Basic Dyes –crystal violet –methylene blue –safranin –basic fuchsin

Acidic Dye Bacteria have slight negative charge on surface, so are not stained by negatively charged stain/ dye Bacteria have slight negative charge on surface, so are not stained by negatively charged stain/ dye Acidic dye are used for negative staining Acidic dye are used for negative staining Common Acidic Dyes Common Acidic Dyes –Nigrocin –Congo red

Mordant  Mordant – increases affinity between stain and cells or coats a structure to make it thicker and easier to see after it is stained  Intensifier – Functionally same as mordent but unlike the mordent which is chemical intensifier are physical agent Example: Flagella - can not normally be seen, but a mordant can be used to increase the diameter of the flagella before it is stained. Gram’s Iodine, Tannic acid are the examples of mordent.

Types of Staining Simple Staining – One step, objective is to determine size, shape and arrangement of organisms. E.g. Monochrome staining, Negative staining. Simple Staining – One step, objective is to determine size, shape and arrangement of organisms. E.g. Monochrome staining, Negative staining. Differential Staining – Multi step, Objective is to differentiate between different group of organisms. E.g. Gram staining, Acid fast staining. Differential Staining – Multi step, Objective is to differentiate between different group of organisms. E.g. Gram staining, Acid fast staining. Structural Staining – Multi step, objective is to see different structures of organisms. E.g. Endo spore staining, Capsule staining, Flagella staining etc. Structural Staining – Multi step, objective is to see different structures of organisms. E.g. Endo spore staining, Capsule staining, Flagella staining etc.

Simple Staining Monochrome Staining – It involves the use of Basic dye (+ve charge) thus bacteria appear colored in a bright background. Also known as positive staining. Monochrome Staining – It involves the use of Basic dye (+ve charge) thus bacteria appear colored in a bright background. Also known as positive staining. Negative staining - It involves the use of Acidic dye (-ve charge) thus bacteria appear colorless in a dark background. Negative staining - It involves the use of Acidic dye (-ve charge) thus bacteria appear colorless in a dark background.

Differential Staining React differently with different types of bacteria React differently with different types of bacteria 2 Most Common 2 Most Common –Gram Staining –Acid-Fast Staining

Gram Staining 1884 Hans Christian Gram 1884 Hans Christian Gram most important stain used in Bacteriology most important stain used in Bacteriology Divides all Bacteria into 2 groups: Divides all Bacteria into 2 groups: –Gram Positive –Gram Negative

Gram Staining

Results Gram PositiveViolet Gram PositiveViolet Gram Negative Pink Gram Negative Pink Difference - due to structure of cell wall Difference - due to structure of cell wall –Gram Positive Thick cell wall –Gram Negative Thin cell wall

Gram positive Gram Negative

Acid - Fast Staining Differential Staining - divides bacteria into 2 groups Differential Staining - divides bacteria into 2 groups Acid - Fast Acid - Fast Non Acid - Fast Non Acid - Fast Used to identify organisms in the Genera Mycobacterium (high lipid and wax content in cell wall) Used to identify organisms in the Genera Mycobacterium (high lipid and wax content in cell wall)

Mycobacterium tuberculosis

Acid - Fast Staining 1. Ziehl Neelsen Carbol Fuchsin - ZNCF (Red) 1. Ziehl Neelsen Carbol Fuchsin - ZNCF (Red) 2. Acid Alcohol 2. Acid Alcohol 3. Counterstain with Malachite green 3. Counterstain with Malachite green Acid - Fast Cells Red Acid - Fast Cells Red Non Acid - Fast Green Non Acid - Fast Green

Structural Staining Capsule Staining

Flagella Staining

Endospore Staining

Thank You Q & A session