Observing Microorganisms through a Microscope

1. Observing Microorganisms through a Microscope Dr. Bhavesh Patel Principal V.P. and R.P.T.P. Science College Vallabh Vidyanagar Email – bhavesh1968@rediffmail.com

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

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

4. Staining Reaction • 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) • Basic Dyes - positively charged • Acid Dyes - negatively charged

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

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

7. 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.

8. Types of 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. • Structural Staining – Multi step, objective is to see different structures of organisms. E.g. Endo spore staining, Capsule staining, Flagella staining etc.

9. 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. • Negative staining - It involves the use of Acidic dye (-ve charge) thus bacteria appear colorless in a dark background.

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

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

12. Gram Staining

13. Results • Gram Positive Violet • Gram Negative Pink • Difference - due to structure of cell wall • Gram Positive Thick cell wall • Gram Negative Thin cell wall

14. Gram positive Gram Negative

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

16. Mycobacterium tuberculosis

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

18. Structural Staining Capsule Staining

19. Flagella Staining

20. Endospore Staining

21. Thank You Q & A session