Observing Microorganisms Through a Microscope Chapter 3 Observing Microorganisms Through a Microscope

Units of Measurement 1 µm = 10-6 m = 10-3 mm 1 nm = 10-9 m = 10-6 mm

Microscopy: The Instruments A simple microscope has only one lens. Figure 1.2b

Microscopy: The Instruments In a compound microscope the image from the objective lens is magnified again by the ocular lens. Figure 3.1b

Microscopy: The Instruments Total magnification = objective lens  ocular lens

Microscopy: The Instruments Resolution is the ability of the lenses to distinguish two points. A microscope with a resolving power of 0.4 nm can distinguish between two points ≥ 0.4 nm. Shorter wavelengths of light provide greater resolution.

Microscopy: The Instruments Refractive index is the light-bending ability of a medium. The light may bend in air so much that it misses the small high-magnification lens. Immersion oil is used to keep light from bending. Figure 3.3

Preparation of Specimens for Light Microscopy A thin film of a solution of microbes on a slide is a smear. A smear is usually fixed to attach the microbes to the slide and to kill the microbes. The smear are fixed by passing it 4-5 times through the flame.

The advantages of fixing a smear are: The smear gets stuck (fixed) to the slide. The penetration of stains into bacteria is better. Vegetative bacteria get killed and become non-infectious. Bacteria retain their original size and shape on staining.

Preparing Smears for Staining Live or unstained cells have little contrast with the surrounding medium. However, researchers do make discoveries about cell behavior looking at live specimens.

Preparing Smears for Staining Stains consist of a positive and negative ion. In a basic dye, the chromophore is a cation. In an acidic dye, the chromophore is an anion. Staining the background instead of the cell is called negative staining.

Simple Stains Use of a single basic dye is called a simple stain. Simple stain is an aqueous or alcoholic solution of a single basic dye. Shape size . Arrangement of bacterial cells. A mordant may be used to hold the stain or coat the specimen to enlarge it.

Differential staining Gram stain Acid fast stain

Gram Stain The Gram stain classifies bacteria into gram-positive and gram-negative. Gram-positive bacteria tend to be killed by penicillin and detergents. Gram-negative bacteria are more resistant to antibiotics.

Gram Stain Color of Gram + cells Gram – cells Primary stain: Crystal violet Purple Mordant: Iodine Decolorizing agent: Alcohol-acetone Colorless Counterstain: Safranin Red

Gram Stain Figure 3.10b

Gram positive and gram negative

Gram positive and Gram negative

Acid-Fast Stain Some of the bacteria take up red color, when stained with carbol fuchsin and withstand decolorization by acid alcohol. These bacteria are called acid fast bacilli. The bacillus responsible for tuberculosis: Mycobacterium tuberculosis belongs to this group Figure 3.11

Acid-Fast Stain Non–acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counter stained (with a different color basic stain methylene blue) to see them.

Acid fast bacteria and non acid fast bacteria

Special staining Negative staining Endospore staining Flagella staining

Negative Staining Negative staining is useful for capsules. Capsule can be detected by India ink staining which is a negative staining in which the capsule stands out as a halo. Capsulated bacteria such as Pneumococci, Klebsiella, Escherichia coli. Production of capsule is better in vivo as compared to in vitro environment. Figure 3.12a-c

Endospore staining Heat is required to drive a stain into endospores. These endospores are capable of survival under adverse conditions such as heat, drying, freezing, radiation and action of toxic chemicals.

Endospore staining

Examples Positive ( spore forming bacteria ) Clostridium perfringens (Gas gangrene ) Clostridium botulinum (Botulism poisoning ) Clostridium tetani (Tetanus) Bacillus anthracis (Anthrax) Negaive ( non spore forming bacteria ) Escherichia coli 

Endospore

Flagella staining Flagella too small to be seen with a light microscope without staining. Flagella staining requires a mordant to make the flagella wide enough to see. Microbiologist use the number and arrangement of flagella as diagnostic aids.

Flagella These are the organ of locomotion and provide motility to the bacterium.

Motile and non motile bacteria Flagella Organisms which are motile by flagella include Vibrio cholerae  Pseudomonas Escherichia coli Organisms which are non motile Klebsiella Shigella

Types of flagellar arrangement