BIO 2246 MICROBIOLOGY LECTURE NOTES

CHAP. 3 MICROBIAL OBSERVATIONS: MICROSCOPY AND STAINING

Prepared by: Alma Theresa S. Cabajes, Ph.D. cand.

(No part of this material may be copied, reproduced, or disseminated in any form or by any means without
written permission from the Instructor and the Biology Department, Ateneo de Davao University)

❖ Microscopy
• use of light or electrons to magnify objects.
• Three branches:
▪ Optical microscopy; Electron microscopy; X-ray microscopy
❖ Importance: essential technique for the identification of microorganisms.

❖ General Principles of Microscopy

• Wave of radiation
➢ Visible light is one part of a spectrum of electromagnetic radiation, including X-rays, microwaves, and radio
waves.
➢ These various forms of radiation differ in wavelength; Using radiation of smaller wavelengths results in
enhanced microscopy.

• Magnification
➢ An apparent increase in an object's size; Results when a beam of radiation refracts (bends) as it passes
through a lens.
➢ A convex lens focuses light on a focal point; The image is enlarged and inverted as light rays pass the focal
point and spread apart.
➢ In a compound microscope, the image from the objective lens is magnified again by the ocular lens.
➢ Magnification - process of producing an enlarged image of a specimen by using a lens system.
➢ Total magnification = objective lens × ocular lens

• Resolution
➢ The ability to distinguish two points that are close together.
➢ The better the resolution, the better the ability to distinguish two objects that are close to one another
➢ Shorter wavelengths of light provide greater resolution.

• Contrast
➢ Differences in intensity between two objects or between an object
and its background
➢ Important to determine the resolution; Staining increases contrast
➢ The use of light that is in phase increases the contrast

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

• Simple light microscope

➢ uses visible light as its source of illumination; Single-lens system for magnification
➢ examine very small specimens and some of their fine detail

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➢ A convex lens is a magnifying lens used in this microscopic system

• Compound Light Microscope

➢ Series of lenses for magnification; Oil immersion lens increases resolution; Have one or two ocular lenses
➢ Total magnification = magnification of objective lens x magnification of ocular lens
➢ Most have condenser lenses (direct light through the specimen)

Oil Immersion Lens

➢ Increases magnification and resolution.
➢ Some of the light passing out of a glass slide is bent so much that it bypasses the lens.
➢ Placing immersion oil between the slide and an oil immersion objective lens enables the lens to capture light
➢ Because light travels at a uniform speed through the slide, the immersion oil and the glass lens do not refract.

❖ Types of Microscope

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❖ Bacterial Stains

❖ Preparation of Specimens
▪ Tools:
▪ Loops and needles
▪ All other preparations require:
▪ 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.

❖ Staining

➢ Coloring microorganisms for better visibility

➢ salts composed of a positive and a negative ion
➢ basic dyes (positive ions like crystal violet)
➢ Acidic dyes (negative ions like India ink)
➢ Staining the background instead of the cell is
called negative staining.

❖ Steps for staining preparations

▪ Smear
▪ Air dry
▪ Fix
▪ Stain
▪ Rinse

❖ Simple Stains
➢ An aqueous or alcohol solution of a single basic dye.
➢ Determine cell shape, size, and arrangement of the microorganisms
➢ A mordant may hold the stain or coat the specimen to enlarge it.
➢ Ex: methylene blue, carbolfuchsin, crystal violet, and safranin.

❖ Differential Stains: Gram Stain

➢ Differential stains are used to differentiate between types of bacteria
➢ 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.

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• Gram Staining Procedure
▪ Reagents:
▪ Crystal violet (primary stain)
▪ Iodine (mordant)
▪ Alcohol (decolorizing agent)
▪ Safranin (counterstain)

Color of Color of
Gram + cells Gram – cells

Primary stain: Purple Purple

Crystal violet

Mordant: Purple Purple

Iodine

Decolorizing agent: Purple Colorless

Alcohol-acetone

Counterstain: Purple Red

Safranin

➢ G (+) cells retain the dye and remain purple.

➢ G (-) cells are colorless until counterstained with a red dye
➢ most consistent when used on young, growing bacteria.
➢ G (+) bacteria – sensitive to penicillins, cephalosporins
➢ G (-) bacteria – more resistant to antibiotics

• Gram Stain Reactions

➢ ALL COCCI are Gram-positive except Neisseria, Branhamella,
Moraxella, Veilonella
➢ ALL BACILLI are Gram-negative except Bacillus, Lactobacillus,
Listeria, Actinomyces, Clostridium, Corynebacterium,
Mycobacterium, Erysiphelothrix, Nocardia
➢ The higher forms of organisms including Actinomyces,
Streptomyces, yeasts and molds are G (+)

➢ Spiral Organisms are not stainable except for some which are G (+)
➢ All cocci are non-motile and non-spore formers
➢ All encapsulated organisms are non-motile
➢ Bacillus and Clostridium are spore-forming organisms

❖ Acid Fast Stain

▪ Identify acid-fast organisms such as members of the genus Mycobacterium.
▪ Cell walls contain mycolic acid and large amounts of fatty acids, waxes, and complex lipids.
▪ Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast.

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❖ Special Stains
❖ Negative staining
➢ Observing overall cell shapes , sizes and capsules
➢ It can also be used to stain cells that are too delicate to be heat-fixed.

❖ Endospore stain
➢ A differential stain used to visualize bacterial endospores
➢ Spores are resistant to heat, desiccation, chemicals, and radiation.
➢ Heat is required to drive a stain into the endospore.

➢ Schaeffer-Fulton endospore stain

▪ Malachite green as primary stain
▪ Heating to penetrate endospore walls
▪ Safranin as counterstain

❖ Capsule stain
▪ A stain is used to reveal negatively charged bacterial capsules. The
encapsulated cells will have a halo appearance under the microscope.

https://www.scienceprofonline.com

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❖ Flagella Staining
➢ Demonstrate the presence of
flagella
➢ Mordant and carbolfuchsin are used

https://biologyease.com/flagella-staining-principle/

❖ Lactophenol Cotton Blue Stain

➢ most widely used method of staining and observing
fungi; Simple to prepare
➢ Phenol: kills any live organisms
➢ Lactic acid: preserves fungal structures
➢ Cotton blue: stains the chitin in the fungal cell walls.

https://link.springer.com/chapter/10.1007/978-3-319-58371-6_11