3 Demonstration of Microorganisms Flashcards
Approximate size ranges for Bacteria: (4)
- Typical Bacteria - range 0.5-5 um, length 1-10 um
- Cocci (Spherical Bacteria) - diameter 0.5-2 um
- Bacilli (Rod-Shaped Bacteria) - diameter 0.5-1 um, length 2-10 um
- Spiral and Helical Bacteria - similar to typical bacteria
- Similar to bacteria, these cells can vary widely in size (from 0.1 μm to over 15 μm).
- Some extremophiles, such as those found in hot springs and deep-sea hydrothermal vents, can have unique shapes(e.g., spherical (cocci), rod-shaped (bacilli), spiral, and even flat or square forms - e.g., Haloquadratum walsbyi) and sizes to adapt to their environments.
Archaea
Bacterial Shapes:
* Coccus –
* Bacillus –
* Spiral
* Vibrio –
* Spirochete -
round
rod
spiral
comma shaped
helical
six common shapes of bacteria:
✔ coccus (meaning “berry”)
✔Bacillus/ rod-shaped (meaning “little staff”)
✔Coccobacillus
✔Vibrio
✔Spirochete
✔spirillum
several arrangements of these cells:
✔single, chains (strepto-)
✔clusters (staphylo-)
✔pairs (diplo-)
- the term for when Most bacteria exhibit only one shape
MONOMORPHIC
Some bacteria can exhibit many shapes
e.g - Members of genus Corynebacterium
PLEOMORPHIC/POLYMORPHIC
TYPES OF FIXATION
- Heat-fixation
- Chemical fixation
- gently flame (heating) an air-dried film of bacteria
- preserve over all morphology but not structures within the cell
Heat-fixation
- used to protect fine cellular substructures and the morphology of larger, more delicate microorganisms
Chemical fixation
2 types of dyes for staining
basic dyes (+charge/cationic)
acid dyes (- charge/anionic)
e.g., methylene blue, basic fuchsin, crystal violet, safranin, malachite green
* most often used in bacteriology
* bind to negatively charged molecules: nucleic acids and proteins; certain organelles
basic dyes (+charge/cationic)
- e.g., eosin, rose bengal, and acid fuchsin
- with carboxyls (-COOH) and phenolic hydroxyls (-OH);
- bind to positively charged cell structures e.g., Histones; cytoplasmic proteins; collagen
acid dyes (- charge/anionic)
- process of applying colored substances, known as stains or dyes, to microbial cells
or structures to enhance their visibility under a microscope
Staining
- is a substance that adheres to a cell, giving the cell color.
- The presence of color gives the cells significant contrast so are much more visible.
- Different stains have different affinities for different organisms, or different parts of organisms
Stain
- Simple, easy to use; single staining agent used; using basic and acid dyes.
- Features of dyes: give coloring of microorganisms; bind specifically to various cell structures.
- involves the application of a single stain solution to a fixed smear
- simple and easy
- Crystal Violet, Methylene Blue, CF (commonly used basic dyes) – determine shape, size and arrangement of bacteria
Simple Staining
- To distinguish different kinds of bacteria into separate groups based on staining properties.
-Two types: Gram stain & Acid-fast stain.
divide bacteria into separate groups based on staining properties
-Gram Stain – developed by Hans Christian Gram in 1884
-Crystal violet-iodine-acetone or alcoholsafranin (CIAS)
Differential Staining
-are designed to visualize specific structures or types of bacteria that are not easily seen with basic stains like the Gram stain.
-These stains can highlight specific features like capsules, spores, flagella,
Special Staining
*Differential stain
- lose crystal violet and
stain red from safranin
counterstain
Gram-negative
*Differential stain
- retain crystal violet and
stain purple
Gram-positive
- Important basis of bacterial classification and identification
- Practical aid in diagnosing infection and guiding drug treatment
-which divides most clinically significant bacteria into two main groups, is the first step in bacterial identification.
The Gram Stain
Bacteria stained purple/blue are Gram ____ - their cell walls have thick petidoglycan and teichoic acid.
Gram +
Bacteria stained red/pink are Gram ____ their cell walls have have thin peptidoglycan and lipopolysaccharides with no ____.
Gram –
teichoic acid
Components of acid-fast staining
- Primary Stain: Strong carbol fuchsin, which is basic fuchsin dissolved in phenol.
- Decolorizer: a solution of 20% sulfuric acid (H₂SO₄) or acid-alcohol.
- Counterstain: Loeffler’s methylene blue or malachite green, used to stain the background and non-acid-fast bacteria
- reveals the presence of diffuse capsules surrounding many bacteria
- ink and dye cannot penetrate the bacterial cell or its capsule
- E.g., Klebsiella pneumoniae
- bacteria are mixed with India ink or Nigrosin dye
- air drying
- bacteria appear lighter bodies with blue black background
Negative staining
type of stain
* * for genus Bacillus and Clostridium Other genera: Desulfotomaculum, Sporosarcina, Sporolactobacillus, Oscillospira,
Thermoactinomyces
* uses the Schaeffer-Fulton Method
Endospore (Spore) staining
*Schaeffer-Fulton Procedure (5)
- Preparation of a Bacterial Smear-Air dry
- Application of Primary Dye (Malachite Green) w/ concurrent application of heat
- Allow slide to cool
- Rinse w/ water
- Application of Counterstain (Safranin) - green endospore resting in a pink to red cell
- Uses mordant and
carbolfuchsin stain to
increase flagellar
diameter until resolved
by light microscope
Flagellar Stain
- provides information and location of flagella
- to observe bacterial flagella with a light microscope, thickness of flagella is increased by coating with mordants like tannic acid and potassium alum and stained with pararosaniline (Leifson method) or basic fuchsin (Gray method)
Flagellar Staining
