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78 terms

Lab Quiz 2

U of MN 3301, exercises 3-5
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Dye
Organic compound containing chromophore and auxochrome chemical groupings attached to benzene rings.
Chromophore
Structural grouping responsible for color.
- examples: -NN(azo), -NO(nitrose), -NO₂ (nitro), -CS (thio)
Auxochrome
Structural groupings that increase the solubility of the molecule and augment the effects of chromophore groups.
- examples: -NH₂ (amino), -OH (hydroxl)
Acidic Dye
Salts dissociate into a negatively charged chromogenic ion (anion) and metal cation. Negatively charged, so they bind to positively charged microbial cell structures.
Basic Dye
Salts dissociate into a positively charged chromogenic ion (cation) and an anion. Bind to negatively charged molecules like nucleic acids and many proteins. Most bacterial cells are negatively charged, so basic dies are used most often in bacteriology.
Benzene
a colorless, cyclic six-carbon compound with no staining properties.
Chromogen
a colored derivative that is not considered a dye because it is insoluble in water.
-example: trinitrobenzene
How a dye is formed
From benzene, replace 3 alternate Hydrogen atoms with a chromophore group (like -NO₂ (nitro)), and replace one more Hydrogen atom with an auxochrome group (like OH (hydroxyl)).
Simple Stain
Consists of one dye that stains a component of the microbial cell. Most often methylene blue.
Yeast used in simple staining experiment
Saccharomyces cerevisiae
Purpose of heat fixing slides
Heat coagulates the protein, causing the organism to adhere to the slide.
Result of over-heating slide
morphology of cells may be distorted
Differential Staining
Uses two or more dyes and can be used to categorize cells into groups.
-example: Gram's technique of staining
Solutions used in Gram Staining
- Crystal violet (basic dye)
- Iodine Solution (mordent)
- 95% Ethyl Alcohol (decolorizer, could also use alcohol and acetone mix)
- Safranin (basic dye counterstain, doesn't necessarily need to be Safranin, but that's what we used in lab)
Gram Positive Bacteria
Retain the crystal violet dye after decolorization and appear purple.
Gram-Negative Bacteria
Do not retain the crystal violet dye after decolorization and take on the red color of the counterstain.
Gram-Variable Bacteria
Both Gram Positive and Gram Negative characteristics- can be a result of using a culture that is too old (more than 12-18 hours)
Gram Positive Cocci used in Gram Staining
Staphylococcus epidermidis
Gram Positive Rod used in Gram Staining
Bacillus subtilis
Gram Negative Rod used in Gram Staining
Escherichia coli
Timing for Gram Staining
- Crystal violet: 1 minute
- Iodine Solution: 1 minute
- Decolorizer: 2-5 seconds
- Counterstain (safranin): 30 seconds
Approximate sizes of organisms
Rod shaped bacterium: 1x3 microns
Coccus shaped bacterium: 1 micron diameter
Spiral shaped bacterium: .5x15 microns
Capsule
Gelatinous material secreted as an envelope around many cells. Usually composed of polysaccharides or polypeptides
Negative Staining
Wet mount stain (not smear, so capsule isn't destroyed) where background is stained so organism appears as clear spaces in a colored background. Uses negatively charged dye for negatively charged organisms so that they repel the dye.

India ink is a dye used for this.
Negative Staining Demo Organism
Capsule stain of Klebsiella
Spore Staining
Usually spores resist dyes, but some basic dyes will penetrate when applied to a smear and heated.
- First, sample is collected on swab and soaked in dye, then boiled in hot water bath for 10 minutes.
- Smear is created
- Dye is then removed with water, but spores retain dye.
- Counterstain is used to dye vegetative cells a different color than spores.
Organism used for Spore Staining Experiment
Bacillis subtilis
- Was gram variable when stained (supposed to be positive)
Stains used for Spore Staining
- 5% malachite green
- .5% safranin O
Spore located in center of cell
central
Spore located between the middle and end of cell
subterminal
Spore located at end of cell
terminal
Spore located in swollen end of cell
Swollen sporangium (diagram looks like wooden spoon with a ball in the middle of spoon part)
Staining of Flagella
Uses Leifson's technique. Difficult due to small diameter of flagella (around 20nm), and that they are easily broken.
- Uses mordant to increase diameter of flagella so it can be viewed with a microscope
Acid-Fast Staining
Ziehl-Neelsen staining method where acid-fast bacteria resist decolorization with acidified alcohol. This helps to differentiate some bacteria from others.
- Also uses boiling water bath
- Acid fast stain red, non acid-fast stain blue
- Mycobacterium and some actinomycetes have acid-fast characteristics
Solutions used in Acid-Fast Staining
- Ziehl's carbol fuchsin
- Loeffler's methylene blue
- Acidified alcohol (3%HCl in 95% ethyl alcohol)
Organisms used in Acid-Fast Staining
- Staphylococcus epidermidis
- Mycobacterium phlei
Granule Staining
In some conditions of growth, granules form from different materials. Based on the type of material, you can determine types of bacteria. We specifically looked at Metachromatic granules.
Metachromatic Granules
Granules made of reserves of polymerized meta phosphates and sometimes called volutin are found in several bacteria, and are a diagnostic features of certain members of the genus Corynebacterium.
- Stain deeply in methylene blue as either blue or purple
Organism used in Granule Staining
Corynebacterium xerosis
Protoplast
Cell with the cell wall removed in an environment with high osmotic pressure so cell contents won't burst
Lysozyme
Enzyme that is most commonly used to remove cell walls of certain bacteria.
- Attacks peptidoglycan by hydrolyzing the glycosidic bond that connects N-acetylmuramic acid with N-Acetylglucosamine.
Organism used for Protoplast Demo
Bacillus subtilis
Tubes in Protoplast Demo
Tube 1: Cell Suspension, Sucrose, and Lysozyme (lysozyme removes cell wall, and sucrose provides high osmotic pressure so cell won't lyse)
Tube 2: Cell Suspension, Saline, and Lysozyme (lack of sucrose and addition of saline causes cell to lyse)
Tube 3: Cell suspension and Saline (cell wall isn't removed with lysozyme, so it can stand up to saline).
Fungi
Large, diverse, and widespread group of eukaryotic organisms that includes the molds, mushrooms, and yeast.
- Fungi are chemoorganoheterotrophs, decomposing organic matter.
- Can be separated into yeasts and molds
Molds
Multicellular, filamentous fungi, that form elaborate branching networks of cells (mycelium)
Yeasts
Unicellular fungi that can reproduce sexually through the formation of spores or asexually through budding.
- Examples: Candida albicans and Saccharomyces cerevisiae
Hyphae
small filaments that form as a fungal spore germinates or as a cell fragment grows out.
Mycelium
Closely interwoven network of hyphae
- Molds commonly form mycelium
Chitin
polysaccharide that often comprises the hyphal cell wall.
Septate
Individual hyphae with regular cross-walls
Aseptate or nonseptate
hyphae that are not interrupted by cross walls, and often have multiple nuclei dispersed throughout the hyphal structure.
Sporangiospores
Type of spore that develop in saclike structures located at the tips of hyphae.
Conidiospores or Conidia
spores that are not enclosed within a sac and are often located at either the tips or the sides of hyphae where they play an important role in fungal dispersal.
Chytridiomycota
Simplest fungi, unique in formation of motile zoospores with flagella, sexual and asexual reproduction. Usually found in water, and implicated in the massing killing of frog populations worldwide.
Zygomycota
Common in soil and decaying plant material, hyphae lack cross-walls (aseptate), sexual and asexual reproduction
- example: bread mold Rhizopus
Ascomycota
Named for sac-like reproductive structure ascus. Major decomposers in a variety of terrestrial environments, sexual and asexual reproduction.
- examples: Aspergillus, and yeast Saccharomyces cerevisiae
Basidiomycota
Club fungi, named from their characteristic spore-producing structure called the basidium, sexual and asexual reproduction.
- examples: human pathogen Cryptococcus neoformans and many mushrooms and toadstools
Organisms used in Fungi Exercise
Rhizopus nigricans (Zygomycota) and Aspergillus (Ascomycota)
Vegetative Hyphae
In plate with agar, they are adhered closely to agar or submerged. Mold obtains nutrients through these structures.
Aerial Hyphae
In plate with agar, the more conspicuous strands that project upward and produce spores.
Rhizoids
attaching vegetative hyphae of Rhizopus
Stolon
aerial hypha that connects adjacent rhizoids
Sporangiophore
fertile hyphal stalk that bears the enlarged, black, spore-containing sporangium of Rhizopus.
Conidiophore
upright spore-bearing stalk in Aspergillus
Conidiospores or Conida
spores that are borne externally in short chains over the whole surface of the slightly enlarged tip of Aspergillus.
Mounting Medium for Fungi Exercise
lactophenol blue
See diagrams on P. 38 of lab manual
Structure of Rhizopus and Aspergillus
Importance of counting cells
Increase in numbers is a convenient index of growth, which is a significant biological parameter. Also the numbers of microbes present in various materials provides a practical means to estimate quality as in water or milk, or to estimate microbial activity as in soil or aquatic environments.
Techniques of cell enumeration in Exercise 5
- Petroff-Hausser counter
- Spectrophotometric determination of the optical density of a cell suspension
- Viable Count
- Dry weight determination
Variables needed to determine direct count
- size of field viewed under microscope
- total area over which a known volume of culture is distributed
- dilution which the culture has undergone
Breed Method
computation of direct count of the bacteria seen in a microscope field.
Organism used for Direct Count
Escherichia coli
Spectrophotometer
used to measure turbidity as a function of the light lost in passage through the uniformly suspended culture, compared to a control medium.
Optical Density
The decrease in the intensity of a light beam due to the scattering of light by a suspension of microorganisms.
- this method is simple, but not sensitive, so is best for very large populations of cells.
See Spectrophotometer diagram on P. 42
Outlines parts of a spectrophotometer.
Turbidimetric Analysis
Use of spectrophotometer for analyzing cell population/growth
Importance of Standard Curve (or calibration curve)
Establishes the accuracy of the spectrophotometer. Most accurate points will be on linear portion of curve. Nonlinear parts of the curve reflect inaccuracy of the instrument.
Paramaters for a countable plate
30-300 colonies