Lab Quiz 2

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U of MN 3301, exercises 3-5

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

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