Quiz 2 Web-Only Edit Flashcards
Simple stain (3A)
Consists of one dye that stains a component of the microbial cell
Staining in general is used to enhance contrast in the normally colorless tissue sections, tissue sections are commonly stained. For light microscopic examinations, colored agents (chromophores) are used.
What is the most commonly used basic dye? (3A)
Methylene blue
(shown: Saccharomyces cerevisiae wet mount stained with methylene blue, prepared in lab 3A)
Why is methylene blue a commonly used dye? (3A)
Because it is a basic dye (a cation when in its dissociated, blue coloured form), it dyes the more acidic (negativly charged) components of the cell like DNA and metachromatic granules.
What are two common things from the lab manual that are smeared for examination with methylene blue? (3A)
- Raw milk before microscopic examination
- Throat smears to diagnose diphtheria
What yeast was used in exercise 3A?
Saccharomyces cerevisiae
Briefly describe how to prepare a simple stain (3A)
- Place a dot of water on the slide
- Smear in a bit of the bacteria/yeast/etc
- Let it air dry, then fix it by passing through flame
- Apply several drops of methylene blue; let sit for about a minute
- Rinse with water, dry, and examine
Was a coverslip used to examine the simple stains prepared with methylene blue in experiment 3A?
No, stained organisms were examined without the use of a coverslip
How is methylene blue used to differentiate certain microbes? What microbes does it test for and give two examples from the lab manual (3A):
Eosin Methylene Blue (EMB, also known as “Levine’s formulation”) is a selective stain for Gram-negative bacteria. EMB contains dyes that are toxic for Gram positive bacteria and bile salt which is toxic for Gram negative bacteria other than coliforms. EMB is the selective and differential medium for coliforms. It is a lactose agar base containing a blend of two stains, eosin and methylene blue in the ratio of 6:1. A common application of this stain is in the preparation of EMB agar, a differential microbiological medium, which slightly inhibits the growth of Gram-positive bacteria and provides a color indicator distinguishing between organisms that ferment lactose (e.g., Escherichia coli, Enterobacter aerogenes) and those that do not. Organisms that ferment lactose display “nucleated colonies” – colonies with dark centers.
About how large is a yeast cell? (3A)
7 x 15 microns
Differential stain (3B)
Uses two or more dyes that can be used to categorize cells into groups
(shown: Gram’s stain)
What are the four chemicals used in a Gram stain? Which are stains? Which are mordants? (3B)
i) Crystal violet (stain)
ii) Gram’s iodine (mordant)
iii) Alcohol wash (generally EtOH, decolorizor)
iv) Safranin (counter-stain)
What color do Gram-positive cells stain? (3B)
Purple – hold onto the crystal violet b/c of the thicker peptidoglycan layer, the decolorizer does not penetrate quickly enough to wash it out
What color do Gram-negative cells strain? (3B)
Pink – Gram-negative organisms appear pink because they are counterstained. Because of presence of higher lipid content, after alcohol-treatment, the porosity of the cell wall increases, hence the CVI complex (crystal violet – iodine) can pass through. Thus, the primary stain is not retained. Also, in contrast to most Gram-positive bacteria, Gram-negative bacteria have only a few layers of peptidoglycan and a secondary cell membrane made primarily of lipopolysaccharide.
What type of cells does safaranin stain?
Both Gram+ and Gram- cells are stained pink by safaranin. The Gram+ cells also retain crystal violet due to their thicker layer of peptidoglycan, the lighter safaranin is masked.
mordant
A substance that binds to the dye and makes it less soluble. Most mordants are polyvalent metal ions. In Gram staining, the iodine acts as a mordant or trapping agent.
Why might a cell be Gram-variable? (3B)
- Depending upon how long the culture has been growing, it might appear Gram-positive or Gram-negative.
- For example, many Gram-positive bacteria will appear Gram-negative in later stages of growth.
- Young growths shouldn’t be Gram-variable (less than 12-18 hrs).
What three bacteria were used in the gram-staining procedure? (3B)
- Staphylococcus epidermis - Gram-positive
- Bacillus subtilis - Gram-variable (technically Gram-positive)
- Escherichia coli - Gram-negative
Briefly describe the Gram staining method (3B)
- Apply water to slide, apply bacterial strains to the water (wet mount), let air-dry, and heat-fix
- Stain with crystal violet for ~1 min, rinse w/ water
- Stain with iodine for ~1 min, rinse w/ water
- Decolorize for 2-5 sec
- Stain w/ Safranin for ~30 sec, rinse, dry; examine
Cell capsule (3C)
- Usually composed of polysaccharides, size varies with environmental conditions
- Protects the cell, but is not essential for cell function, however there may be a correlation in some pathogenic bacteria between virulence and capsule production
- Often destroyed in some methods of staining
- India ink is often used to do a negative stain; stain the background, leaving the cells clear so we can see the capsule
Name a genus that contains a capsule (3C)
- Klebsiella*
(shown: negative stain of Klebsiella from demo 3C)
Name two genus that make spores (3D)
Bacillus; Clostridium
What type of stain would you use to view a capsule?
A negative stain:
Negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid. In this technique, the background is stained, leaving the actual specimen untouched, and thus visible. This contrasts with ‘positive staining’, in which the actual specimen is stained.
For bright field microscopy, negative staining is typically performed using a black ink fluid such as nigrosin or India ink. The specimen, such as a wet bacterial culture spread on a glass slide, is mixed with the negative stain and allowed to dry. When viewed with the microscope the bacterial cells, and perhaps their spores, appear light against the dark surrounding background.
Negative stain
Negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid. In this technique, the background is stained, leaving the actual specimen untouched, and thus visible. This contrasts with ‘positive staining’, in which the actual specimen is stained.
For bright field microscopy, negative staining is typically performed using a black ink fluid such as nigrosin or India ink. The specimen, such as a wet bacterial culture spread on a glass slide, is mixed with the negative stain and allowed to dry. When viewed with the microscope the bacterial cells, and perhaps their spores, appear light against the dark surrounding background.
Schaeffer–Fulton stain
The Schaeffer–Fulton stain is a differential staining technique designed to isolate endospores by staining any present endospores green, and any other bacterial bodies red. The primary stain is malachite green, and the counterstain is safranin, which dyes any other bacterial bodies red.
What are the dormant structures that are made during sporulation called? (3D)
Endospores
An endospore is a dormant, tough, and non-reproductive structure produced by certain bacteria, including Bacillus and Clostridium. The name “endospore” is suggestive of a spore or seed-like form (endo means within), but it is not a true spore (i.e., not an offspring). It is a stripped-down, dormant form to which the bacterium can reduce itself. Endospore formation is usually triggered by a lack of nutrients, and usually occurs in Gram-positive bacteria. In endospore formation, the bacterium divides within its cell wall. One side then engulfs the other. Endospores enable bacteria to lie dormant for extended periods, even centuries. Revival of spores millions of years old has been claimed. When the environment becomes more favorable, the endospore can reactivate itself from the vegetative state.
What triggers the formation of endospores? (3D)
Endospore formation is usually triggered by a lack of nutrients, and usually occurs in Gram-positive bacteria. In endospore formation, the bacterium divides within its cell wall. One side then engulfs the other. Endospores enable bacteria to lie dormant for extended periods, even centuries. Revival of spores millions of years old has been claimed. When the environment becomes more favorable, the endospore can reactivate itself from the vegetative state.
What two genra mentioned in the lab manual could be spore stained? What species is specifially mentioned in the lab manual for experiment 3D?
Both Bacillus and Closridium produce spores and thus may be spore stained.
Experiment 3D (shown):
- Bacillus subtilis*
- Sub-terminal spore
- Gram-positive
Briefly describe the spore-staining technique (3D)
- Swab bacterial culture; place whole swab into malachite green, place in boiling water bath ~10 min
- Rub swab onto a slide, heat fix
- Decolorize with water 2-5 sec
- Stain w/ Safranin ~20 sec-Rinse w/ water, dry; examine
What type of spore stain was used in lab 3D?
A Schaeffer–Fulton stain
The Schaeffer–Fulton stain is a differential staining technique designed to isolate endospores by staining any present endospores green, and any other bacterial bodies red. The primary stain is malachite green, and the counterstain is safranin, which dyes any other bacterial bodies red.
Why would a person want to do a spore stain? (3D)
Can determine size, shape; position of the spore in the cell. The decolorisation will not penetrate the spore, but it will penetrate the vegetative cell; the counterstain will colorize the cell.
What dye is used on the spore stain from lab 3D? What colours does this dye produce?
A Schaeffer–Fulton stain
The Schaeffer–Fulton stain is a differential staining technique designed to isolate endospores by staining any present endospores green, and any other bacterial bodies red. The primary stain is malachite green, and the counterstain is safranin, which dyes any other bacterial bodies red.
What technique was done on the flagellum stain? (3E)
Leifson’s technique
-Uses a mordant to increase cell size
How large are unstained flagella in a bright field microscope (3E)?
Flagella are not visible without a staining technique under standard bright field microscopy. Their diameter is roughly 20nm, making them too small to resolve independently.
What kind of flagella did the microbe in 3E have?
Polar monotrichous
What method was used for the staining of flagella in demo 3E?
Leifson’s Technique
The Leifson flagella stain uses tannic acid (mordant) and basic fuchsin (dye) to form a colloidal (silver-based) precipitate that when absorbed by the flagellum causes it to increase in diameter and become colorized, thus amenable to viewing by light microscopy. Mordant and dye are added simultaneously.
What two types of locomoratory organs do bacteria possess? What type is observable using Leifson’s Technique?
Bacteria have two types of locomotory organs, flagella and pili: Leifson’s Technique studies flagella staining.
Leifson’s Technique
The Leifson flagella stain uses tannic acid and basic fuchsin to form a colloidal (silver-based) precipitate that when absorbed by the flagellum causes it to increase in diameter and become colorized, thus amenable to viewing by light microscopy. Mordant and dye are added simultaneously.
This is not a differential stain! The unique aspect of this stain is the use of tannic acid to actually build up the diameter of the flagellum in order for it to be viewable under bright field microscopy. The basic fuchsin is a basic dye (who would have guessed?), so it would attatch to the amphipathic flagellin protein without tannic acid present. The additional volume added by tannic acid and the subsiquent complex with additional basic fuchsin makes the structure viewable.
What two microbes were used in 3F (acid-fast staining)?
- Mycobacterium phlei
- Acid-fast (resists decolorization with acidified alcohol), Gram-positive
- Staphylococcus epidermis
- Not acid-fast
Briefly describe the technique for acid-fast staining (3F)
- Innoculate Staphylococcus epidermis and Mycobacterium phlei in tube of carbol fuschin dye
- Incubate tube in boiling water bath for 10 minutes.
- Swab contents on slide, heat fix
- Decolorize w/ acid alcohol 2-5 sec, rinse w/ water
- Counterstain w/ methylene blue ~20 sec, rinse, dry; observe
Metachromatic granules (3G)
- Reserves of polymerised metaphosphates; sometimes called volutin
- A feature of Corynebacterium diphtheriae, the causitive agent of diphtheria
- Name comes from reaction with Loffler’s methylene blue, a polychromatic effect is seen in the colouration of these granules (lines in image point to granules).
Give two examples of a bacterium that contains metachromatic granules (3G)
- -Cornyebacterium diptheriae*
- -Cornyebacterium xerosis*
Describe what will happen to a cell without a rigid cell wall in a hypotonic solution (3H)
Solvent (WATER) will flow into the cell and it will swell; lyse due to its inability to resist the high osmotic pressure.
Describe what will happen to a cell without a rigid cell wall in a hypertonic solution (3H)
Solvent (WATER) will flow out of the cell; the cytoplasm will shrivel up and the cell dessicates.
Protoplasts (3H)
A protoplast is a plant, bacterial or fungal cell that had its cell wall completely or partially removed using either mechanical or enzymatic means.
Protoplasts: Have their cell wall entirely removed and are derived from gram-positive
Spheroplasts: Have their cell wall only partially removed (as by the action of penicillin) and are derived from gram-negative
More generally protoplast refers to that unit of biology which is composed of a cell’s nucleus and the surrounding protoplasmic materials.
What is it called when the solute concentration is the same both inside and outside of the cell? (3H)
Isotonic
What does a lysozyme do? (3H)
It’s an enzyme that attacks the peptidoglycan backbone by hydrolyzing the β(1-4) glycosidic bond that connects N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) subunits
What bacteria was used in 3H (protoplast demo)?
- Bacillus subtilis*
(shown: Bacillus subtilis from tube 3 of demo 3H. Hypotonic conditions but no lysozyme was added, cells do not burst due to intact peptidoglycan layer)
What are the two chemical groupings involving benzene rings that define a dye? (3)
Each system of benzene rings (or even linear conjugated double bonds) are the chromophores, and the group(s) attatched are the auxochromes.
Auxochrome (3)
- Augment the effects of the chromophore, can increase the solubility of the molecule.
- Examples include the hydroxyl group (-OH), the amino group (-NH2), the aldehyde group (-CHO), and the methyl mercaptan group (-SMe).
- An auxochrome (Greek auxánein: “to increase” and chrōma: “colour”) is a group of atoms attached to a chromophore which modifies the ability of that chromophore to absorb light. They themselves fail to produce the colour; but when present along with the chromophores in an organic compound intensifies the colour of the chromogen.
Chromophore (3)
-Responsible for the color in dyes
What components of the cell do acidic dyes stain? What components of the cell do basic dyes stain (Addendum 1)?
- Acidic dyes stain the basic components of the cell, such as the cytoplasm (mainly by staining amphoteric proteins suspended in the cytosol).
- Basic dyes stain acidic components of the cell, including nuceleic ribosomes, metachromatic granules, and acidic waxes of acid-fast bacteria.
What is an EMB agar plate? How is it used (Addendum 1)?
EMB agar is a differential microbiological medium, which slightly inhibits the growth of Gram-positive bacteria and provides a color indicator distinguishing between organisms that ferment lactose (e.g., Escherichia coli, Enterobacter aerogenes.) and those that do not (e.g., Salmonella). Organisms that ferment lactose display “nucleated colonies” – colonies with dark centers.
From the lab manual: Eosin Y is an acidic red dye used in conjunction with the basic dye methylene blue; a purple salt is formed between eosin and methylene blue as the pH falls. The salt precipitates in the agar and is taken up by the cells to give a dark purple appearance with green fluroescence.
Methylene blue
Basic, simple stain used widely in bacteriology because it readily binds acidic cellular components such as nucleaic ribosomes and metachromatic granules
What part of the dye is ultimately responsible for the colour? What part of the dye effects the colour and sometimes increases solubility (Addendum 1)?
The chromophore absorbs visible photons and generates the colour, it may be modified or enhanced by the auxochrome.
In modern synthetic dyes, the chromophore is generally made from what substance (Addendum 1)?
Many are made from aniline (C6H5NH2, a phenyl group attached to an amino group), but because most all (including aniline) are made from coal tars, they are called aniline dyes or, more correctly, coal tar dyes.
What differential staining techniques were covered in lab 3?
Gram’s stain (3B), spore stain (3D), acid-fast stain (3F)
Fungi general characteristics (4)
Kingdom Fungi
- Includes yeasts, molds, mushrooms, rusts, and smuts.
- Phenotypically, multicellular and fillamentus fungi are classed molds, while unicellular fungi are classed yeasts
- Chemoorganoheterotrophic eukaryotes
- saprobes (decomposers) that feed by excreting exoenzymes and absorbing digested nutrients
- Mostly terrestrial (aquatic exceptions found)
- Some are pathogenic
What is the fillament of a mold called? A network of them? (4)
Filaments are called hypha (plural hyphae), a network of them are called the mycellium.
Septate vs aseptate fungi (4)
A hypha consists of one or more cells surrounded by a tubular cell wall. In most fungi, hyphae are divided into cells by internal cross-walls called “septa” (singular septum). Septa are usually perforated by pores large enough for ribosomes, mitochondria and sometimes nuclei to flow between cells. The major structural polymer in fungal cell walls is typically chitin, in contrast to plants and oomycetes that have cellulosic cell walls. Some fungi have aseptate hyphae, meaning their hyphae are not partitioned by septa.
Sporangiospores (4)
A sporangium (sac) forms at the tip of the hypae, contains the sporangiospores.
Sporangiospores are spores that are produced in a sporangium (plural: sporangia). A sporangium in fungi (but not mosses and some other organisms) is simply a cell containing spores. We encounter this term in our discussion of sexual reproduction in the Chytridiomycota and Zygomycota, can be meiosis or mitosis.
What basic phylogenic group of fungus is this (4):
Simplest of the fungi, unique in formation of motile zoospores with flagella, sexual and asexual reproduction
Chytridiomycota
Simplest of the fungi, unique in formation of motile zoospores with flagella, sexual and asexual reproduction
What basic phylogenic group of fungus is this (4):
Common in soil and decaying plant material, aseptate hyphae, sexual and asexual reproduction, representative member is bread mold Rhizopus.
Zygomycota
Common in soil and decaying plant material, aseptate hyphae, sexual and asexual reproduction, representative member is bread mold Rhizopus.
(shown: varying phenotypes of sporangia sacs)
What basic phylogenic group of fungus is this (4):
Named for their sac-like reproductive structure, major decomposers of a variety of terrestrial environments, sexual and asexual reproduction, representative members: Aspergillus, Neurospora crassa and the yeast Saccharomyces cerevisiae
Ascomycota
Named for their sac-like reproductive structure, major decomposers of a variety of terrestrial environments, sexual and asexual reproduction, representative members: Aspergillus and the yeast Saccharomyces cerevisiae
What basic phylogenic group of fungus is this (4):
Club fungi, named for their characteristic spore-producing structure called the basidium, sexual and asexual reproduction, representative members: human pathogen Cryptococcus neoformans and many mushrooms and toadstools.
Basidiomycota
Club fungi, named for their characteristic spore-producing structure called the basidium, sexual and asexual reproduction, representative members: human pathogen Cryptococcus neoformans and many mushrooms and toadstools.
Chytridiomycota (4)
Chytridiomycota
Simplest of the fungi, unique in formation of motile zoospores with flagella, sexual and asexual reproduction
Zygomycota (4)
Zygomycota
Common in soil and decaying plant material, aseptate hyphae, sexual and asexual reproduction, representative member is bread mold Rhizopus.
(shown: varying phenotypes of sporangia sacs)
Ascomycota (4)
Ascomycota
Named for their sac-like reproductive structure, the ascus, major decomposers of a variety of terrestrial environments, sexual and asexual reproduction, representative members: Aspergillus and the yeast Saccharomyces cerevisiae
Basidiomycota (4)
Basidiomycota
Club fungi, named for their characteristic spore-producing structure called the basidium, sexual and asexual reproduction, representative members: human pathogen Cryptococcus neoformans and many mushrooms and toadstools.
vegetative hyphae (4)
The portion of the mycelium that obtains nutrients and is adhered to the substrate. In the agar plates on which the fungi from lab 4 was mounted on, the vegetative hyphae were those which were near or submerged in the agar.
aerial hyphae
Strands of hyphae that project up from the medium that regularly produce spores
What is the resource acquiring form of hyphae called? The spore-producing form?
- Vegetative hyphae are attatched to the substrate and collect nutrients
- Aerial hyphae are generally further from the medium, produce spores frequently
Rhizoid
In the fungal genus Rhizopus, rhiziods are small branching hyphae that grow downwards from the stolons that anchor the fungus to the substrate (vegetative), where they release digestive enzymes and absorb digested organic material.
Rhizopus nigricans
- Rhizopus nigricans* is a fungus, a zygomycete commonly known as bread mold and is the most common species of Rhizopus.
(shown: tape mount of Rhizopus nigricans from lab 4)
A zygomycete commonly known as bread mold (4)
Rhizopus nigricans
Stolon
In mycology, a stolon is defined as an occasionally septate hypha, which connects sporangiophores together. Root-like structures called rhizoids may appear on the stolon as well, anchoring the hyphae to the substrate. The stolon is commonly found in bread molds, and are seen as horizontally expanding across the mold.
What are the four parts of the zygomycete Rhizopus?
The sporangiophore is the stalk that bears the large, black, spore containing sporangium at its tip. Stolonsconnect these sporangiophores with therhizoids that attatch the hyphae to the substrate and acquire nutrients.
What is the spore-bearing stalk in Aspergillus called?
Condidiophore
A condidiophore has condidiospores (or just condidia) at its tips and sometimes along its length
Describe the process of making a tape mount
- Take 1 inch of clear tape and hold adhesive side out to blot a fungus sample
- Place 1-2 drops of lactophenol blue on a slide and stick tape sample to slide
- Cut or flatten the excess tape and examine under 10X and 40X
What genus does this represent? What structure is at 1?
- Penicillium*
1) Septate hyphae
What genus does this represent? What structure is at 2?
- Penicillium*
2) Conidia (or conidiaspores)
What genus does this represent? What structure is at 3?
- Penicillium*
3) Phialides
What genus does this represent? What structure is at 4?
- Penicillium*
4) Penicillus
What genus does this represent? What structure is at 5?
- Penicillium*
5) Conidiophone
What genus does this represent? What structure is at 1?
- Aspergillus*
1) Conidia (or conidiaspore)
What genus does this represent? What structure is at 2?
- Aspergillus*
2) Phialides
What genus does this represent? What structure is at 3?
- Aspergillus*
3) Vesicle
What genus does this represent? What structure is at 4?
- Aspergillus*
4) Septate hyphae
What genus does this represent? What structure is at 5?
- Aspergillus*
5) Foot cell
What genus does this represent? What structure is at 6?
- Aspergillus*
6) Conidiophore
What genus does this represent? What structure is at 1?
- Rhizopus*
1) Sporangiospore
What genus does this represent? What structure is at 2?
- Rhizopus*
2) Collapsed sporangium
What genus does this represent? What structure is at 3?
- Rhizopus*
3) Sporangiophore
What genus does this represent? What structure is at 4?
- Rhizopus*
4) Rhizoid
What genus does this represent? What structure is at 5?
- Rhizopus*
5) Node
What genus does this represent? What structure is at 6?
- Rhizopus*
6) Stolon
What genus does this represent? What structure is at 7?
- Rhizopus*
7) Columella
What genus does this represent? What structure is at 8?
- Rhizopus*
8) Sporangium
Lag phase (5)
The first phase of a growth curve, when microbes are introduced to a batch culture and begin to adjust to their new environment, synthesizing needed cellular components. No immediate increase in cell number.
Log phase (5)
The second part of a growth curve, the exponential or log phase is one in which growth is constant and balanced. Cell number double at nearly regular intervals. This is the phase where generation time is often determined.
Stationary phase (5)
The third part of a growth curve, in the stationary phase the growth becomes limited and there is no net increase in cell number due to the depletion of resources, the buildup of toxins, or to a lesser degree, quarum sensing
What is the name of the counting chamber from lab 5A? Describe the principle behind it.
The Petroff-Hauser counting chamber is a specially ruled slide of known area used for visually determining the number of cells in a culture. The depth and grid size gives a number of bacteria per volume of suspension, allowing for total bacteria/volume calculations to be made.
