Quiz 2 Web-Only Edit

Question 1

Simple stain (3A)

Answer 1

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.

Question 2

What is the most commonly used basic dye? (3A)

Answer 2

Methylene blue

(shown: Saccharomyces cerevisiae wet mount stained with methylene blue, prepared in lab 3A)

Question 3

Why is methylene blue a commonly used dye? (3A)

Answer 3

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.

Question 4

What are two common things from the lab manual that are smeared for examination with methylene blue? (3A)

Answer 4

• Raw milk before microscopic examination
• Throat smears to diagnose diphtheria

Question 5

What yeast was used in exercise 3A?

Answer 5

Saccharomyces cerevisiae

Question 6

Briefly describe how to prepare a simple stain (3A)

Answer 6

• 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

Question 7

Was a coverslip used to examine the simple stains prepared with methylene blue in experiment 3A?

Answer 7

No, stained organisms were examined without the use of a coverslip

Question 8

How is methylene blue used to differentiate certain microbes? What microbes does it test for and give two examples from the lab manual (3A):

Answer 8

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.

Question 9

About how large is a yeast cell? (3A)

Answer 9

7 x 15 microns

Question 10

Differential stain (3B)

Answer 10

Uses two or more dyes that can be used to categorize cells into groups

(shown: Gram’s stain)

Question 11

What are the four chemicals used in a Gram stain? Which are stains? Which are mordants? (3B)

Answer 11

i) Crystal violet (stain)
ii) Gram’s iodine (mordant)
iii) Alcohol wash (generally EtOH, decolorizor)
iv) Safranin (counter-stain)

Question 12

What color do Gram-positive cells stain? (3B)

Answer 12

Purple – hold onto the crystal violet b/c of the thicker peptidoglycan layer, the decolorizer does not penetrate quickly enough to wash it out

Question 13

What color do Gram-negative cells strain? (3B)

Answer 13

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.

Question 14

What type of cells does safaranin stain?

Answer 14

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.

Question 15

mordant

Answer 15

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.

Question 16

Why might a cell be Gram-variable? (3B)

Answer 16

• 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).

Question 17

What three bacteria were used in the gram-staining procedure? (3B)

Answer 17

• Staphylococcus epidermis - Gram-positive
• Bacillus subtilis - Gram-variable (technically Gram-positive)
• Escherichia coli - Gram-negative

Question 18

Briefly describe the Gram staining method (3B)

Answer 18

• 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

Question 19

Cell capsule (3C)

Answer 19

• 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

Question 20

Name a genus that contains a capsule (3C)

Answer 20

• Klebsiella*
  (shown: negative stain of Klebsiella from demo 3C)

Question 21

Name two genus that make spores (3D)

Answer 21

Bacillus; Clostridium

Question 22

What type of stain would you use to view a capsule?

Answer 22

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.

Question 23

Negative stain

Answer 23

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.

Question 24

Schaeffer–Fulton stain

Answer 24

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.

Question 25

What are the dormant structures that are made during sporulation called? (3D)

Answer 25

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.

Question 26

What triggers the formation of endospores? (3D)

Answer 26

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.

Question 27

What two genra mentioned in the lab manual could be spore stained? What species is specifially mentioned in the lab manual for experiment 3D?

Answer 27

Both *Bacillus* and *Closridium* produce spores and thus may be spore stained. Experiment 3D (shown): * Bacillus subtilis* - Sub-terminal spore - Gram-positive

Question 28

Briefly describe the spore-staining technique (3D)

Answer 28

- 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

Question 29

What type of spore stain was used in lab 3D?

Answer 29

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.

Question 30

Why would a person want to do a spore stain? (3D)

Answer 30

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.

Question 31

What dye is used on the spore stain from lab 3D? What colours does this dye produce?

Answer 31

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.

Question 32

What technique was done on the flagellum stain? (3E)

Answer 32

Leifson's technique -Uses a mordant to increase cell size

Question 33

How large are unstained flagella in a bright field microscope (3E)?

Answer 33

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.

Question 34

What kind of flagella did the microbe in 3E have?

Answer 34

Polar monotrichous

Question 35

What method was used for the staining of flagella in demo 3E?

Answer 35

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.

Question 36

What two types of locomoratory organs do bacteria possess? What type is observable using Leifson's Technique?

Answer 36

Bacteria have two types of locomotory organs, flagella and pili: Leifson's Technique studies flagella staining.

Question 37

Leifson's Technique

Answer 37

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.

Question 38

What two microbes were used in 3F (acid-fast staining)?

Answer 38

- *Mycobacterium phlei* - Acid-fast (resists decolorization with acidified alcohol), Gram-positive - *Staphylococcus epidermis* - Not acid-fast

Question 39

Briefly describe the technique for acid-fast staining (3F)

Answer 39

- 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

Question 40

Metachromatic granules (3G)

Answer 40

- 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).

Question 41

Give two examples of a bacterium that contains metachromatic granules (3G)

Answer 41

* -Cornyebacterium diptheriae* * -Cornyebacterium xerosis*

Question 42

Describe what will happen to a cell without a rigid cell wall in a hypotonic solution (3H)

Answer 42

Solvent (**WATER**) will flow into the cell and it will swell; lyse due to its inability to resist the high osmotic pressure.

Question 43

Describe what will happen to a cell without a rigid cell wall in a hypertonic solution (3H)

Answer 43

Solvent (**WATER**) will flow out of the cell; the cytoplasm will shrivel up and the cell dessicates.

Question 44

Protoplasts (3H)

Answer 44

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.

Question 45

What is it called when the solute concentration is the same both inside and outside of the cell? (3H)

Answer 45

Isotonic

Question 46

What does a lysozyme do? (3H)

Answer 46

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

Question 47

What bacteria was used in 3H (protoplast demo)?

Answer 47

* 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)

Question 48

What are the two chemical groupings involving benzene rings that define a dye? (3)

Answer 48

Each system of benzene rings (or even linear conjugated double bonds) are the chromophores, and the group(s) attatched are the auxochromes.

Question 49

Auxochrome (3)

Answer 49

- 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**.

Question 50

Chromophore (3)

Answer 50

-Responsible for the color in dyes

Question 51

What type of stain is used to see the shape and size of microbes?

Answer 51

A general stain is used to see the size and shape of microbes, in contrast to a selective stain which is used to see subcellular structures

Question 52

List two reasons microbes are difficult to see under the microscope without a stain?

Answer 52

- Microbes are generally transparent - They have a refractive index close to that of water

Question 53

What type of stain is used to see the subcellular structures of microbes?

Answer 53

A selective stain which is used to see subcellular structures, in contrast to a general stain which is used to see the size and shape of microbes

Question 54

What is the difference between dyes and stains?

Answer 54

Although they act in the same way, dyes and stains differ in their level of purity. Dyes often contain trace amounts of related compounds in a relatively low state of purity compared to stains.

Question 55

What is the difference between modern stains and those used in earlier days of bacteriology?

Answer 55

Modern stains are synthetic in nature, earlier dyes were natural coloring agents

Question 56

What two properties must any dye or stain possess (Addendum 1)?

Answer 56

- It must be colored (i.e. capable of absorbing light in the visible spectrum, the groups that provide this are called **chromophores**) - It must be able to bind to the material to be stained

Question 57

Groups in the chemical structure of a molecule that abosorb light are called (Addendum 1)?

Answer 57

Chromophores A chromophore is the part of a molecule responsible for its color. The color arises when a molecule absorbs certain wavelengths of visible light and transmits or reflects others. The chromophore is a region in the molecule where the energy difference between two different molecular orbitals falls within the range of the visible spectrum. Visible light that hits the chromophore can thus be absorbed by exciting an electron from its ground state into an excited state. In biological molecules that serve to capture or detect light energy, the chromophore is the moiety that causes a conformational change of the molecule when hit by light. Examples are chlorophyll, which is used by plants for photosynthesis and hemoglobin, the oxygen transporter in the blood of vertebrate animals. In these two examples, a metal is complexed at the center of a tetrapyrrole macrocycle ring: the metal being iron in the heme group (iron in a porphyrin ring) of hemoglobin, or magnesium complexed in a chlorin-type ring in the case of chlorophyll. The highly conjugated pi-bonding system of the macrocycle ring absorbs visible light. The nature of the central metal can also influence the absorption spectrum of the metal-macrocycle complex or properties such as excited state lifetime.

Question 58

What components of the cell do acidic dyes stain? What components of the cell do basic dyes stain (Addendum 1)?

Answer 58

- 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.

Question 59

What is an EMB agar plate? How is it used (Addendum 1)?

Answer 59

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.

Question 60

Methylene blue

Answer 60

Basic, simple stain used widely in bacteriology because it readily binds acidic cellular components such as nucleaic ribosomes and metachromatic granules

Question 61

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)?

Answer 61

The chromophore absorbs visible photons and generates the colour, it may be modified or enhanced by the auxochrome.

Question 62

In modern synthetic dyes, the chromophore is generally made from what substance (Addendum 1)?

Answer 62

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**.

Question 63

Why are metachromatic granules so named (Addendum 1)?

Answer 63

The volutin granules found in genus *Corynebacteria* are polymerised metaphosphates and are called metachromatic because when stained with the basic methylene blue dye the resulting colour is somewhere between purple and violet-red.

Question 64

What special stains were covered in lab 3?

Answer 64

India Ink capsule stain (3C), flagellar stain (3E)

Question 65

What differential staining techniques were covered in lab 3?

Answer 65

Gram's stain (3B), spore stain (3D), acid-fast stain (3F)

Question 66

What simple staining techniques were covered in lab 3?

Answer 66

Methylene blue simple stain (3A), granule stain (3G)

Question 67

mycellium (4)

Answer 67

Mycelium is the vegetative part of a fungus, consisting of a mass of branching, thread-like hyphae. Through the mycelium, a fungus absorbs nutrients from its environment via facilitated diffusion and active transport of monomers digested by exoenzymes.

Question 68

Fungi general characteristics (4)

Answer 68

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

Question 69

hyphae (4)

Answer 69

A hypha (plural hyphae, from Greek huphḗ, “web”) is a long, branching filamentous structure of a fungus. In most fungi, hyphae are the main mode of vegetative growth, and are collectively called a **mycelium**. Yeasts are unicellular fungi that _do not grow as hyphae_.

Question 70

What is the fillament of a mold called? A network of them? (4)

Answer 70

Filaments are called hypha (plural **hyphae**), a network of them are called the **mycellium**.

Question 71

What is the cell wall of fungi composed of? (4)

Answer 71

**Chitin** A long-chain polymer of an *N*-acetylglucosamine (NAG) (*huge* oversimplification: Peptidoglycan without the NAM???), a derivative of glucose, found in many places throughout the natural world. It is a characteristic component of the **cell walls of fungi**, the exoskeletons of arthropods such as crustaceans (e.g., crabs, lobsters and shrimps) and insects, the radulae of molluscs, and the beaks and internal shells of cephalopods, including squid and octopuses. The structure of chitin is comparable to the polysaccharide cellulose, forming crystalline nanofibrils or whiskers. In terms of function, it may be compared to the protein keratin.

Question 72

Septate vs aseptate fungi (4)

Answer 72

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._

Question 73

Sporangiospores (4)

Answer 73

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.

Question 74

Four basic groups of fungus, based on phylogenic analysis (4)

Answer 74

_Chytridiomycota_ _Zygomycota_ _Ascomycota_ _Basidiomycota_

Question 75

Conidiospores (4)

Answer 75

Not enclosed in a sac, locaed on tips or side of hyphae. **Conidia**, sometimes termed **conidiospores**, are asexual, non-motile spores of a fungus, from the Greek word for dust, konis. They are generated through the cellular process of mitosis. The two new haploid cells are genetically identical to the haploid parent, and can develop into new organisms if conditions are favorable, and _serve in biological dispersal._

Question 76

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

Answer 76

*Chytridiomycota* Simplest of the fungi, unique in formation of motile zoospores with flagella, sexual and asexual reproduction

Question 77

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*.

Answer 77

*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)

Question 78

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*

Answer 78

*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*

Question 79

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.

Answer 79

*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.

Question 80

*Chytridiomycota* (4)

Answer 80

*Chytridiomycota* Simplest of the fungi, unique in formation of motile zoospores with flagella, sexual and asexual reproduction

Question 81

*Zygomycota* (4)

Answer 81

*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)

Question 82

*Ascomycota* (4)

Answer 82

*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*

Question 83

*Basidiomycota* (4)

Answer 83

*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.

Question 84

vegetative hyphae (4)

Answer 84

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.

Question 85

aerial hyphae

Answer 85

Strands of hyphae that project up from the medium that regularly produce spores

Question 86

What is the resource acquiring form of hyphae called? The spore-producing form?

Answer 86

- **Vegetative** hyphae are attatched to the substrate and collect nutrients - **Aerial** hyphae are generally further from the medium, produce spores frequently

Question 87

Rhizoid

Answer 87

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.

Question 88

*Rhizopus nigricans*

Answer 88

* 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)

Question 89

A zygomycete commonly known as bread mold (4)

Answer 89

*Rhizopus nigricans*

Question 90

Stolon

Answer 90

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.

Question 91

What are the four parts of the zygomycete *Rhizopus*?

Answer 91

The **sporangiophore** is the stalk that bears the large, black, spore containing **sporangium** at its tip. **Stolon****s**connect these sporangiophores with the**rhizoids** that attatch the hyphae to the substrate and acquire nutrients.

Question 92

What is the spore-bearing stalk in *Rhizopus* *nigricans* called?

Answer 92

Sporangiophore A sporangio**phore** holds a sporang**ium** (a sac like structure) at its tip that containes sporangio**spores**

Question 93

What is the spore-bearing stalk in *Aspergillus* called?

Answer 93

**Condidiophore** A condidio**phore** has condidio**spores** (or just **condidia**) at its tips and sometimes along its length

Question 94

Describe the process of making a tape mount

Answer 94

- 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

Question 95

What genus does this represent? What structure is at 1?

Answer 95

* Penicillium* 1) **Septate hyphae**

Question 96

What genus does this represent? What structure is at 2?

Answer 96

* Penicillium* 2) **Conidia** (or conidiaspores)

Question 97

What genus does this represent? What structure is at 3?

Answer 97

* Penicillium* 3) Phialides

Question 98

What genus does this represent? What structure is at 4?

Answer 98

* Penicillium* 4) Penicillus

Question 99

What genus does this represent? What structure is at 5?

Answer 99

* Penicillium* 5) **Conidiophone**

Question 100

What genus does this represent? What structure is at 1?

Answer 100

* Aspergillus* 1) **Conidia** (or **conidiaspore**)

Question 101

What genus does this represent? What structure is at 2?

Answer 101

* Aspergillus* 2) Phialides

Question 102

What genus does this represent? What structure is at 3?

Answer 102

* Aspergillus* 3) Vesicle

Question 103

What genus does this represent? What structure is at 4?

Answer 103

* Aspergillus* 4) **Septate hyphae**

Question 104

What genus does this represent? What structure is at 5?

Answer 104

* Aspergillus* 5) Foot cell

Question 105

What genus does this represent? What structure is at 6?

Answer 105

* Aspergillus* 6) **Conidiophore**

Question 106

What genus does this represent? What structure is at 1?

Answer 106

* Rhizopus* 1) **Sporangiospore**

Question 107

What genus does this represent? What structure is at 2?

Answer 107

* Rhizopus* 2) Collapsed **sporangium**

Question 108

What genus does this represent? What structure is at 3?

Answer 108

* Rhizopus* 3) **Sporangiophore**

Question 109

What genus does this represent? What structure is at 4?

Answer 109

* Rhizopus* 4) **Rhizoid**

Question 110

What genus does this represent? What structure is at 5?

Answer 110

* Rhizopus* 5) Node

Question 111

What genus does this represent? What structure is at 6?

Answer 111

* Rhizopus* 6) **Stolon**

Question 112

What genus does this represent? What structure is at 7?

Answer 112

* Rhizopus* 7) Columella

Question 113

What genus does this represent? What structure is at 8?

Answer 113

* Rhizopus* 8) **Sporangium**

Question 114

Batch culture (5)

Answer 114

A closed culture vessel with a single batch of medium. Because no fresh medium is added during incubation, nutrients decline and waste products accumulate.

Question 115

Lag phase (5)

Answer 115

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.

Question 116

Log phase (5)

Answer 116

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.

Question 117

Stationary phase (5)

Answer 117

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

Question 118

Death phase (5)

Answer 118

The final stage of the growth curve. In the death phase, the number of cells often decline at an exponential rate.

Question 119

What is the name of the counting chamber from lab 5A? Describe the principle behind it.

Answer 119

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.

Question 120

How would you obtain a dry cell sample from a liquid culture for weight determination?

Answer 120

- Centrifuge the suspension then wash to remove any solutes - Dry at 100°C overnight and weigh residue

Question 121

Turbidity (5C)

Answer 121

Turbidity is the cloudiness or haziness of a fluid caused by large numbers of individual particles that are generally invisible to the naked eye (in this case, microbes), similar to smoke in air. The measurement of turbidity is a key test of water quality.

Question 122

What is the relationship between the amount of light scattered by bacteria in suspension and concentration of bacteria?

Answer 122

They are generally proportional

Question 123

What are three common wavelengths for turbidity measurements of a suspended culture on a spectrophotometer (5C)? What wavelength did we actually use in the lab?

Answer 123

540nm (green) 600nm (orange) 660nm (red) We actually used **595nm** (slightly-greener-orange)

Question 124

A photospectrometer measures in units of OD. What is OD, how is it calculated?

Answer 124

OD stands for **optical density**. OD = log( l_entering ) - log( l_exiting ) The difference of the log of the light exiting the suspension taken from the light entering

Question 125

At what concentration is the measurement of microbes most effective using a photospectrometer?

Answer 125

Millions to hundreds of millions per millilitre