Micro 225 Lab Exam #3 final

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Biochemical test methods for bioluminescent marine vibrio & Photobacterium species, Fungus: Yeast + Mold,
updated 6 years ago by YCITLALIC
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1

Lipid Hydrolysis Test is used in biochemical test methods for bioluminescent marine vibrio & Photobacterium species, what medium is used and what are we testing for?

Biochemical test: Lipase
Medium used: Marian fat agar plate, tributyrin is added to agar
Red color of fat agar due to neutral red pH indicator

2
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What would be the results for this Lipid Hydrolysis test?

Positive: colony turns red (may also turn red-pink around & below colony)
-Lipase produced
-free Fatty acids are liberated from hydrolysis of triglyceride
-Neutral red precipitates turn red from acidic pH of fatty acids
Aeromonas hydrophila = +

3
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What would be the results for this Lipid Hydrolysis test?

Negative: no evidence of red color on or around colony
-No lipase production
-Neutral red remains as amber color from neutral pH (no color change)
Photobacterium phosphoreum= -

4

Starch Hydrolysis Test is used in biochemical test methods for bioluminescent marine vibrio & Photobacterium species, what medium is used and what are we testing for?

Biochemical test: presence of Amylase
Medium used: Marine starch agar plate
Flood area of spot inoculation with Gram's Iodine: Chemically reacts with starch =blue-black color

5
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What would be the results for this Starch Hydrolysis Test?

Positive: clearing around colony (halo)
-Amylase produced
-Some starch digested
-NO binding of iodine around colony due to starch breakdown
Aeromonas hydrophila = +

6
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What would be the results for this Starch Hydrolysis Test?

Negative= NO clearing around colony (no halo)
-NO amylase production
-No starch digested
-Iodine binds to starch on all parts of the plate
Photobacterium phosphoreum= -

7

Gelatin Hydrolysis test is used in biochemical test methods for bioluminescent marine vibrio & Photobacterium species, what medium is used and what are we testing for?

Biochemical test: Gelatinase produced
Gelatinase is an enzyme that liquefies gelatin
Medium used:Marin nutrient gelatin agar plate

8
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What would be the results for this Gelatin Hydrolysis test?

Positive: liquefaction of gelatin
-Part or all does not re-solidify
-Gelatinase produced
-Gelatin digested
Aeromonas hydrophila = +

9
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What would be the results for this Gelatin Hydrolysis test?

Negative: Gelatin remains solid
-Re-solidifies with refrigerated
-NO gelatinase produced
-NO Gelatin digested

Photobacterium phosphoreum= -
If NEGATIVE, continue incubation at unknown's preference temperature:
-Repeat refrigeration test each lab for up to 2 weeks
-Do not leave gelatin tubes in refrigerator

10

What biochemical test methods are used for utilization of Amino acids ?

Metabolism of lysine/ornithine by decarboxylation or arginine by dihydroxylation, then decarboxylation
-Lysine (LDC)
-Ornithine (ODC)
-Arginine (ADH)

11

What occurs during decarboxylation?

Removes acid groups- carboxyl groups (COOH) from amino acids & other organic molecules. Only activated by acidic environments: fermentation
-Neutralizes acids: survival mechanism; Amines released increases pH

12

What are the reactions that Lysine (LDC), Ornithine (ODC), and Arginine (ADH) undergo during utilization of amino acids?

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Metabolism of lysine/ornithine by decarboxylation or arginine by dihydroxylation, then decarboxylation

13
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What would be the Decarboxylation results for this utilization of Amino acids test ?

Positive Results:
DC=Yellow
-Glucose fermentation
-Acid produced
-Brom-cresol purple (pH indicator) turns yellow

LDC, ODC, ADH= Purple
-Acids from glucose fermentation stimulated decarboxylase
-Amine neutralizes acids
-pH indicator turns purple

14
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What would be the Decarboxylation results for this utilization of Amino acids test ?

Negative results:
DC=Yellow
-Glucose fermentation
-Acid produced

LDC, ODC, ADH=Yellow
-Acids produced from glucose fermentation
-No Decarboxylase release
-No acid neutralization

15
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What would be the Decarboxylation results for this utilization of Amino acids test ?

Negative results:
DC=purple
-Glucose not fermented
-Acids not produced

LDC, ODC, ADH= Purple
Decarboxylase no produced

16
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What would be the results for this Voges proskauer Biochemical test?

Medium used: Methyl red voges proskauer broth (MRVP) + Marine cations 1559.
Preform test by adding KOH & Alpha mix ever few mins for entire lab
-measure the ability of an organism to produce non-acid products (neutral pH) from glucose

Results:
Voges Proskauer Positive= Butanediol Fermenter
If Reddish pink color slowly develops: Positive
P. phosphoreum= +

NO color change: Negative= Non-fermenter or fermentation pattern
Vibrio fischeri= -

-May take min-hours to develop leave with instructor in rack up front if still negative at the end of lab

17
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What would be the results for this Idole Production Biochemical test?

Medium used: Peptone broth for idone +Marine cation 1559, contains tryptophan and can detect motility of organism
Perform test by Add Kovac's reagent to top

Positive= pink-red color on top
presence of Indole
-tryptophanase produced from tryptophan utilization
-Idole detected

Negative: No color change
Photobacterium phosphoreum= -

18
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What is the function of Poly-B-hydroxybutyrate (PHB) granules?

Medium used: Marine Glucose Fermentation broth, view wet mount under phase contrast microscopy

-energy reserve-> Carbon and energy source
-Produced under high Carbon conditions for later

P. phosphoreum=-
V. logei & V. fischeri= +

19
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Study of fungi:

Mycology

20

What are fungi?

Molds, yeasts, mushroom, relatives

21

Characteristics of fungi?

-Eukaryotes
-Nonphotosynthetic-heterotrophs
-Absorptive metabolism- Exoenzymes

22

Cell structure of Fungi?

-Lack tissue differentiation
-Cell wall of chitin/ other polysaccharides

23

Reproduction of Fungi?

Propagate by spores (sexual/Asexual)
-Deuteromycota division (defunct, term of convenience for non-sexually reproducing fungi) -most now in Ascomycota

24

Habitats of fungi?

Primary terrestrial, a few freshwater and marine

25

Ecology of fungi?

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-Symbionts of plants or animals- commensals, mutualistic, parasitic
-Mutualist:
*Form associations with plant roots (mycorrhizae) - Assist with N & P uptake
* Association with Algae or cyanobacteria (lichens)

26
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How do plants/trees benefit from presence of Mycorrhizae?

Mutualist:
*Form associations with plant roots (mycorrhizae) - Assist with N & P uptake
-Redwood seedling without (left) & with (right) mycorrhizae
-Pine seedling- mycorrhizal roots from one tree spread to inoculate other tree roots

27

Why is fungi important for the environment ?

Decomposers (saprophytes)- break down organic material & return to environment
-Pathogens: mycose of animals & plants

28

Why is fungi important industrially ?

Industrial fermentation- bread, wine, beer, cheese, tofu, soy sauce, steroid manufacture, antibiotic production

29

What is the cell structure of Molds?

multicellular filamentous fungi

30

What are the four major divisions of molds?

1. Zygomycota
2. Ascomycota
-Deuteromycota division (defunct, term of convenience for non-sexually reproducing fungi) -most now in Ascomycota
3. Basidiomycota
4. Chytridiomycota

31

What is the cell structure of Yeast?

-Thallus: fungal body or vegetative structure
-Chitin: N-containing polysaccharide (cell wall of some fungi)

32

What are the four major division of Yeast?

Ascomycota- also contains some molds

33
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fill in the blanks

1. Plasma membrane
2. Chitin microfibrils
3. Cytoplasmic microtubule
4. Cluster of ribosomes
5. Smooth endoplasmic reticulum
6. Mitochondria
7. Rough ER
8. Cell wall
9. Meshwork of microfilaments
10. Plasma membrane invagainations

34
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What is the difference between these hyphae cross walls?

Vegetative (reproductive) hyphae- structures that elongate into branching multicellular filaments
-Hyphae may be septate (contain dividing cross walls) or coenocytic (cross walls absent)

35
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Label mold hyphae structure.

1. Cell wall
2. Pore
3. Nuclei
4. Septum
a) Septum hyphae
b) Coenocytic hyphae

36

What is aerial mycelium and how is it observed?

Hyphae intertwine to form a aerial mycelium on substrate surface- puffy colonies
-Vegetative mycelium seen on underside of plates

37
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Label the diagram

1. Spore: non-motile reproductive units on end of hyphae- useful for ID (development & morphology)
c) Growth of a hypha from a spore

38
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What specialized hyphae & spores is this (label structures)?

Conidiophores bear long chains of asexual conidiophores.
-conidiophores break away & can germinate to form new vegetative hyphae
1. Conidiophores
2. Conidiophore

39
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What specialized hyphae & spores is this (label structures)?

Sporangiophores bear sac (sporangium) that contains sporangiospores.
1. Sporangiospores
2. Sporangium
3. Sporangiophores

40
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What specialized hyphae & spores is this (label structures)?

Vegetative mother cells bear single blastospores that bud off.
1. Blastospores
2. Vegetative mother cell

41
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What specialized hyphae & spores is this (label structures)?1

Arthrospores-hyphae fragment apart & the component function as spores.
a. Arthroconidia (arthrospores)
b. Fragment hypha

42
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What specialized hyphae & spores is this (label structures)?

Transverse tissue forming new cell wall

43
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What specialized hyphae & spores is this (label structures)?

a. Terminal chlamydospores
b. Chlamydospore within a hypha

44
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Identify this cell shape:

Yeast structure; spherical, ellipsoidal, or oval
-Most much larger than bacteria: 3-4 um
-Except for dimorphic yeast (Candida form pseudohyphae) hyphae are not formed

45
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identify these colonies:

Yeast colonies- appear like bacteria, often dull and opaque

46
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Identify and label the structure

Yeast Cell:
1.Nucleus
2. Cytoplasm
3. Polar bud scar
4. Mitochondrion
5. Plasma membrane
6. Golgi apparatus
7. Nuclear envelope
8. Plasma membrane
9. Chromosome
10. Polar bud Scar
11. Cell wall

47

What term is used to describe some fungi alternate between a mold & yeast form: Hyphal cells --> yeast cells?

Dimorphism

48
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label the structure:

a. Spores
b. Hyphae
c. spores
d. Bud
e. Yeast

1. Increased Temperature, Reduced O2, Suboptimal nutrients
1a. When fungal spores from the environment gain entrance to a warm-blooded animal , they germinate into yeast and remain in this phase in the host.
Animal Habitat: Parasitic, Yeast phase
Temp: 35-45 C)
Reproduction through: endospores

2.Decreased temperature, Optimal O2 content, Improved nutrients
2a. Yeast cells leaving the animal host return to the environment and revert to the sporulating hyphal stat. These conversions can be demonstrated on artificial media in the lab.
Natural Habitat:Saprobic (free-living), Mycelial phase
Tem: (<30 C)
Reproduction through: sporulation

49

In animal-infecting fungi:

alternate between a saprophytic mold phase in environment & parasitic yeast phase in host:
-Striking adaptation to permit growth & survival in mammalian host
-Thermal dimorphism-grow as mold at <30C as yeast at 35-40 C
*Candida albicans (thrush), Coccidioides (valley fever)

50

In plan-infecting fungi:

mold form in plant host, yeast form in environment

51
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Identify and label the structure

The Morphology of Coccidioides
a. Free Arthrospores
b. Septate Mycelium
c. Arthrospore formation
d. Disarticulation
e. Immature Spherules
f. Endosporulation spherule (mature)
g. Free endospores
h.Rupture spherule

52
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dentify and label the structures

A. Coccidioides immitis- filamentous colonie isolated from patient's sputum
B. immature and mature spherules in liver tissue from mice inoculated with the isolated filamentous fungus.
C. Histologic section with Gomori-Grocott silver impregnation

53
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Identify and label the structure

a. Gamete release 1N
b. Fertilization 1N+1N
c. Zygote 2N
d. Young sporophyte 2N
e. Zoosporangium (Mitosporangium) 2N
f. Resistant sporangium (Meiosporangium) 2N
g. Meiospores 1N
h. Sporophyte 2N
i. Male gametangium
j. Female gametangium
k. Male gametes
l. Gametophytes 1N
m. Female gametes

54

Reproduction of mold:

-Many have sexual & asexual phases
-Sexual phase: hyphal structures- Gametoangia intertwine or fuse, forming sexual spores
-Asexual phase: form spores or conidia (reproductive units)

55
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Reproduction of Yeast:

-Asexula reproduction by fission (splitting) budding
-Budding: daughter cell formed by parent cell-bud formed by small outpouching of cell
*Bud may separate from original cell or remain attached. Chain of buds may form if they remain attached. forming pseudohyphae (pseudomycelia). Each cell can form up 24 buds.
-SEXUAL reproduction in some yeast. Produce several types of spores (ascospores). Type of spore produced useful for yeast ID.

56

Commensal yeasts in Humans:

-Most yeast nonpathogenic, but opportunistic if host becomes immunosuppressed.
ex: Candida, Malassezia,

57

Candida:

Commensal Yeast of Humans:
normal flora or skin & mucous membranes (mouth)

58

Malassezia:

Dandruff & seborrhea (large flakes & itchy red area around scalp)- Caused by Pityrosporum (malassezia)

59

How is dandruff prevented?

Zinc pyrithione, ketoconazole, Selenium- sulfide in dandruff shampoos inhibit yeast growth & some help inhibit cell turn over.

60

How can Fungi be cultivated?

on plates; molds & yeast colonies cultivated in manner similar to bacteria, but often slower to grow.

61

Why is Sabouraud's dextrose agar (SAB) 4% sugar and pH=5.6 used to grow fungi?

Because fungi and bacteria compete, selective media favoring fungi is used inhibits many bacterial due to osmophilic & acidophilic environment

62

Why are Henrici slides used for to analyze fungi?

mold can be studied during growth, without disturbing vegetative & reproductive structures.

63

Fungi are members of the domain
A) Archaea
B) Bacteria
C) Eukarya
D) Classificationisundetermined

C) Eukarya

64

Fungi reproduce
A) A sexually only
B) Sexuallyonly
C) Bothsexuallyandasexually
D) Type of reproduction is undetermined

C) Bothsexuallyandasexually

65

Which of the following nutritional types best describes the fungi?
A) Heterotrophic
B) Phototrophic
C) Saprophytic
D) Noneoftheabove

C) Saprophytic

66

Fungi are important ecologically because of their ability to
A) Fixatmosphericnitrogenandmakeitavailabletootherorganisms
B) Decompose organic matter to release carbon and nutrients to other organisms
C) Cyclesulfurandiron
D) A ll of the above

B) Decompose organic matter to release carbon and nutrients to other organisms

67

Symbiotic relationships between vascular plant roots and fungi are called
A) Hyphae
B) Mycelium
C) Zooxanthelle
D) Mycorrhizae

D) Mycorrhizae

68

Fungi are producers of which of the following industrial products?
A) Breadsandcheeses
B) Organicacids
C) A ntibiotics and immunosuppressive drugs
D) A ll of the above

D) A ll of the above

69

The simplest group of fungi are the
A) Chytridiomycota
B) Zygomycota
C) Glomeromycota
D) Ascomycota

A) Chytridiomycota

70

When food becomes scarce or environmental conditions become unfavorable, Rhizopus bread mold
A) Reproducessexually
B) Reproducesasexually
C) Doesnotreproduce
D) Noneoftheabove

A) Reproducessexually

71

Which of the following groups of fungi degrade many chemically stable organic compounds including lignin, cellulose, and collagen?
A) Chytridiomycota
B) Zygomycota
C) Glomeromycota
D) Ascomycota

D) Ascomycota

72

A unicellular fungi that reproduces by budding or binary fission is known as a:
A) Mold
B) Ascus
C) Yeast
D) Hyphae

C) Yeast

73

When nutrients are limited, diploid yeast cells undergo
A) Senescence
B) Sporulation
C) Mitosis
D) Meiosis

D) Meiosis

74

Filamentous ascomycetes reproduce by producing
A) Conidia
B) Sclerotia
C) Basidia
D) Asci

A) Conidia

75

Aspergillus mold, the trigger of allergies, asthma, and sinusitis, belongs to the fungal group
A) Chytridiomycota
B) Zygomycota
C) Glomeromycota
D) Ascomycota

D) Ascomycota

76

Ergotism, the toxic condition in humans and animals that eat grain infected with fungus, is caused by an organism from the group
A) Chytridiomycota
B) Zygomycota
C) Glomeromycota
D) Ascomycota

D) Ascomycota

77

Which fungal group includes the stinkhorns, puffballs, toadstools, and mushrooms?
A) Basidiomycota
B) Zygomycota
C) Glomeromycota
D) Ascomycota

A) Basidiomycota

78

Mushroom toxins damage the host by
A) A ttacking liver cells
B) Damagingcellsliningthestomachandintestines
C) Inhibiting RNA polymerase
D) A ll of the above

D) A ll of the above

79

Important plant pathogens causing "rusts" and "smuts" in crop plants are members of which fungal group?
A) Chytridiomycota
B) Basidiomycota
C) Glomeromycota
D) Ascomycota

B) Basidiomycota

80

Fungi can be unicellular or multicellular.
A) True
B) False

A) True

81

Fungi are saphrophytes and use hydrolytic enzymes to degrade organic material.
A) True
B) False

A) True

82

Flagella are absent in all fungi.
A) True
B) False

B) False

83

A fungus in the genus Rhizopus is the causative agent of seedling blight, a rice disease.
A) True
B) False

A) True

84

Mushrooms used in food production belong to the Basidiomycota.
A) True
B) False

A) True

85

Infection by some members of the Basidiomycota triggers tumor formation in crop plants.
A) True
B) False

A) True