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1

Antiseptics

Reduce the concentration of microbes on the skin

2

Steps to safely wash hands

1. Wet your hands with clean running water and apply soap-use warm water if it is available
2.Rub hands together to make a lather and scrub all surfaces
3.Continuing rubbing hands for 20 seconds
4.Rinse well under running water

3

When to wash your hands

Entering the lab
Leaving the lab
Any time in between

4

Media can be either a broth or a

Agar

5

Media can be either undefined or

Defined

6

Undefined media

The exact ingredients and their amounts are not known
Commercially available and easy to prepare

7

Defined media

All of the ingredients and their amounts are known
Preparation is more complicated and time consuming

8

Agar originates from

Seaweed

9

Agar is a better solidifier than gelatin because

It is firmer/stronger and cannot be degraded by bacteria

10

What is the most important thing when preparing media?

Sterility

11

The most effective way to sterilize media is by

Autoclaving

12

What are the three parameters critical in obtaining sterility?

Temperature
Time
Pressure (from steam)

13

Why are all three parameters necessary?

To kill endospores

14

How many ways can steam be released from the chamber of an autoclave?

2

15

When is fast exhaust used?

Dry items like glassware and certain plastics

16

When is slow exhaust used?

when working with liquids

17

Autoclave safety requirements are

Insulated gloves
Face shield
Slow opening of the door
Cart available to place hot items on

18

How can you tell whether something has been autoclave and is sterile?

Autoclave tape will turn striped

19

What kind of media is Tryptic Soy

Undefined

20

What parameters are used for autoclaving (specific)

20 minutes, 121C, 15 psi

21

What do aseptic techniques do?

Prevent media from becoming contaminated

22

What are some tools used in aseptic transfers?

Inoculating loops and needles
Sterile transfer pipette
Glass spreading stick

23

Describe the proper flame for a Bunsen burner

Inner and outer cone

24

What is the goal of isolation

To obtain a pure bacterial culture from a mixed culture

25

What happens when the last quadrant streak overlaps with the 1st quadrant streak?

Very few if any well isolated colonies

26

What happens when the loop is not flamed between quadrants?

No isolated colonies

27

What is ubiquity

Existing or being everywhere

28

What types of microorganisms are out there

Pathogenic
Opportunistic Pathogens
Free-Living Microorganisms

29

Why would you streak an environmental sample differently than a lab sample

Environmental sample will more likely have fewer organisms

30

Environmental Streak

One large zig zag across the entire plate

31

What are the forms a colony can take?

Circular
Irregular
Filamentous
Rhizoids

32

What ways are the elevation of a colony described

Raised
Convex
Flat
Umbonate
Crateriform

33

How are margins defined for colonies?

Entire
Undulate
Filiform
Curled
Locate

34

How can colonies be described?

Form
Elevation
Margin
Pigmentation
Texture
Growth Patterns

35

When making a dilution what are important points to remember

Always add the sample to the saline broth
Always mix each dilution tube after adding the sample
Always change piper tips after each sample transfer

36

Where should you place the bacterial dilution in the agar plate?

The center

37

Colony forming units

Can be calculated when you know the number of colonies at a specific dilution. CFU/mL =#colonies/(dilution factor *volume plated)

38

Biofilm

A complex association that arised from a mixture of microorganisms growing together on the surface of a habitat

39

Stages of biofilm formation

1. Surface conditioning
2. Initial attachment
3. Slime formation
4. Secondary colonization
5. Maturation
6. Dispersal

40

Are bacteria within a biofilm more or less resistant to disinfectants/detergents as well as antibiotics

More

41

What is the media enriched with to help with biofilm formation

Glucose to help with surface conditioning

42

What dye was used to observe the biofilm

Crystal violet

43

What is soil composed of?

40% inorganic material
5% organic material
50% Air and water
5% microorganisms and macroorganism

44

Decomposed plant material lays

Zero to 2 inches into the soil

45

Surface soil

Soil that is 2 to 10 inches and is high in organic matter and microbes

46

Subsoil

10 to 30 inches little organic matter and fewer microbes

47

Soil base

30 to 48 inches with even fewer microbes

48

What roles can microbes play

Carbon cycle - detritivores
Nitrogen cycle-denitrifying bacteria, nitrogen fixing root bacteria
Sulfur cycle
Food web

49

Prokaryotes in Soil

Bacteria

50

Eukaryotes in Soil

Filamentous fungi
Protozoan
Nematode

51

What types of bacteria are in soil?

Chemoautotrophs
Decomposers
Mutualists
Disease suppressors
Pathogens

52

What do chemoautotrophic bacteria do

Oxidize inorganic compounds to yield energy and reduce CO2
Ex.
Nitrosamines europaea
Nitro so after winogradskyi
Thiobacillus
Desulfovibrio

53

What do bacterial decomposed do?

Break down or gain biomass
Ex
Pseudomonas fluorescens
Bacillus subtitles
Actinobacteria

54

Many members of the actinobacteria group produce?

Antibiotics

55

What are bacterial mutualists?

Bacteria that form partnerships with plants
Ex
Rhizobium

56

What re disease suppressors

Bacterial species that can suppress the occurrence of plant disease by secreting anti-fungal or anti-insecticidal compounds
Ex
Bacillus
Rhizoctonia
Bacillus thuringiensis

57

What are bacterial pathogens

Bacterial species that can cause disease in plants and/or animals
Ex
Erwinia
Clostridium tetani

58

What types of eukaryotes are in soil

Decomposers
Mutualists
Pathogens

59

What are fungal decomposers

Species that break down organic biomass
Ex
Aspergillus
Penicillium

60

What are fungal mutualists

Bacteria that form partnerships with plants
Ex
Mycorrhizal fungi

61

What are fungal pathogens

Fungal species that can cause disease in plants and/or animals
Ex
Verticillium

62

Nutrient agar promotes the grow of ______ but not _____

Many types of bacteria; fungi

63

Glycerol yeast extract supports the growth of _____ but not enough to support_______ or _______

Actinobacteria; simple bacteria; fungi

64

Sabouraud Dextrose Agar with antibiotics will allow _____to grow but not ______

Fungi, bacteria

65

What kind of viruses cannot be worked on in this lab?

Animal
Working with animal viruses requires a high level of sterility
Work must be done in biosafety cabinets that have a filtered air supply

66

What is a bacteriophage?

Viruses that only infect bacteria

67

Bacteriophage require a host _______ in which to replicate because they do not have _______,_________ etc, and use the cellular_______ of its host. They contain ______ and are ________ in structure.

Bacteria; enzymes; ribosomes; machinery; DNA; complex

68

Animal viruses require a host _____cell and use the cellular________of its host. They contain _____or_____but never both and are _______in structure.

Eukaryotic; machinery;DNA;RNA;simple

69

A bacteria phage consists of

Caps is or head with dna inside
Sheath
Tail fibers

70

Animal viruses contain

Capsid with DNA

71

Phases of bacteriophage infection

Attachment
Penetration
Synthesis
Assembly
Release

72

Why is soft agar used for plaque assays?

Bacteria cannot move through it but bacteriophage can move short distances

73

What is a plaque

The area of clearing on the bacterial lawn.

74

How do you calculate PFU/mL

#of plaque/(dilution factor * ml of Phage added to e. Coil)

75

Can different phage produce different looking plaques?

Yes

76

How many sets of lenses does a compound microscope have?

2

77

How many hands should be used when carrying a microscope?

2

78

Before you turn on the microscope check the ____ ______.

Lamp control

79

What 4 objectives are there?

4x
10x
40x
100x

80

What is the magnification of the ocular lens?

10x

81

What is a condensor?

The piece that narrows the beam of light that strikes the specimen

82

What must be used when viewing specimen under the 100x objective?

Immersion oil

83

Why must immersion oil be used with the 100x objective lens?

It prevents the light from being refracted.

84

List types of microorganisms

Viruses. Helminths
Bacteria
Archaea
Fungi
Algae
Protozoa

85

Viruses can only be seen using a ______microscope

Electron

86

Most bacteria and archaea can be seen by a light microscope using ______ ________ and the aid of ________.

Oil immersion; staining

87

Bacteria that do not require staining

Cyanobacteria because of photosynthetic pigments
Ex
Gloeocapsa
Anabaena
Oscillatoria

88

Aquatic fungi are from the class

Chytridiomycetes

89

Types of algae in freshwater

Chlorella
Ulothrix
Spirogyra
Diatoms or phytoplankton
Navicula
Synedra
Cocconeis

90

Protozoa include

Euglenids - green flagellated photosynthetic Protozoa
Dinoflagellates - flagellated Protozoa
Ciliates - use cilia for locomotion
Sarcodina- use pseudopods for locomotion

91

Helminths include

Nematodes-unsegmented with a full digestive tract

92

When using a slide cover which objective should you not use?

100X

93

Fungi can be ______ or _______

Macroscopic; microscopic

94

Microscopic fungi have two basic morphological types which are

Yeasts and Molds

95

General characteristics of yeasts

Grow in colonies that are very similar in appearance to bacterial colonies

Yeast cells are round or oval in shape

Reproduce by budding

96

Example of yeast

Candida albicans - oral thrush - opportunistic pathogen

97

General characteristics of molds

Grow as hyphae (long thread-like cells)

Hyphae can be septate(divided by cell walls) or nonseptate(a continuous cell) and vegetative or reproductive.

Primary mode of reproduction is by asexual spore formation

Asexual spore types Nicole sporangiospores (enclosed) and condidospores (free)

Sexual spore formation is some molds involves production of zygospores

98

Examples of mold

Rhizopus- includes common bread mold-sporangium, nonseptate, form zygospore

Aspergillus - opportunistic pathogen, septate, conidiophores

Penicillium - produces penicillin, septate, conidiophores

99

Why do stains need to be used to view most bacteria?

Cytoplasm is transparent

100

What is a stain?

A solution in which a dye or chromogen has been added to a liquid

101

A chromogen consists of __ components. The _____ and the _____.

2; chromosphore; auxochrome

102

The _____ is carried by the auxochrome and determines whether a or not a stain will adhere to a bacterium

Charge

Can be positive or negative depending on the charge carried by the auxochrome

103

Bacteria have a ______charge on their cell surface

Negative

104

A Positive stain has a _______charge so the _______ binds to the bacterial cell surface.

Positive; auxochrome

The bacteria is stained & background is white

105

With a negative stain, the auxochrome has a ________ charge which ______ and the _________ cannot bind to the bacterial cell surface.

Negative; repel; auxochrome

Bacteria left colorless and background is stained.

106

Examples of Positive Stains

Crystal violet
Methylene blue
Safranin

107

Examples of negative stains

India Ink
Nigrosin

108

Positive stains require ____ fixing; negative stains do not.

Heat

109

What are the pros to simple and negative stains?

Only require 1 dye (uncomplicated)

110

What are the cons of simple and negative stains

They cannot differentiate unique cell structures or features between bacteria.

111

All negative stained slides must be _______ in the ______ _______.

Discarded; bleach bucket

112

What are the three general shapes of bacteria?

Bacillus (rods)
Coccus (spheres)
Spirillus (spiral)

113

What are some types of arrangement?

Singles or pairs
Chains
Clusters of cocci
Palisades of bacilli

114

What is pleomorphism

Slight deviations in the overall shape and/or size of a bacteria

115

Who invented gram staining?

Christian Gram

116

What makes the gram stain so important?

It can differentiate between bacteria

117

Gram ______ cells have a thick layer of peptidoglycan; Gram ______ cells have a thin layer of peptidoglycan and an outer membrane.

Positive; Negative

118

Gram staining requires ________ fixation

Heat

119

What is the primary stain in gram staining?

Crystal violet

120

The mordant used is _______ and produces _______-_________ complexes

Iodine;crystal violet;iodine

121

What decolorizer is used?

Ethanol

122

How does the decolorizer work?

Dissolves the lipids in the outer membrane

123

What counterstain is used?

Safranin

124

What bacteria does gram staining not work on?

Nocardia and Mycobacterium

125

What color is a gram positive bacteria?

Purple

126

What color is an acid fast stain positive?

Pink

127

What do acid fast positive bacteria have in their cell walls?

Mycolic acid (waxy lipid)

128

Example of a Mycobacterium

Mycobacterium tuberculosis
Highly communicable
1/3 of the world is infected with it
Leading killer of hiv infected individuals
4.2/100000 people in the US

129

Can acid-fast non-pathogenic mycobacterium and nocardia species exist in the environment?

Yes

130

what is the primary stain in acid fast staining?

Carbolfuchsin
Phenol based that is lipid soluble

131

Carbolfuchsin stain can be enhanced by what two methods?

Applying heat to drive the steam in (steam)

Increase the concentration of phenol

132

If heat is used in the staining procedure, which method is it?

Ziehl-neelsen

133

If a higher concentration of phenol is used in the carbolfuchsin stain which method is it?

Kinyoun Method

134

What is a capsule?

A complex layer of sugars and proteins that tightly surrounds a bacterium

135

Capsule Staining is a _______stain.

Differential

136

What is the role of the capsule?

Protection against environmental conditions and dehydration
Adherence to surfaces
Prevents phagocytosis

137

In a pathogenic bacterial species a capsule is considered a ______ ______.

Virulence factor

138

Can non pathogenic bacteria possess capsules?

Yes

139

Examples of capsule bacteria

Neisseria menigitidis
Many bacillus species including those found in the soil bacillus megaterium, bacillus licheniformis

140

Can a capsule be stained?

No

141

Why wont a capsule stain?

It is water soluble

142

The capsule stain is a differential stain that combines ______ and _____ stains.

Negative; simple

143

Congo red is an ______ dye (ie. it carries a ________charge)

Acidic; negative

144

In a negative stain only the ______ will be stained

Background

145

A capsule stain slide is never _____ _______.

Heat fixed.

146

What is the simple stain used in capsule staining?

Maneval’s stain

147

Maneval’s stain is a ______ dye with a _____ charge.

Basic; positive

148

Capsule stain slides must be discarded in the ______ _______

Bleach; bucket

149

To overcome this constant flux of nutrients in the environment, some bacteria have the capacity to produce _____ ______ or ________.

Inclusion bodies; granules

150

Poly B-hyroxybutyrate (PHB) is an _____ _____ _____

Inclusion body stain

151

If a bacterium is in a nutrient rich environment, it is likely to contain ______ or ______ inclusions

More; larger

152

Why produce inclusion bodies?

Nutrient reservoir

Avoid lyse from an increase in osmotic pressure

153

Do all bacteria produce inclusion bodies?

No

154

Carboxysomes are an ________ that continue the enzyme _______.

Inclusion; ribulose 1,5-diphosphate carboxylase

155

Types of inclusions:

Suffer Granules (Thiobacillus)
Carboxysomes
Polysaccharide Granules - contain glycogen/starch
Metachromatic granules - volutin (inorganic phosphates) for ATP
Corynebacterium diphtheria is a metachromatic granules that causes diphtheria
Magnetosomes contain iron oxide which can act like tiny magnets

Lipid inclusions contain PHB poly B-hyroxybutyrate used as both carbon and energy sources

156

PHB consists of

B-hyroxybutyrate molecules linked by an ester bond between carboxyl group of one an the hydroxyl group of the next

Hydrolysis of the ester bound results in carbon containing molecules and sufficient free energy to be harnessed for atp

157

PHB Inclusions range in diameter from ____ to ____

0.2; 0.7

158

PHB inclusions are contained within a unique membrane that is composed of a _______ lipid layer

Single

159

Can all bacteria contain PHB inclusion?

No

160

What is the primary stain of PHB?

Sudan Black B

161

Sudan Black B is a ______ soluble stain that is ______

Lipid; neutral

162

The PHB counterstain is

Safranin

163

Certain bacteria can survive in extremely unfavorable conditions by forming _______

Endospores

164

Endospores is able to withstand extreme ______, ________, ________, and _______ ________.

Temperature; disinfectants; desiccation; and UV radiation

165

Major characteristics of endospores

Contain dna but are inactive metabolically (dormant)

Consist of a very tough outer cell coat, a cortex, and an inner core (3 C’s)

166

What are the 3 C’s

Coat, cortex, Core

167

Only a few species can form endospores; two of which are _______ and _______

Bacillus; Clostridium

Mostly non pathogenic however bacillus antracis causes anthrax in humans and clostridium tetani cause tetanus and clostridium botulinum cause botulism.

168

The _____, ______, and ________ of the endospores within a bacterium can help in the identification of the organism.

Size; shape; location

169

What is the primary dye in endospores staining?

Malachite green

170

Does endospores staining use steam to drive the stain into the bacteria?

Yes

171

What is the counter stain in endospore staining?

Safranin

172

Flagella may contribute to the _______ of a bacterium since it can allow the bacterium to move to its preferred anatomical site to start the disease process.

Virulence

173

What are two ways to determine whether a bacterium is motile?

Hanging drop slide

Semi-solid motility agar

174

The different arrangements of flagella

Monotrichous = 1 flagella at 1 pole
Lophotrichous = several flagella at 1 pole
Amphitrichous = 1 flagella at each pole
Peritrichous = flagella all over

175

What is used to thicken the flagella so that it can be seen?

Mordant

176

Flagella are thin and ________

Delicate

177

The mordant is _____ meaning it will stick to the flagella as well as any dust or dirt on the slide

Non-specific

178

What is motility media

A semi solid media which has just enough agar added to produce a media with the consistency of sloppy jello (4x less agar)

179

Motility media is thick enough to allow motile bacteria to move but too thick for __________ bacteria to disperse

Non-motile

180

What is added to the motility media to make a motility test easy to read?

Tetrazolium salt

181

TTC reduces to formazan and ______

HCL

182

Formazan is _____ and ______

Red; insoluble

183

TTC is reduced by _______ ______ bacteria

Metabolically active